Individual level microbial communities in the digestive system of the freshwater isopod Asellus aquaticus: Complex, robust and prospective.

The freshwater isopod Asellus aquaticus is an important decomposer of leaf detritus, and its diverse gut microbiome has been depicted as key contributors in lignocellulose degradation as of terrestrial isopods. However, it is not clear whether the individual-level microbiome profiles in the isopod digestive system across different habitats match the implied robust digestion function of the microbiome. Here, we described the bacterial diversity and abundance in the digestive system (hindgut and caeca) of multiple A. aquaticus individuals from two contrasting freshwater habitats. Individuals from a lake and a stream harboured distinct microbiomes, indicating a strong link between the host-associated microbiome and microbes inhabiting the environments. While faeces likely reflected the variations in environmental microbial communities included in the diet, the microbial communities also substantially differed in the hindgut and caeca. Microbes closely related to lignocellulose degradation are found consistently more enriched in the hindgut in each individual. Caeca often associated with taxa implicated in endosymbiotic/parasitic roles (Mycoplasmatales and Rickettsiales), highlighting a complex host-parasite-microbiome interaction. The results highlight the lability of the A. aquaticus microbiome supporting the different functions of the two digestive organs, which may confer particular advantages in freshwater environments characterized by seasonally fluctuating and spatially disparate resource availability.

Environmental DNA/RNA for pathogen and parasite detection, surveillance, and ecology.

Detection of pathogens, parasites, and other symbionts in environmental samples via eDNA/eRNA (collectively eNA) is an increasingly important source of information about their occurrence and activity. There is great potential for using such detections as a proxy for infection of host organisms in connected habitats, for pathogen monitoring and surveillance, and for early warning systems for disease. However, many factors require consideration, and appropriate methods developed and verified, in order that eNA detections can be reliably interpreted and adopted for surveillance and assessment of disease risk, and potentially inclusion in international standards, such as the World Organisation for Animal Health guidelines. Disease manifestation results from host-symbiont-environment interactions between hosts, demanding a multifactorial approach to interpretation of eNA signals.

Food purchase behaviour in a Finnish population: patterns, carbon footprints and expenditures.

OBJECTIVE: To identify food purchase patterns and to assess their carbon footprint and expenditure. DESIGN: Cross-sectional. SETTING: Purchase patterns were identified by factor analysis from the annual purchases of 3435 product groups. The associations between purchase patterns and the total purchases' carbon footprints (based on life-cycle assessment) and expenditure were analysed using linear regression and adjusted for nutritional energy content of the purchases. PARTICIPANTS: Loyalty card holders (n 22 860) of the largest food retailer in Finland. RESULTS: Eight patterns explained 55 % of the variation in food purchases. The Animal-based pattern made the greatest contribution to the annual carbon footprint, followed by the Easy-cooking, and Ready-to-eat patterns. High-energy, Traditional and Plant-based patterns made the smallest contribution to the carbon footprint of the purchases. Animal-based, Ready-to-eat, Plant-based and High-energy patterns made the greatest contribution, whereas the Traditional and Easy-cooking patterns made the smallest contribution to food expenditure. Carbon footprint per euros spent increased with stronger adherence to the Traditional, Animal-based and Easy-cooking patterns. CONCLUSIONS: The Animal-based, Ready-to-eat and High-energy patterns were associated with relatively high expenditure on food, suggesting no economic barrier to a potential shift towards a plant-based diet for consumers adherent to those patterns. Strong adherence to the Traditional pattern resulted in a low energy-adjusted carbon footprint but high carbon footprint per euro. This suggests a preference for cheap nutritional energy rather than environment-conscious purchase behaviour. Whether a shift towards a plant-based pattern would be affordable for those with more traditional and cheaper purchase patterns requires more research.

Diversity and Phylogeny of Novel Cord-Forming Fungi from Borneo.

Cord-forming (CF) fungi are found worldwide; however, tropical CF fungi are poorly documented. They play an essential role in forest ecosystems by interconnecting nutrient resources and aiding in the decomposition of plant matter and woody litter. CF fungi samples were collected from two forest conservation sites in the Sabah region of Malaysian Borneo. Sequencing and phylogenetic analysis of the ribosomal rRNA gene array 18S to 28S region from cords collected placed all of the collected specimens in Agaricomycetes (Basidiomycetes), specifically within the orders Trechisporales, Phallales, Hymenochaetales, Polyporales, and Agaricales. Comparison of the cord-derived sequences against GenBank and UNITE sequence databases, as well as phylogenetic analyses, revealed they were all novel sequences types. Many of these novel lineages were found to be closely related to other basidiomycetes commonly found in tropical forests, suggesting a large undiscovered tropical fungal diversity in Borneo that has been detected independently of sampling fruiting bodies. We show how these sequence types relate to the morphologies of the cords from which they were sampled. We also highlight how rapid, small-scale sampling can be a useful tool as an easy and relatively unbiased way of collecting data on cord-forming fungi in difficult-to-access, complex forest environments, independently of locating and sampling sporophores.

High-throughput sequencing of faeces provides evidence for dispersal of parasites and pathogens by migratory waterbirds.

Examination of faecal material has demonstrated how a broad range of organisms are distributed by bird movements. Such research has largely focused on dispersal of plant seeds by frugivores and of freshwater organisms by waterbirds. However, with few exceptions (e.g. avian influenza, Ebola virus), there is a dearth of evidence for transport of parasites and pathogens. High-throughput sequencing methods now provide a powerful means of addressing this knowledge gap by elucidating faecal contents in unprecedented detail. We collected faeces excreted by a range of migratory waterbirds in south-west Spain and pooled faecal DNA to create libraries reflective of feeding behavior. We created sets of libraries using high-throughput metagenomic and amplicon sequencing. For the latter we employed two sets of primers to broadly target the V4 region of the 18S rRNA gene (one set amplifying the region across all eukaryotes, the other excluding amplification of metazoans). Libraries revealed a wide diversity of eukaryotes, including parasites of the faecal producers themselves, parasites of food items, or those incidentally ingested. We also detected novel microbial eukaryotic taxa and found that parasite assemblage profiles were relatively distinct. Comparing the performance of the methods used supports their joint use for future studies of diversity and abundance. Because viable stages of many parasites are likely to be present in faeces, our results suggest significant levels of bird-mediated dispersal of parasites (both from avian and other hosts). Our methods revealed much hidden biodiversity, and allowed identification of the individuals who produced the faecal samples to species level, facilitating the study of interaction networks.

Gene Duplication and Gain in the Trematode Atriophallophorus winterbourni Contributes to Adaptation to Parasitism.

Gene duplications and novel genes have been shown to play a major role in helminth adaptation to a parasitic lifestyle because they provide the novelty necessary for adaptation to a changing environment, such as living in multiple hosts. Here we present the de novo sequenced and annotated genome of the parasitic trematode Atriophallophorus winterbourni and its comparative genomic analysis to other major parasitic trematodes. First, we reconstructed the species phylogeny, and dated the split of A. winterbourni from the Opisthorchiata suborder to approximately 237.4 Ma (±120.4 Myr). We then addressed the question of which expanded gene families and gained genes are potentially involved in adaptation to parasitism. To do this, we used hierarchical orthologous groups to reconstruct three ancestral genomes on the phylogeny leading to A. winterbourni and performed a GO (Gene Ontology) enrichment analysis of the gene composition of each ancestral genome, allowing us to characterize the subsequent genomic changes. Out of the 11,499 genes in the A. winterbourni genome, as much as 24% have arisen through duplication events since the speciation of A. winterbourni from the Opisthorchiata, and as much as 31.9% appear to be novel, that is, newly acquired. We found 13 gene families in A. winterbourni to have had more than ten genes arising through these recent duplications; all of which have functions potentially relating to host behavioral manipulation, host tissue penetration, and hiding from host immunity through antigen presentation. We identified several families with genes evolving under positive selection. Our results provide a valuable resource for future studies on the genomic basis of adaptation to parasitism and point to specific candidate genes putatively involved in antagonistic host-parasite adaptation.

Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease.

The myxozoan parasite, Tetracapsuloides bryosalmonae has a two-host life cycle alternating between freshwater bryozoans and salmonid fish. Infected fish can develop Proliferative Kidney Disease, characterised by a gross lymphoid-driven kidney pathology in wild and farmed salmonids. To facilitate an in-depth understanding of T. bryosalmonae-host interactions, we have used a two-host parasite transcriptome sequencing approach in generating two parasite transcriptome assemblies; the first derived from parasite spore sacs isolated from infected bryozoans and the second from infected fish kidney tissues. This approach was adopted to minimize host contamination in the absence of a complete T. bryosalmonae genome. Parasite contigs common to both infected hosts (the intersect transcriptome; 7362 contigs) were typically AT-rich (60-75% AT). 5432 contigs within the intersect were annotated. 1930 unannotated contigs encoded for unknown transcripts. We have focused on transcripts encoding proteins involved in; nutrient acquisition, host-parasite interactions, development, cell-to-cell communication and proteins of unknown function, establishing their potential importance in each host by RT-qPCR. Host-specific expression profiles were evident, particularly in transcripts encoding proteases and proteins involved in lipid metabolism, cell adhesion, and development. We confirm for the first time the presence of homeobox proteins and a frizzled homologue in myxozoan parasites. The novel insights into myxozoan biology that this study reveals will help to focus research in developing future disease control strategies.

Developing the collection of statistical food waste data on the primary production of fruit and vegetables.

In order to meet global goals of reducing food waste, feasible monitoring methods to verify the impact of reduction measures are needed. In this study, a method was developed for gathering food waste data related to the primary production of fruit and vegetables using a questionnaire for farmers. A data collection form was planned and tested for this purpose. Data was collected on the volumes of different uses of yields and the reasons why part of the yield does not end up in food use. The crop species that were included in this pilot study were food potatoes, carrots, white cabbage, and strawberries. In primary production, the share of food use of the total yield was highest for potatoes (96%) and lowest for carrots (72%). In the case of strawberries and white cabbage, 86% and 90% were used as food, respectively. In the future, it is recommended that this kind of survey be added to annual crop production surveys that cover agricultural and horticultural enterprises in Finland. To improve the response rate and decrease the reporting load of farmers, it is important that the questionnaire is designed to be as easy as possible to answer.

Broad but restricted detection of malacosporeans in a Neotropical cradle of diversification.

This study undertook the first investigation of malacosporean infections in Neotropical fish. We used polymerase chain reaction detection with a primer set generally targeting known malacosporeans to assay for infection in the kidney of 146 fish in 21 species belonging to 12 families collected from two areas in the Amazon Basin. Infections were found in 13 fish variously belonging to seven species in six families and included the first identification of a malacosporean infection in cartilaginous fish (a freshwater stingray). Based on ssrDNA, all infections represented a single Buddenbrockia species (Buddenbrockia sp. E) that demonstrates an exceptionally broad range of fish species infected, and countered our expectations of high Neotropical malacosporean diversity. Infections were characterized at varying and often high prevalences in fish species but sample sizes were small. Ascertaining whether highly divergent malacosporeans have not been detected by current primers, and more comprehensive sampling may reveal whether malacosporeans are truly as species poor in the Amazon Basin as present data suggest. Our results prompt speculations about evolutionary scenarios including introduction via marine incursions and patterns of host use over time.

Validation of an eDNA-based method for the detection of wildlife pathogens in water.

Monitoring the occurrence and density of parasites and pathogens can identify high infection-risk areas and facilitates disease control and eradication measures. Environmental DNA (eDNA) techniques are increasingly used for pathogen detection due to their relative ease of application. Since many factors affect the reliability and efficacy of eDNA-based detection, rigorous validation and assessment of method limitations is a crucial first step. We evaluated an eDNA detection method using in situ filtration of large-volume water samples, developed to detect and quantify aquatic wildlife parasites by quantitative PCR (qPCR). We assessed method reliability using Batrachochytrium dendrobatidis, a pathogenic fungus of amphibians and the myxozoan Tetracapsuloides bryosalmonae, causative agent of salmonid proliferative kidney disease, in a controlled experimental setup. Different amounts of parasite spores were added to tanks containing either clean tap water or water from a semi-natural mesocosm community. Overall detection rates were higher than 80%, but detection was not consistent among replicate samples. Within-tank variation in detection emphasises the need for increased site-level replication when dealing with parasites and pathogens. Estimated parasite DNA concentrations in water samples were highly variable, and a significant increase with higher spore concentrations was observed only for B. dendrobatidis. Despite evidence for PCR inhibition in DNA extractions from mesocosm water samples, the type of water did not affect detection rates significantly. Direct spiking controls revealed that the filtration step reduced detection sensitivity. Our study identifies sensitive quantification and sufficient replication as major remaining challenges for the eDNA-based methods for detection of parasites in water.

PCR-RFLP: a targeted method to reveal host specific malacosporean infection profiles (Cnidaria: Myxozoa: Malacosporea).

Malacosporeans are a group of endoparasitic cnidarians (Myxozoa) that use freshwater bryozoans and fish as final and intermediate hosts, respectively. The malacosporean Tetracapsuloides bryosalmonae causes proliferative kidney disease (PKD), an emerging disease in aquaculture and wild fish populations, including threatened salmonids in Europe and the USA. Mixed infections of malacosporeans are often encountered, and a monitoring tool for screening of multiple malacosporean species in either their fish or bryozoan hosts is therefore desirable. We describe an inexpensive method that combines PCR amplification of the partial 18S rRNA gene (~260 bp) and a single-step restriction fragment length polymorphism (RFLP) method for identification of 10 malacosporean lineages and species. We demonstrate and test this methodology on a set of DNA extracted from malacosporeans infecting fish kidney and tissues sampled from bryozoan colonies and compare the results with Sanger sequencing of the same parasite DNA isolates. The PCR-RFLP and Sanger sequencing methods agreed in 100% of cases. The PCR-RFLP method offers a number of opportunities, including screening large panels of host tissue samples to gain insights into infection patterns, characterizing mixed infections, and confirming highly pathogenic T. bryosalmonae infections. The method can also be further refined as new sequence data become available for malacosporeans.

Effects of parasite concentrations on infection dynamics and proliferative kidney disease pathogenesis in brown trout (Salmo trutta).

Proliferative kidney disease (PKD) is an emerging disease of salmonids, which is exacerbating with increasing water temperature. Its causative agent, the myxozoan parasite Tetracapsuloides bryosalmonae, exploits freshwater bryozoans as primary hosts and salmonids as intermediate hosts. Our experiments showed that the manipulation of exposure concentrations of infective malacospores had relatively minor impacts for the disease outcomes in the fish host. In this study, brown trout (Salmo trutta) were exposed to three different exposure concentrations of T. bryosalmonae malacospores: (a) a single low parasite concentration (LC), (b) a single high parasite concentration (HC) and (c) three times a low concentration (repeat exposure, RE). Parasite dynamics in the fish host and release of fish malacospores were quantified and fish kidney histopathology was evaluated to determine PKD pathogenesis. Infection prevalence was always lower in the LC group than in the other groups over the course of the study. While the parasite proliferation phase was slower in the LC group, the maximum parasite burden did not differ significantly amongst treatments. The onset of fish malacospore release (day 45 post-exposure), indicated by detection of T. bryosalmonae DNA in the tank water, occurred at the same time point for all groups. Reduced intensity of kidney pathological development was observed in the LC treatment indicating lower disease severity. While the LC treatment resulted in reduced outcomes across several infection parameters (infection prevalence, parasite proliferation, total fish malacospores released), the overall differences were small. The RE and HC treatment outcomes were for most parameters comparable. Our results suggest that repeated exposure, as is likely to occur in the wild during the summer months, might play a more important role in the dynamics of PKD as an emerging infectious disease than the actual concentration of spores.

Estimating species distribution and abundance in river networks using environmental DNA.

All organisms leave traces of DNA in their environment. This environmental DNA (eDNA) is often used to track occurrence patterns of target species. Applications are especially promising in rivers, where eDNA can integrate information about populations upstream. The dispersion of eDNA in rivers is modulated by complex processes of transport and decay through the dendritic river network, and we currently lack a method to extract quantitative information about the location and density of populations contributing to the eDNA signal. Here, we present a general framework to reconstruct the upstream distribution and abundance of a target species across a river network, based on observed eDNA concentrations and hydro-geomorphological features of the network. The model captures well the catchment-wide spatial biomass distribution of two target species: a sessile invertebrate (the bryozoan Fredericella sultana) and its parasite (the myxozoan Tetracapsuloides bryosalmonae). Our method is designed to easily integrate general biological and hydrological data and to enable spatially explicit estimates of the distribution of sessile and mobile species in fluvial ecosystems based on eDNA sampling.

Environmental impacts of food waste in Europe.

Approximately 88 Million tonnes (Mt) of food is wasted in the European Union each year and the environmental impacts of these losses throughout the food supply chain are widely recognised. This study illustrates the impacts of food waste in relation to the total food utilised, including the impact from food waste management based on available data at the European level. The impacts are calculated for the Global Warming Potential, the Acidification Potential and the Eutrophication Potential using a bottom-up approach using more than 134 existing LCA studies on nine representative products (apple, tomato, potato, bread, milk, beef, pork, chicken, white fish). Results show that 186 Mt CO2-eq, 1.7 Mt SO2-eq. and 0.7 Mt PO4-eq can be attributed to food waste in Europe. This is 15 to 16% of the total impact of the entire food supply chain. In general, the study confirmed that most of the environmental impacts are derived from the primary production step of the chain. That is why animal-containing food shows most of the food waste related impacts when it is extrapolated to total food waste even if cereals are higher in mass. Nearly three quarters of all food waste-related impacts for Global Warming originate from greenhouse gas emissions during the production step. Emissions by food processing activities contribute 6%, retail and distribution 7%, food consumption, 8% and food disposal, 6% to food waste related impacts. Even though the results are subject to certain data and scenario uncertainties, the study serves as a baseline assessment, based on current food waste data, and can be expanded as more knowledge on the type and amount of food waste becomes available. Nevertheless, the importance of food waste prevention is underlined by the results of this study, as most of the impacts originate from the production step. Through food waste prevention, those impacts can be avoided as less food needs to be produced.

Rhizarian 'Novel Clade 10' Revealed as Abundant and Diverse Planktonic and Terrestrial Flagellates, including Aquavolon n. gen.

Rhizarian 'Novel Clade 10' (NC10) is frequently detected by 18S rRNA gene sequencing studies in freshwater planktonic samples. We describe a new genus and two species of eukaryovorous biflagellate protists, Aquavolon hoantrani n. gen. n. sp. and A. dientrani n. gen. n. sp., which represent the first morphologically characterized members of NC10, here named Aquavolonida ord. nov. The slightly metabolic cells possess naked heterodynamic flagella, whose kinetosomes lie at a right angle to each other and are connected by at least one fibril. Unlike their closest known relative Tremula longifila, they rotate around their longitudinal axis when swimming and only very rarely glide on surfaces. Screening of a wide range of environmental DNA extractions with lineage-specific PCR primers reveals that Aquavolonida consists of a large radiation of protists, which are most diversified in freshwater planktonic habitats and as yet undetected in marine environments. Earlier-branching lineages in Aquavolonida include less frequently detected organisms from soils and freshwater sediments. The 18S rRNA gene phylogeny suggests that Aquavolonida forms a common evolutionary lineage with tremulids and uncharacterized 'Novel Clade 12', which likely represents one of the deepest lineages in the Rhizaria, separate from Cercozoa (Filosa), Endomyxa, and Retaria.

How effective are messages and their characteristics in changing behavioural intentions to substitute plant-based foods for red meat? The mediating role of prior beliefs.

By means of a population-based survey experiment, we analysed the effectiveness of two message characteristics - message framing and the refutation of misinformation - in persuading respondents to reduce their consumption of red meat and increase that of plant-based alternatives. We also tested whether the effects of those two message characteristics were moderated by prior beliefs about the health and climate impacts of red meat consumption. The data were collected with an online survey of the adult population living in Finland (N = 1279). We found that messages had a small but desired effect on intentions when the effect of prior beliefs was taken into account, but that that effect was strongly moderated by prior beliefs. In particular, messages changed behavioural intentions among the "meat-sceptics" (i.e., those believing relatively strongly in the negative health and climate effects of meat consumption) but not among the "meat believers" (defined symmetrically). The combination of frames and refutation of misinformation were not found to be more effective strategies than the provision of information through single-framed, one-sided messages. We found limited evidence that the way a message was formulated determined its effectiveness in changing behaviours.

Temperature-related parasite infection dynamics: the case of proliferative kidney disease of brown trout.

Climate change, in particular rising temperature, is suspected to be a major driver for the emergence of many wildlife diseases. Proliferative kidney disease of salmonids, caused by the myxozoan Tetracapsuloides bryosalmonae, was used to evaluate how temperature dependence of host-parasite interactions modulates disease emergence. Brown trout (Salmo trutta fario) kept at 12 and 15 °C, were experimentally infected with T. bryosalmonae. Parasite development in the fish host and release of spores were quantified simultaneously to unravel parasite transmission potential from the vertebrate to the invertebrate host. A change to a stable plateau in infection intensity of the kidney coincided with a threshold at which spore shedding commenced. This onset of parasite release was delayed at the low temperature in accordance with reaching this infection intensity threshold, but the amount of spores released was irrespective of temperature. The production of parasite transmission stages declined with time. In conclusion, elevated temperature modifies the parasite transmission opportunities by increasing the duration of transmission stage production, which may affect the spread and establishment of the parasite in a wider range of rivers.

Food waste quantification in primary production - The Nordic countries as a case study.

Our understanding of food waste in the food supply chain has increased, but very few studies have been published on food waste in primary production. The overall aims of this study were to quantify the total amount of food waste in primary production in Finland, Sweden, Norway and Denmark, and to create a framework for how to define and quantify food waste in primary production. The quantification of food waste was based on case studies conducted in the present study and estimates published in scientific literature. The chosen scope of the study was to quantify the amount of edible food (excluding inedible parts like peels and bones) produced for human consumption that did not end up as food. As a result, the quantification was different from the existing guidelines. One of the main differences is that food that ends up as animal feed is included in the present study, whereas this is not the case for the recently launched food waste definition of the FUSIONS project. To distinguish the 'food waste' definition of the present study from the existing definitions and to avoid confusion with established usage of the term, a new term 'side flow' (SF) was introduced as a synonym for food waste in primary production. A rough estimate of the total amount of food waste in primary production in Finland, Sweden, Norway and Denmark was made using SF and 'FUSIONS Food Waste' (FFW) definitions. The SFs in primary production in the four Nordic countries were an estimated 800,000 tonnes per year with an additional 100,000 tonnes per year from the rearing phase of animals. The 900,000 tonnes per year of SF corresponds to 3.7% of the total production of 24,000,000 tonnes per year of edible primary products. When using the FFW definition proposed by the FUSIONS project, the FFW amount was estimated at 330,000 tonnes per year, or 1% of the total production.

Persistence, impacts and environmental drivers of covert infections in invertebrate hosts.

BACKGROUND: Persistent covert infections of the myxozoan, Tetracapsuloides bryosalmonae, in primary invertebrate hosts (the freshwater bryozoan, Fredericella sultana) have been proposed to represent a reservoir for proliferative kidney disease in secondary fish hosts. However, we have limited understanding of how covert infections persist and vary in bryozoan populations over time and space and how they may impact these populations. In addition, previous studies have likely underestimated covert infection prevalence. To improve our understanding of the dynamics, impacts and implications of covert infections we employed a highly sensitive polymerase chain reaction (PCR) assay and undertook the first investigation of covert infections in the field over an annual period by sampling bryozoans every 45 days from three populations within each of three rivers. RESULTS: Covert infections persisted throughout the year and prevalence varied within and between rivers, but were often > 50%. Variation in temperature and water chemistry were linked with changes in prevalence in a manner consistent with the maintenance of covert infections during periods of low productivity and thus poor growth conditions for both bryozoans and T. bryosalmonae. The presence and increased severity of covert infections reduced host growth but only when bryozoans were also investing in the production of overwintering propagules (statoblasts). However, because statoblast production is transitory, this effect is unlikely to greatly impact the capacity of bryozoan populations to act as persistent sources of infections and hence potential disease outbreaks in farmed and wild fish populations. CONCLUSIONS: We demonstrate that covert infections are widespread and persist over space and time in bryozoan populations. To our knowledge, this is the first long-term study of covert infections in a field setting. Review of the results of this and previous studies enables us to identify key questions related to the ecology and evolution of covert infection strategies and associated host-parasite interactions.