Phylogenomics including new sequence data of phytoplankton-infecting chytrids reveals multiple independent lifestyle transitions across the phylum.

Parasitism is the most common lifestyle on Earth and has emerged many times independently across the eukaryotic tree of life. It is frequently found among chytrids (Chytridiomycota), which are early-branching unicellular fungi that feed osmotrophically via rhizoids as saprotrophs or parasites. Chytrids are abundant in most aquatic and terrestrial environments and fulfil important ecosystem functions. As parasites, they can have significant impacts on host populations. They cause global amphibian declines and influence the Earth's carbon cycle by terminating algal blooms. To date, the evolution of parasitism within the chytrid phylum remains unclear due to the low phylogenetic resolution of rRNA genes for the early diversification of fungi, and because few parasitic lineages have been cultured and genomic data for parasites is scarce. Here, we combine transcriptomics, culture-independent single-cell genomics and a phylogenomic approach to overcome these limitations. We newly sequenced 29 parasitic taxa and combined these with existing data to provide a robust backbone topology for the diversification of Chytridiomycota. Our analyses reveal multiple independent lifestyle transitions between parasitism and saprotrophy among chytrids and multiple host shifts by parasites. Based on these results and the parasitic lifestyle of other early-branching holomycotan lineages, we hypothesise that the chytrid last common ancestor was a parasite of phytoplankton.

Climate has contributed to population diversification of Daphnia galeata across Eurasia.

Climate is a fundamental abiotic factor that plays a key role in driving the evolution, distribution and population diversification of species. However, there have been few investigations of genomic signatures of adaptation to local climatic conditions in cladocerans. Here, we have provided the first high-quality chromosome-level genome assembly (~143 Mb, scaffold N50 12.6 Mb) of the waterflea, Daphnia galeata, and investigated genomic variation in 22 populations from Central Europe and Eastern China. Our ecological-niche models suggested that the historic distribution of D. galeata in Eurasia was significantly affected by Quaternary climate fluctuations. We detected pronounced genomic and morphometric divergences between European and Chinese D. galeata populations. Such divergences could be partly explained by genomic signatures of thermal adaptation to distinct climate regimes: a set of candidate single-nucleotide polymorphisms (SNPs) potentially associated with climate were detected. These SNPs were in genes significantly enriched in the Gene ontology terms "determination of adult lifespan" and "translation repressor activity", and especially, mthl5 and SOD1 involved in the IIS pathway, and EIF4EBP2 involved in the target of the rapamycin signalling pathway. Our study indicates that certain alleles might be associated with particular temperature regimes, playing a functional role in shaping the population structure of D. galeata at a large geographical scale. These results highlight the potential role of molecular variation in the response to climate variation, in the context of global climate change.

Responses to predation pressure involve similar sets of genes in two divergent species of Daphnia.

Species that are not closely related can express similar inducible traits, but molecular mechanisms underlying the observed responses are often unknown, nor is it known if these mechanisms are shared between such species. Here, we compared transcriptional profiles of two Daphnia species (D. mitsukuri and D. sinensis) from different subgenera, at both juvenile and adult developmental stages. Both species were exposed to the same predation threat (fish kairomones), and both showed similar induced morphological changes (reduced body length). At the early developmental stage, response to predation risk resulted in similar changes in expression levels of 23 orthologues in both species. These orthologues, involved in 107 GO categories, changed in the same direction in both species (over- or underexpressed), in comparison to non-exposed controls. Several of these orthologues were associated with DNA replication, structural constituents of cuticle or innate immune response. In both species, the differentially expressed (DE) genes on average had higher ω (dN /dS ) values than non-DE genes, suggesting that these genes had experienced greater positive selection or lower purifying selection than non-DE genes. Overall, our results suggest that similar suites of genes, responding in similar ways to predation pressure, have been retained in Daphnia for many millions of years.

Single-cell genomics reveals new rozellid lineages and supports their sister relationship to Microsporidia.

The phylum Rozellomycota has been proposed for a group of early-branching holomycotan lineages representing obligate parasites and hyperparasites of zoosporic fungi, oomycotes or phytoplankton. Given their predominantly intracellular lifestyle, rozellids are typically known from environmental ribosomal DNA data, except for the well-studied Rozella species. To date, the phylogenetic relationship between rozellids and microsporidians (Microsporidia) is not fully understood and most reliable hypotheses are based on phylogenomic analyses that incorporate the only publicly available rozellid genome of Rozella allomycis. Here, we provide genomic data of three new rozellid lineages obtained by single-cell sequencing from environmental samples and show with a phylogenomic approach that rozellids form a monophyletic group that is sister to microsporidians, corroborating the previously proposed phylum Rozellomycota. Whereas no mitochondrial genes coding for the respiratory Complex I could be found, we discovered a gene coding for a nucleotide phosphate transporter in one of the three draft genomes. The scattered absence of Complex I genes and scattered presence of nucleotide transporter genes across diverse microsporidian and rozellid lineages suggest that these adaptations to a parasitic lifestyle, which reduce the parasite's capability to synthesize ATP but enables it to steal ATP from its host, evolved independently in microsporidians and rozellids.

Host-Associated Bacterial Communities Vary Between Daphnia galeata Genotypes but Not by Host Genetic Distance.

Host genotype may shape host-associated bacterial communities (commonly referred to as microbiomes). We sought to determine (a) whether bacterial communities vary among host genotypes in the water flea Daphnia galeata and (b) if this difference is driven by the genetic distance between host genotypes, by using D. galeata genotypes hatched from sediments of different time periods. We used 16S amplicon sequencing to profile the gut and body bacterial communities of eight D. galeata genotypes hatched from resting eggs; these were isolated from two distinct sediment layers (dating to 1989 and 2009) of a single sediment core of the lake Greifensee, and maintained in a common garden in laboratory cultures for 5 years. In general, bacterial community composition varied in both the Daphnia guts and bodies; but not between genotypes from different sediment layers. Specifically, genetic distances between host genotypes did not correlate with beta diversity of bacterial communities in Daphnia guts and bodies. Our results indicate that Daphnia bacterial community structure is to some extent determined by a host genetic component, but that genetic distances between hosts do not correlate with diverging bacterial communities.

Findings in percutaneous trans-hepatic cholecysto-cholangiography in neonates and infants presenting with conjugated hyperbilirubinemia: emphasis on differential diagnosis and cholangiographic patterns.

BACKGROUND: Biliary atresia (BA) is one of the causes of conjugated hyperbilirubinemia in infants which if untreated leads to end-stage liver disease and death. Percutaneous Trans-hepatic Cholecysto-Cholangiography (PTCC) is a minimally invasive study which can be utilized in the diagnostic work-up of these patients. This study's purpose is to describe the experience with PTCC in neonates, the imaging findings encountered, and the abnormal patterns which warrant further investigation. METHODS: A 16-year single-center retrospective study of patients with persistent neonatal cholestasis (suspected BA) undergoing PTCC. Patient demographics, laboratory values, PTCC images, pathology and surgical reports were reviewed. RESULTS: 73 patients underwent PTCC (68% male, mean age 8.7 weeks, mean weight 4.0 Kg). The majority of studies were normal (55%). Abnormal patterns were identified in 33 cases, 79% were diagnosed with BA and 12% with Alagille syndrome. Non-opacification of the common hepatic duct with a narrowed common bile duct (42%) and isolated small gallbladder (38%) were the most common patterns in BA. CONCLUSION: PTCC is a minimally invasive study in the diagnostic work-up of infants presenting with conjugated hyperbilirubinemia (suspected BA). Further invasive investigations or surgery can be avoided when results are normal.

Predictors of Social-Emotional Development and Adaptive Functioning in School-Age Children with Intestinal Failure.

OBJECTIVES: Despite a focus on neurocognition in pediatric intestinal failure (IF) to date, we examined social-emotional and adaptive functioning. METHODS: Children (N = 63) in our IF rehabilitation program underwent neuropsychological assessments including caregiver- and teacher-reported questionnaires. Results were compared to norms using z-tests. Caregiver and teacher reports were compared using t tests. Medical and demographic factors were examined in an exploratory manner using correlation and targeted regression analyses, adjusting for gestational age and full-scale IQ. RESULTS: Caregiver and teacher reports indicated poorer executive, internalizing, behavioral, and adaptive functioning compared to norms. Teachers reported more executive dysfunctions than caregivers. Necrotizing enterocolitis diagnosis predicted internalizing emotional problems. Immigrant status predicted poorer social and practical adaptive functioning. Living with biological parents predicted fewer externalizing emotional and behavioral problems. CONCLUSIONS: The group displayed social-emotional and adaptive functioning concerns. Identifying medical and demographic risks can allow for screening and intervention.

Parasites do not adapt to elevated temperature, as evidenced from experimental evolution of a phytoplankton-fungus system.

Global warming is predicted to impact the prevalence and severity of infectious diseases. However, empirical data supporting this statement usually stem from experiments in which parasite fitness and disease outcome are measured directly after temperature increase. This might exclude the possibility of parasite adaptation. To incorporate the adaptive response of parasites into predictions of disease severity in a warmer world, we undertook an experimental evolution assay in which a fungal parasite of phytoplankton was maintained at elevated or control temperatures for six months, corresponding to 100-200 parasite generations. Host cultures were maintained at the respective temperatures and provided as substrate, but were not under parasite pressure. A reciprocal infection experiment conducted after six-month serial passages revealed no evidence of parasite adaptation. In fact, parasite fitness at elevated temperatures was inferior in parasite populations reared at elevated temperatures compared with those maintained under control temperature. However, this effect was reversed after parasites were returned to control temperatures for a few (approx. 10) generations. The absence of parasite adaptation to elevated temperatures suggests that, in phytoplankton-fungus systems, disease outcome under global warming will be largely determined by both host and parasite thermal ecology.

Genomic regions associated with adaptation to predation in Daphnia often include members of expanded gene families.

Predation has been a major driver of the evolution of prey species, which consequently develop antipredator adaptations. However, little is known about the genetic basis underpinning the adaptation of prey to intensive predation. Here, we describe a high-quality chromosome-level genome assembly (approx. 145 Mb, scaffold N50 11.45 Mb) of Daphnia mitsukuri, a primary forage for many fish species. Transcriptional profiling of D. mitsukuri exposed to fish kairomone revealed that this cladoceran responds to predation risk through regulating activities of Wnt signalling, cuticle pattern formation, cell cycle regulation and anti-apoptosis pathways. Genes differentially expressed in response to predation risk are more likely to be members of expanded families. Our results suggest that expansions of multiple gene families associated with chemoreception and vision allow Daphnia to enhance detection of predation risk, and that expansions of those associated with detoxification and cuticle formation allow Daphnia to mount an efficient response to perceived predation risk. This study increases our understanding of the molecular basis of prey defences, being important evolutionary adaptations playing a stabilizing role in community dynamics.

Sequential infection of Daphnia magna by a gut microsporidium followed by a haemolymph yeast decreases transmission of both parasites.

Over the course of seasonal epidemics, populations of susceptible hosts may encounter a wide variety of parasites. Parasite phenology affects the order in which these species encounter their hosts, leading to sequential infections, with potentially strong effects on within-host growth and host population dynamics. Here, the cladoceran Daphnia magna was exposed sequentially to a haemolymph-infecting yeast (Metschnikowia bicuspidata) and a gut microsporidium (Ordospora colligata), with experimental treatments reflecting two possible scenarios of parasite succession. The effects of single and co-exposure were compared on parasite infectivity, spore production and the overall virulence experienced by the host. We show that neither parasite benefited from coinfection; instead, when hosts encountered Ordospora, followed by Metschnikowia, higher levels of host mortality contributed to an overall decrease in the transmission of both parasites. These results showcase an example of sequential infections generating unilateral priority effects, in which antagonistic interactions between parasites can alleviate the intensity of infection and coincide with maladaptive levels of damage inflicted on the host.

Temporal dynamics of freshwater planktonic parasites inferred using a DNA metabarcoding time-series.

Parasites are important components of biodiversity and contributors to ecosystem functioning, but are often neglected in ecological studies. Most studies examine model parasite systems or single taxa, thus our understanding of community composition is lacking. Here, the seasonal and annual dynamics of parasites was quantified using a 5-year metabarcoding time-series of freshwater plankton, collected weekly. We first identified parasites in the dataset using literature searches of the taxonomic match and using sequence metadata from the National Center for Biotechnology Information (NCBI) nucleotide database. In total, 441 amplicon sequence variants (belonging to 18 phyla/clades) were classified as parasites. The four phyla/clades with the highest relative read abundance and richness were Chytridiomycota, Dinoflagellata, Oomycota and Perkinsozoa. Relative read abundance of total parasite taxa, Dinoflagellata and Perkinsozoa significantly varied with season and was highest in summer. Parasite richness varied significantly with season and year, and was generally lowest in spring. Each season had distinct parasite communities, and the difference between summer and winter communities was most pronounced. Combining DNA metabarcoding with searches of the literature and NCBI metadata allowed us to characterize parasite diversity and community dynamics and revealed the extent to which parasites contribute to the diversity of freshwater plankton communities.

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

Light intensity and spectral distribution affect chytrid infection of cyanobacteria via modulation of host fitness.

Light gradients are an inherent feature in aquatic ecosystems and play a key role in shaping the biology of phytoplankton. Parasitism by chytrid fungi is gaining increasing attention as a major control agent of phytoplankton due to its previously overlooked ubiquity, and profound ecological and evolutionary consequences. Despite this interest, if and how light conditions modulate phytoplankton chytridiomycosis remains poorly studied. We investigated life-history traits of a chytrid parasite, Rhizophydium megarrhizum, under different light intensities and spectral compositions when infecting two closely related planktonic cyanobacteria with different light-harvesting strategies: Planktothrix rubescens and P. agardhii. In general, parasite transmission was highest under light conditions (both intensity and quality) that maximized growth rates for uninfected cyanobacteria. Chytrid encystment on hosts was significantly affected by light intensity and host strain identity. This likely resulted from higher irradiances stimulating the increased discharge of photosynthetic by-products, which drive parasite chemotaxis, and from strain-specific differences at the cell-surface. Comparisons of parasite transmission and host growth rates under different light conditions suggest the potential for epidemic development at higher irradiances, whereas host and parasite could coexist without epidemic outbreaks at lower light levels. These results illustrate the close relationship between parasite transmission and host fitness, which is ultimately modulated by the external environment.

Comparison of sterol and fatty acid profiles of chytrids and their hosts reveals trophic upgrading of nutritionally inadequate phytoplankton by fungal parasites.

Chytrids are ubiquitous fungal parasites in aquatic ecosystems, infecting representatives of all major phytoplankton groups. They repack carbon from inedible phytoplankton hosts into easily ingested chytrid propagules (zoospores), rendering this carbon accessible to zooplankton. Grazing on zoospores may circumvent bottlenecks in carbon transfer imposed by the dominance of inedible or poorly nutritious phytoplankton (mycoloop). We explored qualitative aspects of the mycoloop by analysing lipid profiles (fatty acids, sterols) of two chytrids infecting two major bloom-forming phytoplankton taxa of contrasting nutritional value: the diatom Asterionella formosa and the filamentous cyanobacterium Planktothrix agardhii. The polyunsaturated fatty acid composition of chytrids largely reflected that of their hosts, highlighting their role as conveyors of otherwise inaccessible essential lipids to higher trophic levels. We also showed that chytrids are capable of synthesizing sterols, thus providing a source of these essential nutrients for grazers even when sterols are absent in their phytoplankton hosts. Our findings reveal novel qualitative facets of the mycoloop, showing that parasitic chytrids, in addition to making carbon and essential lipids available from inedible sources, also upgrade their host's biochemical composition by producing sterols de novo, thereby enhancing carbon and energy fluxes in aquatic food webs.

Daphnia galeata and D. dentifera are geographically and ecologically separated whereas their hybrids occur in intermediate habitats: A survey of 44 Chinese lakes.

The idea that hybridization promotes range expansion has received recent attention, but support from field studies is limited. We hypothesized that in the cladoceran waterflea genus Daphnia, parental species are geographically and ecologically separated, whereas hybrids occupy intermediate or occasionally extreme environments, potentially facilitating range expansion of parental species. We assessed the distribution of Daphnia dentifera, Daphnia galeata and their interspecific hybrids across 44 lakes in China (using mitochondrial and microsatellite markers), and related it to geographical and environmental lake descriptors. Parental species were geographically separated: D. dentifera occurred in western and central China, and D. galeata in eastern and central China, whereas hybrids were found in the western and central parts of the country. However, after controlling for geographical differences, the effect of environment on species distribution was strong and significant. Specifically, D. dentifera was present in high-altitude oligotrophic lakes, D. galeata in low-altitude eutrophic lakes and hybrids at intermediate to high altitudes, mainly in mesotrophic lakes. Microsatellite data indicated that hybrids were locally produced rather than having migrated from elsewhere; they probably resulted from encounters between expanding D. galeata and resident D. dentifera. The present study provides evidence that hybrids can survive in habitats that are otherwise suitable for only one of their parental species, emphasizing the importance of hybridization in expansion of species gene pools.

Lineage diversity and reproductive modes of the Daphnia pulex group in Chinese lakes and reservoirs.

Recent studies of the distribution and diversity of freshwater zooplankton have indicated that the previously understudied Eastern Palearctic region is an important biogeographic hotspot. Here, we explored the lineage diversity and reproductive modes of the Daphnia pulex species group across China. Members of this group are often keystone species of standing water bodies and are frequently used as a model system for ecological, evolutionary and, more recently, genomic studies. We found members of the D. pulex group in seven of seventy-six Chinese water bodies examined. We analyzed their phylogenetic position using mitochondrial markers, and explored the genetic structure of six populations using microsatellite markers. Mitochondrial DNA analysis suggested the presence of two distinct species complexes in China: the D. pulex complex that has a global distribution, and an apparently endemic Eastern Palearctic D. mitsukuri complex. Microsatellite analyses of six populations suggested that three of these reproduced by cyclical parthenogenesis, as evidenced by high clonal diversity and the absence of deviations from the Hardy-Weinberg equilibrium. In contrast, three other populations showed remarkably low diversity of multilocus genotypes. This suggests an obligate parthenogenetic reproductive mode, which was confirmed in one of the populations by comparison of genotypes of Daphnia adults and dormant embryos. All presumably obligate parthenogenetic clones were heterozygous at the majority of microsatellite loci, suggesting their hybrid origin. This was further supported by analyses of a small GTPase nuclear gene (rab4), as two alleles within single individuals belonged to different clades. Interestingly, one putatively obligate parthenogenetic clone carried three distinct alleles suggesting higher ploidy and potential gene flow between the D. pulicaria and D. mitsukuri complexes. Our data show that the expansion of the D. pulex complex in the Eastern Palearctic was associated with widespread hybridization.

Cytonuclear diversity and shared mitochondrial haplotypes among Daphnia galeata populations separated by seven thousand kilometres.

BACKGROUND: The zooplanktonic cladocerans Daphnia, present in a wide range of water bodies, are an important component of freshwater ecosystems. In contrast to their high dispersal capacity through diapausing eggs carried by waterfowl, Daphnia often exhibit strong population genetic differentiation. Here, to test for common patterns in the population genetic structure of a widespread Holarctic species, D. galeata, we genotyped two sets of populations collected from geographically distant areas: across 13 lakes in Eastern China and 14 lakes in Central Europe. The majority of these populations were genotyped at two types of markers: a mitochondrial gene (for 12S rRNA) and 15 nuclear microsatellite loci. RESULTS: Mitochondrial DNA demonstrated relatively shallow divergence within D. galeata, with distinct haplotype compositions in the two study regions but one widely distributed haplotype shared between several of the Chinese as well as European populations. At microsatellite markers, clear separation was observed at both large (between China and Europe) and small (within Europe) geographical scales, as demonstrated by Factorial Correspondence Analyses, Bayesian assignment and a clustering method based on genetic distances. Genetic diversity was comparable between the sets of Chinese and European D. galeata populations for both types of markers. Interestingly, we observed a significant association between genetic distance and geographical distance for D. galeata populations in China but not in Europe. CONCLUSIONS: Our results indicate relatively recent spread of D. galeata across wide expanses of the Palaearctic, with one mtDNA lineage of D. galeata successfully establishing over large distances. Despite a clear differentiation of Chinese and European D. galeata at a nuclear level, the pattern of genetic variation is nevertheless similar between both regions. Overall, our findings provide insights into the genetic population structure of a cladoceran species with extremely wide geographical range.

Daphnia galeata responds to the exposure to an ichthyosporean gut parasite by down-regulation of immunity and lipid metabolism.

BACKGROUND: Regulatory circuits of infection in the emerging experimental model system, water flea Daphnia and their microparasites, remain largely unknown. Here we provide the first molecular insights into the response of Daphnia galeata to its highly virulent and common parasite Caullerya mesnili, an ichthyosporean that infects the gut epithelium. We generated a transcriptomic dataset using RNAseq from parasite-exposed (vs. control) Daphnia, at two time points (4 and 48 h) after parasite exposure. RESULTS: We found a down-regulation of metabolism and immunity-related genes, at 48 h (but not 4 h) after parasite exposure. These genes are involved in lipid metabolism and fatty acid biosynthesis, as well as microbe recognition (e.g. c-type lectins) and pathogen attack (e.g. gut chitin). CONCLUSIONS: General metabolic suppression implies host energy shift from reproduction to survival, which is in agreement with the known drastic reduction in Daphnia fecundity after Caullerya infection. The down-regulation of gut chitin indicates a possible interaction between the peritrophic matrix and the evading host immune system. Our study provides the first description of host transcriptional responses in this very promising host-parasite experimental system.

Fitness and eco-physiological response of a chytrid fungal parasite infecting planktonic cyanobacteria to thermal and host genotype variation.

Understanding how individual parasite traits contribute to overall fitness, and how they are modulated by both external and host environment, is crucial for predicting disease outcome. Fungal (chytrid) parasites of phytoplankton are important yet poorly studied pathogens with the potential to modulate the abundance and composition of phytoplankton communities and to drive their evolution. Here, we studied life-history traits of a chytrid parasite infecting the planktonic, bloom-forming cyanobacterium Planktothrix spp. under host genotype and thermal variation. When expressing parasite fitness in terms of transmission success, disease outcome was largely modulated by temperature alone. Yet, a closer examination of individual parasite traits linked to different infection phases, such as (i) the establishment of the infection (i.e. intensity of infection) and (ii) the exploitation of host resources (i.e. size of reproductive structures and propagules), revealed differential host genotype and temperature × host genotype modulation, respectively. This illustrates how parasite fitness results from the interplay of individual parasite traits that are differentially controlled by host and external environment, and stresses the importance of combining multiple traits to gain insights into underlying infection mechanisms.

Temperature modulates the interaction between fungicide pollution and disease: evidence from a Daphnia-microparasitic yeast model.

Temperature is expected to modulate the responses of organisms to stress. Here, we aimed to assess the influence of temperature on the interaction between parasitism and fungicide contamination. Specifically, using the cladoceran Daphnia as a model system, we explored the isolated and interactive effects of parasite challenge (yeast Metschnikowia bicuspidata) and exposure to fungicides (copper sulphate and tebuconazole) at two temperatures (17 and 20 °C), in a fully factorial design. Confirming a previous study, M. bicuspidata infection and copper exposure caused independent effects on Daphnia life history, whereas infection was permanently suppressed with tebuconazole exposure. Here, we show that higher temperature generally increased the virulence of the parasite, with the hosts developing signs of infection earlier, reproducing less and dying at an earlier age. These effects were consistent across copper concentrations, whereas the joint effects of temperature (which enhanced the difference between non-infected and infected hosts) and the anti-parasitic action of tebuconazole resulted in a more pronounced parasite × tebuconazole interaction at the higher temperature. Thus, besides independently influencing parasite and contaminant effects, the temperature can act as a modulator of interactions between pollution and disease.