Parasites driving host diversity: Incidence of disease correlated with Daphnia clonal turnover*

According to the Red Queen hypothesis, clonal diversity in asexual populations could be maintained by negative frequency‐dependant selection by coevolving parasites. If common clones are selected against and rare clones gain a concomitant advantage, we expect that clonal turnover should be faster during parasite epidemics than between them. We tested this hypothesis exploring field data of the Daphnia–Caullerya host–parasite system. The clonal make‐up and turnover of the Daphnia host population was tracked with high temporal resolution from 1998 until 2013, using first allozyme and later microsatellite markers. Significant differences in the clonal composition between random and infected subsamples of Daphnia populations were detected on six of seven tested occasions, confirming genetic specificity of the host–parasite interaction in this system. We used time series analysis to compare the rates of host clonal turnover to the incidence of parasitism, and found that Caullerya prevalence was significantly associated with microsatellite‐based clonal turnover. As alternate hypotheses, we further tested whether turnover was related to a variety of biotic, abiotic, and host demographic parameters. Other significant correlates of turnover were cyanobacterial biomass and (weakly) temperature. Overall, parasitism seems to be a strong driver of host clonal turnover, in support of the Red Queen hypothesis.     

The expression of virulence during double infections by different parasites with conflicting host exploitation and transmission strategies

In many natural populations, hosts are found to be infected by more than one parasite species. When these parasites have different host exploitation strategies and transmission modes, a conflict among them may arise. Such a conflict may reduce the success of both parasites, but could work to the benefit of the host. For example, the less‐virulent parasite may protect the host against the more‐virulent competitor. We examine this conflict using the waterflea Daphnia magna and two of its sympatric parasites: the blood‐infecting bacterium Pasteuria ramosa that transmits horizontally and the intracellular microsporidium Octosporea bayeri that can concurrently transmit horizontally and vertically after infecting ovaries and fat tissues of the host. We quantified host and parasite fitness after exposing Daphnia to one or both parasites, both simultaneously and sequentially. Under conditions of strict horizontal transmission, Pasteuria competitively excluded Octosporea in both simultaneous and sequential double infections, regardless of the order of exposure. Host lifespan, host reproduction and parasite spore production in double infections resembled those of single infection by Pasteuria. When hosts became first vertically (transovarilly) infected with O. bayeri, Octosporea was able to withstand competition with P. ramosa to some degree, but both parasites produced less transmission stages than they did in single infections. At the same time, the host suffered from reduced fecundity and longevity. Our study demonstrates that even when competing parasite species utilize different host tissues to proliferate, double infections lead to the expression of higher virulence and ultimately may select for higher virulence. Furthermore, we found no evidence that the less‐virulent and vertically transmitting O. bayeri protects its host against the highly virulent P. ramosa.     

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence–transmission trade‐off. Our results are relevant in the context of increasing intensive farming, where frequent anti‐parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.     

Genetic architecture of resistance in Daphnia hosts against two species of host-specific parasites

Understanding the genetic architecture of host resistance is key for understanding the evolution of host–parasite interactions. Evolutionary models often assume simple genetics based on few loci and strong epistasis. It is unknown, however, whether these assumptions apply to natural populations. Using a quantitative trait loci (QTL) approach, we explore the genetic architecture of resistance in the crustacean Daphnia magna to two of its natural parasites: the horizontally transmitted bacterium Pasteuria ramosa and the horizontally and vertically transmitted microsporidium Hamiltosporidium tvaerminnensis. These two systems have become models for studies on the evolution of host–parasite interactions. In the QTL panel used here, Daphnia''s resistance to P. ramosa is controlled by a single major QTL (which explains 50% of the observed variation). Resistance to H. tvaerminnensis horizontal infections shows a signature of a quantitative trait based in multiple loci with weak epistatic interactions (together explaining 38% variation). Resistance to H. tvaerminnensis vertical infections, however, shows only one QTL (explaining 13.5% variance) that colocalizes with one of the QTLs for horizontal infections. QTLs for resistance to Pasteuria and Hamiltosporidium do not colocalize. We conclude that the genetics of resistance in D. magna are drastically different for these two parasites. Furthermore, we infer that based on these and earlier results, the mechanisms of coevolution differ strongly for the two host–parasite systems. Only the Pasteuria–Daphnia system is expected to follow the negative frequency-dependent selection (Red Queen) model. How coevolution works in the Hamiltosporidium–Daphnia system remains unclear.     

How a complex life cycle can improve a parasite's sex life

How complex life cycles of parasites are maintained is still a fascinating and unresolved topic. Complex life cycles using three host species, free-living stages, asexual and sexual reproduction are widespread in parasitic helminths. For such life cycles, we propose here that maintaining a second intermediate host in the life cycle can be advantageous for the individual parasite to increase the intermixture of different clones and therefore decrease the risk of matings between genetically identical individuals in the definitive host. Using microsatellite markers, we show that clone mixing occurs from the first to the second intermediate host in natural populations of the eye-fluke Diplostomum pseudospathaceum. Most individuals released by the first intermediate host belonged to one clone. In contrast, the second intermediate host was infected with a diverse array of mostly unique parasite genotypes. The proposed advantage of increased parasite clone intermixture may be a novel selection pressure favouring the maintenance of complex life cycles.     

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.     

GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Background information. The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo‐erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. Results. In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo‐erythrocytic schizogony in vitro, leading to impaired parasite maturation. Conclusions. Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red‐blood‐cell‐infective merozoites.     

Few parasites,and no evidence for Wolbachia infections,in a freshwater ostracod inhabiting temporary ponds

Biological systems with asexual reproduction have often attracted research on parasites and host immune defence, because parasites are expected to be better able to exploit genetically less diverse populations. In addition, maternally inherited parasitic microorganisms such as Wolbachia can directly alter the reproductive systems of their hosts and induce parthenogenesis. In the freshwater ostracod Eucypris virens, both sexual and asexual reproduction is known, and we speculated that parasite pressures might help to explain their co‐existence. This species complex inhabits shallow, often eutrophic temporary water bodies, conditions that should provide ample opportunities for parasite infections. We surveyed natural populations of E. virens throughout its Europe‐wide range for natural parasites, and particularly tested for the presence of intracellular Wolbachia bacteria. Surprisingly, the results indicate that very few E. virens populations support parasite infections. We also found no evidence for the presence of Wolbachia in the populations screened. The results therefore show that parasitic infections do not play a role in the maintenance of sex in this system. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 207–216.     

Variation in life-history traits among Urtica dioica populations with different history in parasitism by the holoparasitic plant Cuscuta europaea

Several studies demonstrate that natural enemies (e.g. parasites) have profound negative effects on the life-history traits of their hosts. If the host can compensate for the negative effects of parasitic infection by altering its life history, these modifications may partly form the basis of resistance or tolerance against parasites. Thus, parasites may be of considerable importance in shaping the evolution of life-history traits of their hosts. To examine if previous parasitism is associated with differences in life-history traits of the host, I conducted a common garden experiment with Urtica dioica plants originating from eight populations of which four were unparasitized, and four parasitized by the holoparasitic plant, Cuscuta europaea. A field survey indicated no differences between unparasitized and parasitized populations in, for example, the number of plant species and nutrient levels in the soil. Thus, it seems reasonable to assume that differences in life-history traits between the two population types in the common garden would reflect the effects of previous selection by the parasite. In the common garden, plants from parasitized populations started to flower later and allocated less biomass to asexual reproduction (measured as the production of stolons, i.e. clonal propagation) compared to plants from unparasitized populations. These results thus indicate that selection by the parasite may have favoured later onset of flowering, and may have selected against asexual reproduction.     

Clonal diversity driven by parasitism in a freshwater snail

One explanation for the widespread abundance of sexual reproduction is the advantage that genetically diverse sexual lineages have under strong pressure from virulent coevolving parasites. Such parasites are believed to track common asexual host genotypes, resulting in negative frequency‐dependent selection that counterbalances the population growth‐rate advantage of asexuals in comparison with sexuals. In the face of genetically diverse asexual lineages, this advantage of sexual reproduction might be eroded, and instead sexual populations would be replaced by diverse assemblages of clonal lineages. We investigated whether parasite‐mediated selection promotes clonal diversity in 22 natural populations of the freshwater snail Melanoides tuberculata. We found that infection prevalence explains the observed variation in the clonal diversity of M. tuberculata populations, whereas no such relationship was found between infection prevalence and male frequency. Clonal diversity and male frequency were independent of snail population density. Incorporating ecological factors such as presence/absence of fish, habitat geography and habitat type did not improve the predictive power of regression models. Approximately 11% of the clonal snail genotypes were shared among 2–4 populations, creating a web of 17 interconnected populations. Taken together, our study suggests that parasite‐mediated selection coupled with host dispersal ecology promotes clonal diversity. This, in return, may erode the advantage of sexual reproduction in M. tuberculata populations.     

Distributions and impacts of microparasites on Daphnia in a rockpool metapopulation

We examined the spatial distributions of the microparasites on two species of Daphnia in rockpools in four areas along the coast of the Gulf of Bothnia, central Sweden. We found five taxa of parasites, of which the microsporidian Larssonia sp. occurred in all the four subareas studied, and in 44% of the pools. Other parasites, such as a microsporidian intestinal parasite of the Glugoides type and the nematode Echinura uncinata, were much less common. Larssonia had a large impact on reproduction in both Daphnia species, although D.␣longispina seemed to be somewhat less affected than D.␣pulex. We used a spatial autocorrelation method (correlogram using Moran's coefficient) to analyse the spatial distribution of the two Daphnia species and the most abundant parasite species Larssonia sp. All three species showed positive spatial autocorrelations over small distances, indicating an aggregated distribution. This suggests that colonization-extinction dynamics may be important in both host species as well as in the parasite. Larssonia showed no relation to environmental factors, while the distributions of the two Daphnia species were significantly related to the water volume of the pools. D. pulex was more often found in small pools, while D. longispina more often inhabited larger pools. Our study suggests that microparasites in rockpools have substantial effects on Daphnia demography and through this they may influence population dynamics and local extinctions. Received: 28 April 1997 / Accepted: 22 January 1998     

Microparasite transmission to <Emphasis Type="Italic">Daphnia magna</Emphasis> 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.     

Effects of maternal parasite load on offspring life-history traits in the common lizard (Lacerta vivipara)

We studied the effect of maternal ectoparasite load (measured at parturition) on the life-history traits of the offspring of the host Lacerta vivipara, the European common lizard. The ectoparasite, a mite belonging to the family Laelapidae, had a detrimental effect on its host: parasite load was associated with increased host mortality, and was negatively correlated with host body mass. Parasite load was persistent over time, suggesting that parasite load can be predictable. Offspring of highly parasitised mothers had higher values of several fitness components early in life than offspring of parasite-free mothers or lightly infested mothers. This was expressed in terms of increased F₁ yearling growth rate, and reproductive investment at first reproduction (measured as F₂ hatchling mass). These results are interpreted as a host adaptation to attenuate the impact of parasites. Indeed, if high parasite loads arise from long exposure time to a constant population of parasites, and if the negative effects of parasites are additive over time, hosts could reduce the impact of parasites simply by investing more during the earlier stages of life. Naturally, having better performance early in life should lead to higher mortality rates and/or lower fecundity later in life.     

Spatial population genetic structure of a bacterial parasite in close coevolution with its host

Knowledge of a species’ population genetic structure can provide insight into fundamental ecological and evolutionary processes including gene flow, genetic drift and adaptive evolution. Such inference is of particular importance for parasites, as an understanding of their population structure can illuminate epidemiological and coevolutionary dynamics. Here, we describe the population genetic structure of the bacterium Pasteuria ramosa, a parasite that infects planktonic crustaceans of the genus Daphnia. This system has become a model for investigations of host–parasite interactions and represents an example of coevolution via negative frequency‐dependent selection (aka “Red Queen” dynamics). To sample P. ramosa, we experimentally infected a panel of Daphnia hosts with natural spore banks from the sediments of 25 ponds throughout much of the species range in Europe and western Asia. Using 12 polymorphic variable number tandem repeat loci (VNTR loci), we identified substantial genetic diversity, both within and among localities, that was structured geographically among ponds. Genetic diversity was also structured among host genotypes within ponds, although this pattern varied by locality, with P. ramosa at some localities partitioned into distinct host‐specific lineages, and other localities where recombination had shuffled genetic variation among different infection phenotypes. Across the sample range, there was a pattern of isolation by distance, and principal components analysis coupled with Procrustes rotation identified congruence between patterns of genetic variation and geography. Our findings support the hypothesis that Pasteuria is an endemic parasite coevolving closely with its host. These results provide important context for previous studies of this model system and inform hypotheses for future research.     

Responses of algae, bacteria, Daphnia and natural parasite fauna of Daphnia to nutrient enrichment in mesocosms

Understanding responses of parasites to changes in nutrient regimes is necessary for prediction of their role in aquatic ecosystems under global change in nutrient loading. We studied the response of the natural parasite fauna of Daphnia longispina to nutrient enrichment in mesocosms in a small humic lake. We measured the concentrations of inorganic phosphorus and nitrogen in the water, total nutrients in the seston, algal and bacterial biomass, Daphnia population dynamics, Daphnia stoichiometry, Daphnia stable isotope values and the presence and abundance of parasites in treated mesocosms as compared to three control ones. Incorporation of the nutrient enrichment in the food web was seen as increased nutrient concentrations in the epilimnion and as a decrease in carbon:nutrient ratios and δ¹⁵N values in Daphnia. Nutrient enrichment did not significantly influence algal, bacterial or Daphnia biomass. One of the four parasite species observed, unidentified small gut parasite, had a higher prevalence (percentage of Daphnia infected) in treated mesocosms, but its intensity (number of parasites per infected host) remained the same among treatments. Our results suggest that the effect of nutrient enrichment on host–parasite dynamics is dependent on complex interactions within food webs and on the epidemiological traits of parasites.     

The Ratchet and the Red Queen: the maintenance of sex in parasites

The adaptive significance of sexual reproduction remains as an unsolved problem in evolutionary biology. One promising hypothesis is that frequency‐dependent selection by parasites selects for sexual reproduction in hosts, but it is unclear whether such selection on hosts would feed back to select for sexual reproduction in parasites. Here we used individual‐based computer simulations to explore this possibility. Specifically, we tracked the dynamics of asexual parasites following their introduction into sexual parasite populations for different combinations of parasite virulence and transmission. Our results suggest that coevolutionary interactions with hosts would generally lead to a stable coexistence between sexual parasites and a single parasite clone. However, if multiple mutations to asexual reproduction were allowed, we found that the interaction led to the accumulation of clonal diversity in the asexual parasite population, which led to the eventual extinction of the sexual parasites. Thus, coevolution with sexual hosts may not be generally sufficient to select for sex in parasites. We then allowed for the stochastic accumulation of mutations in the finite parasite populations (Muller's Ratchet). We found that, for higher levels of parasite virulence and transmission, the population bottlenecks resulting from host–parasite coevolution led to the rapid accumulation of mutations in the clonal parasites and their elimination from the population. This result may explain the observation that sexual reproduction is more common in parasitic animals than in their free‐living relatives.     

Host and parasite life history interplay to yield divergent population genetic structures in two ectoparasites living on the same bat species

Host–parasite interactions are ubiquitous in nature. However, how parasite population genetic structure is shaped by the interaction between host and parasite life history remains understudied. Studies comparing multiple parasites infecting a single host can be used to investigate how different parasite life history traits interplay with host behaviour and life history. In this study, we used 10 newly developed microsatellite loci to investigate the genetic structure of a parasitic bat fly (Basilia nana). Its host, the Bechstein's bat (Myotis bechsteinii), has a social system and roosting behaviour that restrict opportunities for parasite transmission. We compared fly genetic structure to that of the host and another parasite, the wing‐mite, Spinturnix bechsteini. We found little spatial or temporal genetic structure in B. nana, suggesting a large, stable population with frequent genetic exchange between fly populations from different bat colonies. This contrasts sharply with the genetic structure of the wing‐mite, which is highly substructured between the same bat colonies as well as temporally unstable. Our results suggest that although host and parasite life history interact to yield similar transmission patterns in both parasite species, the level of gene flow and eventual spatiotemporal genetic stability is differentially affected. This can be explained by the differences in generation time and winter survival between the flies and wing‐mites. Our study thus exemplifies that the population genetic structure of parasites on a single host can vary strongly as a result of how their individual life history characteristics interact with host behaviour and life history traits.     

Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses

Epichloë endophytes are fungal symbionts of grasses that span a continuum including asexual mutualists that are vertically transmitted, obligately sexual pathogens that are horizontally transmitted, and mixed‐strategy symbionts with both mutualistic and pathogenic capabilities. Here we show that processes of genome evolution differ markedly for the different symbiont types. Genetic and phylogenetic analysis was conducted of a broad taxonomic, ecological and geographical sample of sexual and asexual isolates, in which were identified and sequenced alleles of genes for β‐tubulin (tub2) and translation elongation factor 1‐α (tef1), and microsatellite alleles were identified by length polymorphisms. The majority of asexual isolates had two or three alleles of most loci, but every sexual isolate had only single alleles for each locus. Phylogenetic analysis of tub2 and tef1 indicated that in all instances of multiple alleles in an isolate, the alleles were derived from different sexual species. It is concluded that, whereas horizontally transmissible species had haploid genomes and speciation occurred cladistically, most of the strictly seedborne mutualists were interspecific hybrids with heteroploid (aneuploid or polyploid) genomes. Furthermore, the phylogenetic evidence indicated that, in at least some instances, hybridization followed rather than caused evolution of the strictly seedborne habit. Therefore, the abundance of hybrid species among grass endophytes, and their prevalence in many host populations suggests a selective advantage of hybridization for the mutualistic endophytes.     

The ecology and phylogeny of oomycete infections in Asplanchna rotifers

1. Recently, the potential for parasites to influence the ecology and evolution of their zooplankton hosts has been the subject of increasing study. However, most research to date has focussed on Daphnia hosts, and the potential for parasites to influence other zooplankton taxa remains largely unstudied. 2. During routine sampling of zooplankton in a eutrophic lake, we observed that the rotifer Asplanchna girodi was often infected with a parasitic oomycete. Epidemics of this parasite occurred frequently, with three separate events in a single year. Prevalence at peak infection ranged from 29 to 41% and epidemics lasted from 17 to 56 days. Our data indicate that high densities of the host population are required for epidemics to occur. 3. Our morphological and molecular analyses suggest that this parasite is in the genus Pythium. Most Pythium spp. are plant pathogens, but our study supports recent work on Daphnia, suggesting that Pythium spp. are also important parasites of zooplankton. 4. As the parasite in this study was recalcitrant to cultivation, we developed an alternative method to verify its identity. Our approach used quantitative PCR to show that the ribosomal sequences identified increased with increasing density of infected hosts and, thus, were associated with the parasite. This approach should be generally applicable to other plankton parasites that are difficult to cultivate outside their hosts. 5. Infections significantly reduced host fecundity, lifespan and population growth rate. As a result of the virulence of this parasite, it is likely to influence the population ecology and evolution of its Asplanchna host, and may be a useful model system for studies on host–parasite coevolutionary dynamics.     

Predators can influence the host‐parasite dynamics of their prey via nonconsumptive effects

Ecological communities are partly structured by indirect interactions, where one species can indirectly affect another by altering its interactions with a third species. In the absence of direct predation, nonconsumptive effects of predators on prey have important implications for subsequent community interactions. To better understand these interactions, we used a Daphnia‐parasite‐predator cue system to evaluate if predation risk affects Daphnia responses to a parasite. We investigated the effects of predator cues on two aspects of host–parasite interactions (susceptibility to infection and infection intensity), and whether or not these effects differed between sexes. Our results show that changes in response to predator cues caused an increase in the prevalence and intensity of parasite infections in female predator‐exposed Daphnia. Importantly, the magnitude of infection risk depended on how long Daphnia were exposed to the cues. Additionally, heavily infected Daphnia that were constantly exposed to cues produced relatively more offspring. While males were ~5× less likely to become infected compared to females, we were unable to detect effects of predator cues on male Daphnia–parasite interactions. In sum, predators, prey, and their parasites can form complex subnetworks in food webs, necessitating a nuanced understanding of how nonconsumptive effects may mediate these interactions.