Surface carbohydrate composition of a tapeworm in its consecutive intermediate hosts: individual variation and fitness consequences

Carbohydrates on parasite surfaces have been shown to play an important role in host–parasite coevolution, mediating host non-self recognition and parasite camouflage. Parasites that switch hosts can change their surface molecules to remain undetected by the diverse immune systems of their different hosts. However, the question of individual variation in surface sugar composition and its relation to infectivity, virulence, immune evasion and growth of a parasite in its different hosts is as yet largely unexplored. We studied such fitness consequences of variation in surface sugars in a sympatric host–parasite system consisting of the cestode Schistocephalus solidus and its intermediate hosts, a copepod and the three-spined stickleback. Using lectins to analyse the sugar composition, we show that the tapeworm changes its surface according to the invertebrate or vertebrate host. Importantly, sugar composition seems to be genetically variable, as shown by differences among tapeworm sibships. These differences are related to variation in parasite fitness in its second intermediate host, i.e. infectivity and growth. Surface sugar composition may thus be a proximate correlate of the evolutionarily relevant variability in infectivity and virulence of parasites in different hosts.     

Differential host defense against multiple parasites in ants

Host–parasite interactions are ideal systems for the study of coevolutionary processes. Although infections with multiple parasite species are presumably common in nature, most studies focus on the interactions of a single host and a single parasite. To the best of our knowledge, we present here the first study on the dependency of parasite virulence and host resistance in a multiple parasite system. We evaluated whether the strength of host defense depends on the potential fitness cost of parasites in a system of two Southeast Asian army ant hosts and five parasitic staphylinid beetle species. The potential fitness costs of the parasites were evaluated by their predation behavior on host larvae in isolation experiments. The host defense was assessed by the ants’ aggressiveness towards parasitic beetle species in behavioral studies. We found clear differences among the beetle species in both host–parasite interactions. Particular beetle species attacked and killed the host larvae, while others did not. Importantly, the ants’ aggressiveness was significantly elevated against predatory beetle species, while non-predatory beetle species received almost no aggression. As a consequence of this defensive behavior, less costly parasites are more likely to achieve high levels of integration in the ant society. We conclude that the selection pressure on the host to evolve counter-defenses is higher for costly parasites and, thus, a hierarchical host defense strategy has evolved that depends on the parasites’ impact.     

Reciprocal Interaction Matrix Reveals Complex Genetic and Dose-Dependent Specificity among Coinfecting Parasites

Abstract Understanding genetic specificity in factors determining the outcome of host-parasite interactions is especially important as it contributes to parasite epidemiology, virulence, and maintenance of genetic variation. Such specificity, however, is still generally poorly understood. We examined genetic specificity in interactions among coinfecting parasites. In natural populations, individual hosts are often simultaneously infected by multiple parasite species and genotypes that interact. Such interactions could maintain genetic variation in parasite populations if they are genetically specific so that the relative fitness of parasite genotypes varies across host individuals depending on (1) the presence/absence of coinfections and/or (2) the genetic composition of the coinfecting parasite community. We tested these predictions using clones of fish eye flukes Diplostomum pseudospathaceum and Diplostomum gasterostei. We found that interactions among parasites had a strong genetic basis and that this modified genetic variation in infection success of D. pseudospathaceum between single and multiple infections as well as across multiply infected host individuals depending on the genetic identity of the coinfecting D. gasterostei. The relative magnitude of these effects, however, depended on the exposure dose, suggesting that ecological factors can modify genetic interactions between parasites.     

ON THE EVOLUTION OF SEXUAL REPRODUCTION IN HOSTS COEVOLVING WITH MULTIPLE PARASITES

Host–parasite coevolution has been studied extensively in the context of the evolution of sex. Although hosts typically coevolve with several parasites, most studies considered one‐host/one‐parasite interactions. Here, we study population‐genetic models in which hosts interact with two parasites. We find that host/multiple‐parasite models differ nontrivially from host/single‐parasite models. Selection for sex resulting from interactions with a single parasite is often outweighed by detrimental effects due to the interaction between parasites if coinfection affects the host more severely than expected based on single infections, and/or if double infections are more common than expected based on single infections. The resulting selection against sex is caused by strong linkage‐disequilibria of constant sign that arise between host loci interacting with different parasites. In contrast, if coinfection affects hosts less severely than expected and double infections are less common than expected, selection for sex due to interactions with individual parasites can now be reinforced by additional rapid linkage‐disequilibrium oscillations with changing sign. Thus, our findings indicate that the presence of an additional parasite can strongly affect the evolution of sex in ways that cannot be predicted from single‐parasite models, and that thus host/multiparasite models are an important extension of the Red Queen Hypothesis.     

Coinfecting parasites can modify fluctuating selection dynamics in host–parasite coevolution

Genetically specific interactions between hosts and parasites can lead to coevolutionary fluctuations in their genotype frequencies over time. Such fluctuating selection dynamics are, however, expected to occur only under specific circumstances (e.g., high fitness costs of infection to the hosts). The outcomes of host–parasite interactions are typically affected by environmental/ecological factors, which could modify coevolutionary dynamics. For instance, individual hosts are often infected with more than one parasite species and interactions between them can alter host and parasite performance. We examined the potential effects of coinfections by genetically specific (i.e., coevolving) and nonspecific (i.e., generalist) parasite species on fluctuating selection dynamics using numerical simulations. We modeled coevolution (a) when hosts are exposed to a single parasite species that must genetically match the host to infect, (b) when hosts are also exposed to a generalist parasite that increases fitness costs to the hosts, and (c) when coinfecting parasites compete for the shared host resources. Our results show that coinfections can enhance fluctuating selection dynamics when they increase fitness costs to the hosts. Under resource competition, coinfections can either enhance or suppress fluctuating selection dynamics, depending on the characteristics (i.e., fecundity, fitness costs induced to the hosts) of the interacting parasites.     

Differential virulence in a multiple‐host parasite of bumble bees: resolving the paradox of parasite survival?

Parasite virulence determines both the impact that parasites have on their hosts and parasite fitness. While most studies of virulence have involved single-species host–parasite interactions, the majority of parasites are likely to use multiple concurrent host species. Our understanding of how this impacts on parasite epidemiology and virulence is limited. Using the bumble bee Bombus lucorum , which exists in sympatry with B. terrestris in multi-species assemblages, and their generalist micosporidian parasite Nosema bombi , we tested whether the apparent paradox of parasite maintenance due to parasite virulence in a single host, B. terrestris , could be resolved through understanding the parasite's virulence in this sympatric host species. Nosema bombi significantly impacted colony growth, individual longevity, and individual development in B. lucorum . However, these effects were different both qualitatively and quantitatively to the parasite's impact in B. terrestris . Infected colonies of B. lucorum successfully produced both male and female reproductives, and infected female reproductives were capable of successful mating. Variation in life-history across host species may explain differences in the virulence, or impact of the parasite in B. terrestris and B. lucorum , with species with shorter life-cycles being more likely to transfer the parasite from one annual generation to the next. These results suggest that to understand the virulence and epidemiology of multi-host parasites we need to examine their ecological interactions across their various host species.     

Effects of malaria double infection in birds: one plus one is not two

Avian malaria parasites are supposed to exert negative effects on host fitness because these intracellular parasites affect host metabolism. Recent advances in molecular genotyping and microscopy have revealed that coinfections with multiple parasites are frequent in bird-malaria parasite systems. However, studies of the fitness consequences of such double infections are scarce and inconclusive. We tested if the infection with two malaria parasite lineages has more negative effects than single infection using 6 years of data from a natural population of house martins. Survival was negatively affected by both types of infections. We found an additive cost from single to double infection in body condition, but not in reproductive parameters (double-infected had higher reproductive success). These results demonstrate that malaria infections decrease survival, but also have different consequences on the breeding performance of single- and double-infected wild birds.     

Linking community assembly and structure across scales in a wild mouse parasite community

Understanding what processes drive community structure is fundamental to ecology. Many wild animals are simultaneously infected by multiple parasite species, so host–parasite communities can be valuable tools for investigating connections between community structures at multiple scales, as each host can be considered a replicate parasite community. Like free‐living communities, within‐host–parasite communities are hierarchical; ecological interactions between hosts and parasites can occur at multiple scales (e.g., host community, host population, parasite community within the host), therefore, both extrinsic and intrinsic processes can determine parasite community structure. We combine analyses of community structure and assembly at both the host population and individual scales using extensive datasets on wild wood mice (Apodemus sylvaticus) and their parasite community. An analysis of parasite community nestedness at the host population scale provided predictions about the order of infection at the individual scale, which were then tested using parasite community assembly data from individual hosts from the same populations. Nestedness analyses revealed parasite communities were significantly more structured than random. However, observed nestedness did not differ from null models in which parasite species abundance was kept constant. We did not find consistency between observed community structure at the host population scale and within‐host order of infection. Multi‐state Markov models of parasite community assembly showed that a host's likelihood of infection with one parasite did not consistently follow previous infection by a different parasite species, suggesting there is not a deterministic order of infection among the species we investigated in wild wood mice. Our results demonstrate that patterns at one scale (i.e., host population) do not reliably predict processes at another scale (i.e., individual host), and that neutral or stochastic processes may be driving the patterns of nestedness observed in these communities. We suggest that experimental approaches that manipulate parasite communities are needed to better link processes at multiple ecological scales.     

Predicting the similarity of parasite communities in freshwater fishes using the phylogeny,ecology and proximity of hosts

Parasite communities tend to be dissimilar in hosts that are geographically, phylogenetically, ecologically and developmentally distant from one another. The decay of community similarity is a powerful and increasingly common method of studying parasite beta diversity, but most studies have examined only a single type of distance. Here, we evaluate distances based on the phylogeny, ecology, spatial proximity and size of hosts, as predictors of the similarity of parasite communities in individual hosts, host populations and host species. We surveyed parasites in six species of fish collected simultaneously from six localities in the St. Lawrence River, Canada, and species in a common group of larval parasites were discriminated using DNA sequences from barcode region of cytochrome c oxidase I. Distances based on the habitat use patterns of host species were good predictors of short‐term, ecological similarity of parasite communities, such as that operating at the scale of the individual host. The genetic distance between host species was associated with almost all types of similarity at all scales, particularly qualitative and phylogenetic similarity of parasite communities at the level of populations and meta‐populations of hosts. The trophic level, diet, spatial proximity and size of hosts were poor predictors of parasite community similarity. The increased taxonomic resolution provided by molecular data increased the explanatory power of regression models, and different factors were implicated when parasite species were distinguished with DNA barcodes than when larval parasites were lumped into morphospecies, as is commonly practiced.     

Minimal selfing, few clones, and no among-host genetic structure in a hermaphroditic parasite with asexual larval propagation

Little is known about actual mating systems in natural populations of parasites or about what constitutes the limits of a parasite deme. These parameters are interesting because they affect levels of genetic diversity, opportunities for local adaptation, and other evolutionary processes. We expect that transmission dynamics and the distribution of parasites among hosts should have a large effect on mating systems and demic structure, but currently we have mostly speculation and very few data. For example, infrapopulations (all the parasites in a single host) should behave as demes if parasite offspring are transmitted as a clump from host to host over several generations. However, if offspring are well mixed, then the parasite component population (all the parasites among a host population) would function as the deme. Similarly, low mean intensities or a high proportion of worms in single infections should increase the selfing rate. For species having an asexual amplification stage, transmission between intermediate and definitive (final) hosts will control the variance in clonal reproductive success, which in turn could have a large influence on effective sizes and rates of inbreeding. We examined demic structure, selfing rates, and the variance in clonal reproductive success in natural populations of Plagioporus shawi, a hermaphroditic trematode that parasitizes salmon. Overall levels of genetic diversity were very high. An a posteriori inference of population structure overwhelmingly supports the component population as the deme, rather than individual infrapopulations. Only a single pair of 597 adult individuals was identified as clones. Thus, the variance in clonal reproductive success was almost zero. Despite being hermaphroditic, P. shawi appears to be almost entirely outcrossing. Genetic estimates of selfing (<5%) were in accordance with the proportion of parasites from single infections. Thus, it appears that individual flukes outcross whenever possible and only resort to selfing when alone. Finally, our data support the hypothesis that aquatic transmission and the use of several intermediate hosts promotes high genetic diversity and well-mixed infrapopulations.     

The influence of a parasite community on the dynamics of a host population: a longitudinal study on willow ptarmigan and their parasites

Despite the fact that most host populations are infected by a community of different parasite species, the majority of empirical studies have focused on the interaction between the host and a single parasite species. Here, we explore the hypothesis that host population dynamics are affected both by single parasite species and by the whole parasite community. We monitored population density and breeding productivity of two populations of willow ptarmigan ( Lagopus lagopus ) in northern Norway for 8 and 11 years, respectively, and sampled eukaryotic endoparasites. We found that increasing abundances of the cestode Hymenolepis microps was associated with increased breeding mortality and reduced annual growth rate of the host population in both areas, and reduced host body mass and body condition in the area where such data were available. In one of the areas, the abundance of the nematode Trichostrongylus tenuis was associated with reductions in host body mass, body condition and breeding mortality and the filaroid nematode Splendidofilaria papillocerca was negatively related to host population growth rates. The parasite community was also negatively related to host fitness parameters, suggesting an additional community effect on host body mass and breeding mortality, although none of the parasites had a significant impact on their own. The prevalence of parasites with very different taxonomical origins tended to covary within years, suggesting that variability in the parasite community was not random, but governed by changes in host susceptibility or environmental conditions that affected exposure to parasites in general. Other variables including climate, plant production and rodent densities were not associated with the recorded demographic changes in the host population.     

Priority effects within coinfected hosts can drive unexpected population‐scale patterns of parasite prevalence

Organisms are frequently coinfected by multiple parasite strains and species, and interactions between parasites within hosts are known to influence parasite prevalence and diversity, as well as epidemic timing. Importantly, interactions between coinfecting parasites can be affected by the order in which they infect hosts (i.e. within‐host priority effects). In this study, we use a single‐host, two‐pathogen, SI model with environmental transmission to explore how within‐host priority effects scale up to alter host population‐scale infection patterns. Specifically, we ask how parasite prevalence changes in the presence of different types of priority effects. We consider two scenarios without priority effects and four scenarios with priority effects where there is either an advantage or a disadvantage to being the first to infect in a coinfected host. Models without priority effects always predict negative relationships between the prevalences of both parasites. In contrast, models with priority effects can yield unimodal prevalence relationships where the prevalence of a focal parasite is minimized or maximized at intermediate prevalences of a coinfecting parasite. The mechanism behind this pattern is that as the prevalence of the coinfecting parasite increases, most infections of the focal parasite change from occurring as solo infections, to first arrival coinfections, to second arrival coinfections. The corresponding changes in parasite fitness as the focal parasite moves from one infection class to another then map to changes in focal parasite prevalence. Further, we found that even when parasites interact negatively within a host, they still can have positive prevalence relationships at the population scale. These results suggest that within‐host priority effects can change host population‐scale infection patterns in systematic (and initially counterintuitive) ways, and that taking them into account may improve disease forecasting in coinfected populations.     

Potential Parasite Transmission in Multi-Host Networks Based on Parasite Sharing

Epidemiological networks are commonly used to explore dynamics of parasite transmission among individuals in a population of a given host species. However, many parasites infect multiple host species, and thus multi-host networks may offer a better framework for investigating parasite dynamics. We investigated the factors that influence parasite sharing – and thus potential transmission pathways – among rodent hosts in Southeast Asia. We focused on differences between networks of a single host species and networks that involve multiple host species. In host-parasite networks, modularity (the extent to which the network is divided into subgroups of rodents that interact with similar parasites) was higher in the multi-species than in the single-species networks. This suggests that phylogeny affects patterns of parasite sharing, which was confirmed in analyses showing that it predicted affiliation of individuals to modules. We then constructed “potential transmission networks” based on the host-parasite networks, in which edges depict the similarity between a pair of individuals in the parasites they share. The centrality of individuals in these networks differed between multi- and single-species networks, with species identity and individual characteristics influencing their position in the networks. Simulations further revealed that parasite dynamics differed between multi- and single-species networks. We conclude that multi-host networks based on parasite sharing can provide new insights into the potential for transmission among hosts in an ecological community. In addition, the factors that determine the nature of parasite sharing (i.e. structure of the host-parasite network) may impact transmission patterns.     

Parasites contribute to ecologically dependent postmating isolation in the adaptive radiation of three-spined stickleback

Spatial variation in parasitic infections is common, and has the potential to drive population divergence and the reproductive isolation of hosts. However, despite support from theory and model laboratory systems, little strong evidence has been forthcoming from the wild. Here, we show that parasites are likely to cause reproductive isolation in the adaptive radiation of three-spined stickleback. Adjacent wild populations on the Scottish island of North Uist differ greatly and consistently in the occurrence of different parasites that have substantial effects on fitness. Laboratory-reared fish are more resistant to experimental infection by parasite species from their own population. Furthermore, hybrid backcrosses between the host populations are more resistant to parasites from the parental population to which they are more closely related. These patterns provide strong evidence that parasites can cause ecological speciation, by contributing to selection against migrants and ecologically dependent postmating isolation.     

Of mice and worms: are co-infections with unrelated parasite strains more damaging to definitive hosts?

Intraspecific competition between co-infecting parasites can influence the amount of virulence, or damage, they do to their host. Kin selection theory dictates that infections with related parasite individuals should have lower virulence than infections with unrelated individuals, because they benefit from inclusive fitness and increased host longevity. These predictions have been tested in a variety of microparasite systems, and in larval stage macroparasites within intermediate hosts, but the influence of adult macroparasite relatedness on virulence has not been investigated in definitive hosts. This study used the human parasite Schistosoma mansoni to determine whether definitive hosts infected with related parasites experience lower virulence than hosts infected with unrelated parasites, and to compare the results from intermediate host studies in this system. The presence of unrelated parasites in an infection decreased parasite infectivity, the ability of a parasite to infect a definitive host, and total worm establishment in hosts, impacting the less virulent parasite strain more severely. Unrelated parasite co-infections had similar virulence to the more virulent of the two parasite strains. We combine these findings with complementary studies of the intermediate snail host and describe trade-offs in virulence and selection within the life cycle. Damage to the host by the dominant strain was muted by the presence of a competitor in the intermediate host, but was largely unaffected in the definitive host. Our results in this host–parasite system suggest that unrelated infections may select for higher virulence in definitive hosts while selecting for lower virulence in intermediate hosts.     

Host ecology and life-history traits associated with blood parasite species richness in birds

Identifying host traits associated with the number of different parasite species or strains harboured by a particular host species can have important implications for understanding the impact of parasitism on hosts. We investigated associations between host ecology and life history, and parasite richness and prevalence of the four major avian blood parasite genera. We used an extensive data on blood parasite infections and host ecology in 263 bird species from the Western Palearctic, combining species-specific data with a comparative approach to control for similarity in phenotype among host species due to the effects of common phylogenetic descent. Adult survival rate negatively correlated with the number of parasite species infecting a host species when controlling for similarity due to common descent and body mass. In addition, the prevalence of Haemoproteus, Plasmodium and Leucocytozoon was higher in species harbouring a richer parasite assemblage. These results suggest that the impact on host fitness caused by avian haematozoa may be underestimated in natural populations if the exacerbated virulence associated with exposure to multiple parasites is not taken into account.     

The costs of evolving resistance in heterogeneous parasite environments

The evolution of host resistance to parasites, shaped by associated fitness costs, is crucial for epidemiology and maintenance of genetic diversity. Selection imposed by multiple parasites could be a particularly strong constraint, as hosts either accumulate costs of multiple specific resistances or evolve a more costly general resistance mechanism. We used experimental evolution to test how parasite heterogeneity influences the evolution of host resistance. We show that bacterial host populations evolved specific resistance to local bacteriophage parasites, regardless of whether they were in single or multiple-phage environments, and that hosts evolving with multiple phages were no more resistant to novel phages than those evolving with single phages. However, hosts from multiple-phage environments paid a higher cost, in terms of population growth in the absence of phage, for their evolved specific resistances than those from single-phage environments. Given that in nature host populations face selection pressures from multiple parasite strains and species, our results suggest that costs may be even more critical in shaping the evolution of resistance than previously thought. Furthermore, our results highlight that a better understanding of resistance costs under combined control strategies could lead to a more 'evolution-resistant' treatment of disease.     

Host lifespan and the evolution of resistance to multiple parasites

Hosts are typically challenged by multiple parasites, but to date theory on the evolution of resistance has mainly focused on single infections. We develop a series of models that examine the impact of multiple parasites on the evolution of resistance under the assumption that parasites coexist at the host population scale as a consequence of superinfection. In this way, we are able to explicitly examine the impact of ecological dynamics on the evolutionary outcome. We use our models to address a key question of how host lifespan affects investment in resistance to multiple parasites. We show that investment in costly resistance depends on the specificity of the immune response and on whether or not the focal parasite leads to more acute infection than the co‐circulating parasite. A key finding is that investment in resistance always increases as the immune response becomes more general independently of whether it is the focal or the co‐circulating parasite that exploits the host most aggressively. Long‐lived hosts always invest more than short‐lived hosts in both general resistance and resistance that is specific to relatively acute focal parasites. However, for specific resistance to parasites that are less acute than co‐circulating parasites it is the short‐lived hosts that are predicted to invest most. We show that these results apply whatever the mode of defence, that is whether it is through avoidance or through increased recovery, with or without acquired immunity, or through acquired immunity itself. As a whole, our results emphasize the importance of considering multiple parasites in determining optimal immune investment in eco‐evolutionary systems.     

Larval helminths in intermediate hosts: does competition early in life determine the fitness of adult parasites?

Density-dependent effects on parasite fitness have been documented from adult helminths in their definitive hosts. There have, however, been no studies on the cost of sharing an intermediate host with other parasites in terms of reduced adult parasite fecundity. Even if larval parasites suffer a reduction in size, caused by crowding, virtually nothing is known about longer-lasting effects after transmission to the definitive host. This study is the first to use in vitro cultivation with feeding of adult trematodes to investigate how numbers of parasites in the intermediate host affect the size and fecundity of adult parasites. For this purpose, we examined two different infracommunities of parasites in crustacean hosts. Firstly, we used experimental infections of Maritrema novaezealandensis in the amphipod, Paracalliope novizealandiae, to investigate potential density-dependent effects in single-species infections. Secondly, we used the crab, Macrophthalmus hirtipes (Ocypodidae), naturally infected by the trematodes, M. novaezealandensis and Levinseniella sp., the acanthocephalan, Profilicollis spp., and an acuariid nematode. These four helminths all develop and grow in their crustacean host before transmission to their bird definitive host by predation. In experimental infections, we found an intensity-dependent establishment success, with a decrease in the success rate of cercariae developing into infective metacercariae with an increasing dose of cercariae applied to each amphipod. In natural infections, we found that M. novaezealandensis-metacercariae achieved a smaller volume, on average, when infrapopulations of this parasite were large. Small metacercariae produced small in vitro-adult worms, which in turn produced fewer eggs. Crowding effects in the intermediate host thus were expressed at the adult stage in spite of the worms being cultured in a nutrient-rich medium. Furthermore, excystment success and egg-production in M. novaezealandensis in naturally infected crabs were influenced by the number of co-occurring Profilicollis cystacanths, indicating interspecific interactions between the two species. Our results thus indicate that the infracommunity of larval helminths in their intermediate host is interactive and that any density-dependent effect in the intermediate host may have lasting effects on individual parasite fitness.     

Determinants of haemosporidian single- and co-infection risks in western palearctic birds

Understanding the drivers of infection risk helps us to detect the most at-risk species in a community and identify species whose intrinsic characteristics could act as potential reservoirs of pathogens. This knowledge is crucial if we are to predict the emergence and evolution of infectious diseases. To date, most studies have only focused on infections caused by a single parasite, leaving out co-infections. Yet, co-infections are of paramount importance in understanding the ecology and evolution of host-parasite interactions due to the wide range of effects they can have on host fitness and on the evolutionary trajectories of parasites. Here, we used a multinomial Bayesian phylogenetic modelling framework to explore the extent to which bird ecology and phylogeny impact the probability of being infected by one genus (hereafter single infection) or by multiple genera (hereafter co-infection) of haemosporidian parasites. We show that while nesting and migration behaviours influenced both the probability of being single- and co-infected, species position along the slow-fast life-history continuum and geographic range size were only pertinent in explaining variation in co-infection risk. We also found evidence for a phylogenetic conservatism regarding both single- and co-infections, indicating that phylogenetically related bird species tend to have similar infection patterns. This phylogenetic signal was four times stronger for co-infections than for single infections, suggesting that co-infections may act as a stronger selective pressure than single infections. Overall, our study underscores the combined influence of hosts’ evolutionary history and attributes in determining infection risk in avian host communities. These results also suggest that co-infection risk might be under stronger deterministic control than single infection risk, potentially paving the way toward a better understanding of the emergence and evolution of infectious diseases.