Pathways linking nutrient enrichment, habitat structure, and parasitism to host-resource interactions.

Human activities simultaneously alter nutrient levels, habitat structure, and levels of parasitism. These activities likely have individual and joint impacts on food webs. Furthermore, there is particular concern that nutrient additions and changes to habitat structure might exacerbate the size of epidemics and impacts on host density. We used a well-studied zooplankton-fungus host-parasite system and experimental whole water column enclosures to factorially manipulate nutrient levels, habitat structure (specifically: mixing), and presence of parasites. Nutrient addition increased infection prevalence, density of infected hosts, and total host density. We hypothesized that nutrients, mixing, and parasitism were linked in multiple ways, including via their combined effects on phytoplankton (resource) abundance, and we used structural equation modeling to disentangle these pathways. In the absence of the parasite, both nutrients and mixing increased abundance of phytoplankton, whereas host density was negatively related to phytoplankton abundance, suggesting a mixture of bottom-up and top-down control of phytoplankton. In the presence of the parasite, nutrients still increased phytoplankton abundance but mixing no longer did, and there was no longer a significant relationship between host density and phytoplankton. This decoupling of host-resource dynamics may have resulted from reduced grazing due to illness-mediated changes in feeding behavior. Overall, our results show that the impact of one human activity (e.g., altered habitat structure) might depend on other human impacts (e.g., parasite introduction). Fortunately, carefully designed experiments and analyses can help tease apart these multifaceted relationships, allowing us to understand how human activities alter food webs, including interactions between hosts and their parasites and resources.

Pathogens stabilize or destabilize depending on host stage structure.

A common assumption is that pathogens more readily destabilize their host populations, leading to an elevated risk of driving both the host and pathogen to extinction. This logic underlies many strategies in conservation biology and pest and disease management. Yet, the interplay between pathogens and population stability likely varies across contexts, depending on the environment and traits of both the hosts and pathogens. This context-dependence may be particularly important in natural consumer-host populations where size- and stage-structured competition for resources strongly modulates population stability. Few studies, however, have examined how the interplay between size and stage structure and infectious disease shapes the stability of host populations. Here, we extend previously developed size-dependent theory for consumer-resource interactions to examine how pathogens influence the stability of host populations across a range of contexts. Specifically, we integrate a size- and stage-structured consumer-resource model and a standard epidemiological model of a directly transmitted pathogen. The model reveals surprisingly rich dynamics, including sustained oscillations, multiple steady states, biomass overcompensation, and hydra effects. Moreover, these results highlight how the stage structure and density of host populations interact to either enhance or constrain disease outbreaks. Our results suggest that accounting for these cross-scale and bidirectional feedbacks can provide key insight into the structuring role of pathogens in natural ecosystems while also improving our ability to understand how interventions targeting one may impact the other.

Global trends in antimicrobial resistance on organic and conventional farms.

The important hypothesis that organic livestock management reduces the prevalence of antimicrobial resistance is either fiercely supported or bitterly contested. Yet, empirical evidence supporting this view remains fragmentary, in part because relationships between antimicrobial use and drug resistance vary dramatically across contexts, hosts, pathogens, and country-specific regulations. Here, we synthesize global policies and definitions of 'organic' and ask if organic farming results in notable reductions in the prevalence of antimicrobial resistance when directly examined alongside conventional analogs. We synthesized the results of 72 studies, spanning 22 countries and five pathogens. Our results highlight substantial variations in country-specific policies on drug use and definitions of 'organic' that hinder broad-scale and generalizable patterns. Overall, conventional farms had slightly higher levels of antimicrobial resistance (28%) relative to organic counterparts (18%), although we found significant context-dependent variation in this pattern. Notably, environmental samples from organic and conventional farms often exhibited high levels of resistance to medically important drugs, underscoring the need for more stringent and consistent policies to control antimicrobial contaminants in the soil (particularly on organic farms, where the application of conventional manure could faciliate the spread antimicrobial resistance). Taken together, these results emphasize the challenges inherent in understanding links between drug use and drug resistance, the critical need for global standards governing organic policies, and greater investment in viable alternatives for managing disease in livestock.

Coinfection with chytrid genotypes drives divergent infection dynamics reflecting regional distribution patterns.

By altering the abundance, diversity, and distribution of species-and their pathogens-globalization may inadvertently select for more virulent pathogens. In Brazil's Atlantic Forest, a hotspot of amphibian biodiversity, the global amphibian trade has facilitated the co-occurrence of previously isolated enzootic and panzootic lineages of the pathogenic amphibian-chytrid (Batrachochytrium dendrobatidis, 'Bd') and generated new virulent recombinant genotypes ('hybrids'). Epidemiological data indicate that amphibian declines are most severe in hybrid zones, suggesting that coinfections are causing more severe infections or selecting for higher virulence. We investigated how coinfections involving these genotypes shapes virulence and transmission. Overall, coinfection favored the more virulent and competitively superior panzootic genotype, despite dampening its transmission potential and overall virulence. However, for the least virulent and least competitive genotype, coinfection increased both overall virulence and transmission. Thus, by integrating experimental and epidemiological data, our results provide mechanistic insight into how globalization can select for, and propel, the emergence of introduced hypervirulent lineages, such as the globally distributed panzootic lineage of Bd.

Molecular Surveillance Detects High Prevalence of the Neglected Parasite Mansonella ozzardi in the Colombian Amazon.

BACKGROUND: Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions. METHODS: We deployed molecular and classical approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. RESULTS: Loop-mediated isothermal amplification (LAMP) assays on whole-blood samples detected a much higher prevalence of Mansonella ozzardi infection (approximately 40%) compared to blood smear microscopy or LAMP performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias and occult infections. Mansonella infection rates increased with age and were higher among men. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. CONCLUSION: These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

Natural variation in host feeding behaviors impacts host disease and pathogen transmission potential.

Animals ranging from mosquitoes to humans often vary their feeding behavior when infected or merely exposed to pathogens. These so-called "sickness behaviors" are part of the innate immune response with many consequences, including avoiding orally transmitted pathogens. Fully understanding the role of this ubiquitous behavior in host defense and pathogen evolution requires a quantitative account of its impact on host and pathogen fitness across environmentally relevant contexts. Here, we use a zooplankton host and fungal pathogen as a case study to ask if infection-mediated feeding behaviors vary across pathogen exposure levels and natural genetic variation in susceptibility to infection. Then, we connect these changes in behavior to pathogen transmission potential (spore yield) and fitness and growth costs to the host. Our results validate a protective effect of altered feeding behavior during pathogen exposure while also revealing significant variation in the magnitude of this response across host susceptibility and pathogen exposure levels. Across all four host genotypes, feeding rates were negatively correlated with susceptibility to infection and transmission potential. The most susceptible genotypes exhibited either strong anorexia, reducing food intake by 26%-42%, ("Standard") or pronounced hyperphagia, increasing food intake by 20%-54% ("A45"). Together, these results suggest that infection-mediated changes in host feeding behavior-which are traditionally interpreted as immunopathology- may in fact serve as crucial components of host defense strategies and warrant further investigation.

Circadian rhythms mediate infection risk in Daphnia dentifera.

Biological rhythms mediate important within-host processes such as metabolism, immunity, and behavior which are often linked to combating disease exposure. For many hosts, exposure to pathogens occurs while feeding. However, the link between feeding rhythms and infection risk is unclear because feeding behavior is tightly coupled with immune and metabolic processes which may decrease susceptibility to infection. Here, we use the Daphnia dentifera-Metschnikowia bicuspidata host-pathogen system to determine how rhythms in feeding rate and immune function mediate infection risk. The host is known to have a nocturnal circadian rhythm in feeding rate, yet we found that they do not exhibit a circadian rhythm in phenoloxidase activity. We found that the time of day when individuals are exposed to pathogens affects the probability of infection with higher infection prevalence at night, indicating that infection risk is driven by a host's circadian rhythm in feeding behavior. These results suggest that the natural circadian rhythm of the host should be considered when addressing epidemiological dynamics.

Transient disease dynamics across ecological scales.

Analyses of transient dynamics are critical to understanding infectious disease transmission and persistence. Identifying and predicting transients across scales, from within-host to community-level patterns, plays an important role in combating ongoing epidemics and mitigating the risk of future outbreaks. Moreover, greater emphases on non-asymptotic processes will enable timely evaluations of wildlife and human diseases and lead to improved surveillance efforts, preventive responses, and intervention strategies. Here, we explore the contributions of transient analyses in recent models spanning the fields of epidemiology, movement ecology, and parasitology. In addition to their roles in predicting epidemic patterns and endemic outbreaks, we explore transients in the contexts of pathogen transmission, resistance, and avoidance at various scales of the ecological hierarchy. Examples illustrate how (i) transient movement dynamics at the individual host level can modify opportunities for transmission events over time; (ii) within-host energetic processes often lead to transient dynamics in immunity, pathogen load, and transmission potential; (iii) transient connectivity between discrete populations in response to environmental factors and outbreak dynamics can affect disease spread across spatial networks; and (iv) increasing species richness in a community can provide transient protection to individuals against infection. Ultimately, we suggest that transient analyses offer deeper insights and raise new, interdisciplinary questions for disease research, consequently broadening the applications of dynamical models for outbreak preparedness and management. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12080-021-00514-w.

A high-throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia.

Food ingestion is one of the most basic features of all organisms. However, obtaining precise-and high-throughput-estimates of feeding rates remains challenging, particularly for small, aquatic herbivores such as zooplankton, snails, and tadpoles. These animals typically consume low volumes of food that are time-consuming to accurately measure.We extend a standard high-throughput fluorometry technique, which uses a microplate reader and 96-well plates, as a practical tool for studies in ecology, evolution, and disease biology. We outline technical and methodological details to optimize quantification of individual feeding rates, improve accuracy, and minimize sampling error.This high-throughput assay offers several advantages over previous methods, including i) substantially reduced time allotments per sample to facilitate larger, more efficient experiments; ii) technical replicates; and iii) conversion of in vivo measurements to units (mL-1 hr-1 ind-1) which enables broad-scale comparisons across an array of taxa and studies.To evaluate the accuracy and feasibility of our approach, we use the zooplankton, Daphnia dentifera, as a case study. Our results indicate that this procedure accurately quantifies feeding rates and highlights differences among seven genotypes.The method detailed here has broad applicability to a diverse array of aquatic taxa, their resources, environmental contaminants (e.g., plastics), and infectious agents. We discuss simple extensions to quantify epidemiologically relevant traits, such as pathogen exposure and transmission rates, for infectious agents with oral or trophic transmission.

Starving the Enemy? Feeding Behavior Shapes Host-Parasite Interactions.

The loss of appetite that typically accompanies infection or mere exposure to parasites is traditionally considered a negative byproduct of infection, benefitting neither the host nor the parasite. Numerous medical and veterinary practices directly or indirectly subvert this 'illness-mediated anorexia'. However, the ecological factors that influence it, its effects on disease outcomes, and why it evolved remain poorly resolved. We explore how hosts use anorexia to defend against infection and how parasites manipulate anorexia to enhance transmission. Then, we use a coevolutionary model to illustrate how shifts in the magnitude of anorexia (e.g., via drugs) affect disease dynamics and virulence evolution. Anorexia could be exploited to improve disease management; we propose an interdisciplinary approach to minimize unintended consequences.

Genotypic variation in parasite avoidance behaviour and other mechanistic, nonlinear components of transmission.

Traditional epidemiological models assume that transmission increases proportionally to the density of parasites. However, empirical data frequently contradict this assumption. General yet mechanistic models can explain why transmission depends nonlinearly on parasite density and thereby identify potential defensive strategies of hosts. For example, hosts could decrease their exposure rates at higher parasite densities (via behavioural avoidance) or decrease their per-parasite susceptibility when encountering more parasites (e.g. via stronger immune responses). To illustrate, we fitted mechanistic transmission models to 19 genotypes of Daphnia dentifera hosts over gradients of the trophically acquired parasite, Metschnikowia bicuspidata. Exposure rate (foraging, F) frequently decreased with parasite density (Z), and per-parasite susceptibility (U) frequently decreased with parasite encounters (F×Z). Consequently, infection rates (F×U×Z) often peaked at intermediate parasite densities. Moreover, host genotypes varied substantially in these responses. Exposure rates remained constant for some genotypes but decreased sensitively with parasite density for others (up to 78%). Furthermore, genotypes with more sensitive foraging/exposure also foraged faster in the absence of parasites (suggesting 'fast and sensitive' versus 'slow and steady' strategies). These relationships suggest that high densities of parasites can inhibit transmission by decreasing exposure rates and/or per-parasite susceptibility, and identify several intriguing axes for the evolution of host defence.

Parasite-Mediated Anorexia and Nutrition Modulate Virulence Evolution.

Temporary but substantial reductions in voluntary food intake routinely accompany parasite infection in hosts ranging from insects to humans. This "parasite-mediated anorexia" drives dynamic nutrient-dependent feedbacks within and among hosts, which should alter the fitness of both hosts and parasites. Yet, few studies have examined the evolutionary and epidemiological consequences of this ubiquitous but overlooked component of infection. Moreover, numerous biomedical, veterinary, and farming practices (e.g., rapid biomass production via high-calorie or high-fat diets, low-level antibiotics to promote growth, nutritional supplementation, nonsteroidal anti-inflammatory drugs like Ibuprofen) directly or indirectly alter the magnitude of host anorexia-while also controlling host diet and therefore the nutrients available to hosts and parasites. Here, we show that anorexia can enhance or diminish disease severity, depending on whether the current dietary context provides nutrients that bolster or inhibit immune function. Feedbacks driven by nutrition-mediated competition between host immune function and parasite production can create a unimodal relationship between anorexia and parasite fitness. Subsequently, depending on the host's diet, medical or husbandry practices that suppress anorexia could backfire, and inadvertently select for more virulent parasites and larger epidemics. These findings carry implications for the development of integrated treatment programs that consider links between host feeding behavior, nutrition, and disease severity.

Parasite rearing and infection temperatures jointly influence disease transmission and shape seasonality of epidemics.

Seasonal epidemics erupt commonly in nature and are driven by numerous mechanisms. Here, we suggest a new mechanism that could determine the size and timing of seasonal epidemics: rearing environment changes the performance of parasites. This mechanism arises when the environmental conditions in which a parasite is produced impact its performance-independently from the current environment. To illustrate the potential for "rearing effects", we show how temperature influences infection risk (transmission rate) in a Daphnia-fungus disease system through both parasite rearing temperature and infection temperature. During autumnal epidemics, zooplankton hosts contact (eat) fungal parasites (spores) reared in a gradually cooling environment. To delineate the effect of rearing temperature from temperature at exposure and infection, we used lab experiments to parameterize a mechanistic model of transmission rate. We also evaluated the rearing effect using spores collected from epidemics in cooling lakes. We found that fungal spores were more infectious when reared at warmer temperatures (in the lab and in two of three lakes). Additionally, the exposure (foraging) rate of hosts increased with warmer infection temperatures. Thus, both mechanisms cause transmission rate to drop as temperature decreases over the autumnal epidemic season (from summer to winter). Simulations show how these temperature-driven changes in transmission rate can induce waning of epidemics as lakes cool. Furthermore, via thermally dependent transmission, variation in environmental cooling patterns can alter the size and shape of epidemics. Thus, the thermal environment drives seasonal epidemics through effects on hosts (exposure rate) and the infectivity of parasites (a rearing effect). Presently, the generality of parasite rearing effects remains unknown. Our results suggest that they may provide an important but underappreciated mechanism linking temperature to the seasonality of epidemics.

Resource-driven changes to host population stability alter the evolution of virulence and transmission.

What drives the evolution of parasite life-history traits? Recent studies suggest that linking within- and between-host processes can provide key insight into both disease dynamics and parasite evolution. Still, it remains difficult to understand how to pinpoint the critical factors connecting these cross-scale feedbacks, particularly under non-equilibrium conditions; many natural host populations inherently fluctuate and parasites themselves can strongly alter the stability of host populations. Here, we develop a general model framework that mechanistically links resources to parasite evolution across a gradient of stable and unstable conditions. First, we dynamically link resources and between-host processes (host density, stability, transmission) to virulence evolution, using a 'non-nested' model. Then, we consider a 'nested' model where population-level processes (transmission and virulence) depend on resource-driven changes to individual-level (within-host) processes (energetics, immune function, parasite production). Contrary to 'non-nested' model predictions, the 'nested' model reveals complex effects of host population dynamics on parasite evolution, including regions of evolutionary bistability; evolution can push parasites towards strongly or weakly stabilizing strategies. This bistability results from dynamic feedbacks between resource-driven changes to host density, host immune function and parasite production. Together, these results highlight how cross-scale feedbacks can provide key insights into the structuring role of parasites and parasite evolution.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'.

Temperature Drives Epidemics in a Zooplankton-Fungus Disease System: A Trait-Driven Approach Points to Transmission via Host Foraging.

Climatic warming will likely have idiosyncratic impacts on infectious diseases, causing some to increase while others decrease or shift geographically. A mechanistic framework could better predict these different temperature-disease outcomes. However, such a framework remains challenging to develop, due to the nonlinear and (sometimes) opposing thermal responses of different host and parasite traits and due to the difficulty of validating model predictions with observations and experiments. We address these challenges in a zooplankton-fungus (Daphnia dentifera-Metschnikowia bicuspidata) system. We test the hypothesis that warmer temperatures promote disease spread and produce larger epidemics. In lakes, epidemics that start earlier and warmer in autumn grow much larger. In a mesocosm experiment, warmer temperatures produced larger epidemics. A mechanistic model parameterized with trait assays revealed that this pattern arose primarily from the temperature dependence of transmission rate (ß), governed by the increasing foraging (and, hence, parasite exposure) rate of hosts (f). In the trait assays, parasite production seemed sufficiently responsive to shape epidemics as well; however, this trait proved too thermally insensitive in the mesocosm experiment and lake survey to matter much. Thus, in warmer environments, increased foraging of hosts raised transmission rate, yielding bigger epidemics through a potentially general, exposure-based mechanism for ectotherms. This mechanistic approach highlights how a trait-based framework will enhance predictive insight into responses of infectious disease to a warmer world.

Rapid evolution rescues hosts from competition and disease but-despite a dilution effect-increases the density of infected hosts.

Virulent parasites can depress the densities of their hosts. Taxa that reduce disease via dilution effects might alleviate this burden. However, 'diluter' taxa can also depress host densities through competition for shared resources. The combination of disease and interspecific competition could even drive hosts extinct. Then again, genetically variable host populations can evolve in response to both competitors and parasites. Can rapid evolution rescue host density from the harm caused by these ecological enemies? How might such evolution influence dilution effects or the size of epidemics? In a mesocosm experiment with planktonic hosts, we illustrate the joint harm of competition and disease: hosts with constrained evolutionary ability (limited phenotypic variation) suffered greatly from both. However, populations starting with broader phenotypic variation evolved stronger competitive ability during epidemics. In turn, enhanced competitive ability-driven especially by parasites-rescued host densities from the negative impacts of competition, disease, and especially their combination. Interspecific competitors reduced disease (supporting dilution effects) even when hosts rapidly evolved. However, this evolutionary response also elicited a potential problem. Populations that evolved enhanced competitive ability and maintained robust total densities also supported higher densities of infections. Thus, rapid evolution rescued host densities but also unleashed larger epidemics.

Allocation, not male resistance, increases male frequency during epidemics: a case study in facultatively sexual hosts.

Why do natural populations vary in the frequency of sexual reproduction? Virulent parasites may help explain why sex is favored during disease epidemics. To illustrate, we show a higher frequency of males and sexually produced offspring in natural populations of a facultative parthenogenetic host during fungal epidemics. In a multi-year survey of 32 lakes, the frequency of males (an index of sex) was higher in populations of zooplankton hosts with larger epidemics. A lake mesocosm experiment established causality: experimental epidemics produced a higher frequency of males relative to disease-free controls. One common explanation for such a pattern involves Red Queen (RQ) dynamics. However, this particular system lacks key genetic specificity mechanisms required for the RQ, so we evaluated two other hypotheses. First, individual females, when stressed by infection, could increase production of male offspring vs. female offspring (a tenant of the "Abandon Ship" theory). Data from a life table experiment supports this mechanism. Second, higher male frequency during epidemics could reflect a purely demographic process (illustrated with a demographic model): males could resist infection more than females (via size-based differences in resistance and mortality). However, we found no support for this resistance mechanism. A size-based model of resistance, parameterized with data, revealed why: higher male susceptibility negated the lower exposure (a size-based advantage) of males. These results suggest that parasite-mediated increases in allocation to sex by individual females, rather than male resistance, increased the frequency of sex during larger disease epidemics.

Joint effects of habitat, zooplankton, host stage structure and diversity on amphibian chytrid.

Why does the severity of parasite infection differ dramatically across habitats? This question remains challenging to answer because multiple correlated pathways drive disease. Here, we examined habitat-disease links through direct effects on parasites and indirect effects on parasite predators (zooplankton), host diversity and key life stages of hosts. We used a case study of amphibian hosts and the chytrid fungus, Batrachochytrium dendrobatidis, in a set of permanent and ephemeral alpine ponds. A field experiment showed that ultraviolet radiation (UVR) killed the free-living infectious stage of the parasite. Yet, permanent ponds with more UVR exposure had higher infection prevalence. Two habitat-related indirect effects worked together to counteract parasite losses from UVR: (i) UVR reduced the density of parasite predators and (ii) permanent sites fostered multi-season host larvae that fuelled parasite production. Host diversity was unlinked to hydroperiod or UVR but counteracted parasite gains; sites with higher diversity of host species had lower prevalence of infection. Thus, while habitat structure explained considerable variation in infection prevalence through two indirect pathways, it could not account for everything. This study demonstrates the importance of creating mechanistic, food web-based links between multiple habitat dimensions and disease.

Parasites destabilize host populations by shifting stage-structured interactions.

Should parasites stabilize or destabilize consumer-resource dynamics? Recent theory suggests that parasite-enhanced mortality may confer underappreciated stability to their hosts. We tested this hypothesis using disease in zooplankton. Across both natural and experimental epidemics, bigger epidemics correlated with larger--not smaller--host fluctuations. Thus, we tested two mechanistic hypotheses to explain destabilization or apparent destabilization by parasites. First, enrichment could, in principle, simultaneously enhance both instability and disease prevalence. In natural epidemics, destabilization was correlated with enrichment (indexed by total phosphorous). However, an in situ (lake enclosure) experiment did not support these links. Instead, field and experimental results point to a novel destabilizing mechanism involving host stage structure. Epidemics pushed hosts from relatively more stable host dynamics with less-synchronized juveniles and adults to less stable dynamics with more-synchronized juveniles and adults. Our results demonstrate how links between host stage structure and disease can shape host/consumer-resource stability.

Consequences of induced hatching plasticity depend on predator community.

Many prey species face trade-offs in the timing of life history switch points like hatching and metamorphosis. Costs associated with transitioning early depend on the biotic and abiotic conditions found in the subsequent life stage. The red-eyed treefrog, Agalychnis callidryas, faces risks from predators in multiple, successive life stages, and can hatch early in response to mortality threats at the egg stage. Here we tested how the consequences of life history plasticity, specifically early hatching in response to terrestrial egg predators, depend on the assemblage of aquatic larval predators. We predicted that diverse predator assemblages would impose lower total predation pressure than the most effective single predator species and might thereby reduce the costs of hatching early. We then conducted a mesocosm experiment where we crossed hatchling phenotype (early vs. normal hatching) with five larval-predator environments (no predators, either waterbugs, dragonflies, or mosquitofish singly, or all three predator species together). The consequences of hatching early varied across predator treatments, and tended to disappear through time in some predation treatments, notably the waterbug and diverse predator assemblages. We demonstrate that the fitness costs of life history plasticity in an early life stage depend critically on the predator community composition in the next stage.