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Host–parasite interactions, while often studied in isolation, are affected by a multitude of direct and indirect effects from other members of the community (Omacini et al. 2001; Lafferty 2004; Lafferty et al. 2006). Predators are one notable community member that can strongly impact infectious diseases. In some cases, predators can increase infection in their prey (e.g., through impacts on host immune function or host traits; Ramirez and Snyder 2009; Duffy et al. 2011). In other cases, predators can decrease disease risk in their prey (e.g., by decreasing prey population sizes or directly consuming infected hosts; Packer et al. 2003; Keesing et al. 2006; Duffy et al. 2005). Predators can also impact disease risk in non-prey species via consumption of disease vectors or free-living stages of parasites (Grutter 1996; Nelson and Jackson 2006; Orlofske et al. 2012). Therefore, predators have the potential to alter rates of infection in both prey and nonprey species through a variety of routes.
The potential role of predation in reducing infectious disease risk is of particular interest when applied to medicine and conservation. Indeed, manipulation of predator densities has been suggested as a potential conservation measure (Packer et al. 2003). In some cases, the focus is on the potential for predators to reduce density of vectors. For example, augmentation of populations of mosquito predators has been suggested as a way to control mosquito-borne parasites such as malaria (Nelson and Jackson 2006; Howard et al. 2007). In other cases, the focus has been on the potential for predators to directly prey upon the parasites. It is this latter scenario that is the focus of the experiments reported here. We studied the potential for predation upon the fungus Batrachochytrium dendrobatidis (Bd), which has caused population declines and extirpations of amphibians around the globe (Bosch et al. 2001; Lips et al. 2006; Skerratt et al. 2007; Wake and Vredenburg 2008). Predation by zooplankton on free-swimming Bd zoospores has been suggested as a possible method for biocontrol of this fungus (Buck et al. 2011). Our study further evaluates this possibility.
There are reasons to expect that free-swimming Bd might be vulnerable to predation. First, Bd is generally transmitted through an aquatic zoospore stage that swims through water to infect new hosts (Longcore et al. 1999). The length of time that zoospores can remain infectious is context-dependent; Piotrowski et al. (2004) found that 95% of zoospores stop moving after just 24 h in distilled water, while Johnson and Speare (2003) reported motile zoospores in lake water after 7 weeks. Given the potential for a long free-swimming stage, Bd zoospores may be at risk of direct predation during this infectious period. Second, many bodies of water contain numerous microcrustaceans that have the potential to consume Bd zoospores. For example, Daphnia are generalist grazers of algae, bacteria, cyanobacteria, protozoans, fungi, and detritus. One species of Daphnia (D. galeata hyalina) has been shown to consume zoospores of a pathogenic chytrid of diatoms, reducing infection in the hosts (Kagami et al. 2004). Bd zoospores are generally 3−5 μm in diameter (Longcore et al. 1999), which is within the preferred range of food size for many Daphnia (Burns 1968; Geller and Muller 1981). Therefore, Daphnia are good candidates for predators of Bd.
Three previous studies have directly investigated the potential for Daphnia to impact Bd. In a laboratory experiment, Buck et al. (2011) demonstrated that Daphnia can consume Bd zoospores. However, the ability of Daphnia to digest those zoospores was not tested, and previous studies have shown that passage through a Daphnia gut can actually increase growth of some organisms (Porter 1976). Therefore, it is possible that Bd zoospores can be ingested by Daphnia but not digested. Two additional laboratory studies demonstrated that Daphnia reduce the number of zoospores detected in water samples (Woodhams et al. 2011; Hamilton et al. 2012), but a mesocosm experiment did not find changes in infection rates in tadpoles (Hamilton et al. 2012). Each of these studies only investigated one species of Daphnia, but Daphnia species vary in body size, which can influence feeding preferences and rates (Burns 1968; Hall et al. 2007). Additionally, the laboratory studies testing for Daphnia predation upon Bd (Woodhams et al. 2011; Hamilton et al. 2012) combined Daphnia and Bd zoospores in clean water without the presence of other food sources for Daphnia. Other studies have demonstrated that gut passage time and food assimilation efficiency in Daphnia change with food concentration (DeMott et al. 2010). Therefore, the presence of alternative food resources (as is the case in natural communities containing Bd and Daphnia) may alter the consumption and digestion rates of Daphnia on Bd zoospores.
In this study, we tested the ability of Daphnia to consume Bd zoospores and reduce both environmental levels of Bd and infection in tadpoles. In a series of laboratory experiments, we varied density of two species of Daphnia (D. magna and D. dentifera) to compare the effectiveness of each species at consuming Bd. Additionally, as algal levels can vary greatly between water bodies, we manipulated the density of suspended algae (food for Daphnia) to determine its effects on Bd consumption by D. dentifera. Our goal was to understand the impact of zooplankton predation on Bd levels in the environment and hosts.
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Our results demonstrate that direct predation upon parasites can reduce density of parasites in the environment and infection in hosts. Specifically, we found that Daphnia can reduce Bd levels in water and infection in tadpoles, but this effect was context-dependent. Daphnia abundance, Daphnia species identity, food concentration, and grazing period all affected the ability of Daphnia to reduce Bd in water and tadpole samples. Therefore, caution is warranted in assuming that Daphnia can successfully reduce infection in amphibians in natural systems.
In experiment 1, we found that high densities of both Daphnia species reduced Bd in water samples (see Fig. 2C, 3A), as previously demonstrated by Hamilton et al. (2012) and Woodhams et al. (2011). We also show that Daphnia can reduce tadpole infection (see Fig. 2B), but this effect only occurred for one of the two species we used in this study; direct comparison between the two Daphnia species (see Table 2) showed that only D. magna were able to reduce infection in tadpoles. As both species reduced the amount of Bd detected in water samples, this indicates that these species have similar rates of Bd consumption. However, zoospores may survive gut passage but be damaged and unable to infect tadpole hosts. Daphnia magna are larger than D. dentifera, and therefore are able to filter more water in a given time period (Burns 1969). Thus, zoospores are more likely to be consumed multiple times by D. magna than by D. dentifera. Therefore, D. magna may reduce infectiousness of zoospores at a greater rate than D. dentifera, even if the relative rates of Bd digestion are similar. Alternatively, Bd zoospores do not have thick cell walls or sheaths (Longcore et al. 1999), which suggests that they should not be particularly digestion resistant. Therefore, other mechanisms may drive the different effects of these two Daphnia species. Our results suggest that, when studying the effects of Daphnia on Bd in the field, it is important to consider the species identity and size of the Daphnia that are present.
High densities of Daphnia were able to reduce Bd in water and tadpole samples, but not in all circumstances. While the Daphnia densities we used in this experiment are within the range of densities found in natural systems, our highest densities (25 Daphnia per beaker; 100 Daphnia per L) and the highest densities in previous studies (1400 Daphnia per L; Hamilton et al. 2012; 1600 Daphnia per L; Woodhams et al. 2011) were likely above most natural densities. Field densities of Daphnia can occasionally reach over 100 individuals per L (e.g., ~150: Luecke et al. 1990; up to 104: DeMott and Gulati 1999), but many field surveys have reported maximum densities below 50 individuals per L (Kwik and Carter 1975; DeMott 1983; Dawes et al. 1987). Thus, densities as high as those found in our 25 Daphnia treatments and as those used in previous studies are unlikely to be commonly found in nature. Limitations on Daphnia abundance from competition or predation may decrease the likelihood of Daphnia reducing Bd infection in natural systems.
In the absence of Daphnia, we found that the amount of Bd detected in water was lower when concentrations of algae were higher (see Fig. 4C). This pattern suggests a direct negative interaction between algae and Bd zoospores. This could occur if high concentrations of algae interfere with the ability of zoospores to swim through water through physical interference. Alternatively, some green algae exhibit allelopathy (Wolfe and Rice 1979), so A. falcatus may release chemicals that directly kill or impair Bd. Future studies are necessary to understand the direct impacts of algae on Bd zoospores. We also found that when Daphnia were present, the amount of Bd detected in water samples showed a different pattern; in this case, Bd was higher when densities of algae were higher (see Fig. 4C). It is possible that this pattern is driven by food saturation in the high-food treatments, where Daphnia were unable to consume all the algae and Bd in the water. However, individual D. dentifera under these conditions can filter over 10 mL water per day (Hall et al. 2010), so it is likely that the entire contents of our beakers would have been filtered at least once during the 24-h experiment. Gut passage time and food assimilation in Daphnia vary with food density; when food densities are low, gut passage time increases and assimilation efficiency of field-collected algae increases (DeMott et al. 2010). Therefore, even if Daphnia in the low-food treatments consumed the same number of zoospores as in the high-food treatments, a greater proportion of those zoospores may have been digested. This indicates that zoospores may be better able to survive passage through the gut of a Daphnia in high-food conditions. Alternatively, Daphnia can exhibit selective grazing (Burns 1968; Porter 1976; Haney 1987), so high densities of algae could have led to Daphnia consuming fewer Bd zoospores if A. falcatus is their preferred food. These results have implications for Bd disease risk in natural systems. In eutrophic lakes, for example, high densities of algae may have direct negative effects on Bd zoospores, reducing disease risk for amphibians. However, high densities of algae may reduce digestion of Bd zoospores, which would create an indirect positive effect of algae on Bd. It is unknown how these conflicting forces will affect Bd levels in eutrophic environments.
In both experiments 1 and 2, we found that treatments with longer grazing periods almost always reduced Bd in our samples. As we saw this pattern across all Daphnia densities (including treatments with no Daphnia), this is unlikely due to effects of Daphnia grazing. Bd zoospores were only added once at the beginning of the grazing period and have a limited lifespan in water (Piotrowski et al. 2004). Therefore, it is likely that we detected less Bd in water samples after 72 h due to increased time for zoospore mortality compared with the 5-h grazing periods. When zoospores die, their cells and DNA degrade, resulting in lower qPCR values. Additionally, when tadpoles were added after the beakers after 72 h of grazing, there were fewer surviving zoospores able to infect the tadpoles. It is possible that we would have found different results had we exposed tadpoles to Bd while simultaneously allowing Daphnia to graze. The ability of Daphnia to consume Bd in natural systems is likely affected by the length of time that zoospores remain in the water before finding hosts. Thus, if zoospores are able to find hosts quickly, then the effects of Daphnia grazing may be limited.
Another notable trend we observed is that patterns found in water samples were not necessarily similar to the observed patterns in tadpoles from the same experiment. For example, in both experiments 1 and 2, D. dentifera reduced Bd in water samples, but had no effect on tadpole samples. This indicates that infection in tadpoles is not necessarily dose-dependent. A previous experimental study demonstrated that only one of three amphibian species tested exhibited a dose-dependent response to Bd (Gervasi et al. 2013). Multiple factors may be involved in determining infection in tadpoles. For example, there is variation within amphibian species in anti-Bd microbial defenses (Harris et al. 2006; Lam et al. 2010), and different species exhibit behaviors that affect their chances of becoming infected (Rowley and Alford 2007). These factors may have large effects on Bd infection in tadpoles and sometimes outweigh the effects of zoospore densities. Therefore, it is essential to monitor Bd in both water bodies and amphibian hosts.
Our study demonstrated that direct predation on parasites can reduce infection of a deadly fungal parasite responsible for amphibian population declines and extirpations around the globe. However, this effect was context-dependent and varied with predator species, predator density and resource availability. Therefore, it cannot be assumed that predators will successfully act as biocontrol agents for infectious diseases, even if they have the ability to consume parasites. When attempting to understand the effects predation upon parasites, numerous biotic and abiotic conditions must be considered.