HOST DIET QUALITY ALTERS PRIORITY EFFECTS OF COINFECTING PARASITES
ABSTRACT
Separate bodies of literature suggest that the ecology and evolution of host-parasite systems are influenced by the quality of a host’s diet and priority effects (i.e., when the order of exposure to multiple parasites influences the outcome of the infection). Although resource competition is invoked as a mechanism responsible for priority effects, the effect of host diet quality on priority effects is unexplored. We tested the hypothesis that low-quality diets reduce priority effects between the bacterial parasite Pasteuria ramosa and the microsporidian Hamiltosporidium tvaerminnensis in their daphnid host. We found that P. ramosa benefits from prior residency when hosts are fed high phosphorus (i.e., high-quality) but not low phosphorus diets. We were unable to detect priority effects for H. tvaerminnensis. These results are consistent with evidence from single-infection experiments on the same parasites, suggesting that low phosphorus diets could reduce resource competition between these parasites. Our study shows how the host’s environment can influence infection outcomes by moderating parasite interactions.
Understanding how host diet quality influences the outcome of host-parasite interactions is challenging (Smith, 2007; Aalto et al., 2015; Pike et al., 2019). Both the complexity and value of meeting this challenge are heightened by the routine occurrence of more than 1 parasite at a time in a single host (Cressler et al., 2016). Host diet quality could influence the relative success of coinfecting parasites (Lange et al., 2014), especially when the diet affects the strength of priority effects, i.e., when the order of exposure to multiple parasites influences the outcome of the infection (Jackson et al., 2006; Hoverman et al., 2013). If host diet quality mediates priority effects, it is likely to play an important role in the ecology and evolution of many host-parasite systems because both the quality of a host’s diet and the order in which hosts are exposed to parasites are influenced by seasonal endemic cycles and location (Søndergaard et al., 1999; Karvonen et al., 2019). However, the role of host diet quality in mediating priority effects has not, to our knowledge, been explored.
Shifts in host diet quality could enhance or ameliorate priority effects. A depletion of host resources by the prior resident is often expected to place parasites encountering infected hosts at a disadvantage relative to those encountering an uninfected host (Jackson et al., 2006; Mideo, 2009). If so, low-quality host diets might enhance the advantage of the prior resident by further depleting the resources available to a second parasite. However, low-quality diets can also limit the immune response of hosts (Chandra, 1996). Because host immune responses can produce antagonistic (Read and Taylor, 2001) or synergistic (Graham, 2008) interactions between coinfecting parasites, the effects of low-quality diets on priority effects are likely dependent on specific features of the host-parasite system. Differences in the virulence (e.g., decreases in host fecundity and survival) produced by simultaneous and sequential infections can also influence the relative success of coinfecting parasites (Manzi et al., 2021; O’Keeffe et al., 2024). Although simultaneous infections can be more virulent than sequential infections (Lohr et al., 2010), a reduction in parasite load caused by low-quality diets (Frost et al., 2008) could reduce the virulence experienced by all hosts, reducing the role of prior residency in dictating outcomes for the host. However, the effects of host diet quality on virulence can be difficult to predict (Pike et al., 2019).
In the present study, the effect of diet quality on priority effects was quantified in a well-studied coinfection system. The consumer Daphnia has well-documented negative responses to dietary phosphorus (P) limitation that include reductions in growth rate (Sterner et al., 1993), fecundity (Hessen, 1992), and in extreme cases survival (Prater et al., 2016). Host diet P content can influence the abundance of parasites in primary consumer hosts (Frost et al., 2008; Narr and Krist, 2015), but the precise mechanism responsible for these effects is unclear and differs across host-parasite systems (Bernot and Poulin, 2018; Sanders and Taylor, 2018; Frenken et al., 2021). Both the bacterial parasite Pasteuria ramosa (Metchnikoff, 1888) and the microsporidian parasite Hamiltosporidium tvaerminnensis (formerly Octosporea bayeri, Jirovec 1936) infect Daphnia magna Strauss, 1820 in rock pools on skerry islands in the Baltic Sea (Ebert, 2005; Haag et al., 2011), and both produce lower spore loads when D. magna are fed low P diets (Frost et al., 2008; Narr et al., 2025). Pasteuria ramosa and H. tvaerminnensis transmit horizontally through the ingestion of spores released from dead hosts, but H. tvaerminnensis also transmits vertically to the offspring of its host (Ebert, 2005). Although H. tvaerminnensis is more abundant in these rock pools (Ebert et al., 2001), previous work suggests that in both simultaneous and sequential infections, P. ramosa competitively excluded H. tvaerminnensis (Ben-Ami et al., 2011). We hypothesized that the relative availability of P in D. magna diets, which was inversely related to the prevalence and spore load of H. tvaerminnensis in rock pools (Narr et al., 2019), contributes to the success of H. tvaerminnensis in rock pools despite the observation of its competitive exclusion in experiments.
To test this hypothesis, we compared the spore loads, fecundity, and mortality rates of D. magna horizontally infected either simultaneously or sequentially with both parasites when D. magna were fed high or low P diets. Given that single infection experiments showed that low P host diets decrease the spore loads of both of these parasites in D. magna (Frost et al., 2008; Narr et al., 2025), we expected that low P diets would decrease the resource demand of each parasite, decreasing the potential for priority effects and limiting the virulence experienced by coinfected hosts. We also expected that low P D. magna diets would decrease the potential for priority effects in this system because low P diets altered the carbon (C):P ratios of P. ramosa and H. tvaerminnensis spores in different ways (Narr et al., 2024). If these changes in spore C:P ratio reflect shifts in P demand, we expect low P host diets to reduce resource competition (and the benefits of prior residency) for both parasites. Given that other studies have documented the competitive dominance of P. ramosa over H. tvaerminnensis in lab experiments (where D. magna is presumably fed a high-quality diet) and an inverse relationship between host diet P and H. tvaerminnensis prevalence in rock pools, we anticipated that the effect of prior residency in hosts fed high P diets would be stronger for P. ramosa than for H. tvaerminnensis.
MATERIALS AND METHODS
Study system
The aquatic crustacean D. magna is a cyclic parthenogen frequently used to study parasite-host and producer-grazer interactions (Sterner and Elser, 2002; Ebert, 2005). We infected D. magna (clone FI-OER3-3) with H. tvaerminnensis, a parasite of D. magna fat cells and ovaries (Haag et al., 2011), and P. ramosa, an endospore-forming bacterium that infects host hemolymph (Ebert et al., 1996). Both H. tvaerminnensis and P. ramosa infect D. magna in rock pools on islands in the Baltic Sea in southwestern Finland. Hamiltosporidium tvaerminnensis spores can be detected in D. magna as early as 11 days post-infection, but, especially for horizontal infections, low spore loads may prevent detection until a few days later (Vizoso and Ebert, 2004). Mature P. ramosa spores can be detected within 15–18 days, but immature spores can be detected a few days before this (Ebert et al., 2016). Expected horizontal transmission success for both parasites is approximately 100% at the spore loads administered in this experiment (150,000; Ebert et al., 2000; Vizoso et al., 2005).
Experimental design
To address our hypothesis that host diet P content influences priority effects, we manipulated the amount of P in algal diets by altering the amount of P (NaH2PO4) in the growth media (COMBO; Kilham et al., 1998) of continuously grown Scenedesmus obliquus algal cultures (Sterner et al., 1993). Then we mixed the cultures to achieve nominal molar C:P ratios of approximately 100 and 400. We verified the P concentrations of our diets throughout the experiment by filtering a known amount of algae onto pre-weighed filters, drying them, and then using the ascorbic acid molybdenum blue method following persulfate digestion (Greenberg et al., 1992). We calculated nominal C:P ratios based on the assumption that the algal dry mass was 50% C (the approximate percentage C of the same algal cultures measured during a concurrent experiment; Narr et al., 2024).
The H. tvaerminnensis isolate used in our experiment was collected from rock pools in southwestern Finland with our D. magna host clone and maintained in the lab for over a decade (D. Ebert, pers. comm.). The P. ramosa clone used was C19, which was cloned from an isolate (P1) from a farm field in Germany (54.282°N, 10.966°E). C19 has been used in many previous studies (Fredericksen et al., 2021), including recent work examining diet effects on P. ramosa and H. tvaerminnensis nutrient content (Narr et al., 2024), and the isolate it was derived from (P1) was used in a seminal coinfection study with H. tvaerminnensis in D. magna from southwestern Finland (Ben-Ami et al., 2011). Our infection treatments included 3 scenarios of parasite exposure: (1) H. tvaerminnensis first: hosts exposed to the microsporidian parasite H. tvaerminnensis on day 0 (early) and P. ramosa on day 6 (late); (2) P. ramosa first: hosts exposed to P. ramosa on day 0 (early) and H. tvaerminnensis on day 6 (late); and (3) simultaneous infection: hosts exposed to both parasites on day 0 (early). The timing of this exposure sequence is similar to that in other experiments quantifying priority effects in parasites of Daphnia (Ben-Ami et al., 2011; O’Keeffe et al., 2024). Spore solutions were obtained from homogenized infected individuals, diluted to a concentration of 150,000 spores/individual, and pipetted into the medium containing each D. magna. Simultaneous infection treatments consisted of 150,000 spores of each parasite. All 3 treatments and a control group of uninfected D. magna were replicated under 2 separate diets (high and low P), for a total of 8 experimental treatments. We added a homogenized uninfected D. magna solution to uninfected individuals (on days 0 and 6) and the simultaneously infected individuals (on day 6).
To maximize our sample size, D. magna neonates (<24 hr) were collected from the third and fourth clutch of adult uninfected D. magna clones born within 24 hr of each other and distributed into individual 20 ml containers of Daphnia COMBO medium. Daphnia magna were grown in individual containers for the duration of the experiment (starting on day 0). Approximately half of each treatment comprised individuals from each clutch for a total of 24 or 25 D. magna per treatment. The Daphnia were fed 2 mg C/L on days 0, 2, and 4 and 4 mg C/L on days 6, 8, and 10. On day 6, they were transferred to 20 ml of fresh COMBO and exposed to their second infection treatment. On day 12, all D. magna were transferred into 40 ml of fresh COMBO, fed 8 mg C/L every other day, and transferred to new media every 4 days until the end of our experimental trial period on day 18 (individuals from the fourth clutch) and 22 (individuals from the third clutch). The experiment ended earlier for D. magna taken from the fourth clutch because of high levels of mortality among individuals from both clutches in the simultaneous and H. tvaerminnensis first treatments (Table I). Mortality was recorded, and offspring were removed and counted every other day. On the final day of the experimental period, all living D. magna were rinsed in ultrapure water, measured, and frozen for spore counts. Spores of each parasite were distinguished based on characteristic differences in morphology visible at ×400 magnification (Ebert, 2005). Spore counts were conducted using 4 aliquot samples (11 µl) on a Neubauer improved hemocytometer and then divided by the weight of D. magna (calculated using length-weight regression from Ku et al., 2022).
Statistical analysis
We tested for the presence of priority effects on rank-transformed spore loads of hosts by using separate linear mixed effects models for each parasite with the fixed effect of infection sequence (sequential vs. simultaneous) nested within the fixed effect of diet treatment with the package lme4 (Bates et al., 2015). Because our experimental design did not allow us to compare (and control for) the effect of the time at which D. magna were exposed to parasites (early vs. late) on spore load and to ensure spores had sufficient time to develop before their hosts were harvested, we did not include the parasite load of the parasite in the late exposure treatment in these comparisons. Thus, we detected priority effects on spore load by comparing the spore loads of hosts infected by the focal parasite alone on day 0 (when the focal parasite was given priority) with those of hosts infected by both the focal parasite and its competitor on day 0 (when the focal parasite was not given priority). We included a random intercept to account for differences caused by the clutch origin of D. magna, and D. magna with no visible spores were excluded from this comparison.
We assessed the assumptions of these models by evaluating the relationship between the Pearson residuals and fitted values of each model. We evaluated the impact of infection sequence and diet on fecundity by fitting a negative binomial regression to account for overdispersion (Zuur et al., 2009) with the package MASS (Venables and Ripley, 2002). Diet and infection were added as interactions. Because the experiment was ended earlier for D. magna taken from the fourth clutch, we included the duration of the experiment for individuals from each clutch (18 vs. 22 days) as a covariate in these models. A mixed effects Cox proportional hazards model was used to assess the impact of diet and infection sequence on survival (Therneau and Grambsch, 2000). We first limited our analyses to the first 18 days of the lives of individuals from the fourth clutch and then used a right-censored design with time at risk based on the time (days) since the beginning of the experiment. We included clutch as a random factor in this model, and we assessed whether the proportional hazard assumption was met with model diagnostics, based on Schoenfeld residuals (Murray and Bastille-Rousseau, 2020). We used the survival package in R for this part of this analysis (Therneau and Grambsch, 2000; Therneau, 2024). To ease the interpretation of the fecundity and survival models, we calculated estimated marginal means of each treatment. We determined the ratio of expected marginal means (on the log scale) and used the Tukey method for estimating the significance of any differences between treatments. The emmeans package (Lenth, 2024) was used for this part of the analysis. We visualized differences in survival between treatments by plotting Kaplan-Meier survival curves with 95% confidence intervals.
RESULTS
The average P concentration of our diets throughout the experiment was 1.88% (SE = 0.16) and 0.37% (SD = 0.042) for the high and low P diets, respectively, which equate to nominal C:P values of 74 and 398, respectively. When D. magna were exposed to both parasites simultaneously, P. ramosa spores were detected in 75% (9 of 12 Daphnia alive at the end of the experiment) and 83% (5 of 6) of individuals fed the high and low P diets, respectively, whereas H. tvaerminnensis spores were detected in 42% (5 of 12) and 67% (4 of 6) of individuals fed the high and low P diets, respectively. All D. magna exposed to P. ramosa first and H. tvaerminnensis second (21 D. magna in the high P treatment and 22 in the low P treatment) that were alive at the end of the experiment had detectable P. ramosa spore loads. When D. magna were exposed to H. tvaerminnensis before P. ramosa, H. tvaerminnensis spores were detected in 50% (6 of 12 in the high P diet) and 40% (2 of 5 in the low P diet) of individuals.
When D. magna were fed high P diets, P. ramosa spore loads were higher when it was given priority over H. tvaerminnensis compared with when it was exposed simultaneously with H. tvaerminnensis (priority coefficient = 18.94, t = 3.0, df = 53, P = 0.004; Fig. 1). We did not detect any priority effects based on the spore loads of H. tvaerminnensis for hosts fed high P diets (4.0, t = 1.25, df = 14, P = 0.23) or either parasite in D. magna fed the low P diet treatments (H. tvaerminnensis: −1.25, t = −0.26, df = 14, P = 0.80; P. ramosa: −3.10, t = −0.40, df = 53, P = 0.69). All coefficients from each model are presented in Suppl. Data, Tables S1, S2.
Citation: The Journal of Parasitology 111, 6; 10.1645/24-144
Across diet treatments, D. magna in the H. tvaerminnensis first and simultaneous infection treatments were less fecund than were D. magna in both the uninfected and P. ramosa first treatments, but simultaneous infections had a particularly strong effect on the fecundity of D. magna fed low P diets (Fig. 2). The fecundity ratio of uninfected/simultaneously individuals fed the high P diet was 4.08, and the ratio for individuals fed the low P diet was 21.76 (Tables I, S3). Similarly, when D. magna were fed low P diets, D. magna in the H. tvaerminnensis first and simultaneous infection treatments had greater mortality than did D. magna in both the uninfected and P. ramosa first treatments (Fig. 3; Tables I, S4). These infection treatments had no effect on D. magna mortality when D. magna was fed high P diets.
Citation: The Journal of Parasitology 111, 6; 10.1645/24-144
Citation: The Journal of Parasitology 111, 6; 10.1645/24-144
DISCUSSION
Our experiment contributes to the growing body of literature indicating that prior residency of 1 parasite influences outcomes of coinfection (Karvonen et al., 2019) by showing how these effects can be moderated by host diet quality. We found that P. ramosa spore loads were higher when it had prior residency in hosts but only when hosts were fed high P diets (Fig. 1). Conversely, hosts exposed to P. ramosa first (and H. tvaerminnensis later) experienced lower levels of virulence than did hosts exposed to H. tvaerminnensis first or to both parasites simultaneously, especially when hosts were fed low P diets (Table I). The moderation of these effects by host diet quality is consistent with the effects of diet on this host-parasite system observed in single infection experiments and support the hypothesis that resource competition contributes to antagonistic effects between coinfecting parasites (Mideo, 2009).
We expected that high P diets for D. magna would enhance the benefits of prior residency for both parasites because previous work has shown that low P host diets could limit the overall resource demand and resource overlap between these 2 parasites. Diets low in P reduce Daphnia growth and fecundity (Sterner et al., 1993) and influence the success of both of the parasites studied here by decreasing their spore loads (Frost et al., 2008; Narr et al., 2025). Low P diets for D. magna also influenced the C:P ratios of P. ramosa and H. tvaerminnensis spores in different ways (Narr et al., 2024). Consistent with our predictions, our results show that P. ramosa benefited from prior residency when hosts were fed high P diets, but we did not detect a significant priority effect benefiting H. tvaerminnensis under either diet treatment. In other experiments, including an experiment on our host-parasite system (Ben-Ami et al., 2011), no priority effects were found among parasites (Lohr et al., 2010; Hoverman et al., 2013; O’Keeffe et al., 2024), possibly because of species-specific (rather than cross-reactive) immune responses (Hoverman et al., 2013). In our experiment, the smaller number of confirmed H. tvaerminnensis infections present at the end of the experiment (9 and 8 hosts for the simultaneous and H. tvaerminnensis first treatments, respectively) likely limited our ability to detect priority effects for this parasite. However, this low sample size was the result of intrinsic characteristics of horizontal H. tvaerminnensis infection, i.e., high host mortality and low spore loads, both of which could prevent detection of H. tvaerminnensis infection. High mortality would likely also reduce priority effects in natural systems. Other differences between these 2 parasites (e.g., differences in within host density dependence; Ebert et al., 2000; Vizoso and Ebert, 2004) may also enhance the benefits of prior residency for P. ramosa relative to H. tvaerminnensis when hosts are fed high-quality diets.
We expected that by reducing the spore loads of both parasites low P diets would decrease the virulence experienced by all hosts. However, we found that low P diets enhanced the reduction in fecundity caused by simultaneous infections and increased the mortality rates of hosts exposed to H. tvaerminnensis before or simultaneously with P. ramosa (Table I). This enhanced virulence is consistent with the effects of low P diets on D. magna with single H. tvaerminnensis infections (Narr et al., 2025). Low P diets enhance the ingestion rates of this D. magna clone (Narr and Frost, 2016), which likely increases the rate at which these crustaceans ingest spores. Increased ingestion of spores is, in turn, expected to increase cell damage during the infection process (Vizoso and Ebert, 2005). Why late exposure to H. tvaerminnensis did not induce this virulence is unclear. Early exposure to P. ramosa may buffer the host against the virulence induced by H. tvaerminnensis infection. Coinfection with P. ramosa decreased the virulence experienced by D. magna infected by white fat cell disease and actually increased the fecundity of hosts relative to uninfected D. magna (Reyserhove et al., 2017). However, reduced virulence in hosts exposed later to H. tvaerminnensis may also have been caused by the effect of host age on the virulence exerted by this parasite. So-called age effects have not, to our knowledge, been previously documented for H. tvaerminnensis, but the virulence of the microsporidian Ordospora colligata was higher in younger D. magna (O’Keeffe et al., 2024). If younger D. magna are more affected than older animals by H. tvaerminnensis, this would explain the reduced virulence experienced by hosts infected with P. ramosa first (and H. tvaerminnensis second) in our experiment. Our study design did not allow us to distinguish between a potential buffering effect of P. ramosa and age effects on the interaction between D. magna and H. tvaerminnensis.
Because of logistical constraints, we did not include single-infection controls in our experiment, so our inference is limited to the effects of host diet on prior residency in coinfected hosts. This design differs from that of other studies in which the effects of prior residency in coinfected hosts was quantified and these findings were compared with those from individuals infected with 1 parasite during the first and/or second infection period. The most obvious drawback of our simplified experimental design is that it does not allow us to distinguish between effects caused by the timing of the infection (i.e., age effects) and those caused by the presence of the other parasite. However, by limiting our comparison of parasite load to hosts exposed to the focal parasite on day 1, our experimental design enables us to assess how an additional factor (host diet) mediates the strength of priority effects on spore load. This simplified experimental design may provide a useful template for a first step in investigating the effects of other aspects of a host’s environment on prior residency. This design is particularly appropriate for studies in which (1) the effects of the mediating environmental factor on single infections are well documented and (2) the additional replication required to quantify environmental effects prohibits more conventional experimental designs.
For our host-parasite system, the reduced advantage of prior residency by P. ramosa when host diets were low in P may help explain why H. tvaerminnensis is so abundant in the natural rock pools in which it is native (especially those with high nitrogen:P ratios; Narr et al., 2019), despite previous work indicating that P. ramosa dominates H. tvaerminnensis in the lab (Ben-Ami et al., 2011). Although H. tvaerminnensis is capable of both vertical and horizontal transmission, horizontal transmission plays a critical role in maintaining the prevalence of this parasite during the host population’s asexual growth phase (Lass and Ebert, 2006). If low P host diets help even the playing field between horizontal infections of these parasites in rock pools where they co-occur, we might expect P. ramosa to dominate pools with consistently higher P inputs, especially when hosts are exposed to P. ramosa before H. tvaerminnensis. Given that both parasites transmit horizontally through spore banks that build up over time, this priority effect may serve primarily to prevent the establishment of H. tvaerminnensis in pools where P. ramosa spore banks have already been established. Although P. ramosa and H. tvaerminnensis co-occur in D. magna in rock pools in southwestern Finland and our diets reflect the C:P ratios of particulate matter in these pools, the P. ramosa clone used in this experiment is from Germany. As a result, inferences regarding these interactions in the field should be made with caution. Our experiment quantifies the effects of host diet quality and infection sequence on the early within-host growth period of these parasites, and at present very little is known about how parasite responses to these factors are governed by genotype-genotype or genotype-environment interactions.
Our experimental results indicate that host diet quality can influence priority effects between coinfecting parasites, suggesting the need for a better understanding of how parasite characteristics dictate the outcome of resource competition within hosts. Given that competition for resources likely plays a central role in dictating the outcome of sequential infection in many hosts and anthropogenic modification of host diet quality likely coincides with shifts in the timing of host exposure to parasites in many habitats, we suspect that interactions between host diet quality and priority effects help shape the ecology and evolution of many parasites.
Spore loads (spores per microgram of Daphnia magna) of Hamiltosporidium tvaerminnensis (A) and Pasteuria ramosa (B) in D. magna fed high and low phosphorus (P) diets and exposed to the 2 parasites simultaneously (dots), H. tvaerminnensis first (stripes), or P. ramosa first (crosshatch). Asterisk denotes a significant difference (α = 0.05). The scale for H. tvaerminnensis is nearly an order of magnitude lower than that for P. ramosa.
Fecundity (total number of offspring produced per Daphnia magna over the duration of the experiment) for individuals fed high or low phosphorus (P) diets and exposed to the 2 parasites simultaneously (dots), Hamiltosporidium tvaerminnensis first (stripes), Pasteuria ramosa first (crosshatch), or neither parasite (uninfected, open).
Kaplan-Meier survival curves for individual Daphnia magna fed (A) high or (B) low phosphorus (P) diets and exposed to Hamiltosporidium tvaerminnensis first (dotted line), Pasteuria ramosa first (dot-dash line), the 2 parasites simultaneously (dashed line), or neither parasite (solid line). Shaded lines indicate 95% confidence intervals for each treatment. The survival curves for P. ramosa–infected D. magna and uninfected D. magna are nearly identical for D. magna fed the low P diet.
Contributor Notes