BACKGROUND
the production and biomass of organisms are affected by the abundance and quality of available resources  and predators   <cit> .
in aquatic ecosystems, fish and invertebrate predators have attracted the most interest in food web studies, while the role of parasites in food webs has been largely neglected despite their pervasiveness and importance.
parasite “predation” and interaction with its host’s nutritional status can be far subtler than the direct impact of fish predation.
parasites drain energy from their hosts, thereby affecting their hosts’ growth, reproduction and survival and, consequently, the population dynamics of the hosts  <cit> .
traditionally, heavy parasite burdens have been interpreted as a sign of poor host condition, while hosts in good condition may escape serious infections because they can afford to invest in immune responses or other defences against parasites .
this view is, however, challenged by experiments demonstrating that parasites can perform better in well-fed hosts .
generally, the resources acquired by the parasite are considered in terms of energy, i.e., the resource quantity in terms of carbon   <cit> , while the role of elemental nutrients such as nitrogen  or phosphorus  as limiting resources for parasite growth has received less attention  <cit> .
the inclusion of elemental ratios  in models may generate substantially different population dynamics in prey and predator systems compared with previous models that considered only resource quantity  <cit> .
when the relative element content of the food  is below the threshold elemental ratio  of the consumer, there will be c in relative excess, and consumer c-use efficiency and growth rate will decrease even when food is abundant  <cit> .
thus, at high food abundance but low food quality, consumers will suffer from “quality starvation”  <cit> .
interactions with other consumers, such as predators or parasites, may further complicate the consumer population response to food quality, and, in fact, resource competition theory has been suggested as a basis for predicting the outcomes of infectious diseases  <cit> .
according to this theory, hosts and pathogens compete for the resources  that the host uses for its own growth and maintenance in a pathogen-free situation  <cit> .
“quality starvation” of hosts would be expected to increase the strength of competition for limiting elements between hosts and parasites, and the net outcome of this enhanced competition would depend on the demands of the host and the parasite for the limiting nutrient  <cit> .
thus, a low quality diet might, despite reducing the fitness of the host, also decrease the relative effect of parasites.
alternatively, the increased drain of nutrients caused by parasites might further amplify the effect of nutrient limitation on hosts and lead to decreased host reproduction, as well as increased mortality  <cit> .
correspondingly, parasite demands will determine the effect of host resources on parasite growth.
understanding the interactions between parasites and food quality is essential to predict the outcomes of infectious diseases in the face of changing nutrient loading  <cit>  and whether focus at the individual or population level may yield different predictions.
we studied how host nutrient limitation combined with parasite infection affected the host and the parasite populations in a model system consisting of a freshwater crustacean, daphnia magna, and its microparasite.
in aquatic environments, daphnia often play a key role in mediating effects between primary producers and secondary consumers  <cit>  and, consequently, in the recycling of nutrients  <cit> .
daphnia have a high demand for p  <cit>  and have an ability to maintain a high degree of homeostatic control  <cit> .
because daphnia commonly feed on p-limited seston , they are likely to face p limitations on growth in nature .
daphnia populations in nature are frequently parasitised by various species of bacterial, fungal and helminth parasites, with negative effects on population density .
at the individual level, host nutrient limitation has been shown to decrease growth and production of infective propagules for bacterial  <cit>  and fungal  <cit>  parasites of daphnia.
however, effects detected at individual level might not always predict the outcome of host-parasite interaction at population level  <cit> .
at present, knowledge is sparse on how nutrient limitation affects parasite performance at the host population level or how this affects the host populations.
RESULTS
population dynamics
during the 32-day experiment, the population numbers peaked at approximately  <dig> individuals after 28 days in the uninfected populations fed with the p-sufficient algae .
for the infected populations fed with the p-sufficient algae, the numbers reached a lower density of approximately  <dig> individuals compared with the uninfected populations  and infection status ; table  <dig>  within subject contrasts for d × i).
the uninfected populations fed the p-limited food reached a maximum density of approximately  <dig> individuals, while the infected p-limited populations remained at a lower level of fewer than  <dig> animals .
in the p-sufficient treatments, the numbers of adults increased towards the end of the experiment.
in the infected populations, this occurred sooner but ended at a lower level compared with the uninfected populations .
juvenile abundance in the p-sufficient treatments peaked between days  <dig> and  <dig> and decreased thereafter, and this decline started sooner in the infected populations .
in the p-limited treatments, the numbers of adults did not differ between the infected and the uninfected populations .
the abundance of juveniles in the p-limited populations was at first higher in the infected than in the uninfected populations.
in the uninfected p-limited populations the numbers of juveniles declined to zero, followed by a new increase at day  <dig>  while in the infected populations the numbers increased more steadily .figure  <dig>
abundances and biomasses in different treatments in the 32 day experiment.
mean total  adult and  juvenile abundances  of daphnia magna in uninfected control populations fed with p-sufficient  or p-limited  green algae and in populations infected with a microsporidian glugoides intestinalis ,  mean carbon biomass mg c l- <dig>  in all four treatments and relative biomass  of the infected populations for the p-sufficient  and p-limited  treatments during the experiment,  mean individual size of adult, and  juvenile daphnia μg c .
the populations were started with  <dig> juveniles each born to uninfected mothers fed with p-sufficient algae, and half of the animals were infected before allocation to feeding treatments.
see materials and methods for calculation of relative biomass.
note that the abundances are given per population in  <dig>  l vials to emphasise the actual densities, while biomass is given per l to aid comparison with previously published results.
the legend in panel  applies to treatments in all panels  - , while the legend in panel  indicates the relative biomass in panel .
results of the repeated measures anovas for
daphnia
population data
p
-
sufficient
p
-
limited
df
f
p
df
f
p
tested variables are population abundance , abundances of adults and juveniles, population biomass  and individual sizes  of adults and juveniles for uninfected and infected daphnia magna in p-sufficient and p-limited populations in the 32-day experiment.
a)between subjects effects, b)within subject effects, c)ln-transformed, d)transformation y = -1/^ <dig>  e)greenhouse geisser corrected statistics, f)square root-transformed, g)transformation y = ln.
within-subject contrasts for repeated measures anova
abundance
individual size
total
adult
juv.
biom.
adult
juv.
within-subject contrasts for the interaction between day  and infection  for the repeated measures anova on daphnia population data presented in table  <dig>  the contrast is “difference”, which compares each level to the mean of previous levels.
a carbon content of 44 ±  <dig> %  dry weight was found for uninfected animals in the p-sufficient treatment at the end of the 32 day population experiment and was used in subsequent biomass calculations.
the populations fed with p-sufficient algae yielded maximum biomasses of  <dig>  to  <dig>  mg c-1 l- <dig>  with no significant interaction detected between the uninfected and the infected populations over time .
in the populations fed with the p-limited algae, biomasses remained below  <dig>  mg c-1 l- <dig> , and there was a significant difference between the uninfected and the infected populations in biomass accumulation over time; the infected populations had increasingly lower biomasses than the uninfected populations from day  <dig> until day  <dig> .
the overall effect of infection on population biomass is illustrated in the relative biomasses of the infected populations compared to those of the uninfected ones .
for the p-sufficient treatment, the relative biomass remained at approximately  <dig>  to  <dig>  for the entire experimental period .
the p-limited treatment experienced higher variability , and at the end of the experiment, infection led to a dramatic decline to less than half of the biomass of the uninfected populations .
in the p-sufficient treatment, adults were larger in the uninfected than in the infected populations , but significant d × i interactions indicated that this difference became apparent only after the second generation became adults .
juvenile size in the p-sufficient populations was lower for the uninfected than the infected animals on days  <dig> and  <dig> .
in the p-limited populations, both adults and juveniles were at first larger in the infected than in the uninfected populations , but the situation reversed after day  <dig> .
in the 14-day experiment, population growth was slightly higher than in the 32-day experiment , but the differences among treatments confirmed the pattern observed in the 32-day experiment.
at the end of the 14-day experiment, the mean abundances of animals did not differ between the infected and the uninfected populations in either of the feeding treatments , but the numbers were significantly higher in the p-sufficient populations  for uninfected and infected daphnia, respectively) than in the p-limited treatments .
the mean size of the founder animals fed with p-sufficient food was higher than for those fed with p-limited food and higher for uninfected than infected animals .
the difference in size between the uninfected and the infected animals was larger for animals fed with the p-sufficient food .figure  <dig>
the mean sizes of founder animals in the 14 day experiment.
the mean carbon biomasses mg c l- <dig>  of the uninfected and infected founder animals of equal age fed with the p-sufficient  or the p-limited  algae at the end of the 14 day experiment.
population abundance and mean size in the 14 day population experiment
df
f
p
two-way anovas for the population abundance and the biomass  of the uninfected and infected founder animals at the end of the 14-day experiment for daphnia magna in p-sufficient and p-limited populations.
parasite load
at the end of the 32-day experiment, the mean number of parasite clusters in adult daphnia was 569 ±  <dig>  for daphnia fed with the p-limited food and was significantly greater than the mean of 241 ±  <dig> for daphnia fed with the p-sufficient food .
the mean parasite load per carbon biomass of daphnia was significantly higher for the animals fed with the p-limited food than for animals fed with the p-sufficient food .
animals dissected from uninfected populations were found to be free of parasites.figure  <dig>
parasite load in the different feeding treatments.
the number of glugoides intestinalis spore clusters in the gut of daphnia magna  for animals fed with the p-sufficient food  and for animals fed with the p-limited food  at the end of 32 day and 14 day population experiments, and in animals exposed individually before allocation to the two feeding treatments.
the number of spore clusters  per daphnia biomass  in the p-sufficient  and the p-limited  treatments at the end of 32 day and 14 day population experiments.
pairwise comparisons between treatments within different experiments: *p < <dig> , **p < <dig> , ns not statistically significant.
at the end of the 14-day experiment, in founder animals of the same age, the mean number of parasite clusters was significantly higher for daphnia fed with the p-limited food  than for daphnia fed with the p-sufficient food .
the mean parasite load per carbon biomass of daphnia was significantly higher for the animals fed with p-limited food than for animals fed with p-sufficient food .
no parasites were detected from animals dissected from uninfected populations.
in animals that were exposed pairwise to the same donors and allocated singly to different food treatments, the mean numbers of parasite clusters did not differ between animals fed with the p-limited  or the p-sufficient  diet .
the number of spore clusters in animals fed with the p-sufficient or the p-limited algae did not correlate with each other .
the number of spore clusters in donor females did not correlate with the number of spore clusters in the experimental daphnia fed with the p-limited algae, but the correlation was positive for animals fed with the p-sufficient algae .
discussion
the results of our experiments demonstrated that infection by the microsporidian glugoides intestinalis exerted a strong check on the population density of daphnia, regardless of food quality, in agreement with the results of earlier studies  <cit> .
over time, however, population biomass was more severely affected by the parasite under p-limited conditions, clearly suggesting that the infection posed an extra burden on animals suffering from dietary nutrient deficiency.
contrary to expectations based on results from other daphnia-parasite systems  <cit> , animals collected from the p-limited populations had higher numbers of parasite spore clusters than animals from the p-sufficient populations.
an additional experiment indicated that food quality did not affect the development of spores.
this result suggests that the greater number of spore clusters in the p-limited populations is the result of population level phenomena arising from feedback effects between spore production and exposure in host populations.
in the early phase of the experiment, the infected populations produced more offspring than the uninfected populations.
for daphnia, a life history shift towards producing more offspring earlier in life could compensate for decreases later in life caused by parasitism.
such a plastic response has been previously demonstrated for d. magna exposed to g. intestinalis  <cit> .
however, during the course of the experiment, densities in the infected populations decreased due to hampered reproduction  and/or increased juvenile mortality.
in the p-limited populations, the population biomass decreased to 50% in the infected populations relative to the uninfected populations by the end of the experiment.
our results suggest that under severe p limitation the additional burden from parasites intensifies the negative population impacts of nutrient limitation, agreeing with previous reports on the combined effects of nutritional stress and parasite infection on hosts  <cit> .
according to the individual level experiments, where individual daphnia juveniles were first exposed to a single dose of parasite spores and then reared either under p-sufficient of p-limited diet, host food quality did not directly affect the development of g. intestinalis spore clusters within the hosts.
in other host-parasite systems, e.g.
a fungal parasite in a diatom host  <cit>  and a bacterial parasite in daphnia  <cit> , host p-limitation has been shown to affect parasite growth negatively.
g. intestinalis does not seem to be limited by p, but possibly by some other nutrient or c  <cit> .
as parasite within-host growth did not differ between p-sufficient and p-limited hosts, the difference in parasite loads between the two feeding treatments might have simply reflected differences in the per capita exposure rates to spores because of differences in population densities.
the transmission of g. intestinalis is density-dependent, and increased per capita exposure to spores increased the probability of infection  <cit> .
because of the 10- <dig> day lag between exposure and the onset of spore production  <cit> , most of the spores in the populations during the first three weeks were produced by the founder animals, which were exposed in the initial phase of the experiment.
in the p-limited populations, the density of juveniles that were not yet producing spores themselves but filtering spores from the water was lower than in the p-sufficient populations.
consequently, in the p-limited populations, spores were shared among fewer animals than in p-sufficient populations, leading to higher number of spore clusters per animal in the p-limited populations.
the p limitation thus led to higher parasite numbers by increasing the per capita exposure rates to parasite spores via density effects at the host population level .figure  <dig>
conceptualisation of population responses and per capita parasite infection related to diet treatment.
the p-limited diet  led to a five- to six-fold decrease in population numbers of daphnia magna compared to populations receiving p-sufficient food  but did not affect the spore production in infected founder animals .
because of a 10- <dig> day time lag in parasite development, the density of juveniles, not yet producing spores but filtering spores from water, remained lower in the p-limited than in the p-sufficient populations, leading to a higher per capita spore load in the p-limited daphnia populations.
additionally, changes in filtration rates could affect the intake of spores from the water  <cit> , as well as their release into the water from the gut  <cit> .
crowding in the p-sufficient populations might have decreased their filtration rates  <cit> , while low food quality in the p-limited population might have increased their filtration rates  <cit> .
indeed, water from the crowded daphnia populations has earlier been shown to reduce transmission of g. intestinalis  <cit> .
on the other hand, previous experiments with the daphnia clone used in this study found five-fold lower ingestion rates for animals grown with the p-limited algae than for those fed with the p-sufficient algae  <cit> ; thus, the responses of animals subjected continuously to food quality limitations might differ from those obtained with short-term changes in the diet as performed in the ingestion rate experiments  <cit> .
while the differences in the spore loads in the 32-day experiment may have been confounded by age and size, in the 14-day experiment all daphnia screened for parasites were the founders of populations of the same age and had equal exposure time for infection.
our results are in agreement with studies showing a “dilution” effect, where high host density or high diversity of hosts and/or non-hosts can remove parasite propagules, which can lead to a decrease in parasite spread .
to reveal the complex interactions between host and parasite under various p-regimes, pools and mass balance for c and p should be studied for both the host and the parasite.
resolving the effect of p on parasite fitness would require measurements of transmission success as well as infectivity of the spores to the next host.
additionally, the relationship between parasite loads and available nutrients should be further studied using a wider array of host clones to reveal possible genotype x environment interactions  <cit> .
the population experiments were performed within a confined space with donor-controlled algal dynamics, i.e., the algal stock was replenished in cultures every second day.
this setup differs from a situation where the algal c:p dynamic is governed by the interaction between grazing and recycling of nutrients by herbivores  <cit> .
the uninfected daphnia dynamics in our batch-type setup resemble those obtained in two-step flow-through chemostats  <cit> .
however, parasite spore availability might have been different in a flow-through system, where a wash out of spores is possible.
although the phenomenon described here might be transient, it could operate within populations in closed systems, such as in experimental settings, and could be important in natural ecosystems, especially in ponds and small lakes.
previously, frost et al.
<cit>  reported reduced pathogen production, and penczykowski et al.
<cit>  reported reduced transmission potential in individual hosts receiving low-quality food; both reports suggested that reductions in disease incidence could occur in ecosystems limited by poor resource quality.
our results indicate that through its effects on population dynamics, nutrient limitation may increase parasite exposure and aggravate the negative effects of parasite infection at the population level.
CONCLUSIONS
for the host-parasite combination used in the current study, the additional burden from parasites intensified the negative population impacts of nutrient limitation.
furthermore, our results suggest that nutrient limitation affected parasite transmission and exposure via its effect on host population dynamics rather than by directly affecting parasite growth within the host.

 the production and biomass of organisms are affected by the abundance and quality of available resources and predators. in aquatic ecosystems, fish and invertebrate predators have attracted the most interest in food web studies, while the role of parasites in food webs has been largely neglected despite their pervasiveness and importance. parasite “predation” and interaction with its host’s nutritional status can be far subtler than the direct impact of fish predation. parasites drain energy from their hosts, thereby affecting their hosts’ growth, reproduction and survival and, consequently, the population dynamics of the hosts. traditionally, heavy parasite burdens have been interpreted as a sign of poor host condition, while hosts in good condition may escape serious infections because they can afford to invest in immune responses or other defences against parasites. this view is, however, challenged by experiments demonstrating that parasites can perform better in well-fed hosts. generally, the resources acquired by the parasite are considered in terms of energy, i.e., the resource quantity in terms of carbon, while the role of elemental nutrients such as nitrogen or phosphorus as limiting resources for parasite growth has received less attention. the inclusion of elemental ratios in models may generate substantially different population dynamics in prey and predator systems compared with previous models that considered only resource quantity. when the relative element content of the food is below the threshold elemental ratio of the consumer, there will be c in relative excess, and consumer c-use efficiency and growth rate will decrease even when food is abundant. thus, at high food abundance but low food quality, consumers will suffer from “quality starvation”. interactions with other consumers, such as predators or parasites, may further complicate the consumer population response to food quality, and, in fact, resource competition theory has been suggested as a basis for predicting the outcomes of infectious diseases. according to this theory, hosts and pathogens compete for the resources that the host uses for its own growth and maintenance in a pathogen-free situation. “quality starvation” of hosts would be expected to increase the strength of competition for limiting elements between hosts and parasites, and the net outcome of this enhanced competition would depend on the demands of the host and the parasite for the limiting nutrient. thus, a low quality diet might, despite reducing the fitness of the host, also decrease the relative effect of parasites. alternatively, the increased drain of nutrients caused by parasites might further amplify the effect of nutrient limitation on hosts and lead to decreased host reproduction, as well as increased mortality. correspondingly, parasite demands will determine the effect of host resources on parasite growth. understanding the interactions between parasites and food quality is essential to predict the outcomes of infectious diseases in the face of changing nutrient loading and whether focus at the individual or population level may yield different predictions. we studied how host nutrient limitation combined with parasite infection affected the host and the parasite populations in a model system consisting of a freshwater crustacean, daphnia magna, and its microparasite. in aquatic environments, daphnia often play a key role in mediating effects between primary producers and secondary consumers and, consequently, in the recycling of nutrients. daphnia have a high demand for p and have an ability to maintain a high degree of homeostatic control. because daphnia commonly feed on p-limited seston, they are likely to face p limitations on growth in nature. daphnia populations in nature are frequently parasitised by various species of bacterial, fungal and helminth parasites, with negative effects on population density. at the individual level, host nutrient limitation has been shown to decrease growth and production of infective propagules for bacterial and fungal parasites of daphnia. however, effects detected at individual level might not always predict the outcome of host-parasite interaction at population level. at present, knowledge is sparse on how nutrient limitation affects parasite performance at the host population level or how this affects the host populations.  population dynamics during the 32-day experiment, the population numbers peaked at approximately individuals after 28 days in the uninfected populations fed with the p-sufficient algae. for the infected populations fed with the p-sufficient algae, the numbers reached a lower density of approximately individuals compared with the uninfected populations and infection status; table  within subject contrasts for d × i). the uninfected populations fed the p-limited food reached a maximum density of approximately individuals, while the infected p-limited populations remained at a lower level of fewer than animals. in the p-sufficient treatments, the numbers of adults increased towards the end of the experiment. in the infected populations, this occurred sooner but ended at a lower level compared with the uninfected populations. juvenile abundance in the p-sufficient treatments peaked between days and and decreased thereafter, and this decline started sooner in the infected populations. in the p-limited treatments, the numbers of adults did not differ between the infected and the uninfected populations. the abundance of juveniles in the p-limited populations was at first higher in the infected than in the uninfected populations. in the uninfected p-limited populations the numbers of juveniles declined to zero, followed by a new increase at day while in the infected populations the numbers increased more steadily.figure abundances and biomasses in different treatments in the 32 day experiment. mean total adult and juvenile abundances of daphnia magna in uninfected control populations fed with p-sufficient or p-limited green algae and in populations infected with a microsporidian glugoides intestinalis, mean carbon biomass mg c l- in all four treatments and relative biomass of the infected populations for the p-sufficient and p-limited treatments during the experiment, mean individual size of adult, and juvenile daphnia μg c. the populations were started with juveniles each born to uninfected mothers fed with p-sufficient algae, and half of the animals were infected before allocation to feeding treatments. see materials and methods for calculation of relative biomass. note that the abundances are given per population in  l vials to emphasise the actual densities, while biomass is given per l to aid comparison with previously published results. the legend in panel applies to treatments in all panels -, while the legend in panel indicates the relative biomass in panel. results of the repeated measures anovas for daphnia population data p - sufficient p - limited df f p df f p tested variables are population abundance, abundances of adults and juveniles, population biomass and individual sizes of adults and juveniles for uninfected and infected daphnia magna in p-sufficient and p-limited populations in the 32-day experiment. a)between subjects effects, b)within subject effects, c)ln-transformed, d)transformation y = -1/^ e)greenhouse geisser corrected statistics, f)square root-transformed, g)transformation y = ln. within-subject contrasts for repeated measures anova abundance individual size total adult juv. biom. adult juv. within-subject contrasts for the interaction between day and infection for the repeated measures anova on daphnia population data presented in table  the contrast is “difference”, which compares each level to the mean of previous levels. a carbon content of 44 ±  % dry weight was found for uninfected animals in the p-sufficient treatment at the end of the 32 day population experiment and was used in subsequent biomass calculations. the populations fed with p-sufficient algae yielded maximum biomasses of to  mg c-1 l- with no significant interaction detected between the uninfected and the infected populations over time. in the populations fed with the p-limited algae, biomasses remained below  mg c-1 l-, and there was a significant difference between the uninfected and the infected populations in biomass accumulation over time; the infected populations had increasingly lower biomasses than the uninfected populations from day until day. the overall effect of infection on population biomass is illustrated in the relative biomasses of the infected populations compared to those of the uninfected ones. for the p-sufficient treatment, the relative biomass remained at approximately to for the entire experimental period. the p-limited treatment experienced higher variability, and at the end of the experiment, infection led to a dramatic decline to less than half of the biomass of the uninfected populations. in the p-sufficient treatment, adults were larger in the uninfected than in the infected populations, but significant d × i interactions indicated that this difference became apparent only after the second generation became adults. juvenile size in the p-sufficient populations was lower for the uninfected than the infected animals on days and. in the p-limited populations, both adults and juveniles were at first larger in the infected than in the uninfected populations, but the situation reversed after day. in the 14-day experiment, population growth was slightly higher than in the 32-day experiment, but the differences among treatments confirmed the pattern observed in the 32-day experiment. at the end of the 14-day experiment, the mean abundances of animals did not differ between the infected and the uninfected populations in either of the feeding treatments, but the numbers were significantly higher in the p-sufficient populations for uninfected and infected daphnia, respectively) than in the p-limited treatments. the mean size of the founder animals fed with p-sufficient food was higher than for those fed with p-limited food and higher for uninfected than infected animals. the difference in size between the uninfected and the infected animals was larger for animals fed with the p-sufficient food.figure the mean sizes of founder animals in the 14 day experiment. the mean carbon biomasses mg c l- of the uninfected and infected founder animals of equal age fed with the p-sufficient or the p-limited algae at the end of the 14 day experiment. population abundance and mean size in the 14 day population experiment df f p two-way anovas for the population abundance and the biomass of the uninfected and infected founder animals at the end of the 14-day experiment for daphnia magna in p-sufficient and p-limited populations. parasite load at the end of the 32-day experiment, the mean number of parasite clusters in adult daphnia was 569 ±  for daphnia fed with the p-limited food and was significantly greater than the mean of 241 ±  for daphnia fed with the p-sufficient food. the mean parasite load per carbon biomass of daphnia was significantly higher for the animals fed with the p-limited food than for animals fed with the p-sufficient food. animals dissected from uninfected populations were found to be free of parasites.figure parasite load in the different feeding treatments. the number of glugoides intestinalis spore clusters in the gut of daphnia magna for animals fed with the p-sufficient food and for animals fed with the p-limited food at the end of 32 day and 14 day population experiments, and in animals exposed individually before allocation to the two feeding treatments. the number of spore clusters per daphnia biomass in the p-sufficient and the p-limited treatments at the end of 32 day and 14 day population experiments. pairwise comparisons between treatments within different experiments: *p <, **p <, ns not statistically significant. at the end of the 14-day experiment, in founder animals of the same age, the mean number of parasite clusters was significantly higher for daphnia fed with the p-limited food than for daphnia fed with the p-sufficient food. the mean parasite load per carbon biomass of daphnia was significantly higher for the animals fed with p-limited food than for animals fed with p-sufficient food. no parasites were detected from animals dissected from uninfected populations. in animals that were exposed pairwise to the same donors and allocated singly to different food treatments, the mean numbers of parasite clusters did not differ between animals fed with the p-limited or the p-sufficient diet. the number of spore clusters in animals fed with the p-sufficient or the p-limited algae did not correlate with each other. the number of spore clusters in donor females did not correlate with the number of spore clusters in the experimental daphnia fed with the p-limited algae, but the correlation was positive for animals fed with the p-sufficient algae. discussion the results of our experiments demonstrated that infection by the microsporidian glugoides intestinalis exerted a strong check on the population density of daphnia, regardless of food quality, in agreement with the results of earlier studies. over time, however, population biomass was more severely affected by the parasite under p-limited conditions, clearly suggesting that the infection posed an extra burden on animals suffering from dietary nutrient deficiency. contrary to expectations based on results from other daphnia-parasite systems, animals collected from the p-limited populations had higher numbers of parasite spore clusters than animals from the p-sufficient populations. an additional experiment indicated that food quality did not affect the development of spores. this result suggests that the greater number of spore clusters in the p-limited populations is the result of population level phenomena arising from feedback effects between spore production and exposure in host populations. in the early phase of the experiment, the infected populations produced more offspring than the uninfected populations. for daphnia, a life history shift towards producing more offspring earlier in life could compensate for decreases later in life caused by parasitism. such a plastic response has been previously demonstrated for d. magna exposed to g. intestinalis. however, during the course of the experiment, densities in the infected populations decreased due to hampered reproduction and/or increased juvenile mortality. in the p-limited populations, the population biomass decreased to 50% in the infected populations relative to the uninfected populations by the end of the experiment. our results suggest that under severe p limitation the additional burden from parasites intensifies the negative population impacts of nutrient limitation, agreeing with previous reports on the combined effects of nutritional stress and parasite infection on hosts. according to the individual level experiments, where individual daphnia juveniles were first exposed to a single dose of parasite spores and then reared either under p-sufficient of p-limited diet, host food quality did not directly affect the development of g. intestinalis spore clusters within the hosts. in other host-parasite systems, e.g. a fungal parasite in a diatom host and a bacterial parasite in daphnia, host p-limitation has been shown to affect parasite growth negatively. g. intestinalis does not seem to be limited by p, but possibly by some other nutrient or c. as parasite within-host growth did not differ between p-sufficient and p-limited hosts, the difference in parasite loads between the two feeding treatments might have simply reflected differences in the per capita exposure rates to spores because of differences in population densities. the transmission of g. intestinalis is density-dependent, and increased per capita exposure to spores increased the probability of infection. because of the 10- day lag between exposure and the onset of spore production, most of the spores in the populations during the first three weeks were produced by the founder animals, which were exposed in the initial phase of the experiment. in the p-limited populations, the density of juveniles that were not yet producing spores themselves but filtering spores from the water was lower than in the p-sufficient populations. consequently, in the p-limited populations, spores were shared among fewer animals than in p-sufficient populations, leading to higher number of spore clusters per animal in the p-limited populations. the p limitation thus led to higher parasite numbers by increasing the per capita exposure rates to parasite spores via density effects at the host population level.figure conceptualisation of population responses and per capita parasite infection related to diet treatment. the p-limited diet led to a five- to six-fold decrease in population numbers of daphnia magna compared to populations receiving p-sufficient food but did not affect the spore production in infected founder animals. because of a 10- day time lag in parasite development, the density of juveniles, not yet producing spores but filtering spores from water, remained lower in the p-limited than in the p-sufficient populations, leading to a higher per capita spore load in the p-limited daphnia populations. additionally, changes in filtration rates could affect the intake of spores from the water, as well as their release into the water from the gut. crowding in the p-sufficient populations might have decreased their filtration rates, while low food quality in the p-limited population might have increased their filtration rates. indeed, water from the crowded daphnia populations has earlier been shown to reduce transmission of g. intestinalis. on the other hand, previous experiments with the daphnia clone used in this study found five-fold lower ingestion rates for animals grown with the p-limited algae than for those fed with the p-sufficient algae; thus, the responses of animals subjected continuously to food quality limitations might differ from those obtained with short-term changes in the diet as performed in the ingestion rate experiments. while the differences in the spore loads in the 32-day experiment may have been confounded by age and size, in the 14-day experiment all daphnia screened for parasites were the founders of populations of the same age and had equal exposure time for infection. our results are in agreement with studies showing a “dilution” effect, where high host density or high diversity of hosts and/or non-hosts can remove parasite propagules, which can lead to a decrease in parasite spread. to reveal the complex interactions between host and parasite under various p-regimes, pools and mass balance for c and p should be studied for both the host and the parasite. resolving the effect of p on parasite fitness would require measurements of transmission success as well as infectivity of the spores to the next host. additionally, the relationship between parasite loads and available nutrients should be further studied using a wider array of host clones to reveal possible genotype x environment interactions. the population experiments were performed within a confined space with donor-controlled algal dynamics, i.e., the algal stock was replenished in cultures every second day. this setup differs from a situation where the algal c:p dynamic is governed by the interaction between grazing and recycling of nutrients by herbivores. the uninfected daphnia dynamics in our batch-type setup resemble those obtained in two-step flow-through chemostats. however, parasite spore availability might have been different in a flow-through system, where a wash out of spores is possible. although the phenomenon described here might be transient, it could operate within populations in closed systems, such as in experimental settings, and could be important in natural ecosystems, especially in ponds and small lakes. previously, frost et al. reported reduced pathogen production, and penczykowski et al. reported reduced transmission potential in individual hosts receiving low-quality food; both reports suggested that reductions in disease incidence could occur in ecosystems limited by poor resource quality. our results indicate that through its effects on population dynamics, nutrient limitation may increase parasite exposure and aggravate the negative effects of parasite infection at the population level.  for the host-parasite combination used in the current study, the additional burden from parasites intensified the negative population impacts of nutrient limitation. furthermore, our results suggest that nutrient limitation affected parasite transmission and exposure via its effect on host population dynamics rather than by directly affecting parasite growth within the host. 
because host diet quality did not affect parasite numbers at the individual level, we suggest that the higher parasite load in the p-limited populations is a result of feedback effects arising at the population level. the increases in exposure led to a higher parasite load and aggravated the negative effects of parasite infection at the population level. so far, knowledge of how nutrient limitation affects parasite performance at the host population level and how this affects the host populations is limited. we followed the population growth of daphnia magna that were uninfected or experimentally infected with a microsporidian, glugoides intestinalis. daphnia magnaecological stoichiometryepidemiologyglugoides intestinalisparasitetransmissionzooplanktonissue-copyright-statement© the author 2014 in the p-sufficient populations, infection with the parasite reduced the population density but not the biomass of daphnia, while in the p-limited populations, both the density and biomass of daphnia decreased toward the end of the 32 day experiment compared with the uninfected controls. nutrient deficiency affects the growth and population dynamics of consumers. this effect may have been further reinforced by decreases in filtration rates caused by crowding in the p-sufficient populations and/or increases in filtration rates as a response to poor food quality in the p-limited populations. the p-limited diet decreased the population density and biomass compared with the populations fed with the p-sufficient algae. because of the density-dependent transmission of the parasite and the time lag between exposure and transmission, the lower host population density in the p-limited populations led to a higher spore:host ratio. depending on the relative demands of the host and the parasite for the limiting nutrient, intensified resource competition under nutrient limitation can either reduce the parasite’s effect on the host or further reduce the fitness of the nutrient-limited host.
 nutrient deficiency affects the growth and population dynamics of consumers. endoparasites can be seen as consumers that drain carbon or energy from their host while simultaneously competing for limiting resources such as phosphorus. depending on the relative demands of the host and the parasite for the limiting nutrient, intensified resource competition under nutrient limitation can either reduce the parasite’s effect on the host or further reduce the fitness of the nutrient-limited host. so far, knowledge of how nutrient limitation affects parasite performance at the host population level and how this affects the host populations is limited.  we followed the population growth of daphnia magna that were uninfected or experimentally infected with a microsporidian, glugoides intestinalis. the daphnia were fed either p-sufficient or p-limited algae. the p-limited diet decreased the population density and biomass compared with the populations fed with the p-sufficient algae. in the p-sufficient populations, infection with the parasite reduced the population density but not the biomass of daphnia, while in the p-limited populations, both the density and biomass of daphnia decreased toward the end of the 32 day experiment compared with the uninfected controls. the infected animals from the p-limited populations had higher parasite spore cluster counts, while, in a separate experiment, host diet quality did not affect the number of parasites in individually kept daphnia.  because host diet quality did not affect parasite numbers at the individual level, we suggest that the higher parasite load in the p-limited populations is a result of feedback effects arising at the population level. because of the density-dependent transmission of the parasite and the time lag between exposure and transmission, the lower host population density in the p-limited populations led to a higher spore:host ratio. this effect may have been further reinforced by decreases in filtration rates caused by crowding in the p-sufficient populations and/or increases in filtration rates as a response to poor food quality in the p-limited populations. the increases in exposure led to a higher parasite load and aggravated the negative effects of parasite infection at the population level. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords daphnia magnaecological stoichiometryepidemiologyglugoides intestinalisparasitetransmissionzooplanktonissue-copyright-statement© the author 2014 