Oligosaccharides derived from dragon fruit modulate gut microbiota, reduce oxidative stress and stimulate toll-pathway related gene expression in freshwater crustacean Daphnia magna.

Dragon fruit oligosaccharide (DFO) is an indigestible prebiotic. In this study, we aimed to investigate the effects of DFO on gut microbiota, oxidative stress and immune-related gene expression in Daphnia magna. The 10-day-old D. magna were treated with 0, 9, and 27 mg l-1 DFO for 85 h. The gut bacterial communities, superoxide dismutase (SOD) activity, lipid peroxidation and the expressions of genes in Toll signaling pathway were observed. The results showed that D. magna treated with 9 and 27 mg l-1 DFO altered gut microbiota composition by increasing Limnohabitans and Lactobacillus, and significantly increased SOD activity and reduced lipid peroxidation. Moreover, the expressions of Toll2, Toll3, Toll5, Toll7 and Pelle genes were significantly increased in D. magna treated with 9 and 27 mg l-1 DFO. Our results suggested that DFO changed the composition of the gut microbiota of D. magna by increasing the beneficial bacteria. DFO also had the ability to stimulate innate immunity in D. magna by increasing SOD activity, reducing lipid peroxidation, and increasing the expression of immune-related genes.

Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model.

Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions.

Does infection tilt the scales? Disease effects on the mass balance of an invertebrate nutrient recycler.

While parasites are increasingly recognized as important components of ecosystems, we currently know little about how they alter ecosystem nutrient availability via host-mediated nutrient cycling. We examined whether infection alters the flow of nutrients through hosts and whether such effects depend upon host diet quality. To do so, we compared the mass specific nutrient (i.e., nitrogen and phosphorus) release rates, ingestion rates, and elemental composition of uninfected Daphnia to those infected with a bacterial parasite, P. ramosa. N and P release rates were increased by infection when Daphnia were fed P-poor diets, but we found no effect of infection on the nutrient release of individuals fed P-rich diets. Calculations based on the first law of thermodynamics indicated that infection should increase the nutrient release rates of Daphnia by decreasing nutrient accumulation rates in host tissues. Although we found reduced nutrient accumulation rates in infected Daphnia fed all diets, this reduction did not increase the nutrient release rates of Daphnia fed the P-rich diet because infected Daphnia fed this diet ingested nutrients more slowly than uninfected hosts. Our results thus indicate that parasites can significantly alter the nutrient use of animal consumers, which could affect the availability of nutrients in heavily parasitized environments.

An ancient immunity gene duplication in Daphnia magna: RNA expression and sequence analysis of two nitric oxide synthase genes.

NO (nitric oxide) is a highly reactive free radical gas thought to play a major role in the invertebrate immune response by harming pathogens and limiting their growth. Here we report on studies of nitric oxide synthase (NOS) genes in the crustacean Daphnia, one of the few non-insect arthropod models used to study host-pathogen interactions. While the NOS gene is found as a single copy in other invertebrates, we found two copies (NOS1 and NOS2), which a phylogenetic reconstruction showed to be the result of an ancient duplication event. Both genes bear features commonly found in invertebrate NOS, however, the two genes differ in their rate of evolution, intraspecific polymorphism and expression level. We tested whether the more rapid evolution of NOS2 could be due to positive selection, but found the rate of amino-acid substitutions between Daphnia species to be compatible with a neutral model. To associate NOS or NO activity with infection, we performed infection experiments with Daphnia magna and one of its natural pathogens (the bacterium Pasteuria ramosa). In one set of experimental infections, we supplemented D. magna with L-arginine, the NOS substrate, or with L-NAME, a NOS antagonist, and found this to result in lower and higher infection levels, respectively, which is at least compatible with the notion that NO may aid defence against Pasteuria. A second experiment indicated that NOS transcription does not increase following exposure to Pasteuria. Thus, the function of NOS in Daphnia immunity remains uncertain, but the pattern of gene duplication and subsequent divergence suggests evolution via neo- or subfunctionalization.

Festering food: chytridiomycete pathogen reduces quality of Daphnia host as a food resource.

When parasitic infections are severe or highly prevalent among prey, a significant component of the predator's diet may consist of parasitized hosts. However, despite the ubiquity of parasites in most food webs, comparisons of the nutritional quality of prey as a function of infection status are largely absent. We measured the nutritional consequences of chytridiomycete infections in Daphnia, which achieve high prevalence in lake ecosystems (>80%), and tested the hypothesis that Daphnia pulicaria infected with Polycaryum laeve are diminished in food quality relative to uninfected hosts. Compared with uninfected adults, infected individuals were smaller, contained less nitrogen and phosphorus, and were lower in several important fatty acids. Infected zooplankton had significantly shorter carapace lengths (8%) and lower mass (8-20%) than uninfected individuals. Parasitized animals contained significantly less phosphorus (16-18% less by dry mass) and nitrogen (4-6% less) than did healthy individuals. Infected individuals also contained 26-34% less saturated fatty acid and 31-42% less docosahexaenoic acid, an essential fatty acid that is typically low in cladocera, but critical to fish growth. Our results suggest that naturally occurring levels of chytrid infections in D. pulicaria populations reduce the quality of food available to secondary consumers, including planktivorous fishes, with potentially important effects for lake food webs.

Bacterial infection changes the elemental composition of Daphnia magna.

1. An animal's elemental composition can be an important indicator of its physiological state and role in ecosystem nutrient cycling. We examined the interactive effects of bacterial (Pasteuria ramosa) infection and phosphorus (P)-poor food on the body stoichiometry of Daphnia magna. Daphnia were exposed to or held free of a bacterial parasite and fed algal food of different C:P ratios (100-500) over a 28-day period. 2. To assess the effects of exposure and infection on Daphnia stoichiometry, we measured their whole body content of carbon (C), nitrogen (N), and P on four different days (4, 8, 15, and 28) during the experiment. 3. We found strong effects of infection, food quality, and/or their interactions on the C, N, and P content of Daphnia, especially as the infectious disease progressed. At the end of the experiment, infected animals had significantly more C and less P in their bodies than uninfected conspecifics. Body N content of Daphnia consuming P-rich food was reduced by bacterial infection whereas Daphnia consuming P-poor algae showed increased body N content from infection. 4. Using a mass-balance model, we found that changes in N and P content of host bodies were largely accountable by disease-induced alterations to Daphnia reproduction (i.e. bacterial induced sterility) and the accumulation of Pasteuria spores in the body cavity. Our calculations also show that the observed increase in host C content could not be accounted for by loss of eggs or accumulation of bacterial spores. This instead must result from unidentified changes to underlying daphnid tissue C content. 5. These results demonstrate that intraspecific variation in zooplankton body stoichiometry can be caused by exposure to and infection by bacterial parasites. In addition, these effects were found to depend both upon the stage of the disease and upon the food quality consumed by the host.

Responses of a bacterial pathogen to phosphorus limitation of its aquatic invertebrate host.

Host nutrition is thought to affect the establishment, persistence, and severity of pathogenic infections. Nutrient-deficient foods possibly benefit pathogens by constraining host immune function or benefit hosts by limiting parasite growth and reproduction. However, the effects of poor elemental food quality on a host's susceptibility to infection and disease have received little study. Here we show that the bacterial microparasite Pasteuria ramosa is affected by the elemental nutrition of its aquatic invertebrate host, Daphnia magna. We found that high food carbon:phosphorus (C:P) ratios significantly reduced infection rates of Pasteuria in Daphnia and led to lower within-host pathogen multiplication. In addition, greater virulent effects of bacterial infection on host reproduction were found in Daphnia-consuming P-deficient food. Poor Daphnia elemental nutrition thus reduced the growth and reproduction of its bacterial parasite, Pasteuria. The effects of poor host nutrition on the pathogen were further evidenced by Pasteuria's greater inhibition of reproduction in P-limited Daphnia. Our results provide strong evidence that elemental food quality can significantly influence the incidence and intensity of infectious disease in invertebrate hosts.