Increased virulence due to multiple infection in Daphnia leads to limited growth in 1 of 2 co-infecting microsporidian parasites

Abstract

Recent outbreaks of various infectious diseases have highlighted the ever-present need to understand the drivers of the outbreak and spread of disease. Although much of the research investigating diseases focuses on single infections, natural systems are dominated by multiple infections. These infections may occur simultaneously, but are often acquired sequentially, which may alter the outcome of infection. Using waterfleas (Daphnia magna) as a model organism, we examined the outcome of sequential and simultaneous multiple infections with 2 microsporidian parasites (Ordospora colligata and Hamiltosporidium tvaerminnensis) in a fully factorial design with 9 treatments and 30 replicates. We found no differences between simultaneous and sequential infections. However, H. tvaerminnensis fitness was impeded by multiple infection due to increased host mortality, which gave H. tvaerminnensis less time to grow. Host fecundity was also reduced across all treatments, but animals infected with O. colligata at a younger age produced the fewest offspring. As H. tvaerminnensis is both horizontally and vertically transmitted, this reduction in offspring may have further reduced H. tvaerminnensis fitness in co-infected treatments. Our findings suggest that in natural populations where both species co-occur, H. tvaerminnensis may evolve to higher levels of virulence following frequent co-infection by O. colligata.

Keywords: Daphnia; Hamiltosporidium tvaerminnensis; Ordospora colligata; age effects; multiple infection; prior residency.

Graphical abstract

Figure 1.

Illustration of the experimental set up. The left panel shows the preparation and generation of the juveniles used in the experiment, carried out between 28 and 4 days before infection. The central panel shows the infection process carried out between 0 and 5 days after infection. Nine treatments were included in the experiment: 4 single infection treatments (‘OC Early’: only exposed to O. colligata on day 0; ‘HT Early’: only exposed to H. tvaerminnensis on day 0; ‘OC Late’: only exposed to O. colligata on day 5; ‘HT Late’: only exposed to H. tvaerminnensis on day 5), 2 simultaneous co-infection treatments (‘Both Early’: exposed to both O. colligata and H. tvaerminnensis on day 0; ‘Both Late’: exposed to both O. colligata and H. tvaerminnensis on day 5), 2 sequential co-infection treatments (‘OC Early & HT Late’: exposed to O. colligata on day 0 and then exposed to H. tvaerminnensis on day 5; ‘HT Early & OC Late’: exposed to H. tvaerminnensis on day 0 and then exposed to O. colligata on day 5), and 1 placebo treatment (Control), which was not exposed to any parasite but given equal volumes of crushed up uninfected D. magna. The right panel shows the maintenance and measurements taken during the experiment carried out between 9 and 115 days after infection. Figure created on Biorender.com.

Figure 2.

Impacts of treatment on parasite fitness. Panels A and B show the infection rates of H. tvaerminnensis and O. colligata respectively. Panels C and D show the number of spores present in infected animals for H. tvaerminnensis and O. colligata, respectively. Treatments may be single infections, sequential co-infections (Sequen.) or simultaneous co-infections (Simul.) and are split into early and late exposures. Error bars represent 95% confidence intervals for infection rates and standard error for spore burdens. Sample sizes for each treatment are indicated on each bar but partially omitted from panel C for legibility. Omitted sample sizes for H. tvaerminnensis early-exposed sequential, early-exposed simultaneous, late-exposed sequential and late-exposed simultaneous are n = 23, n = 16, n = 18 and n = 26, respectively. Statistical significance is indicated through letters visible above each bar which represent the results of Tukey's post-hoc tests. Panel E summarizes the GLM statistics for parasite infectivity and spore burden of H. tvaerminnensis and O. colligata and significance was obtained through a χ² analysis of deviance.

Figure 3.

Impacts of treatment on host fitness. Panel A shows projected survival over time for all treatments using a Kaplan–Meier survival analysis. Panels B and C show host survival for treatments exposed to H. tvaerminnensis and O. colligata, respectively. Panels D and E show host fecundity for animals exposed to H. tvaerminnensis and O. colligata, respectively. Treatments may be single infections, sequential co-infections (Sequen.) or simultaneous co-infections (Simul.) and are split into early and late exposure timepoints. Error bars represent standard error for panels B–E. Sample sizes for each treatment are indicated on each bar. Blue horizontal lines in panels B–E represent the control treatment. Statistical significance is indicated through letters visible above each bar which represent the results of Tukey's post-hoc tests. Panel F summarizes the GLM analyses carried out for host mortality and fecundity when exposed to H. tvaerminnensis and O. colligata and shows the results of a χ² analysis of deviance.