Introduction
Parasites are ubiquitous across all ecosystems (Karvonen et al., Reference Karvonen, Jokela and Laine2019), with conservative estimates predicting that there must be at least 1 parasite species for each known free-living species (Poulin, Reference Poulin1999). Pathogens and the diseases they cause can impact human and animal health (Hilgenfeld and Peiris, Reference Hilgenfeld and Peiris2013), as they may influence host fitness (Johnson and Hoverman, Reference Johnson and Hoverman2012), behaviour (Milne et al., Reference Milne, Webster and Walker2020) and physiology (Corbin et al., Reference Corbin, Heyworth, Ferrari and Hurst2017). Furthermore, parasites may affect evolutionary processes (Lafferty et al., Reference Lafferty, Dobson and Kuris2006) and lead to major economic losses (Sachs and Malaney, Reference Sachs and Malaney2002; Nicola et al., Reference Nicola, Alsafi, Sohrabi, Kerwan, Al-Jabir, Iosifidis, Agha and Agha2020). In addition, despite parasitic organisms representing a relatively small proportion of total animal biomass (Kuris et al., Reference Kuris, Hechinger, Shaw, Whitney, Aguirre-Macedo, Boch, Dobson, Dunham, Fredensborg and Huspeni2008; Paseka, Reference Paseka2017; Preston et al., Reference Preston, Layden, Segui, Falke, Brant and Novak2021), parasites may have large ecological effects such as causing tropic cascades (Schultz et al., Reference Schultz, Cloutier and Côté2016) and impacting nutrient cycling (Mischler et al., Reference Mischler, Johnson, McKenzie and Townsend2016). Gaining a better understanding of which factors facilitate or impede disease spread is crucial to prevent the many negative impacts of disease, especially given that the frequency of epidemics is increasing due to anthropogenic change and greater host mobility (Smith et al., Reference Smith, Goldberg, Rosenthal, Carlson, Chen, Chen and Ramachandran2014). However, although many studies investigating the effects of parasites focus on interactions between a single disease and its host(s), co-infections are common in real-world systems. Indeed, under natural conditions, pathogens rarely occur in isolation (Lively et al., Reference Lively, de Roode, Duffy, Graham and Koskella2014), with multiple infections accounting for up to 80% of parasite infections in some human populations (Petney and Andrews, Reference Petney and Andrews1998). Moreover, interactions among multiply infecting pathogens can either have synergistic or antagonistic effects (Vaumourin et al., Reference Vaumourin, Vourc'h, Gasqui and Vayssier-Taussat2015; Cressler et al., Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016) and may cause more variation in the outcome of infection than commonly studied environmental and host factors (e.g. seasonality or host age) (Telfer et al., Reference Telfer, Lambin, Birtles, Beldomenico, Burthe, Paterson and Begon2010). This highlights the importance of studying and identifying the principles and mechanisms that drive interactions among parasites.
Numerous mechanisms, traits and factors may affect the outcome of multiple infections. For example, virulence may depend on spatial structure, host longevity, competition for resources, host availability (Godinho et al., Reference Godinho, Rodrigues, Lefèvre, Delteil, Mira, Fragata, Magalhães and Duncan2023) and the immune response, among other factors (see for review Cressler et al. (Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016)). Studies have shown that co-infection may drive changes in parasite virulence (Alizon, Reference Alizon2008), which may either increase (Taylor et al., Reference Taylor, Walliker and Read1997; Vojvodic et al., Reference Vojvodic, Boomsma, Eilenberg and Jensen2012) or decrease (Hood, Reference Hood2003; Schürch and Roy, Reference Schürch and Roy2004). Moreover, virulence of co-infecting parasites may be dependent on the transmission modes of the parasites (Cressler et al., Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016). Indeed, parasites which are vertically transmitted (from mother to offspring) are generally less virulent than those that rely on horizontal transmission (from host to host through the environment), as these maintain host fecundity at a higher level (Ebert, Reference Ebert2013). This may lead to mismatches in virulence levels between co-infecting parasites that have differing transmission modes, which can result in 1 parasite outcompeting the other. Furthermore, many studies have highlighted the importance of priority effects, suggesting that the order of exposure of the host to multiple parasites may determine the outcome of multiple infection (Hood, Reference Hood2003; Ben-Ami et al., Reference Ben-Ami, Rigaud and Ebert2011). Prior residency, where 1 parasite infects earlier than the second, may lead to a competitive advantage for the first parasite when compared to cases of simultaneous infection where both parasites invade the host at the same time (Hood, Reference Hood2003; Karvonen et al., Reference Karvonen, Jokela and Laine2019). For example, a study investigating multiple infection of genetically distinct clones of the protist Plasmodium chabaudi infecting mice found that prior residency dictated which clone would succeed at infecting the host, regardless of which strain would typically gain a competitive advantage (De Roode et al., Reference De Roode, Helinski, Anwar and Read2005). In addition, the timing of exposure may have implications for the outcome of co-infection, as effects of infection may be different depending on the age of the host at infection (Ben-Ami, Reference Ben-Ami2019). Indeed, many studies have observed differences in host immunity between juvenile and adult animals (Davidar and Morton, Reference Davidar and Morton1993; Cote et al., Reference Cote, Korba, Miller, Jacob, Baldwin, Hornbuckle, Purcell, Tennant and Gerin2000). One notable example of this phenomenon has been observed during the Covid-19 pandemic, with older individuals being more susceptible to the virus (Davies et al., Reference Davies, Klepac, Liu, Prem, Jit and Eggo2020). Overall, the many factors which may influence co-infection make it difficult to predict its outcome which is often specific to the host–parasite system in question.
Here, we investigate the effects of co-infection and prior residency using Daphnia magna and 2 of its microsporidian parasites, Ordospora colligata and Hamiltosporidium tvaerminnensis, as a model system. Ordospora colligata is a horizontally transmitted gut parasite while H. tvaerminnensis is found in fat and ovarian cells and is transmitted both vertically (mother to offspring) and horizontally. Animals were exposed to either single, sequential or simultaneous infections at 2 timepoints (early and late, days 0 and 5 respectively) in a fully factorial experimental design. We collected measures of parasite fitness (infection rates and spore burden) and host fitness (survival and fecundity rates) to examine the effects of co-infection and determine whether prior residency plays a role in the outcome of infection in this system. Given previous studies using these parasites, which showed that O. colligata is less virulent than H. tvaerminnensis (Ebert, Reference Ebert2005), we expected that H. tvaerminnensis would outcompete O. colligata when competing over host resources. However, these parasites have previously only been studied individually, and given the complexity of the evolution of virulence, other outcomes including additive and synergistic interactions are a possibility.
Materials and methods
Study system
Daphnia magna (order: Cladocera) has been widely utilized as a model organism in ecological and evolutionary studies (Ebert, Reference Ebert2005), due to its role as a keystone species in freshwater ecosystems (Altshuler et al., Reference Altshuler, Demiri, Xu, Constantin, Yan and Cristescu2011; Jansen et al., Reference Jansen, Geerts, Rago, Spanier, Denis, De Meester and Orsini2017) and ease of use in the laboratory (Ebert, Reference Ebert2022). Daphnia magna is ubiquitous throughout the Northern Hemisphere (Lange et al., Reference Lange, Kaufmann and Ebert2015) and can be infected by a multitude of parasites in natural systems (Ebert, Reference Ebert2005), often leading to multiple infection (Decaestecker et al., Reference Decaestecker, Declerck, De Meester and Ebert2005). Daphnia magna is known to encounter the microsporidian parasites O. colligata and H. tvaerminnensis throughout its range, with the distribution of these 2 parasites overlapping in Western and Northern Europe (Ebert, Reference Ebert2005; Krebs et al., Reference Krebs, Routtu and Ebert2017) allowing co-infection to occur in natural populations. Furthermore, both pathogens are microsporidians and host resitance loci are known to overlap (Keller et al., Reference Keller, Kirk and Luijckx2019), therefore they may trigger a similar immune reaction in the host (Stirnadel and Ebert, Reference Stirnadel and Ebert1997). Ordospora colligata and H. tvaerminnensis spores are ingested by the host D. magna during filter feeding (Ebert, Reference Ebert2005), but spores are shed differently for each of these 2 parasites. Ordospora colligata is an obligate gut parasite whose spores are shed continuously into the enviroment through the feces of the host, leading to horizontal transmission (Ebert et al., Reference Ebert, Lipsitch and Mangin2000). In contrast, H. tvaerminnensis is transmitted both vertically and horizontally (upon death of the host) and exclusively infects the fat and ovarian cells of D. magna (Vizoso and Ebert, Reference Vizoso and Ebert2005; Urca and Ben-Ami, Reference Urca and Ben-Ami2018). Both O. colligata and H. tvaerminnensis are considered to be relatively avirulent (Ebert, Reference Ebert2005; Narr and Frost, Reference Narr and Frost2016), therefore reducing the risk of competitive exclusion as seen when Pasteuria ramosa co-infects with H. tvaerminnensis (Regoes et al., Reference Regoes, Hottinger, Sygnarski and Ebert2003; Ben-Ami et al., Reference Ben-Ami, Rigaud and Ebert2011). The host and parasite isolates used in this study were originally collected from the Tvärminne Archipelago in Finland (D. magna genotype Fi-Oer-3-3, O. colligata isolate OC3 and H. tvaerminnensis), and both parasites are known to infect this host genotype in natural populations (Haag et al., Reference Haag, Pombert, Sun, de Albuquerque, Batliner, Fields, Lopes and Ebert2020; Zukowski et al., Reference Zukowski, Kirk, Wadhawan, Shea, Start and Krkošek2020).
Experimental procedure
Host preparation
To obtain sufficient juvenile females for the experiment and minimize maternal effects, ~180 D. magna adult females from clonal line Fi-Oer-3-3 were grown under standardized conditions for 4 weeks in advance of the experiment (10–12 female animals per 400 mL microcosm with 200 mL of medium, transferred to a new microcosm with fresh medium twice a week, kept at 20°C with continuous light and fed ad libitum with Scenedesmus algae). Four days prior to the beginning of the experiment, animals were transferred into new microcosms to ensure no juveniles were present. Juveniles born within the next 4 days (96 h) were collected, sexed (using a dissecting microscope at 8× to 12× magnification), placed individually in a 100 mL microcosm containing 60 mL of medium and were randomly assigned to the different treatments.
Experimental design
We exposed individual juvenile female D. magna to either spores of O. colligata, H. tvaerminnensis, both parasites or a placebo comprising of crushed uninfected D. magna individuals at 2 different timepoints (at day 0, ‘early exposure’ and at day 5, ‘late exposure’) resulting in 9 different combinations covering both sequential infection and simultaneous infection. Treatments in this fully factorial design included 4 single infections (each of the parasites early and late), 2 simultaneous co-infections (early and late), 2 sequential co-infections (O. colligata early and H. tvaerminnensis late; H. tvaerminnensis early and O. colligata late) and 1 control treatment (2 placebo doses at early and late timepoints, Fig. 1). Each treatment included 30 replicates, for a total of 270 animals. Animals were kept under a 16:8 light–dark photoperiod at a constant temperature of 20°C and were transferred to new microcosms with fresh artificial Daphnia medium ADaM (Ebert et al., Reference Ebert, Zschokke-Rohringer and Carius1998) twice per week (after the initial infection period where transfers occurred 4 days post infection, on days 4 and 9) to avoid build-up of waste products and offspring produced during the experiment. Each animal was fed with 1 mL of batch cultured Scenedesmus algae grown in WC medium (Kilham et al., Reference Kilham, Kreeger, Lynn, Goulden and Herrera1998) 4 times a week during the experiment, with the density of algae increasing in increments from 5 to 12 million per mL in week 1, then remaining constant at 12 million per mL for the remainder of the experiment. Daphnia were monitored until natural death, and host fecundity and survival, and parasite infection and burden (number of spores within the host) were recorded.
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.
Parasite spore preparation and exposure
On infection days (days 0 and 5 of the experiment), Daphnia were exposed to either 50 000 spores of O. colligata, 35 000 spores of H. tvaerminnensis or a placebo dose. Appropriate spore doses were selected based on previous experiments conducted with these parasites. The spore dose of O. colligata was prepared by crushing O. colligata-infected individuals with known prevalence and burden (determined by phase-contrast microscopy on a subsample of the stock population) using a mortar and pestle and diluting this suspension of spores to 50 000 spores per mL. Similarly, the H. tvaerminnensis dose was generated by crushing H. tvaerminnensis-infected individuals, counting the resulting spore suspension using a haemocytometer [Neubauer improved] and phase-contrast microscopy and diluting down the suspension to 35 000 spores per mL. The placebo dose was prepared by crushing uninfected D. magna (of the Fi-Oer-3-3 strain which was also used to grow both parasites). On days 0 and 5, each Daphnia received a total of 2 mL of either placebo, infective doses or a combination of placebo and infective doses depending on the treatment. For single infections, this included 1 mL of the appropriate infective dose and 1 mL of the placebo dose. For simultaneous co-infection, this meant 1 mL of O. colligata infective dose and 1 mL of H. tvaerminnensis infective dose. Daphnia received 2 mL of placebo doses if no infection was required at the timepoint (Fig. 1). At both infection timepoints, Daphnia were transferred 4 days after infection (days 4 and 9 respectively) to allow sufficient exposure time to the spores of the parasites. The same doses were used at both timepoints for O. colligata, H. tvaerminnensis and the placebo to keep the numbers of spores per mL as consistent as possible over the 2 infection timepoints, as previous experiments have shown that O. colligata and H. tvaerminnensis can both persist in the environment for extended periods of time (Vizoso et al., Reference Vizoso, Lass and Ebert2005; Pombert et al., Reference Pombert, Haag, Beidas, Ebert and Keeling2015).
Measurements
Animals were checked for mortality daily, and any animals which died were subsequently examined for infection. Status of infection and spore burden were checked by placing the animals on a slide in 100 μL of water. Ordospora colligata infection was determined by dissecting the animals under a stereo microscope and counting the number of spore clusters (as O. colligata spores are known to form clusters of up to 64 spores) in the gut of the animal using phase-contrast microscopy at 400× magnification. Hamiltosporidium tvaerminnensis infection was measured by crushing and homogenizing the dissected animal on the slide and examining 12.5 μL of this liquid on a haemocytometer using phase-contrast microscopy at 400× magnification. Lifetime reproductive success of the host was calculated by summing the offspring counts which were performed twice a week following transfers. Once experimental animals were transferred to fresh microcosms, any remaining offspring were counted in a Petri dish which was placed on a light box to ensure all individuals were visible.
Statistical analyses
All statistical analyses were performed using R (version 4.0.2). All animals were included to determine infection rates as there were no deaths until 2 weeks after infection, by which point infections could be reliably scored (Larsson et al., Reference Larsson, Ebert and Vavra1997; Haag et al., Reference Haag, Larsson, Refardt and Ebert2011). Similarly, all spore counts were included as animals were followed until their natural death and therefore spore burden represents the lifetime reproductive success of H. tvaerminnensis (only transmitted upon death of the host) and abundance upon death for O. colligata (continuous transmission throughout the lifetime of the host). Analyses on host fitness measures (mortality and fecundity) were done for both animals that were exposed to the pathogens and those that became successfully infected to examine whether exposure or establishment of infection was driving the patterns observed in the data. The first analyses using exposure to the parasite(s) as the explanatory variable can be found in the result section, while the second set of analyses where the data were filtered to include only true infections (infections by 1 parasite in the single exposure treatments and infections by both parasites in the double exposure treatments) can be found in Supplementary Fig. S1. Analyses of infection rates, spore burden, fecundity and average survival used a generalized linear model (GLM) with appropriate error distributions which was compared to the null model using the χ 2 distribution and Tukey's post-hoc tests were used to determine differences between treatments. Response variables measuring parasite success (infection rates and spore burden) were analysed for each of the 6 treatments (3 exposure treatments [single, sequential and simultaneous] at 2 timepoints [early and late], i.e. y ~ treatment) for each parasite. Response variables measuring host success (average survival and fecundity) were analysed in the same manner but also included the double-placebo-exposed control animals. Survival over time was assessed through a Kaplan–Meier survival analysis and a log-rank test, where survival was analysed for all treatments (single O. colligata, single H. tvaerminnensis, sequential and simultaneous, at each of the 2 timepoints) and was also compared to double-placebo-exposed control animals.
Results
Parasite fitness
Exposure to both parasites and timing of exposure did not influence infection rates across most of the treatments (Fig. 2A, B and E). Ordospora colligata displayed high infection rates across all affected treatments (between 73 and 93%) (Fig. 2B), regardless of whether animals were single or double exposed and timing of exposure (GLM, analysis of deviance, d.f. = 5, P = 0.324). We observed a similar pattern in H. tvaerminnensis, where 5 of the 6 treatments had comparable infection rates (between 40 and 77%), although the final treatment (late-exposed simultaneous infection, 90% infection rate) differed from the early simultaneous treatments and late single H. tvaerminnensis treatment (Tukey's post-hoc test, P = 0.049 and P = 0.00428, respectively). Similarly to O. colligata infection rates, there was no effect of treatment on O. colligata spore burden, with average spore burden between 301 and 464 spore clusters per animal observed across all treatments (Fig. 2D) (GLM, analysis of deviance, d.f. = 5, P = 0.715). Hamiltosporidium tvaerminnensis spore burden in single infections (~1.6 million spores per ml), however, was 10-fold higher than those observed in double-exposed treatments (~0.2 million spores per mL), regardless of whether they were exposed early or late (Fig. 2C) (GLM, analysis of deviance, d.f. = 5, P < 0.001). Furthermore, H. tvaerminnensis produced fewer spores in both early co-infected treatments (early sequential and early simultaneous) compared to their late-exposed co-infected counterparts (late sequential and late simultaneous) (Fig. 2C) (GLM, analysis of deviance, d.f. = 5, P < 0.001).
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 χ 2 analysis of deviance.
Host fitness
Animals in single-exposed treatments lived longer than those in double-exposed treatments regardless of timing of exposure (Fig. 3B, C and F), and survival analysis by means of the Kaplan–Meier method detected differences in survival over time (Fig. 3A). Single-exposed animals lived on average 18 days longer than their double-exposed counterparts (Fig. 3B and C) (GLM, analysis of deviance, d.f. = 6, P < 2.2 × 1016), except for the early-exposed single O. colligata treatment which showed no difference in mortality to the early co-infected treatments (Fig. 3C). In contrast to increased mortality being driven by co-exposure, patterns observed in the fecundity data are driven by age effects caused by early O. colligata exposure. Indeed, single- and double-exposed animals which encountered the O. colligata parasite earlier produced fewer offspring than their late-exposed counterparts (Fig. 3E and F) (GLM, analysis of deviance, d.f. = 6, P < 2.2 × 10−16), suggesting that early exposure results in an increase in O. colligata virulence. Similarly, H. tvaerminnensis-exposed animals had consistent fecundity across all treatments, except in those where animals were exposed to O. colligata at the early timepoint, where offspring production was reduced (Fig. 3D and F) (GLM, analysis of deviance, d.f. = 6, P < 2.2 × 10−16). Results shown here include animals that were infected by at least 1 parasite at the end of the experiment (although animals with spores of neither parasite were excluded). Analysis using only double-infected animals in co-exposed treatments showed similar results although in some cases with reduced significance due to lower replication (Fig. S1).
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 χ 2 analysis of deviance.
Discussion
We observed age effects on the host with regards to fecundity, where animals which were exposed early to O. colligata produced fewer offspring (both from H. tvaerminnensis and O. colligata perspectives) than animals which were exposed at the later timepoint, regardless of whether exposures were single, sequential or simultaneous. In addition, we found that multiple exposure to O. colligata and H. tvaerminnensis led to increased mortality in the Daphnia host, with Daphnia survival being higher in single-exposure treatments (except in hosts exposed early to O. colligata where the difference was not significant). These higher rates of mortality in multiple-infected treatments are likely to be responsible for the lower spore burden observed in H. tvaerminnensis, as in these treatments this parasite had reduced time to grow.
Age effects
We observed age effects in the fecundity of O. colligata-exposed animals, where early-exposed animals experienced higher levels of virulence. Regardless of whether animals were double or single exposed, those that were exposed to O. colligata at the early timepoint produced fewer offspring than those that were exposed later in life, despite similar average survival. The decreased fecundity in earlier infected animals may have been caused by a trade-off between investment in the immune response and reproductive output (Gustafsson et al., Reference Gustafsson, Nordling, Andersson, Sheldon and Qvarnström1994; Sheldon and Verhulst, Reference Sheldon and Verhulst1996), as immune function requires a high-energy expenditure (Demas et al., Reference Demas, Chefer, Talan and Nelson1997; Rauw, Reference Rauw2012), or may be mechanistically linked to reproductive function as genes involved in the immune response may also play a role in reproductive function (i.e. pleiotropy) (Fuchs et al., Reference Fuchs, Behrends, Bundy, Crisanti and Nolan2014). Additionally, differences in host immunity between juvenile and adult animals, which have been observed in numerous organisms (e.g. in rodents [Cote et al., Reference Cote, Korba, Miller, Jacob, Baldwin, Hornbuckle, Purcell, Tennant and Gerin2000], purple martin birds [Davidar and Morton, Reference Davidar and Morton1993] and honeybees [Roberts and Hughes, Reference Roberts and Hughes2014]), may have contributed to the observed differences. Changes in disease transmission have also been linked to age in humans, with older individuals being more susceptible to Covid-19 (Davies et al., Reference Davies, Klepac, Liu, Prem, Jit and Eggo2020), exhibiting more symptoms (Davies et al., Reference Davies, Klepac, Liu, Prem, Jit and Eggo2020) and suffering higher fatality rates (Levin et al., Reference Levin, Hanage, Owusu-Boaitey, Cochran, Walsh and Meyerowitz-Katz2020). Although immune responses vary depending on the pathogen encountered (Shoham and Levitz, Reference Shoham and Levitz2005), host susceptibility may be dependent on age of infection (Hamley and Koella, Reference Hamley and Koella2021), and previous research in D. magna found that younger animals may be less efficient at dealing with parasitic infection (Izhar and Ben-Ami, Reference Izhar and Ben-Ami2015; Ben-Ami, Reference Ben-Ami2019), potentially explaining the higher costs to fecundity we observe. The existence of age effects in the D. magna–O. colligata system may have implications for studies using this host–parasite pairing as a model, including studies creating a predictive framework for the effects of climate change on parasitism (Kirk et al., Reference Kirk, Jones, Peacock, Phillips, Molnár, Krkošek and Luijckx2018, Reference Kirk, Luijckx, Jones, Krichel, Pencer, Molnár and Krkošek2020; Kunze et al., Reference Kunze, Luijckx, Jackson and Donohue2022), as many of these studies often only infect juvenile animals and do not consider age-mediated changes. Indeed, studies have found that transmission of certain diseases is dependent on age structure of the affected population (Cook et al., Reference Cook, McMeniman and O'Neill2008; Brooks-Pollock et al., Reference Brooks-Pollock, Cohen and Murray2010; Laskowski et al., Reference Laskowski, Mostaço-Guidolin, Greer, Wu and Moghadas2011), and the inclusion of population age structure in some predictive models is critical to their accuracy (Castillo-Chavez et al., Reference Castillo-Chavez, Hethcote, Andreasen, Levin and Liu1989; Dowd et al., Reference Dowd, Andriano, Brazel, Rotondi, Block, Ding, Liu and Mills2020). In conclusion, while there are clear effects of age of exposure on reproductive output in the Daphnia–Ordospora system, the epidemiological consequences of these effects need be studied in more detail.
Host effects
Concurrent exposure to both H. tvaerminnensis and O. colligata resulted in higher virulence, with animals which were exposed to both parasites generally dying earlier than those that were exposed to single parasites. Although host mortality may be exacerbated by multiple infection (Knowles, Reference Knowles2011; Chu et al., Reference Chu, Zhang, Zhang, Han, Zhao, Khan, He and Wu2016; Zilio and Koella, Reference Zilio and Koella2020), virulence in co-infection scenarios has frequently been observed to be determined by just one of the co-infecting parasites (De Roode et al., Reference De Roode, Helinski, Anwar and Read2005; Ben-Ami et al., Reference Ben-Ami, Mouton and Ebert2008; Manzi et al., Reference Manzi, Halle, Seemann, Ben-Ami and Wolinska2021). However, this does not seem to be the case for our study system with single infections of both pathogens living longer than multiple infections. Similar results where both parasites contribute to mortality have been found in several other systems (Su et al., Reference Su, Segura, Morgan, Loredo-Osti and Stevenson2005; Louhi et al., Reference Louhi, Sundberg, Jokela and Karvonen2015), although the mechanism behind this increased mortality may depend on the host–parasite system in question. Indeed, within-host competition for resources (Choisy and de Roode, Reference Choisy and de Roode2010), interactions with the host immune system (Cressler et al., Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016) or unrelatedness of concurrent parasites (Gleichsner et al., Reference Gleichsner, Reinhart and Minchella2018) may all lead to increased virulence. However, while both parasites in our system contribute to increased average host mortality, early mortality and fecundity patterns seem more strongly affected by O. colligata.
Despite both parasites contributing to overall virulence, O. colligata is more virulent in the earlier stages of the infection process, as seen by increased early mortality and decreased offspring production. This may be explained by the difference in life histories of the 2 parasites. Ordospora colligata spores are horizontally transmitted (Ebert et al., Reference Ebert, Lipsitch and Mangin2000) while H. tvaerminnensis spores are transmitted vertically from parent to offspring, as well as horizontally upon death of the host (Vizoso and Ebert, Reference Vizoso and Ebert2005; Haag et al., Reference Haag, Larsson, Refardt and Ebert2011). Given that vertically transmitted parasites increase their fitness by allowing their host to reproduce, they often evolve towards lower virulence (Ebert, Reference Ebert2013; Cressler et al., Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016). Moreover, despite O. colligata being a horizontally transmitted parasite, the large reduction in fecundity following exposure to O. colligata (−49% when compared to control) is at odds with previously published work (Ebert et al., Reference Ebert, Lipsitch and Mangin2000) where O. colligata has been described as a benign parasite only reducing fecundity by up to 20%. These differences may be attributed to experimental design and/or conditions under which experiments were conducted. Indeed, previous work on microsporidians has shown that they require high levels of phosphorous to grow (Aalto and Pulkkinen, Reference Aalto and Pulkkinen2013) and that parasite spore loads within a host can be dependent on the genotype of both the host and the parasite (Refardt and Ebert, Reference Refardt and Ebert2007). However, it is clear that under some circumstances O. colligata can have higher virulence than previously reported and that this, when combined with H. tvaerminnensis exposure, can reduce survival in multiply exposed treatments which can have consequences for parasite fitness and the evolution of virulence.
Parasite fitness
While the fitness of O. colligata was not affected by multiple infection, the spore burden of H. tvaerminnensis decreased in multiply exposed animals, and infection rates of H. tvaerminnensis differed when co-exposed with O. colligata. Previous studies investigating co-infection have frequently observed that 1 parasite may impede the establishment or development of the other (Gold et al., Reference Gold, Giraud and Hood2009; Ben-Ami et al., Reference Ben-Ami, Rigaud and Ebert2011; Manzi et al., Reference Manzi, Halle, Seemann, Ben-Ami and Wolinska2021). In our system, the reduction of H. tvaerminnensis spore burden in multiply infected treatments is likely due to the increased mortality in these treatments, giving H. tvaerminnensis less time to develop leading to lower spore burdens. In addition, being a gut parasite (Ebert, Reference Ebert2005), O. colligata may have monopolized host resources, leading to reduced resource availability for H. tvaerminnensis. Indeed, several studies have suggested that parasitic infection may modify host resource levels (Dobson, Reference Dobson1988; Tocque and Tinsley, Reference Tocque and Tinsley1994; Ebert, Reference Ebert2005), impacting host resource quality, and potentially reducing the fitness of a second parasite (Norton et al., Reference Norton, Rollinson, Richards and Webster2008; Randall et al., Reference Randall, Cable, Guschina, Harwood and Lello2013), especially in cases where the parasites have conflicting interests with respect to resource allocation (Karvonen et al., Reference Karvonen, Jokela and Laine2019). While O. colligata may, by its position in the host gut, have easier access to host resources, we found no impact of prior residency, as sequential and simultaneous infections led to consistent infection rates and spore burden across the affected treatments. Absence of prior residency has been reported in several studies (Ben-Ami et al., Reference Ben-Ami, Mouton and Ebert2008; Lohr et al., Reference Lohr, Yin and Wolinska2010; Ben-Ami et al., Reference Ben-Ami, Rigaud and Ebert2011; Hoverman et al., Reference Hoverman, Hoye and Johnson2013), although it has also been observed numerous times (De Roode et al., Reference De Roode, Helinski, Anwar and Read2005; Ezenwa et al., Reference Ezenwa, Etienne, Luikart, Beja-Pereira and Jolles2010; Karvonen et al., Reference Karvonen, Jokela and Laine2019). Another Daphnia study found that prior residency both benefitted and disadvantaged co-infecting parasites, depending on which parasite infected first (Lohr et al., Reference Lohr, Yin and Wolinska2010). Interactions among parasites combined with prior residency effects can thus lead to complex infection patterns, and despite the lack of prior residency in our study system, there is some evidence that interactions between both parasites could impact parasite fitness.
Interactions between both parasites may explain why late simultaneous infections of H. tvaerminnensis have higher spore burden and infection levels than early simultaneous infections, and higher infection rates than late single infections. This pattern could be explained by stronger immunity in younger animals, which would explain the lower H. tvaerminnensis infection rate and burden. However, this is at odds with previous research which found that younger Daphnia are less efficient at fighting off parasitic infection (Izhar and Ben-Ami, Reference Izhar and Ben-Ami2015; Ben-Ami, Reference Ben-Ami2019) and with our own finding that young animals experience higher virulence. Alternatively, H. tvaerminnensis may elude the immune response when co-infecting with O. colligata, therefore causing H. tvaerminnensis infection rates to be lower in the late single treatment than in the late simultaneous treatment. However, if co-exposure with O. colligata is indeed driving this pattern, it must be age specific given that the pattern is not visible in the early simultaneous treatment when compared to the early single treatment. While the mechanism underlying these differences remains unclear, an interaction between host age and immunity could potentially be present. Indeed, given that we observed that exposure of the host to the parasite without successful establishment of infection was sufficient to cause the same patterns in host fitness (survival and fecundity) as we observed in infected animals, it seems likely that the immune system did play a role. Immune-mediated competition is known to play a role in within-host interactions of co-infecting parasites, and may facilitate or hinder the existence of co-infection (Read and Taylor, Reference Read and Taylor2001). While immunity may thus modify the outcome of the interaction between H. tvaerminnensis and O. colligata, the observed reduction in H. tvaerminnensis spore burden is due in large part to the increased mortality in multiply exposed treatments, which may in turn influence the evolution of virulence in this system.
Virulence
Multiple infection may alter the evolutionary trajectories of co-occurring parasites (Tollenaere et al., Reference Tollenaere, Susi and Laine2016), which in turn may affect population dynamics (van Baalen and Sabelis, Reference van Baalen and Sabelis1995; Heesterbeek et al., Reference Heesterbeek, Anderson, Andreasen, Bansal, De Angelis, Dye, Eames, Edmunds, Frost and Funk2015) and species coexistence (Escribano et al., Reference Escribano, Williams, Goulson, Cave, Chapman and Caballero2001). Indeed, both modelling work and empirical data suggest that multiple infection can lead to altered levels of virulence which can change host–parasite interactions (Alizon, Reference Alizon2008; Alizon et al., Reference Alizon, de Roode and Michalakis2013), whether due to increasing (Taylor et al., Reference Taylor, Walliker and Read1997; López-Villavicencio et al., Reference López-Villavicencio, Courjol, Gibson, Hood, Jonot, Shykoff and Giraud2011; Vojvodic et al., Reference Vojvodic, Boomsma, Eilenberg and Jensen2012; Susi et al., Reference Susi, Barrès, Vale and Laine2015) or decreasing virulence (Hood, Reference Hood2003; Massey et al., Reference Massey, Buckling and ffrench–Constant2004; Schürch and Roy, Reference Schürch and Roy2004). In addition, transmission modes may have strong implications for parasite virulence (Cressler et al., Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016), as horizontally transmitted parasites are generally expected to be more virulent than vertically transmitted parasites due to the latter's need to keep their host alive for longer to effectively reproduce (Ebert, Reference Ebert2013). In our system, given that O. colligata seems to be the more virulent of the 2 parasites (contrary to expectation), as it inhibits H. tvaerminnensis transmission through reduced spore growth and offspring production (vertical transmission), in areas where both parasites co-occur O. colligata may reduce the prevalence of H. tvaerminnensis. In response, when it is frequently co-infecting with O. colligata, H. tvaerminnensis may evolve increased levels of growth and horizontal transmission, and therefore higher virulence, to offset the earlier mortality and reduced fecundity caused by O. colligata. Indeed, higher virulence is a frequent outcome when parasites compete (Taylor et al., Reference Taylor, Walliker and Read1997; Vojvodic et al., Reference Vojvodic, Boomsma, Eilenberg and Jensen2012), although reduced virulence has been observed when parasites cooperate (Hood, Reference Hood2003; Schürch and Roy, Reference Schürch and Roy2004), while in other cases patterns of virulence can be obscured by phenotypic plasticity (Choisy and de Roode, Reference Choisy and de Roode2010).
Shifts in virulence, in other systems, have been reported to lead to extinction of the host population (Rafaluk et al., Reference Rafaluk, Gildenhard, Mitschke, Telschow, Schulenburg and Joop2015) and can affect disease dynamics (van Baalen and Sabelis, Reference van Baalen and Sabelis1995). Moreover, co-infecting parasites may alter the outcome of apparent competition (Rovenolt and Tate, Reference Rovenolt and Tate2022), affect host behaviour (Haine et al., Reference Haine, Boucansaud and Rigaud2005) and may explain a larger proportion of variation than commonly studied environmental factors (Telfer et al., Reference Telfer, Lambin, Birtles, Beldomenico, Burthe, Paterson and Begon2010). Thus, interactions among co-infecting parasites can have far-reaching consequences and can impact evolutionary trajectories (Cressler et al., Reference Cressler, McLeod, Rozins, Van Den Hoogen and Day2016), population dynamics (Laurenson et al., Reference Laurenson, Norman, Gilbert, Reid and Hudson2003), human health (De Roode et al., Reference De Roode, Helinski, Anwar and Read2005) and animal health (Graham, Reference Graham2008). Indeed, given their key role in aquatic ecosystems, alterations to Daphnia population dynamics may have far-reaching consequences (Carpenter et al., Reference Carpenter, Cole, Hodgson, Kitchell, Pace, Bade, Cottingham, Essington, Houser and Schindler2001; Sarnelle, Reference Sarnelle2005).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182023001130.
Data availability
Data from this study are available at https://github.com/florianeok/multiple_infection.
Acknowledgements
We would like to thank Dieter Ebert and Jürgen Hottinger for the provision of the Daphnia clone and parasite isolates, and Alison Boyce for technical assistance.
Author contributions
All authors contributed to the experimental design, F. E. O.’K. and R. C. P. conducted the experiment with assistance of P. L. F. E. O.’K. wrote the first draft of the manuscript and all authors contributed to revisions. P. L. and C. V. H. supervised the project.
Financial support
This work was supported by the Irish Research Council (F. E. O.’K., Government of Ireland Postgraduate Scholarship Programme 2019/3448) and Science Foundation Ireland (P. L., Frontiers for the Future 19/FFP/6839).
Competing interests
None.
Ethical standards
Not applicable.
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