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Keywords:

  • Daphnia magna;
  • Octosporea bayeri;
  • Pasteuria ramosa;
  • within-host competition

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

In many natural populations, hosts are found to be infected by more than one parasite species. When these parasites have different host exploitation strategies and transmission modes, a conflict among them may arise. Such a conflict may reduce the success of both parasites, but could work to the benefit of the host. For example, the less-virulent parasite may protect the host against the more-virulent competitor. We examine this conflict using the waterflea Daphnia magna and two of its sympatric parasites: the blood-infecting bacterium Pasteuria ramosa that transmits horizontally and the intracellular microsporidium Octosporea bayeri that can concurrently transmit horizontally and vertically after infecting ovaries and fat tissues of the host. We quantified host and parasite fitness after exposing Daphnia to one or both parasites, both simultaneously and sequentially. Under conditions of strict horizontal transmission, Pasteuria competitively excluded Octosporea in both simultaneous and sequential double infections, regardless of the order of exposure. Host lifespan, host reproduction and parasite spore production in double infections resembled those of single infection by Pasteuria. When hosts became first vertically (transovarilly) infected with O. bayeri, Octosporea was able to withstand competition with P. ramosa to some degree, but both parasites produced less transmission stages than they did in single infections. At the same time, the host suffered from reduced fecundity and longevity. Our study demonstrates that even when competing parasite species utilize different host tissues to proliferate, double infections lead to the expression of higher virulence and ultimately may select for higher virulence. Furthermore, we found no evidence that the less-virulent and vertically transmitting O. bayeri protects its host against the highly virulent P. ramosa.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Host–parasite interactions rarely involve a one-to-one association in nature. Instead, in many populations, hosts are commonly found to be multiply infected by two or more parasite species (Lello et al., 2004; Decaestecker et al., 2005; Rutrecht & Brown, 2008; reviewed by Petney & Andrews, 1998; Cox, 2001; Read & Taylor, 2001; Rigaud et al., 2010) or by different strains of the same parasite species (Werren et al., 1995; Lagrue et al., 2007; López-Villavicencio et al., 2007). Numerous theoretical and empirical studies have highlighted that interactions between genetically diverse parasites may influence both within- and between-host selection and consequently shape the evolution of parasite traits. In particular, under conditions of frequent multiple infections, lineages of virulent parasites are predicted to be more competitive than those exploiting their host more prudently, leading to an overall increase in virulence (Antia et al., 1994; Bonhoeffer & Nowak, 1994; van Baalen & Sabelis, 1995; Frank, 1996; Mosquera & Adler, 1998). These theoretical predictions received empirical support, notably from studies of multiple infections involving several strains of a single parasite species (Davies et al., 2002; Wille et al., 2002; Hood, 2003; Hodgson et al., 2004; Hughes et al., 2004; Vizoso & Ebert, 2005a; Jäger & Schjørring, 2006; Wargo et al., 2007; Ben-Ami et al., 2008) and to a lesser extent from studies of multiple infections by different parasite species (Thomas et al., 2003; Lohr et al., 2010). The latter are at the centre of this study and hereafter referred to as double infections.

Virulent effects of parasites can take diverse forms, with very different consequences for potential conflicts with other parasites or the host. Among the most fitness-devastating parasites for the host are castrating parasites, here defined as parasites whose primary effect on the host is to curtail host reproduction, while having relatively little effect on host mortality (Baudoin, 1975). Castrating parasites convert the resources the host invests into its offspring into their own growth and reproduction (Baudoin, 1975; Obrebski, 1975; Jokela et al., 1993; Jaenike, 1996; O’Keefe & Antonovics, 2002). By doing so they can gain a substantial biomass (Baudoin, 1975). The high needs of castrators for resources put them in conflict with other parasites. This may select castrators to eliminate competitors, because sacrificing resources to other parasites may have strongly detrimental effects. Castrators rely solely on horizontal transmission, and rapid castration was suggested as a means to maximize their fitness (Obrebski, 1975; O’Keefe & Antonovics, 2002; Ebert et al., 2004). This life history strategy places castrators in direct conflict with vertically transmitted (VT) parasites, which rely on host reproduction.

Like horizontally transmitted (HT) parasites, VT parasites have been shown to exhibit diverse effects on their hosts. For example, VT microbes may feminize their host, alter the sex ratio of host offspring (Werren et al., 1995; Bouchon et al., 1998; Weeks et al., 2003; Terry et al., 2004) and manipulate or ‘sabotage’ host behaviour (Thomas et al., 2002; Haine et al., 2005). In recent years, an increasing number of examples have been presented, which show that VT microbes protect their hosts against other parasites (Gil-Turnes et al., 1989; Tsuchida et al., 2002; Oliver et al., 2003; Ferrari et al., 2004; Zchori-Fein & Perlman, 2004), and there is growing appreciation that VT defensive symbionts play an important role in the ecology of host–parasite interactions (Scarborough et al., 2005; Hedges et al., 2008; Jaenike et al., 2010). This protection can render the parasitic effect of a VT microbe into a mutualistic effect, potentially explaining the persistence of exclusive VT parasites (Haine, 2008; Brownlie & Johnson, 2009). The advantage for the VT microbe in protecting its host from other parasites is the increased lifetime reproductive success of the host and the VT parasite.

The conflict between coinfecting parasites is most extreme when the two parasites employ strategies that exclude each other. This is the case when a VT parasite encounters a castrating parasite: whereas the former transmits via host offspring, the latter suppresses host reproduction. As outlined previously, there is considerable evidence demonstrating that VT parasites protect their host against other parasites, whereas at the same time virulent parasites, and in particular castrators, were suggested to be superior competitors. These contrasting predictions of the outcome of the conflict between VT parasites and castrators are at the centre of this study.

The study system

The present study examines double infections in Daphnia magna, a cyclical parthenogenetic crustacean parasitized by a wide variety of bacterial, microsporidial and fungal parasites (Green, 1974; Ebert, 2005), with severe impact on host fitness (Stirnadel & Ebert, 1997; Ebert et al., 2000). In field populations, many parasites may coexist in the same pond and multiple infections of host individuals are often observed (Stirnadel & Ebert, 1997; Decaestecker et al., 2005). A common and widely spread parasite of D. magna is the bacterium Pasteuria ramosa Metchnikoff 1888, an obligate endoparasite with strict horizontal transmission, in which infective stages (i.e. spores) are released from the decaying cadaver of the host (Ebert et al., 1996; Ebert, 2005). It has a strong castrating effect on the host, which rarely produces any offspring after infection (Jensen et al., 2006). It is also capable of infecting other Daphnia species (e.g. D. dolichocephala; Duneau et al., 2011). The microsporidium Octosporea bayeri Jirovec 1936 is host specific to D. magna and is very common in rock pool habitats of the Baltic Sea (Ebert et al., 2001). It has a mixed transmission strategy, because infection can be acquired horizontally via waterborne spores released from a host cadaver or vertically from a mother to her offspring (Vizoso et al., 2005). Vertical transmission is 100% efficient to asexual host offspring, but lower to sexually produced host eggs (Ebert et al., 2007). Vertical transmission to sexual eggs is important for the parasite to survive host diapause (Ebert et al., 2007). Following infection, O. bayeri develops in the host’s fat cells and ovaries. Infected hosts are able to reproduce nearly normal, but suffer about 20% loss in competitive ability (Vizoso & Ebert, 2004; Bieger & Ebert, 2009).

The present study investigates a clearly identifiable conflict: P. ramosa requires host resources to grow and therefore castrates its host, whereas O. bayeri needs host offspring for vertical transmission. We examine differences between the parasite species and their associated transmission strategies. Both parasites coexist in the same D. magna metapopulation in southwestern Finland, although O. bayeri is much more abundant in these D. magna rock pool populations (Ebert et al., 2001). As the virulence and the expected lifetime transmission success of the two parasite species can be accurately quantified in the laboratory, we are able to test for conflicts between the parasites over when should the Daphnia host be castrated and when should it be killed. We also attempt to identify possible benefits incurred on D. magna by O. bayeri during its horizontal and vertical transmission phases. Taken together, these tests will determine whether (i) the concurrently transmitted Octosporea can protect the Daphnia host against Pasteuria and whether (ii) Pasteuria, being a castrator, is a stronger competitor than Octosporea.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Host and parasite collections

We used a single D. magna clone (SP1-2-3) originally collected from a rock pool in the Tvärminne archipelago of southwestern Finland, by isolating parthenogenetic eggs from the brood chamber of an uninfected adult female and raising the clonal offspring in isolation under standardized laboratory conditions. In preparation to the experiment, we stock-cultured D. magna in 400-mL glass beakers, each containing eight individuals with artificial medium (Klüttgen et al., 1994; Ebert et al., 1998), where they were fed daily with 1.5 × 105 cells mL−1 medium of the chemostat-cultured unicellular algae Scenedesmus gracilis.

The two parasite species used in this experiment were each obtained from a singly infected D. magna individual (P. ramosa P1 isolate from Germany and O. bayeri FUNR-8-5 isolate from Finland), different from the host clone used for the actual experiment. In the case of P. ramosa, the infected individual was well fed until it died, upon which its parasite spores were used to repeatedly propagate infection via the highly susceptible D. magna clone HO2 from Hungary. In the case of O. bayeri, the parthenogenetic offspring of the ‘initial’D. magna clone were used to repeatedly propagate infection, until there were enough spore-carrying cadavers to produce sufficient amounts of spore suspensions for the experiment. All cadavers were carefully homogenized, and spore concentrations were determined using a haemocytometer (Thoma ruling).

Experimental design and setup

The experiment had two phases: in the first phase, we examined double infections that resulted from horizontal infections by both parasite species, whereas in the second phase, we exposed vertically infected D. magna (by O. bayeri) to horizontally infecting P. ramosa. In the first phase, we followed a cohort of 448 D. magna individuals and examined the outcome of single infections as well as of double infections, both simultaneously and sequentially. In total, there were eight treatments, each with 56 replicates as listed in Table 1A. In the second phase, we followed a cohort of 224 D. magna individuals, half of which were already vertically infected by O. bayeri and the other half was naïve. These vertically infected offspring were obtained from 24 mothers exposed as juveniles to horizontally infecting O. bayeri. Our aim was to assess the effects of horizontal infection (by P. ramosa) on hosts previously infected vertically by O. bayeri. In this phase, we had four treatments, each with 56 replicates as described in Table 1B.

Table 1.   Overview of the treatments in the (A) first and (B) second phase of the experiment. The use of different dose levels (50 000 in single infections vs. 100 000 spores in double infections) has been previously found to be insufficient to produce significant effects on any of the variables in this study, i.e. host longevity, host reproduction and parasite spore production (Ben-Ami et al., 2008).
Treatment AType of infectionFirst infection (day 5)Second infection (day 12)
PSingle50 000 spores of Pasteuria ramosaNone
OSingle50 000 spores of Octosporea bayeriNone
N+PdSingle, delayedNone50 000 spores of P. ramosa
N+OdSingle, delayedNone50 000 spores of O. bayeri
P+ODouble, simultaneous50 000 spores of each parasite speciesNone
P+OdDouble, sequential50 000 spores of P. ramosa50 000 spores of O. bayeri
O+PdDouble, sequential50 000 spores of O. bayeri50 000 spores of P. ramosa
ControlNoneNoneNone
Treatment BType of infectionFirst infectionSecond infection (day 5)
PSingleNoneHorizontal infection using 50 000 spores of P. ramosa
OvSingleVertical infection by O. bayeri from motherNone
Ov+PDouble, sequentialVertical infection by O. bayeri from motherHorizontal infection using 50 000 spores of P. ramosa
ControlNoneNoneNone

Throughout both phases of the experiment and on a daily basis, we monitored D. magna survival, release of offspring and the amount of P. ramosa and O. bayeri spores following the host’s death. We defined virulence as time-to-host-death-since-first-exposure (i.e. host longevity). This can be time-to-host-death-since-age-12 days for delayed single infections; time-to-host-death-since-age-5 days for single, simultaneous and sequential infections; and time-to-host-death-since-birth for control and vertically infected hosts. Host fitness was defined as the lifetime number of offspring produced. Pasteuria fitness was estimated from the number of spores at the time of host death, which is equal to the lifetime spore production of an infection. Octosporea fitness was estimated from host reproductive success (vertical transmission) and from spores counted in killed hosts (horizontal transmission success).

In both phases of the experiment, we used third clutch offspring of the D. magna clone line. To start the experiment, we separated newborns from the D. magna clone line (0–24 h old) into four 400-mL beakers and fed them daily with 1.5 × 105 algae cells mL−1 medium. On day four, we singly placed female-only Daphnia in 100-mL jars, filled with 20 mL of artificial medium, and initially fed them 2 × 106 algae cells per animal per day. The first infection treatment was performed on day 5. A week later, on day 12, we replaced the medium of all animals with 20 mL of fresh medium and exposed the appropriate treatment groups to P. ramosa or O. bayeri spores (those with a delayed challenge). A week after the second infection, we replaced the medium of all animals with 100 mL of fresh medium and thereafter medium was replaced on a weekly basis. To accommodate the growing food demands of the growing animals, on days 9, 15, 18, 22, 27, 30 and 37, we increased the daily food level for all individuals to 3 × 106, 5 × 106, 6 × 106, 7 × 106, 8 × 106, 9 × 106 and 10 × 106 algae cells per day, respectively.

The temperature was 20 ± 0.5 °C and the light/dark cycle was 16 h : 8 h. All treatments were randomly distributed across the shelves of the incubator, and their position was rearranged frequently to avoid position effects. Offspring counts and dead animals were recorded daily. Animals that had died after day 16 (since birth) were dissected and checked for disease using phase-contrast microscopy (300–600×). Animals that had died earlier could not be reliably checked for infection and were thus excluded from the analyses. The experiment was terminated after all animals had died. The dead Daphnia were then frozen in 0.1 mL of medium at −20 °C for subsequent parasite spore counting with a haemocytometer.

Statistical analyses

All statistical tests were performed using SPSS for Windows release 15.0.1.1 (SPSS Inc., 2005). Host longevity was analysed using Cox regression with the relevant treatments dummy-coded as categorical covariables. No censoring was required because all Daphnia had died during the experiment. Offspring data failed to meet the normality and equality-of-variances assumptions and were thus analysed using nonparametric Kruskal–Wallis anova. For multiple comparisons, the Mann–Whitney U-test was used after adjusting the P-value with the Bonferroni method. Although this method is over conservative, it did not cause a statistically significant result to become nonsignificant. Spore production was analysed using parametric anova and t-tests, except when the data set was too small, in which case we used nonparametric tests. Infection rates were compared using Fisher’s exact test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Phase 1 – horizontal infection of previously uninfected hosts

During the first 2 weeks of the experiment, 49 of 448 Daphnia individuals died for unknown reasons (10.9%). This mortality was unrelated to treatments (inline image, = 0.12). None of the control Daphnia became infected. Controls were excluded from the analyses of infection rates and parasite spore production. Uninfected Daphnia in the infection treatments were excluded from all analyses except infection rates.

Host reproduction

Host control animals produced as many offspring as hosts singly infected with Octosporea (control, O, N+Od; Kruskal–Wallis inline image, = 0.92; abbreviations as in Table 1, Fig. 1a), but considerably more than hosts singly infected with Pasteuria (control, P, N+Pd; Kruskal–Wallis inline image, < 0.001). Hosts exposed to Pasteuria on day 12 produced significantly more offspring than those exposed on day 5 (P, N+Pd; Mann–Whitney U = 387.5, = 0.001). This day-of-exposure effect was not observed in Octosporea (O, N+Od; Mann–Whitney U = 68.5, = 0.64). In simultaneous and sequential double infections when Pasteuria was first to infect, offspring production followed largely Pasteuria single infection (P, P+O, P+Od; Kruskal–Wallis inline image, = 0.11). However, in sequential double infections when Octosporea infected first, the number of offspring was similar to delayed single infection by Pasteuria (N+Pd, O+Pd; Mann–Whitney U = 781.5, = 0.86).

image

Figure 1.  Mean ± SE of host lifetime offspring production in the treatments of the (a) first and (b) second phase of the experiment. Means with the same letter are not significantly different.

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Host longevity

Host-longevity-since-exposure in treatments involving Pasteuria with or without Octosporea did not differ significantly (P, N+Pd, P+O, P+Od, O+Pd; Cox regression inline image, = 0.15; Fig. 2a). Host-longevity-since-birth in the control group and in treatments involving only Octosporea was also similar (control, O, N+Od; Cox regression inline image, = 0.77). However, as can be clearly seen in Fig. 2a, we found considerable differences between the Pasteuria treatments (with or without Octosporea) and the remaining Octosporea-only treatments pooled within each group (Cox regression inline image, < 0.001).

image

Figure 2.  Mean ± SE of time-to-host-death-since-first-exposure (or since-birth for controls) in the treatments of the (a) first and (b) second phase of the experiment (see Materials and methods for details). Means with the same letter are not significantly different.

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Infection rates

The infection rate in treatments involving Pasteuria was similar (varying from 80.0% to 92.7%; Fisher’s exact test, = 0.39) and independent of the presence of Octosporea. In contrast, infection rates significantly differed among Octosporea treatments (Fisher’s exact test, < 0.001) and they were strongly influenced by the presence of Pasteuria. No Daphnia were infected with Octosporea when Pasteuria was first to infect (P+Od), and only two individuals were infected by Octosporea (but also by Pasteuria) in simultaneous double infections (P+O). Because of these low infection rates, these treatments were not further analysed with regard to Octosporea fitness. The infection rate in the remaining Octosporea treatments (O, N+Od, O+Pd) varied between 14.0% and 33.3%.

Parasite spore production

Parasite spore production varied considerably between parasite species and among treatments. Spore production by Pasteuria was relatively similar in single, delayed single, simultaneous and sequential double infections (P, N+Pd, P+O, P+Od, O+Pd; F4,183 = 2.19, = 0.072; Fig. 3a). Spore production by Octosporea was much higher in single and delayed single infection than in sequential double infections when Octosporea was first to infect (O, N+Od, O+Pd; Kruskal–Wallis inline image, = 0.001). In summary, Pasteuria fitness is largely unaffected by the presence of Octosporea, whereas Octosporea strongly loses fitness when Pasteuria is present.

image

Figure 3.  Mean ± SE of parasite spore production in treatments of the (a) first and (b) second phase of the experiment.

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Phase 2 – horizontal infection of vertically infected hosts (by Octosporea)

In all four treatments (including control), 11 of 224 Daphnia individuals died for unknown reasons during the first 2 weeks of the experiment (4.9%). This mortality was unrelated to treatments as there were no differences among treatments (inline image, = 0.18). All controls remained uninfected, and uninfected Daphnia (belonging to infection treatments) were not included in the analyses of host reproduction, host longevity and parasite spore production.

Host reproduction

Host control animals produced the highest number of offspring per individual, followed by single infection by Octosporea, then by single infection by Pasteuria, and finally by double infections by both parasite species (Kruskal–Wallis inline image, < 0.001; Fig. 1b). All Mann–Whitney pairwise comparisons were significant at < 0.001.

Host longevity

Both parasites substantially reduced host longevity relative to the control group (Table 2, Fig. 2b), although there were no differences in virulence between the two single infection treatments. Double infections were even more virulent than single infection by either parasite.

Table 2.   Results of survival analysis (Cox regression) showing treatment effects on the survival of Daphnia in the second phase of the experiment. We used repeated contrasts whereby each category of the predictor variable except the first category is compared to the category that precedes it. The negative sign of all estimated regression coefficients indicates that the hazard of mortality increases in comparison with the preceding category, e.g. the hazard of mortality in the Ov treatment is higher than that of the control treatment. exp(B) is the relative risk, e.g. the risk of death in the control treatment is less than a fifth of the risk of death in the Ov treatment. Bold typeface indicates significant effects. See Fig. 2b for comparison.
Contrastd.f.BWaldPexp (B)
Ov vs. Control1−1.6643.70< 0.0010.19
P vs. Ov1−0.030.020.88 
Ov+P vs. P1−0.8313.68< 0.0010.48
Infection rates

The infection rate of Pasteuria was 97.6% in previously uninfected hosts and 90.9% in hosts vertically infected by Octosporea (Fisher’s exact test, = 0.19). At birth, all newborn hosts (except controls) were vertically infected by Octosporea. This was confirmed by examining clonal siblings of offspring used in all infection treatments – these extra control siblings were always found to be infected by Octosporea. Moreover, upon death, all individuals in the Ov treatment were still infected, and the infection rate of Octosporea after exposure to Pasteuria decreased to 86.4% (Fisher’s exact test, = 0.01), indicating that P. ramosa clears O. bayeri in some cases.

Parasite spore production

Parasite spore production in double infections was considerably lower than in single infection by either parasite species. In the case of Octosporea, single infection produced more than three-fold Octosporea spores than double infections (Ov, Ov+P; unequal variances t65.6 = 10.82, < 0.001; Fig. 3b), whereas single infection by Pasteuria produced twice as much Pasteuria spores as double infections (P, Ov+P; unequal variances t60.9 = −4.84, < 0.001).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

We found that under conditions of strict horizontal transmission, P. ramosa competitively excluded O. bayeri in both simultaneous and sequential double infections, regardless of the order of inoculation. Consistent with this finding, host longevity, host reproduction and parasite spore production in double infections resembled those of single infection by P. ramosa. Only when O. bayeri is VT, it is able to resist to some degree competition by P. ramosa and produce a considerable proportion of transmission stages that can be used for horizontal transmission. In this case, both parasites and the host suffer from the competition in comparison with single infections: the host D. magna suffers from lower fecundity and longevity, and the two parasites suffer from reduced spore production. Despite the slightly lower susceptibility of D. magna to P. ramosa under conditions of double infections, and the substantially lower Pasteuria and Octosporea spore production in vertically infected hosts, it did not translate into a relative increase in host fitness. Therefore, O. bayeri does not appear to confer protection to D. magna against P. ramosa.

These results suggest that on a population level, Pasteuria may outcompete Octosporea in the long run. The current geographical distribution of the two species overlaps only in the rock pool metapopulation of D. magna on the Baltic Sea. There, Octosporea is found in about 50% of the D. magna rock pool populations, whereas Pasteuria in about 1% (Green, 1957; Ebert et al., 2001). In regions where Pasteuria is common (most of Central, Eastern and Western Europe), Octosporea is absent. Whether these distributions are shaped by the relative competitiveness of the two parasites or by other factors such as habitat quality or history is unclear. As both Pasteuria and Octosporea can persist outside the host in sediments under dry and wet conditions, vertical transmission (via parthenogenetic and sexual offspring) certainly provides Octosporea an advantage in the highly dynamic rock pools along the Baltic Sea (Vizoso et al., 2005), possibly explaining the dominance there (Ebert et al., 2001).

The interactions between a strictly HT parasite and a concurrently transmitted parasite (i.e. HT+VT) have not been modelled, especially when the transmission of the HT parasite is density dependent (a function of the absolute density of infected hosts in the population). Under conditions of frequency-dependent transmission (transmission success is a function of the frequency of infected hosts in the population, as in vector and sexually transmitted parasites), Altizer & Augustine (1997) showed that adding vertical transmission capabilities to an HT parasite significantly broadens the conditions for parasite invasion. When both HT and VT parasite strains are allowed to coexist within a host population, Lipsitch et al. (1996) showed that a VT strain can persist if it provides protection against a more-virulent HT strain. Similar predictions have been made by Lively et al. (2005) and Faeth et al. (2007). These models, however, assume that an HT parasite cannot superinfect a host that is already infected by a VT parasite – an assumption that does not hold for many host–parasite systems (reviewed in Haine, 2008) including the here used DaphniaPasteuriaOctosporea system. If one allows for superinfection by the HT parasite, and assuming that the VT parasite reduces the transmission ability of the HT parasite (as O. bayeri did via a two-fold reduction of P. ramosa spore production in double infections), Jones et al. (2007) showed that VT parasites are more likely to persist with HT parasites that prevent host reproduction (such as P. ramosa) than with those that allow it. Jones et al. (2010) also suggested that competition between HT and VT parasites critically depends on life history trade-offs the two parasites face, which may be highly specific to their particular biology (e.g. feminization vs. virulence, transmission efficiency vs. virulence). To explore these conjectures further, one could, for instance, compare the present results with studies of double infections by O. bayeri and a noncastrating HT parasite of Daphnia.

In several studies of double infections, the parasite species employed were not in conflict over transmission. For example, using the desert locust and two HT fungal entomopathogen species, Thomas et al. (2003) found that the avirulent parasite can alter the virulence and reproduction of the virulent parasite, depending on the order of infection and environmental conditions. Consistent with our findings, Lohr et al. (2010) showed that double infections of D. galeata by an intestinal protozoan and a haemolymph fungus (both HT) were more virulent than single infections and that prior residency does not always provide a competitive advantage. Given that sympatric coexistence of different parasite species is common in nature, these diverse results and in particular the role of abiotic factors emphasize the importance of studying double infections with the same ‘vigour’ as studies of multiple infections involving several strains of a single parasite species.

Another aspect of double infections that has often been overlooked concerns the mechanisms employed by each parasite to penetrate and infect the host. In the case of P. ramosa, Duneau et al. (2011) showed that spore penetration and activation in single infections are independent of genetic and environmental factors, whereas parasite attachment to the oesophagus is strongly influenced by host and parasite genotypes. Possible dependencies of O. bayeri infection steps on genetic or environmental factors have not been examined. Vizoso et al. (2005) found that in single infections, O. bayeri spores accumulate not only in fat cells but also in the ovary, eventually spreading throughout the entire body cavity. It remains to be determined how P. ramosa and O. bayeri interact with each other and/or with the host immune system. For instance, it is unknown whether double infections increase D. magna phenoloxidase (PO) activity beyond already-higher PO levels found in single infections (Pauwels et al., 2011).

The widespread persistence of strictly VT parasites is often attributed to their ability to kill males or induce feminization and thus alter host population sex ratio (Bandi et al., 2001), as well as to protect their host against more-virulent HT parasites (Haine, 2008; Brownlie & Johnson, 2009). This is because vertical transmission alone does not allow a virulent parasite to persist (Lipsitch et al., 1995). Despite the somewhat lower susceptibility to Pasteuria of hosts vertically infected by Octosporea, when compared to single infection by Pasteuria (P vs. Ov+P: 97.6% vs. 90.9%), VT O. bayeri kill their host around the same time as P. ramosa. Additionally, double infections (i.e. P+O, P+Od, O+Pd, Ov+P; Fig. 1a, b) considerably reduced host reproduction regardless of the mode of transmission. Animals vertically infected with Octosporea indeed carried a significantly lower sporeload of Pasteuria, probably due to the prior residency of Octosporea, but it remains to be determined whether such a reduction is sufficient to offset the harm caused by Octosporea. Hence, although VT O. bayeri does not appear to protect D. magna against P. ramosa, VT O. bayeri at least produces spores that can propagate via HT, which emphasizes the importance of horizontal transmission for Octosporea. It could perhaps be argued that concurrent transmission evolved as an alternative mechanism for some strictly VT parasites to offset their inability to confer resistance to their host against HT parasites. To attest this hypothesis, one would need (i) to repeat the present experiments with different combinations of Daphnia/Pasteuria/Octosporea clones, (ii) to demonstrate that O. bayeri does not benefit D. magna in other ways (e.g. reduced susceptibility to predation) and (iii) to investigate potential long-term effects of O. bayeri on P. ramosa in doubly infected Daphnia.

Consistent with previous studies of the DaphniaOctosporea system (Vizoso and Ebert, 2005a, b), sporeloads were higher in vertically vs. horizontally infected hosts. However, we found that a VT O. bayeri kills its host earlier and reduces fecundity more strongly than an HT O. bayeri, whereas previously it was found that horizontally infected hosts died earlier than vertically infected ones and that the infection route did not influence host fecundity (Vizoso and Ebert, 2005a,b). There are several possible explanations for these differences. First, in our study, the host clone was naïve to the O. bayeri isolate, whereas in previous studies, the parasite had been adapted to the host clone for several host generations. Previous work on O. bayeri indicated rapid adaptation of O. bayeri to its host clones (Altermatt et al., 2007). The increased virulence during the second phase of our experiment may thus be a result of the parasite adapting to the new host clone. O. bayeri adaptation may also ‘purify’ the parasite isolate by eliminating less-competitive or less-infective strains from the cocktail. Passaging and purifying prior to an experiment could result in more infective and virulent strains (spores) for horizontal infections (Ebert, 1998; Luijckx et al., 2011). Second, O. bayeri strains may differ in their within-host replication rates, insofar that some strains may benefit from increased virulence after being transmitted vertically, because their within-host replication rate is very high. In accordance with this prediction, O. bayeri sporeloads in vertically infected Daphnia (single infection) were more than two-fold higher in the present study than in previous Octosporea experiments. Third, horizontal transmission success is sensitive to the environmental conditions of the experiment. In particular, spore dose and culture conditions, which differed strongly between Vizoso & Ebert (2005a,b) and the current study, may influence the results and thus may explain part of the differences. Fourth, the Daphnia clone used in this experiment was different from that used in previous experiments. Strong host genotype × parasite genotype interactions, which have been observed in the DaphniaPasteuria system (Carius et al., 2001), could also account for the differences.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Our study demonstrated that within-host competition between different parasite species can be severe, even when the two parasites utilize different host tissues to proliferate (Pasteuria is an extracellular blood parasite and Octosporea is an intracellular parasite of fat cells and ovaries). Frequent competition of this form may select for higher virulence of any of the two parasites. We have also shown a strong asymmetry in competitive success; though, it remains to be determined whether this asymmetry is general or isolate specific. Our data do not support the idea that VT parasites may prevent later-infecting HT parasites from establishing successful infections. Neither do our results suggest that prior residency predicts the competitive outcome. Future work should focus on reconfirming our results using a wider range of Daphnia genotypes and parasite clones, as well as the long-term implications of double infections on the persistence of the two parasites and on the evolution of their virulences.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

We thank two anonymous reviewers for their helpful comments. We are in debt to Jürgen Hottinger for laboratory assistance and support. Urs Stiefel provided help during the experiment. This study was supported by the Swiss National Fonds.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References