Infection, Genetics and Evolution 11 (2011) 399–406

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Infection, Genetics and Evolution

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A virulent parasite can provide protection against a lethal

Eleanore D. Sternberg *, Thierry Lefe`vre, Amanda H. Rawstern, Jacobus C. de Roode

Biology Department, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA

ARTICLE INFO ABSTRACT

Article history: Hosts often become infected with multiple parasite strains or species. Previous work has shown that the Received 16 September 2010 outcome of infections with multiple parasite strains or species often differs significantly from that of Received in revised form 24 November 2010 single infections, making them a potentially important factor in determining the prevalence and spread Accepted 25 November 2010 of disease. Here we show that infection with a virulent parasite increases host survival during later Available online 9 December 2010 exposure to a lethal parasitoid. Specifically, when monarch butterfly larvae (Danaus plexippus) are inoculated with the virulent protozoan parasite Ophryocystis elektroscirrha and then attacked by the Keywords: lethal parasitoid fly archippivora, survival is higher than when the larvae are exposed to the Multiple infection parasitoid only. This is potentially a result of the protozoan’s requirement for host survival to obtain Co-infection Parasites between-host transmission. Our findings suggest that a virulent parasite can play a protective role for its Monarch butterflies (Danaus plexippus) host and indicate that parasites can act as mutualists depending on the presence of other parasites. We Ophryocystis elektroscirrha emphasize the importance of considering infection in an ecological context, including the presence of competing parasites. ß 2010 Elsevier B.V. All rights reserved.

1. Introduction From the parasite’s perspective, there can be benefits to infecting a host with other parasites, e.g., if one of the parasites In laboratory studies of infectious diseases, experimental dampens the host immune response (Cattadori et al., 2007; infections typically consist of a single parasite strain or species. Graham, 2008; Su et al., 2005) or if one of the parasites facilitates However, infection of a host with multiple parasite strains or infection or transmission of the other parasite (Friedli and Bacher, species occurs frequently outside of the laboratory (Cox, 2001; 2001; Hughes and Boomsma, 2004; Poulin et al., 2003). However, Petney and Andrews, 1998; Rigaud et al., 2010). In natural there can also be costs if the parasites overlap extensively in their populations, the parasites infecting a host can range from multiple host resource use (Hochberg, 1991; Ishii et al., 2002) or if one strains of the same species (Bharaj et al., 2008; Lord et al., 1999)to species alters the host environment in a way that makes it different species with varying degrees of taxonomical distance inhospitable to another parasite, for example through the (Cattadori et al., 2007; Craig et al., 2008; Petney and Andrews, activation of the host immune response (Dobson and Barnes, 1998; Rutrecht and Brown, 2008). Previous work has demonstrat- 1995; Lello et al., 2004). Negative interactions can occur between ed that the effect of co-occurring parasites on the host is not and microparasites (e.g., bacteria and viruses), for necessarily the additive effect of each single infection (Druilhe example when parasite-induced host death occurs too quickly for et al., 2005; Haine et al., 2005; Malakar et al., 1998; Pedersen and the second parasite to develop fully, thereby blocking the Fenton, 2006; Thomas et al., 2002). For example, infection with transmission of the second parasite (Chilcutt and Tabashnik, multiple parasite strains or species may benefit the host if the 1997; Escribano et al., 2000). In these examples, the amount of parasites use similar host resources, thus resulting in the time between infections often determines whether the second competitive suppression of parasite growth (Berchieri and Barrow, parasite is able to transmit upon host death. In addition to the 1990; Dobson and Barnes, 1995; Ishii et al., 2002; Read and Taylor, temporal spacing of infection, the order of infection can modify the 2001). Alternatively, the host may incur a greater cost from effect of multiple infections on host and parasites. Simultaneous infection with multiple parasites strains or species, for example infections may have a different outcome than sequential infections due to collateral damage from intense competition between and in some systems (but not all: Lohr et al., 2010), the effect of parasites (Read and Taylor, 2001; Roper et al., 1998). multiple infection on the parasites depends on which parasite has prior residency in the host (De Roode et al., 2005). All of the examples above illustrate that the outcome of infection with multiple parasites can diverge greatly from that of * Corresponding author. Tel.: +1 404 727 9344. single infections, and that the different biological characteristics of E-mail addresses: [email protected] (E.D. Sternberg), [email protected] (T. Lefe`vre), [email protected] (A.H. Rawstern), [email protected] the host and parasite species determine the outcome of infection. (J.C. de Roode). Furthermore, theoretical work has shown that the impact of

1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.11.017 400 E.D. Sternberg et al. / Infection, Genetics and Evolution 11 (2011) 399–406 co-occurring parasites extends to the evolution of hosts and outside of the developing butterfly. The production of these spores parasites (Rigaud et al., 2010), for example, by affecting the reduces monarch adult lifespan, mating ability and fecundity (De selection pressures that drive parasite virulence (Brown et al., Roode et al., 2009; De Roode et al., 2007; De Roode et al., 2008b). O. 2002; Choisy and De Roode, 2010; Van Baalen and Sabelis, 1995), elektroscirrha does not continue to replicate once the adult facilitating the emergence of novel pathogen strains (Lloyd-Smith monarch emerges, so the number of spores present on a newly et al., 2008), and altering the antagonistic co-evolutionary emerged adult monarch represents the entire transmission feedback between host and parasite (Mostowy et al., 2010). potential of that infection. Importantly, O. elektroscirrha requires Even with increasing awareness of both the ubiquity and its host to reach the adult stage, when the host can lay eggs and importance of multi-parasite infections, there are relatively few transfer these spores to hatching (De Roode et al., examples of studies in systems where transmission of one parasite 2009). is completely blocked by the successful transmission of a second The parasitoid fly (L. archippivora) co-occurs with O. elekroscir- parasite. In the literature that does exist on this subject, the rha in the monarch populations inhabiting North America and examples predominantly come from systems in which one parasite Hawaii (Altizer et al., 2000; Etchegaray and Nishida, 1975; Leong is transmitted trophically (through predation by a definitive host et al., 1997; Oberhauser et al., 2007). Female L. archippivora deposit on the intermediate host) while a second parasite is either not their eggs on the cuticle of larval monarchs and when these eggs trophically transmitted or requires a different definitive host hatch, the maggots burrow into the host. Previous work on L. species (Cezilly et al., 2000; Haine et al., 2005; Rigaud and Haine, archippivora indicates that this species typically limits its brood 2005; Thomas et al., 2002). In these cases, there is evidence that size to one to three offspring per host (Etchegaray and Nishida, parasites can rescue their hosts from the potentially lethal effects 1975; Oberhauser et al., 2007; Stapel et al., 1997). When the host of a second parasite if host survival is required for transmission of reaches its final instar or soon after it pupates, the fly maggots kill the first parasite (Cezilly et al., 2000; Haine et al., 2005). For the host as they emerge to form pupae (Oberhauser et al., 2007; example, the amphipod Gammarus roeseli serves as a host for both Stapel et al., 1997; Stireman et al., 2006). Thus, in contrast with the the trophically transmitted acanthocephalan Polymorphus minutus protozoan O. elektroscirrha, the parasitoid fly L. archippivora kills and the vertically transmitted (from parent to offspring) micro- the host during the pre-adult stage. sporidium Dictyocoela sp. (roeselum). P. minutus induces behav- ioral changes in G. roeseli to increase predation by its definitive 2.2. Host and parasite collection and care host; however, in the presence of the microsporidium, this behavioral manipulation is reduced (Haine et al., 2005). This study consisted of two experiments conducted approxi- Based on these results, we expect to find a similar outcome in mately one year apart. Experimental designs were similar, except other systems where transmission of one parasite is blocked by a for some minor differences as outlined below. All of the monarchs second parasite causing extensive damage to their shared host. belonged to the migratory eastern North American population, and This type of interaction occurs between the monarch butterfly all of the parasites were isolated from wild-caught monarchs (Danaus plexippus) and two of its most common parasites, the belonging to this population. Thus, both experiments used virulent protozoan Ophryocystis elektroscirrha and the lethal sympatric host and parasite combinations. The protozoan parasite parasitoid fly Lespesia archippivora. Transmission of the protozoan used for both studies (denoted C1E3-P3-1) was isolated from a O. elektroscirrha occurs exclusively through the transfer of spores monarch belonging to the eastern North American population from adult butterflies to larvae, and hence depends on the survival (Cape May, New Jersey, 2001). A clonal line of the parasite was used of infected monarchs to the adult stage (De Roode et al., 2008b; to prevent mixed-genotype infections and to provide consistency McLaughlin and Myers, 1970). In contrast, the fly L. archippivora in protozoan parasite genotype across experiments. To establish lays eggs onto monarch larvae, after which the eggs hatch and the the line, a monarch was inoculated with a single spore to produce maggots penetrate the larvae, consume them from the inside out, an infection with genetically identical parasites. The parasite was and emerge at the monarchs’ pre-pupal or pupal stage, killing the then passaged through three monarchs and held at 12 8C between monarchs in the process. These differences in life cycle would infections. suggest that L. archippivora prevents transmission of O. elektroscir- The monarchs used in this study were the grand-progeny of rha when it kills the host during the pre-adult stages. However, as monarchs collected either as overwintering adults in Central an alternative hypothesis, it is possible that the protozoan parasite Mexico (March 2008; experiment 1) or as larvae in Georgia, USA reduces the infection success of the lethal fly, and thereby (September 2009; experiment 2). Unrelated females and males alleviates its own fitness loss as well as that of the host. Here, were mated in a design that produced independent families of half- we address these hypotheses by studying single and multiple or full siblings. Adults were held in mesh cages at 26 8C on a 16L:8D infections of these two parasite species in laboratory experiments. cycle and fed with a 10% honey water solution. After mating, males were removed and females were provided with greenhouse-grown 2. Materials and methods Asclepias incarnata (swamp milkweed) for ovipositing. The plants were checked daily and those with eggs were replaced with fresh 2.1. The host–parasites system plants. Hatching larvae were pooled by hatch date and family, and transferred into plastic containers (739 mL) with fresh A. incarnata The protozoan O. elektroscirrha is a parasite that infects cuttings. Individuals from each of the families were randomly monarch butterflies across their natural range (Altizer et al., distributed across all treatment groups for both experiments. 2000; Leong et al., 1997; McLaughlin and Myers, 1970). Infection The parasitoid flies used for this study came from a laboratory occurs when adult female butterflies shed parasite spores on their colony descended from maggots that emerged from monarchs eggs or milkweed foliage during oviposition, after which these collected as larvae in Ohio, USA (June 2008; experiment 1) or Georgia, spores are ingested by hatching larvae; as a result, transmission USA (September 2009; experiment 2). Upon emergence from the occurs often from mother to offspring but may also occur from monarch larvae, the maggots were transferred either into 1L plastic adult butterflies to unrelated larvae (Altizer et al., 2004; containers (experiment 1) or into a 50.8 cm  27.9 cm  33.0 cm McLaughlin and Myers, 1970). Upon ingestion, parasite spores glass terrarium fitted with a screened lid (experiment 2). When adult lyse in the larval gut to release sporozoites that invade the flies eclosed, they were provided with sugar, dehydrated milk and a hypoderm, replicate asexually, and then form sexual spores on the moistened cotton ball for water. To establish and maintain the colony, E.D. Sternberg et al. / Infection, Genetics and Evolution 11 (2011) 399–406 401

flies were given 24 h to mate, then provided with 3rd instar larger sample sizes were used for groups exposed to the parasitoid monarch larvae for ovipositing. Once the monarch larvae had been fly to enable the detection of differences in the low survival rates of attacked by flies, they were pooled into plastic containers and fly-exposed monarchs. provided with fresh A. incarnata cuttings. When maggots emerged Procedures for experiment 2 were similar to those for from these monarch larvae, they were collected and added to the experiment 1, except for the following. First, to infect monarchs existing colony. with parasitoid flies, 3rd instar monarch larvae were placed in the terrarium housing the L. archippivora colony and observed until a 2.3. Experiment 1 fly was seen approaching a larva and performing characteristic ovipositing behaviors, at which point the larva was removed from Monarchs (n = 292) received one of four treatments: (1) the terrarium. The larva was then examined under a dissecting uninfected control (n = 18); (2) exposure to only the protozoan microscope and the number of parasitoid fly eggs present recorded. parasite O. elektroscirrha (n = 38); (3) exposure to only the Unlike experiment 1, larvae were not returned to the container parasitoid fly L. archippivora (n = 117); and (4) exposure to both with the flies even if there were no visible eggs. The results from the protozoan parasite and the parasitoid fly (n = 119). Larger experiment 1 had shown to us that we often missed eggs, as sample sizes were used for the monarchs that were exposed to the evidenced by the fact that some monarchs produced more maggots parasitoid fly so that differences in low survival rates could be than the recorded number of eggs. This modified protocol was used detected, even in the presence of the highly lethal parasitoid. to more accurately mimic the natural numbers of flies per infected To infect monarchs with the protozoan parasite, 2-day-old 2nd monarch (in experiment 1 they exceeded those numbers). Second, instar monarch larvae were placed in individual 10 cm petri dishes larvae were reared as in experiment 1, except that Asclepias with moist filter paper and a disc of A. incarnata leaf (0.8 cm in curassavica (tropical milkweed) was used in addition to A. incarnata diameter) on which 10 O. elektroscirrha spores had been deposited to feed later stage caterpillars; the use of a second species of manually (De Roode et al., 2007; De Roode et al., 2008a); milkweed at this stage has no effects on the protozoan parasite (De uninfected controls and monarchs that were infected with the Roode et al., in press) and parasitoid fly post-inoculation (M. parasitoid fly only were fed a leaf disk without parasite spores. To Solensky, unpublished data). The host plant species that was infect monarchs with the parasitoid fly, we placed 4-day-old 3rd provided was randomly allocated across treatment groups. Third, if instar monarch larvae in the plastic containers housing the fly maggots emerged from monarch larvae or pupae, the number of parasitoid flies and observed until a fly was seen approaching a maggots per host and the mass of each maggot once it pupated larva and performing ovipositing behaviors. At this point the larva were recorded, after which the fly pupae were placed in individual was removed and examined under a dissecting microscope for fly plastic containers. If a fly eclosed from its , it was recorded to eggs. If fly eggs were not observed, the larva was returned to the measure the proportion of flies that survived to adulthood. Adult container with flies. Once eggs were visible, the number of eggs flies were provided with sugar, dehydrated milk, and a moist present was recorded and the larva was not returned to the plastic cotton ball. were checked daily for mortality to measure adult container with the flies. In the multiple infection treatment, longevity. Fourth, after monarchs died, their bodies were vortexed monarchs were first inoculated with the protozoan parasite (2 days and the protozoan parasite spore load was measured with a post-hatching) and then exposed to the parasitoid (4 days post- haemocytometer as described in De Roode et al. (2007, 2008a). hatching). Since the protozoan does not replicate on the adult monarch, this After treatment, monarch larvae were transferred into individ- was used as a measure of parasite replication and transmission ual plastic containers covered with mesh tops. They were provided potential. with fresh cuttings of greenhouse-grown A. incarnata in florist tubes. These containers were kept in a climate-controlled room 2.5. Statistics (26 8C, 16L:8D) and checked daily. Fresh plant cuttings were added as needed. Monarch larvae that died prior to pupation were All analyses were carried out in R version 2.7.1 (R Development monitored for signs of parasitoid maggots. If maggots emerged, the Core Team, 2006). Logistic regression by Generalized Linear Model number of maggots per host was recorded. If the monarch larvae (GLM, binomial error distribution, logit link) was used to survived to pupation, they were transferred into clean plastic investigate the effect of treatment, number of parasitoid fly eggs, containers and kept in the climate controlled room for an and experimental block (experiment 1 vs. 2) on the proportion of additional 7 days. Monarch pupae were also checked daily for monarch hosts that survived to adulthood. A multiway Analysis of signs of fly maggots. After 7 days, the monarch pupae were Variance (ANOVA) was used to analyze the effect of treatment and transferred to a separate room to prevent cross-contamination experimental block on monarch host longevity and in experiment with spores from the protozoan parasite. When the adult monarchs 2 protozoan parasite spore load. Logistic regression (GLM, quasi- eclosed, they were sexed and weighed and then transferred into binomial distribution) was used to assess the effect of treatment individual glassine envelopes and kept at 12 8C. Adult monarchs and experimental block on the proportion of monarch hosts that were checked daily for mortality to measure post-eclosion produced fly parasitoid maggots. A GLM with a quasi-Poisson error longevity. This measure of lifespan provides a combined index distribution was used to analyze the effect of treatment on the of adult monarch life span and starvation resistance and responds number of fly parasitoid maggots that emerged from monarch to parasite infection and increasing parasite numbers in a similar hosts. For the data from experiment 2, a Generalized Linear Mixed way as lifespan under more natural conditions (De Roode et al., Model (GLMM) with a normal error distribution was used to 2009). analyze the effect of treatment (fixed effect) and monarch host (random effect) on fly parasitoid mass and longevity. An Analysis of 2.4. Experiment 2 Covariance (ANCOVA) was used to test for a relationship between fly parasitoid pupal mass, adult longevity, and for an effect of As with experiment 1, monarchs (n = 351) received one of four treatment on this relationship. Protozoan parasite load and treatments: (1) uninfected control (n = 25); (2) exposure to only monarch host longevity were Log10-transformed prior to analyses the protozoan parasite O. elektroscirrha (n = 32); (3) exposure to and models were checked for homogeneity of variance by using the only the parasitoid fly L. archippivora (n = 155); and (4) exposure to Fligner–Killeen test (Crawley, 2007). In these analyses, treatment both the protozoan parasite and the parasitoid fly (n = 139). Again, and experimental block were treated as categorical explanatory 402 E.D. Sternberg et al. / Infection, Genetics[()TD$FIG] and Evolution 11 (2011) 399–406 variables. Full models included treatment, experimental block and the interaction between them as explanatory variables. Minimal models were derived by removing model terms followed by model comparison. Only terms for which removal significantly (P < 0.05) reduced the explanatory power of the model were retained in the minimal model (Crawley, 2007).

3. Results

3.1. Monarch host pre-adult survival

Parasitoid flies dramatically reduced the survival to adulthood of their monarch hosts, both in the presence of the protozoan parasite (Fig. 1; Odds Ratio (OR) = 25.0, 95% Confidence Interval (CI) = [24.1, 26.0], P < 0.001) and in single infections (OR = 41.9, CI = [40.9, 42.9], P < 0.001). The protozoan parasite alone did not affect monarch host survival to adulthood; however, it did increase survival of monarchs that were also infected with the parasitoid fly (Fig. 1; OR = 1.8, CI = [1.28, 2.27], P = 0.0239). Only 12% and 17% (experiments 1 and 2 respectively) of monarchs survived to Fig. 1. Monarch survival to adulthood by experiment and treatment group. The adulthood when infected with the parasitoid fly alone, but when presence of the parasitoid fly resulted in a large decrease in survival for both the the protozoan parasite was also present, survival increased to 18% singly and multiply infected monarchs, but there was a higher percent survival for and 27% (experiments 1 and 2 respectively). The number of eggs monarchs infected with the fly and the protozoan together compared to those that were infected with the fly only. Data are presented as percent survival ÆSE. laid by the parasitoid fly also had a significant effect on survival in monarchs that were exposed to the parasitoid fly (OR = 4.12, CI = [3.72, 4.52], P < 0.001), with higher numbers resulting in 3.4. Parasitoid fly emergence lower survival. There were no significant interactions between the number of eggs and the treatment group. Overall, monarch pre- Although the protozoan parasite lowered the pre-adult adult survival differed significantly between experiments mortality of monarchs infected with the parasitoid fly, this did (OR = 1.9, CI = [1.34, 2.50], P = 0.0281) but there were no significant not result in a significant reduction of parasitoid fly fitness. Thus, interactions between experiment and treatment or the number of although slightly fewer fly-infected monarchs produced viable eggs present. maggots when the protozoan parasite was present (Fig. 3; single infection vs. multiple infection: 82% vs. 78% in experiment 1 and 3.2. Monarch host adult longevity 58% vs. 54% in experiment 2), these reductions were not significant (OR = 1.11, CI = [0.71, 1.52], P = 0.602). As expected (based on Analysis of monarch host longevity was performed on the lower inoculation doses in experiment 2), fewer monarchs monarchs that survived to adulthood. For experiment 1, there were produced maggots in experiment 2 (OR = 2.45, CI = [1.51, 3.38], 89 surviving monarchs (18 of 18 in the control group, 34 of 38 in P[()TD$FIG]= 0.0609). There was a significant interaction between number of the protozoan only group, 15 of 117 in the fly only group, and 22 of 119 in the protozoan and fly group). For experiment 2, there were 111 surviving monarchs (20 of 25 in the control group, 26 of 32 in the protozoan only group, 27 of 155 in the fly only group, and 38 of 139 in the protozoan and fly group). The presence of the protozoan parasite had a significant effect on mean longevity (Fig. 2; F1,185 = 695, P < 0.001): both singly protozoan parasite-infected and multiply-infected monarchs lived much shorter as adults than control monarchs and monarchs that had survived single infection with the parasitoid fly. There was no significant effect of larval exposure to the parasitoid fly on adult longevity across all treatment groups (F1,186 = 1.2, P = 0.271). A significant interaction between experiment and treatment

(F1,185 = 62.0, P < 0.001) arose from the fact that the protozoan parasite reduced adult monarch lifespan more strongly in experiment 1 than 2.

3.3. Protozoan parasite spore load

Analysis of the protozoan parasite spore load was carried out for the monarchs in experiment 2 that survived to adulthood (n = 64). All of the surviving monarchs that were inoculated with the protozoan parasite were infected. There was no Fig. 2. Mean adult longevity in days for monarchs that survived the larval stage, by experiment and treatment group. The presence of the protozoan parasite had significant difference in the mean spore loads of monarchs that a significant effect on host longevity in both single and multiple infection survived attack by the parasitoid fly compared to monarchs that groups; however, in monarchs that survived attack by the parasitoid fly there had been exposed to the protozoan parasite only (F1,62 =0.036, was no additional decrease in adult longevity. Data are presented as mean adult P = 0.85). longevity ÆSE. [()TD$FIG] E.D. Sternberg et al. / Infection, Genetics and[()TD$FIG] Evolution 11 (2011) 399–406 403

Fig. 3. Percent of monarchs attacked by flies that produced maggots, by experiment and treatment group. There was a small trend with fewer monarchs producing maggots when also infected with the protozoan parasite, but this trend was not Fig. 5. Relationship between parasitoid fly pupal mass and parasitoid adult significant. Data are presented as percent of monarchs that produced at least one longevity; experiment 2 only. Data points represent individual parasitoids. There maggot ÆSE. was a significant positive relationship between pupal mass and longevity; however, this relationship was not significantly different between single and multiple infection groups. eggs and experiment, indicating that the proportion of monarchs that produced maggots increased more quickly with increasing numbers of eggs in experiment 1 than experiment 2 (OR = 3.78, the protozoan and fly multiple infection group were removed from CI = [3.06, 4.50], P < 0.001). The interaction between treatment analysis due to incomplete data. and experiment was not significant. Comparison between fly single infections and protozoan and fly As with the proportion of monarchs that produced maggots, the multiple infections showed that the protozoan parasite had no protozoan parasite did not significantly reduce the numbers of effect on the mean fly pupal mass (mean Æ SE; 26.2 Æ 1.1 and maggots that emerged from monarchs exposed to the parasitoid fly 27.0 Æ 1.2 mg in single and multiple infections respectively; d.f. = 1,

(Fig. 4; F1,525 = 0.423, P = 0.52). As expected, however, monarchs P = 0.851), on the proportion of fly pupae that successfully eclosed as produced fewer maggots in experiment 2 than experiment 1 adults (73.9% and 78.8% from single and multiple infection 2 (F1,526 = 231, P < 0.001). There was no significant interaction respectively; X = 0.517, d.f. = 1, P = 0.472), or on the adult longevity between treatment and experiment. of flies that eclosed successfully (18 Æ 1 days for single and multiple infections; d.f. = 1, P = 0.83). Higher fly pupal mass resulted in greater 2 3.5. Parasitoid fly pupal mass and adult longevity fly adult longevity (Fig. 5; F1,185 = 22.4, R = 0.103, P < 0.001) but there was no significant interaction with treatment group. Data on fly pupal mass and adult longevity were obtained for 249 flies (142 maggots from monarchs singly infected with the fly 4. Discussion and 107 maggots from multiply-infected monarchs); 3 maggots in [()TD$FIG] Our results clearly demonstrate that infection of hosts with multiple parasite species may have important consequences for host and parasite fitness. Overall, the parasitoid fly L. archippivora had strong detrimental effects on its monarch host as well as on the co-occurring protozoan parasite O. elektroscirrha. L. archippivora caused dramatic pre-adult mortality of monarch butterflies and thereby strongly reduced O. elektroscirrha’s fitness as the latter parasite requires the monarch to reach adulthood for its transmission. However, despite the parasitoid’s overwhelming effects, we found that the protozoan parasite reduced the mortality caused by the parasitoid fly, with more monarchs surviving multiple infection with the protozoan and fly than infection with the fly alone (Fig. 1). This effect is beneficial to the host and resembles the protective effects that mutualists and commensals can confer. For example, previous research has shown that mutualistic bacterial symbionts of aphids can protect their host against parasitoid wasps and pathogenic fungi (Oliver et al., 2003; Scarborough et al., 2005; Vorburger et al., 2010), that Wolbachia bacteria can protect Drospophila melanogaster against viral infections (Hedges et al., 2008) and that Spiroplasma bacteria Fig. 4. Mean number of maggots emerging per host for all monarchs exposed to the confer protection against a sterilizing nematode in D. neotestacea parasitoid, by experiment and treatment group. There was no significant difference between treatments in the mean number of maggots that emerged from monarchs. (Jaenike et al., 2010). Like symbionts, transmission of the There was a significantly higher parasitoid burden in experiment 1 compared to monarch’s protozoan parasite depends on the survival of its host experiment 2. Data are presented as mean number of maggots per host ÆSE. but unlike these bacterial symbionts, O. elektroscirrha is highly 404 E.D. Sternberg et al. / Infection, Genetics and Evolution 11 (2011) 399–406 virulent to monarchs, reducing adult longevity, mating ability, parasites, such as parasitoid flies. If the parasitoid fly elicits a fecundity and flight ability (De Roode et al., 2007, 2008b, 2009; monarch immune response that damages both monarch and fly, Bradley and Altizer, 2005). Thus, our results suggest that infection and if the protozoan parasite decreases this response, then this with a virulent parasite can be beneficial for a host when it confers may result in increased host survival. Clearly, we must advance our protection against a parasite that is even more detrimental. understanding of the monarch’s immune system to test these However, this protection appears to be weaker than that provided hypotheses. by beneficial symbionts, perhaps because parasites face different Our results showed that multiple infection with the protozoan constraints than symbionts. For instance, it is likely that a and parasitoid fly affected host and parasite fitness only on the beneficial symbiont and host cooperate to resist lethal parasitoids; basis of parasite interactions in the monarch’s larval stage, and that in contrast, although it may be beneficial to one parasite to reduce these interactions did not lead to further effects during the adult the virulence induced by another, it is in the host’s interest to resist stage. From the monarch host’s perspective, adult longevity was both. not significantly different between uninfected monarchs and The increased survival of monarchs infected with both parasites monarchs that survived attack by the parasitoid fly, and there was is not only beneficial to the host, but also to the protozoan parasite, no difference in longevity between monarchs infected with the suggesting that this effect may be an adaptive parasite trait. protozoan alone or those who were also exposed to the parasitoid Parasites are well known to change the phenotypes of their hosts in fly. Thus, the parasitoid fly does not appear to have a long-lasting ways that serve a specific adaptive function for the parasite effect on the monarch if the monarch is able to successfully defend (Lefe`vre et al., 2009; Moore, 2002; Poulin, 2010; Thomas et al., itself against fly infection. 2005). For example, parasites often modify the behaviors of their As for the protozoan parasite, infecting a host that was later host to prevent infection with a competing parasite (Brodeur and infected with the parasitoid fly severely reduced its fitness, since McNeil, 1992), to prevent predation (Grosman et al., 2008), or to most monarchs were killed before they reached adulthood and increase transmission to the definitive host (Lagrue et al., 2007). transmitted the protozoan. However, we found no significant Based on these studies, it is possible that the increase in host differences between the mean spore loads of surviving monarchs survival that we observed may also serve an adaptive function for infected only with the protozoan compared to monarchs that were the protozoan parasite due to the protozoan parasite’s requirement also attacked by the parasitoid fly. Spore load is a measure of that the monarch host survive to adulthood for transmission to replication for the protozoan and it is also associated with occur. If this is the case, we would expect that the parasite’s ability virulence and transmission probability, which makes it an to increase host survival is selected for in populations where the appropriate measure of parasite fitness (De Roode et al., 2008b). parasitoid occurs and is highly prevalent. Since this study tested These results further support the conclusion that if the monarch the effect of a single genotype of the protozoan parasite, future succeeds in defending itself against the parasitoid, there are no experiments are necessary to determine if there is variation among long-term costs to either the host or the protozoan parasite protozoan parasite genotypes in their protective effect. However, resulting from exposure to the parasitoid fly. Finally, we also found since all genotypes of the protozoan parasite require host survival no evidence for an effect of multiple infection on the parasitoid fly to the adult stage for transmission to occur, we expect to see a beyond its development inside of the host. When we compared the similar effect with other parasite genotypes. flies that developed in the presence of the protozoan to those that One challenge to understanding the protozoan parasite’s developed alone, we found no significant difference in the pupal protective effect will be to explain how the protozoan reduced mass of the flies, the proportion of flies that successfully eclosed or monarch pre-adult mortality without also affecting the parasitoid the longevity of the flies that survived to adulthood. We did find a fly’s fitness. Although the protozoan reduced the mortality of fly- positive correlation between pupal mass and longevity in the infected monarchs (Fig. 1), this did not reduce the proportion of fly- parasitoid fly, but this relationship did not differ between flies that infected monarchs that produced maggots (Fig. 3), nor did it reduce developed from monarchs infected or not infected with the the average number of maggots produced per monarch (Fig. 4). It is protozoan parasite (Fig. 5). Thus, while the protozoan parasite can known that in some infections, fly maggots do not develop reduce the host mortality caused by exposure to the parasitoid fly, successfully into pupae, but still end up killing the monarch it does not appear to do so in a way that affects any of the fly fitness butterfly larva (Oberhauser et al., 2007). This finding, in combina- components that we measured. tion with our results, suggests that the protozoan parasite reduced Overall, our results underscore the need to consider infection monarch mortality caused by unsuccessful, rather than successful, with multiple parasites as a major determinant of parasite and host parasitoid flies. There are two potential explanations for such a fitness, particularly in systems where one parasite’s success results scenario. First, the protozoan parasite may enhance the monarch’s in a loss of transmission for another one. We emphasize the clearance of dying parasitoids inside its body. Previous research on importance of the host and parasite life cycles and biological multiple infections has suggested that parasites can limit the characteristics in determining how multiple infection differs from growth and transmission of a competing parasite by eliciting a host single infection. Finally we suggest that in systems where one immune response (Lello et al., 2004; Meister et al., 2009; Pedersen parasite is extremely deadly, the host may actually benefit from a and Fenton, 2006; Ra˚ berg et al., 2006), and it is possible that the second, more benign parasite if that parasite can increase its own protozoan parasite increases the immune response against the transmission by increasing host survival. As such, our results parasitoid fly. It is certainly likely that the monarch host mounts an support the view that parasitism is a context-dependent phenom- immune response against the fly since in our experiments there enon (Fellous and Salvaudon, 2009; Michalakis et al., 1992; were several cases in which monarchs were infected with the fly, Thomas et al., 2000; Vale et al., 2008; Wolinska and King, 2009), yet did not produce any maggots. As an alternative explanation, it and that parasites can act as mutualists in the presence of more is possible that the protozoan parasite increases host survival by deadly natural enemies. dampening the host immune response, and that this reduced immune response results in a lower amount of immunopathology. Acknowledgements Immunopathology occurs in many vertebrate and invertebrate species (Brandt et al., 2004; Graham et al., 2005; Sadd and Siva- We thank Michelle Solensky, Rachel Rarick, Brandon Hedrick, Jothy, 2006; Shi et al., 2001) and may be especially relevant in Carlos Lopez, Emily Toriani-Moura and the Cobb County Water systems where an host must defend itself against insect System for assistance with experiments and obtaining flies and E.D. Sternberg et al. / Infection, Genetics and Evolution 11 (2011) 399–406 405 butterflies. We also thank members of the Invertebrate Host– Graham, A.L., 2008. Ecological rules governing helminth-microparasite coinfection. 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