INTRODUCTION
The environment organisms experience is rarely uniform; it varies over time and space. This applies to both free-living organisms and their parasites. Environmental stressors can reshape host–parasite interactions in various ways depending on the species and stressors involved (Lafferty and Kuris, Reference Lafferty and Kuris1999). Many parasites enhance their fitness by changing the behaviour of their host (reviewed by Holmes and Bethel, Reference Holmes and Bethel1972; Poulin and Thomas, Reference Poulin and Thomas1999; Moore, Reference Moore2002, Reference Moore2013; Poulin, Reference Poulin2010). Such host manipulation is well-known in complex life cycle parasites, where it not only serves to enhance transmission to a target host (predation enhancement), but also to avoid fatal predation before the parasite reaches infectivity (predation suppression) (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Parker et al. Reference Parker, Ball, Chubb, Hammerschmidt and Milinski2009; Dianne et al. Reference Dianne, Perrot-Minnot, Bauer, Elsa and Rigaud2011; Thomas et al. Reference Thomas, Brodeur, Maure, Franceschi, Blanchet and Rigaud2011; Weinreich et al. Reference Weinreich, Benesh and Milinski2013). Such host manipulation could also be influenced by the host's environment (Thomas et al. Reference Thomas, Rigaud, Brodeur, Hughes, Brodeur and Thomas2012). Biotic factors such as predator cues (e.g. Jakobsen and Wedekind, Reference Jakobsen and Wedekind1998; Baldauf et al. Reference Baldauf, Thünken, Frommen, Bakker, Heupel and Kullmann2007; Durieux et al. Reference Durieux, Rigaud and Médoc2012; Dianne et al. Reference Dianne, Perrot-Minnot, Bauer, Guvenatam and Rigaud2014) or the presence of other parasites (Cézilly et al. Reference Cézilly, Grégoire and Bertin2000, Reference Cézilly, Perrot-Minnot and Rigaud2014; Haine et al. Reference Haine, Boucansaud and Rigaud2005; Dianne et al. Reference Dianne, Rigaud, Léger, Motreuil, Bauer and Perrot-Minnot2010; Hafer and Milinski, Reference Hafer and Milinski2015) can influence host manipulation. Differences in abiotic factors might also play a role. In isopods infected by an acanthocephalan parasite, host manipulation changes between seasons, but this does not seem to be caused by either temperature or lighting conditions but might rather be related to host or parasite age (Benesh et al. Reference Benesh, Hasu, Seppälä and Valtonen2009). Resource availability, too, could affect host manipulation. Hungry and satiated hosts can differ in the extent of parasite-altered behaviour they show (e.g. Giles, Reference Giles1987; Barber et al. Reference Barber, Huntingford and Crompton1995; Jakobsen and Wedekind, Reference Jakobsen and Wedekind1998). If the existence and efficacy of host manipulation varies with particular environmental factors, then parasite transmission rates will differ between environments. An evolutionary consequence of this is that the benefits of and selection for host manipulation will vary across time and/or space (Franceschi et al. Reference Franceschi, Cornet, Bollache, Dechaume-Moncharmont, Bauer, Motreuil and Rigaud2010b ).
Host manipulation can result in energetically costly behaviour such as increased activity (reviewed by Poulin, Reference Poulin1994a ). Resource limitation will reduce a host's ability to perform such behaviours (Thomas et al. Reference Thomas, Brodeur, Maure, Franceschi, Blanchet and Rigaud2011, Reference Thomas, Rigaud, Brodeur, Hughes, Brodeur and Thomas2012). Limited resources could also reduce host manipulation, if parasites need to use energy to manipulate and are limited by the available resources of their host. Energetic costs of host manipulation have often been assumed, because parasites might use some substance they have to produce and emit to manipulate their hosts (Poulin, Reference Poulin1994b , Reference Poulin2010; Biron et al. Reference Biron, Moura, Marché, Hughes and Thomas2005; Thomas et al. Reference Thomas, Adamo and Moore2005, Reference Thomas, Brodeur, Maure, Franceschi, Blanchet and Rigaud2011; Vickery and Poulin, Reference Vickery and Poulin2009); but these have yet to be convincingly demonstrated. Potential trade-offs between the level of host manipulation and other important parasite traits could hint at such a cost (Franceschi et al. Reference Franceschi, Bollache, Cornet, Bauer, Motreuil and Rigaud2010a ; Maure et al. Reference Maure, Brodeur, Ponlet, Doyon, Firlej, Elguero and Thomas2011). Alternatively, host manipulation could increase under resource limitation. For example, lack of resources could impair a host's defences against manipulation (Roitberg, Reference Roitberg, Hughes, Brodeur and Thomas2012). Another way for manipulation to increase under resource limitation is if parasites affect host behaviour by draining energy from the host (reviewed by Adamo, Reference Adamo, Hughes, Brodeur and Thomas2012; Lafferty and Shaw, Reference Lafferty and Shaw2013). Energy drain shifts the trade-off between predator avoidance and feeding towards feeding and might hence resemble host manipulation that serve to increase a host's predation susceptibility (Milinski, Reference Milinski, Barnard and Behnke1990).
Schistocephalus solidus has a three host life cycle consisting of a cyclopoid copepod, a three-spined stickleback and a piscivorous bird (Clarke, Reference Clarke1954; Dubinina, Reference Dubinina1980). At 18 °C it spends about 2 weeks in copepods before it is ready for transmission (i.e. infective) to the next host, which takes place when the copepod is consumed by a stickleback. During this time, it reduces the activity (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Benesh, Reference Benesh2010a ; Hafer and Milinski, Reference Hafer and Milinski2015) and predation susceptibility (Weinreich et al. Reference Weinreich, Benesh and Milinski2013) of its host. In a previous study (Benesh and Hafer, Reference Benesh and Hafer2012), we investigated the effect of the performance of S. solidus in its copepod host (i.e. growth and development) on performance in the fish host (e.g. infection success and growth). To create variation in copepod quality, we used two different feeding treatments which differed in food quantity, but not quality. Here, we report the impact of these feeding treatments on the behaviour of infected and uninfected copepods. In addition, we explored potential trade-offs between host manipulation and other life history traits that could hint at an energetic cost to host manipulation.
METHODS
Schistocephalus solidus and infection
Schistocephalus solidus originated from Lake Skogseidvatnet, Norway. They were obtained by dissecting wild-caught three-spined sticklebacks (Gasterosteus aculeatus) and breeding the adult parasites in pairs in an in vitro system (Smyth, Reference Smyth1946). Tapeworm eggs were stored in the fridge (4 °C) until use, incubated for 3 weeks at 20 °C, and then exposed to light over night to induce hatching (Dubinina, Reference Dubinina1980). Infection took place by exposing copepods to one coracidium each. Copepods that were used as uninfected controls received no coracidia but were otherwise treated the same.
Copepod maintenance
We used copepods (Macrocyclops albidus) from a laboratory stock that originated from the same population as S. solidus (Lake Skogseidvatnet, Norway). On the day prior to infection they were distributed on 24-well microtitre plates with about 1 mL of water. Copepods were maintained at 18 °C in a 16/8 h light/dark cycle and fed with Artemia sp. naupili every other day. Copepods in the high food treatment (H) received four Artemia at each feeding, while copepods in the low food treatment (L) received two (Fig. 1). These treatments were sufficient to affect copepod mortality and parasite growth in other experiments (Benesh, Reference Benesh2010b ). In our experiment, the high food treatment led to faster parasite development, faster growth and higher infection success in one experimental group (day-11 copepods). In a second experimental group (day-17 copepods), the high food treatment only significantly improved parasite growth but not development or infection success (See Supplementary information).
Fig. 1. Overview of the experimental setup. Copepods were either exposed to S. solidus coracidia or not, subjected to a high or a low food treatment and exposed to fish at two different time points, 11 dpi (day-11 copepods) or 17 dpi (day-17 copepods). We measured three aspects of parasite performance: development and size in copepods and infection success in fish. We measured two aspects of copepod behaviour: activity (i.e. proportion of time copepods spent moving) and, when moving, the distance moved.
Measurements of parasite performance
Copepods are transparent making it possible to view and measure a parasite in vivo (Wedekind et al. Reference Wedekind, Christen, Schärer and Treichel2000; Benesh et al. Reference Benesh, Weinreich and Kalbe2012). We checked copepods for infection 6–8 days post infection (dpi). Parasites were additionally checked inside their hosts for the presence or absence of a cercomer 9 dpi. Although the function of the cercomer is unknown, it is a good indicator for parasite development and the ability to infect fish (Smyth and McManus, Reference Smyth and McManus1989). The parasites used in this study were more likely to infect fish 11 dpi if they had a cercomer 9 dpi than if they did not (Benesh and Hafer, Reference Benesh and Hafer2012). On the day prior to exposure to fish (10 or 16 dpi), we measured the size of the parasite. This took place by photographing each parasite twice within its host under a microscope. From these photographs, we measured the area of the parasite (without cercomer) using image J (Rasband, Reference Rasband2008) and took the average from these two measurements. Three-spined sticklebacks (G. aculeatus) were individually exposed to a single copepod, either 11 (day-11 copepods) or 17 (day-17 copepods) dpi. Any potential trade-off between manipulation and the ability to infect fish should be especially crucial 11 dpi, since at this time only the fastest developing S. solidus might be ready to infect the next host (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Benesh and Hafer, Reference Benesh and Hafer2012). By 17 dpi, nearly all S. solidus are morphologically well-developed (Benesh, Reference Benesh2010b ) and ready to infect fish (Benesh and Hafer, Reference Benesh and Hafer2012). Approximately 4 weeks after exposure, we dissected the fish to determine infection success (see Benesh and Hafer, Reference Benesh and Hafer2012). Fish experiments were conducted with the permission of the ‘Ministry of Energy, Agriculture, the Environment and Rural Areas’ of the state of Schleswig-Holstein, Germany (reference number: V 313-72241·123-34).
Copepod behavioural measurements
Schistocephalus solidus has to spend between 10 days and 2 weeks at 18 °C in its copepod host before it becomes infective to sticklebacks, at which time host manipulation switches from predation suppression to predation enhancement (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Hafer and Milinski, Reference Hafer and Milinski2015). This switch is not obvious in all populations and some populations show no clear predation enhancement, including the population used in this study (Benesh, Reference Benesh2010a ). We measured behaviour on multiple days and grouped the observations into two age groups: behaviour 7, 8 and 9 dpi (parasite is not yet infective to fish) and 13, 14 and 15 dpi (infective parasite, only recorded for the day-17 copepods). We mostly focus on predation suppression, which should occur 7, 8 and 9 dpi when parasites should not yet be infective. Multiple observations help to more accurately quantify an individual's typical behaviour and make correlations between host manipulation and other parasite traits more robust (Benesh et al. Reference Benesh, Valtonen and Seppälä2008).
Each well plate with copepods was gently placed on an apparatus that dropped it by 3 mm in a standardized way to simulate a predator attack (see Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009). Once the plate had been on the apparatus for a 1 min acclimation period, we started recording copepod behaviour using a video camera (Panasonic Super DynamicWV-BP550, Panasonic Corporation, Osaka, Japan). Copepods were recorded for 90 s, then the plate was dropped and copepods were recorded for an additional 90 s. We split the recordings into 2 s intervals (90 observations per copepod per recording event). Preliminary analyses found that little extra movement was recorded with shorter intervals. Using the manual tracking plugin within image J (Rasband, Reference Rasband2008), we recorded whether or not each copepod moved within each of these 2 s intervals (activity) and, if so, how far it moved (distance). We only analysed the behaviour of exposed and infected and unexposed and uninfected copepods that survived until the day that its treatment group was used to infect fish (11 or 17 dpi). We did not record the behaviour of exposed but uninfected copepods, because previous studies indicated they do not differ from unexposed controls (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Benesh, Reference Benesh2010a ). In total we recorded the behaviour of 382 copepods (day-11 copepods: infected: H: 65, L: 68, uninfected: H: 45, L: 44; day-17 copepods: infected: H: 51, L: 51, uninfected: H: 30, L: 28).
Statistical analysis
We analysed copepod behaviour at a fine scale, i.e. each 2 s observation. Thus, we had repeated observations on the same copepod at two levels: within a recording event (i.e. each copepod on a given day) and across recording events on different days. To analyse copepod activity (proportion of 2 s intervals spent moving), we used generalized linear mixed models with binomial error family in the lme4 package (Bates et al. Reference Bates, Maechler, Bolker and Walker2014) in R (R Development Core Team, 2010). To analyse the distance copepods moved, we included only data from when movement had occurred. We used linear mixed models (lme4 package, Bates et al. Reference Bates, Maechler, Bolker and Walker2014) in R (R Development Core Team, 2010) after log transforming the distance. To account for variation between individual copepods over days we included copepod identity into the random effects. Additionally, we included the recording event to account for variation within days during the recording event together with the time interval in the recording (before vs after the simulated predator attack) to account for differences before and after the simulated predator attack. For day-17 copepods, we included an additional term, ‘age’, in the ‘copepod identity’ random effect. Age distinguished behavioural observations made early (7–9 dpi, i.e. parasites were not yet infective to fish) from those made later (13–15 dpi, i.e. when parasites were likely to be infective to fish). This term quantifies how the behaviour of individual copepods changes over time and as such applies to both, infected and uninfected copepods. Specifically for infected copepods it shows how behaviour changes as their parasites mature (see Table 1 for more details on the model). Both, time interval (i.e. before vs after the simulated predator attack) and age (only day-17 copepods) were also included as fixed effects. We stepwise added infection, feeding treatment and their pairwise interactions with each other and with time interval and age (only day-17 copepods). We compared models using likelihood ratio tests. We accepted a model as having a better fit than a less complicated one if it explained the data significantly better as judged by likelihood ratio tests. See Table 1 for the details and outputs of the models. We fitted separate models for day-11 and day-17 copepods since only the later included the behaviour of copepods, that, if they were infected, harboured infective parasites (age 13–15 dpi). If we found any significant interactions between infection and feeding, we conducted Tukey post hoc tests. For post hoc tests, we used the same models described above but combined infection and feeding into a single factor whose levels comprised all possible combinations of these two factors and removed all other interactions involving feeding or infection from the model. On these models we applied general linear hypotheses within the multcomp package in R (Hothorn et al. Reference Hothorn, Bretz and Westfall2008). For all other statistics and more detailed information on the models used please refer to Table S1.
Table 1. Outcome of likelihood ratio tests for generalized linear models of copepod activity (i.e. proportion of time spent moving) and the distance copepods moved when moving
Whether or not a copepod moved within a two second interval (activity) or, if it moved, how far it moved (distance, log transformed) were our response variables. Copepod identity (ID), the recording event (RE, i.e. a combination of copepod identity and the day of the recording), and the time interval in the recording (i.e. before vs after the simulated predator attack, INTERVAL) were used to construct the random effects. INTERVAL was additionally included as a fixed effect. For day-17 copepods we additionally included an AGE effect, which was, how long after exposure or sham-exposure the behaviour was recorded (7–9 dpi, parasites not yet infective, vs 13–15 dpi, parasites infective), as both fixed and random effect (together with ID). Subsequently, we added whether or not copepods were infected (INFECTION) and which feeding treatment they received (FEED) and all their pairwise interactions with INTERVAL and AGE (day-17 copepods). Test statistics and MCMC-estimated P-values are for the comparison with the preceding model. Null model: day-11 copepods: INTERVAL + (INTERVAL|RE) + (1|ID), day-17 copepods: INTERVAL + AGE + INTERVAL: AGE + (INTERVAL|RE) + (AGE|ID). Significant P-values have been marked in bold.
To investigate a potential association between copepod behaviour and parasite size, development (i.e. presence or absence of a cercomer on day 9) and infection success in fish, we used the same mixed models described above except we limited them to infected copepods. We added the parasite performance traits (size, development and infection success) to the models, as well as their interactions with the other fixed factors, and performed likelihood ratio tests (see Table S2). Since this involved multiple comparisons we took adjusted P-values (after bonferroni) into account when we found any significant differences.
RESULTS
Confirmation of the effect of parasite infection on copepod behaviour
Schistocephalus solidus reduces the predation susceptibility of its copepod host before reaching infectivity (Weinreich et al. Reference Weinreich, Benesh and Milinski2013). This is marked by a reduction in activity (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Benesh, Reference Benesh2010a ; Hafer and Milinski, Reference Hafer and Milinski2015) which we also found in our study (Fig. 2A and B, P < 0·0002, Table 1). In day-17 copepods there was an interaction between infection and age (i.e. 7–9 dpi vs 13–15 dpi, P = 0·0141, Table 1). Differences between infected and uninfected copepods were more pronounced at an earlier age, i.e. before the parasites reached infectivity (Fig. 2B). This is in line with previous predictions (Parker et al. Reference Parker, Ball, Chubb, Hammerschmidt and Milinski2009) and findings that any predation suppression at least decreases as S. solidus becomes infective (Hammerschmidt et al. Reference Hammerschmidt, Koch, Milinski, Chubb and Parker2009; Benesh, Reference Benesh2010a ; Hafer and Milinski, Reference Hafer and Milinski2015). The distance which copepods moved was also significantly affected by infection with S. solidus (Fig. 2C andD, P < 0·0001, Table 1); infected copepods moved shorter distances. Age did not significantly interact with infection (Table 1). There was some interaction between infection and how copepods responded to the predator attack (i.e. time interval, before vs after a simulated predator attack) for activity in day-11 copepods (P = 0·0033, Table 1) and the distance copepods moved in day-17 copepods (P = 0·0325, Table 1). In day-11 copepods, infected copepods reacted more strongly while in day-17 copepods infected copepods reacted less strongly than uninfected ones. However, both uninfected copepods and infected ones always showed the same, clear reaction to the simulated predator attack; they reduced their activity and the distance they moved (Figure S1). Accordingly, our results were not very sensitive to the time interval in the recording, i.e. differences between experimental treatments were not strongly contingent on whether copepods were observed before or after the simulated attack. Overall we can confirm that S. solidus alters the behaviour of its copepod host by decreasing its activity and the distance it moves.
Fig. 2. Activity (A, B) and distance (C, D) as response to infection and feeding treatment. Error bars present means +/−95% CI. Feeding treatment: H: high food treatment, L: low food treatment. N: day-11 copepods: not infected: H: 45, L: 44, infected: H: 65, L: 68, day-17 copepods: 7–9 dpi (parasites not yet infective): not infected: H: 30, L: 28, infected: H: 51, L: 49, day-17 copepods: 13–15 dpi (parasites infective): not infected: H: 30, L: 27, infected: H: 51, L:51. Dotted lines connecting groups are to aid comparison of feeding treatments, whereas the bar colours differentiate infection status. Age represents copepod behaviour 7–9 dpi (parasites not yet infective) vs 13–15 dpi (parasites infective).
The effect of feeding on host behaviour and host manipulation
If feeding treatment affects host manipulation, a significant interaction between feeding treatment and infection should occur. Neither this interaction nor feeding alone had any significant effect on copepod activity (Fig. 2A and B, Table 1). However, distance was affected by both feeding treatment (Fig. 2C and D, P < 0·03, Table 1) and its interaction with infection (Fig. 2C and D, P < 0·05, Table 1). Feeding treatment affected uninfected copepods more strongly than infected ones (Fig. 2C and D). Post hoc tests reveal that uninfected copepods moved significantly slower if they were in the low food treatment than if they were in the high food treatment (P < 0·02, Table S1). In infected copepods distance did not differ between feeding treatments (Table S1, Fig. 2C and D). Consequently, differences between infected and uninfected copepods were larger in the high food treatment than in the low food treatment (Table S2, Fig. 2C and D). This was not due to an effect of feeding treatments on parasites, but because of an effect on uninfected hosts.
Associations between host manipulation and other fitness-related traits
If host manipulation is costly and those costs lead to trade-offs with other fitness-related traits, parasites that manipulate less (i.e. behave more similar to uninfected hosts) should do better with regards to other traits such as growth or development.
Parasite size and development
Parasite size 10 dpi in day-11 copepods did not correlate with copepod activity (Table S2, Fig. 3A). However, there was a positive correlation between size and distance in day-11 copepods (P = 0·0017, adjusted P value = 0·0204, Table S1, Fig. 3C). Parasite size 16 dpi in day-17 copepods correlated positively with activity and distance (P < 0·003, adjusted P value < 0·04, Fig. 3B–D, Table S1). Copepods with larger parasites moved more often and further (Fig. 3B–D).
Fig. 3. The relationship between parasite size and host activity (A, B) and distance moved (C, D). Solid lines indicate the trend line. Dotted horizontal lines indicate mean behaviour of uninfected copepods. The shaded areas around the lines indicate 95% CI. Each dot represents the mean behaviour of one copepod calculated over all days on which its behaviour was recorded. N: day-11 copepods: 122, day-17 copepods: 102.
Development (presence or absence of a cercomer 9 dpi) was associated positively with activity in day-11 copepods (more active, i.e. less manipulated, copepods are more likely to have a cercomer 9 dpi, P = 0·0103, Table S1), but this association disappeared when correcting for multiple testing (P = 0·1236). Neither distance in day-11 copepods nor activity nor distance in day-17 copepods showed any association with parasite development (Table S2). Overall, the developmental rate did not seem to be associated with host behaviour.
Infection probability in fish
Parasites in day-11 copepods that moved further were more likely to have successfully infected fish 11 dpi (P = 0·0049, Table S2, Fig. 4C). When we adjusted for multiple testing, this finding was only marginally significant (P = 0·0588). However, host activity was not associated with the parasite's probability to infect fish (Table S2, Fig. 4A). In day-17 copepods, we found no significant correlation between infection probability in fish and host behaviour (Table S2, Fig. 4). It appears that parasites that successfully infected fish came from hosts that moved larger distances, and hence were possibly less manipulated, but only if parasites were exposed to fish at the earlier time point (11 dpi).
Fig. 4. Differences in activity (A, B) and distance moved (C, D) of copepods that successfully infected fish and those that did not. Error bars indicate 95% CI. Dotted horizontal lines indicate mean behaviour of uninfected copepods. The shaded areas around the lines indicate 95% CI. N (fish infection successful/failed): day-11 copepods: 11/110, day-17 copepods: 81/18.
DISCUSSION
Copepods infected with not yet infective S. solidus moved less and slower than uninfected copepods, which is compatible with predation suppression manipulation (Parker et al. Reference Parker, Ball, Chubb, Hammerschmidt and Milinski2009). This behavioural alteration seemed unaffected by resource availability, as infected copepods behaved similarly in low and high food treatments. Differences in feeding treatments also have no effect on host manipulation in acanthocephalan-infected gammarids (Labaude et al. Reference Labaude, Cézilly, Tercier and Rigaud2015). In uninfected copepods, activity (i.e. proportion of time spent moving) is not affected by resource availability either, but they move less far in a low food than in a high food environment. As a result, behavioural differences between infected and uninfected copepods are less pronounced in a low food environment. Rather than changes in how much parasites manipulate, such an effect could be produced by differences in the condition of uninfected copepods between feeding treatments. Copepods that are in worse condition (e.g. because they have been limited in their resources) may be less able to invest in muscle tissue, limiting the distance they are able to move. Copepods that die sooner have less muscle tissue (Franz and Kurtz, Reference Franz and Kurtz2002). Infected copepods already move less than uninfected ones, so it might not be practical for them to reduce their movement even further in a low food environment, since they still have to be able to e.g. forage.
If reduced physical condition is responsible for the reduced distance travelled by uninfected copepods under food constraints, could the modified behaviour of infected copepods also be caused by a decline in host condition, in this case due to parasite infection (see McElroy and de Buron, Reference McElroy and de Buron2014)? Infection with S. solidus has no significant effect on either lipid storage or the amount of muscle tissue in the host (Franz and Kurtz, Reference Franz and Kurtz2002). Other trophically-transmitted parasites have even been found to increase the energy reservoirs of their intermediate host (Amat et al. Reference Amat, Gozalbo, Navarro, Hontoria and Varo1991; Plaistow et al. Reference Plaistow, Troussard and Cézilly2001; Ponton et al. Reference Ponton, Biron, Joly, Helluy, Duneau and Thomas2005). Schistocephalus solidus not only reduces how far but also how often its host moves. The latter is not affected by feeding regime. Furthermore, an adverse effect of a parasite infection should increase with infection intensity. Host activity is not affected by the number of not yet infective S. solidus and even increases with the number of infective S. solidus (Hafer and Milinski, Reference Hafer and Milinski2015).
We find no clear connection between cercomer presence 9 dpi (development) and host behaviour. However, host activity in day-17 copepods and distance in day-11 and day-17 copepods correlates positively with size 10 dpi (day-11 copepods) or 16 dpi (day-17 copepods). This is puzzling since Benesh (Reference Benesh2010a ) found no such correlations. On a family level, there was even a negative correlation between parasite size on day 11 and development and activity before reaching infectivity (Benesh, Reference Benesh2010a ). Our food treatments possibly introduced more variation in parasite size and/or copepod behaviour that enhanced co-variation between them. A positive correlation between size and activity after reaching infectivity is in line with the finding that two infective S. solidus enhance the activity of their host more than one (Hafer and Milinski, Reference Hafer and Milinski2015). Maybe this is not so much an effect of number as of size. However, there need not be any causal relationship between parasite size and host activity. Copepods in better physical condition might at the same time be able to move further and allow their parasites to grow to a larger size. Indeed, parasites whose hosts are in a high food treatment do grow larger (Benesh, Reference Benesh2010a , Supplementary information).
We found a trend for an association between infection rates in fish 11 dpi and the distance copepods with not yet infective parasites moved. Parasites that successfully infected fish on day 11 came from copepods that had moved further (i.e. less ‘predation suppression’ manipulation) than those that failed to infect fish. This, however, need not imply any causal relationship between host manipulation and infection success. Previous studies have also reported a negative correlation between parasite fitness components and host manipulation, suggestive of trade-offs (Franceschi et al. Reference Franceschi, Bollache, Cornet, Bauer, Motreuil and Rigaud2010a ; Maure et al. Reference Maure, Brodeur, Ponlet, Doyon, Firlej, Elguero and Thomas2011). Since these studies are also correlational, alternative explanations cannot be ruled out. In our case, a trade-off may not be the best explanation. As discussed above, the distance which copepods move might be affected by their physical condition. It seems reasonable to assume that copepods that are in better physical condition can move further and can harbour rapidly growing parasites that are better at infecting fish. This will be especially crucial for parasites that are transmitted to fish early (i.e. 11 dpi) when they are just starting to become infective and there is still variation in developmental stage, and only the fastest developing parasites succeed in infecting fish. By 17 dpi even parasites in copepods in worse condition should have been able to catch up in their development and hence be able to infect fish (Benesh and Hafer, Reference Benesh and Hafer2012).
Host manipulation can have important ecological consequences (Thomas et al. Reference Thomas, Renaud, Demee and Poulin1998, Reference Thomas, Poulin and de Meeüs1999, Reference Thomas, Adamo and Moore2005; Lefèvre et al. Reference Lefèvre, Lebarbenchon, Gauthier-Clerc, Missé, Poulin and Thomas2009; Lafferty and Kuris, Reference Lafferty, Kuris, Hughes, Brodeur and Thomas2012), such as changes in food webs (Lefèvre et al. Reference Lefèvre, Lebarbenchon, Gauthier-Clerc, Missé, Poulin and Thomas2009; Lafferty and Kuris, Reference Lafferty, Kuris, Hughes, Brodeur and Thomas2012). Differences between infected and uninfected copepods seem more pronounced in a high food environment. Infected copepods are less likely to be consumed by sticklebacks before reaching infectivity, i.e. when S. solidus reduces activity (Weinreich et al. Reference Weinreich, Benesh and Milinski2013), and more likely to be consumed thereafter, i.e. when S. solidus enhances activity (Wedekind and Milinski, Reference Wedekind and Milinski1996). While we do not know if the differences between feeding treatments are large enough to influence predation susceptibility, our results nonetheless indicate that behavioural differences between infected and uninfected copepods can depend on resource availability. This raises the possibility that S. solidus transmission to fish might be environmentally-dependent.
SUPPLEMENTARY MATERIAL
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/pao.2015.3.
ACKNOWLEDGEMENTS
We would like to thank R. Leipnitz and M. Schwarz for technical assistance, I. Salmonte-Padilla for providing fish and two anonymous reviewers for helpful comments on an earlier version of the manuscript.
FINANCIAL SUPPORT
This work was supported by the Max Planck Society. N. H. received her funding through the International Max Planck Research School (IMPRS) for Evolutionary Biology. D. P. B. was partially supported by a DFG fellowship (BE 5336/1-1).
CONFLICT OF INTEREST
None.
ETHICAL STANDARDS
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
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