Nathalie Franceschi*, Loic Bollache, Lucile Dianne, Alexandre Bauer, Thierry Rigaud
Université de Bourgogne, Laboratoire Biogéosciences, UMR CNRS 5561, Equipe Ecologie Evolutive, 6 Boulevard Gabriel, 21000 Dijon, France
*Corresponding author. Equipe Ecologie Evolutive, UMR CNRS Biogéosciences 5561, Université de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France.
Tel.: + 33 380 39 62 28; fax: + 33 380 39 62 31.
E-mail address: nathalie.franceschi@u-bourgogne.fr
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Abstract
In acanthocephalan parasites, larval size is an important parameter since it determines to some extent the adult body size, and thus influences the individual fitness. However, competition between larval parasites within a host can highly influence parasite larval size, inducing a negative relationship between size and intensity. Parker et al. (2003) proposed a Life History Strategy (LHS) model of growth, which predicts that, if larval stages are able to detect the presence of competing conspecifics and to adjust their growth pattern in consequence, even if the volume of each individual parasite should decrease when infection intensity increases, the total mass of parasites should increase with infection intensity. Using experimental infection of G. pulex by P. laevis, a manipulative acanthocephalan parasite, we first compared parasite growth rates between different parasite families and investigated intraspecific competition in order to test the model of Parker et al. (2003). In addition, we examined the relationship between parasite size and the ability to manipulate the behaviour of the intermediate host. Our results revealed that individual cystacanth size was highly variable between parasite families, suggesting that a genetic component could account for these differences. Moreover, individual cystacanth size was density-dependent, decreasing with raising infection intensity, which reveals an intraspecific competition between P. laevis cystacanths within G. pulex. The total parasite volume within a host increased linearly with infection intensity, thus supporting the LHS model of Parker et al. (2003). Concerning behavioural manipulation, we only found a weak positive relationship between size and manipulation at ‘old cystacanth stage’, in some parasite families. Therefore, the hypothesis of a trade-off between parasite growth rate and manipulation is not supported.
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1. Introduction
In many invertebrates, adult body size is a key determinant of numerous fitness components (Blueweiss et al. 1978, Peters 1983, Schmidt-Nielsen 1984, Stearns 1992).
Complex life-cycle parasites can reproduce in their definitive host only after having exploited one or several intermediate hosts supplying resources for parasite growth. In different parasites species, a greater larval size has been demonstrated to be associated with fitness benefits like better establishment success and survival in the final host (Rosen & Dick 1983, Steinauer & Nickol 2003) or higher adult fecondity (Fredensborg & Poulin 2005).
Moreover, Poulin et al. (2003a) showed that, in acanthocephalans, the size of the cystacanth (final larval stage of the parasite, infective for the definitive host) can determine to some extent the adult body size. Selection should thus promote a greater larval size. However, complex-life cycle parasites face a trade-off between a high growth rate and the prudent exploitation of the host, in order to keep the intermediate host alive until the transmission to the final host. The directional selection towards a large larval stage is thus stabilized by the necessity to maintain the viability of the intermediate host (Lafferty & Kuris 2002). In some circumstances, in particular when host viability is essential for parasite transmission, lowering their own exploitation of the host (i.e. lowering their virulence and growth) may be a beneficial and evolutionary stable strategy to increase the chance of transmission to the next host. On the other hand, larval parasite growth might also be modulated by tradeoffs with other life history traits.
An important parameter likely to influence parasite larval size is the presence of other parasites sharing the same individual host, the competition for host resources inducing a negative relationship between size and intensity. Several studies have shown such a density-dependent reduction in parasite size (Gordon & Whitfield 1985, Wedekind et al. 2000, Dezfuli et al. 2001, Brown et al. 2003, Fredensborg & Poulin 2005). In case of multiple-infections, size might thus be modulated by inter- and/or intraspecific competition in an
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individual host. However, unless studies on interspecific competition between parasites are common, both in intermediate and definitive hosts (e.g. Dezfuli et al. 2002, Fredensborg &
Poulin 2005, Poulin et al. 2003b, 2003c, Thomas et al. 2002), very few ones have been undertaken on intraspecific competition between larval macro-parasites sharing the same intermediate host (Dezfuli et al. 2001, Fredensborg & Poulin 2005, Benesh & Valtonen 2007). As regards to acanthocephalan parasites, we have very few information about their growth strategies in their intermediate host and, even if several studies have indeed shown that the presence of competitors influenced acanthocephalan development (e.g., Dezfuli et al.
2001, Steinauer & Nickol 2003), the magnitude of this phenomenon is still poorly known.
Understanding the effects of between-parasites competition is of great importance, since inter- and/or intra-competition is believed to increase parasite virulence, natural selection acting against the less competitive parasites (Fredensborg & Poulin 2005). Parker et al.
(2003) proposed a model of growth strategies of parasites under competition in their intermediate hosts. Counter to the classic interpretation considering that growth patterns are only a function of resources available in the host (Resource Constraints (RC) model), they proposed that larval stages are able to detect the presence of competing conspecifics and thus to adjust their growth pattern in order to avoid host over-exploitation and to maintain its viability (Life History Strategy (LHS) model). According to their predictions, the volume of each individual parasite should decrease in both cases when infection intensity increases.
Under the RC model the total volume of larval parasites in a single intermediate host should be constant whatever the number of parasites in the host, because of limited available resources. On the contrary, under the LHS model, the total mass of parasites should increase with infection intensity. Very few studies have experimentally validated this model. Michaud et al. (2006), using the cestode Schistocephalus solidus in its copepod intermediate host, have shown that the parasite used a LHS growth strategy under competition.
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Numerous complex life-cycle parasites have developed the ability to manipulate their intermediate host behaviour, thus increasing their trophic transmission to the definitive host (Bakker et al. 1997, Lafferty 1999, Moore 2002, Tain et al. 2006, Perrot-Minnot et al. 2007, Franceschi et al. 2008). However, this manipulation is supposed to be energetically costly (Thomas et al. 2005), and if it is, it can interfere with other life history traits requiring energy, such as growth. We already showed that, in an acanthocephalan parasite species, the rapidity of development (i.e. the time to reach the infective stage) traded-off with the behavioural manipulation, the slower the development time, the higher the intensity of phototaxis reversal (Franceschi et al. 2009, voir chapitre XX). Thus, if we just consider growth and behavioural manipulation, we should expect a trade-off between these two mechanisms, with parasites reaching larger sizes having lower manipulation ability, and good manipulators reaching a reduced final size.
Pomphorhynchus laevis is a fish acanthocephalan parasite using amphipod crustaceans as intermediate hosts. In the gammarid Gammarus pulex, P. laevis is known to induce an alteration of phototactic behaviour (Brown & Thompson 1986, Bauer et al. 2000, Tain et al.
2006, Franceschi et al. 2008). However, the intensity of this manipulation is highly variable – some hosts show a complete phototaxis reversal while others are almost not affected by infection – and the causes of these variations are still incompletely understood, even if some factors such as day-time and age of the infection have been demonstrated to affect behavioural manipulation (Lagrue et al. 2007, Franceschi et al. 2008). To our knowledge, only one study has investigated growth patterns in this parasite. Indeed, in natural infections in another amphipod species, Echinogammarus stammeri, Dezfuli et al. (2001) have shown that the size of P. laevis cystacanths could be modulated by intraspecific competition in the intermediate host. The growth strategy and its interaction with behavioural manipulation was nevertheless not investigated.
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The aims of our study were thus, using experimental infection of G. pulex by P. laevis, 1) to compare parasite growth rates between different parasite families, 2) to investigate intraspecific competition by comparing the sizes of cystacanths in single and multiple infections and to discriminate between the RC and LSH models described by Parker et al.
(2003), and 3) to examine the relationship between size of parasites and their ability to manipulate the behaviour of their intermediate host.
2. Materials and methods 2.1. Origin of hosts and parasites
Gammarus pulex were collected in May and October 2007 in a small tributary of the Suzon River (Burgundy, eastern France; N 47° 24’12.6’’ ; E 4° 52’58.2’’). This population is known to be totally free of P. laevis, and can therefore be considered naïve for this parasite (see Franceschi et al. 2008). In the laboratory, the gammarids were acclimated for 4 weeks prior to infection experiments, in well-aerated aquaria of 37 ¯ 55 ¯ 10 cm containing dechlorinated, UV-treated tap water at 15 ± 1°C and elm leaves for food, under a 12:12 h light:dark cycle.
Naturally-parasitized chubs (Leuciscus cephalus) were sampled by electrofishing in the Ouche River, a tributary of the Saône River. Fish were anaesthetized, killed and dissected, and adult parasites were collected from their intestines. Eggs were obtained by dissecting female worms and placed in 400 µL of water. Parasite tissues were preserved in 300 µL of alcohol for species molecular identification. Indeed, two closely-related species of acanthocephalan parasites, Pomphorhynchus laevis and P. tereticollis co-occur in the rivers of Burgundy. Since these two species cannot be reliably distinguished based on morphology, we used a molecular method for parasite identification (see details in Franceschi et al. 2008).
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2.2. Infection procedure
Experimental infections have been carried out in May 2007 and October 2007, following the same protocol, detailed below (see Franceschi et al. 2009).
Parasite eggs from each female were examined under a Nikon microscope (20 x) to evaluate their maturity and then counted in three optical areas, in order to carry out the experiments with clutches having approximately the same number of mature eggs. For each infection, we selected ten clutches of P. laevis, coming from ten different fish. We considered each clutch as a “parasite family”, since all eggs are supposed to be full-sibs (see Franceschi et al. 2009) The number of eggs was then estimated by averaging the counts made under a microscope in 10 samples of 1 μL. After dilution with water, suitable egg exposure doses were obtained. We finally used 8 parasite families in May and 10 in October.
Prior to infection, gammarids were deprived of food for 24 h. The infection was then carried out as described in Franceschi et al. (2008). Two gammarids were placed in a dish of 6 cm diameter, filled with water at 15 ± 1 °C, and the egg suspension at suitable concentration (100 parasite eggs per gammarid, see Franceschi et al. 2008) was deposited on a 1cm² dry elm leaf placed in the dish. The gammarids were allowed to feed on it for 48 h. For each treatment (one treatment corresponding to an infection with one parasite eggs clutch), 108 male gammarids were used. Uninfected leaves were provided to control groups. At the end of the exposure period, the gammarids were rinsed and placed in 0.5 L aquaria, and maintained in standard conditions (water at 15 ± 1 °C, 12:12 h light:dark cycle). Eighteen individuals undergoing the same treatment (exposed to eggs from the same parasite female) were randomly assigned to each aquarium.
From the sixth week after infection, all gammarids were inspected once a week under a binocular microscope to detect the presence of parasites (see Franceschi et al. 2008). Infected individuals were isolated as soon as a larval parasitic stage was detected through the cuticle, and they were then followed twice a week to note when the parasite reached the cystacanth
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stage (final stage in the gammarid, infective for the definitive host). The day after the parasite had reached this stage and then fifteen days later, the reaction to light of the host was measured as described in Franceschi et al. (2008). A single gammarid was introduced into a horizontal tube filled with well-aerated water, with a dark zone and a light zone of equal size.
After a 5 min period of acclimatization, the position of the gammarid was recorded each 30 s for 5 min. At each observation, a score of 0 was given if the individual was located in the dark area and a score of 1 was given if it resided in the lighted area. At the end of each trial, summed scores ranged from 0 (always in the dark, strongly photophobic) to 10 (always in the light, strongly photophilic). The gammarid was then measured (body height at the level of the fourth coxal plate basis) using a Nikon SMZ 1500 stereoscopic microscope and Lucia G 4.81 software. After dissection of the amphipod, each cystacanth was measured (length and width) with the same device. The volume of each cystacanth was then calculated as the volume of an ellipsoid, using the following formula: (πLW²)/6 with L and W being respectively length and width of the cystacanth (Poulin et al. 2003a).
2.3 Statistics
All tests were performed using JMP 6.0 Software (SAS Institute Inc.) and were two-tailed. P values < 0.05 were considered significant.
Two analyses were carried out in order to analyse the effects of different variables on the average volume of cystacanths and on their total volume within the host. Average cystacanth volumes were analysed with mixed linear model including the following explicatory factors: ‘replicate’ (May or October), ‘parasite family’ treated as a random factor nested within ‘replicate’, ‘infection intensity’ (number of cystacanth per host, Log10
transformed), and the interaction between these two last factors. Host size may also explain a part of parasite size (e.g. Dezfuli et al. 2001, Steinauer & Nickol 2003). However, in our case, host size was not entered in the model as covariate, because since some parasites may share
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the same host, they are not independent and some gammarids (e.g. a gammarids harbouring 10 parasites) may appear to have more weight in the analysis than others (e.g. gammarids harbouring only one parasite). To take this into account, the volume of each cystacanth was thus divided by the size of its host. Therefore, we did not analyse the absolute size of the parasites, but their size relative to that of the host. These ratios did not meet either normality or homoscedasticity conditions, and were thus Log10 transformed prior to analysis. For this analysis, we removed 3 parasite families in which the number of infected hosts was <13, because the interaction between parasite family and parasite intensity was impossible to analyse accurately. Total cystacanth sizes were analysed with mixed linear model including the following explicatory variables: ‘replicate’ (May or October), ‘parasite family’ treated as a random factor nested within ‘replicate’, ‘infection intensity’ (number of cystacanth per host, Log10 transformed), ‘host size’ and the interaction between these three last variables.
Finally, we investigated the relation between cystacanth volume and phototaxis scores using nonparametric Spearman correlations, because data distribution of phototaxis scores never met conditions allowing parametric statistics. To avoid the confounding effect of parasite numbers on size (see results), this was done for singly-infected hosts only. We first calculated the mean cystacanth volume corrected for host size (see before) and the median phototaxis score for each parasite family, and the relationship between these average values was investigated across families. We then investigated the individual relationship between volume and behavioural modification, first globally (i.e. all parasite families grouped), and then within parasite families, for those where more than 10 infected hosts were obtained.
3. Results
3.1. Individual average cystacanth size
Our experimental infections yielded from 1 to 13 parasites per host. The individual size of P. laevis cystacanths infecting G. pulex, relative to their host size, varied from single to
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double according to the parasite family (Table 1, figure 1). The number of cystacanths per host also influenced their size: the higher the parasite intensity, the smaller the cystacanth (Table 1). This result remained the same if the extreme intensity (n = 13) was removed from the analysis (not showed). Lastly, the significant interaction revealed that parasite intensity did not influenced parasite size in the same way according to parasite families (Table 1). The negative relationship between parasite size and parasite intensity was significant in 12 families, whereas it was not in 3 families (Figure 1). Even in series where this negative relationship was very strong, parasite size in cases of multiple infections was always higher than the theoretical size of 1/N predicted by a Resource Constraint model (Parker et al. 2003, Figure 1).
3.2. Total parasite volume within the host
The total parasitic volume within infected hosts was also variable between parasite families (Table 2, Figure 2). It was also strongly linearly increasing with an increasing number of cystacanths (Table 2, Figure 3), almost doubling when the number of parasites was doubled. Only in case of the extreme intensity of 13 parasites the total volume seems to reach a plateau (Figure 3), but this was obtained in a single individual host, so this data was not strongly supported. Host size did not significantly influence total parasite volume, either alone or in interaction with other factors (Table 2).
3.3. Consequences on behavioural manipulation
For the analysis of behavioural manipulation at ‘young cystacanth stage’, there was a significant difference between the average phototaxis scores between the two replicates (May and October; Wilcoxon test: Z = 5.38, P < 0.0001), with a median score of 4 in May (interquartile range = 1 – 6, N = 63) and a median score of 1 in October (range = 0 – 3, N = 99). These replicates were therefore analyzed separately. Across parasite families, we found
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no correlation between average parasite size and average parasite ability to modify the host phototactic behaviour (Spearman ρ = 0.19, P = 0.66, N = 8 in May and ρ = -0.14, P = 0.69, N
= 10 in October). At individual level, no global correlation between parasite size and behavioural manipulation was found, neither in May (ρ = 0.22, p = 0.08, N = 62) or in October (ρ = 0.01, p = 0.91, N = 97). This absence of significant relationship was confirmed within parasite families (Table 4a).
For the analysis of behavioural manipulation at ‘old cystacanth stage’, there was no significant difference between the average phototaxis scores between the two replicates (May and October; Wilcoxon test: Z = 0.15, P = 0.87). Since these replicates were also homogenous for the average size of cystacanths (Table 1), they were analyzed simultaneously. Across parasite families, we found no correlation between average parasite size and average parasite ability to modify the host phototactic behaviour (Spearman ρ = -0.13, P = 0.61, Figure 4a). At individual level, a global weak positive correlation between parasite size and behavioural manipulation was found (ρ = 0.25, p = 0.001, N = 159, Figure 4b). However, this relationship was variable between parasite families, some of them showing a correlation coefficient close to 0 (Table 4b).
Discussion
Our study revealed that individual cystacanth size within the intermediate host was under the control of different parameters.
First of all, we found a significant difference between the various parasite families used in the experimental infections, suggesting a genetic component in the size variation, in interaction with the environment (see below), as already shown for several invertebrates (e.g.
Mousseau & Roff 1995, Ryder & Syva-Jothy 2001). However, because it was not possible to control for parasite mating, we can not ignore the possibility that these differences could be due to maternal effects. Individual cystacanth size was also density-dependent, with a
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negative relationship between parasite size and infection intensity, and this effect was significant in the majority of the studied parasite families. This is in agreement with Dezfuli et al. (2001), who also found that P. laevis cystacanths attained a smaller size when co-occurring with cystacanths of other acanthocephalan species, and with Benesh & Valtonen (2007) who found that intraspecific crowding limited the growth of Acanthocephalus lucii in its isopod intermediate host. Our study thus revealed intraspecific competition between P.
laevis cystacanths within G. pulex. The amount of resources available in the parasite environment (the host) being limited, individual larval growth is decreased when theses resources have to be shared with competitors. Since larval size greatly influences adult fitness (Rosen & Dick 1983, Steinauer & Nickol 2003, Poulin et al. 2003a, Fredensborg & Poulin 2005), selection should favour single infections in the field. Interestingly, naturally P. laevis-infected G. pulex are indeed generally singly-infected, thus confirming this prediction. The other natural infections found in the field consist in gammarids infected by two, rarely three cystacanths. Franceschi et al. (2008) showed that the level of behavioural manipulation induced by two cystacanths was higher than that induced by only one parasite. Therefore, it is possible that the fitness benefits related to the transmission to the next host obtained in double-infections could be sufficient to compensate the costs on cystacanth size, thus maintaining some double-infections in the field.
This competition between parasites was found despite the fact that all co-infecting
This competition between parasites was found despite the fact that all co-infecting