Geographic variation and trade-offs in parasitoid virulence

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This is the published version of a paper published in Journal of Animal Ecology.

Citation for the original published paper (version of record):

Fors, L., Markus, R., Theopold, U., Ericson, L., Hamback, P A. (2016) Geographic variation and trade-offs in parasitoid virulence.

Journal of Animal Ecology, 85(6): 1595-1604 https://doi.org/10.1111/1365-2656.12579

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Geographic variation and trade-offs in parasitoid virulence

Lisa Fors

¹

*, Robert Markus

²

, Ulrich Theopold

³

, Lars Ericson

⁴

and Peter A. Hamb€ack

¹

1Department of Ecology, Environment and Plant Sciences, Stockholm University, 10691 Stockholm, Sweden;²School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK;³Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden; and⁴Department of Ecology and Environmental Science, Umea University, 90187 Umea, Sweden

Summary

1. Host–parasitoid systems are characterized by a continuous development of new defence strategies in hosts and counter-defence mechanisms in parasitoids. This co-evolutionary arms race makes host–parasitoid systems excellent for understanding trade-offs in host use caused by evolutionary changes in host immune responses and parasitoid virulence. However, knowledge obtained from natural host–parasitoid systems on such trade-offs is still limited.

2. In this study, the aim was to examine trade-offs in parasitoid virulence in Asecodes parviclava (Hymenoptera: Eulophidae) when attacking three closely related beetles: Galerucella pusilla, Galerucella calmariensis and Galerucella tenella (Coleoptera: Chrysomelidae). A second aim was to examine whether geographic variation in parasitoid infectivity or host immune response could explain differences in parasitism rate between northern and southern sites.

3. More specifically, we wanted to examine whether the capacity to infect host larvae differed depending on the previous host species of the parasitoids and if such differences were connected to differences in the induction of host immune systems. This was achieved by combining controlled parasitism experiments with cytological studies of infected larvae.

4. Our results reveal that parasitism success in A. parviclava differs both depending on previous and current host species, with a higher virulence when attacking larvae of the same species as the previous host. Virulence was in general high for parasitoids from G. pusilla and low for parasitoids from G. calmariensis. At the same time, G. pusilla larvae had the strongest immune response and G. calmariensis the weakest. These observations were linked to changes in the larval hemocyte composition, showing changes in cell types important for the encapsulation process in individuals infected by more or less virulent parasitoids.

5. These findings suggest ongoing evolution in parasitoid virulence and host immune response, making the system a strong candidate for further studies on host race formation and speciation.

Key-words: Asecodes, cellular defence, ecological immunology, Galerucella, host–parasitoid interactions, host–pathogen evolution

Introduction

Co-evolution, the process of reciprocal evolutionary change driven by natural selection in interacting species, has a central role in explaining the high species diversity in many systems (Thompson 2005). In situations with antagonistic interactions, such as host–parasitoid systems, there is often a constant development of resistance and

counter-resistance mechanisms (Fytrou et al. 2006; Kraai- jeveld & Godfray 2009) making these systems excellent models for co-evolutionary studies. Host insects use several strategies to escape parasitoid attack, such as enemy- free space, concealment or physical counter-attack. Even when successfully parasitized, the host can defend itself through a potent immune defence, most commonly by encapsulation of the parasitoid eggs by aggregating cells (Nappi 1981; Rizki & Rizki 1990; Schmidt, Theopold &

Strand 2001; Lavine & Strand 2002). In the fruit fly Dro- sophila melanogaster, parasitoid eggs initially attract

*Correspondence author. E-mail: lisa.fors@su.se

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Journal of Animal Ecology2016,85, 1595–1604 doi: 10.1111/1365-2656.12579

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plasmatocytes, which spread around the egg surface. This is followed by the differentiation of a specialized larger class of hemocytes (lamellocytes), which form a second cellular layer. Ultimately, the capsule melanizes through the release and activation of phenoloxidase from specialized cells (crystal cells in Drosophila and oenocytoids in many other insect species (Honti et al. 2014)). These resistance mechanisms have imposed strong selection on the parasitoid to evolve counter-resistance strategies, such as the injection of venom, teratocytes or viruses into the host, thereby protecting the progeny from being rejected (Dahlman 1991; Firlej et al. 2007; Beckage 2008; Strand 2010; Burke & Strand 2014).

Parasitoid adaptations to specific hosts often come at a cost, sometimes resulting in a reduced ability in the parasitoid to infest other host species (Van Veen et al. 2008;

Jones et al. 2015). In the extreme case, as in the matching-alleles model, an exact genetic match is necessary for successful attack by the parasitoid (Agrawal & Lively 2002). According to this model, which is based on self/

non-self recognition systems (Grosberg & Hart 2000;

Honti et al. 2014), the parasitoid may completely lose the ability to infect the ancestral host when successfully adapting to a novel host. The alternative infection model, gene-for-gene matching, rather assumes that adaptations to different hosts involve different genes (Agrawal &

Lively 2002). Due to costs in developing a higher virulence, the parasitoid will not evolve high infectivity to all hosts and may still vary in their virulence to different hosts (Antolin, Bjorksten & Vaughn 2006).

According to the geographic mosaic theory of co-evolution, interactions between two species vary spatially, causing spatial differences in the direction and strength of selection (Thompson 1994, 2005). In some areas, reciprocal selection may be strong (‘hot spots’ of co-evolution), whereas selection in other areas may be reduced or unidirectional (‘cold spots’). As a consequence, host resistance and parasitoid counter-resistance in a specific host–parasitoid system can vary geographically. One mechanism

causing such geographic mosaics of selection is variation in the availability of different host species, as for instance in the interactions between D. melanogaster and one of its main parasitoids, Asobara tabida (Kraaijeveld & van Alphen 1994, 1995; Kraaijeveld, Nowee & Najem 1995).

In this system, large population variations have been observed both in the ability of the host to encapsulate parasitoid eggs and in parasitoid ability to prevent the encapsulation response. In southern Europe where D. melanogaster is the only host available, the counter- resistance of A. tabida is stronger than in northern Eur- ope, where the additional host Drosophila subobscura (lacking the encapsulation ability) is also present (Kraai- jeveld & Godfray 2009). A similar variation in parasitoid counter-adaptations has also been observed in another parasitoid (Leptopilina boulardi) commonly attacking D. melanogaster(Dupas & Boscaro 1999), but few similar studies exist from non-Drosophila systems.

In this study, we investigated parasitism success (i.e. the development of parasitoid larvae in infected host individuals) of the parasitoid Asecodes parviclava (Hymenoptera:

Eulophidae), attacking three closely related species of Galerucellaleaf beetles: Galerucella pusilla, G. calmariensis and Galerucella tenella (Coleoptera: Chrysomelidae) (Figs 1 and 2). Previous genetic studies indicate that A. parviclava could be at an early stage of further speciation, making Galerucella–Asecodes a useful model system for investigating evolution of resistance and counter-resistance mechanisms. The most recent divergence date for Galerucellais estimated to 77 000 years ago for G. pusilla and G. calmariensis, and the speciation of Asecodes seems to be following the speciation of the hosts (Hamb€ack et al. 2013; Hansson & Hamb€ack 2013). Differences in immune response in G. pusilla and G. calmariensis have previously been observed (Fors et al. 2014), as well as among population differences in parasitism rate (i.e. the proportion of Galerucella larvae killed by parasitism). In northern localities of Sweden, where only G. calmariensis and G. tenella occur (Fig. 2), the parasitism rate is

G. calmariensis G. pusilla G. tenella

(a) (c) (e)

(b) (d) (f)

Fig. 1. The three beetle species (Coleop- tera: Chrysomelidae): Galerucella calmariensis (a) adult and (b) larva, Galerucella pusilla (c) adult and (d) larva and Galerucella tenella (e) adult and (f) larva. Scale bars: 1 mm.

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generally much higher (>70%) compared with southern localities (<10%) (Hamb€ack, Stenberg & Ericson 2006;

Stenberg et al. 2007; Hamb€ack 2010).

The current study had two aims. The first aim was to examine trade-offs connected to parasitoid virulence, or more specifically, whether parasitoids hatching from one host had an equal or reduced capacity to infect larvae of other host species, and if such differences were connected to difference in the induction of host immune systems.

The second aim was to examine whether observed differences in parasitism rate between southern and northern localities may be caused by geographic variation in either the ability of parasitoids to infect hosts or in hosts to mount an efficient immune response against parasitoid

eggs. The study was performed by combining controlled parasitism experiments in the laboratory with an investi- gation of hemocyte composition in infected larvae. Our results revealed differences in the strength of the immune response of Galerucella and indicated a possible hierarchi- cal division of the Asecodes parasitoids depending on their former host species. We found an overall stronger immune defence in G. pusilla compared with both G. calmariensis and G. tenella when infected by A. parviclava.

In accordance with this, parasitoids from G. pusilla successfully infect larvae of all species, parasitoids from G. tenella successfully infect larvae of G. tenella and G. calmariensis, whereas parasitoids from G. calmariensis are successful mainly when infecting larvae of 9 8

76 5 4

13 12 11

10

0 25 50 km 1

2

3

1 2 km

0 (a)

(b)

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Fig. 2. Map of Sweden showing the distribution of the study species and the field sites used for collection of Galerucella adults and larvae. All species are present in the south of Sweden, whereas Galerucella pusilla does not occur north of the dashed line. The species were collected from the following localities, indicated by numbers: Galerucella calmariensis (1–4, 6–8, 10–11), G. pusilla (4, 6–8, 10–13) and Galerucella tenella(1–3, 5–9). Subfigures show the distribution of field sites in the northern (a) and southern (b) region respectively.

Trade-offs in parasitoid virulence 1597

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G. calmariensis. We also found indications that parasitoid virulence varied among sites, whereas no indications of geographic variation in host immune response were found. Cytological studies strengthen our observations, where the induction of certain cell types important for the encapsulation process is suppressed when individuals are infected by a highly virulent parasitoid.

Materials and methods

s t u d y s p e c i e s

Galerucella pusilla Duftschmid, G. calmariensis L. and G. tenella L. (Coleoptera: Chrysomelidae) are closely related beetle species with similar life cycles. The adult beetles emerge in early spring when mating takes place on the host plant. Eggs are deposited directly onto the leaves or stem of host plants, hatch after a few weeks and the larvae pupate in the ground 3–4 weeks later; 2–

3 weeks after pupation, the next generation emerges. The adult morphology is similar between the species, but larvae are easily distinguished based on colour (Hamb€ack 2004) (Fig. 1). Galeru- cella pusillaand G. calmariensis use Lythrum salicaria L. (Lythra- ceae) exclusively as host plant, for feeding and oviposition, whereas the most common host plant of the oligophagous G. tenella is Filipendula ulmaria L. (Rosaceae). In Sweden, G. pusilla occurs from the south up until Sundsvall (N 62°, E17°), whereas G. tenella and G. calmariensis are common also further north (Fig. 2).

Asecodes parviclavaThompson (Hymenoptera: Eulophidae) is a small (<1 mm) parasitoid known to attack G. calmariensis, G. pusilla and G. tenella in the larval stage, laying one or more eggs inside the larva (Hamb€ack et al. 2013). The parasitoid larvae consume their host from within, eventually preventing pupation.

Instead of forming a pupa, the infected larva turns into a mum- mified black shell from which the adult parasitoids subsequently hatch, usually during the next summer.

Adult beetles of all three species were collected in the field in May–June each year of the experiments (2012–2014). To avoid the risk of larvae being parasitized prior to the tests, the beetles were held in the laboratory for mating and oviposition and both eggs and larvae were kept enclosed before the experiments.

G. pusilla was collected at eight field sites in the south-east of Sweden: one in the county of G€avleborg (N61°, E17°) and seven in the county of Uppland (N60°, E18°). G. calmariensis was collected in six of the southern sites and in three sites in the county of V€asterbotten in the north of Sweden (N64°, E21°). Galerucella tenellawas collected at the same three sites in the north and at five sites in Uppland (Fig. 2). The parasitoids used for the parasitism experiments were all females, deriving from previous sea- son’s parasitized beetle larvae, collected at the same sites as the adult beetles. All larvae used for the parasitism tests were in the second instar and of approximately the same size.

c o n t r o l l e d p a r a s i t i s m e x p e r i m e n t s

To study the immune response in the beetles, 6, 9 or 12 laboratory-reared larvae of G. calmariensis, G. pusilla and G. tenella were put in 200-mL transparent plastic containers together with mated A. parviclava females. The number of parasitoids in each test was one-third of the number of larvae, which has previously

proven to be a sufficient amount in order to get all larvae parasitized during 24 h. Asecodes parviclava hatching from all three beetle species were used with each host separately, in order to investigate whether the level of successful parasitism might be affected by the former host species of the parasitoid. Larvae and parasitoids of northern and southern populations were used separately, to be able to study possible geographic variation. How- ever, due to limitation in locating parasitoids in southern localities, caused by the low parasitism rates, some intended host–parasitoid combinations were not possible (see Table S1, Supporting Information for details). After 24 h, the parasitoids were removed and the larvae were thereafter kept for an additional 96 h before dissection, to provide sufficient time for encapsulation processes to be completed and for parasitoid larvae to develop enough for easy detection. All beetle larvae were provided with fresh leaves of L. salicaria or F. ulmaria each day of the experiment. At dissection, the percentage of successfully parasitized beetle larvae (containing live parasitoid larvae) and the percentage of larvae showing a successful immune response (containing exclusively melanized eggs) were determined.

p r e p a r a t i o n o f h e m o c y t e s a n d c e l l u l a r s t u d y

Dissections and preparations of hemocyte samples were performed according to the method described in Fors et al. (2014).

Only individuals that proved to be infected at dissection were included in the cellular study. Three hemocyte samples were pre- pared from each individual larva. To reveal the nuclei, the cells were treated with blue-fluorescent nucleic acid stain DAPI (4⁰,6- diamidino-2-phenylindole). All samples were studied in a Zeiss Axioplan2, phase contrast, epifluorescent microscope connected to a Hamamatsu camera withAXIO VISION4.6 (Carl Zeiss Vision GmbH, Munchen, Germany). For the differential hemocyte counts, nine images per individual were taken at random. On average, 800 hemocytes per individual were counted and grouped into six cell types: granulocytes, phagocytes, lamellocyte precursors, lamellocytes, prohemocytes and oenocytoids, according to morphology as described in Fors et al. (2014).

s t a t i s t i c a l a n a l y s i s

The data from the controlled parasitism tests were analysed in a generalized linear model with binomial distribution, with the number of infected individuals showing either a successful immune response (containing only melanized eggs) or an unsuc- cessful immune response (containing only live parasitoid larvae) as response variables. To examine the ability of parasitoids to infect different hosts, we used the previous host species (from where the parasitoid individual hatched) as explanatory variable and performed separate analyses for the three species. To examine the geographic variation in the ability of parasitoids to infect the same host, we used previous host species and region (south vs. north) as explanatory variables when possible.

In order to investigate effects on hemocyte composition in the host, we performed separate MANOVAS for the host species with all cell types as response variables. When aMANOVA was significant, we performed separateANOVASfor the six cell types, with a sequential Bonferroni adjustment on P-values (Holm 1979).

When applicable, based on the tests of parasitism rates, we exam- ined the effect of geographic origin of parasitoids on hemocyte

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composition. The data also allowed for one comparison on the geographic variation in immune response for larvae, comparing G. tenellalarvae from north and south using parasitoids from the north. Other comparisons on geographic variation in immune response for larvae were not possible as parasitoid origin was not sufficiently replicated. Since the cell counts were estimated as pro- portions, and theMANOVA requires normally distributed data, all values were logit-transformed prior to analysis. In addition, the same tests were performed on ln-transformed numbers of counted cells for each cell type (as a measure of absolute numbers) (Tables S2 and S3, Figs S1–S4). All analyses were performed usingR2.15.2 (R Development Core Team 2012).

Results

c o n t r o l l e d p a r a s i t i s m e x p e r i m e n t s a n d i m m u n e r e s p o n s e

Galerucella calmariensismounted an overall poor immune defence, which confirms our previous results (Fors et al.

2014). In addition, the study shows that the weak defence was independent of the former host species of the parasitoid (Table 1). When infected by parasitoids deriving from the same species or from G. tenella, 0% of the G. calmariensis larvae showed a successful immune response at dissection (containing exclusively melanized eggs). When attacked by parasitoids from G. pusilla, only 3% (one individual) showed a successful immune response.

When combining data from all infected individuals independent of parasitoid origin, larvae of G. pusilla and G. tenella mounted a stronger immune defence than G. calmariensis (z= 46, P < 0001), and G. pusilla showed a stronger defence than G. tenella (z= 45, P< 00001). However, the immune responses were affected by parasitoid origin. Galerucella pusilla larvae were less able to encapsulate eggs when infected by parasitoids from the same species than when infected by parasitoids from either G. calmariensis (v²= 454, d.f. = 1, P< 0001) or G. tenella (v²= 688, d.f. = 1, P < 0001) (Table 1). Conversely, G. tenella larvae had a lower ability to encapsulate eggs when infected by parasitoids from the same species than when infected by parasitoids from G. pusilla (v²= 47, d.f.= 1, P< 003) or

G. calmariensis(v²= 241, d.f. = 1, P < 00001). However, the encapsulation ability was also lower when infected by parasitoids obtained from G. pusilla compared with parasitoids from G. calmariensis (v²= 82, d.f. = 1, P < 0005) (Table 1). Taken together, this confirms previous findings on the differences in immune competence between G. calmariensis and G. pusilla and places the competence of G. tenella between the two other species. In addition, species-specific adaptations in parasitization strategy are observed showing that parasitoids deriving from the same species are generally most successful.

Due to the overall weak immune response in G. calmariensis, it was not meaningful to compare variation among regions for this species. In G. pusilla, which only occurs in the south of Sweden, the immune response was compared between larvae infected with parasitoids deriving from all host species but only from southern localities.

The overall results were still the same, with the weakest response towards parasitoids from the same species and the strongest response towards parasitoids from G. tenella. However, the strength of the immune response towards parasitoids from G. pusilla and G. calmariensis was no longer significantly different (v²= 29, d.f. = 1, P= 009), suggesting a higher infectivity in parasitoids deriving from southern populations of G. calmariensis.

For G. tenella, no differences in the immune response of the northern and southern population could be found upon infection with parasitoids from G. tenella (v²= 07, d.f.= 1, P = 041) or G. calmariensis (v²= 19, d.f. = 1, P= 017).

c e l l u l a r s t u d y

In all three Galerucella species, the general cell composition was in accordance with our previous finding (Fors et al. 2014), consisting of six hemocyte types: granulocytes, phagocytes, prohemocytes, oenocytoids, lamellocytes and lamellocyte precursors, with granulocytes being the most common. Notable was, however, that oenocytoids, which are in general very rare, were more common in G. tenella than in the other two species.

Moreover, in addition to the regular hemocyte types, we also observed some large cell aggregates in G. tenella, which we had not previously seen in Galerucella. These structures looked like long ribbons of connected cells, containing several nuclei (Fig. S5). The multinucleated cells were observed exclusively in G. tenella and only in some of the infected individuals (6% of the infected individuals).

As the immune response in G. calmariensis did not differ between larvae attacked by parasitoids of different origin, or between individuals from different areas, this species was not included in any further cellular study. For the other two species, tests were run on the relative numbers from the hemocyte counts. For G. pusilla, twoMANO- VAS were performed, one with all parasitoids included (Fig. 3) and a second with only parasitoids from southern Table 1. The percentage of Galerucella calmariensis, Galerucella

pusilla and Galerucella tenella larvae showing a successful immune response at dissection (containing exclusively melanised eggs) when infected by Asecodes parviclava deriving from different host species. N refers to the total number of individuals infected in each group (containing either melanised eggs or live parasitoid larvae)

Parasitoid origin

G. calmariensis G. tenella G. pusilla

Larval species

G. calmariensis 0 (N= 82) 0 (N= 69) 3 (N= 39) G. tenella 49 (N= 39) 4 (N= 45) 8 (N= 13) G. pusilla 78 (N= 37) 92 (N= 38) 12 (N= 59)

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localities (Fig. 4), since the parasitism tests indicated a geographic difference in infectivity for G. calmariensis parasitoids attacking G. pusilla larvae. The first MANOVA

(with all data included) indicated that the cell composition was affected by the parasitoid origin (MANOVA: F2,44= 41, P˂ 0001), and the post hoc ^ANOVAs revealed that this effect was due to differences in the relative levels of phagocytes (F2,44= 118, P˂ 0005), lamellocytes (F2,44= 51, P˂ 005) and lamellocyte precursors (F2,44= 51, P ˂ 005) (Fig. 3). The second^MANOVA (with only southern parasitoids) suggested that the cell composition was different in G. pusilla larvae infected by parasitoids from the same species or G. calmariensis compared with larvae infected by parasitoids from G. tenella (MAN- OVA: F_1,21= 36, P < 002). The post hoc ^ANOVAs revealed that the relative levels of phagocytes (F_1,21= 189, P< 0002), lamellocytes (F1,21= 73, P < 005) and lamellocyte precursors (F1,21= 116, P < 002) were higher and the levels of granulocytes were lower (F1,21= 212 P< 0001) in G. pusilla larvae infected by parasitoids from G. tenella compared with larvae infected by parasitoids from the other two species (Fig. 4).

Similarly for G. tenella, two MANOVAs were performed, one with all parasitoids included (Fig. 5) and a second with parasitoids only from northern populations of

G. tenella and G. calmariensis (Fig. S6). The firstMANOVA

indicated an effect of former parasitoid host on the cell composition (MANOVA: F2,48= 45, P ˂ 0001). The post hocANOVAs suggested that the relative levels of phagocytes (F2,48= 62, P< 002), lamellocytes (F2,48= 128, P< 00002) and oenocytoids (F2,48= 140, P < 00001) differed depending on origin of the parasitoids. The relative levels of lamellocytes and oenocytoids were higher in larvae infected by parasitoids from G. calmariensis compared with the other parasitoids, while there was also a lower level of phagocytes in larvae infected by parasitoids from G. pusilla (Fig. 5). The second MANOVA (with parasitoids only from northern populations) indicated that both the former host of the parasitoid (MANOVA: F_1,29= 90, P < 00001) and the geographic origin of the larvae (MANOVA: F_1,29= 48, P < 0003) had an effect on the cell composition in G. tenella, but there was no inter- active effect of the two variables (MANOVA: F1,29= 15, P> 02). The post hoc ^ANOVAs revealed that the relative levels of lamellocytes were higher in G. tenella larvae from the north (F1,31= 78, P = 005) and that the levels of lamellocytes and oenocytoids were higher when larvae were infected by parasitoids from G. calmariensis (lamellocytes: F1,31= 149, P < 0004, oenocytoids: F1,31= 114, P< 002) (Fig. S6).

0 5 10 15 20 25 30

Phagocytes Lam precursors Lamellocytes Prohemocytes Oenocytoids G. pusilla G. calmariensis G. tenella

(12%) (78%) (92%)

0 10 20 30 40 50 60 70 80 90

Granulocytes

setycomehfo%

*

G. pusilla larvae infected with parasitoids deriving from:

*

***

Fig. 3. Back-transformed hemocyte levels (SE) in Galerucella pusilla larvae when infected by parasitoids deriving from G. pusilla, Galerucella calmariensis and Galerucella tenella (Ninf Gp-parasitoid= 8, Ninf Gc-parasitoid= 23, Ninf Gt-parasitoid= 16). The percentages in brackets refer to the level of successful immune response towards each parasitoid, as shown in Table 1. Asterisks denoting significant difference:*P < 005; ***P < 0001.

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Similar results were obtained for all tests when comparing the number of counted cells (as a measure of absolute numbers) from the hemocyte counts (results presented as supporting information: Tables S2 and S3, Figs S1–S4).

Taken together, our results show a correlation between the strength of the immune response in Galerucella and the appearance of the cell types that have been implicated in encapsulation, in particular lamellocytes.

Discussion

Despite an increasing interest to study immunity-related traits in relation to evolution and ecology (Rolff & Siva- Jothy 2003; Schulenburg et al. 2009), there is still limited knowledge on the connections between host immunity, parasitoid virulence (and costs thereof), host race formation and speciation in natural host–parasitoid systems. In this study, we investigated parasitism success (i.e. developed parasitoid larvae in infected hosts) in the eulophid parasitic wasp A. parviclava when attacking three beetle species: G. pusilla, G. calmariensis and G. tenella. We found that the infectivity of Asecodes parasitoids is dependent on their former host species. The main pattern was that parasitoids deriving from G. pusilla have much higher success rates when attacking G. pusilla larvae than parasitoids from any of the other species. Similarly, parasitoids from G. tenella had much higher infectivity than parasitoids from G. calmariensis when attacking G. tenella

larvae. However, parasitoids from G. pusilla had similar success rates to parasitoids from G. tenella when attacking G. tenella larvae. As G. pusilla larvae have the overall strongest immune defence of the three beetle species, parasitoids that have developed an ability to overcome this defence might also be more effective when infecting larvae of the other two species. In concordance with this, parasitoids deriving from G. calmariensis, with the overall poorest immune defence, generally show low success rates when attacking larvae of the other two species. The excep- tion was for parasitoids deriving from southern populations of G. calmariensis, which showed a similar virulence when attacking G. pusilla larvae as parasitoids deriving from G. pusilla (i.e. higher virulence than parasitoids from G. tenella). Accordingly, the hemocyte composition did not differ in larvae infected by parasitoids from G. pusilla and G. calmariensis. This suggests geographic differences in parasitoid infectivity with higher virulence in parasitoids deriving from southern populations of G. calmariensis compared with parasitoids from the north (where G. pusilla is not present).

Our observations in the Asecodes–Galerucella system could be an example of Thompson’s theory of ‘hot spots’

and ‘cold spots’ of co-evolution, with strong reciprocal selection in some areas and reduced or unidirectional selection in others, resulting in geographic variation in host resistance and parasitoid counter-resistance in the system (Thompson 1994, 2005). The variation in the

0 5 10 15 20 25 30

Phagocytes Lam precursors Lamellocytes Prohemocytes Oenocytoids

G. pusilla G. calmariensis G. tenella

G. pusilla larvae infected with parasitoids deriving from southern populations of:

0 10 20 30 40 50 60 70 80 90

Granulocytes

setycomehfo%

*

**

*

***

Fig. 4. Back-transformed hemocyte levels (SE) in Galerucella pusilla larvae when infected by parasitoids deriving from southern populations of G. pusilla, Galerucella calmariensis and Galerucella tenella (Ninf Gp-parasitoid = 8, Ninf Gc-parasitoid South= 7, Ninf Gt-parasitoid South= 8). Asterisks denoting significant difference:*P < 005; **P < 001, ***P < 0001.

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availability of host species (in this case the presence of G. pusilla in the south of Sweden but not in the north) could be one reason for such geographic mosaic of selection. In a previous study by Kraaijeveld & van Alphen (1994), southern European populations of the polypha- gous Drosophila parasitoid A. tabida were found to have a better counter-resistance to the encapsulation defence in D. melanogaster, the most common host species in the south, compared with northern European populations.

Due to a ‘stickiness’ of the parasitoid eggs, the encapsulation was only partial or absent, and the parasitoid larvae could develop successfully despite the immune response of D. melanogaster (Kraaijeveld & van Alphen 1994). Dupas and Boscaro similarly investigated the geographic variation of counter-resistance in the parasitoid L. boulardi, which is specific on D. melanogaster but able to develop on several species of the D. melanogaster subgroup. Their study showed that the immunosuppressive ability against D. melanogaster was reduced in areas where the parasitoid was also exposed to Drosophila yakuba (Dupas & Boscaro 1999). In a later study by Dubuffet et al. (2007), variation in resistance patterns in D. melanogaster and D. yakuba towards L. boulardi were showed, as well as differences in the specificity levels in the parasitoid.

In the current study, field data suggest that parasitism rates are much higher on G. calmariensis in northern (>70%) than in southern (<10%) localities. When starting this study, we suspected that this difference was due to differences in the strength of the immune response. How- ever, our study suggests that G. calmariensis has a very weak immune defence irrespective of origin and that the different parasitism rates must have some other causality.

One possible reason is that the presence of G. pusilla in southern localities also affects the parasitoid population size, but further data are needed to examine this possibil- ity. Moreover, the proportion of G. calmariensis and G. pusilla show large variation among sites and selection strengths on parasitoid virulence may thus vary. We have some indications that the virulence of parasitoids from G. pusilla varies geographically also within the southern area, but firm proof necessitates a larger sampling effort.

The differences we found among the beetle species in overall immune responses match differences in cell composition, and the general pattern in hemocyte composition after wasp infestation was very similar to previous studies within the Galerucella system. As previously shown, lamellocytes, phagocytes and granulocytes all participate in the encapsulation process in Galerucella, with

0 2 4 6 8 10 12 14 16 18 20

Phagocytes Lam precursors Lamellocytes Prohemocytes Oenocytoids G. pusilla G. calmariensis G. tenella G. tenella larvae infected with parasitoids deriving from:

(8%) (44%) (4%)

0 10 20 30 40 50 60 70 80 90

Granulocytes

setycomehfo%

*

***

Fig. 5. Back-transformed hemocyte levels (SE) in Galerucella tenella larvae infected by parasitoids deriving from Galerucella pusilla, Galerucella calmariensisand G. tenella (Ninf Gp-parasitoid= 9, Ninf Gc-parasitoid= 17, Ninf Gt-parasitoid= 26). The percentages in brackets refer to the level of successful immune response towards each parasitoid, as shown in Table 1. Asterisks denoting significant difference:

*P < 005; ***P < 0001.

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lamellocytes being crucial for the capsule formation to be completed (Fors et al. 2014). In this study, we find the same pattern in a third species (G. tenella). In larvae of G. pusilla, the relative levels of both lamellocytes and phagocytes were higher when infected by parasitoids from G. calmariensisor G. tenella, towards which the strongest immune response is observed, than when infected by parasitoids from the same species. In larvae of G. tenella, the relative level of lamellocytes was again much higher when infected by parasitoids from G. calmariensis, towards which the strongest immune response is mounted, whereas the level of phagocytes was equally high in individuals infected by parasitoids deriving from both G. calmariensis and G. tenella. The fact that the increase in lamellocytes and in some cases phagocytes coincides with a drop in both prohemocytes and granulocytes (Figs 3–5) is com- patible with the idea that upon wasp infestation effector cells, in particular lamellocytes, are recruited from the pool of undifferentiated prohemocytes (Honti et al. 2014).

Similarly, the increase in the number of cells that perform phagocytic functions may be due to some granulocytes obtaining phagocytic activity.

Our study suggests that also oenocytoids may be involved in the immune response, at least in G. tenella.

The level of oenocytoids was much higher in G. tenella infected by parasitoids deriving from G. calmariensis, towards which the strongest response was seen, suggesting an active role in the immune defence. We propose that oenocytoids contribute to the capsule melanization in Galerucella since they are known to be involved in the melanization process in other species (Jiang et al. 1997;

Shrestha & Kim 2008). In infected individuals of G. tenella, we also observed a type of multinucleated cells which we had not previously seen in Galerucella. We believe these cells to be similar to the multinucleated giant hemocytes described recently in Drosophila, where they have a function in parasite elimination (Markus et al.

2015). However, at this point no functional tests have been performed on the multinucleated cells in G. tenella to prove their possible role in the immune response.

In conclusion, our studies reveal that parasitism success in A. parviclava is affected by former parasitoid host species and to some extent also the geographic origin of the parasitoid, whereas we found no indications of geographic variation in host immune response. In accordance, the cytological studies showed that the induction of certain cell types important for the encapsulation process, in particular lamellocytes, is suppressed when individuals are infected by a highly virulent parasitoid. Our findings suggest ongoing evolution in both parasitoid virulence and host immune response in this system. Given these patterns, we believe that the Asecodes–Galerucella system is a useful model system for understanding the evolutionary processes underlying host race formation and speciation in host–parasitoid systems. To elucidate these patterns, a better understanding both on the phylogeographic patterns underlying the current distribution of virulence and

immune response traits and the genes involved will be required.

Acknowledgements

This work was supported by grants VR-2009-4943 and VR-2012-3578 from the Swedish Research Council Vetenskapsradet (to PAH), VR-2010- 5988 from the Swedish Research Council Vetenskapsradet (to U.T.) and (IG2011-2042) from the Swedish Foundation for International Coopera- tion in Research and Higher Education (to U.T.).

Data accessibility

The raw data from the cell counts are available in the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.7h554 (Fors et al. 2016).

References

Agrawal, A. & Lively, C.M. (2002) Infection genetics: gene-for-gene versus matching-alleles models and all points in between. Evolutionary Ecology Research,4, 79–90.

Antolin, M.F., Bjorksten, T.A. & Vaughn, T.T. (2006) Host-related fitness trade-offs in a presumed generalist parasitoid, Diaeretiella rapae (Hyme- noptera: Aphidiidae). Ecological Entomology,31, 242–254.

Beckage, N.E. (2008) Parasitoid polydnaviruses and insect immunity.

Insect Immunology(ed. N.E. Beckage), pp. 243–270. Academic Press, Oxford, UK.

Burke, G.R. & Strand, M.R. (2014) Systematic analysis of a wasp parasitism arsenal. Molecular Ecology,23, 890–901.

Dahlman, D.L. (1991) Teratocytes and host/parasitoid interactions.

Biological Control,1, 118–126.

Dubuffet, A., Dupas, S., Frey, F., Drezen, J.M., Poirie, M. & Carton, Y.

(2007) Genetic interactions between the parasitoid wasp Leptopilina bou- lardiand its Drosophila hosts. Heredity,98, 21–27.

Dupas, S. & Boscaro, R. (1999) Geographic variation and evolution of immunosuppressive genes in a Drosophila parasitoid. Ecography, 22, 284–291.

Firlej, A., Lucas, E., Coderre, D. & Boivin, G. (2007) Teratocytes growth pattern reflects host suitability in a host-parasitoid assemblage. Physio- logical Entomology,32, 181–187.

Fors, L., Markus, R., Theopold, U. & Hamb€ack, P.A. (2014) Differences in cellular immune competence explain parasitoid resistance for two coleopteran species. PLoS ONE,9, e108795.

Fors, L., Markus, R., Theopold, U., Ericson, L. & Hamb€ack, P.A.

(2016) Data from: Geographic variation and trade-offs in parasitoid virulence. Dryad Digital Repository, http://dx.doi.org/10.5061/

dryad.7h554.

Fytrou, A., Schofield, P.G., Kraaijeveld, A.R. & Hubbard, S.F. (2006) Wolbachiainfection suppresses both host defence and parasitoid counter-defence. Proceedings of the Royal Society B: Biological Sciences,273, 791–796.

Grosberg, R.K. & Hart, M.W. (2000) Mate selection and the evolution of highly polymorphic self/nonself recognition genes. Science,289, 2111–

2114.

Hamb€ack, P. (2004) Why purple loosestrife in sweet gale shrubs are less attacked by herbivorous beetles? Entomol Tidskr,125, 93–102.

Hamb€ack, P.A. (2010) Density-dependent processes in leaf beetles feeding on purple loosestrife: aggregative behaviour affecting individual growth rates. Bulletin of Entomological Research,100, 605–611.

Hamb€ack, P.A., Stenberg, J.A. & Ericson, L. (2006) Asymmetric indirect interactions mediated by a shared parasitoid: connecting species traits and local distribution patterns for two chrysomelid beetles. Oecologia, 148, 475–481.

Hamb€ack, P.A., Weingartner, E., Ericson, L., Fors, L., Cassel-Lundhagen, A., Stenberg, J.A. et al. (2013) Bayesian species delimitation reveals generalist and specialist parasitic wasps on Galerucella beetles (Chrysomelidae): sorting by herbivore or plant host. BMC Evolutionary Biology,13, 92.

Hansson, C. & Hamb€ack, P.A. (2013) Three cryptic species in Asecodes (Forster) (Hymenoptera, Eulophidae) parasitizing larvae of Galerucella spp. (Coleoptera, Chrysomelidae), including a new species. J Hymenopt Res,30, 51–64.

(11)

Holm, S. (1979) A simple sequentially rejective multiple test procedure.

Scandinavian Journal of Statistics,6, 65–70.

Honti, V., Csordas, G., Kurucz, E., Markus, R. & Ando, I. (2014) The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation. Developmental and Comparative Immunology,42, 47–56.

Jiang, H.B., Wang, Y., Ma, C.C. & Kanost, M.R. (1997) Subunit composition of pro-phenol oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1. Insect Biochemistry and Molecular Biology,27, 835–

850.

Jones, T.S., Bilton, A.R., Mak, L. & Sait, S.M. (2015) Host switching in a generalist parasitoid: contrasting transient and transgenerational costs associated with novel and original host species. Ecology and Evolution, 5, 459–465.

Kraaijeveld, A.R. & Godfray, H.C.J. (2009) Evolution of host resistance and parasitoid counter-resistance. Advances in Parasitology, Parasitoids of Drosophila(ed. G. Prevost), pp. 257–280. Elsevier, London, UK.

Kraaijeveld, A.R., Nowee, B. & Najem, R.W. (1995) Adaptive variation in host-selection behavior of Asobara tabida, a parasitoid of Drosophila larvae. Functional Ecology,9, 113–118.

Kraaijeveld, A.R. & van Alphen, J.J. (1994) Geographical variation in resistance of the parasitoid A. tabida against encapsulation by D. melanogaster larvae: the mechanism explored. Physiological Entomology, 19, 9–14.

Kraaijeveld, A.R. & van Alphen, J.J.M. (1995) Geographical variation in encapsulation ability of Drosophila melanogaster larvae and evidence for parasitoid-specific components. Evolutionary Ecology,9, 10–17.

Lavine, M.D. & Strand, M.R. (2002) Insect hemocytes and their role in immunity. Insect Biochemistry and Molecular Biology, 32, 1295–1309.

Markus, R., Lerner, Z., Honti, V., Csordas, G., Zsamboki, J., Cinege, G.

et al.(2015) Multinucleated giant hemocytes are effector cells in cell- mediated immune responses of Drosophila. Journal of Innate Immunity, 7, 340–353.

Nappi, A.J. (1981) Cellular immune response of Drosophila melanogaster against Asobara tabida. Parasitology,83, 319–324.

R Development Core Team (2012) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.

Rizki, R.M. & Rizki, T.M. (1990) Encapsulation of parasitoid eggs in phenoloxidase-deficient mutants of Drosophila melanogaster. Journal of Insect Physiology,36, 523–529.

Rolff, J. & Siva-Jothy, M.T. (2003) Invertebrate ecological immunology.

Science,301, 472–475.

Schmidt, O., Theopold, U. & Strand, M. (2001) Innate immunity and its evasion and suppression by hymenopteran endoparasitoids. BioEssays, 23, 344–351.

Schulenburg, H., Kurtz, J., Moret, Y. & Siva-Jothy, M.T. (2009) Ecologi- cal immunology. Philosophical Transactions of the Royal Society of Lon- don. Series B, Biological sciences,364, 3–14.

Shrestha, S. & Kim, Y. (2008) Eicosanoids mediate prophenoloxidase release from oenocytoids in the beet armyworm Spodoptera exigua.

Insect Biochemistry and Molecular Biology,38, 99–112.

Stenberg, J.A., Heijari, J., Holopainen, J.K. & Ericson, L. (2007) Presence of Lythrum salicaria enhances the bodyguard effects of the parasitoid Asecodes mentofor Filipendula ulmaria. Oikos,116, 482–490.

Strand, M.R. (2010) Polydnaviruses. Insect Virology (eds S. Asgari &

K.N. Johnson), pp. 171–197. Caister Academic Press, Norwich, UK.

Thompson, J.N. (1994) The Coevolutionary Process. The University of Chicago Press, Chicago, IL, USA.

Thompson, J.N. (2005) The Geographic Mosaic of Coevolution. The University of Chicago Press, Chicago, IL, USA.

Van Veen, F.J.F., Mueller, C.B., Pell, J.K. & Godfray, H.C.J. (2008) Food web structure of three guilds of natural enemies: predators, parasitoids and pathogens of aphids. Journal of Animal Ecology,77, 191–200.

Received 5 February 2016; accepted 21 July 2016 Handling Editor: Sheena Cotter

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Fig. S1. Absolute hemocyte numbers observed in G. pusilla larvae when infected by parasitoids deriving from G. pusilla, G. calmariensisand G. tenella.

Fig. S2. Absolute hemocyte numbers observed in G. pusilla larvae when infected by parasitoids deriving from southern populations of G. pusilla, G. calmariensis and G. tenella.

Fig. S3. Absolute hemocyte numbers observed in G. tenella larvae infected by parasitoids deriving from G. pusilla, G. calmariensis and G. tenella.

Fig. S4. Absolute hemocyte numbers observed in G. tenella larvae from northern and southern localities, infected with parasitoids deriving from northern populations of G. calmariensis and G. tenella.

Fig. S5. Multinucleated cell structures in G. tenella larvae infected with A. parviclava.

Fig. S6. Back-transformed hemocyte levels (SE) in G. tenella larvae from northern and southern localities.

Table S1. All possible combinations of Galerucella spp. larvae and Asecodes parasitoids deriving from the different host species, divided into northern and southern populations.

Table S2. Output from analyses of variance on cell composition [F-value (P-value)] for G. pusilla and G. tenella larvae, infected with parasitoids deriving from either G. pusilla, G. tenella or G. calmariensis.

Table S3. Output from analyses of variance on cell composition [F-value (P-value)] for G. tenella larvae of two regional origins and infected with parasitoids deriving from either G. tenella or G. calmariensis.