Geographic Variation and Trade&#X2010;Offs in Parasitoid Virulence

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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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Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-128966 Journal of Animal Ecology 2016, 85, 1595–1604 doi: 10.1111/1365-2656.12579 Geographic variation and trade-offs in parasitoid virulence

Lisa Fors1*, Robert Markus2, Ulrich Theopold3, Lars Ericson4 and Peter A. Hamback€ 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; and 4Department 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

counter-resistance mechanisms (Fytrou et al. 2006; Kraai- Introduction jeveld & Godfray 2009) making these systems excellent Co-evolution, the process of reciprocal evolutionary models for co-evolutionary studies. Host insects use sev- change driven by natural selection in interacting species, eral strategies to escape parasitoid attack, such as enemy- has a central role in explaining the high species diversity free space, concealment or physical counter-attack. Even in many systems (Thompson 2005). In situations with when successfully parasitized, the host can defend itself antagonistic interactions, such as host–parasitoid systems, through a potent immune defence, most commonly by there is often a constant development of resistance and 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- *Correspondence author. E-mail: [email protected] sophila melanogaster, parasitoid eggs initially attract

© 2016 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society. 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. 1596 L. Fors et al. plasmatocytes, which spread around the egg surface. This causing such geographic mosaics of selection is variation is followed by the differentiation of a specialized larger in the availability of different host species, as for instance class of hemocytes (lamellocytes), which form a second in the interactions between D. melanogaster and one of its cellular layer. Ultimately, the capsule melanizes through main parasitoids, Asobara tabida (Kraaijeveld & van the release and activation of phenoloxidase from special- Alphen 1994, 1995; Kraaijeveld, Nowee & Najem 1995). ized cells (crystal cells in Drosophila and oenocytoids in In this system, large population variations have been many other insect species (Honti et al. 2014)). These resis- observed both in the ability of the host to encapsulate tance mechanisms have imposed strong selection on the parasitoid eggs and in parasitoid ability to prevent parasitoid to evolve counter-resistance strategies, such as the encapsulation response. In southern Europe where the injection of venom, teratocytes or viruses into the D. melanogaster is the only host available, the counter- host, thereby protecting the progeny from being rejected resistance of A. tabida is stronger than in northern Eur- (Dahlman 1991; Firlej et al. 2007; Beckage 2008; Strand ope, where the additional host Drosophila subobscura 2010; Burke & Strand 2014). (lacking the encapsulation ability) is also present (Kraai- Parasitoid adaptations to specific hosts often come at a jeveld & Godfray 2009). A similar variation in parasitoid cost, sometimes resulting in a reduced ability in the para- counter-adaptations has also been observed in another sitoid to infest other host species (Van Veen et al. 2008; parasitoid (Leptopilina boulardi) commonly attacking Jones et al. 2015). In the extreme case, as in the match- D. melanogaster (Dupas & Boscaro 1999), but few similar ing-alleles model, an exact genetic match is necessary for studies exist from non-Drosophila systems. successful attack by the parasitoid (Agrawal & Lively In this study, we investigated parasitism success (i.e. the 2002). According to this model, which is based on self/ development of parasitoid larvae in infected host individu- non-self recognition systems (Grosberg & Hart 2000; als) of the parasitoid Asecodes parviclava (Hymenoptera: Honti et al. 2014), the parasitoid may completely lose the Eulophidae), attacking three closely related species of ability to infect the ancestral host when successfully Galerucella leaf beetles: Galerucella pusilla, G. calmariensis adapting to a novel host. The alternative infection model, and Galerucella tenella (Coleoptera: Chrysomelidae) gene-for-gene matching, rather assumes that adaptations (Figs 1 and 2). Previous genetic studies indicate that to different hosts involve different genes (Agrawal & A. parviclava could be at an early stage of further specia- Lively 2002). Due to costs in developing a higher viru- tion, making Galerucella–Asecodes a useful model system lence, the parasitoid will not evolve high infectivity to all for investigating evolution of resistance and counter-resis- hosts and may still vary in their virulence to different tance mechanisms. The most recent divergence date for hosts (Antolin, Bjorksten & Vaughn 2006). Galerucella is estimated to 77 000 years ago for G. pusilla According to the geographic mosaic theory of co-evolu- and G. calmariensis, and the speciation of Asecodes seems tion, interactions between two species vary spatially, caus- to be following the speciation of the hosts (Hamback€ ing spatial differences in the direction and strength of et al. 2013; Hansson & Hamback€ 2013). Differences in selection (Thompson 1994, 2005). In some areas, recipro- immune response in G. pusilla and G. calmariensis have cal selection may be strong (‘hot spots’ of co-evolution), previously been observed (Fors et al. 2014), as well as whereas selection in other areas may be reduced or unidi- among population differences in parasitism rate (i.e. the rectional (‘cold spots’). As a consequence, host resistance proportion of Galerucella larvae killed by parasitism). In and parasitoid counter-resistance in a specific host–para- northern localities of Sweden, where only G. calmariensis sitoid system can vary geographically. One mechanism and G. tenella occur (Fig. 2), the parasitism rate is

(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) G. calmariensis G. pusilla G. tenella larva. Scale bars: 1 mm.

(a)

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6 10 7 12 9 8 13 (b) 11

0 25 50 km

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. generally much higher (>70%) compared with southern eggs. The study was performed by combining controlled localities (<10%) (Hamback,€ Stenberg & Ericson 2006; parasitism experiments in the laboratory with an investi- Stenberg et al. 2007; Hamback€ 2010). gation of hemocyte composition in infected larvae. Our The current study had two aims. The first aim was to results revealed differences in the strength of the immune examine trade-offs connected to parasitoid virulence, or response of Galerucella and indicated a possible hierarchi- more specifically, whether parasitoids hatching from one cal division of the Asecodes parasitoids depending on their host had an equal or reduced capacity to infect larvae of former host species. We found an overall stronger other host species, and if such differences were connected immune defence in G. pusilla compared with both G. cal- to difference in the induction of host immune systems. mariensis and G. tenella when infected by A. parviclava. The second aim was to examine whether observed differ- In accordance with this, parasitoids from G. pusilla suc- ences in parasitism rate between southern and northern cessfully infect larvae of all species, parasitoids from localities may be caused by geographic variation in either G. tenella successfully infect larvae of G. tenella and the ability of parasitoids to infect hosts or in hosts to G. calmariensis, whereas parasitoids from G. calmariensis mount an efficient immune response against parasitoid are successful mainly when infecting larvae of

G. calmariensis. We also found indications that parasitoid proven to be a sufficient amount in order to get all larvae para- virulence varied among sites, whereas no indications of sitized during 24 h. Asecodes parviclava hatching from all three geographic variation in host immune response were beetle species were used with each host separately, in order to found. Cytological studies strengthen our observations, investigate whether the level of successful parasitism might be where the induction of certain cell types important for the affected by the former host species of the parasitoid. Larvae and parasitoids of northern and southern populations were used sepa- encapsulation process is suppressed when individuals are rately, to be able to study possible geographic variation. How- infected by a highly virulent parasitoid. ever, due to limitation in locating parasitoids in southern localities, caused by the low parasitism rates, some intended Materials and methods host–parasitoid combinations were not possible (see Table S1, Supporting Information for details). After 24 h, the parasitoids study species were removed and the larvae were thereafter kept for an additional 96 h before dissection, to provide sufficient time for encap- Galerucella pusilla Duftschmid, G. calmariensis L. and G. tenella sulation processes to be completed and for parasitoid larvae to L. (Coleoptera: Chrysomelidae) are closely related beetle species develop enough for easy detection. All beetle larvae were pro- with similar life cycles. The adult beetles emerge in early spring vided with fresh leaves of L. salicaria or F. ulmaria each day of when mating takes place on the host plant. Eggs are deposited the experiment. At dissection, the percentage of successfully para- directly onto the leaves or stem of host plants, hatch after a few sitized beetle larvae (containing live parasitoid larvae) and the weeks and the larvae pupate in the ground 3–4 weeks later; 2– percentage of larvae showing a successful immune response (con- 3 weeks after pupation, the next generation emerges. The adult taining exclusively melanized eggs) were determined. morphology is similar between the species, but larvae are easily € distinguished based on colour (Hamback 2004) (Fig. 1). Galeru- preparation of hemocytes and cellular cella pusilla and G. calmariensis use Lythrum salicaria L. (Lythra- study ceae) exclusively as host plant, for feeding and oviposition, whereas the most common host plant of the oligophagous Dissections and preparations of hemocyte samples were per- G. tenella is Filipendula ulmaria L. (Rosaceae). In Sweden, formed according to the method described in Fors et al. (2014). ° G. pusilla occurs from the south up until Sundsvall (N 62 , Only individuals that proved to be infected at dissection were ° E17 ), whereas G. tenella and G. calmariensis are common also included in the cellular study. Three hemocyte samples were pre- further north (Fig. 2). pared from each individual larva. To reveal the nuclei, the cells Asecodes parviclava Thompson (Hymenoptera: Eulophidae) is a were treated with blue-fluorescent nucleic acid stain DAPI (40,6- small (<1 mm) parasitoid known to attack G. calmariensis, diamidino-2-phenylindole). All samples were studied in a Zeiss G. pusilla and G. tenella in the larval stage, laying one or more Axioplan2, phase contrast, epifluorescent microscope connected € eggs inside the larva (Hamback et al. 2013). The parasitoid larvae to a Hamamatsu camera with AXIO VISION 4.6 (Carl Zeiss Vision consume their host from within, eventually preventing pupation. GmbH, Munchen, Germany). For the differential hemocyte Instead of forming a pupa, the infected larva turns into a mum- counts, nine images per individual were taken at random. On mified black shell from which the adult parasitoids subsequently average, 800 hemocytes per individual were counted and grouped hatch, usually during the next summer. into six cell types: granulocytes, phagocytes, lamellocyte precur- Adult beetles of all three species were collected in the field in sors, lamellocytes, prohemocytes and oenocytoids, according to May–June each year of the experiments (2012–2014). To avoid morphology as described in Fors et al. (2014). 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. statistical analysis G. pusilla was collected at eight field sites in the south-east of The data from the controlled parasitism tests were analysed in a Sweden: one in the county of Gavleborg€ (N61°, E17°) and seven generalized linear model with binomial distribution, with the in the county of Uppland (N60°, E18°). G. calmariensis was col- number of infected individuals showing either a successful lected in six of the southern sites and in three sites in the county immune response (containing only melanized eggs) or an unsuc- of Vasterbotten€ in the north of Sweden (N64°, E21°). Galerucella cessful immune response (containing only live parasitoid larvae) tenella was collected at the same three sites in the north and at as response variables. To examine the ability of parasitoids to five sites in Uppland (Fig. 2). The parasitoids used for the para- infect different hosts, we used the previous host species (from sitism experiments were all females, deriving from previous sea- where the parasitoid individual hatched) as explanatory variable son’s parasitized beetle larvae, collected at the same sites as the and performed separate analyses for the three species. To exam- adult beetles. All larvae used for the parasitism tests were in the ine the geographic variation in the ability of parasitoids to infect second instar and of approximately the same size. the same host, we used previous host species and region (south vs. north) as explanatory variables when possible. controlled parasitism experiments In order to investigate effects on hemocyte composition in the host, we performed separate MANOVAS for the host species with To study the immune response in the beetles, 6, 9 or 12 labora- all cell types as response variables. When a MANOVA was signifi- tory-reared larvae of G. calmariensis, G. pusilla and G. tenella cant, we performed separate ANOVAS for the six cell types, with a were put in 200-mL transparent plastic containers together with sequential Bonferroni adjustment on P-values (Holm 1979). mated A. parviclava females. The number of parasitoids in each When applicable, based on the tests of parasitism rates, we exam- test was one-third of the number of larvae, which has previously ined the effect of geographic origin of parasitoids on hemocyte

© 2016 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society., Journal of Animal Ecology, 85, 1595–1604 Trade-offs in parasitoid virulence 1599 composition. The data also allowed for one comparison on the G. calmariensis (v2 = 241, d.f. = 1, P < 00001). However, geographic variation in immune response for larvae, comparing the encapsulation ability was also lower when infected by G. tenella larvae from north and south using parasitoids from the parasitoids obtained from G. pusilla compared with para- north. Other comparisons on geographic variation in immune sitoids from G. calmariensis (v2 = 82, d.f. = 1, P < 0005) response for larvae were not possible as parasitoid origin was not (Table 1). Taken together, this confirms previous findings sufficiently replicated. Since the cell counts were estimated as pro- on the differences in immune competence between G. cal- portions, and the MANOVA requires normally distributed data, all mariensis and G. pusilla and places the competence of values were logit-transformed prior to analysis. In addition, the same tests were performed on ln-transformed numbers of counted G. tenella between the two other species. In addition, spe- cells for each cell type (as a measure of absolute numbers) cies-specific adaptations in parasitization strategy are (Tables S2 and S3, Figs S1–S4). All analyses were performed observed showing that parasitoids deriving from the same using R 2.15.2 (R Development Core Team 2012). species are generally most successful. Due to the overall weak immune response in G. calmariensis, it was not meaningful to compare variation Results among regions for this species. In G. pusilla, which only controlled parasitism experiments and occurs in the south of Sweden, the immune response was immune response compared between larvae infected with parasitoids deriving from all host species but only from southern localities. Galerucella calmariensis mounted an overall poor immune The overall results were still the same, with the weakest defence, which confirms our previous results (Fors et al. response towards parasitoids from the same species and 2014). In addition, the study shows that the weak defence the strongest response towards parasitoids from was independent of the former host species of the para- G. tenella. However, the strength of the immune response sitoid (Table 1). When infected by parasitoids deriving towards parasitoids from G. pusilla and G. calmariensis from the same species or from G. tenella, 0% of the G. cal- was no longer significantly different (v2 = 29, d.f. = 1, mariensis larvae showed a successful immune response at P = 009), suggesting a higher infectivity in parasitoids dissection (containing exclusively melanized eggs). When deriving from southern populations of G. calmariensis. attacked by parasitoids from G. pusilla, only 3% (one indi- For G. tenella, no differences in the immune response of vidual) showed a successful immune response. the northern and southern population could be found When combining data from all infected individuals upon infection with parasitoids from G. tenella (v2 = 07, independent of parasitoid origin, larvae of G. pusilla and d.f. = 1, P = 041) or G. calmariensis (v2 = 19, d.f. = 1, G. tenella mounted a stronger immune defence than P = 017). G. calmariensis (z = 46, P < 0001), and G. pusilla = showed a stronger defence than G. tenella (z 4 5, cellular study P < 00001). However, the immune responses were affected by parasitoid origin. Galerucella pusilla larvae In all three Galerucella species, the general cell composi- were less able to encapsulate eggs when infected by para- tion was in accordance with our previous finding (Fors sitoids from the same species than when infected by para- et al. 2014), consisting of six hemocyte types: granulo- sitoids from either G. calmariensis (v2 = 454, d.f. = 1, cytes, phagocytes, prohemocytes, oenocytoids, lamel- P < 0001) or G. tenella (v2 = 688, d.f. = 1, P < 0001) locytes and lamellocyte precursors, with granulocytes (Table 1). Conversely, G. tenella larvae had a lower being the most common. Notable was, however, that ability to encapsulate eggs when infected by parasitoids oenocytoids, which are in general very rare, were more from the same species than when infected by parasitoids common in G. tenella than in the other two species. from G. pusilla (v2 = 47, d.f. = 1, P < 003) or Moreover, in addition to the regular hemocyte types, we also observed some large cell aggregates in G. tenella, Table 1. The percentage of Galerucella calmariensis, Galerucella which we had not previously seen in Galerucella. These pusilla and Galerucella tenella larvae showing a successful structures looked like long ribbons of connected cells, immune response at dissection (containing exclusively melanised containing several nuclei (Fig. S5). The multinucleated eggs) when infected by Asecodes parviclava deriving from different host species. N refers to the total number of individuals cells were observed exclusively in G. tenella and only in infected in each group (containing either melanised eggs or live some of the infected individuals (6% of the infected indi- parasitoid larvae) viduals). As the immune response in G. calmariensis did not dif- Parasitoid origin fer between larvae attacked by parasitoids of different ori- G. calmariensis G. tenella G. pusilla gin, or between individuals from different areas, this species was not included in any further cellular study. For Larval species the other two species, tests were run on the relative num- G. calmariensis 0(N = 82) 0 (N = 69) 3 (N = 39) bers from the hemocyte counts. For G. pusilla, two MANO- G. tenella 49 (N = 39) 4 (N = 45) 8 (N = 13) VAS were performed, one with all parasitoids included G. pusilla 78 (N = 37) 92 (N = 38) 12 (N = 59) (Fig. 3) and a second with only parasitoids from southern

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f 40 o % G. pusilla larvae infected 30 10 with parasitoids deriving from: G. pusilla (12%) 20 * G. calmariensis (78%) 5 G. tenella (92%)

0 0 Granulocytes Phagocytes Lam precursors Lamellocytes Prohemocytes Oenocytoids

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.

localities (Fig. 4), since the parasitism tests indicated a G. tenella and G. calmariensis (Fig. S6). The ﬁrst MANOVA geographic difference in infectivity for G. calmariensis indicated an effect of former parasitoid host on the cell parasitoids attacking G. pusilla larvae. The ﬁrst MANOVA composition (MANOVA: F2,48 = 45, P ˂ 0001). The post (with all data included) indicated that the cell composition hoc ANOVAs suggested that the relative levels of phagocytes was affected by the parasitoid origin (MANOVA: F2,44 = 41, (F2,48 = 62, P < 002), lamellocytes (F2,48 = 128,

P ˂ 0001), and the post hoc ANOVAs revealed that this P < 00002) and oenocytoids (F2,48 = 140, P < 00001) effect was due to differences in the relative levels of differed depending on origin of the parasitoids. The rela- phagocytes (F2,44 = 118, P ˂ 0005), lamellocytes tive levels of lamellocytes and oenocytoids were higher in (F2,44 = 51, P ˂ 005) and lamellocyte precursors larvae infected by parasitoids from G. calmariensis com-

(F2,44 = 51, P ˂ 005) (Fig. 3). The second MANOVA (with pared with the other parasitoids, while there was also a only southern parasitoids) suggested that the cell composi- lower level of phagocytes in larvae infected by parasitoids tion was different in G. pusilla larvae infected by para- from G. pusilla (Fig. 5). The second MANOVA (with parasitoids from the same species or G. calmariensis compared sitoids only from northern populations) indicated that with larvae infected by parasitoids from G. tenella (MAN- both the former host of the parasitoid (MANOVA:

OVA: F1,21 = 36, P < 002). The post hoc ANOVAs revealed F1,29 = 90, P < 00001) and the geographic origin of the that the relative levels of phagocytes (F1,21 = 189, larvae (MANOVA: F1,29 = 48, P < 0003) had an effect on P < 0002), lamellocytes (F1,21 = 73, P < 005) and lamel- the cell composition in G. tenella, but there was no inter- locyte precursors (F1,21 = 116, P < 002) were higher and active effect of the two variables (MANOVA: F1,29 = 15, the levels of granulocytes were lower (F1,21 = 212 P > 02). The post hoc ANOVAs revealed that the relative P < 0001) in G. pusilla larvae infected by parasitoids levels of lamellocytes were higher in G. tenella larvae from from G. tenella compared with larvae infected by para- the north (F1,31 = 78, P = 005) and that the levels of sitoids from the other two species (Fig. 4). lamellocytes and oenocytoids were higher when larvae Similarly for G. tenella, two MANOVAs were performed, were infected by parasitoids from G. calmariensis (lamel- one with all parasitoids included (Fig. 5) and a second locytes: F1,31 = 149, P < 0004, oenocytoids: F1,31 = 114, with parasitoids only from northern populations of P < 002) (Fig. S6).

90 30 ***

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m 15 G. pusilla larvae infected e

h with parasitoids deriving f 40 o from southern

% populations of: G. pusilla 30 10 ** G. calmariensis G. tenella 20

5 * 10

0 0 Granulocytes Phagocytes Lam precursors Lamellocytes Prohemocytes Oenocytoids

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.

Similar results were obtained for all tests when compar- larvae. However, parasitoids from G. pusilla had similar ing the number of counted cells (as a measure of absolute success rates to parasitoids from G. tenella when attacking numbers) from the hemocyte counts (results presented as G. tenella larvae. As G. pusilla larvae have the overall supporting information: Tables S2 and S3, Figs S1–S4). strongest immune defence of the three beetle species, par- Taken together, our results show a correlation between asitoids that have developed an ability to overcome this the strength of the immune response in Galerucella and defence might also be more effective when infecting larvae the appearance of the cell types that have been implicated of the other two species. In concordance with this, para- in encapsulation, in particular lamellocytes. sitoids 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- Discussion tion was for parasitoids deriving from southern popula- Despite an increasing interest to study immunity-related tions of G. calmariensis, which showed a similar virulence traits in relation to evolution and ecology (Rolff & Siva- when attacking G. pusilla larvae as parasitoids deriving Jothy 2003; Schulenburg et al. 2009), there is still limited from G. pusilla (i.e. higher virulence than parasitoids from knowledge on the connections between host immunity, G. tenella). Accordingly, the hemocyte composition did parasitoid virulence (and costs thereof), host race forma- not differ in larvae infected by parasitoids from G. pusilla tion and speciation in natural host–parasitoid systems. In and G. calmariensis. This suggests geographic differences this study, we investigated parasitism success (i.e. devel- in parasitoid infectivity with higher virulence in para- oped parasitoid larvae in infected hosts) in the eulophid sitoids deriving from southern populations of G. cal- parasitic wasp A. parviclava when attacking three beetle mariensis compared with parasitoids from the north species: G. pusilla, G. calmariensis and G. tenella.We (where G. pusilla is not present). found that the infectivity of Asecodes parasitoids is depen- Our observations in the Asecodes–Galerucella system dent on their former host species. The main pattern was could be an example of Thompson’s theory of ‘hot spots’ that parasitoids deriving from G. pusilla have much higher and ‘cold spots’ of co-evolution, with strong reciprocal success rates when attacking G. pusilla larvae than para- selection in some areas and reduced or unidirectional sitoids from any of the other species. Similarly, para- selection in others, resulting in geographic variation in sitoids from G. tenella had much higher infectivity than host resistance and parasitoid counter-resistance in the parasitoids from G. calmariensis when attacking G. tenella system (Thompson 1994, 2005). The variation in the

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o 40 G. tenella larvae

% infected with parasitoids 8 deriving from: G. pusilla (8%) 30 *** 6 G. calmariensis (44%) G. tenella (4%) 20 4

10 2 ***

0 0 Granulocytes Phagocytes Lam precursors Lamellocytes Prohemocytes Oenocytoids

Fig. 5. Back-transformed hemocyte levels (SE) in Galerucella tenella larvae infected by parasitoids deriving from Galerucella pusilla,

Galerucella calmariensis and 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.

availability of host species (in this case the presence of In the current study, field data suggest that parasitism G. pusilla in the south of Sweden but not in the north) rates are much higher on G. calmariensis in northern could be one reason for such geographic mosaic of selec- (>70%) than in southern (<10%) localities. When starting tion. In a previous study by Kraaijeveld & van Alphen this study, we suspected that this difference was due to (1994), southern European populations of the polypha- differences in the strength of the immune response. How- gous Drosophila parasitoid A. tabida were found to have ever, our study suggests that G. calmariensis has a very a better counter-resistance to the encapsulation defence in weak immune defence irrespective of origin and that the D. melanogaster, the most common host species in the different parasitism rates must have some other causality. south, compared with northern European populations. One possible reason is that the presence of G. pusilla in Due to a ‘stickiness’ of the parasitoid eggs, the encapsula- southern localities also affects the parasitoid population tion was only partial or absent, and the parasitoid larvae size, but further data are needed to examine this possibil- could develop successfully despite the immune response of ity. Moreover, the proportion of G. calmariensis and D. melanogaster (Kraaijeveld & van Alphen 1994). Dupas G. pusilla show large variation among sites and selection and Boscaro similarly investigated the geographic varia- strengths on parasitoid virulence may thus vary. We have tion of counter-resistance in the parasitoid L. boulardi, some indications that the virulence of parasitoids from which is specific on D. melanogaster but able to develop G. pusilla varies geographically also within the southern on several species of the D. melanogaster subgroup. Their area, but firm proof necessitates a larger sampling effort. study showed that the immunosuppressive ability against The differences we found among the beetle species in D. melanogaster was reduced in areas where the parasitoid overall immune responses match differences in cell com- was also exposed to Drosophila yakuba (Dupas & Boscaro position, and the general pattern in hemocyte composition 1999). In a later study by Dubuffet et al. (2007), variation after wasp infestation was very similar to previous studies in resistance patterns in D. melanogaster and D. yakuba within the Galerucella system. As previously shown, towards L. boulardi were showed, as well as differences in lamellocytes, phagocytes and granulocytes all participate the specificity levels in the parasitoid. in the encapsulation process in Galerucella, with

© 2016 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society., Journal of Animal Ecology, 85, 1595–1604 Trade-offs in parasitoid virulence 1603 lamellocytes being crucial for the capsule formation to be immune response traits and the genes involved will be completed (Fors et al. 2014). In this study, we find the required. same pattern in a third species (G. tenella). In larvae of G. pusilla, the relative levels of both lamellocytes and Acknowledgements phagocytes were higher when infected by parasitoids from G. calmariensis or G. tenella, towards which the strongest This work was supported by grants VR-2009-4943 and VR-2012-3578 from the Swedish Research Council Vetenskapsradet (to PAH), VR-2010- immune response is observed, than when infected by para- 5988 from the Swedish Research Council Vetenskapsradet (to U.T.) and sitoids from the same species. In larvae of G. tenella, the (IG2011-2042) from the Swedish Foundation for International Coopera- relative level of lamellocytes was again much higher when tion in Research and Higher Education (to U.T.). infected by parasitoids from G. calmariensis, towards which the strongest immune response is mounted, whereas Data accessibility the level of phagocytes was equally high in individuals infected by parasitoids deriving from both G. calmariensis 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). 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- References patible with the idea that upon wasp infestation effector Agrawal, A. & Lively, C.M. (2002) Infection genetics: gene-for-gene versus cells, in particular lamellocytes, are recruited from the matching-alleles models and all points in between. Evolutionary Ecology – pool of undifferentiated prohemocytes (Honti et al. 2014). Research, 4,79 90. Antolin, M.F., Bjorksten, T.A. & Vaughn, T.T. (2006) Host-related fitness Similarly, the increase in the number of cells that perform trade-offs in a presumed generalist parasitoid, Diaeretiella rapae (Hyme- phagocytic functions may be due to some granulocytes noptera: Aphidiidae). Ecological Entomology, 31, 242–254. obtaining phagocytic activity. Beckage, N.E. (2008) Parasitoid polydnaviruses and insect immunity. Insect Immunology (ed. N.E. Beckage), pp. 243–270. Academic Press, Our study suggests that also oenocytoids may be Oxford, UK. involved in the immune response, at least in G. tenella. Burke, G.R. & Strand, M.R. (2014) Systematic analysis of a wasp – The level of oenocytoids was much higher in G. tenella parasitism arsenal. Molecular Ecology, 23, 890 901. Dahlman, D.L. (1991) Teratocytes and host/parasitoid interactions. infected by parasitoids deriving from G. calmariensis, Biological Control, 1, 118–126. towards which the strongest response was seen, suggesting Dubuffet, A., Dupas, S., Frey, F., Drezen, J.M., Poirie, M. & Carton, Y. an active role in the immune defence. We propose that (2007) Genetic interactions between the parasitoid wasp Leptopilina boulardi and its Drosophila hosts. Heredity, 98,21–27. oenocytoids contribute to the capsule melanization in Dupas, S. & Boscaro, R. (1999) Geographic variation and evolution of Galerucella since they are known to be involved in the immunosuppressive genes in a Drosophila parasitoid. Ecography, 22, – melanization process in other species (Jiang et al. 1997; 284 291. Firlej, A., Lucas, E., Coderre, D. & Boivin, G. (2007) Teratocytes growth Shrestha & Kim 2008). In infected individuals of pattern reflects host suitability in a host-parasitoid assemblage. Physio- G. tenella, we also observed a type of multinucleated cells logical Entomology, 32, 181–187. € which we had not previously seen in Galerucella.We Fors, L., Markus, R., Theopold, U. & Hamback, P.A. (2014) Differences in cellular immune competence explain parasitoid resistance for two believe these cells to be similar to the multinucleated giant coleopteran species. PLoS ONE, 9, e108795. hemocytes described recently in Drosophila, where they Fors, L., Markus, R., Theopold, U., Ericson, L. & Hamback,€ P.A. have a function in parasite elimination (Markus et al. (2016) Data from: Geographic variation and trade-offs in parasitoid virulence. Dryad Digital Repository, http://dx.doi.org/10.5061/ 2015). However, at this point no functional tests have dryad.7h554. been performed on the multinucleated cells in G. tenella Fytrou, A., Schofield, P.G., Kraaijeveld, A.R. & Hubbard, S.F. (2006) to prove their possible role in the immune response. Wolbachia infection suppresses both host defence and parasitoid counter-defence. Proceedings of the Royal Society B: Biological Sciences, 273, In conclusion, our studies reveal that parasitism success 791–796. in A. parviclava is affected by former parasitoid host spe- Grosberg, R.K. & Hart, M.W. (2000) Mate selection and the evolution of – cies and to some extent also the geographic origin of the highly polymorphic self/nonself recognition genes. Science, 289, 2111 2114. parasitoid, whereas we found no indications of geographic Hamback,€ P. (2004) Why purple loosestrife in sweet gale shrubs are less variation in host immune response. In accordance, the attacked by herbivorous beetles? Entomol Tidskr, 125,93–102. € cytological studies showed that the induction of certain Hamback, P.A. (2010) Density-dependent processes in leaf beetles feeding on purple loosestrife: aggregative behaviour affecting individual growth cell types important for the encapsulation process, in par- rates. Bulletin of Entomological Research, 100, 605–611. ticular lamellocytes, is suppressed when individuals are Hamback,€ P.A., Stenberg, J.A. & Ericson, L. (2006) Asymmetric indirect infected by a highly virulent parasitoid. Our findings sug- interactions mediated by a shared parasitoid: connecting species traits and local distribution patterns for two chrysomelid beetles. Oecologia, gest ongoing evolution in both parasitoid virulence and 148, 475–481. host immune response in this system. Given these pat- Hamback,€ P.A., Weingartner, E., Ericson, L., Fors, L., Cassel-Lundhagen, terns, we believe that the Asecodes–Galerucella system is a A., Stenberg, J.A. et al. (2013) Bayesian species delimitation reveals generalist and specialist parasitic wasps on Galerucella beetles useful model system for understanding the evolutionary (Chrysomelidae): sorting by herbivore or plant host. BMC Evolutionary processes underlying host race formation and speciation Biology, 13, 92. € in host–parasitoid systems. To elucidate these patterns, a Hansson, C. & Hamback, P.A. (2013) Three cryptic species in Asecodes (Forster) (Hymenoptera, Eulophidae) parasitizing larvae of Galerucella better understanding both on the phylogeographic pat- spp. (Coleoptera, Chrysomelidae), including a new species. J Hymenopt terns underlying the current distribution of virulence and Res, 30,51–64.

Holm, S. (1979) A simple sequentially rejective multiple test procedure. Thompson, J.N. (2005) The Geographic Mosaic of Coevolution. The Scandinavian Journal of Statistics, 6,65–70. University of Chicago Press, Chicago, IL, USA. Honti, V., Csordas, G., Kurucz, E., Markus, R. & Ando, I. (2014) The Van Veen, F.J.F., Mueller, C.B., Pell, J.K. & Godfray, H.C.J. (2008) Food cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, web structure of three guilds of natural enemies: predators, parasitoids immune compartments, microanatomy and regulation. Developmental and pathogens of aphids. Journal of Animal Ecology, 77, 191–200. and Comparative Immunology, 42,47–56. Jiang, H.B., Wang, Y., Ma, C.C. & Kanost, M.R. (1997) Subunit compo- Received 5 February 2016; accepted 21 July 2016 sition of pro-phenol oxidase from Manduca sexta: molecular cloning of Handling Editor: Sheena Cotter 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 Supporting Information generalist parasitoid: contrasting transient and transgenerational costs associated with novel and original host species. Ecology and Evolution, Additional Supporting Information may be found in the online version – 5, 459 465. of this article. Kraaijeveld, A.R. & Godfray, H.C.J. (2009) Evolution of host resistance and parasitoid counter-resistance. Advances in Parasitology, Parasitoids Fig. S1. Absolute hemocyte numbers observed in G. pusilla larvae of Drosophila (ed. G. Prevost), pp. 257–280. Elsevier, London, UK. when infected by parasitoids deriving from G. pusilla, G. cal- Kraaijeveld, A.R., Nowee, B. & Najem, R.W. (1995) Adaptive variation in host-selection behavior of Asobara tabida, a parasitoid of Drosophila mariensis and G. tenella. larvae. Functional Ecology, 9, 113–118. Kraaijeveld, A.R. & van Alphen, J.J. (1994) Geographical variation in Fig. S2. Absolute hemocyte numbers observed in G. pusilla larvae resistance of the parasitoid A. tabida against encapsulation by D. me- when infected by parasitoids deriving from southern populations lanogaster larvae: the mechanism explored. Physiological Entomology, 19,9–14. of G. pusilla, G. calmariensis and G. tenella. Kraaijeveld, A.R. & van Alphen, J.J.M. (1995) Geographical variation in encapsulation ability of Drosophila melanogaster larvae and evidence for Fig. S3. Absolute hemocyte numbers observed in G. tenella larvae parasitoid-speciﬁc components. Evolutionary Ecology, 9,10–17. infected by parasitoids deriving from G. pusilla, G. calmariensis Lavine, M.D. & Strand, M.R. (2002) Insect hemocytes and their role in and G. tenella. immunity. Insect Biochemistry and Molecular Biology, 32, 1295–1309. Markus, R., Lerner, Z., Honti, V., Csordas, G., Zsamboki, J., Cinege, G. Fig. S4. Absolute hemocyte numbers observed in G. tenella larvae et al. (2015) Multinucleated giant hemocytes are effector cells in cell- from northern and southern localities, infected with parasitoids mediated immune responses of Drosophila. Journal of Innate Immunity, deriving from northern populations of G. calmariensis and 7, 340–353. G. tenella. 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 Fig. S5. Multinucleated cell structures in G. tenella larvae infected Statistical Computing. R Foundation for Statistical Computing, Vienna, with A. parviclava. Austria. Rizki, R.M. & Rizki, T.M. (1990) Encapsulation of parasitoid eggs in phenoloxidase-deﬁcient mutants of Drosophila melanogaster. Journal of Fig. S6. Back-transformed hemocyte levels ( SE) in G. tenella Insect Physiology, 36, 523–529. larvae from northern and southern localities. Rolff, J. & Siva-Jothy, M.T. (2003) Invertebrate ecological immunology. Science, 301, 472–475. Table S1. All possible combinations of Galerucella spp. larvae Schmidt, O., Theopold, U. & Strand, M. (2001) Innate immunity and its and Asecodes parasitoids deriving from the different host species, evasion and suppression by hymenopteran endoparasitoids. BioEssays, 23, 344–351. divided into northern and southern populations. Schulenburg, H., Kurtz, J., Moret, Y. & Siva-Jothy, M.T. (2009) Ecologi- cal immunology. Philosophical Transactions of the Royal Society of Lon- Table S2. Output from analyses of variance on cell composition – don. Series B, Biological sciences, 364,3 14. [F-value (P-value)] for G. pusilla and G. tenella larvae, infected Shrestha, S. & Kim, Y. (2008) Eicosanoids mediate prophenoloxidase release from oenocytoids in the beet armyworm Spodoptera exigua. with parasitoids deriving from either G. pusilla, G. tenella or Insect Biochemistry and Molecular Biology, 38,99–112. G. calmariensis. Stenberg, J.A., Heijari, J., Holopainen, J.K. & Ericson, L. (2007) Presence of Lythrum salicaria enhances the bodyguard effects of the parasitoid Table S3. Output from analyses of variance on cell composition – Asecodes mento for Filipendula ulmaria. Oikos, 116, 482 490. [F-value (P-value)] for G. tenella larvae of two regional origins Strand, M.R. (2010) Polydnaviruses. Insect Virology (eds S. Asgari & K.N. Johnson), pp. 171–197. Caister Academic Press, Norwich, UK. and infected with parasitoids deriving from either G. tenella or Thompson, J.N. (1994) The Coevolutionary Process. The University of G. calmariensis. Chicago Press, Chicago, IL, USA.