Chemical Defence in a Sawfly

Chemical defence in a sawﬂy: genetic components of variation in relevant life-history traits

CMu¨ ller1, BJ Zwaan, H de Vos and PM Brakeﬁeld Institute of Biology, Leiden University, PO Box 9516, NL-2300 RA Leiden, The Netherlands

Larvae of several tenthredinid sawfly species readily release negative phenotypic correlation was found between the two droplets of haemolymph through their integument when traits directly related to the defence mechanism: integument attacked by predators. This defence mechanism via ‘bleed- resistance and haemolymph deterrence. However, the ing’ is characterised by a low integument resistance and a significant heritabilities found for these traits in the full-sib high haemolymph deterrence. Both traits are variable, and analysis (0.39 and 0.35, respectively, for males in the Swiss negatively correlated among species. We sought to deter- population) show that the variation has a genetic component. mine if such differences in the propensity to bleed also occur While full-sib analysis revealed highly significant heritabilities intraspecifically by studying the heritability of traits potentially for most traits in all the three populations, parent–offspring associated with the bleeding phenomenon in the turnip regression revealed little or no evidence of heritable sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: variation. Effects of common environment for siblings and Tenthredinidae, Allantinae). For three European populations, variation in the host-plant quality between insect generations heritabilities were estimated in the laboratory in a parent– are likely to be the main factors explaining these differences. offspring and a full-sib design for haemolymph deterrence A consequence of such host-plant variation in the wild might (measured as concentration of sequestered glucosinolate), be that genetic variation of such chemical defensive traits is integument resistance, body mass of eonymph and adult, largely invisible to natural selection. and developmental time. Within A. rosae, no significant Heredity (2003) 90, 468–475. doi:10.1038/sj.hdy.6800265

Keywords: chemical defence; heritability; life history; quantitative genetics; sawﬂy

Introduction example, pointed mandibles of a predator, their integument will disrupt readily at this spot and a droplet of Although traits related to chemical defence mechanisms haemolymph is released. This droplet has been shown to of insect herbivores are directly subjected to natural have deterrent effects on predators such as birds and selection, their genetic architecture is poorly understood. ants (Ohara et al, 1993; Schaffner et al, 1994; Mu¨ ller et al, A few studies have measured genetic and phenotypic 2002). The deterrent component is often derived from variation of chemical defences, for example, in species of bioactive host-plant metabolites that are sequestered in beetles (eg Eggenberger and Rowell-Rahier, 1992; Hollo- the haemolymph of the sawflies (Schaffner et al, 1994; way et al, 1993) or butterflies (Camara, 1997). Two Mu¨ ller et al, 2001; Boeve´ and Schaffner, 2003). Two arguments might explain the lack of more investigations quantitative traits characterise this bleeding phenomen- of this type. Firstly, as the chemistry of the insect is often on, namely the resistance of the integument and the very closely linked to that of the host plant, chemical haemolymph deterrence. These traits vary in different variation in the latter might overshadow genetic varia- tenthredinid species and have been shown to be tion in the former. Secondly, the effort required to negatively correlated across 22 species (Boeve´ and measure chemical variation qua time and technique is Schaffner, 2003). A less-resistant integument results in a usually higher than for measurements of morphological more rapid contact between the exuding haemolymph of or developmental traits, thus tending to lead to more the prey and an attacking predator, whereby this limited sample sizes. Nevertheless, the intriguingly high haemolymph apparently needs to be more toxic to deter diversity of chemical defence mechanisms utilised in a predator efficiently. sawflies through self-synthesised or sequestered toxins As tenthredinid species differ in their propensity to (Codella and Raffa, 1993) offers an interesting field for ‘bleed’, we sought to determine whether variation in investigations. Several species of the tenthredinid saw- traits associated with this defence mechanism occurs flies exhibit a so-called ‘easy bleeding’ defence (Boeve´ intraspecifically and whether it is heritable. Such and Schaffner, 2003); when the larvae are touched by, for quantitative genetic variation could then be the basis for macroevolutionary patterns (Schluter, 1996; Merila¨ and Bjo¨rklund, 1999). The Turnip sawfly, Athalia rosae Correspondence: Caroline Mu¨ller, Institute of Biology, Leiden University, ruficornis Jakovlev (Hymenoptera: Tenthredinidae, Al- PO Box 9516, NL-2300 RA Leiden, The Netherlands. lantinae), was chosen for our study. A. rosae sequesters E-mail: [email protected] 1Current address: University of Wu¨rzburg, Institute of Botany II, Julius- glucosinolates of its cruciferous host plants into von-Sachs-Platz 3, D-97082 Wu¨rzburg, Germany. the haemolymph (Mu¨ ller et al, 2001). A deterrent Received 6 August 2002; accepted 10 January 2003 effect on the ant species Myrmica rubra (Hymenoptera: Genetic variation of a chemical defence CMu¨ller et al 469 Formicidae) is at least partly caused by this plant were collected from the resulting wound, weighed in secondary metabolite (Mu¨ller et al, 2002). A. rosae is a Eppendorf caps (1.5 ml) and frozen at À801C for later haplodiploid insect and thus, unfertilised eggs develop analysis of glucosinolate concentration (see below). into haploid males while fertilised eggs produce diploid When larvae reached their eonymph stage, they were females. An advantage of haplodiploids for studies of weighed and placed in plastic cups (diameter: 5 cm, heritability is that there is no effect of dominance in the height: 6.5 cm) filled to 2 cm height with a mixture of production of male offspring, and thus only additive sieved sand and soil in which they formed cocoons to genetic variance is estimated in sib analysis of brothers pupate. After 1 week, cocoons were carefully removed (Liu and Smith, 2000). Parent–offspring and sib analyses and placed in Petri dishes lined with moistened filter were carried out in the laboratory for potential traits paper. Adult mass was measured on the day of related to the defence mechanism, and the phenotypic and emergence. Developmental times both from egg to genetic correlations between all the traits were examined. eonymph and from eonymph to adult were recorded. Sawflies from three different populations were studied to P-adults were set up in a half-sib design. Males from a attempt to obtain a wider range of variation. range of families were each mated to two to three individual females from different families. Matings were Material and methods made assortatively based on high (43 g) and low (o3g) integument resistance values to increase the precision of the heritability estimates (Falconer and Mackay, 1996). Plants Mated females, as well as individual virgin females, were Sinapis alba Plants of L. (cultivar: Salvo, seeds from allowed to oviposit on individual S. alba shoots for 1 day, Advanta Seeds B.V., The Netherlands) were grown in a 1 3–4 days after emergence. After 14 days, seven to 10 F1 greenhouse (20 C, 70% relative humidity, L16:D8). larvae per family were taken randomly and placed Preflowering plants at 4–5 weeks were used. individually into Petri dishes. Quantitative traits were measured sequentially as in the parent generation. As the Insects number of female offspring in the F1 was very low, an A. rosae were collected as fertilised adult females while additional generation of mated females from the French nectaring on Dipsacaceae at Autreppes, France (F). and Swiss populations was set up for oviposition, and Larvae were collected from mustard fields both at the same traits measured in the F2. Dele´mont, Switzerland (CH) and Mu¨ hlheim, Germany (D). Adults were provided with honeywater-soaked cottonwool. Larvae were reared on shoots or leaves of Measurement of integument resistance S. alba. Eonymphs (the final, nonfeeding larval instar) An advanced force gauge (Mecmism, AFG 2.5 N) with an were offered a mixture of sand and soil for pupation. iron needle (diameter: 0.2 mm; tip cut straight, but with a Offspring of individual females from France were fine rounded border) was attached to a micromanipu- reared as separate families. In the next generation, lator. A larva was placed laterally in a curled position crosses of females were made to males of unrelated under a gauze frame (3.5 cm diameter, mesh width: families of the same population to minimise inbreeding. 1.6 mm). The force gauge was moved perpendicular at The resulting adults and those that emerged from the 0.1 mm/s towards the larva, until the needle disrupted larvae collected at Switzerland and Germany were used the integument. The maximum force applied to the larva as the grandparent (GP) generation for the heritability was recorded from a digital display. studies in which all the three populations were analysed To obtain information about the reliability of the data, separately. Insects were reared at 221C, 70% relative four groups of 50 larvae from our stock were measured humidity, and L16:D8 in the Leiden laboratory. Plant repeatedly. The first measurement was taken in all the material for all families of one generation was of the larvae when they had reached the second day of the final same age, and shoots or leaves were picked at random feeding instar. A second measurement was taken at the from plants. opposite lateral body side of the same individuals either after 0, 3, 6, or 18 h. The maximum force was always Heritability studies measured left to the centre of the larva (in the first measurement the larva’s head was placed on the left In the GP generation, one-half of the females was not side, in the second measurement on the right side, or the mated to produce male offspring. The other half were other way round in half of the larvae measured per mated individually to males, by placing couples together treatment). in Petri dishes for 1 day. Virgin and mated females were allowed to oviposit for 24 h on individual shoots of S. alba at 3–4 days after emergence from pupa when the Analysis of glucosinolate concentration number of mature eggs reaches a maximum in females Haemolymph samples were lyophilised. Glucosinolates (Sawa and Oishi, 1989). After 14 days of common were extracted in 70% boiling methanol after adding development on the shoot, larval offspring (parent- 20 ml of benzylglucosinolate as internal standard. Gluco- generation P) were counted and five to 10 larvae of each sinolate analysis was elaborated by conversion to family placed individually in Petri dishes lined with desulfoglucosinolates as described elsewhere (Agerbirk moist filter paper. Larvae were provided daily with et al, 2001). For analysis of desulfoglucosinolates, a high- freshly picked S. alba leaves. performance liquid chromatograph coupled to a photo- Integument resistance was measured (see below) on diode array UV detector (Waters 990) was equipped with the second day of the final feeding larval instar (males an ODS 2 column (Alltech: Allsphere, length 150 mm, ID 4th, females 5th instar). For larvae of the Swiss P and 4.6 mm). Methanol (15%) in water was used as solvent, at male F1-generation, haemolymph samples of about 2 ml a flow rate of 1 ml/min. Peaks were quantified by the

Heredity Genetic variation of a chemical defence CMu¨ller et al 470 peak area at 226 nm (bandwidth 4 nm) relative to the area Results of the internal standard peak. The relative response factors reported by McGregor (1985), as cited by Buchner Repeatability of integument measurement data (1987), were used for sinalbin (p-hydroxybenzylglucosi- The repeatability of measurements of the integument nolate) (0.50), and benzylglucosinolate (0.86). Identifica- resistance was 89% after 0 h (Po0.001, n ¼ 48), 62% after tion of glucosinolates in haemolymph samples of A. rosae 3h(P ¼ 0.01, n ¼ 49), and 96% after 6 h (Po0.001, n ¼ 50). had been confirmed previously (Mu¨ ller et al, 2001). In These values indicate that most of the variance was several samples, trace amounts of two unidentified peaks among individuals rather than within individuals, and were found, one possibly 1-methylpropylglucosinolate that, therefore, both measurement accuracy (Becker, (Mu¨ ller et al, 2001) and the other with a close retention 1992) and potential heritability was high. When the time and similar spectrum to sinalbin. For both, a second measurement was made on the following day response factor of 1 was assumed. Glucosinolate con- after 18 h, data were not repeatable (r ¼À0.11, P ¼ 0.12, centration was expressed as total glucosinolate concen- n ¼ 50) indicating the necessity of careful standardisation tration in mmol/g fresh weight, summing the of timing of integument measurements with respect to concentrations of sinalbin and the two unknown gluco- developmental stage. sinolates (concentration of the latter always o1 mmol/g fresh weight). Heritability estimates – male offspring Mother–son regressions revealed no statistically signifi- Allozyme analysis cant heritabilities for the traits glucosinolate concentration, To compare the populations, male and female adults of integument resistance, eonymph mass, and adult mass in the the P-generation were frozen and analysed for allozyme three populations (glucosinolate concentration only polymorphism using electrophoresis on cellulose–acetate tested for the Swiss population) (Table 1). The lack of gels. Staining and buffer recipes were taken from significance was partly due to high standard errors (eg in Richardson et al (1986) and Hebert and Beaton (1989). two traits of the French population) and partly because 2 Allozyme data were analysed using GDA, version D12 the estimates of h were close to zero (eg all traits in the (Lewis and Zaykin, 1999). Swiss population, Table 1). The developmental time from egg to eonymph and eonymph to adult was significantly heritable only in the German population. The sole trait Statistical analysis with no significant differences in phenotype between Repeatability of integument resistance measurements populations was integument resistance. For all other traits, was calculated using one-way analysis of variance there were significant effects of populations (Po0.01, (ANOVA) (Lessels and Boag, 1987). Heritabilities (h2) ANOVA): for eonymph mass, differences in phenotype were estimated using offspring-on-parent regression were significant between the French and Swiss, and analysis (Falconer and Mackay, 1996) adapted to haplo- Swiss and German populations (Po0.001, Scheffe´-test); diploid insects by assuming gene dosage compensation for adult mass, differences were significant between the in the haploid males (Liu and Smith, 2000). Sexes were French and Swiss (Po0.001), and French and German analysed in different ways: h2 ¼ b Â 2(b: regression populations (P ¼ 0.04); for developmental times, differ- coefficient) for mother–son regression and h2 ¼ b for ences were significant between the Swiss and German mid-parent–daughter regression. Linear regressions of populations (P ¼ 0.03), and for time from eonymph to parents to mean values of offspring were weighted for adult additionally between the French and German family size. populations (Po0.001). Sib analysis was carried out by ANOVA with restricted In contrast to mother–son regressions, full-sib analysis maximum likelihood estimation and with ‘family’ as a of sons revealed highly significant heritabilities in all random factor. Heritabilities were estimated for male sibs traits and populations, except body masses of eonymphs as h2 ¼ 2 Â Var(family)/[Var(family)+Var(error)] and for and adults which were only significantly heritable in the female sibs as h2 ¼ 4/3 Â Var(family)/[Var(family)+Var Swiss population. Mean heritabilites differed between (error)]. Several P females had probably not mated populations but the ranges were overlapping (Table 1). successfully as they only produced sons. Thus, data did not allow the partition of variance into components due Heritability estimates – female offspring to sire and dam nested within sires for female offspring. Parent–offspring regressions for female offspring were Heritability estimates of the trait integument resistance examined over two generations due to a lack of a were corrected for assortative mating by the formula sufficient number of female offsprings in one generation. 2 2 h ¼ [À1+O(1+4rh0)]/2r (where r is the correlation be- There was a significant effect of generation for the 2 tween the father and mother value and h0 is the phenotypic values of all traits measured (Po0.05, uncorrected h2 estimate) (Falconer and Mackay, 1996). ANOVA; there were significant differences between F1 ANOVA was used to test for effects (phenotypic and F2 for all traits). Furthermore, only three females of differences) of population and generation. Data were the German population produced female offspring and only pooled when there were no significant effects in the thus, data from this population for this sex could not be latter analysis. analysed. Phenotypic correlations between different traits were Mid-parent–daughter regression revealed a statisti- estimated by Pearson product–moment correlation of cally significant heritability only for one trait, integument individual measurements. For genetic correlations, resistance, in one generation (F2) of the French population means of offspring per family were weighted and (Table 2). On the other hand, once again, full-sib analysis analysed by Pearson product–moment correlation. of daughters revealed significant heritabilities for all

Heredity Genetic variation of a chemical defence CMu¨ller et al 471 Table 1 Narrow-sense heritability estimates for male A. rosae of three populations from France (F), Switzerland (CH), and Germany (D), calculated by parent–offspring regression (linear regression) of quantitative traits between mothers and mean value of sons, weighted for number offspring per family, and by full-sib analysis (ANOVA)

Trait Pop. Mean7SD Mean7SD Ind. (n) Fam. (n) h27SE parent-offspring h27SE full-sib mothers sons

Gls. Conc. CH 19.2378.51 13.7177.22 186 41 À0.00170.16 n.s. 0.3570.19***

Integument F 3.0970.77 2.5870.45 90 19 0.3470.27 n.s. 0.4470.29** resistance CH 2.9270.53 2.6170.44 260 41 0.0970.26 n.s. 0.3970.17*** (g) D 2.8070.88 2.8470.50 72 9 0.4770.39 n.s. 0.5070.52** F+CH+D 2.9370.66 2.6470.46 422 69 0.2470.17 n.s. 0.4070.14***

Mass F 49.9273.70 30.9371.50 89 19 À0.0870.20 n.s. 0.1370.19 n.s. eonymph CH 49.3475.82 28.5372.62 256 41 0.0370.14 n.s. 0.7570.24*** (mg) D 46.6173.21 31.7171.00 70 9 0.0170.24 n.s. 0.1170.18 n.s.

Mass F 22.1372.58 11.1470.75 88 19 À0.005 70.14 n.s. 0.2270.22 n.s. adult CH 21.8072.36 10.1871.01 253 41 0.00370.14 n.s. 0.6970.22*** (mg) D 21.0870.85 10.3770.85 58 9 À0.1470.43 n.s. 0.9770.88 n.s.

Dev. F 18.7570.68 17.9570.80 89 19 0.3470.56 n.s. 1.3370.62*** egg–eon. CH 19.4770.79 18.1171.53 256 41 0.0770.62 n.s. 1.6070.40*** (d) D 18.7070.42 17.4470.86 70 9 3.9770.41*** 1.7070.96***

Dev. F 14.1570.52 16.3570.81 88 19 1.25 70.70 n.s. 1.2070.61*** eon.Àadult CH 15.4270.72 15.8070.53 246 41 À0.2470.23 n.s. 0.6670.21*** (d) D 14.1470.35 15.2471.68 58 9 7.2772.39* 0.9070.57***

Significant differences from zero: *Po0.05; **Po0.01; ***Po0.001; n.s. not significant. Statistically significant heritability estimates are highlighted in bold. ind.=individuals; fam. = families; gls-glucosinolate, conc. = concentration (mmol/g fresh weight); eon. = eonymph; dev. = developmental time. Differences in phenotype between populations were examined by ANOVA. The only trait without a significant population effect is the integument resistance (P = 0.058). For all other traits Po0.01.

Table 2 Broad-sense heritability estimates for female A. rosae of two populations from France (F), and Switzerland (CH) for two generations (F1, F2), calculated by mid-parent–offspring regression (linear regression) of quantitative traits between mean value of parents and mean value of daughters, weighted for number offspring per family, and by full-sib analysis (analysis of variance)

Trait Pop./gen. Mean7SD Mean7SD Ind. Fam. h27SE h27SE mid-parents daughters (n) (n) parent-offspring full-sib

Integument F F1 2.6670.76 2.6370.82 59 12 0.1770.27 n.s. 0.4370.28** resistance F F2 2.7071.16 3.1270.61 61 12 0.3370.07*** 0.4870.31*** (g) CH F1 2.8170.53 2.4470.64 49 10 À0.0470.30 n.s. 0.4370.41** CH F2 2.4070.90 3.0370.64 81 13 À0.00670.07 n.s. 0 n.s.

Mass F F1 40.9972.59 51.99573.996 59 12 0.4770.32 n.s. 0.3670.26** eonymph F F2 40.2272.35 51.7975.12 61 12 0.0770.37 n.s. 0.1770.17 n.s. (mg) CH F1 38.5172.34 45.9574.72 48 10 0.2170.40 n.s. 0.2470.30 n.s. CH F2 37.4373.25 49.6776.79 81 13 0.0270.26 n.s. 0.04 70.10 n.s.

Mass F F1 17.1672.17 21.5571.58 55 12 0.0570.16 n.s. 0.3270.27* adult F F2 15.9871.25 22.1872.35 60 12 0.3770.34 n.s. 0.2270.20* (mg) CH F1 16.6071.80 19.8571.67 45 10 À0.0270.16 n.s. 0 n.s. CH F2 14.9771.02 21.5073.03 81 13 0.5570.33 n.s. 0.0770.12 n.s.

Dev. F F1 17.7570.45 19.0571.65 59 12 À0.7470.12 n.s. 1.1370.55*** eggÀeon. F F2 18.3370.91 16.6270.93 61 12 À0.1470.26 n.s. 0.8370.44*** (d) CH F1 18.6570.97 19.2571.18 48 10 À0.1270.34 n.s. 0.9570.57*** CH F2 18.2371.09 16.3371.26 81 13 0.1170.30 n.s. 1.0670.49***

Dev. F F1 14.0070.83 15.2570.97 55 12 0.3270.31 n.s. 0.8970.48*** eon.Àadult F F2 15.5870.70 14.7771.00 60 12 0.3070.37 n.s. 0.6670.39*** (d) CH F1 15.8570.47 14.2871.03 46 10 À0.2870.66 n.s. 0.7870.50*** CH F2 15.1970.56 14.5870.72 81 13 0.0970.27 n.s. 0.5670.30***

Heredity Genetic variation of a chemical defence CMu¨ller et al 472 Table 3 Phenotypic (P) and genetic (G) correlations (Pearson product–moment correlation) between quantitative traits, measured in males (F2) of the Swiss population of A. rosae

Trait Integument resistance Glucosinolate conc. (Gc) Mass eonymph (Me) Mass adult (Ma) Dev. Egg-eon. (De)

PG P G PGPGPG

Gc À0.13N.S. 0.02N.S.

Me À0.03N.S. 0.01N.S. 0.15* 0.37***

Ma À0.04N.S. À0.09N.S. 0.12N.S. 0.25*** 0.71*** 0.77***

De 0.16* 0.37*** 0.13N.S. 0.14N.S. 0.01N.S. 0.04N.S. À0.21** À0.29***

Da À0.06N.S. À0.13N.S. À0.16* À0.31*** À0.04N.S. À0.17N.S. À0.20N.S. À0.07N.S. 0.02N.S. 0.12N.S.

Significant differences from zero: *Po0.05; **Po0.01; ***Po0.001; n.s. not significant. Statistically significant correlations are highlighted in bold. conc. = concentration; dev. = developmental time; eon. = eonymph; Da: dev. eon.-adult.

traits measured in the French population in both analysing full-sib data but not when regressing offspring generations except the eonymph mass of the F2. In the on parents. Full-sib analysis often overestimates herit- Swiss population, integument resistance was heritable in ability compared to parent–offspring regression, because one generation (F1) and developmental times in both of both dominance variance and common environments generations (Table 2). for sibs. However, as highly significant values were also obtained for male full-sibs, where no dominance var- Correlations among different traits iance is present, the common environment issue is more Significantly positive phenotypic correlations between important. Such common environment conditions result the mass of the eonymphs and of the adults were found in a small within-family variation and thus heritabilities in all the generations and populations (r40.5, Po 0.01). for the full-sib-analysis are inflated as within-family A detailed overview of the correlations is given for the variation forms the denominator in the estimates. male offspring of the Swiss population (Table 3 and Detailed explanations for possible causes of the differ- Figure 1) since this population is also representative of ences between heritability estimates in A. rosae are given the other two. Glucosinolate concentration showed a in the following discussion of the individual traits. negative phenotypic relation with integument resistance The chemical trait of glucosinolate concentration in the although the correlation did not reach significance. haemolymph of A. rosae showed significant genetic 2 7 Furthermore, this chemical trait was positively correlated variation (h ¼ 0.35 0.19) when analysing full-sib data with the eonymph mass, but negatively with develop- (Table 1). The glucosinolate concentration serves as an mental time from eonymph to adult (Figure 1), although indicator for the deterrence of the haemolymph towards again these correlations were rather low and levels of predators such as ants (Mu¨ ller et al, 2002). In the leaf significance weak. The genetic correlations were of the beetle Oreina gloriosa (Coleoptera: Chrysomelidae), the same sign but stronger than the phenotypic correlations concentration of defensive compounds secreted by in several cases, for example, those between glucosino- adults was significantly heritable in estimates of full-sib late concentration and either body mass or develop- analysis (h2 ¼ 0.57), and of mother–offspring regression mental time (Table 3). However, there was no indication (h2 ¼ 0.51) (Eggenberger and Rowell-Rahier, 1992). The of any genetic correlation between the two defensive defensive secretion of O. gloriosa contains a complex traits. mixture of cardenolides and ethanolamines that are not sequestered but synthesised de novo (Rowell-Rahier et al, Allozyme analysis 1995) and thus are unlikely to be dependent on host- plant quality. In contrast, for A. rosae the host-plant The FST values for males and females of the investigated populations are low and indicate ‘little’ genetic differ- quality is crucial for the concentration of the defence entiation (according to the classification of Hartl and compound in the haemolymph. Analysis of experiments Clark, 1997) (Table 4). involving switching larvae to host plants with other glucosinolate profiles during the last feeding of larval instar revealed that the glucosinolate composition of the Discussion larval haemolymph corresponds to that of the new leaf in a very short time: after 20 h it closely resembles the new This study is the first to examine the quantitative host (Mu¨ ller, unpublished). In the full-sib experiment, genetics of an insect chemical defence mechanism that we minimised the effects of common environment for involves several traits, namely the concentration of a families by placing larvae individually at least 2 days deterrent compound and an integument adaptation. before sampling of haemolymph on randomised leaves Although because of the fact that some females produced in Petri dishes. After this time, the glucosinolate no or only male offspring, sample sizes are lower than concentration of the haemolymph will be more influ- we had planned, some clear results were obtained. Most enced by the quality of that particular leaf than the heritability estimates for both defence and life-history previous food supply. Furthermore, it might be affected traits in the sawfly A. rosae were significant when by the ability of the larva to concentrate glucosinolates

Heredity Genetic variation of a chemical defence CMu¨ller et al 473 a Table 4 FST-values for A. rosae populations from Switzerland (CH), France (F) and Germany (D) obtained by allozyme electrophoresis ] 5 Sex overall CH/F CH/D F/D n Total

Male 0.03 0.07 0.01 0.05 49 Female — 0.05 N.D. N.D. 94

Analysis of nine different loci from the enzymes (malate dehydrogenase, MDH; 1.1.1.37), (malic enzyme, ME; 1.1.1.40), (isocitrate dehydrogenase, IDH; 1.1.1.42), (aspartate aminotransferase, AAT; 2.6.1.1), (b-esterase, b-est.; 3.1.1.1), (glucose-6-phosphate isomerase, GPI; 5.3.1.9), and (phosphoglucomutase, PGM; 5.4.2.2) (nomencla-

Integument resistance [g ture following Murphy et al, 1996, EC numbers given). Presence of 0 private alleles: two in Swiss, and two in French females. No linkage 010203040 disequilibrium was detected. N.D., not detected. b 40 moderate heritability estimates of about 0.4 (0.38–0.50). 35 The similarity between female and male heritability estimates suggests that dominance variance is of minor inﬂuence on this trait (brother–brother analysis estimates 30

mph [mg] narrow-sense heritability only). This may in turn indicate that selection on this particular trait is low and may be 25 over-riden by environmental variance (Crnokrak and Roff, 1995). The integument resistance measurements on living larvae are a sum of the integument strength and

Mass eony 20 the hydraulic pressure of the haemolymph. Hydration has a marked effect on the integument properties 15 (Hepburne and Joffe, 1976) which is dependent on 010203040 environmental conditions such as humidity and host- c plant quality. Variation in these factors during the 20 experiment might again account for the absence of 19 significant correlations between measurements from different generations. For the integument resistance, the 18 timing of measurement is critical and closely linked to 17 the moulting cycle of the larvae. Our measurement in the mid-phase of the last feeding larval instar is highly 16 repeatable, while measurements closer to the end of the 15 phase are no longer comparable probably because the mph-adult [d] new integument of the eonymph is already formed 14 under the larval cuticula. To our knowledge, heritabil- eony Developmental time Developmental 13 ities of integument resistance have not been estimated for other insects. 12 The life-history traits of body mass of eonymphs and of 010203040 adults were only significantly heritable in some popula- Glucosinolate concentration tions/generations. Where significant, heritabilities varied [mol / g fresh weight] between 0.2270.20 (Table 2) and 0.7570.24 (Table 1). In other haplodiploid sawflies, heritabilities for body mass Figure 1 Regression of different traits on the glucosinolate estimated by half-sib analysis were comparable [eg 0.38, concentration (as measurement for deterrence) in males of the 7 Swiss population of A rosae. For r- and P-values refer to the SE not given, Sequeira and Mackauer, 1992; 0.55 0.15, phenotypic correlations in Table 3. Kause et al, 2001]. For developmental time, very high values for heritabilites were found for full-sib analysis of all A. rosae populations and generations. However, some up to a certain threshold level. Although our S. alba caution is needed in interpreting our estimates because plants were all grown from the same seed source and they have rather high and overlapping standard errors. under the same environmental conditions, their glucosi- Furthermore, those for developmental time are likely to nolate content and overall quality may have differed be even more sensitive to common environment effects, between batches used for subsequent generations, as it especially in early development where larvae shared does in many Brassicaceae due to seasonal variation and environments. A high variation of estimated heritabilities induction events (Rosa, 1997; Agrawal et al, 1999; for morphological and life-history traits is typical for Agerbirk et al, 2001). This could explain the observed many studies including other sawflies (eg Chenot and lack of correlation between haemolymph glucosinolate Raffa, 1998). Furthermore, for several tree feeding sawfly concentrations for parents and their offspring in A. rosae. species, it has been shown that plant chemistry can have For integument resistance, the full-sib analysis revealed a strong effect, particularly on developmental time (Geri for male and female siblings in all the three populations et al, 1993).

Heredity Genetic variation of a chemical defence CMu¨ller et al 474 Differences in heritabilities were found between the A. mental effects. These are likely to arise from the tight rosae populations, most strikingly in the trait body mass. coupling of the insect herbivore traits to host-plant Such differences could indicate different histories of quality and chemistry. In the field, sawfly larvae are selection, for example, due to regional climates and confronted with an even wider range of plant nutrients habitat diversity. A high variation in heritability esti- and plant secondary compounds than in our laboratory mates for body size of wild populations along a conditions. Our findings imply that genetic variation is latitudinal gradient has been demonstrated for Drosophila unlikely to be visible in the field as it will be readily (van ‘t Land et al, 1999). Differences between populations overshadowed by environmental variation. However, could also be because of different founder effects or there is clearly some potential for adaptive evolution in bottleneck events resulting in loss of genetic variation or A. rosae, for example, in response to progressive change inbreeding depression (eg Saccheri et al, 1996). Molecular in environmental conditions. Such genetic variation markers are a practical tool for investigating such effects within species must have been the basis for evolution (eg Brown et al, 1997; de Jong et al, 2001). Allozyme of phenotypic differences found across species. The analyses of A. rosae revealed little genetic variation apparent profound effects of a variable host plant on between the three populations (Table 4). Thus, the the chemical defence trait may have also led to differences found in heritability estimates might be maintenance of genetic variation in the sawfly popula- partly explained by differences in genetic history. tions for traits that are potentially closely related to However, molecular variability has only a limited ability fitness. to predict quantitative genetic variability (Reed and Frankham, 2001), making a different history of selection Acknowledgements the most likely reason for the differences between populations. We thank A Barker (Switzerland) for providing us with A high phenotypic and genetic correlation between the larvae of A. rosae, J Prijs and M Heijmans for technical body mass of eonymph and adult was consistently found assistance; N Wu¨ rzer and M Lavrijsen for the supply of in all the three A. rosae populations. However, all the mustard plants; A Kause (Finland), K Fischer (Leiden), other phenotypic correlations between the quantitative and J-L Boeve´ (Belgium) for useful improvements on the traits were weak (Table 3). Body mass and develop- manuscript. This research was supported by the Eur- mental time of larvae have been shown to be clearly opean Community’s Improving Human Potential Pro- positively correlated in a comparative study on six birch- gramme under contract HPRN-CT-1999-00054, feeding sawfly species when food was of stable high INCHECO. quality and constraints on long development were relaxed (Kause and Morin, 2001; Kause et al, 2001). References Seasonal variation of host-plant quality has a major influence on the phenotypic and genotypic architecture Agerbirk N, Olsen CE, Nielsen JK (2001). Seasonal variation in of this insect guild (Kause et al, 2001). The influence of leaf glucosinolates and insect resistance in two types of host-plant quality on the performance of A. rosae is the Barbarea vulgaris ssp. arcuata. Phytochemistry 58: 91–100. subject of further studies. Agrawal AA, Gorski PM, Tallamy DW (1999). Polymorphism in Within A. rosae, a high concentration of glucosinolates plant defense against herbivory: constitutive and induced in the haemolymph seems to be linked to a high resistance in Cucumis sativus. J Chem Ecol 25: 2285–2304. eonymph weight and a short developmental time from Becker WA (1992). Manual of Quantitative Genetics. Academic eonymph to the adult stage, as indicated by the Enterprises; Pullman, WA. Boeve´ J-L, Schaffner U (2003). Why does the larval integument respective signs for the phenotypic and genetic correla- of some sawfly species disrupt so easily? The harmful tions between these traits, Table 3, Figure 1). Thus, there hemolymph hypothesis. Oecologia 134: 104–111. is no obvious trade-off involved in sequestration of the Brown RJ, Malcolm CA, Mason PL, Nichols RA (1997). Genetic chemical defence compound. There was no significant differentiation between and within strains of the saw-toothed phenotypic correlation between the two defence traits grain beetle, Oryzaephilus surinamensis (Coleoptera: Silvani- integument resistance and haemolymph deterrence; dae) at RAPD loci. Insect Mol Biol 6: 285–289. however, the sign of this correlation was negative Buchner R (1987). Approach to determination of HPLC (Figure 1). A clearly significant negative correlation response factors for glucosinolates. In: Wathelet J-P (ed) between these traits has been demonstrated among Glucosinolates in Rapeseeds: Analytical Aspects. Martinus Nijh- off Publishers: Dordrecht. pp 50–58. tenthredinid species where haemolymph deterrence Camara MD (1997). Predator responses to sequestered plant was measured in bioassays with M. rubra (Boeve´ and toxins in buckeye caterpillars: Are tritrophic interactions Schaffner, 2003). The link between these traits is thought locally variable? J Chem Ecol 23: 2093–2106. to be functionally, rather than physiologically based, and Chenot AB, Raffa KF (1998). Heritability estimates of to have evolved under the selection pressure of preda- development time and size characters in the gypsy moth tion (Boeve´ and Schaffner, 2003). The correlation might (Lepidoptera: Lymantriidae) parasitoid Cotesia melanoscela represent an emergent property, becoming more obvious (Hymenoptera: Braconidae). Environ Entomol 27: 415–418. only when a wide range of differences in the traits are Codella SG, Raffa KF (1993). Defense strategies of folivorous considered, as has been reported recently for egg size in sawflies. In: Wagner MR, Raffa KF (eds) Sawfly Life History. relation to adult body size between and within butterfly Adaptations to Woody Plants. Academic Press: San Diego. pp 261–294. species (Fischer et al, 2002). Crnokrak P, Roff DA (1995). Dominance variance: associations Heritabilities in A. rosae both for life-history traits and with selection and fitness. Heredity 75: 530–540. defence traits of a chemical nature appeared to depend de Jong PW, de Vos H, Nielsen JK (2001). Demic structure and on the level of relatedness used to estimate them, its relation with the distribution of an adaptive trait in probably because of their high sensitivity to environ- Danish flea beetles. Mol Ecol 10: 1323–1332.

Heredity Genetic variation of a chemical defence CMu¨ller et al 475 Eggenberger F, Rowell-Rahier M (1992). Genetic component of lates in the defensive hemolymph of the sawfly Athalia variation in chemical defense of Oreina gloriosa (Coleoptera, rosae. J Chem Ecol 27: 2505–2516. Chrysomelidae). J Chem Ecol 18: 1375–1404. Mu¨ ller C, Boeve´ J-L, Brakefield PM (2002). Host plant derived Falconer DS, Mackay TFC (1996). Introduction to Quantitative feeding deterrence towards ants in the turnip sawfly Athalia Genetics, 4th edn. Pearson Education: Harlow. rosae. Entomol Exp Appl 104: 153–157. Fischer K, Zwaan BJ, Brakefield PM (2002). How does egg size Murphy Jr RW, Sites JW, Buth DG, Haufler CH (1996). Proteins: relate to body size in butterflies? Oecologia 131: 375–379. isozyme electrophoresis. In: Hillis DM, Moritz C, Mable BK Geri C, Allais JP, Auger M-A (1993). Effects of plant chemistry (eds) Molecular Systematics, 2nd edn. Simauer: Sunderland, and phenology on sawfly behavior and development. In: MA. pp 51–120. Wagner MR, Raffa KF (eds) Sawfly Life History. Adaptations to Ohara Y, Nagasaki K, Ohsaki N (1993). Warning coloration in Woody Plants. Academic Press: San Diego. pp 173–210. sawfly Athalia rosae larva and concealing coloration in Hartl DL, Clark AG (1997). Principles of Population Genetics. butterfly Pieris rapae larva on similar plants evolved through Sinauer Associates, Inc.: Sunderland, MA. individual selection. Res Popul Ecol (Kyoto) 19: 223–230. Hebert PDN, Beaton MJ (1989). Methodologies for Allozyme Reed DH, Frankham R (2001). How closely correlated are Analysis Using Cellulose Acetate Electrophoresis: A Practical molecular and quantitative measures of genetic variation? A Handbook. Helena laboratories: Texas. meta-analysis. Evolution 55: 1095–1103. Hepburn HR, Joffe I (1976). On the material properties of Richardson BJ, Baverstock PR, Adams M (1986). Allozyme insect exoskeletons. In: Hepburn HR (ed) The Insect Integu- electrophoresis. A Handbook For Animal Systematics and Popula- ment, Elsevier Scientific Publishing Company: Amsterdam. tion Studies. Academic Press: Sydney. pp 207–235. Rosa EAS (1997). Daily variation in glucosinolate concentrations Holloway GJ, de Jong PW, Ottenheim M (1993). The genetics in the leaves and roots of cabbage seedlings in two constant and cost of chemical defense in the two-spot ladybird (Adalia temperature regimes. J Sci Food Agric 73: 364–368. bipunctata L.). Evolution 47: 1229–1239. Rowell-Rahier M, Pasteels JM, Alonsomejia A, Brower LP Kause A, Morin J-P (2001). Seasonality and genetic architecture (1995). Relative unpalatability of leaf beetles with either of development time and body size of the birch feeding biosynthesized or sequestered chemical defense. Anim Behav sawfly Priophorus pallipes. Genet Res Camb 78: 31–40. 49: 709–714. Kause A, Saloniemi I, Morin JP, Haukioja E, Hanhimaki S, Saccheri IJ, Brakefield PM, Nichols RA (1996). Severe inbreed- Ruohomaki K (2001). Seasonally varying diet quality and the ing depression and rapid fitness rebound in the butterfly quantitative genetics of development time and body size in Bicyclus anynana (Satyridae). Evolution 50: 2000–2013. birch feeding insects. Evolution 55: 1992–2001. Sawa M, Oishi K (1989). Studies on the sawfly, Athalia rosae Lessels CM, Boag PT (1987). Unrepeatable repeatabilities: a (Insecta, Hymenoptera, Tenthredinidae) II. Experimental common mistake. Auk 104: 116–121. activation of mature unfertilized eggs. Zool Sci (Tokyo) 6: Lewis PO, Zaykin DZ (1999). Genetic Data Analysis: Computer 549–556. program for the analysis of allelic data. Version 1.0 (D12). Schaffner U, Boeve´ J-L, Gfeller H, Schlunegger UP (1994). [WWW Document]. Free program distributed by the authors Sequestration of Veratrum alkaloids by specialist Rhadino- over the internet from the GDA home page at URL http:// ceraea nodicornis Konow (Hymenoptera, Tenthredinidae) alleyn.eeb.uconn.edu/gda/. and its ecoethological implications. J Chem Ecol 20: 3233– Liu F-H, Smith SM (2000). Estimating quantitative genetic 2250. parameters in haplodiploid organisms. Heredity 85: 373–382. Schluter D (1996). Adaptive radiation along genetic lines of least McGregor DI (1985). Determination of glucosinolates in Brassica resistance. Evolution 50: 1766–1774. seed. EUCARPIA Cruciferae Newslett 10: 132–136. Sequeira R, Mackauer M (1992). Quantitative genetics of body Merila¨ J, Bjo¨rklund M (1999). Population divergence and size and developmental time in the parasitoid wasp Aphidius morphometric integration in the greenfinch (Carduelis chloris) ervi (Hymenoptera, Aphidiidae). Can J Zool 70: 1102–1108. – evolution against the trajectory of least resistance. J Evol van’t Land J, van Putten P, Zwaan BJ, Kamping A, van Delden Biol 12: 103–112. W (1999). Latitudinal variation in wild populations of Mu¨ ller C, Agerbirk N, Olsen CE, Boeve´ J-L, Schaffner U, Drosophila melanogaster: heritabilities and reaction norms. Brakefield PM (2001). Sequestration of host plant glucosino- J Evol Biol 12: 222–232.

Heredity