Development of Issoria Lathonia (Lepidoptera: Nymphalidae) on Zinc- Accumulating and Nonaccumulating Viola Species (Violaceae)

Environmental Toxicology and Chemistry, Vol. 26, No. 3, pp. 565–571, 2007 ᭧ 2007 SETAC Printed in the USA 0730-7268/07 $12.00 ϩ .00

DEVELOPMENT OF ISSORIA LATHONIA (LEPIDOPTERA: NYMPHALIDAE) ON ZINC- ACCUMULATING AND NONACCUMULATING VIOLA SPECIES (VIOLACEAE)

NAUSICAA NORET,*†‡ GUY JOSENS,§ JOSE´ ESCARRE´ ,‡ CLAUDE LEFE` BVRE,† STEVE PANICHELLI,§ and PIERRE MEERTS† †Universite´ Libre de Bruxelles, Laboratoire de Ge´ne´tique et d’Ecologie Ve´ge´tales, CP 320, 1850 chausse´e de Wavre, B-1160 Bruxelles, Belgium ‡Centre d’Ecologie Fonctionnelle et Evolutive (CNRS), 1919 Route de Mende, F-34293 Montpellier Cedex 05, France §Universite´ Libre de Bruxelles, Laboratoire d’Ecologie et de Syste´matique Animales CP 160/13, 50 Avenue F.D. Roosevelt, B-1050 Bruxelles, Belgium

(Received 14 August 2006; Accepted 10 October 2006)

Abstract—The larvae of Issoria lathonia L. feed in natural conditions on several Viola spp., among which are the zinc-accumulating Viola calaminaria (Gingins) Lej. and the nonmetal-accumulating Viola tricolor L. To examine how I. lathonia caterpillars cope with the naturally high foliar zinc concentration of V. calaminaria, we compared the growth of caterpillars reared on leaves varying in zinc concentration. Larvae were fed in controlled conditions with V. calaminaria and V. tricolor grown on noncontaminated soil (i.e., two low-Zn diets) and with V. calaminaria grown on zinc-enriched soil (i.e., one high-Zn diet). Larvae had a higher growth rate when fed with noncontaminated V. calaminaria compared to zinc-enriched V. calaminaria, suggesting that zinc slows down larval growth. However, larvae consumed more leaves of zinc-enriched V. calaminaria (ϩ45%; estimated from fecal mass) compared with noncontaminated V. calaminaria, suggesting that zinc accumulation would not be advantageous to plants. Caterpillars reared on high-zinc leaves regulate their internal zinc concentration through excretion of highly metal-concentrated feces. When kinetics of growth on both low-zinc diets were compared, it appeared that larval development was faster on noncontaminated V. calaminaria than on V. tricolor. This suggests that more nutrients or less feeding inhibitors in V. calaminaria account for fastest growth. Developmental rates on V. tricolor and on zinc-enriched V. calaminaria were similar, despite the high leaf zinc concentration of the latter species. Together with the abundance of V. calaminaria on calamine soils, this may explain why the largest populations of I. lathonia develop on V. calaminaria in Belgium.

Keywords—Metal tolerance Metal excretion Insect Host-plant relationship

INTRODUCTION Zinc is an essential trace element for terrestrial invertebrates [10]. However, high-zinc diets slow down the growth of Lep- Calamine soils, which occurred as natural outcrops and have largely been exploited by humans, contain high concen- idoptera larvae and often decrease pupal mass [10–15]. Metals trations of zinc, lead, and cadmium, which are toxic to most are known to disturb protein synthesis [16], carbohydrate con- living organisms. Accordingly, the vegetation of calamine soils tent [17], and lipid content [18] and to increase respiration in Central and Western Europe comprises specialized plant rate [15]. However, all published studies examining the effects species (metallophytes) that have evolved high tolerance to of zinc on caterpillar growth have been conducted with arti- heavy metals [1]. Compared to plants growing on normal soils, ficial diets [11–13,15] or with normal, non–metal tolerant metallophytes often show higher foliar concentrations of heavy plants subject to toxic concentrations of heavy metals [19]. In metals. Viola calaminaria (Gingins) Lej., the zinc violet, is addition to the changes of their elemental composition caused one of the most characteristic species of the so-called calamine by metals, the primary and secondary metabolisms of these flora. The foliar zinc concentration of V. calaminaria ranges nontolerant plants also may be modified [20,21]. from 1,000 to 10,000 ␮g/g dry weight [1–5], or more than 20- Metalliferous sites may represent unfavorable habitats for fold higher than that of normal plants (20–100 ␮g/g dry wt) herbivores, and possibly because of relaxed herbivory pres- [6]. Despite its high zinc content, V. calaminaria can be used sure, metallophytes have evolved lower investment in physical as food plant by the butterfly Issoria lathonia L. (Nymphal- [22] and chemical (Noret et al., Universite´ Libre de Bruxelles, idae). The caterpillar can complete its whole life cycle on this Bruxelles, Belgium, unpublished data) defenses compared to plant, which should, however, be a toxic diet [7,8]. Interest- that of nonmetal-tolerant taxa. In addition, metal accumulation ingly, the largest populations of I. lathonia in Belgium are in leaves has been suggested to protect metallophytes against found in calamine sites, where V. calaminaria is abundant and herbivores [23,24]. the only suitable food plant [8]. However, this butterfly also Few studies have examined the biology of insects able to occurs out of calamine sites, and larvae then develop on other complete their whole life cycle on leaves of metal-accumu- Viola spp., among which is the widespread Viola tricolor,a lating plants. A Chrysomelid beetle accomplishing its whole non–metal accumulating species that mainly grows on fallow life cycle on the nickel-accumulating Berkheya coddii has lands or on waysides and that is closely related to V. cal- been found in South Africa [25]. Boyd et al. [26] found another aminaria [9]. Chrysomelid beetle as well as an orthopteran feeding on B. coddii. In California, a plant bug restricted to the nickel-ac- * To whom correspondence may be addressed ([email protected]). cumulating Streptanthus polygaloides was identified [27]. The

565 566 Environ. Toxicol. Chem. 26, 2007 N. Noret et al. adaptive features of these metal-tolerant insects to the partic- thonia is protected by law in Belgium, we obtained an autho- ular mineral composition of their host plant remain largely rization to capture 20 butterflies from the calamine site of unexplored. According to Posthuma and van Straalen [28] as Prayon in May and June of 2005. These butterflies therefore well as Boyd [29], two main resistance mechanisms allow come from the same site as the V. calaminaria seeds. In the animals to avoid a high metal body burden: A low metal uptake field, the captured butterflies were distributed among several (avoidance or diet dilution), and a high detoxication rate. This cages containing Viola plants, and cages were brought back may be achieved by selectively eating less toxic parts of the to the laboratory. A few days later, at least seven females had plant and/or by evacuating metals via the feces (solid and laid eggs in separate cages; after one week, the eggs had liquid excrements) and/or by molting. hatched. First-instar larvae, which probably presented the sec- The present study aimed at understanding how I. lathonia ond generation, were collected for the experiments. Because cope with the high foliar zinc concentration of V. calaminaria, our aim was to examine how I. lathonia larvae withstand a which is the natural food plant of the larvae on calamine sites. high-zinc diet and not to investigate genetic variation for this To investigate the effects of zinc on larval growth, we com- behavior, larvae from all cages were randomly used without pared the development of I. lathonia larvae feeding on leaves distinguishing between maternal progenies. of V. calaminaria with high- and low-zinc concentrations. The zinc concentrations of leaves, insect bodies, feces, and exuviae Growth of I. lathonia on Viola spp. were measured to test whether zinc is concentrated and/or The first experiment (experiment 1) was conducted in May excreted by larvae. In addition, we compared larval growth to verify that I. lathonia larvae can develop normally on V. on low-zinc leaves of V. calaminaria to that on V. tricolor, calaminaria despite its high foliar zinc concentration. Larvae which is a common food plant for I. lathonia larvae out of (n ϭ 15–30) were individually reared in Petri dishes (diameter, calamine sites. This allowed us to evaluate the quality of V. 5 cm) with detached leaves of five-month-old V. calaminaria calaminaria as a food plant for I. lathonia independently of NC or V. calaminaria ZN. Larvae received only one sort of its zinc concentration. leaves (V. calaminaria NC or ZN), provided once a day until pupation. At the end of the experiment, uneaten leaves of V. MATERIALS AND METHODS calaminaria NC and V. calaminaria ZN as well as four ran- Plant material domly selected pupae were dried to be analyzed later for their zinc concentrations. Two Viola spp., V. calaminaria (Gingins) Lej. and V. tri- The second experiment (experiment 2) was conducted in color L., were grown from seeds in a nonheated glasshouse. June to compare the developmental rates of larvae reared on Seeds were collected in the field (V. calaminaria, population V. tricolor, V. calaminaria NC, and V. calaminaria ZN. Lar- of Prayon, province of Lie`ge, Belgium) from at least 20 dif- vae (n ϭ 29–30 for each leaf diet) were individually reared ferent plants or were obtained from botanical gardens (V. tri- in Petri dishes with detached leaves of six-month-old plants. color). Viola calaminaria is a perennial species restricted to Dishes were kept under natural light conditions (June 25th to calamine soils in eastern Belgium, southern portions of The July 8th) at a temperature of 21.4 Ϯ 0.8ЊC (mean Ϯ standard Netherlands, and western Germany, where it forms large pop- error). To maintain the water content of leaves near saturation, ulations [9]. Viola tricolor is a widespread annual or perennial petioles were kept between two water-saturated pieces of filter plant growing on waysides and fallow lands. Molecular mark- paper. Each larva received only one sort of leaves (V. tricolor, ers have shown that V. calaminaria and V. tricolor are closely V. calaminaria NC, or V. calaminaria ZN) during its whole related species, with both belonging to the same Melanium development until pupation. Leaves were supplied to cater- subgenus [9]. pillars twice a day to ensure that these organisms were not Seeds of both species were germinated on compost; seed- limited by food. Each time, leaves were collected from at least lings were then transplanted into individual pots (diameter, 9 10 plants, pooled together, and then distributed to larvae. The cm) filled with a 50:50 mixture of leaf compost and arable mass of provided leaves was not assessed; however, the mass soil. To obtain plants with a high-zinc concentration, V. cal- of leaves consumed by larvae on each diet could be estimated aminaria also was grown on the same soil amended with zinc from the fecal mass. To follow their developmental rate, cat- (9,000 mg/kg dry soil, supplied as ZnSO4·7H2O). Neither tox- erpillars were weighed (Ϯ0.01 mg) every 2 to 3 d until pu- icity symptoms nor growth delays were observed in zinc-en- pation. Feces were removed daily and collected individually riched V. calaminaria plants. Viola tricolor, which is not a in small tubes to be weighed and analyzed later. Larval skins metal-tolerant species, cannot grow on this zinc-enriched soil. and pupal exuviae also were kept for analysis. Adult mass was Thus, we had three sorts of plants (V. tricolor, noncontami- recorded immediately after the emergence of butterflies. nated V. calaminaria [NC], and zinc-enriched V. calaminaria [ZN]), hereafter referred to as three diets. Plants (n Ն 40 Elemental composition individuals/diet) were watered with deionized water when nec- Uneaten parts of leaves as well as entire leaves that were essary. not yet consumed by larvae were removed from the Petri dishes when new leaves were supplied. No visual differences were Butterflies observed between eaten and uneaten leaves or parts of leaves Issoria lathonia L. (1758), the Queen of Spain fritillary, (no apparent food selection). Uneaten leaves or parts of leaves belongs to the Nymphalidae family (Argynninae subfamily). collected over two consecutive days were pooled into one leaf It has a wide distribution, from North Africa to southern Nor- sample. These were analyzed to assess the elemental com- way and to China. This butterfly has two or three generations position of the food provided to larvae (experiments 1 and 2). per year in Belgium, where it flies from April to September. In addition, individual V. calaminaria plants grown in the field The usual host plants of larvae are species of the genera Viola, in the Prayon population also were collected and analyzed. Onobrychis, Borago, and Cynoglossum [7]. Because I. la- This allowed us to compare the mineral composition of field- Development of Issoria lathonia larvae on Viola sp. Environ. Toxicol. Chem. 26, 2007 567

the method was tested using a reference material (Brassica rapa); for insect material, results were compared with those obtained using another method (ﬂame atomic absorption spec- troscopy). Elemental concentrations were determined by in- ductively coupled plasma–optical emission spectrometry. Re- sults of three emission lines were averaged to avoid interfer- ence errors. The leaf nitrogen concentration of leaves in experiment 2 was measured with an elemental analyzer (CHN; Carlo Erba Instruments, Milan, Italy).

Statistics The kinetics of growth were analyzed by a multivariate Wilk’s lambda statistic using the PROC GLM procedure of SAS (1999; SAS Institute, Cary, North Carolina, USA), because the sphericity condition of repeated-measures analysis of variance (ANOVA) was not fulfilled. Individual adult mass and fecal mass, as well as duration of the larval period, were analyzed by one-way ANOVA (factor: leaf diet) followed by least-squares-mean tests (Tukey’s). A one-way ANOVA also was performed to analyze the variation in elemental composition (Ca, Fe, K, Mg, Na, P, and Zn) of leaves, pupae, feces, larval skins, pupal exuviae, and field-caught butterflies. The Fig. 1. Growth kinetics of Issoria lathonia larvae fed with three nitrogen concentration of glasshouse-grown plants was simi- different leaf diets (n ϭ 29–30 per diet): Viola tricolor, Viola cal- larly analyzed. Logarithmic transformations were performed aminaria grown on noncontaminated soil (NC), and V. calaminaria when necessary. When transformed data did not fit the as- grown on zinc-enriched soil (ZN). Data are presented as the mean Ϯ sumption of homoscedasticity of the ANOVA, a Kruskal–Wal- standard error. Growth was affected by the diet (Wilk’s lambda sta- lis one-way nonparametric ANOVA was used, followed by a tistic: F ϭ 10.23; df ϭ 2, 86; p Ͻ 0.001). Growth was significantly faster from the fifth day for larvae reared with V. calaminaria NC Kruskal–Wallis all-pairwise comparisons test (Statistix 7.0; (least-squares-mean tests: **p Ͻ 0.01, ***p Ͻ 0.001). Analytical Software, Tallahassee, FL, USA). The bioaccumulation factor (BAF) for zinc was calculated by dividing the zinc concentration of pupae by that of the leaves (experiments and glasshouse-grown V. calaminaria to estimate the simi- 1 and 2). Linear regression was used to test the significance larity of the food provided in controlled experiments to the of the relationship between the mean zinc concentrations of I. food in natural conditions. lathonia and V. calaminaria (experiments 1 and 2) (Statistix Four (experiment 1) or five (experiment 2) pupae of each 7.0). leaf diet were randomly chosen for elemental analyses. Feces corresponding to the five pupae selected for elemental analyses RESULTS in each leaf diet were analyzed (experiment 2). For exuviae, Suitability of V. calaminaria as a food plant for I. lathonia larval skins of all individuals (experiment 2) were collected and pooled in two samples to have sufficient material for el- The first experiment permitted us to confirm that V. cal- emental analyses. The same procedure was applied for the aminaria is a suitable food plant for I. lathonia larvae, which analysis of pupal exuviae. Nine butterflies caught in the field pupate successfully on both V. calaminaria NC and V. cal- in June (parents of experiment 2 larvae) were analyzed after aminaria ZN. Because the aim of this first experiment was to egg laying to assess the elemental composition of butterflies verify that I. lathonia larvae can develop on V. calaminaria, that had fed on V. calaminaria grown in the field. The com- we did not measure parameters other than the zinc concentra- parison of the elemental compositions of butterflies that fed tions of leaves and pupae. on field-grown V. calaminaria with that of pupae reared on glasshouse-grown V. calaminaria allowed us to check our Caterpillar development according to diet experimental results for their relevance to natural conditions. Concerning growth kinetics, a clear effect of the leaf diet All samples were oven-dried (48 h, 45ЊC) and mineralized on the developmental rate (Fig. 1) and on the duration of the by dry ashing in porcelain crucibles (16 h, 450ЊC). Ashes were larval period (Table 1) was observed in the second experiment. dissolved with 1 ml of suprapur HCl. This solution was brought When growth curves of larvae on V. calaminaria NC and on to a final volume of 50 ml. For plant analyses, accuracy of V. calaminaria ZN are compared, it clearly appears that a

Table 1. Development of Issoria lathonia on three different diets: Viola tricolor, Viola calaminaria grown on noncontaminated soil (V. cal. NC), and V. calaminaria grown on zinc-enriched soil (V. cal. ZN)a

V. tricolor V. cal.NC V. cal.ZN ndfF p

Adult mass (mg fresh wt) 103 Ϯ 26 94 Ϯ 22 93 Ϯ 28 65 2, 62 1.12 0.33 Fecal mass (mg dry wt) 200 Ϯ 44 B 140 Ϯ 38 A 204 Ϯ 49 B 88 2, 85 23.7 0.00 Duration of the larval period (d) 13.1 Ϯ 1.6 B 11.2 Ϯ 1.8 A 13.2 Ϯ 1.5 B 87 2, 84 13.3 0.00

a Data are presented as the mean Ϯ standard deviation. The number of samples (n), degrees of freedom (df ), F statistic, and p value of the one- way analysis of variance are shown. Values with identical capital letters are not signiﬁcantly different (Tukey’s post hoc test, ␣Ͻ0.05). 568 Environ. Toxicol. Chem. 26, 2007 N. Noret et al. a 60 51 35 40 75 25 60 325 50 45 55 100 235 125 170 205 g/g) ␮ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 1.81.02.12.0 515 1.9 385 4.9 380 2.91.5 480 3.2 — 355 0.9 345 0.7 635 660 0.4 480 1,330 0.6 880 845 0.060.19 735 3.16 575 520 0.72 575 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ . ZN). Elemental compositions of V. cal 1.2 20.6 0.780.86 17.4 0.34 23.5 0.940.77 27.3 0.61 21.0 0.15 32.3 0.41 32.9 0.09 15.3 0.12 17.4 0.05 16.3 0.23 11.0 0.20 17.7 0.06 19.4 0.06 17.9 0.16 4.04 2.8 4.04 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 2.92.22.9 4.82 7.39 1.6 9.06 1.51.1 7.19 0.1 12.7 12.4 1.56 0.70.1 0.30 0.38 0.6 2.17 0.190.140.36 1.76 0.02 1.64 1.30 0.55 0.76 0.01 0.99 0.34 0.43 0.87 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Fig. 2. Individual Issoria lathonia adult mass in relation to fecal mass grown on zinc-enriched soil ( of the larval stage according to the three different leaf diets on which larvae fed: Viola tricolor, Viola calaminaria grown on noncontaminated soil (NC), and V. calaminaria grown on zinc-enriched soil (ZN). The highest ratio between adult mass and larval fecal mass was 5 21.3 253065 14.9 40 11.9 14.1 1020 6.2 21.3 23.5 951535 1.22 35 2.72 55 2.5 3.33 2 12.0 11.0 10.6 achieved on V. calaminaria NC. 162010 1.49 1.62 1.69 g/g) Ca (mg/g) Mg (mg/g) K (mg/g) Na ( V. calaminaria ␮ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ standard deviation expressed in terms of dry weight.

high-zinc diet slows down larval growth (Fig. 1). Comparing Ϯ . NC), or growth curves of larvae on the two low-zinc diets reveals that V. calaminaria NC resulted in faster development of larvae V. cal 5 190 9504353405 115 135 16 165 185 190 140 200 60 200 1515 60 130 227 295 523 1020125 55 70 153 10 23

than V. tricolor did. The fastest development was therefore g/g) Fe ( ␮ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ achieved on V. calaminaria NC, whereas the developmental Ϯ Ϯ Ϯ Ϯ rates were similar on V. tricolor and on V. calaminaria ZN (Table 1). larvae and elemental concentrations in feces, pupae, larval skins, and pupal exuviae of the Lepidopteran No mortality was observed in any of the three leaf diets. Adult mass was not affected by the leaf diet, despite the dif-

ferences of developmental rates among the diets (Table 1). 2.11.62.4 62 120 1,740 Fecal mass was signiﬁcantly inﬂuenced by the diet, with lower Ϯ Ϯ Ϯ

values for V. calaminaria NC (Table 1). Figure 2 shows the Issoria lathonia relation between adult and fecal masses. This ratio was sig-

niﬁcantly inﬂuenced by the diet: Insects feeding on V. cal- grown on noncontaminated soil ( aminaria NC, which had the shortest larval period, also had 0.2 — 440 the highest insect to fecal mass ratio. 0.350.290.36 37.6 0.56 37.1 0.43 37.8 0.770.130.59 —1.26 — —0.36 —0.04 — 1,300 0.44 —0.05 83 107 0.02 — 2,285 0.08 —0.50 — 68 — 90 — 440 — 125 — 135 285 25 26 30 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Elemental composition 2.38 2.51 7.38 0.57 0.56 Experiment 1. The foliar zinc concentrations of V. calam- Viola calaminaria

inaria NC and V. calaminaria ZN were significantly different , from one another (Tukey’s post hoc test, p Ͻ 0.01) and reached 260 Ϯ 90 and 3,840 Ϯ 190 ␮g/g dry weight (mean Ϯ standard deviation [SD], n ϭ 6), respectively. Issoria lathonia pupae .NC.ZN. field 2.98 .NC 2.47 .ZN 3.80 .NC 2.18 .ZN 2.22 8.65 .NC 8.31 .ZN.NC 0.93 .ZN 0.60 0.26 0.58 Diet P (mg/g) N (mg/g) Zn ( fed with V. calaminaria NC had a body zinc concentration collected in the field also are reported. Data are presented as the mean V. tricolor V. cal V. cal V. cal V. tricolor V. cal V. cal V. tricolor V. cal V. cal V. tricolor V. cal V. cal V. tricolor V. cal V. cal significantly lower (135 Ϯ 20 ␮g/g dry wt, mean Ϯ SD, n ϭ Viola tricolor 4) than pupae fed with V. calaminaria ZN (493 Ϯ 50 ␮g/g Ϯ ϭ Ͻ

dry wt, mean SD, n 4) (Tukey’s post hoc test, p 0.01). I. lathonia 2)

Experiment 2. The elemental composition of leaves, feces, 2) ϭ and 9) Field 7.49 ϭ pupae, butterﬂies, larval skins, and pupal exuviae of experi- n n ϭ 4)

ment 2 is presented in Table 2. The zinc concentration of the 5) 5) n ϭ

ϭ ϭ ϭ

three leaf diets was signiﬁcantly different (F 135; df 2, ϭ n n 9; p Ͻ 0.001), with higher values in V. calaminaria ZN (Table n Table 2. Elemental concentrations in leaves provided as food to 2). The leaf zinc concentration of V. calaminaria ZN (1,740

Ϯ ␮ Ϯ Larvae were fed eitherV. with calaminaria Leaves ( a Pupal exuviae ( Feces ( 435 g/g dry wt, mean SD) was not signiﬁcantly different Pupae ( Butterﬂies ( Larval skins ( Development of Issoria lathonia larvae on Viola sp. Environ. Toxicol. Chem. 26, 2007 569

herbivores feeding on them remain scarce [28,30]. To our knowledge, the present study is the first to investigate the development of an insect naturally feeding on a plant that accumulates toxic amounts of zinc. Our results show that I. lathonia larvae develop normally and pupate successfully when fed exclusively with high-zinc leaves of Viola calaminaria. However, by comparing the growth of larvae fed with high- and low-zinc leaves (ϳ1,700 and ϳ120 ␮g/g dry wt, respectively, in experiment 2) of V. calaminaria, it appears that a high-zinc diet slows down insect growth and protracts the duration of the larval period by ap- proximately2d(ϩ18%). This is in agreement with previous data showing that a delayed growth generally is observed in Lepidoptera larvae fed with artificial diets enriched with zinc [11,13–15] or with other metals, such as cadmium, copper, and lead [10,13,14]. Metals have toxic effects by entering into biochemical reactions in which they are not normally involved [10], and larvae feeding on high-metal diets probably need more energy for metal-detoxication processes [15]. However, Fig. 3. Relation between the zinc concentration of Issoria lathonia in contrast with other data, the high-zinc leaves of V. cal- and that of its food plant, Viola calaminaria. Closed circles are for aminaria in the present study did not affect adult mass [13– data obtained in controlled conditions (pupae and plants), and the 15] or survival [31]. In these latter studies, a toxic effect of open circle refers to data obtained for field-collected butterflies and zinc on pupal mass was observed even at much lower zinc plants. Data are presented as means (n ϭ 4–5 for pupae, 9 for but- ␮ terflies, and 4–6 for plants). concentrations (e.g., 200 g/g dry wt in Kramarz and Kafel [15]) than in the present study (ϳ1,700 ␮g/g dry wt). Such discrepancies may be explained by the use of artificial diets from that of field-grown individuals (1,300 Ϯ 340 ␮g/g dry compared to leaf diets [13,14], by different anionic ligands wt, mean Ϯ SD) (F ϭ 2.3; df ϭ 1, 6; p ϭ 0.18). influencing metal toxicity [10], or by a species-specific toxicity Pupae show a higher concentration of phosphorus and lower threshold [32]. Toxicity thresholds obtained from artificial di- concentrations of calcium, iron, potassium, and magnesium ets therefore seem to be difficult to extrapolate to natural sys- than plants did. In line with this result, feces were enriched tems [10]. with these elements (Ca, Fe, K, and Mg) (Table 2). Compared Our results show how I. lathonia larvae develop success- with pupae, larval skins were particularly poor in Mg and P, fully across a broad range of dietary concentrations of zinc. and pupal exuviae were particularly impoverished in potas- Pupae feeding on the low-zinc diets (V. tricolor and V. cal- sium, iron, and zinc. aminaria NC in experiment 2, 60–120 ␮g/g dry wt) had a Just as the zinc concentration of field- and glasshouse- body zinc concentration similar to the concentration in their grown V. calaminaria plants (experiment 2) did not differ, the food (BAFpupae-leaves close to 1). Zinc is an essential micronu- zinc concentration of field-caught butterflies was not signifi- trient used to strengthen larval mandibles [33], which explains cantly different from that of pupae reared in controlled con- its high concentration in larval skins for both low-zinc diets. ditions on V. calaminaria ZN (F ϭ 0.00; df ϭ 1, 12; p ϭ When larvae were fed on higher zinc concentrations (1,740 0.96) (Table 2). and 3,840 ␮g/g dry wt in V. calaminaria ZN in experiment

2 and experiment 1, respectively), the BAFpupae-leaves decreased Relation between Zn concentrations of I. lathonia and to 0.3 and 0.1, whereas the concentration of zinc in feces V. calaminaria increased (experiment 2). On the other hand, as the zinc con- The zinc BAF of pupae decreased as the zinc concentration centration of larval exuviae (285 ␮g/g dry wt) was much lower of dietary leaves increased. Pupae fed on V. tricolor and V. compared to that of leaves (1,740 ␮g/g dry wt), molting does calaminaria NC (experiment 2) had a zinc concentration close not appear to be an efficient pathway for metal detoxication to that of their diet (BAF ϭ 1.1 and 0.8, respectively). In [10,12,34]. contrast, pupae reared on V. calaminaria ZN maintained a low Some nonessential metals may be accumulated by Lepi- body metal burden (BAF ϭ 0.3 and 0.1 in experiment 2 and doptera larvae (e.g., Cd [10,12]) or hardly assimilated (e.g., experiment 1, respectively). Linear regression of log-trans- Pb [12,14]). The concentration of essential metals, such as formed values indicates a significant relationship between the zinc, nickel, and with more variability, copper, generally is mean zinc concentration of V. calaminaria leaves and that of controlled by regulation mechanisms (e.g., Zn [14,19] as well I. lathonia pupae (p Ͻ 0.05, adjusted r2 ϭ 0.97) (Fig. 3). The as Ni and Cu [35]). This regulation is achieved through lysis slope parameter for the regression relationship is less than one of gut cells (where metals are accumulated) into the lumen of (ϳ0.5), which indicates that zinc concentration is regulated in the digestive system for excretion in the feces [10] and/or I. lathonia. The elemental composition of field-caught butter- through excretion of metals via the Malpighian tubules flies and field-collected V. calaminaria also are reported on [10,32,36]. the graph (Fig. 3). In our results, I. lathonia therefore appears to be a ‘‘metal deconcentrator’’ insect [32] that is able to regulate effectively DISCUSSION its internal zinc level through metal excretion in the feces, as Even though the occurrence of plant populations adapted suggested by studies using synthetic diets [12,14] or normal to heavy metals has been reported for a long time, studies of plants subject to toxic concentrations of heavy metals [19]. In 570 Environ. Toxicol. Chem. 26, 2007 N. Noret et al. the present study, the zinc concentration of approximately 450 as by an Agence de l’Environnement et de la Maıˆtrise de l’Energie ␮g/g dry weight in I. lathonia was found for pupae reared in contract (04.72.C.0037). controlled conditions on V. calaminaria ZN and for field- REFERENCES caught butterflies, both originating from the same population of Prayon. It would be interesting to test if the zinc-detoxifi- 1. Ernst WHO. 1974. Schwermetallvegetation der Erde. 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