Biological Control 26 (2003) 128–138 www.elsevier.com/locate/ybcon

Non-target host acceptance and by brassicae Bezdenko (: ) in the laboratory

D. Babendreier,* S. Kuske, and F. Bigler

Swiss Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, CH-8046 Zurich, Switzerland

Received 7 December 2001; accepted 11 September 2002

Abstract

As part of a risk assessment study, we exposed eggs of 23 non-target lepidopteran species including nine butterflies endangered in Switzerland to individual Bezdenko females under no-choice conditions in the laboratory. We could show that Papilio machaon L. (Papilionidae), Artogeia ( ¼ Pieris) napi L. (Pieridae), Argynnis adippe Denis & Schiffermuuller,€ Clossiana titania Esper, (), Aphantopus hyperanthus L., Maniola jurtina L., Coenonympha pamphilus L., Melanargia galathea L., ligea L., Hipparchia alcyone Denis & Schiffermuuller€ (Satyridae), Polyommatus icarus Rottemburg and Plebejus idas L. () were well accepted and not parasitized significantly different (range 73–94%) than the target, Ostinia nubilalis Huubner€ (81%). Virtually all eggs of Vanessa atalanta L., Argynnis niobe L., Clossiana selene L. (Nymphalidae), and semiargus Rottemburg (Lycaenidae) were accepted for oviposition resulting in significantly higher parasitism rates of 94–97% compared with the target. parthenoides Keferstein, M. diamina Lang, and nearly 50% of Mellicta athalia Rottemburg (Nymphalidae) eggs were rejected early in the host selection process. Ovipositional success on eggs of Zygaena filipendula L. (Zygaenidae), Hesperia comma L. (Hesperidae), Sphinx ligustri L., and Deilephila elpenor L. (Sphingidae) was less than 30%. The number of times a female left a host egg before acceptance as well as the time from first host egg contact to acceptance was not related to parasitism rate on the tested non-targets. Offspring emerging from non-target hosts was of similar or even larger size compared to offspring emerging from the target, and in all cases larger compared to individuals emerging from the factitious host, Ephestia kuehniella Zeller. We found that large T. brassicae individuals had significantly higher success in penetrating the chorion and parasitizing eggs of S. ligustri than smaller adults. The results show that T. brassicae parasitizes a number of non-target lepidopteran eggs belonging to different families. Host range and impact under field conditions have yet to be determined. Ó 2002 Elsevier Science (USA). All rights reserved.

Keywords: Trichogramma brassicae; Biological control; Non-target effect; Risk assessment; Host specificity; Egg ; Endangered butterflies

1. Introduction biological control worldwide (Li, 1994; van Lenteren, 2000). While in some cases native species were mass Since the beginning of biological control, more than reared and released, in many cases the utilized Tricho- 5200 agent introductions against economically impor- gramma species were exotic. Trichogramma brassicae tant pests have been made (Greathead and Bezdenko was introduced 30 years ago from Moldavia Greathead, 1992, updated 2000; Waage, 1990). Egg (former Soviet Union) to control the European corn parasitoids of the genus Trichogramma have been used borer, Ostrinia nubilalis Huubner€ (: Crambi- successfully as inundative biological control agents dae), in several parts of Western . This was a against a range of agricultural pests, mainly lepidopt- complete success which markedly reduced the pesticide erans and are the most widely used natural enemies in levels applied in maize in those countries (Bigler, 1986; Hassan, 1988). In other parts of the world, the use of * Corresponding author. Fax: +41-1-377-72-01. Trichogramma spp. has also led to occasional specta- E-mail address: [email protected] (D. Babendreier). cular successes (Li, 1994; Smith, 1996).

1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S1049-9644(02)00121-4 D. Babendreier et al. / Biological Control 26 (2003) 128–138 129

Biological control of insect pests has been viewed as US. However, very few butterflies and no rare species environmentally safe for much of its history. Over the were included in the analyses. last two decades, concerns about possible detrimental Virtually no information on parasitism by Tricho- effects for native fauna have been raised by ecologists gramma spp. of non-target butterflies, especially endan- (Howarth, 1983). Several papers have since reviewed the gered species, is available. Therefore, and because cases where non-target effects have been observed but, conservationists are much concerned about this group of more importantly, highlighted the fact that few studies non-target , we did not select host species from all on non-target effects were conducted and effects due to potential taxa but rather focused on butterflies. As a first exotic biological control agents may have been over- step of a general risk assessment procedure, we investi- looked (Howarth, 1991; Lynch et al., 2001; Simberloff gated whether eggs of potentially susceptible non-target and Stiling, 1996; Stiling and Simberloff, 2000). species were attacked by T. brassicae in the laboratory. One of the key parameters determining non-target We tested T. brassicae on butterfly species from all major effects is the range of species an agent is able to attack. It families occuring in Europe and included species re- is generally accepted that the release of agents with re- corded on the Swiss Red List of Endangered Species stricted host ranges should be favored in order to min- (Duelli, 1994). In addition, the performance of imize potential non-target effects. Therefore, host offspring within non-target host eggs was investigated specificity testing has become a central issue in projects because this might have important implications for the on biological control of insect pests. However, methods risk of butterfly populations. This study should provide a to test the host specificity of biological control agents first indication as to which species of butterflies are po- have been refined significantly for weeds (McEvoy, tentially at risk and should also serve to identify species 1996) but not so for insects. There is an ongoing debate that have to be further evaluated under field conditions. on this issue with the aim to improve the predictability of non-target attack after introduction (Barratt et al., 1999; Kuhlmann et al., 2000). 2. Materials and methods The vast majority of Trichogramma species are known to be fairly polyphagous, attacking a wide range 2.1. Trichogramma brassicae strain of lepidopterans and even species of other insect orders (e.g., Clausen, 1940; Pinto and Stouthamer, 1994; Trichogramma brassicae used in this study originated Thomson and Stinner, 1989). Information on the host from a colony that was maintained at the Swiss Federal range of T. brassicae in general is scarce and virtually Research Station for Agroecology and Agriculture for absent regarding economically unimportant species. In about 60 generations on European corn borer eggs. Prior contrast with the current practice of biological control to experiments, parasitoids were reared for 4–5 genera- of insect pests, the host range of T. brassicae had not tions on the Mediterranean flour moth, Ephestia ku- been evaluated prior to introduction to Western Europe ehniella Zeller, at 16:8 (L:D) h, 25 °C, and 60–70% RH. because non-target effects were not considered an im- O. nubilalis egg masses laid on wax paper were obtained portant issue at that time. from the French Agricultural Research, Lambert (US), Because most Trichogramma are known to be highly every four weeks and then stored at 4 °C, 85% RH. E. polyphagous and are currently released annually on kuehniella eggs were provided by Biotop, Valbonne, several million ha worldwide, information on potential France, fortnightly and stored under the same condi- non-target effects due to Trichogramma is in demand. tions. In order to verify the identity of our T. brassicae Andow et al. (1995), using a theoretical approach rearing strain, material from our colony was sent to based partially on empirical data, could show that the Wageningen Agricultural University, NL, periodically , an endangered butterfly in the USin- where it was identified by PCR. About 5000 parasitized habiting oak savanna, faces only a small risk due to eggs were put in a ventilated container of 1.3 L from mass releases of Trichogramma nubilale Ertle & Davis which T. brassicae adults could emerge. Adult parasi- in corn. Preliminary data provided by Suverkropp toids were stored at 25 Æ 0:5°C and 70 Æ 5% RH and (unpublished OECD report, 1997) showed that T. provided with small droplets of honey. One day-old brassicae parasitized some non-target host eggs (one mated females were used for the experiments. undetermined nymphalid and Pterostoma palpinum Clerck (Lepipoptera: Notodontidae)) in the vicinity of 2.2. Selection of butterfly species release fields in Switzerland, but the rate of parasitism was 4% or less. Only recently Orr et al. (2000) pub- Non-target host species selection was based mainly lished data on the potential host range of T. brassicae. on ecological criteria as well as on habitat and temporal In laboratory trials, these authors found that this overlap of hosts and inundatively released T. brassicae. parasitoid is able to attack several Lepidopterans po- Eggs of the following 23 non-target lepidopteran species tentially occurring near cornfields in the Midwestern were tested in the present study, including nine species 130 D. Babendreier et al. / Biological Control 26 (2003) 128–138 on the list of endangered species in Switzerland (marked proached the egg, they were carefully removed before with an asterisk throughout the text): Papilio machaon any interaction could occur. The arena was not covered L. (Papilionidae), Artogeia ( ¼ Pieris) napi L. (Pieridae), by a lid at any time and thus the female was free to Vanessa atalanta L., Argynnis adippe Denis & Schif- leave. Behavioral parameters measured were host in- fermuuller*,€ A. niobe L.*, Clossiana titania Esper*, C. spection (contact with the egg and drumming with the selene L.*, Mellicta athalia Rottemburg*, Melitaea antennae) and contact of the host egg with the ovipos- parthenoides Keferstein*, M. diamina Lang*, (Nymp- itor. In addition, we recorded whether and how often halidae), Aphantopus hyperanthus L., Maniola jurtina L., females left the egg during the host inspection process. If Melanargia galathea L., Coenonympha pamphilus L., the egg was abandoned for more than 60 s the trial was Erebia ligea L., Hipparchia alcyone Denis & Schif- terminated and the egg was regarded as being rejected. fermuuller*€ (Satyridae), Polyommatus icarus Rottem- An egg was regarded as being accepted if the female burg, Cyaniris semiargus Rottemburg, Plebejus idas L.* drilled and/or penetrated the egg continuously with her (Lycaenidae), Zygaena filipendula L. (Zygaenidae), ovipositor for longer than 60 s (cf., Schmidt, 1994). Hesperia comma L. (Hesperidae), Sphinx ligustri L., and Subsequently, the cardboard strip with the host egg and Deilephila elpenor L. (Sphingidae). In addition, we tested the female parasitoid were carefully removed to prevent egg masses of the target O. nubilalis as well as individual the attack of conspecifics. This setup was checked every eggs and egg masses of E. kuehniella. Most of these 5–10 min and as soon as the female was not found species were obtained from field collections. However, anymore on the host egg or the cardboard strip, the egg A. niobe*, A. adippe*, M. parthenoides*,andH. alcyone* was transferred into a glass vial of 2.5 ml and stored at adults were reared from eggs and S. ligustri as well as D. 25 °C and 70% RH until parasitoid emergence. elpenor from pupae that were commercially obtained. This experimental design enabled us to calculate the Adult butterflies were kept in gauze cages together with host inspection time (time that females contacted the host their respective host plant. Various flowers were pro- egg before it was accepted) as well as the number of times vided as a food source together with a mixture of yeast, a female left the egg before its acceptance. Both mea- honey, and water smeared in patches on the cage wall. surements will give an indication about the acceptance of Both food sources were replenished every second day. eggs of non-target species. According to Van Dijken et al. The size of the cages varied depending on the size and (1987), we calculated a ratio between the number of times behavior of the butterfly in the range of 20 Â 20 Â 30 cm a host egg was contacted and the number of eggs that (e.g., for M. athalia*)to50Â 60 Â 80 cm (e.g., for S. were accepted (a/c ratio). Between 18 and 56 females were ligustri). Cages were placed in a greenhouse at 25 Æ 2°C tested on each of the tested species. and 65 Æ 10% RH. Most tested host species attached Parasitism was calculated from blackened eggs after 5 their eggs on the gauze of the rearing cage. These were days and emergence of parasitoid offspring assessed. For carefully removed with a brush between 08:00 and each tested lepidopteran species, a similar number of non- 12:00 h daily and stored at 6 °C for a maximum of 7 days target host eggs were reared under identical conditions until the start of experiments. However, P. machaon laid without exposure to T. brassicae to check for emerging their eggs on carrot leaves and the three lycaenids (P. larvae. On each day that a non-target species was tested, icarus, C. semiargus, and P. idas*) on blossoms or leaves we tested T. brassicae females on batches of approxi- of red . Because these eggs were difficult to remove mately 30 E. kuehniella eggs glued on cardboard. If a fe- from the plant, small pieces of carrot leaves or clover male contacted an egg batch she was allowed to parasitize blossoms were used in the experiments for the above- for 15 min. Subsequently, the females were removed and mentioned species. the eggs reared under the same conditions as eggs of the test species (25 °C, 70% RH). E. kuehniella eggs were 2.3. No-choice tests checked for parasitism after 5–7 days. We performed data analyses for the corresponding non-target species only if In the present study, we used no-choice tests because at least 75% of females successfully parasitized within the these would reflect the chance of parasitism in the field first 15 min. For species that lay their eggs in batches (e.g., more accurately than choice tests, because it is unlikely Melitaea spp.) and that were hardly accepted, we also that the eggs of the target host O. nubilalis are in very tested T. brassicae on naturally laid egg batches to eluci- close proximity to eggs of non-targets. Individual host date potential confounding effects due to the fact that eggs were attached to cardboard strips of 5 Â 10 mm, single eggs were offered to females. All experiments were placed in a petri dish of 40 mm diameter, and small carried out at 25 Æ 1°C. The number of emerged offspring numbers of 5–10 T. brassicae females that had not been was counted and the sex determined under a microscope exposed to host eggs prior to the experiments were in- (magnification 24Â or 50Â). In addition, we measured troduced. The egg was continuously observed and the maximum length and width of the eggs (magnification behavior of the female recorded as soon as she came in 125Â) and calculated a volume using a sphere as a model. contact with the host egg. If additional females ap- Because the size of Trichogramma offspring may affect D. Babendreier et al. / Biological Control 26 (2003) 128–138 131 host acceptance and thus non-target effects, the hind tibia ella, were exposed to individual T. brassicae females. length of T. brassicae emerged from several representative Most T. brassicae females accepted egg masses of the species (including those with very small and very large target species O. nubilalis (84%) and 81.3% of the egg eggs) was measured: from each parasitized egg where fe- masses were successfully parasitized. E. kuehniella was male offspring had emerged we measured one hind tibia of only well accepted if eggs were offered as clusters to a randomly selected female under a dissecting microscope parasitoid females whereas single eggs showed a low (magnification 125Â) to the nearest 0.01 mm (see Olson acceptance rate and were significantly less parasitized and Andow, 1998). (Fig. 1). Eggs of the majority of non-target species were readily accepted with acceptance rates between 75 and 2.4. Penetration of egg chorion 100% (Fig. 1). Only Melitaea parthenoides* and M. di- amina* were rejected by T. brassicae females very early For the two sphingid species, D. elpenor and S. ligustri, in the host selection process after drumming the egg for where we obtained a high rate of acceptance, but a low a few seconds or even at 1–3 mm distance without rate of successful parasitization, we carried out trials with coming into contact with the egg. Only 8.9% of M. continuous observation throughout the whole oviposi- parthenoides* eggs and no M. diamina* eggs were ac- tion event to observe whether females were able to pene- cepted during the host selection process by T. brassicae trate the egg chorion and whether any eggs were deposited females. This behavior did not change if egg masses were in the host. Oviposition was noted to have occurred if the provided instead of single eggs (n ¼ 10 for each species). typical movements with the abdomen (as described in The eggs of Mellicta athalia* were accepted at an in- detail by Suzuki et al., 1984) were observed. Under the termediate level of 61%. same experimental conditions as above, single T. brassi- cae females were observed on single eggs continuously as 3.2. Parasitism soon as she came in contact with the host egg. We ob- served whether females penetrated the chorion, the time Twelve species had parasitism rates not significantly this took the female and whether oviposition occurred. different from that of the target, O. nubilalis (Fig. 1). In addition, we investigated whether there was a Four species (Vanessa atalanta, Argynnis niobe*, Clos- difference between large and small females in their siana selene*, and Cyaniris semiargus) were accepted by ability to penetrate the thick chorion of S. ligustri. For T. brassicae females at rates close to 100%, and para- this experiment, we reared T. brassicae on eggs of sitism rates observed were significantly higher than that Papilio machaon, which resulted in significantly larger of the target. Significantly lower parasitism rates com- adult . Small Trichogramma wasps were obtained pared with the target were obtained for Mellicta atha- from rearing on E. kuehniella. Again, we observed lia*, Melitaea parthenoides*, M. diamina*, Zygaena whether females penetrated the chorion, the time this filipendula, Hesperia comma, Sphinx ligustri,andDeile- took the female and whether oviposition occurred in phila elpenor (Fig. 1). With the exception of M. athalia* continuous observations of single females. and Z. filipendula having intermediate values of 50 and 30% parasitism, respectively, all these species were par- 2.5. Statistical analyses asitized at a very low level of 0–13.5%. Interestingly, we obtained parasitoid offspring from all host species where Non-target parasitism was compared with that of the any acceptance could be observed. Although many fe- target using a v2 test. Hind tibia length was analyzed males rejected eggs of M. athalia* and only 8.9% of M. using a single factorial ANOVA and the Newman–Ke- parthenoides* eggs were attacked by female wasps, these uls test. Regression analysis was carried out to deter- were suitable hosts as well because nearly all accepted mine whether egg volume and host inspection time are eggs gave viable offspring. In contrast, low parasitism related to non-target host acceptance or parasitism. All rates were obtained for H. comma and the two sphing- percentages were arcsin transformed prior to analysis. ids, S. ligustri and D. elpenor, despite high acceptance Handling times were compared using the non-paramet- rates. ric Mann–Whitney U test. All statistical analyses were carried out with the Statistica software package. 3.3. Penetration of the egg chorion

Continuous observations of T. brassicae females on 3. Results eggs of S. ligustri and D. elpenor revealed that only 40% of the females were able to penetrate the chorion of D. 3.1. Acceptance elpenor eggs and only 12% actually oviposited ðn ¼ 25Þ. The remaining females either did not penetrate the cho- Overall, 25 lepidopteran species, including the target, rion or withdrew the ovipositor after 1–2 min. The fe- O. nubilalis, and the factitious rearing host, E. kuehni- males needed 2305 Æ 410 s SD ðn ¼ 10Þ to penetrate the 132 D. Babendreier et al. / Biological Control 26 (2003) 128–138

Fig. 1. Host acceptance (drilling the host egg for > 60 s) and parasitism (calculated from blackened eggs after 5 days) for 23 tested non-target le- pidopterans by Trichogramma brassicae. The number of tested females is given in brackets for each species. Bars marked with asterisks indicate a significant difference between parasitism rate of the respective non-target host and the target, Ostrinia nubilalis (v2 test procedure; *P < 0:05; **P < 0:01; ***P < 0:001). chorion of D. elpenor. Females eventually tried to attack 3.4. Rejection rate the egg at another location (mean: 1:5ÂÆ0:7), but in no case did we observe a female penetrating the egg chorion In addition to the acceptance rates, we calculated an successfully during the second drilling period and fe- acceptance/contact ratio (Table 1). We hypothesized males never tried to penetrate the egg for longer than 60 s that females may leave less preferred eggs more often at the third attempt. In all cases where no oviposition than preferred ones which would lead to a low a/c ratio was observed, healthy host larvae emerged. and to a lower risk for non-target effects. Again, as with Hind tibia of T. brassicae females reared from E. acceptance, species can be divided into distinct groups. kuehniella measured 0:138 Æ 0:0085 mm ðn ¼ 25Þ and were significantly smaller than those reared from Papilio machaon (0:174 Æ 0:0125 mm, n ¼ 22, t ¼ 11:6, df ¼ 1, P < 0:001). The proportion of females successfully pe- netrating the thick chorion of S. ligustri eggs was 28% for those reared on E. kuehniella. Significantly more eggs (86%) were successfully penetrated by females reared from P. machaon (v2 ¼ 4:69, df ¼ 1, P < 0:05, Fig. 2). Similarly, a significant difference was found between the oviposition success of these two groups of females: while only 16% of the females reared on E. kuehniella suc- cessfully parasitized eggs of S. ligustri, 82% of the fe- males reared from P. machaon successfully oviposited (v2 ¼ 7:54, df ¼ 1, P < 0:01, Fig. 2). No difference was found between the time which females needed to pene- trate the chorion of S. ligustri eggs (2882 Æ 1041 s; n ¼ 7) for T. brassicae reared on E. kuehniella and for T. Fig. 2. Success of smaller Trichogramma brassicae females (reared from brassicae reared on P. machaon (2477 Æ 1032 s; n ¼ 19) Ephestia kuehniella) and larger females (reared from Papilio machaon) (U19;7 ¼ 49; P ¼ 0:31). In all cases where no oviposition to penetrate and oviposit into eggs of Sphinx ligustri (v2 test procedure; had been observed, the host larvae emerged. *P < 0:05, **P < 0:01). D. Babendreier et al. / Biological Control 26 (2003) 128–138 133

Table 1 Acceptance/contact ratio ( ¼ a/c ratio) of non-target host eggs (egg mass in the case of the target, Ostrinia nubilalis) and host inspection time together with the number of replicatesa Species a/c ratio N 1 Host inspection time Æ SD (s) N 2 Ostrinia nubilalis 0.45 32 160.8 Æ 59.0 27 Ephestia kuehniella * 30 39.8 Æ 9.7 4 Papilio machaon 0.97 29 50.0 Æ 19.3 29 Artogeia napi 0.67 22 50.5 Æ 17.7 20 Vanessa atalanta 0.96 52 41.7 Æ 16.0 50 Argynnis adippe 1.00 30 53.7 Æ 21.8 29 A. niobe 1.00 34 48.3 Æ 21.4 33 Clossiana titania 1.00 23 34.8 Æ 9.1 22 C. selene 0.89 31 41.7 Æ 15.6 31 Mellicta athalia 0.76 36 23.1 Æ 7.3 22 Melitaea parthenoides * 56 33.0 Æ 11.5 5 Aphantopus hyperanthus 0.88 30 38.7 Æ 16.7 30 Maniola jurtina 0.91 33 45.2 Æ 17.8 32 Hipparchia alcyone 0.85 32 66.0 Æ 25.6 29 Melanargia galathea 0.92 45 55.6 Æ 24.4 43 Coenonympha pamphilus 0.92 27 38.8 Æ 15.4 24 Erebia ligea 0.95 18 56.4 Æ 28.7 18 Polyommatus icarus 0.94 32 61.5 Æ 26.2 30 Cyaniris semiargus 0.97 31 47.2 Æ 25.6 30 Plebejus idas 0.93 28 35.0 Æ 13.4 25 Zygaena filipendula 0.90 46 51.0 Æ 22.8 36 Hesperia comma 0.89 32 45.1 Æ 16.8 31 Sphinx ligustri 1.00 37 84.2 Æ 23.8 34 Deilephila elpenor 1.00 40 64.5 Æ 19.2 37 *, number of accepted eggs too low for calculation. a N 1, number of Trichogramma brassicae females tested; N 2, number of females that had accepted an egg.

For the majority of species that showed high acceptance F1;15 ¼ 2:22; P > 0:05) and inspection time and parasit- 2 and parasitism rates, parasitoid females rarely left a host ism (r ¼ 0:014; F1;15 ¼ 0:216; P > 0:05). egg even once until acceptance and showed a/c ratios of 1 or close to 1 (Table 1). The lowest a/c ratio was ob- 3.5. Performance on host eggs served in the target, O. nubilalis; 66% of females that eventually accepted an egg mass rejected it at the first Emergence rate of parasitoid offspring based on encounter. A. napi also showed a low a/c ratio and a blackened ( ¼ parasitized) eggs was 100% or very close to parasitism rate similar to that of the target. M. athalia* 100% for all tested non-target species as well as for showed a moderately low a/c ratio and low parasitism O. nubilalis and E. kuehniella (range 96–100%). Wing rates. In contrast, virtually all T. brassicae females ac- cepted eggs of H. comma, S. ligustri, and D. elpenor at first encounter but had very low oviposition success (i.e., parasitism rates of 3–13.5%). Consequently, we did not find any relationship between initial rejection and par- 2 asitism (r ¼ 0:009; F1;15 ¼ 0:134; P > 0:05). Time from the first contact until acceptance (ovipos- itor in contact with host egg for >60 s) of an egg ranged from 23 s for M. athalia* to 84 s for S. ligustri, a sphingid which laid the largest eggs tested (Table 1). Incidently, T. brassicae females needed 160 s until acceptance of eggs of the target, but this value was not included in further analyses because O. nubilalis was the only species where eggs were offered to females in the form of egg-masses. We could show a significant linear relationship between 2 Fig. 3. Hind tibia length (+SD) of Trichogramma brassicae females egg-size and host inspection time (r ¼ 0:616; F1;15 ¼ reared from eggs of various lepidopteran species. The number of fe- 24:1; P < 0:001): the larger an egg, the longer females males measured is given in brackets for each species. Different letters took until acceptance. No relationship was found be- above the bars indicate significant differences (one-way ANOVA with tween host inspection time and acceptance (r2 ¼ 0:129; Newman–Keuls test procedure; P < 0:05). 134 D. Babendreier et al. / Biological Control 26 (2003) 128–138

Table 2 Host egg volume, number of Trichogramma brassicae per host egg and sex ratio (% females) of offspring (N, number of parasitized eggs) Species Egg-volume ðmm3ÞÆSD No. of adults emerged Æ SD Females (%) N Ostrinia nubilalis * 1.6 Æ 0.25 66.4 27 Ephestia kuehniella 0:056 Æ 0:007 1.0 Æ 0 63.3 30 Papilio machaon 0:91 Æ 0:058 11.6 Æ 2.1 70.9 27 Artogeia napi *4:1 Æ 1:2 66.6 16 Vanessa atalanta 0:21 Æ 0:028 4.1 Æ 1.8 65.9 47 Argynnis niobe 0:33 Æ 0:039 5.1 Æ 1.6 69.2 33 Clossiana titania 0:30 Æ 0:045 4.4 Æ 0.85 69.1 22 C. selene 0:30 Æ 0:044 5.3 Æ 1.7 68.9 30 Mellicta athalia 0:20 Æ 0:014 3.4 Æ 0.49 65.1 17 Melitaea parthenoides 0:16 Æ 0:006 2.3 Æ 0.50 ** 4 Aphantopus hyperanthus 0:26 Æ 0:026 5.2 Æ 1.6 69.9 26 Maniola jurtina 0:23 Æ 0:025 4.0 Æ 0.83 62.5 24 Hipparchia alcyone 0:99 Æ 0:111 14.7 Æ 3.8 67.0 26 Melanargia galathea 1:14 Æ 0:097 17.6 Æ 3.6 71.6 33 Coenonympha pamphilus 0:32 Æ 0:027 4.25 Æ 1.1 60.3 16 Erebia ligea 0:55 Æ 0:076 8.3 Æ 2.9 69.9 18 Polyommatus icarus 0:10 Æ 0:006 1.8 Æ 0.43 54.5 27 Plebejus idas 0:10 Æ 0:008 2.0 Æ 0.47 56.3 19 Sphinx ligustri 4:54 Æ 0:245 38.9 Æ 10.7 81.1 15 Deilephila elpenor 1:90 Æ 0:227 20.9 Æ 4.3 79.0 13 *, not measured; **, number of parasitized eggs too low for calculation, egg size based on n ¼ 20 host eggs. deformations were observed only in E. kuehniella where 11% of emerging adults had abnormally short wings. Size measurement of emerging adults from a range of species, including the smallest and the largest host eggs tested, revealed a discontinuous pattern (Fig. 3): the smallest adults were reared from the factitious host, E. kuehniella (egg volume: 0.057 mm3), slightly but significantly larger adults emerged from hosts of rather small to intermediate egg volume (egg volume: 0.1–0.3 mm3) and again signif- icantly larger adults emerged from the largest host-eggs (egg volume: 0.9–4.5 mm3, see Fig. 3). Not only were larger offspring produced on larger eggs but, in addition, we found a strong linear and significant relationship Fig. 4. The relationship between the egg size of non-target lepidopt- between egg volume and number of Trichogramma adults erans and the sex ratio of offspring that emerged from parasitized host 2 eggs. Each measurement is based on 13–47 females (see Table 2). per egg (r ¼ 0:955; F1;15 ¼ 316:2; P < 0:001). The sex ratio measured as proportion females varied from 0.55 for P. icarus (the smallest non-target host 1964) although exceptions to this rule have also been tested) and 0.82 for S. ligustri which laid the largest eggs found (Battisti, 1991; Bourchier et al., 2000). To assess (Table 2). Consequently, we found a significant rela- the complete host range for polyphagous parasitoids is tionship between egg volume and the sex ratio. The lar- difficult and laborious (Kuhlmann et al., 2000), but nec- ger an egg, the more female-biased was the resulting sex cessary for a risk analysis. Lepidoptera are the main hosts ratio, and we obtained a significant logarithm relation- for Trichogramma (Thomson and Stinner, 1989) and fo- ship that accounted for 76.3% of the variance in the data cussing on Switzerland only, there are 180 potential (r2 ¼ 0:763; Fig. 4). No relationship was found either butterfly hosts which could be tested and several hundred between egg volume and acceptance rate (r2 ¼ 0:049; moth species (Schweizerischer Bund fuur€ Naturschutz, F1;15 ¼ 0:77; P > 0:05) or between egg volume and par- 1987, 1997, 2000). Obviously, this number would increase 2 asitism rate (r ¼ 0:126; F1;15 ¼ 2:16; P > 0:05). if other geographical regions were to be included. Virtually nothing is known about butterflies as po- tential hosts of T. brassicae despite the fact that con- 4. Discussion servation biologists are much concerned about this group. In the present study, we therefore tested 21 Trichogramma egg parasitoids are regarded as being non-target butterfly species including nine species listed highly polyphagous (Fulmek, 1955; Hirai, 1988; Kot, on the Red List (Duelli, 1994) from seven different D. Babendreier et al. / Biological Control 26 (2003) 128–138 135 families as well as two sphingids. We focussed on the from the target in corn and higher than offspring last two steps in the host selection process (cf. Vinson, emerging from the factitious host, E. kuehniella, which is 1976; Vinson, 1991) (i.e., host acceptance and host used for commercial mass releases. Larger females have suitability) and could show that T. brassicae attacks also been shown to walk faster than their smaller and is able to develop inside eggs of many butterfly counterparts (Bigler, 1989) which might translate into species. From the nine rare species that were included higher searching abilities (Kazmer and Luck, 1995). in our analysis, we found that six were parasitized at a Furthermore, the present study has shown that larger similar or even higher level than the target. Our results females have a higher oviposition success on eggs with a show that T. brassicae may parasitize several species thick chorion. Altogether, larger females can be ex- within a family or subfamily of butterflies while certain pected to find and parasitize more non-target butterflies species of the same group are not parasitized. For outside release fields, which has to be taken into account instance, M. diamina was not attacked at all while a for a risk analysis. close relative, M. athalia, sustained 50% parasitism and Acceptance of hosts is a subtle process and results other members of the family were attacked to nearly obtained in the laboratory with confined adult wasps 100%. can lead to an overestimation of the parasitoidÕs host Orr et al. (2000) have tested T. brassicae on several range. We propose that observation of female behavior lepidopterans, but focussed mainly on moths. Their re- on potential non-target host eggs is important and sults are in part consistent with ours, but there are also confinement should be avoided. As an example, obser- some differences. For instance, Orr et al. (2000) found vations on A. napi showed that a high rate of females left that a lycaenid, Everes comyntas Godart, was a poor the egg before final acceptance indicating that this host host while all lycaenids in our study were high quality will be less parasitized under natural conditions, where hosts. The satyrid they tested was a high quality host many more stimuli are present. This would be in like all but one of the satyrid species tested in our study. agreement with the findings of Orr et al. (2000) where Performance of adults was high for all of the non- pierids were less suitable hosts for T. brassicae. It was target species where emergence of parasitoids was ob- also important to note that some butterfly hosts (e.g., served. Offspring reared from all non-target species was M. parthenoides) were not attractive, although they were larger than those reared from the factitious host, E. suitable for Trichogramma development. Other hosts kuehniella, which will be released in maize fields. Off- were attractive, but the parasitization success was low spring raised from non-targets were of similar size or (e.g., S. ligustri). We conclude from our data that direct larger than offspring that emerged from the European observational experiments provide additional informa- corn borer. Such T. brassicae adults will be present in tion to simple no-choice tests in the laboratory, which the field later in the season after successful development could be overestimating acceptance of non-target hosts in parasitized eggs of the corn borer. Adult size is gen- (cf. Sands, 1997). erally believed to be a predictor for female fecundity and Most eggs of M. parthenoides and M. diamina and longevity, the most important parameters for female part of M. athalia eggs were rejected after only a few fitness (Godfray, 1994). This has been demonstrated for seconds of drumming or even before females contacted Trichogramma in laboratory (e.g., Kuhlmann and Mills, the host. All the butterflies mentioned above feed on 1999; Olson and Andow, 1998; Waage and Ming, 1984) species during their larval development and and field studies (Kazmer and Luck, 1995). According this plant genus is known to contain iridoid glycosides. to the data of Waage and Ming (1984) for T. evanescens, Camara (1997) has shown that sequestration of these a close relative of T. brassicae, the differences in hind chemicals by larvae of the buckeye butterfly, Junonia tibia length found in the present study would translate coenia Huubner,€ resulted in significantly lower pre- to a three-fold higher fecundity for the offspring of the dation and van Nouhuys and Hanski (1999) showed for largest non-target host egg (Sphinx ligustri) compared iridoid glycoside-containing larvae of the Glanville with offspring from the rearing host, E. kuehniella (see fritillary butterfly, Melitaea cinxia L., a reduced risk of also Mansfield and Mills, 2002; Mills and Kuhlmann, parasitization by a larval specialist parasitoid. Assuming 2000). The sex ratio was female biased in all tested non- that eggs also contain iridoid glycosides, this could be target species and this bias increased significantly with responsible for the deterring effect on T. brassicae fe- host egg size in a nonlinear manner. This result is in males. agreement with predictions of local mate competition Direct observations furthermore revealed that the (Colazza and Wajnberg, 1998; Hardy et al., 1998; Wa- chorion of certain non-target hosts is a rather strong age and Ming, 1984), and was also found in other barrier for probing Trichogramma females. Suzuki et al. studies on Trichogramma (Schmidt, 1994; Waage, 1986). (1984) already noticed the prolonged period of drilling From the data on size and sex ratio, we expect that the of about 400 s by Trichogramma minutum Riley on eggs fitness of parasitoid offspring emerging from non-target of Papilio xuthus L., which is less than we observed on species is in general similar or higher than that emerging P. machaon by T. brassicae. Similarly, in our study T. 136 D. Babendreier et al. / Biological Control 26 (2003) 128–138 brassicae needed longer for the penetration of sphingid cluding species endangered in Switzerland are readily eggs than was observed for T. minutum on eggs of attacked by female T. brassicae should be recognized as Manduca sexta L. (Schmidt and Pak, 1991). Recently, a cautionary signal although the findings of Babendreier Mansfield and Mills (2002) reasoned that chorion et al. (2002) under semifield and field conditions indicate strength was an important factor limiting successful low risks for the Swiss butterfly fauna. oviposition by T. platneri Nagarkatti. Some sphingid eggs were rejected after females had inserted their ovi- positor into the host egg. One possible explanation is Acknowledgments provided by Olson (1998) who found that Trichogramma nubilale females preferentially oviposited near the em- We thank Heinz Rothacher, Richard Baltensperger bryo of O. nubilalis eggs and that survival of eggs laid and David Jutzeler for providing some of the eggs tested far from the embryo was low. Possibly, T. brassicae fe- in this study. The help of Mario Waldburger with the males rejected sphingid eggs because they could not lo- rearing of butterflies was greatly appreciated and Ste- cate the embryo in such large eggs. phan Bosshart provided the parasitoids at every point in How indicative are laboratory host specificity tests time. We also thank Joorg€ Romeis, Heiri Klein, Sabine and can these be used for evaluating what might happen Keil and Michael Winzeler (Zurich, Switzerland) for in the field? The degree of host specificity per se is im- helpful comments on earlier versions of the manuscript. portant and results from laboratory studies might be This study was funded by BBW/EU-FAIR project more easy to interpret for parasitoids with a narrow host number FAIR5-CT97-3489. range than for polyphagous parasitoid species like most Trichogramma spp., given that not all host species can be tested. Barratt et al. (1997) have shown for braconid References parasitoids with both a broad host range (Microctonus aethiopoides Loan) and a narrow host range (M. hy- Andow, D.A., Lane, C.P., Olson, D.M., 1995. Use of Trichogramma in perodae Loan) that laboratory host range testing is in- maize—estimating environmental risks. In: Lynch, J.M., Hokka- dicative of non-target parasitism in the field. nen, H.H. (Eds.), Benefits and Risks of Introducing Biocontrol Babendreier et al. (2002) have recently shown that hosts Agents. Cambridge University Press, New York, pp. 101–118. attacked in the laboratory are also parasitized under Babendreier, D., Kuske, S. and Bigler F., 2002. Parasitism of non- target butterflies by Trichogramma brassicae Bezdenko (Hymenop- field cage and field conditions, but parasitism rates were tera: Trichogrammatidae) under field cage and field conditions. found to be low. Similar results were obtained by Orr Biol. Control, in press. et al. (2000) showing that parasitism of non-target le- Barratt, B.I.P., Evans, A.A., Ferguson, C.M., Barker, G.M., McNeill, pidopterans by T. brassicae was low if parasitoids are M.R., Phillips, C.B., 1997. Laboratory nontarget host range of the released into non-target habitats despite the fact that the introduced parasitoids Microctonus aethiopoides and M. hyperodae (Hymenoptera: ) compared with field parasitism in New same hosts were well accepted in the laboratory. The Zealand. Environ. Entomol. 26, 694–702. latter two studies well document that it is neccessary to Barratt, B.I.P., Ferguson, C.M., McNeill, M.R., Goldson, S.L., 1999. distinguish between the fact that a non-target host is Parasitoid host specificity testing to predict field host range. In: attacked in the field and impact on non-target host Withers, T.M., Barton Browne, L., Stanley, J.N. (Eds.), Host populations. Specificity Testing in Australasia: Towards Improved Assays for Biological Control. CRC for Tropical Pest Management, Brisbane, Higher parasitization success might occur, however, Australia, pp. 70–83. if T. brassicae is present in a particular habitat for more Battisti, A., 1991. Accounts on the bio-ecology of Trichogramma than one generation because learning and adaptation cephalciae Hochmuth and Martinek in N-E Italy (Hym.: Tricho- processes are well described for Trichogramma spp. grammatidae). Redia 74, 179–184. Rearing a parasitoid on a particular host has shown to Bigler, F., 1986. Mass production of Trichogramma maidis Pint. et Voeg. and its field application against Ostrinia nubilalis Hbn. in be important (Kaiser et al., 1989; Taylor and Stern, Switzerland. J. Appl. Entomol. 101, 23–29. 1971). Also, the experience of oviposition on a host may Bigler, F., 1989. Quality assessment and control in entomophagous influence preference behavior of adult wasps (Bjorksten insects used for biological control. J. Appl. Entomol. 108, 390–400. and Hoffmann, 1998a; Brodeur and Rosenheim, 2000; Bjorksten, T.A., Hoffmann, A.A., 1998a. Persistence of experience Kaiser et al., 1989). Bjorksten and Hoffmann (1998b) effects in the parasitoid Trichogramma nr. brassicae. 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