Non-target host acceptance and parasitism by Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) in the laboratory

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

Non-target host acceptance and parasitism by Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) 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 Trichogramma brassicae 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 & Schifferm€ uller, Clossiana titania Esper, (Nymphalidae), Aphantopus hyperanthus L., Maniola jurtina L., Coenonympha pamphilus L., Melanargia galathea L., Erebia ligea L., Hipparchia alcyone Denis & Schifferm€ uller (Satyridae), Polyommatus icarus Rottemburg and Plebejus idas L. (Lycaenidae) were well accepted and not parasitized significantly different (range 73–94%) than the target, Ostinia nubilalis H€ ubner (81%). Virtually all eggs of Vanessa atalanta L., Argynnis niobe L., Clossiana selene L. (Nymphalidae), and Cyaniris semiargus Rottemburg (Lycaenidae) were accepted for oviposition resulting in significantly higher parasitism rates of 94–97% compared with the target. Melitaea 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 parasitoids; Endangered butterflies

1. Introduction Since the beginning of biological control, more than 5200 agent introductions against economically important insect pests have been made (Greathead and Greathead, 1992, updated 2000; Waage, 1990). Egg parasitoids of the genus Trichogramma have been used successfully as inundative biological control agents against a range of agricultural pests, mainly lepidopterans and are the most widely used natural enemies in * Corresponding author. Fax: +41-1-377-72-01. E-mail address: [email protected] (D. Babendreier).

biological control worldwide (Li, 1994; van Lenteren, 2000). While in some cases native species were mass reared and released, in many cases the utilized Trichogramma species were exotic. Trichogramma brassicae Bezdenko was introduced 30 years ago from Moldavia (former Soviet Union) to control the European corn borer, Ostrinia nubilalis H€ ubner (Lepidoptera: Crambidae), in several parts of Western Europe. This was a complete success which markedly reduced the pesticide levels applied in maize in those countries (Bigler, 1986; Hassan, 1988). In other parts of the world, the use of Trichogramma spp. has also led to occasional spectacular successes (Li, 1994; Smith, 1996).

1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 9 - 9 6 4 4 ( 0 2 ) 0 0 1 2 1 - 4

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Biological control of insect pests has been viewed as environmentally safe for much of its history. Over the last two decades, concerns about possible detrimental effects for native fauna have been raised by ecologists (Howarth, 1983). Several papers have since reviewed the cases where non-target effects have been observed but, more importantly, highlighted the fact that few studies on non-target effects were conducted and effects due to exotic biological control agents may have been overlooked (Howarth, 1991; Lynch et al., 2001; Simberloff and Stiling, 1996; Stiling and Simberloff, 2000). One of the key parameters determining non-target effects is the range of species an agent is able to attack. It is generally accepted that the release of agents with restricted host ranges should be favored in order to minimize potential non-target effects. Therefore, host specificity testing has become a central issue in projects on biological control of insect pests. However, methods to test the host specificity of biological control agents have been refined significantly for weeds (McEvoy, 1996) but not so for insects. There is an ongoing debate on this issue with the aim to improve the predictability of non-target attack after introduction (Barratt et al., 1999; Kuhlmann et al., 2000). The vast majority of Trichogramma species are known to be fairly polyphagous, attacking a wide range of lepidopterans and even species of other insect orders (e.g., Clausen, 1940; Pinto and Stouthamer, 1994; Thomson and Stinner, 1989). Information on the host range of T. brassicae in general is scarce and virtually absent regarding economically unimportant species. In contrast with the current practice of biological control of insect pests, the host range of T. brassicae had not been evaluated prior to introduction to Western Europe because non-target effects were not considered an important issue at that time. Because most Trichogramma are known to be highly polyphagous and are currently released annually on several million ha worldwide, information on potential non-target effects due to Trichogramma is in demand. Andow et al. (1995), using a theoretical approach based partially on empirical data, could show that the Karner blue, an endangered butterfly in the US inhabiting oak savanna, faces only a small risk due to mass releases of Trichogramma nubilale Ertle & Davis in corn. Preliminary data provided by Suverkropp (unpublished OECD report, 1997) showed that T. brassicae parasitized some non-target host eggs (one undetermined nymphalid and Pterostoma palpinum Clerck (Lepipoptera: Notodontidae)) in the vicinity of release fields in Switzerland, but the rate of parasitism was 4% or less. Only recently Orr et al. (2000) published data on the potential host range of T. brassicae. In laboratory trials, these authors found that this parasitoid is able to attack several Lepidopterans potentially occurring near cornfields in the Midwestern

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US. However, very few butterflies and no rare species were included in the analyses. Virtually no information on parasitism by Trichogramma spp. of non-target butterflies, especially endangered species, is available. Therefore, and because conservationists are much concerned about this group of non-target insects, we did not select host species from all potential taxa but rather focused on butterflies. As a first step of a general risk assessment procedure, we investigated whether eggs of potentially susceptible non-target species were attacked by T. brassicae in the laboratory. We tested T. brassicae on butterfly species from all major families occuring in Europe and included species recorded on the Swiss Red List of Endangered Species (Duelli, 1994). In addition, the performance of parasitoid offspring within non-target host eggs was investigated because this might have important implications for the risk of butterfly populations. This study should provide a first indication as to which species of butterflies are potentially at risk and should also serve to identify species that have to be further evaluated under field conditions.

2. Materials and methods 2.1. Trichogramma brassicae strain Trichogramma brassicae used in this study originated from a colony that was maintained at the Swiss Federal Research Station for Agroecology and Agriculture for about 60 generations on European corn borer eggs. Prior to experiments, parasitoids were reared for 4–5 generations on the Mediterranean flour moth, Ephestia kuehniella Zeller, at 16:8 (L:D) h, 25 °C, and 60–70% RH. O. nubilalis egg masses laid on wax paper were obtained from the French Agricultural Research, Lambert (US), every four weeks and then stored at 4 °C, 85% RH. E. kuehniella eggs were provided by Biotop, Valbonne, France, fortnightly and stored under the same conditions. In order to verify the identity of our T. brassicae rearing strain, material from our colony was sent to Wageningen Agricultural University, NL, periodically where it was identified by PCR. About 5000 parasitized eggs were put in a ventilated container of 1.3 L from which T. brassicae adults could emerge. Adult parasitoids were stored at 25  0:5 °C and 70  5% RH and provided with small droplets of honey. One day-old mated females were used for the experiments. 2.2. Selection of butterfly species Non-target host species selection was based mainly on ecological criteria as well as on habitat and temporal overlap of hosts and inundatively released T. brassicae. Eggs of the following 23 non-target lepidopteran species were tested in the present study, including nine species

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on the list of endangered species in Switzerland (marked with an asterisk throughout the text): Papilio machaon L. (Papilionidae), Artogeia ( ¼ Pieris) napi L. (Pieridae), Vanessa atalanta L., Argynnis adippe Denis & Schifferm€ uller*, A. niobe L.*, Clossiana titania Esper*, C. selene L.*, Mellicta athalia Rottemburg*, Melitaea parthenoides Keferstein*, M. diamina Lang*, (Nymphalidae), Aphantopus hyperanthus L., Maniola jurtina L., Melanargia galathea L., Coenonympha pamphilus L., Erebia ligea L., Hipparchia alcyone Denis & Schifferm€ uller* (Satyridae), Polyommatus icarus Rottemburg, Cyaniris semiargus Rottemburg, Plebejus idas L.* (Lycaenidae), Zygaena filipendula L. (Zygaenidae), Hesperia comma L. (Hesperidae), Sphinx ligustri L., and Deilephila elpenor L. (Sphingidae). In addition, we tested egg masses of the target O. nubilalis as well as individual eggs and egg masses of E. kuehniella. Most of these species were obtained from field collections. However, A. niobe*, A. adippe*, M. parthenoides*, and H. alcyone* adults were reared from eggs and S. ligustri as well as D. elpenor from pupae that were commercially obtained. Adult butterflies were kept in gauze cages together with their respective host plant. Various flowers were provided as a food source together with a mixture of yeast, honey, and water smeared in patches on the cage wall. Both food sources were replenished every second day. The size of the cages varied depending on the size and behavior of the butterfly in the range of 20  20  30 cm (e.g., for M. athalia*) to 50  60  80 cm (e.g., for S. ligustri). Cages were placed in a greenhouse at 25  2 °C and 65  10% RH. Most tested host species attached their eggs on the gauze of the rearing cage. These were carefully removed with a brush between 08:00 and 12:00 h daily and stored at 6 °C for a maximum of 7 days until the start of experiments. However, P. machaon laid their eggs on carrot leaves and the three lycaenids (P. icarus, C. semiargus, and P. idas*) on blossoms or leaves of red clover. Because these eggs were difficult to remove from the plant, small pieces of carrot leaves or clover blossoms were used in the experiments for the abovementioned species. 2.3. No-choice tests In the present study, we used no-choice tests because these would reflect the chance of parasitism in the field more accurately than choice tests, because it is unlikely that the eggs of the target host O. nubilalis are in very close proximity to eggs of non-targets. Individual host eggs were attached to cardboard strips of 5  10 mm, placed in a petri dish of 40 mm diameter, and small numbers of 5–10 T. brassicae females that had not been exposed to host eggs prior to the experiments were introduced. The egg was continuously observed and the behavior of the female recorded as soon as she came in contact with the host egg. If additional females ap-

proached the egg, they were carefully removed before any interaction could occur. The arena was not covered by a lid at any time and thus the female was free to leave. Behavioral parameters measured were host inspection (contact with the egg and drumming with the antennae) and contact of the host egg with the ovipositor. In addition, we recorded whether and how often females left the egg during the host inspection process. If the egg was abandoned for more than 60 s the trial was terminated and the egg was regarded as being rejected. An egg was regarded as being accepted if the female drilled and/or penetrated the egg continuously with her ovipositor for longer than 60 s (cf., Schmidt, 1994). Subsequently, the cardboard strip with the host egg and the female parasitoid were carefully removed to prevent the attack of conspecifics. This setup was checked every 5–10 min and as soon as the female was not found anymore on the host egg or the cardboard strip, the egg was transferred into a glass vial of 2.5 ml and stored at 25 °C and 70% RH until parasitoid emergence. This experimental design enabled us to calculate the host inspection time (time that females contacted the host egg before it was accepted) as well as the number of times a female left the egg before its acceptance. Both measurements will give an indication about the acceptance of eggs of non-target species. According to Van Dijken et al. (1987), we calculated a ratio between the number of times a host egg was contacted and the number of eggs that were accepted (a/c ratio). Between 18 and 56 females were tested on each of the tested species. Parasitism was calculated from blackened eggs after 5 days and emergence of parasitoid offspring assessed. For each tested lepidopteran species, a similar number of nontarget host eggs were reared under identical conditions without exposure to T. brassicae to check for emerging larvae. On each day that a non-target species was tested, we tested T. brassicae females on batches of approximately 30 E. kuehniella eggs glued on cardboard. If a female contacted an egg batch she was allowed to parasitize for 15 min. Subsequently, the females were removed and the eggs reared under the same conditions as eggs of the test species (25 °C, 70% RH). E. kuehniella eggs were checked for parasitism after 5–7 days. We performed data analyses for the corresponding non-target species only if at least 75% of females successfully parasitized within the first 15 min. For species that lay their eggs in batches (e.g., Melitaea spp.) and that were hardly accepted, we also tested T. brassicae on naturally laid egg batches to elucidate potential confounding effects due to the fact that single eggs were offered to females. All experiments were carried out at 25  1 °C. The number of emerged offspring was counted and the sex determined under a microscope (magnification 24 or 50). In addition, we measured maximum length and width of the eggs (magnification 125) and calculated a volume using a sphere as a model. Because the size of Trichogramma offspring may affect

D. Babendreier et al. / Biological Control 26 (2003) 128–138

host acceptance and thus non-target effects, the hind tibia length of T. brassicae emerged from several representative species (including those with very small and very large eggs) was measured: from each parasitized egg where female offspring had emerged we measured one hind tibia of a randomly selected female under a dissecting microscope (magnification 125) to the nearest 0.01 mm (see Olson and Andow, 1998). 2.4. Penetration of egg chorion For the two sphingid species, D. elpenor and S. ligustri, where we obtained a high rate of acceptance, but a low rate of successful parasitization, we carried out trials with continuous observation throughout the whole oviposition event to observe whether females were able to penetrate the egg chorion and whether any eggs were deposited in the host. Oviposition was noted to have occurred if the typical movements with the abdomen (as described in detail by Suzuki et al., 1984) were observed. Under the same experimental conditions as above, single T. brassicae females were observed on single eggs continuously as soon as she came in contact with the host egg. We observed whether females penetrated the chorion, the time this took the female and whether oviposition occurred. In addition, we investigated whether there was a difference between large and small females in their ability to penetrate the thick chorion of S. ligustri. For this experiment, we reared T. brassicae on eggs of Papilio machaon, which resulted in significantly larger adult wasps. Small Trichogramma wasps were obtained from rearing on E. kuehniella. Again, we observed whether females penetrated the chorion, the time this took the female and whether oviposition occurred in continuous observations of single females. 2.5. Statistical analyses Non-target parasitism was compared with that of the target using a v2 test. Hind tibia length was analyzed using a single factorial ANOVA and the Newman–Keuls test. Regression analysis was carried out to determine whether egg volume and host inspection time are related to non-target host acceptance or parasitism. All percentages were arcsin transformed prior to analysis. Handling times were compared using the non-parametric Mann–Whitney U test. All statistical analyses were carried out with the Statistica software package.

3. Results 3.1. Acceptance Overall, 25 lepidopteran species, including the target, O. nubilalis, and the factitious rearing host, E. kuehni-

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ella, were exposed to individual T. brassicae females. Most T. brassicae females accepted egg masses of the target species O. nubilalis (84%) and 81.3% of the egg masses were successfully parasitized. E. kuehniella was only well accepted if eggs were offered as clusters to parasitoid females whereas single eggs showed a low acceptance rate and were significantly less parasitized (Fig. 1). Eggs of the majority of non-target species were readily accepted with acceptance rates between 75 and 100% (Fig. 1). Only Melitaea parthenoides* and M. diamina* were rejected by T. brassicae females very early in the host selection process after drumming the egg for a few seconds or even at 1–3 mm distance without coming into contact with the egg. Only 8.9% of M. parthenoides* eggs and no M. diamina* eggs were accepted during the host selection process by T. brassicae females. This behavior did not change if egg masses were provided instead of single eggs (n ¼ 10 for each species). The eggs of Mellicta athalia* were accepted at an intermediate level of 61%. 3.2. Parasitism Twelve species had parasitism rates not significantly different from that of the target, O. nubilalis (Fig. 1). Four species (Vanessa atalanta, Argynnis niobe*, Clossiana selene*, and Cyaniris semiargus) were accepted by T. brassicae females at rates close to 100%, and parasitism rates observed were significantly higher than that of the target. Significantly lower parasitism rates compared with the target were obtained for Mellicta athalia*, Melitaea parthenoides*, M. diamina*, Zygaena filipendula, Hesperia comma, Sphinx ligustri, and Deilephila elpenor (Fig. 1). With the exception of M. athalia* and Z. filipendula having intermediate values of 50 and 30% parasitism, respectively, all these species were parasitized at a very low level of 0–13.5%. Interestingly, we obtained parasitoid offspring from all host species where any acceptance could be observed. Although many females rejected eggs of M. athalia* and only 8.9% of M. parthenoides* eggs were attacked by female wasps, these were suitable hosts as well because nearly all accepted eggs gave viable offspring. In contrast, low parasitism rates were obtained for H. comma and the two sphingids, S. ligustri and D. elpenor, despite high acceptance rates. 3.3. Penetration of the egg chorion Continuous observations of T. brassicae females on eggs of S. ligustri and D. elpenor revealed that only 40% of the females were able to penetrate the chorion of D. elpenor eggs and only 12% actually oviposited ðn ¼ 25Þ. The remaining females either did not penetrate the chorion or withdrew the ovipositor after 1–2 min. The females needed 2305  410 s SD ðn ¼ 10Þ to penetrate the

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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 lepidopterans 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 the egg at another location (mean: 1:5 0:7), but in no case did we observe a female penetrating the egg chorion successfully during the second drilling period and females never tried to penetrate the egg for longer than 60 s at the third attempt. In all cases where no oviposition was observed, healthy host larvae emerged. Hind tibia of T. brassicae females reared from E. kuehniella measured 0:138  0:0085 mm ðn ¼ 25Þ and were significantly smaller than those reared from Papilio machaon (0:174  0:0125 mm, n ¼ 22, t ¼ 11:6, df ¼ 1, P < 0:001). The proportion of females successfully penetrating 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 successfully parasitized eggs of S. ligustri, 82% of the females 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 penetrate the chorion of S. ligustri eggs (2882  1041 s; n ¼ 7) for T. brassicae reared on E. kuehniella and for T. brassicae reared on P. machaon (2477  1032 s; n ¼ 19) (U19;7 ¼ 49; P ¼ 0:31). In all cases where no oviposition had been observed, the host larvae emerged.

3.4. Rejection rate In addition to the acceptance rates, we calculated an acceptance/contact ratio (Table 1). We hypothesized that females may leave less preferred eggs more often than preferred ones which would lead to a low a/c ratio and to a lower risk for non-target effects. Again, as with acceptance, species can be divided into distinct groups.

Fig. 2. Success of smaller Trichogramma brassicae females (reared from Ephestia kuehniella) and larger females (reared from Papilio machaon) to penetrate and oviposit into eggs of Sphinx ligustri (v2 test procedure; *P < 0:05, **P < 0:01).

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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

N1

Host inspection time  SD (s)

N2

Ostrinia nubilalis Ephestia kuehniella Papilio machaon Artogeia napi Vanessa atalanta Argynnis adippe A. niobe Clossiana titania C. selene Mellicta athalia Melitaea parthenoides Aphantopus hyperanthus Maniola jurtina Hipparchia alcyone Melanargia galathea Coenonympha pamphilus Erebia ligea Polyommatus icarus Cyaniris semiargus Plebejus idas Zygaena filipendula Hesperia comma Sphinx ligustri Deilephila elpenor

0.45 * 0.97 0.67 0.96 1.00 1.00 1.00 0.89 0.76 * 0.88 0.91 0.85 0.92 0.92 0.95 0.94 0.97 0.93 0.90 0.89 1.00 1.00

32 30 29 22 52 30 34 23 31 36 56 30 33 32 45 27 18 32 31 28 46 32 37 40

160.8  59.0 39.8  9.7 50.0  19.3 50.5  17.7 41.7  16.0 53.7  21.8 48.3  21.4 34.8  9.1 41.7  15.6 23.1  7.3 33.0  11.5 38.7  16.7 45.2  17.8 66.0  25.6 55.6  24.4 38.8  15.4 56.4  28.7 61.5  26.2 47.2  25.6 35.0  13.4 51.0  22.8 45.1  16.8 84.2  23.8 64.5  19.2

27 4 29 20 50 29 33 22 31 22 5 30 32 29 43 24 18 30 30 25 36 31 34 37

*, number of accepted eggs too low for calculation. a 1 N , 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 and parasitism rates, parasitoid females rarely left a host egg even once until acceptance and showed a/c ratios of 1 or close to 1 (Table 1). The lowest a/c ratio was observed in the target, O. nubilalis; 66% of females that eventually accepted an egg mass rejected it at the first encounter. A. napi also showed a low a/c ratio and a parasitism rate similar to that of the target. M. athalia* showed a moderately low a/c ratio and low parasitism rates. In contrast, virtually all T. brassicae females accepted 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 parasitism (r2 ¼ 0:009; F1;15 ¼ 0:134; P > 0:05). Time from the first contact until acceptance (ovipositor 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 egg-size and host inspection time (r2 ¼ 0:616; F1;15 ¼ 24:1; P < 0:001): the larger an egg, the longer females took until acceptance. No relationship was found between host inspection time and acceptance (r2 ¼ 0:129;

F1;15 ¼ 2:22; P > 0:05) and inspection time and parasitism (r2 ¼ 0:014; F1;15 ¼ 0:216; P > 0:05). 3.5. Performance on host eggs Emergence rate of parasitoid offspring based on blackened ( ¼ parasitized) eggs was 100% or very close to 100% for all tested non-target species as well as for O. nubilalis and E. kuehniella (range 96–100%). Wing

Fig. 3. Hind tibia length (+SD) of Trichogramma brassicae females reared from eggs of various lepidopteran species. The number of females measured is given in brackets for each species. Different letters above the bars indicate significant differences (one-way ANOVA with Newman–Keuls test procedure; P < 0:05).

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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 ðmm3 Þ  SD

No. of adults emerged  SD

Females (%)

N

Ostrinia nubilalis Ephestia kuehniella Papilio machaon Artogeia napi Vanessa atalanta Argynnis niobe Clossiana titania C. selene Mellicta athalia Melitaea parthenoides Aphantopus hyperanthus Maniola jurtina Hipparchia alcyone Melanargia galathea Coenonympha pamphilus Erebia ligea Polyommatus icarus Plebejus idas Sphinx ligustri Deilephila elpenor

* 0:056  0:007 0:91  0:058 * 0:21  0:028 0:33  0:039 0:30  0:045 0:30  0:044 0:20  0:014 0:16  0:006 0:26  0:026 0:23  0:025 0:99  0:111 1:14  0:097 0:32  0:027 0:55  0:076 0:10  0:006 0:10  0:008 4:54  0:245 1:90  0:227

1.6  0.25 1.0  0 11.6  2.1 4:1  1:2 4.1  1.8 5.1  1.6 4.4  0.85 5.3  1.7 3.4  0.49 2.3  0.50 5.2  1.6 4.0  0.83 14.7  3.8 17.6  3.6 4.25  1.1 8.3  2.9 1.8  0.43 2.0  0.47 38.9  10.7 20.9  4.3

66.4 63.3 70.9 66.6 65.9 69.2 69.1 68.9 65.1 ** 69.9 62.5 67.0 71.6 60.3 69.9 54.5 56.3 81.1 79.0

27 30 27 16 47 33 22 30 17 4 26 24 26 33 16 18 27 19 15 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 significantly 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 between egg volume and number of Trichogramma adults per egg (r2 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 tested) and 0.82 for S. ligustri which laid the largest eggs (Table 2). Consequently, we found a significant relationship between egg volume and the sex ratio. The larger an egg, the more female-biased was the resulting sex ratio, and we obtained a significant logarithm relationship that accounted for 76.3% of the variance in the data (r2 ¼ 0:763; Fig. 4). No relationship was found either between egg volume and acceptance rate (r2 ¼ 0:049; F1;15 ¼ 0:77; P > 0:05) or between egg volume and parasitism rate (r2 ¼ 0:126; F1;15 ¼ 2:16; P > 0:05).

4. Discussion Trichogramma egg parasitoids are regarded as being highly polyphagous (Fulmek, 1955; Hirai, 1988; Kot,

Fig. 4. The relationship between the egg size of non-target lepidopterans and the sex ratio of offspring that emerged from parasitized host eggs. Each measurement is based on 13–47 females (see Table 2).

1964) although exceptions to this rule have also been found (Battisti, 1991; Bourchier et al., 2000). To assess the complete host range for polyphagous parasitoids is difficult and laborious (Kuhlmann et al., 2000), but neccessary for a risk analysis. Lepidoptera are the main hosts for Trichogramma (Thomson and Stinner, 1989) and focussing on Switzerland only, there are 180 potential butterfly hosts which could be tested and several hundred moth species (Schweizerischer Bund f€ ur Naturschutz, 1987, 1997, 2000). Obviously, this number would increase if other geographical regions were to be included. Virtually nothing is known about butterflies as potential hosts of T. brassicae despite the fact that conservation biologists are much concerned about this group. In the present study, we therefore tested 21 non-target butterfly species including nine species listed on the Red List (Duelli, 1994) from seven different

D. Babendreier et al. / Biological Control 26 (2003) 128–138

families as well as two sphingids. We focussed on the last two steps in the host selection process (cf. Vinson, 1976; Vinson, 1991) (i.e., host acceptance and host suitability) and could show that T. brassicae attacks and is able to develop inside eggs of many butterfly species. From the nine rare species that were included in our analysis, we found that six were parasitized at a similar or even higher level than the target. Our results show that T. brassicae may parasitize several species within a family or subfamily of butterflies while certain species of the same group are not parasitized. For instance, M. diamina was not attacked at all while a close relative, M. athalia, sustained 50% parasitism and other members of the family were attacked to nearly 100%. Orr et al. (2000) have tested T. brassicae on several lepidopterans, but focussed mainly on moths. Their results are in part consistent with ours, but there are also some differences. For instance, Orr et al. (2000) found that a lycaenid, Everes comyntas Godart, was a poor host while all lycaenids in our study were high quality hosts. The satyrid they tested was a high quality host like all but one of the satyrid species tested in our study. Performance of adults was high for all of the nontarget species where emergence of parasitoids was observed. Offspring reared from all non-target species was larger than those reared from the factitious host, E. kuehniella, which will be released in maize fields. Offspring raised from non-targets were of similar size or larger than offspring that emerged from the European corn borer. Such T. brassicae adults will be present in the field later in the season after successful development in parasitized eggs of the corn borer. Adult size is generally believed to be a predictor for female fecundity and longevity, the most important parameters for female fitness (Godfray, 1994). This has been demonstrated for Trichogramma in laboratory (e.g., Kuhlmann and Mills, 1999; Olson and Andow, 1998; Waage and Ming, 1984) and field studies (Kazmer and Luck, 1995). According to the data of Waage and Ming (1984) for T. evanescens, a close relative of T. brassicae, the differences in hind tibia length found in the present study would translate to a three-fold higher fecundity for the offspring of the largest non-target host egg (Sphinx ligustri) compared with offspring from the rearing host, E. kuehniella (see also Mansfield and Mills, 2002; Mills and Kuhlmann, 2000). The sex ratio was female biased in all tested nontarget species and this bias increased significantly with host egg size in a nonlinear manner. This result is in agreement with predictions of local mate competition (Colazza and Wajnberg, 1998; Hardy et al., 1998; Waage and Ming, 1984), and was also found in other studies on Trichogramma (Schmidt, 1994; Waage, 1986). From the data on size and sex ratio, we expect that the fitness of parasitoid offspring emerging from non-target species is in general similar or higher than that emerging

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from the target in corn and higher than offspring emerging from the factitious host, E. kuehniella, which is used for commercial mass releases. Larger females have also been shown to walk faster than their smaller counterparts (Bigler, 1989) which might translate into higher searching abilities (Kazmer and Luck, 1995). Furthermore, the present study has shown that larger females have a higher oviposition success on eggs with a thick chorion. Altogether, larger females can be expected to find and parasitize more non-target butterflies outside release fields, which has to be taken into account for a risk analysis. Acceptance of hosts is a subtle process and results obtained in the laboratory with confined adult wasps can lead to an overestimation of the parasitoidÕs host range. We propose that observation of female behavior on potential non-target host eggs is important and confinement should be avoided. As an example, observations on A. napi showed that a high rate of females left the egg before final acceptance indicating that this host will be less parasitized under natural conditions, where many more stimuli are present. This would be in agreement with the findings of Orr et al. (2000) where pierids were less suitable hosts for T. brassicae. It was also important to note that some butterfly hosts (e.g., M. parthenoides) were not attractive, although they were suitable for Trichogramma development. Other hosts were attractive, but the parasitization success was low (e.g., S. ligustri). We conclude from our data that direct observational experiments provide additional information to simple no-choice tests in the laboratory, which could be overestimating acceptance of non-target hosts (cf. Sands, 1997). Most eggs of M. parthenoides and M. diamina and part of M. athalia eggs were rejected after only a few seconds of drumming or even before females contacted the host. All the butterflies mentioned above feed on Plantago species during their larval development and this plant genus is known to contain iridoid glycosides. Camara (1997) has shown that sequestration of these chemicals by larvae of the buckeye butterfly, Junonia coenia H€ ubner, resulted in significantly lower ant predation and van Nouhuys and Hanski (1999) showed for iridoid glycoside-containing larvae of the Glanville fritillary butterfly, Melitaea cinxia L., a reduced risk of parasitization by a larval specialist parasitoid. Assuming that eggs also contain iridoid glycosides, this could be responsible for the deterring effect on T. brassicae females. Direct observations furthermore revealed that the chorion of certain non-target hosts is a rather strong barrier for probing Trichogramma females. Suzuki et al. (1984) already noticed the prolonged period of drilling of about 400 s by Trichogramma minutum Riley on eggs of Papilio xuthus L., which is less than we observed on P. machaon by T. brassicae. Similarly, in our study T.

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brassicae needed longer for the penetration of sphingid eggs than was observed for T. minutum on eggs of Manduca sexta L. (Schmidt and Pak, 1991). Recently, Mansfield and Mills (2002) reasoned that chorion strength was an important factor limiting successful oviposition by T. platneri Nagarkatti. Some sphingid eggs were rejected after females had inserted their ovipositor into the host egg. One possible explanation is provided by Olson (1998) who found that Trichogramma nubilale females preferentially oviposited near the embryo of O. nubilalis eggs and that survival of eggs laid far from the embryo was low. Possibly, T. brassicae females rejected sphingid eggs because they could not locate the embryo in such large eggs. How indicative are laboratory host specificity tests and can these be used for evaluating what might happen in the field? The degree of host specificity per se is important and results from laboratory studies might be more easy to interpret for parasitoids with a narrow host 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 parasitoids with both a broad host range (Microctonus aethiopoides Loan) and a narrow host range (M. hyperodae Loan) that laboratory host range testing is indicative of non-target parasitism in the field. Babendreier et al. (2002) have recently shown that hosts attacked in the laboratory are also parasitized under field cage and field conditions, but parasitism rates were found to be low. Similar results were obtained by Orr et al. (2000) showing that parasitism of non-target lepidopterans by T. brassicae was low if parasitoids are released into non-target habitats despite the fact that the same hosts were well accepted in the laboratory. The latter two studies well document that it is neccessary to distinguish between the fact that a non-target host is attacked in the field and impact on non-target host populations. Higher parasitization success might occur, however, if T. brassicae is present in a particular habitat for more than one generation because learning and adaptation processes are well described for Trichogramma spp. Rearing a parasitoid on a particular host has shown to be important (Kaiser et al., 1989; Taylor and Stern, 1971). Also, the experience of oviposition on a host may influence preference behavior of adult wasps (Bjorksten and Hoffmann, 1998a; Brodeur and Rosenheim, 2000; Kaiser et al., 1989). Bjorksten and Hoffmann (1998b) found that even the plant on which the parasitoid emerges could effect searching for hosts. Considering these factors, extrapolation of results on host range from the laboratory to impact in the field is difficult for polyphagous Trichogramma. Nevertheless such experiments in the laboratory are a necessary first step to assess potential non-target impacts (cf., Mansfield and Mills, 2002). Our observation that many butterflies in-

cluding species endangered in Switzerland are readily attacked by female T. brassicae should be recognized as a cautionary signal although the findings of Babendreier et al. (2002) under semifield and field conditions indicate low risks for the Swiss butterfly fauna.

Acknowledgments We thank Heinz Rothacher, Richard Baltensperger and David Jutzeler for providing some of the eggs tested in this study. The help of Mario Waldburger with the rearing of butterflies was greatly appreciated and Stephan Bosshart provided the parasitoids at every point in time. We also thank J€ org Romeis, Heiri Klein, Sabine Keil and Michael Winzeler (Zurich, Switzerland) for helpful comments on earlier versions of the manuscript. This study was funded by BBW/EU-FAIR project number FAIR5-CT97-3489.

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