WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY

Hitch-hiking behavior of Trichogramma wasps on cabbage white butterflies and moths

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No: 08.02 Name: Joop Woelke Period: July 2007 – January 2008 Supervisors: Ties Huigens Bonne Beerda Abstract Many animals use chemical cues for communication and those used within a species are called pheromones. Pheromones can, however, also be eavesdropped (“overheard”) by natural enemies. Some species of cabbage white butterflies are known to use species specific anti-aphrodisiac pheromones: when a couple is mating the male transfers an anti- aphrodisiac to the female, to render her less attractive to other males. Fatouros et al. (2005) found that the small egg parasitoid Trichogramma brassicae eavesdrops on the anti- aphrodisiac benzyl cyanide emitted by mated females of the large cabbage white butterfly Pieris brassicae and hitch-hikes with them to parasitize freshly laid butterfly eggs. This study investigates if eavesdropping on an anti-aphrodisiac pheromone and hitch-hiking with mated female hosts by Trichogramma wasps occurs on multiple species of butterflies and moths in the laboratory and the field. Results from laboratory two-choice bioassays showed that naïve T. brassicae wasps do not only eavesdrop on the anti-aphrodisiac of P. brassicae but also on the anti-aphrodisiac odor (a combination of methyl salicylate and indole) of mated females of the small cabbage white butterfly Pieris rapae. Trichogramma brassicae wasps where shown to hitch-hike with mated female P. rapae butterflies though they prefer mated female P. brassicae butterflies when compared to mated female P. rapae butterflies. On which this preference is based remains unclear as the wasps did not discriminate between the odor profiles of both types of butterflies. Finally, one-choice bioassays revealed that hitch-hiking with mated butterflies of P. rapae and P. brassicae is a female specific strategy in T. brassicae wasps. In the field 777 moths were collected during night catches and 35 moths during day time in and around Wageningen, The Netherlands. Moths were monitored for the presence of parasitic wasps. Only one female Trichogramma brassicae was found, i.e. on a female Xestia c-nigrum moth at night and at a temperature of 7.2 degrees Celsius. To test whether Trichogramma wasps can parasitize moth eggs in the dark at such low temperatures, the parasitism behavior of females of T. brassicae and T. evanescens was tested at different hours of darkness and temperatures. Results indicate that T. brassicae can parasitize moth eggs in the dark during day time, but not at night. Trichogramma evanescens can parasitize moth eggs in the dark during day time and at night. In all cases T. evanescens parasitize more than T. brassicae. For both species the temperature is important: the higher the temperature, the higher the parasitizing rate. Overall, the results indicate that females of a single Trichogramma species can hitch-hike and spy on an anti-aphrodisiac pheromone of multiple host species. This strategy is expected to have evolved frequently in egg-parasitoids.

Key words: Host finding, anti-aphrodisiac pheromone, phoresy, Trichogramma brassicae, Trichogramma evanescens, Pieris rapae, Pieris brassicae, moths, night parasitism

2 Table of Contents

1 Introduction 4

2 Material and methods 7 2.1 Does Trichogramma brassicae eavesdrop on the anti-aphrodisiac odor 7 of Pieris rapae butterflies and specifically hitch-hike with mated P. rapae females? 2.1.1 Olfactory bioassays 7 2.1.2 Mounting bioassays 8 2.2 Does Trichogramma brassicae prefer to hitch-hike with Pieris brassicae 8 butterflies (that lay groups of eggs) compared to Pieris rapae butterflies (that lay single eggs)? 2.2.1 Olfactory bioassays 8 2.2.2 Mounting bioassays 9 2.3 Is hitch-hiking a specific strategy of female Trichogramma brassicae wasps? 9 2.4 Do Trichogramma wasps hitch-hike with moths in the field? 9 2.4.1 Moth selection 9 2.4.2 Night catches 10 2.4.3 Day catches 10 2.4.4 Trichogramma species identification 11 2.4.4.1 DNA extraction 11 2.4.4.2 PCR amplification 11 2.4.4.3 Cloning and sequencing 11 2.5 Do Trichogramma wasps parasitize moth eggs at low temperatures in 12 the dark? 2.6 Statistical analysis 12

3 Results 13 3.1 Does Trichogramma brassicae eavesdrop on the anti-aphrodisiac odor 13 of Pieris rapae butterflies and specifically hitch-hike with mated P. rapae females? 3.2 Does Trichogramma brassicae prefer to hitch-hike with Pieris brassicae 14 butterflies (that lay groups of eggs) compared to Pieris rapae butterflies (that lay single eggs)? 3.3 Is hitch-hiking a specific strategy of female Trichogramma brassicae wasps? 15 3.4 Do Trichogramma wasps hitch-hike with moths in the field? 16 3.5 Do Trichogramma wasps parasitize moth eggs at low temperatures in 17 the dark?

4 Discussion 19

Acknowledgements 22

References 23

Appendix 1: Research locations of the day and night moth catches 26

Appendix 2: List of caught moths at night per research location 27

Appendix 3: List of caught moths during day time per research location 49

3 1 Introduction Chemical cues play an important role in bio-interactions between organisms. Chemical cues that are involved in animal communication are also called semiochemicals (Law & Regnier, 1971). A subclass of semiochemicals are pheromones. Pheromones are used for communication within a species (also called intraspecific chemical signals), like for example sex-pheromones (aphrodisiacs) that are produced by one sex to attract the other or aggregation pheromones that are produced by one sex and attract individuals of both sexes or the same sex as the emitter (Stowe et al., 1995; Waytt, 2003).

Those signals that are broadcasted to the wider world can, however, also be detected by individuals from other species. Semiochemicals that act between individuals of different species are called allelochemicals (Nordlund, 1981). There are three classifications of allelochemicals. Synomones are signals that benefit both the emitter and the receiver. For example, in case of the mutualism between the anemone fish and the sea anemones (Wyatt, 2003). There are also species that emit signals that is costly for the receiving species. These signals are called allomones. An example are bolas spiders that emit a pheromone that is almost chemically identical to the sex pheromone of female moths. Male moths are attracted by this signal and will fly into the web of the spiders (Stowe et al., 1995; Wyatt, 2003). Pheromones can be eavesdropped (“overheard”) by natural enemies. If a predator or a parasitoid, for example, spies on a pheromone and benefits from it at the cost of the emitting species, the signal is called a kairomone (Nordlund, 1981; Wyatt, 2003).

Many parasitoids exploit hosts’ pheromonal signals (i.e. kairomones) while foraging. This strategy is termed “chemical espionage” and is only effective when the pheromones of the target host of the parasitoid indicate the life stage and the location of the host, otherwise other strategies are used (Vinson, 1984). Parasitoids and predatory insects can use signals of plants that are induced by herbivorous insects to locate them. In that case the plant benefits as well as the parasitoid or predator (Dicke et al., 1990; Nordlund et al., 1988; Turlings et al., 1990; Vet & Dicke, 1992). There are also predators that emit mimetic pheromone signals to attract and capture their prey (Stowe, 1988).

Many predators and parasitoids are able to intercept sex pheromone signals emitted by their prey and hosts. For example, hexadecanal and a (Z)-7-, (Z)-9-, and (Z)-11-hexadecenal blend is emitted by females of the corn earworm moth Helicoverpa zea (Klun et al., 1980). This blend is very attractive for H. zea males. However, field applications of the above mentioned sex pheromone increased rates of parasitism of H. zea eggs by wasps of the genus Trichogrammatidae (Lewis et al., 1982). Also, it is known from laboratory olfactometer experiments that female Trichogramma evanescens wasps respond to the sex pheromones emitted by females of Pieris brassicae and Mamestra brassicae (Noldus & Van Lenteren, 1985). This seems surprising as M. brassicae moths release the sex pheromones at night and the wasps are only active during day time (Noldus & Van Lenteren, 1985). Probably, the wasps can detect traces of the sex pheromone that are adsorbed onto the leaf surfaces (Noldus & Van Lenteren, 1985). The wasps respond to the pheromone odor by landing nearby and then rely on visual and short-range chemical cues to indicate where the eggs can be found. Recognizing sex pheromone scents could then be useful cues to find moth eggs (Stowe et al., 1995).

Volatile kairomones are widely used by specialist predators to locate their pray. This is also the case in egg-parasitoids (Fatouros et al., in press). Till now there are 4 known families of egg-parasitoids which have members that spy on volatile kairomones of adult hosts. Those are Trichogrammatidae with 12 species, then Scelionidae with 10 species, Eulophidae with 3 species and Encyrtidae with 2 species. Most of these species spy on the sex pheromone or on the aggregation pheromone of their adult host (Fatouros et al., in press).

4 Eggs of the bug Podisus maculiventris are parasitized by female wasps of Telenomus calvus. Female wasps wait in the neighbourhood of male bugs releasing pheromone. When the bugs are mating the wasps become phoretic on (hitch-hike with) female bugs. For their development, T. calvus need freshly laid eggs that are not older than 12 hours. By hitch- hiking with just mated female bugs and waiting until the bugs are ovipositing, female wasps have access to freshly laid eggs that are not yet discovered by other competing parasitoids or predators (Aldrich, 1995; Aldrich et al., 1985; Orr, et al., 1986). The strategy of hitch-hiking with hosts is not only known from Telonomus calvus but also from 30 other phoretic egg- parasitoids (Fatouros, 2006; N.E. Fatouros & M.E. Huigens, unpublished data). There are 6 families of egg-parasitoids that contain phoretic species. Those are Scelionidae with 17 known phoretic egg-parasitoid species, Trichogrammatidae with 8 species, Eulophidae with 3 species and Encyrtidae, Eupelmidae and Torymidae with both 1 species (Fatouros, 2006; N.E. Fatouros & M.E. Huigens, unpublished data).

A variety of devices have evolved in male insects to prevent or inhibit further mating by females. Some male butterflies evolved the production of a proteinaceous plug or sphragus (Labine, 1968). The injection of male accessory gland substances binds to sites in the nervous system of the female. After this the female remains monogamous. This is shown in certain flies, mosquitoes and moths (Barth & Lester, 1973; Gilbert, 1976). Also it is known that chemical methods are used by male Tenebrio beetles to enforce monogamy. The male Tenebrio beetles do this by using an anti-aphrodisiac pheromone to stop females from re- mating. Transferring the anti-aphrodisiac is done when the beetles are mating. Males inject females with a pheromone that is repellent to other males (Kukuk, 1985). In 1976, Gilbert suggested that Heliconius erato males transport an anti-aphrodisiac pheromone to female butterflies during mating to render them less attractive to other males. He did, however, not identify the chemical compound(s) (Gilbert, 1976).

From butterflies it is known that female receptivity is signaled by a combination of visual and olfactory signals (Silberglied, 1984). At a distance the visual stimuli are most important, from close-range the olfactory stimuli (Silberglied, 1984). Andersson et al. (2000) found that an anti-aphrodisiac pheromone is used by males of the green-veined white butterfly, Pieris napi. They found that the male butterflies transfer the chemical compound methyl salicylate (MeS) to females during mating. This substance makes P. napi females unattractive and unlikely to be mated by other males. Andersson et al. (2003) also found the chemical compounds that render female butterflies unattractive to other males of two other closely relatives of P. napi, Pieris rapae (small cabbage white butterfly) and Pieris brassicae (large cabbage white butterfly). The anti-aphrodisiac of P. rapae exists of two compounds, MeS and indole (Andersson et al., 2003). The anti-aphrodisiac of P. brassicae is the chemical compound benzyl cyanide (BC) (Andersson et al., 2003). The males of P. napi and P. rapae transfer a volatile anti-aphrodisiac that is completely different from the male odor, whereas the males of P. brassicae transfer an anti-aphrodisiac to the females that is identical with the male odor. In the latter case the females become chemical mimics of the males (Andersson et al., 2000; Andersson et al., 2003).

Trichogramma wasps are egg parasitoids and are considered efficient biological control agents. They are used worldwide for the control of lepidopteran pests in many crops, especially corn borers, sugarcane borers and cotton bollworm (Delpuech et al., 2001; Fatouros et al., 2007; Herz et al., 2007). Trichogramma wasps are regarded as relatively polyphagous, so they are less host specific than specialist egg parasitoids like for example some Telenomus spp. (Pinto & Stouthamer, 1994). There are more than 150 species described worldwide and those species attack more than 400 pest species (Delpuech et al., 2001; Pinto, 1999; Pinto & Stouthamer, 1994).Trichogramma wasps are from the family Trichogrammatidae (Pinto & Stouthamer, 1994). They are 0.2 to 1.5 mm ‘large’ (Knutson, 2000; Pinto & Stouthamer, 1994). Due to their limited size Trichogramma wasps have a low capacity for active flights (Romeis et al., 2005). They occur in almost every terrestrial habitat.

5 Trichogramma wasps parasitize freshly laid insect eggs, especially those laid by butterflies and moths (Herz et al., 2007; Keasar et al., 2000; Knutson, 2000). To find their hosts Trichogramma are believed to use both short-range and long-range cues. Volatile chemical signals such as plant synomones or moth sex pheromones are used as long-range cues. Chemical signals emitted through moth scales or trail pheromones are used as intermediate- and short-range cues. Also visual cues, i.e. objects approximating the size and shape of host eggs, are used at intermediate- and short-range (Lobdell et al. 2005). From Trichogramma species the circadian rhythm of locomotor activity varies during the day (Allemand et al., 1994). For Trichogramma brassicae this varies according to genetic and non-genetic factors. The males are most active in the morning and females in the afternoon (Pompanon et al., 1999). At a constant temperature of 22°C, T. brassicae wasps are not active in the dark period of 12 hours (Pompanon et al., 1999). They start to become active one hour before it turns light and remain active until the dark period starts. Pompanon et al. (1999) did not describe if female wasps parasitize eggs in the dark.

Recently, Fatouros et al. (2005) found that the parasitic wasp T. brassicae can detect the anti-aphrodisiac pheromone BC that is passed from P. brassicae males to P. brassicae females. The wasp prefers the odor of mated P. brassicae females, which they can discriminate from odors associated with males and virgins. Interestingly, the wasp T. brassicae, exploits the anti-aphrodisiac of P. brassicae by specifically climbing on a mated P. brassicae female. When the butterfly lays her eggs, the wasp parasitizes them by laying its own eggs into the butterfly eggs at the cost of caterpillar development (Fatouros et al., 2005). Mated P. brassicae females lays their eggs in clusters with sometimes more than hundred eggs together (Bellmann, 2003; Bos et al., 2006). Pieris rapae lays their eggs solitary and P. napi lays their eggs solitary or in small groups (Bellmann, 2003; Bos et al., 2006). This fascinating strategy of spying on an anti-aphrodisiac pheromone in combination with specifically hitch-hiking with mated female hosts is expected to be widespread in nature in egg-parasitoids that have limited flight control, need to parasitize freshly laid eggs, that suffer from competition with other (conspecific) parasitoids, and that parasitize eggs of hosts that lay their eggs in groups. This strategy could then severely constrain the use of sexual communication between hosts (Fatouros et al., 2005). The goal of this research was to investigate if eavesdropping on an anti-aphrodisiac pheromone and hitch-hiking with mated female hosts by Trichogramma wasps occurs on multiple species of butterflies and moths. More specifically I tried to answer the following more specific research questions: ƒ Does Trichogramma brassicae eavesdrop on the anti-aphrodisiac odor of Pieris rapae butterflies and specifically hitch-hike with mated P. rapae females? ƒ Does Trichogramma brassicae prefer to hitch-hike with Pieris brassicae butterflies compared to Pieris rapae butterflies? ƒ Is hitch-hiking a specific strategy of female Trichogramma brassicae wasps? ƒ Do Trichogramma wasps hitch-hike with moths in the field? ƒ Do Trichogramma wasps parasitize moth eggs at low temperatures in the dark?

6 2 Material and methods In this part the material and methods are described per research question. Experiments were done in the field and in the laboratory under room conditions. For the laboratory experiments two experimental settings were used: two-choice olfactory bioassays and mounting bioassays (Fatouros et al., 2005). The female and male Trichogramma brassicae wasps that were used were 2 to 5 days old. The wasps where reared by a temperature of 22°C and with a light period of 16 hours and 8 hours darkness. The wasps were sexed under a microscope. Males and mated Pieris rapae females were collected in the afternoon as mating couples and used the next day. Virgin females were collected by separating pupae or collecting freshly emerged female butterflies from the breeding boxes in the afternoon and were also used the next day (in the morning all boxes were emptied). Pieris brassicae females were collected in the same way.

2.1 Does Trichogramma brassicae eavesdrop on the anti-aphrodisiac odor of Pieris rapae butterflies and specifically hitch-hike with mated P. rapae females?

2.1.1 Olfactory bioassays This experiment was carried out in a static two-chamber olfactometer (figure 2.1). Female T. brassicae wasps were released in the middle of the gauze. The time that the female wasps spent in one of the two odor fields was determined for 300 seconds (5 minutes). Four butterflies were introduced per chamber as odor source. A number of 10 naïve wasps were tested per day. A total number of 40 wasps per combination were tested. The test combinations were: 1. Mated females vs. air (control combination) 2. Mated females vs. males 3. Mated females vs. virgin females 4. Males vs. virgin females 5. Virgin females painted with 2 μl solution of 20 μg methyl salicylate (MeS) + indole (I) in hexane vs. virgin females painted with2 μl hexane only 6. Virgin females painted with 2 μl solution of 2 μg MeS + I in hexane vs. virgin females painted with 2 μl hexane only 7. Virgin females painted with 2 μl solution of 1 μg MeS + I in hexane vs. virgin females painted with 2 μl hexane only After each 2 wasps the olfactometer was rotated for 180 degrees to assure, that the light had no influence on the wasp’s choice. Also a box with a lamp above was placed over the two- chamber olfactometer to avoid influences of the environment.

Figure 2.1 Two-chamber olfactometer with a test combination of Pieris rapae. In the middle of the gauze of the olfactometer a female Trichogramma brassicae wasp was released so it can choose which odor it prefers to go to. The olfactometer is placed in a box with a lamp above so there will be no influence of the environment on the female T. brassicae wasps.

7 The test combination virgin females painted with synthetic anti-aphrodisiac blend (MeS + I = Methyl salicylate + Indole) vs. virgin controls was done by applying 2 μl of solution in a fume hood. The concentration 20 μg MeS + I works like an anti-aphrodisiac for the P. rapae butterflies (Andersson et al., 2003).

2.1.2 Mounting bioassays The selective mounting behaviour of the female wasp was tested in a two-choice bioassay with two adult butterflies (figure 2.2). A female T. brassicae wasp was released at the bottom of the arena at a distance of ± 3 cm of each butterfly (figure 2.2, white dot). The wasp was observed for 300 seconds (5 minutes). When the wasp climbed on a butterfly, the duration of phoresy (= time that the wasp stayed on the butterfly) was measured. A phoresy was reported if the wasp stays longer than 2 seconds on the butterfly. Also the butterfly’s body part where the wasp climbed on was reported. Whenever a wasp did not climb on a butterfly after the five minutes time, the observation was reported as ‘no response’. When a butterfly flew within the 5 minutes without a wasp on it the experiment was redone. A number of 10 naïve wasps were tested per day. A total number of 40 wasps that climbed per combination were measured. The test combinations were: 1. Mated females vs. males 2. Mated females vs. virgin females 3. Males vs. virgin females 4. Virgin females painted with 2 μl solution of 2 μg MeS + I in hexane vs. virgin females painted with 2 μl hexane only Just before the experiment, the butterflies were cooled down in a refrigerator (4˚C) for ±10 minutes, in order to decrease their activity. The butterflies were exchanged with new ones after 2 wasps or when they were started to become active. Also after 2 wasps the position of the butterflies were changed to avoid preference for a left or right side of the mounting bioassay. Furthermore, the plate was cleaned to make sure that the odor of the previous butterfly sexes did not had any influence on the wasp’s choice.

Figure 2.2 Two-choice bioassay with a test combination of Pieris rapae where a female Trichogramma brassicae wasp is released at a distance of ± 3 cm of each butterfly (white dot) and can choose on which butterfly it likes to climb on.

2.2 Does Trichogramma brassicae prefer to hitch-hike with Pieris brassicae butterflies compared to Pieris rapae butterflies?

2.2.1 Olfactory bioassay This experiment was done in a two-chamber olfactometer. Here, the test combination of four mated P. brassicae females vs. four mated P. rapae females was tested. Also the odor of two mated P. brassicae females were tested against the odor of four mated P. rapae females. The proportion 2 against 4 was chosen because P. brassicae is almost 2 times bigger than P. rapae and maybe P. brassicae release more anti-aphrodisiac, so to avoid that

8 this had influence on the wasp’s choice this combination was chosen. The olfactometer experiment was performed as described above.

2.2.2 Mounting bioassay In this experiment, the mounting behaviour of female T. brassicae wasps were tested in a two-choice bioassay of a mated P. brassicae female versus a mated P. rapae female. The mounting experiment was performed as described above.

2.3 Is hitch-hiking a specific strategy of female Trichogramma brassicae wasps? To test if hitch-hiking is a specific strategy of female T. brassicae wasps a one-choice bioassay was used. 40 naïve female and 40 naïve male wasps were tested on mated P. brassicae females and on mated P. rapae females. A total number of 5 wasps of both sexes were tested per day. In the middle of the arena a butterfly was placed (see figure 2.3). The wasp was released at the bottom of the arena (± 3 cm from the butterfly). The butterflies were first cooled down in a refrigerator (4˚C) for ±10 minutes, in order to decrease there activity. The wasp was observed for 300 seconds (5 minutes). When the wasp climbed on the butterfly, the duration of phoresy (must be longer than 2 seconds) was measured and the body part where the wasp climbed on was reported. When a wasp did not climbed after 5 minutes on the butterfly the observation was reported as ‘no response’. The experiment was redone when a butterfly flew within the 5 minutes without a wasp on it. The butterfly was exchanged by a new one after 2 tested wasps or when she was starting to be active.

Figure 2.3 One-choice bioassay with a mated female Pieris rapae butterfly in the middle. A male or female Trichogramma brassicae wasp is released at a distance of ± 3 cm of the butterfly so it can choice if it wants to climb on the butterfly.

2.4 Do Trichogramma wasps hitch-hike with moths in the field?

2.4.1 Moth selection Here it was tested whether Trichogramma wasps also hitch-hike with moths. Moths can be divided in 2 groups (Waring & Townsend, 2003): macro-moths and micro-moths. Micro- lepidoptera or ‘micros’ are generally smaller in size than macro-moths. They have a forewing length of 1 cm or less (there is overlap between the macro- and micro-moths). Macro-moths are better described so the identification of them is less difficult than for micro-species (except some macro-moth species that are difficult to discriminate) (Waring & Townsend, 2003). During this research only macro-moths were collected. The moth families that were selected for this research are all common macro-moth families representative for The Netherlands. The selected families were: Hepialidae, Cossidae, Zygaenidae, Limacodidae, Sisiidae, Lasiocampidae, Saturniidae, Drepanidae, Geometridae, Sphingidae, Notodontidae, Thaumetopoeidae, Lymantriidae, Arctiidae, Nolidae and Noctuidae (Waring & Townsend, 2006).

9 2.4.2 Night catches The goal here was to collect moths that fly during night. When the weather conditions were good (meaning no rain) moths were collected twice a week at night. The moths were collected in different habitats: Groot arboretum (urban park, X:17570 Y:44195), Pabstsendam (river plain meadow with shrubs, X:17389 Y:44108), Marijkeweg (student compound surrounded by shrubs, X:17311 Y:44232) and Entomology garden (meadow of flowering plants, X:17316 Y:44258), all located in Wageningen and Natuurcentrum Veluwe (butterfly garden in mixed forest, X:17855 Y:45057) located in Ede (see appendix 1 for map research locations). By doing this, more moth species were collected and the chance of finding Trichogramma wasps was higher. For collecting moths a big white cloth (6 m2) tied up onto 2 sticks, a lamp, a generator, butterfly nets, and plastic pots were used (see figure 2.4).

Figure 2.4 Used elaboration for collecting moths. A big white cloth tied up onto 2 sticks and rope for tight stretching the cloth, a lamp powered by a generator, butterfly nets, and plastic pots for catching moths are used.

The moths were attracted by the light and most of the time they sat down on the cloth and could be caught in a plastic pot. For those moths that only fly in circles a butterfly net with plastic pot inside (attached with a rubber band) was used. Using a plastic pot inside the butterfly net avoids wasps to escape through the net. After collecting moths, they were placed in a refrigerator (4˚C) in order to decrease their activity. After a cooling down period of at least 15 minutes the species were determined and they were monitored for Trichogramma wasps under a microscope. If there was a wasp it was put in alcohol (95%) and the species was determined by molecular methods described below in the part “Trichogramma species identification” (Gonçalves et al., 2006; Stouthamer et al., 1999). The moths were released again in the field after checking them for Trichogramma wasps.

2.4.3 Day catches Moths do not only fly at night. There are also species that only fly during day time (for example Euclidia gyphica) and there are species that fly both at night and during day time (for example Autographa gamma) (Waring & Townsend, 2006). The goal here was to collect the moths that fly during day time. Catching procedures were as described for the night catches. For collecting these moths, places with a lot of flowers were visited (figure 2.5). Those were the Pabstsendam (river plain meadow with shrubs, X:17389 Y:44108), Marijkeweg (student compound surrounded by shrubs, X:17311 Y:44232), Entomology garden (meadow of flowering plants, X:17316 Y:44258) and Klein arboretum (urban park with a lot of flowering plants and shrubs, X:17497 Y:44223), all located in Wageningen (see appendix 1 for map research locations). Some moths that only fly at night were caught because they flew away when being disturbed by the experimenter searching the vegetation for day active moths. All insects were thereafter treated in the same way as described above.

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Figure 2.5 Entomology garden, moth searching place with a lot of flowers.

2.4.4 Trichogramma species identification Each single wasp was put in alcohol (95%). To identify which species was collected, a Trichogramma identification method was used as described in Gonçalves et al. (2006) and Stouthamer et al. (1999).

2.4.4.1 DNA extraction The wasps that where put in alcohol were first dried on a filter paper. When a wasp was dry it was put in a 0.5 ml Eppendorf tube and crushed with a closed pasteur pipet tip. After crushing, 50 μl of Chelex solution (5%) and 4 μl of proteinase K (20 mg/ml) were added. This was incubated overnight at 56°C and finally the samples stayed at 95°C for 10 minutes.

2.4.4.2 PCR amplification After the DNA extraction a PCR reaction was performed for every sample. In a 0.2 ml Eppendorf tube 25 μl consisting of 18.43 μl of distilled water, 2.5 μl 10x PCR reaction buffer, 2.5 μl DNA template, 0.5 μl dNTP (10 mM), 0.5 μl forward primer, 0.5 μl reverse primer and 0.07 μl Taq polymerase (5 units/μl) was added. To amplify the ITS-2 region the forward primer 5’-TGTGAACTGCAGGACACATG-3’ and the reverse primer 5’- GTCTTGCCTGCTCTGAG-3’ were used. The used PCR cycling program was 3 minutes at 94°C, 33 cycles of 40 seconds at 94°C, 45 seconds at 53°C and 45 seconds at 72°C, followed by 10 minutes at 72°C after the last cycle. After this the PCR products were run for 1.5 hours on a 1.5% agarose gel and stained with ethidium bromide.

2.4.4.3 Cloning and sequencing Each amplified ITS-2 gene was cloned and sequenced. The ITS-2 products were excised from the gel using the kit “QIAquick Gel Extraction Kit” and using the protocol “MinElute Gel Extraction Kit Protocol” (QIAGEN GmbH, Hilden, Germany) for DNA fragment purification. The PCR fragment was ligated to a pGEM-T easy vector and transformed into Escherichia coli x12 cells. To confirm if the insertion of the ITS-2 fragments was correct an ITS2-PCR was done. To purify the plasmid the “GenElute Plasmid Miniprep Kit” (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used. By using an Applied Biosystems automatic sequencer the ITS-2 fragments were sequenced. The ITS-2 sequences were finally aligned and matched against sequences present in GenBank.

11 2.5 Do Trichogramma wasps parasitize moth eggs at low temperatures in the dark? It is known that Trichogramma wasps parasitize a wide range of moth eggs (Herz et al., 2007; Keasar et al., 2000; Knutson, 2000) but do they also parasitize them at night? To test this, naïve female wasps of T. brassicae and Trichogramma evanescens of the same age (2 days old) were tested in climate rooms. In this experiment female wasps were offered the opportunity to parasitize an overamount of Ephestia kuehniella eggs at two different periods of darkness and four different temperatures (table 2.1). Subsequently, their parasitism behavior was measured (number of parasitizing females, the number of parasitized eggs and offspring production). All wasps where reared at the same conditions. They were reared at a temperature of 22°C and with a light period of 16 hours and 8 hours darkness. The light period was from 6 AM till 10 PM. Tested were two lines of T. brassicae, line Y175 (collected from self made Mamestra brassicae egg cards in a vegetable garden in Lierop, The Netherlands, 1999) and J007 (collected from an adult female Xestia c-nigrum moth at 7.2°C (weather station Den Bilt)) in Ede, The Netherlands, during this study), and two lines of T. evanescens, line GD011 and GD025 (both collected from Pieris rapae eggs in Wageningen, The Netherlands, 2004), all reared on E. kuehniella eggs at the Laboratory of Entomology of Wageningen University.

Table 2.1 Test combinations per Trichogramma line in the dark. Line Temperature Hours darkness Y175 20°C, 15°C, 10°C and 5°C 6 and 12 J007 20°C, 15°C, 10°C and 5°C 6 and 12 GD011 20°C, 15°C, 10°C and 5°C 6 and 12 GD025 20°C, 15°C, 10°C and 5°C 6 and 12

Of each line 40 wasps were tested per test combination (table 2.1). After the test the wasps were removed from the vials with E. kuehniella eggs. After 7 days the parasitized eggs were counted because parasitized eggs should become black after 5 days (Knutson, 2000). After 12 days the emerged wasps were counted on numbers and sexes after a cooling down period in a freezer. Normally wasps emerge after 10 days at 22°C. The experiments with 12 hours of darkness started at 9 PM and were finished at 9 AM the next day. The experiments with 6 hours of darkness started at 11 AM and were finished at 5 PM the same day. A temperature range between 5-20 degrees was chosen because this is rather typical for summer and autumn nights in The Netherlands. The same goes for the hours of darkness.

2.6 Statistical analysis In the olfactory bioassays the time that a wasp spends (residence time) in the two odor fields were compared with a Wilcoxon’s matched pairs signed rank test with α < 0.05. The percentages of first mounts in the two-choice mounting bioassays were analyzed with a binomial test, α < 0.05. The percentages of climbing female and male wasps in the one- choice mounting bioassays were analyzed with a Chi-square test with 2x2 contingency table using α < 0.05. For the “night parasitism” experiment, the percentages of parasitizing female wasps were analyzed with a generalized linear model with a logit link function and a binomial distribution for errors, again using α < 0.05.

12 3 Results

3.1 Does Trichogramma brassicae eavesdrop on the anti-aphrodisiac odor of Pieris rapae butterflies and specifically hitch-hike with mated P. rapae females? The results of the olfactometer experiment are shown in figure 3.1. The wasps showed a significant preference for the odor of the mated Pieris rapae females compared to clean air (P=0.020, α < 0.05). Also in the test combinations mated females vs. males and mated females vs. virgin females, the wasps showed a significant preference for the odor of mated females (P=0.004 and P=0.013, α < 0.05). The wasps did not discriminate between the odor of males and virgin females (P=0.767, α < 0.05). The test on the attractiveness of the synthetic anti-aphrodisiac blend of methyl salicylate (MeS) and indole (I) showed that the concentration of 20 μg MeS + I was repellent for the female Trichogramma brassicae wasps (P=0.001, α < 0.05). The wasps significantly preferred the odor of virgin females painted with two lower concentrations (2 μg MeS + I and 1 μg MeS + I, P=0.007 and P=0.049, α < 0.05) over the odor of virgin females painted with the solvent only.

Trichogramma brassicae Pieris rapae50% 50% Pieris rapae

AIR P=0.020 *

P=0.004 **

P=0.026 *

P=0.767 (NS)

P=0.008 **

+ 20μg MeS and I

P=0.007 ** + 2μg MeS and I

P=0.049 *

+ 1μg MeS and I

240 200 160 120 80 40 04080 120 160 200 240 Residence time (s)

Mated Virgin

Figure 3.1 Odor preference of female Trichogramma brassicae wasps on Pieris rapae sexes and on virgin Pieris rapae butterflies treated with and without methyl salicylate (MeS) and indole (I) (anti-aphrodisiac) in a two- chamber olfactometer. Measured was the time that a wasp spends in the odor field of a butterfly (residence time, maximum 300 seconds). Per combination 40 wasps were tested. Error bars indicate standard error means.

Figure 3.2 shows the results of the two-choice bioassays. The wasps significantly preferred to climb onto mated P. rapae females compared to virgin P. rapae females (P=0.010, α < 0.05). Similarly, the wasps also preferred to climb onto virgin females painted with the synthetic anti-aphrodisiac pheromone concentration of 2 μg MeS + I over the virgin control females (P=0.002, α < 0.05). The wasps did, however, not distinguish between mated P. rapae females and males (P=0.161, α < 0.05). There was also no discrimination between P. rapae males and virgin females (P=0.206, α < 0.05).

13 In total 123 wasps climbed on the wings of the butterflies, 26 on the legs and 11 on the abdomen.

Trichogramma brassicae Pieris rapae Pieris rapae 50% 50% 28 12 P=0.010 *

30 10 P=0.002 **

+ 2μg MeS and I

23 17 P=0.161 (NS)

18 22 P=0.206 (NS)

80 70 60 50 40 30 20 10 0 1020304050607080 Proportion of first mounts (%)

Mated Virgin

Figure 3.2 Hitch-hike preferences of female Trichogramma brassicae wasps on Pieris rapae sexes and on virgin Pieris rapae butterflies treated with and without methyl salicylate (MeS) and indole (I) (anti-aphrodisiac) in a two- choice bioassay. In the bars the numbers of climbing wasps are shown per butterfly sex. In total 40 wasps were tested per combination.

3.2 Does Trichogramma brassicae prefer to hitch-hike with Pieris brassicae butterflies compared to Pieris rapae butterflies? The female T. brassicae wasps did not have a significant preference for the odor of one of the 2 tested Pieris species (P=0.828 and P=0.778, α < 0.05, figure 3.3).

Trichogramma brassicae Pieris rapae Pieris brassicae

4x 2x P=0.829 (NS)

4x 4x P=0.778 (NS)

200 150 100 50 0 50 100 150 200 Residence time (s)

Mated Mated Pieris Pieris rapae brassicae Figure 3.3 Odor preference of female Trichogramma brassicae wasps on mated Pieris rapae females (single egg laying butterfly) and mated Pieris brassicae females (gregarious egg laying butterfly) in a two-chamber olfactometer. Tested are 4 P. rapae females against 2 P. brassicae females and 4 P. rapae females against 4 P. brassicae females. Measured was the time that a wasp spends in the odor field of a butterfly (residence time, maximum 300 seconds). Per combination 40 wasps were tested. Error bars indicate standard error means.

However, when tested against each other in a two-choice bioassay, female wasps significantly preferred to climb onto mated Pieris brassicae females (gregarious egg laying species) over mated P. rapae females (solitary egg laying species) (P=0.022, α < 0.05, figure 3.4). In totally the wasps climbed 31 times on the wings, 7 times on the legs and 2 times on the abdomen.

14

Pieris rapaeTrichogramma brassicae Pieris brassicae

13 27 P=0.022 *

80 70 60 50 40 30 20 10 0 1020304050607080

Proportion of first mounts (%)

Mated Mated

Pieris Pieris rapae brassicae

Figure 3.4 Hitch-hike preferences of female Trichogramma brassicae wasps on mated Pieris brassicae females (gregarious egg laying butterfly) and mated Pieris rapae females (solitary egg laying butterfly) in a two-choice bioassay. In the bars the numbers of climbed wasps are shown per butterfly sex. In total 40 wasps were tested per combination.

3.3 Is hitch-hiking a specific strategy of female Trichogramma brassicae wasps? In the one-choice experiments, 40 T. brassicae wasps of each sex were tested. Of the tested wasps 14 male wasps (33.3%) and 28 female wasps (66.7%) climb onto a mated female P. brassicae butterfly. On mated P. rapae female butterflies, 8 male wasps (24.4%) and 25 female wasps (75.8%) climbed. On both butterfly species female wasps climbed significantly more often than male wasps (P. brassicae P=0.0017, P. rapae P=0.0001, α < 0.05, figure 3.5). The body part where the wasps climb on was for P. brassicae most of the times the wing (37 times) followed by leg (4 times) and abdomen (1 time). For P. rapae this was almost the same, 31 times on the wings, 1 time on the leg and 1 time on the abdomen.

80 P=0.0017 ** 70 P=0.0001 *** 60

50 male wasps 40 female wasps 30 climbing% 20 10

0

P. brassicae P. rapae

Figure 3.5 Climbing strategy of male and female Trichogramma brassicae wasps on mated female Pieris brassicae butterflies and mated female Pieris rapae butterflies in a one-choice bioassay. Blue bars indicate the proportions of climbing male wasps and the white bars indicate the proportions of climbing female wasps. Of each sex 40 wasps were tested on each butterfly species.

Also tested is if the female wasps have a preference for climbing onto one of the two Pieris species over the other. The wasps did not significantly climb onto one of the two butterfly species more frequently in these one-choice tests (P=0.478, α < 0.05, figure 3.6).

15 P=0.478 (NS) 30

25

20 Wasp climbed

N 15 Wasp not climbed 10

5

0

P. brassicae P. rapae

Figure 3.6 Climbing preference of female Trichogramma brassicae wasps in a one-choice bioassay on mated female Pieris rapae butterflies and mated female Pieris brassicae butterflies. The blue bars indicate how many female wasps climbed and the white bars indicate how many female wasps did not climbed. Forty female wasps were tested on each butterfly species.

3.4 Do Trichogramma wasps hitch-hike with moths in the field? In total, 777 moths were caught during the night catches in the period from 26 April to 27 October 2007. These belong to 112 different species. Table 3.1 shows the top ten of most caught moth species. The whole list of caught moths is shown in appendix 2.

Table 3.1 Top ten of at night caught moth species from the night catches research locations. Top Species Number of moths 1 Ochropleura plecta 104 2 Xestia c-nigrum 66 3 Noctua pronuba 51 4 Epirrhoe alternata 43 5 Xestia xanthographa 34 6 Diachrysia chrysitis 27 7 Diarsia rubi 22 8 Hypena proboscidalis 19 9 Axylia putris, Opisthograptis luteolata 18 10 Amphipyra pyramidea, Rivula sericealis 14

From the 777 moths, only 1 carried a female Trichogramma brassicae wasp (figure 3.7b). This moth was a female Xestia c-nigrum (figure 3.7a) (sex determined by R. de Vos) and was caught on 13 September 2007 at the research location Natuurcentrum Veluwe, Ede when the temperature was 7.2 degrees Celsius (weather station Den Bilt). This moth is a gregarious egg laying species (J. Voogd, personal communication). With the female wasp a culture was started to get more wasps for future experiments. At the Laboratory of Entomology of Wageningen University this T. brassicae line is still in rearing under the name J007. All other moths did not carry Trichogramma wasps.

a) b) Figure 3.7 a) One of the Xestia c-nigrum moths that were caught. b) The female Trichogramma brassicae wasp that was found on a female X. c-nigrum moth.

16 During the day catches 35 moths were caught in the period of 1 May to 21 November 2007. In total, 20 species were caught (appendix 3). There were no Trichogramma wasps caught during the day catches.

During the moth catches (day and night) there were more parasitoid wasp species found in the vials (table 3.2).

Table 3.2 List of other parasitoid wasps that were found during the moth catches (day and night). Date Place Moth species Parasitoid species 26-08-2007 Pabstsendam Cabera exanthemata Anaprostocetus acuminatus 28-08-2007 Groot arboretum Opisthograptis luteolata Anaprostocetus acuminatus 06-09-2007 Klein arboretum Eupithecia absinthiata Eulophidae spp. 06-09-2007 Natuurcentrum Veluwe Noctua pronuba Eulophidae spp.

Twice the parasitoid wasp Anaprostocetus acuminatus was caught on moths. This wasp is a member of the family Eulophidae tetrastichinae and parasitizes the eggs of Euura atra and Euura laeta (both sawflies). The two other caught parasitoid wasps were only identified to family level and are part of the family Eulophidae (all four wasps were determined by Y. Jongema).

Also caterpillars of Hoplodrina ambigua (figure 3.8) were found in vials of two moth species that were caught during the night catches. One caterpillar was found in a vial of a female Watsonalla binaria (6 September 2007 Natuurcentrum Veluwe, Ede) and 5 in a vial of a female Peribatodes rhomboidaria (7 September 2007, Groot arboretum, Wageningen).

Figure 3.8 Different stages of Hoplodrina ambigua that was found in vials of different moth species during the night catches.

3.5 Do Trichogramma wasps parasitize moth eggs at low temperatures in the dark? Figures 3.9a and 3.9b shown how many female wasps had parasitized the Ephestia kuehniella eggs. When female T. brassicae wasps had the opportunity to parasitize eggs at 6 hours of darkness, only few parasitized the eggs. Females of line J007 start parasitizing at 10°C and of line Y175 at 20°C. Female T. brassicae wasps did not parasitize eggs when it was 12 hour dark. Female Trichogramma evanescens wasps parasitized eggs at 6 and 12 hours of darkness. Both lines start with parasitizing eggs at 5°C at 6 hours of darkness. At 12 hours of darkness line GD011 start at 5°C but line GD025 at 15°C. They parasitize more and more when temperature increasing.

17

a) 6 hours of darkness

20

15

10

females 5

Number of parasitizing 0 T. evanescens line GD011 A

5°C 10°C 15°C 20°C T. evanescens line GD025 A Temperature b) T. brassicae line Y175 B 12 hours of darkness T. brassicae line J007 B 20

15

10

females 5

Number parasitizing of 0 5°C 10°C15°C 20°C Temperature

Figure 3.9 a) and b) Number of female wasps that parasitized at different temperatures for 6 and 12 hours of darkness. The experiments with 12 hours of darkness started at 9 PM and were finished at 9 AM the next day. The experiments with 6 hours of darkness started at 11 AM and were finished at 5 PM the same day. In total 40 wasps per line per temperature were tested. Lines with an A are not significantly different from each other in the graphs but they are different from the lines indicated with a B. Lines with a B are not significantly different from each other.

There was no significant difference in the frequency of parasitizing females within T. brassicae (P=0.6539, α < 0.05) and T. evanescens (P=0.5342, α < 0.05). When comparing the frequency of parasitizing females of T. brassicae and T. evanescens, significantly more female T. evanescens wasps parasitized in the dark than female T. brassicae wasps (P<0.0001, α < 0.05). 6 hours of darkness, there were significantly more eggs parasitized than at 12 hours of darkness (P<0.0001, α < 0.05). Overall, the higher the temperature, the more Trichogramma females had been parasitizing (P<0.0001, α < 0.05). There was only no significant difference between 5°C and 10°C (P=0.4291, α < 0.05).

18 4 Discussion This study on egg parasitoids investigated whether the strategy of eavesdropping on an anti- aphrodisiac pheromone and specifically hitch-hiking with mated female hosts, as was recently found for the parasitoid wasp Trichogramma brassicae on the large cabbage white butterfly Pieris brassicae (Fatouros et al., 2005), is more common and occurs on multiple butterflies and moths. The present findings support that female T. brassicae wasps also hitch-hike with mated females of the small cabbage white butterfly Pieris rapae and establish this by eavesdropping on their anti-aphrodisiac pheromone. Furthermore it was found that T. brassicae wasps do not only hitch-hike with butterflies but also with moths.

The results demonstrate that a single Trichogramma species has evolved this same strategy on multiple host species. Similarly, it is known from other egg-parasitoids that they can spy on volatile kairomones of multiple host species; for example, Telenomus busseolae can spy on volatiles of Sesamia nonagriodes (Colazza et al., 1997) and Sesamia calamistis (Fiaboe et al., 2003) and Chrysonotomyia ruforum can spy on synthetic sex pheromones of Diprion pini and Neodiprion sertifer (Hilker et al., 2000).

The laboratory experiments have shown that female T. brassicae wasps can detect the anti- aphrodisiac odor methyl salicylate and indole of mated female P. rapae butterflies. The first concentration that was tested was 20 μg. This concentration was chosen because Andersson et al. (2003) found this concentration act as an anti-aphrodisiac for the butterflies. This concentration was however repellent towards the female T. brassicae wasps. Only two lower concentrations (2 μg and 1 μg) were attractive for the wasps. Fatouros et al. (2005; unpublished data) found similar results when testing the attractiveness of the anti- aphrodisiac benzyl cyanide of the large cabbage white butterfly P. brassicae towards female T. brassicae wasps.

The two-choice mounting bioassay showed that female T. brassicae wasps do not prefer to hitch-hike with mated P. rapae females compared to P. rapae males. This indicates that there are may be other short-range cues that the wasps use to discriminate between mated females and males. In nature, female T. brassicae wasps will almost never be in a situation where they have to choose between the P. rapae sexes. They will mostly be attracted from a distance to mated female P. rapae butterflies that are emitting the anti-aphrodisiac pheromone.

When female T. brassicae wasps can choose between mated female P. brassicae butterflies (gregarious egg laying species; Bellmann, 2003; Bos et al., 2006) and female P. rapae butterflies (solitary egg laying species; Bellmann, 2003; Bos et al., 2006), they prefer to hitch- hike with mated P. brassicae females. The wasps benefit more from hitch-hiking with P. brassicae than with P. rapae because they gain more offspring from parasiting a clutch of P. brassicae eggs than a single P. rapae egg. Female T. brassicae wasps did not discriminate between the anti-aphrodisiac odors of both Pieris species. This suggests that short-distance cues are used by T. brassicae while discriminating between the butterfly species. Maybe they prefer to hitch-hike more with mated P. brassicae females because they are bigger than P. rapae females. To exclude that butterfly size plays a major role, an experiment has to be done with for example on one side of the two-choice mounting bioassay a mated P. brassicae butterfly and on the other side a virgin female P. brassicae butterfly treated with anti-aphrodisiac blend of P. rapae.

From the comparison between the climbing responses of male and female wasps it can be concluded that hitch-hiking is a female strategy within T. brassicae wasps. In the field only female wasps have been found on several butterfly species in The Netherlands (M.E. Huigens, unpublished data). This is to be expected because only female wasps parasitize butterfly and moth eggs. Maybe female T. brassicae wasps need freshly laid eggs for successful oviposition (Herz et al., 2007; Keasar et al., 2000; Knutson, 2000). An easy way

19 to find those eggs is to hitch-hike with a mated female butterfly. Some male T. brassicae wasps also climbed onto mated female butterflies in the experiments. Maybe male wasps occasionally do this to find females to mate with.

Earlier and present findings show that T. brassicae can hitch-hike with P. brassicae (Fatouros et al., 2005), Maniola jurtina (M.E. Huigens, unpublished data) and P. rapae (this study). Trichogramma brassicae wasps obviously hitch-hike with different butterfly species. During this research it was found that they can also hitch-hike with moths. One female wasp was caught on a female Xestia c-nigrum moth. Previous work has shown that Trichogramma wasps also occur on adults of different moth species in The Netherlands. Wasps were found on Xestia baja and Phlogophora meticulosa (J. Voogd, unpublished data). Unfortunately, those Trichogramma wasps were not identified to species level.

Only one Trichogramma wasp was found during the moth catches. This probably is a strong underestimation of the actual frequency of hitch-hiking wasps on adult moths. Wasps may stay on moths/butterflies only for a short time, which decreases the chance of detecting them. Also, wasps may have fallen off the moths when the moths landed onto the white cloth or crashed against the lamp that was used during the night catches. Most of the moths first crashed onto the cloth or lamp before they were sat down on the cloth. Also a lot of moths fell down onto the ground. It is recommended to also place a white cloth on the ground in future catches.

Also other parasitoid wasps were found during the moth catches (day and night). It is still unclear why they hitch-hike with moths. More research is needed to understand why these parasitoids were found in the vials and if they really hitch-hike with moths and butterflies in the field, and not ended up in all the vials accidently. These wasp species should be tested in a mounting bioassay and they should be offered eggs of the moth species on which they were found.

There were also caterpillars of Hoplodrina ambigua found during the night catches. The egg laying behavior of H. ambigua is unknown (T. Muus, personal communication). From this species is known that the caterpillars are herbivorous and polyphagous (Waring & Townsend, 2006). Maybe this moth species lays its eggs on other moths that are resting, to distribute their offspring over a greater region, and the resulting caterpillars have no intraspecific competition.

The female T. brassicae wasp that was found on a moth was collected during a night catch when the temperature was low (i.e. 7.2°C). This raises the question whether Trichogramma wasps can parasitize moth eggs in the dark at low temperatures. Experimental findings indicate that T. brassicae can parasitize eggs in the dark at temperatures as low as 10 °C. Trichogramma brassicae, however, only parasitizes in the dark during day time, not in the dark at night. Trichogramma evanescens can parasitize moth eggs in the dark during day time and at night. Present results show that time of day is maybe even more important for these wasps than whether it is light or dark because they parasitized more during the 6 hours period in the afternoon than during the 12 hours period overnight. Pompanon et al. (1999) reported for T. brassicae wasps that they have a day-night rhythm. Trichogramma brassicae is only active during day time (when it was light in their experiments) and not at night (darkness in their experiments). This can explain why female T. brassicae wasps parasitized more eggs in the 6 hours of darkness experiment than in the 12 hours of darkness experiment. In the latter experiment even none of the female T. brassicae wasps parasitized eggs. This can be due to the fact that this experiment was carried out at a time when it is normally dark for the wasps and so they are inactive. Pompanon et al. (1999) only tested one strain (line). Here, two lines were studied per species and it was shown that there was no variation within a species.

20 At almost all temperatures T. evanescens parasitized more than T. brassicae, regardless of the time of day. The day/night rhythm of this species is unknown but it is known that this species has a wide range of hosts (Lepidopteran) including many moths (Fatouros et al., 2007; Jabob & Boivin, 2005). Because most of the moth species lay their eggs at night (Waring & Townsend; 2003), and Trichogramma wasps need freshly laid eggs (Herz et al., 2007; Keasar et al., 2000; Knutson, 2000), T. evanescens wasps must also be active at night to find host eggs.

The work of Fatouros et al. (2005), present findings and preliminary results by F. Pashalidou and M.E. Huigens (unpublished data) now indicate that another parasitic wasp species, T. evanescens, has to learn to spies on an anti-aphrodisiac pheromone and specifically hitch- hike with mated female cabbage white butterflies. This suggests that eavesdropping on an anti-aphrodisiac and hitch-hiking with mated female butterflies is a common strategy within Trichogramma wasps. Obviously more Trichogramma and host species have to be tested in laboratory tests and field surveys as described in this work to determine whether this strategy is as or more important than other host finding strategies like the use of chemical cues that are emitted by plants (Lobdell et al. 2005). It is also important to understand the hitch-hiking behavior of certain species to improve the efficiency of mass releases of Trichogramma wasps in biological control. Timing of release is crucial when wasps would rely on hitch- hiking to find host eggs.

21 Acknowledgments The following persons I want to thank for there support and help: ƒ Ties Huigens for supervising, help with laboratory experiments, field work and borrowing the car for night catches. ƒ Bonne Beerda for supervising. ƒ Foteini Pashalidou for help with the laboratory experiments and field work. ƒ Silja Tribuhl for help with laboratory experiments and field work. ƒ Marion Munneke for help with field work. ƒ Patrick Verbaarschot for help with molecular laboratory experiments. ƒ Nina Fatouros for information about laboratory experiments. ƒ Tibor Bukovinszky for helping with statistical analysis of the night parasitism experiment. ƒ Hans Smid for taking pictures of the caterpillar species Hoplodrina ambigua. ƒ Jeroen Spitzen for arranging the climate chambers. ƒ Yde Jongema for the identification of small insects. ƒ Rob de Vos for identification of the sexes of the moth Xestia c-nigrum. ƒ Jeroen Voogd for information about the moth Xestia c-nigrum. ƒ Tymo Muus for information about the moth Hoplodrina ambigua. ƒ The Dutch Butterfly Conservation for moth catches and for second opinions on moths that were difficult to identify. ƒ Theo Damen for permission to catch moths at Groot Arboretum, Wageningen. ƒ Ben Mol for permission to catch moths at Natuurcentrum Veluwe, Ede. ƒ Gradus Leenders for borrowing the van for night catches.

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25 Appendix 1: Research locations of the day and night moth catches

Area map of all research locations

1 = Entomology garden, Wageningen 4 = Klein arboretum, Wageningen 2 = Marijkeweg, Wageningen 5 = Groot arboretum, Wageningen 3 = Pabstsendam, Wageningen 6 = Natuurcentrum Veluwe, Ede

Area map of the research locations in Wageningen

1 = Entomology garden, Wageningen 4 = Klein arboretum, Wageningen 2 = Marijkeweg, Wageningen 5 = Groot arboretum, Wageningen 3 = Pabstsendam, Wageningen

26 Appendix 2: List of caught moths at night per research location Reported are the research locations and their coordinates, caught moths, gender of caught moths, found eggs in the vials, number of caught Trichogramma wasps, which Trichogramma species was on the moth and where it was found in the vial.

No Date Research location Coordinates Moth species Gender Eggs N Trichogramm Body Tricho a species part 1 26/4/07 Entomology garden X:17570 Ligdia adustata male 0 Wageningen Y:44195 2 7/9/07 Groot aboretum X:17570 Abrostola 0 Wageningen Y:44195 triplasia 3 7/9/07 Groot aboretum X:17570 Agrotis ipsilon 0 Wageningen Y:44195 4 7/9/07 Groot aboretum X:17570 Agrotis ipsilon 0 Wageningen Y:44195 5 2/8/07 Groot aboretum X:17570 Agrotis puta male 0 Wageningen Y:44195 6 2/8/07 Groot aboretum X:17570 Agrotis puta male 0 Wageningen Y:44195 7 8/8/07 Groot aboretum X:17570 Agrotis puta male 0 Wageningen Y:44195 8 8/8/07 Groot aboretum X:17570 Agrotis puta male 0 Wageningen Y:44195 9 8/8/07 Groot aboretum X:17570 Agrotis puta male 0 Wageningen Y:44195 10 8/8/07 Groot aboretum X:17570 Agrotis puta female 0 Wageningen Y:44195 11 7/9/07 Groot aboretum X:17570 Agrotis puta male 0 Wageningen Y:44195 12 8/8/07 Groot aboretum X:17570 Agrotis segetum 0 Wageningen Y:44195 13 8/8/07 Groot aboretum X:17570 Agrotis segetum 0 Wageningen Y:44195 14 28/8/07 Groot aboretum X:17570 Agrotis segetum female single 0 Wageningen Y:44195 15 8/8/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 16 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 17 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 18 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 19 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 20 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 21 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 22 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 23 7/9/07 Groot aboretum X:17570 Amphipyra 0 Wageningen Y:44195 pyramidea 24 7/9/07 Groot aboretum X:17570 Atethmia 0 Wageningen Y:44195 centrago 25 7/9/07 Groot aboretum X:17570 Atethmia 0 Wageningen Y:44195 centrago 26 8/8/07 Groot aboretum X:17570 Autographa 0 Wageningen Y:44195 gamma 27 8/8/07 Groot aboretum X:17570 Autographa 0 Wageningen Y:44195 gamma 28 7/9/07 Groot aboretum X:17570 Autographa 0 Wageningen Y:44195 gamma 29 8/8/07 Groot aboretum X:17570 Cabera pusaria 0 Wageningen Y:44195 30 8/8/07 Groot aboretum X:17570 Cabera pusaria 0 Wageningen Y:44195 31 2/8/07 Groot aboretum X:17570 Campaea 0 Wageningen Y:44195 margaritata 32 8/8/07 Groot aboretum X:17570 Campaea 0 Wageningen Y:44195 margaritata

27 33 8/8/07 Groot aboretum X:17570 Campaea 0 Wageningen Y:44195 margaritata 34 8/8/07 Groot aboretum X:17570 Campaea 0 Wageningen Y:44195 margaritata 35 8/8/07 Groot aboretum X:17570 Camptogramma 0 Wageningen Y:44195 bilineata 36 8/8/07 Groot aboretum X:17570 Colostygia 0 Wageningen Y:44195 pectinataria 37 2/8/07 Groot aboretum X:17570 Cosmia 0 Wageningen Y:44195 trapezina 38 2/8/07 Groot aboretum X:17570 Craniophora 0 Wageningen Y:44195 ligustri 39 8/8/07 Groot aboretum X:17570 Craniophora 0 Wageningen Y:44195 ligustri 40 8/8/07 Groot aboretum X:17570 Cyclophora 0 Wageningen Y:44195 albipunctata 41 8/8/07 Groot aboretum X:17570 Diachrysia 0 Wageningen Y:44195 chrysitis 42 28/8/07 Groot aboretum X:17570 Diachrysia 0 Wageningen Y:44195 chrysitis 43 7/9/07 Groot aboretum X:17570 Diachrysia 0 Wageningen Y:44195 chrysitis 44 8/8/07 Groot aboretum X:17570 Diarsia rubi 0 Wageningen Y:44195 45 8/8/07 Groot aboretum X:17570 Diarsia rubi 0 Wageningen Y:44195 46 8/8/07 Groot aboretum X:17570 Diarsia rubi 0 Wageningen Y:44195 47 8/8/07 Groot aboretum X:17570 Diarsia rubi 0 Wageningen Y:44195 48 7/9/07 Groot aboretum X:17570 Ecliptopera 0 Wageningen Y:44195 silaceata 49 2/8/07 Groot aboretum X:17570 Eilema 0 Wageningen Y:44195 complana 50 8/8/07 Groot aboretum X:17570 Eilema 0 Wageningen Y:44195 complana 51 2/8/07 Groot aboretum X:17570 Epirrhoe 0 Wageningen Y:44195 alternata 52 8/8/07 Groot aboretum X:17570 Epirrhoe 0 Wageningen Y:44195 alternata 53 8/8/07 Groot aboretum X:17570 Epirrhoe 0 Wageningen Y:44195 alternata 54 2/8/07 Groot aboretum X:17570 Eupithecia 0 Wageningen Y:44195 succenturiata 55 8/8/07 Groot aboretum X:17570 Hypena 0 Wageningen Y:44195 proboscidalis 56 8/8/07 Groot aboretum X:17570 Hypena 0 Wageningen Y:44195 proboscidalis 57 2/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 58 2/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 59 2/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 60 2/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 61 8/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 62 8/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 63 8/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 64 8/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 65 8/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 66 8/8/07 Groot aboretum X:17570 Idaea aversata 0 Wageningen Y:44195 67 8/8/07 Groot aboretum X:17570 Lacanobia 0 Wageningen Y:44195 oleracea 68 8/8/07 Groot aboretum X:17570 Lacanobia female 0 Wageningen Y:44195 oleracea

28 69 8/8/07 Groot aboretum X:17570 Lacanobia 0 Wageningen Y:44195 oleracea 70 8/8/07 Groot aboretum X:17570 Lacanobia 0 Wageningen Y:44195 oleracea 71 8/8/07 Groot aboretum X:17570 Lymantria dispar male 0 Wageningen Y:44195 72 8/8/07 Groot aboretum X:17570 Macaria 0 Wageningen Y:44195 alternata 73 8/8/07 Groot aboretum X:17570 Macaria 0 Wageningen Y:44195 alternata 74 8/8/07 Groot aboretum X:17570 Macaria notata 0 Wageningen Y:44195 75 8/8/07 Groot aboretum X:17570 Macaria notata 0 Wageningen Y:44195 76 8/8/07 Groot aboretum X:17570 Macaria notata 0 Wageningen Y:44195 77 8/8/07 Groot aboretum X:17570 Macaria notata 0 Wageningen Y:44195 78 2/8/07 Groot aboretum X:17570 Macuria 0 Wageningen Y:44195 alternata 79 2/8/07 Groot aboretum X:17570 Macuria 0 Wageningen Y:44195 alternata 80 2/8/07 Groot aboretum X:17570 Macuria 0 Wageningen Y:44195 alternata 81 2/8/07 Groot aboretum X:17570 Macuria 0 Wageningen Y:44195 alternata 82 2/8/07 Groot aboretum X:17570 Macuria 0 Wageningen Y:44195 alternata 83 8/8/07 Groot aboretum X:17570 Memestra 0 Wageningen Y:44195 brassicae 84 7/9/07 Groot aboretum X:17570 Mesapamea 0 Wageningen Y:44195 secalis 85 2/8/07 Groot aboretum X:17570 Mesoligia 0 Wageningen Y:44195 furuncula 86 8/8/07 Groot aboretum X:17570 Mesoligia 0 Wageningen Y:44195 furuncula 87 2/8/07 Groot aboretum X:17570 Mythimna 0 Wageningen Y:44195 albipuncta 88 8/8/07 Groot aboretum X:17570 Mythimna 0 Wageningen Y:44195 albipuncta 89 8/8/07 Groot aboretum X:17570 Mythimna 0 Wageningen Y:44195 albipuncta 90 8/8/07 Groot aboretum X:17570 Mythimna 0 Wageningen Y:44195 albipuncta 91 7/9/07 Groot aboretum X:17570 Noctua comes 0 Wageningen Y:44195 92 7/9/07 Groot aboretum X:17570 Noctua interjecta 0 Wageningen Y:44195 93 7/9/07 Groot aboretum X:17570 Noctua interjecta 0 Wageningen Y:44195 94 28/8/07 Groot aboretum X:17570 Noctua janthina 0 Wageningen Y:44195 95 7/9/07 Groot aboretum X:17570 Noctua janthina 0 Wageningen Y:44195 96 7/9/07 Groot aboretum X:17570 Noctua janthina 0 Wageningen Y:44195 97 2/8/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 98 8/8/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 99 8/8/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 100 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 101 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 102 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 103 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 104 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195

29 105 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 106 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 107 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 108 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 109 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 110 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 111 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 112 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 113 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 114 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 115 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 116 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 117 7/9/07 Groot aboretum X:17570 Noctua pronuba male 0 Wageningen Y:44195 118 7/9/07 Groot aboretum X:17570 Noctua pronuba female 0 Wageningen Y:44195 119 8/8/07 Groot aboretum X:17570 Notodonta 0 Wageningen Y:44195 dromedarius 120 8/8/07 Groot aboretum X:17570 Notodonta 0 Wageningen Y:44195 dromedarius 121 2/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 122 2/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 123 2/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 124 2/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 125 2/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 126 8/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 127 8/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 128 8/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 129 8/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 130 8/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 131 8/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 132 28/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 133 28/8/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 134 7/9/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 135 7/9/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 136 7/9/07 Groot aboretum X:17570 Ochropleura 0 Wageningen Y:44195 plecta 137 2/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 138 2/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 139 8/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 140 8/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata

30 141 8/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 142 8/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 143 8/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 144 28/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 145 28/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 146 28/8/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 147 7/9/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 148 7/9/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 149 7/9/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 150 7/9/07 Groot aboretum X:17570 Opisthograptis 0 Wageningen Y:44195 luteolata 151 8/8/07 Groot aboretum X:17570 Paradrina 0 Wageningen Y:44195 clavipalpis 152 8/8/07 Groot aboretum X:17570 Peribatodes 0 Wageningen Y:44195 rhomboidaria 153 7/9/07 Groot aboretum X:17570 Peribatodes female 0 Wageningen Y:44195 rhomboidaria 154 8/8/07 Groot aboretum X:17570 Perizoma 0 Wageningen Y:44195 alchemillata 155 8/8/07 Groot aboretum X:17570 Perizoma 0 Wageningen Y:44195 alchemillata 156 8/8/07 Groot aboretum X:17570 Perizoma 0 Wageningen Y:44195 alchemillata 157 28/8/07 Groot aboretum X:17570 Perizoma 0 Wageningen Y:44195 alchemillata 158 8/8/07 Groot aboretum X:17570 Pheosia tremula 0 Wageningen Y:44195 159 8/8/07 Groot aboretum X:17570 Phlogophora 0 Wageningen Y:44195 meticulosa 160 7/9/07 Groot aboretum X:17570 Phlogophora 0 Wageningen Y:44195 meticulosa 161 7/9/07 Groot aboretum X:17570 Phlogophora female single/ 0 Wageningen Y:44195 meticulosa group 162 7/9/07 Groot aboretum X:17570 Phragmatobia 0 Wageningen Y:44195 fuliginosa 163 7/9/07 Groot aboretum X:17570 Phragmatobia 0 Wageningen Y:44195 fuliginosa 164 2/8/07 Groot aboretum X:17570 Ptilodon 0 Wageningen Y:44195 capucina 165 2/8/07 Groot aboretum X:17570 Ptilodon 0 Wageningen Y:44195 capucina 166 2/8/07 Groot aboretum X:17570 Ptilodon 0 Wageningen Y:44195 capucina 167 2/8/07 Groot aboretum X:17570 Ptilodon 0 Wageningen Y:44195 capucina 168 2/8/07 Groot aboretum X:17570 Ptilodon 0 Wageningen Y:44195 capucina 169 8/8/07 Groot aboretum X:17570 Ptilodon 0 Wageningen Y:44195 capucina 170 7/9/07 Groot aboretum X:17570 Rivula sericealis 0 Wageningen Y:44195 171 7/9/07 Groot aboretum X:17570 Rivula sericealis 0 Wageningen Y:44195 172 28/8/07 Groot aboretum X:17570 Thaumetopoea 0 Wageningen Y:44195 processionea 173 7/9/07 Groot aboretum X:17570 Thaumetopoea 0 Wageningen Y:44195 processionea 174 7/9/07 Groot aboretum X:17570 Thaumetopoea 0 Wageningen Y:44195 processionea 175 7/9/07 Groot aboretum X:17570 Thera britannica 0 Wageningen Y:44195 176 7/9/07 Groot aboretum X:17570 Timandra comae male 0 Wageningen Y:44195

31 177 2/8/07 Groot aboretum X:17570 Watsonalla female 0 Wageningen Y:44195 binaria 178 7/9/07 Groot aboretum X:17570 Xanthia citrago 0 Wageningen Y:44195 179 24/9/07 Groot aboretum X:17570 Xanthia citrago 0 Wageningen Y:44195 180 8/8/07 Groot aboretum X:17570 Xanthorhoe 0 Wageningen Y:44195 designata 181 8/8/07 Groot aboretum X:17570 Xanthorhoe 0 Wageningen Y:44195 designata 182 28/8/07 Groot aboretum X:17570 Xanthorhoe 0 Wageningen Y:44195 ferrugata 183 7/9/07 Groot aboretum X:17570 Xanthorhoe 0 Wageningen Y:44195 ferrugata 184 7/9/07 Groot aboretum X:17570 Xanthorhoe 0 Wageningen Y:44195 fluctuata 185 8/8/07 Groot aboretum X:17570 Xanthorhoe 0 Wageningen Y:44195 spadicearia 186 7/9/07 Groot aboretum X:17570 Xestia baja 0 Wageningen Y:44195 187 2/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 188 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 189 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 190 8/8/07 Groot aboretum X:17570 Xestia c-nigrum female 0 Wageningen Y:44195 191 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 192 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 193 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 194 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 195 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 196 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 197 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 198 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 199 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 200 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 201 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 202 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 203 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 204 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 205 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 206 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 207 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 208 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 209 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 210 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 211 8/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 212 28/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195

32 213 28/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 214 28/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 215 28/8/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 216 7/9/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 217 7/9/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 218 7/9/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 219 7/9/07 Groot aboretum X:17570 Xestia c-nigrum 0 Wageningen Y:44195 220 7/9/07 Groot aboretum X:17570 Xestia 0 Wageningen Y:44195 xanthographa 221 7/9/07 Groot aboretum X:17570 Xestia 0 Wageningen Y:44195 xanthographa 222 7/9/07 Groot aboretum X:17570 Xestia 0 Wageningen Y:44195 xanthographa 223 7/9/07 Groot aboretum X:17570 Xestia 0 Wageningen Y:44195 xanthographa 224 7/9/07 Groot aboretum X:17570 Xestia 0 Wageningen Y:44195 xanthographa 225 27/10/07 Marijkeweg X:17311 Colotois male 0 Wageningen Y:44232 pennaria 226 6/9/07 Natuurcentrum Ede X:17855 Amphipyra 0 Y:45057 pyramidea 227 6/9/07 Natuurcentrum Ede X:17855 Amphipyra 0 Y:45057 pyramidea 228 6/9/07 Natuurcentrum Ede X:17855 Amphipyra 0 Y:45057 pyramidea 229 6/9/07 Natuurcentrum Ede X:17855 Amphipyra 0 Y:45057 pyramidea 230 6/9/07 Natuurcentrum Ede X:17855 Autographa 0 Y:45057 gamma 231 6/9/07 Natuurcentrum Ede X:17855 Campaea female group 0 Y:45057 margaritata 232 6/9/07 Natuurcentrum Ede X:17855 Campaea female group 0 Y:45057 margaritata 233 6/9/07 Natuurcentrum Ede X:17855 Campaea female group 0 Y:45057 margaritata 234 6/9/07 Natuurcentrum Ede X:17855 Campaea 0 Y:45057 margaritata 235 6/9/07 Natuurcentrum Ede X:17855 Campaea 0 Y:45057 margaritata 236 6/9/07 Natuurcentrum Ede X:17855 Campaea 0 Y:45057 margaritata 237 13/9/07 Natuurcentrum Ede X:17855 Campaea female 0 Y:45057 margaritata 238 6/9/07 Natuurcentrum Ede X:17855 Camptogramma female single 0 Y:45057 bilineata 239 6/9/07 Natuurcentrum Ede X:17855 Cerapteryx female group 0 Y:45057 graminis 240 6/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 citrata 241 6/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 truncata 242 6/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 truncata 243 6/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 truncata 244 6/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 truncata 245 6/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 truncata 246 13/9/07 Natuurcentrum Ede X:17855 Chloroclysta 0 Y:45057 truncata 247 6/9/07 Natuurcentrum Ede X:17855 Colostygia male 0 Y:45057 pectinataria 248 6/9/07 Natuurcentrum Ede X:17855 Cyclophora 0 Y:45057 linearia

33 249 13/9/07 Natuurcentrum Ede X:17855 Cyclophora 0 Y:45057 linearia 250 6/9/07 Natuurcentrum Ede X:17855 Diachrysia 0 Y:45057 chrysitis 251 6/9/07 Natuurcentrum Ede X:17855 Diarsia rubi 0 Y:45057 252 6/9/07 Natuurcentrum Ede X:17855 Epione 0 Y:45057 repandaria 253 6/9/07 Natuurcentrum Ede X:17855 Epirrhoe 0 Y:45057 alternata 254 6/9/07 Natuurcentrum Ede X:17855 Epirrhoe 0 Y:45057 alternata 255 6/9/07 Natuurcentrum Ede X:17855 Epirrhoe 0 Y:45057 alternata 256 6/9/07 Natuurcentrum Ede X:17855 Epirrhoe 0 Y:45057 alternata 257 6/9/07 Natuurcentrum Ede X:17855 Erannis 0 Y:45057 defoliaria 258 6/9/07 Natuurcentrum Ede X:17855 Eupithecia female single 0 Y:45057 centaureata 259 6/9/07 Natuurcentrum Ede X:17855 Eupithecia 0 Y:45057 centaureata 260 13/9/07 Natuurcentrum Ede X:17855 Eupithecia 0 Y:45057 centaureata 261 6/9/07 Natuurcentrum Ede X:17855 Eupithecia 0 Y:45057 indigata 262 6/9/07 Natuurcentrum Ede X:17855 Eupithecia 0 Y:45057 indigata 263 6/9/07 Natuurcentrum Ede X:17855 Hoplodrina female single 0 Y:45057 blanda 264 13/9/07 Natuurcentrum Ede X:17855 Idaea biselata 0 Y:45057 265 6/9/07 Natuurcentrum Ede X:17855 Macaria liturata 0 Y:45057 266 6/9/07 Natuurcentrum Ede X:17855 Macaria liturata 0 Y:45057 267 6/9/07 Natuurcentrum Ede X:17855 Macaria liturata 0 Y:45057 268 6/9/07 Natuurcentrum Ede X:17855 Macaria liturata 0 Y:45057 269 6/9/07 Natuurcentrum Ede X:17855 Macaria liturata 0 Y:45057 270 6/9/07 Natuurcentrum Ede X:17855 Macaria liturata 0 Y:45057 271 6/9/07 Natuurcentrum Ede X:17855 Mesapamea 0 Y:45057 secalis 272 6/9/07 Natuurcentrum Ede X:17855 Mesapamea 0 Y:45057 secalis 273 6/9/07 Natuurcentrum Ede X:17855 Noctua comes female 0 Y:45057 274 13/9/07 Natuurcentrum Ede X:17855 Noctua comes female single 0 Y:45057 275 6/9/07 Natuurcentrum Ede X:17855 Noctua pronuba female 0 Y:45057 276 6/9/07 Natuurcentrum Ede X:17855 Noctua pronuba female 0 Y:45057 277 6/9/07 Natuurcentrum Ede X:17855 Noctua pronuba male 0 Y:45057 278 13/9/07 Natuurcentrum Ede X:17855 Noctua pronuba female 0 Y:45057 279 6/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta 280 6/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta 281 6/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta 282 6/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta 283 6/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta 284 6/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta

34 285 13/9/07 Natuurcentrum Ede X:17855 Ochropleura 0 Y:45057 plecta 286 6/9/07 Natuurcentrum Ede X:17855 Opisthograptis 0 Y:45057 luteolata 287 6/9/07 Natuurcentrum Ede X:17855 Opisthograptis 0 Y:45057 luteolata 288 6/9/07 Natuurcentrum Ede X:17855 Peribatodes female single 0 Y:45057 rhomboidaria 289 6/9/07 Natuurcentrum Ede X:17855 Peribatodes female single 0 Y:45057 rhomboidaria 290 6/9/07 Natuurcentrum Ede X:17855 Peribatodes male 0 Y:45057 rhomboidaria 291 6/9/07 Natuurcentrum Ede X:17855 Peribatodes female single 0 Y:45057 rhomboidaria 292 6/9/07 Natuurcentrum Ede X:17855 Peribatodes male 0 Y:45057 rhomboidaria 293 6/9/07 Natuurcentrum Ede X:17855 Phlogophora 0 Y:45057 meticulosa 294 13/9/07 Natuurcentrum Ede X:17855 Rivula sericealis 0 Y:45057 295 6/9/07 Natuurcentrum Ede X:17855 Thaumetopoea 0 Y:45057 processionea 296 6/9/07 Natuurcentrum Ede X:17855 Thaumetopoea 0 Y:45057 processionea 297 6/9/07 Natuurcentrum Ede X:17855 Thaumetopoea 0 Y:45057 processionea 298 6/9/07 Natuurcentrum Ede X:17855 Thera britannica 0 Y:45057 299 13/9/07 Natuurcentrum Ede X:17855 Thera britannica 0 Y:45057 300 6/9/07 Natuurcentrum Ede X:17855 Tholera 0 Y:45057 decimalis 301 6/9/07 Natuurcentrum Ede X:17855 Tholera 0 Y:45057 decimalis 302 6/9/07 Natuurcentrum Ede X:17855 Tholera 0 Y:45057 decimalis 303 6/9/07 Natuurcentrum Ede X:17855 Timandra comae male 0 Y:45057 304 6/9/07 Natuurcentrum Ede X:17855 Watsonalla male 0 Y:45057 binaria 305 6/9/07 Natuurcentrum Ede X:17855 Watsonalla male 0 Y:45057 binaria 306 6/9/07 Natuurcentrum Ede X:17855 Watsonalla male 0 Y:45057 binaria 307 6/9/07 Natuurcentrum Ede X:17855 Watsonalla male 0 Y:45057 binaria 308 6/9/07 Natuurcentrum Ede X:17855 Watsonalla female 0 Y:45057 binaria 309 13/9/07 Natuurcentrum Ede X:17855 Watsonalla 0 Y:45057 binaria 310 13/9/07 Natuurcentrum Ede X:17855 Watsonalla 0 Y:45057 binaria 311 6/9/07 Natuurcentrum Ede X:17855 Watsonalla 0 Y:45057 cultraria 312 6/9/07 Natuurcentrum Ede X:17855 Xanthorhoe 0 Y:45057 biriviata 313 13/9/07 Natuurcentrum Ede X:17855 Xanthorhoe 0 Y:45057 ferrugata 314 6/9/07 Natuurcentrum Ede X:17855 Xestia baja 0 Y:45057 315 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 316 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 317 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 318 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 319 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 320 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057

35 321 6/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 322 13/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 1 T. brassicae lid Y:45057 female 323 13/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 324 13/9/07 Natuurcentrum Ede X:17855 Xestia c-nigrum 0 Y:45057 325 6/9/07 Natuurcentrum Ede X:17855 Xestia 0 Y:45057 xanthographa 326 13/9/07 Natuurcentrum Ede X:17855 Xestia 0 Y:45057 xanthographa 327 11/9/07 Pabstsendam X:17389 Abrostola 0 Wageningen Y:44108 triplasia 328 15/10/07 Pabstsendam X:17389 Agrochola lota 0 Wageningen Y:44108 329 15/10/07 Pabstsendam X:17389 Agrochola female 0 Wageningen Y:44108 macilenta 330 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 331 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 332 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 333 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 334 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 335 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 336 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 337 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 338 6/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 339 21/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 340 21/6/07 Pabstsendam X:17389 Agrotis 0 Wageningen Y:44108 exclamationis 341 31/7/07 Pabstsendam X:17389 Agrotis puta male 0 Wageningen Y:44108 342 31/7/07 Pabstsendam X:17389 Agrotis puta male 0 Wageningen Y:44108 343 5/8/07 Pabstsendam X:17389 Agrotis puta female 0 Wageningen Y:44108 344 31/7/07 Pabstsendam X:17389 Agrotis segetum 0 Wageningen Y:44108 345 31/7/07 Pabstsendam X:17389 Agrotis segetum 0 Wageningen Y:44108 346 5/8/07 Pabstsendam X:17389 Agrotis segetum 0 Wageningen Y:44108 347 11/9/07 Pabstsendam X:17389 Amphipyra 0 Wageningen Y:44108 pyramidea 348 11/9/07 Pabstsendam X:17389 Atethmia female single 0 Wageningen Y:44108 centrago 349 11/9/07 Pabstsendam X:17389 Atethmia 0 Wageningen Y:44108 centrago 350 31/7/07 Pabstsendam X:17389 Autographa 0 Wageningen Y:44108 gamma 351 31/7/07 Pabstsendam X:17389 Autographa 0 Wageningen Y:44108 gamma 352 26/8/07 Pabstsendam X:17389 Autographa 0 Wageningen Y:44108 gamma 353 15/10/07 Pabstsendam X:17389 Autographa 0 Wageningen Y:44108 gamma 354 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 355 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 356 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108

36 357 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 358 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 359 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 360 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 361 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 362 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 363 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 364 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 365 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 366 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 367 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 368 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 369 6/6/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 370 13/7/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 371 13/7/07 Pabstsendam X:17389 Axylia putris 0 Wageningen Y:44108 372 31/7/07 Pabstsendam X:17389 Cabera 0 Wageningen Y:44108 exanthemata 373 5/8/07 Pabstsendam X:17389 Cabera 0 Wageningen Y:44108 exanthemata 374 26/8/07 Pabstsendam X:17389 Cabera male 0 Wageningen Y:44108 exanthemata 375 26/8/07 Pabstsendam X:17389 Cabera male 0 Wageningen Y:44108 exanthemata 376 26/8/07 Pabstsendam X:17389 Cabera male 0 Wageningen Y:44108 exanthemata 377 26/8/07 Pabstsendam X:17389 Cabera female single 0 Wageningen Y:44108 exanthemata 378 5/8/07 Pabstsendam X:17389 Cabera pusaria 0 Wageningen Y:44108 379 26/8/07 Pabstsendam X:17389 Cabera pusaria male 0 Wageningen Y:44108 380 31/7/07 Pabstsendam X:17389 Campaea 0 Wageningen Y:44108 margaritata 381 5/8/07 Pabstsendam X:17389 Camptogramma 0 Wageningen Y:44108 bilineata 382 11/9/07 Pabstsendam X:17389 Chloroclysta 0 Wageningen Y:44108 truncata 383 11/9/07 Pabstsendam X:17389 Chloroclysta 0 Wageningen Y:44108 truncata 384 11/9/07 Pabstsendam X:17389 Chloroclysta 0 Wageningen Y:44108 truncata 385 11/9/07 Pabstsendam X:17389 Chloroclysta female single 0 Wageningen Y:44108 truncata 386 11/9/07 Pabstsendam X:17389 Chloroclysta 0 Wageningen Y:44108 truncata 387 26/8/07 Pabstsendam X:17389 Cosmorhoe 0 Wageningen Y:44108 ocellata 388 31/7/07 Pabstsendam X:17389 Cossus cossus 0 Wageningen Y:44108 389 31/7/07 Pabstsendam X:17389 Cucullia 0 Wageningen Y:44108 umbratica 390 5/8/07 Pabstsendam X:17389 Cyclophora 0 Wageningen Y:44108 punctaria 391 6/6/07 Pabstsendam X:17389 Deilephila 0 Wageningen Y:44108 elpenor 392 6/6/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis

37 393 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 394 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 395 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 396 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 397 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 398 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 399 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 400 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 401 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 402 31/7/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 403 5/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 404 5/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 405 26/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 406 26/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 407 26/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 408 26/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 409 26/8/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 410 11/9/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 411 11/9/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 412 11/9/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 413 11/9/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 414 11/9/07 Pabstsendam X:17389 Diachrysia 0 Wageningen Y:44108 chrysitis 415 5/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 416 5/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 417 5/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 418 5/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 419 5/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 420 5/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 421 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 422 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 423 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 424 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 425 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 426 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 427 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 428 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108

38 429 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 430 26/8/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 431 11/9/07 Pabstsendam X:17389 Diarsia rubi 0 Wageningen Y:44108 432 31/7/07 Pabstsendam X:17389 Earias clorana 0 Wageningen Y:44108 433 11/9/07 Pabstsendam X:17389 Ectropis 0 Wageningen Y:44108 crepuscularia 434 13/7/07 Pabstsendam X:17389 Epione 0 Wageningen Y:44108 repandaria 435 13/7/07 Pabstsendam X:17389 Epione 0 Wageningen Y:44108 repandaria 436 11/9/07 Pabstsendam X:17389 Epione 0 Wageningen Y:44108 repandaria 437 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 438 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 439 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 440 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 441 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 442 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 443 13/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 444 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 445 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 446 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 447 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 448 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 449 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 450 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 451 31/7/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 452 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 453 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 454 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 455 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 456 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 457 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 458 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 459 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 460 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 461 5/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 462 26/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 463 26/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 464 26/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata

39 465 26/8/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 466 11/9/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 467 11/9/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 468 11/9/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 469 11/9/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 470 11/9/07 Pabstsendam X:17389 Epirrhoe female single 0 Wageningen Y:44108 alternata 471 11/9/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 472 11/9/07 Pabstsendam X:17389 Epirrhoe 0 Wageningen Y:44108 alternata 473 31/7/07 Pabstsendam X:17389 Eupithecia 0 Wageningen Y:44108 centaureata 474 5/8/07 Pabstsendam X:17389 Eupithecia 0 Wageningen Y:44108 centaureata 475 31/7/07 Pabstsendam X:17389 Eupithecia 0 Wageningen Y:44108 succenturiata 476 11/9/07 Pabstsendam X:17389 Eupithecia 0 Wageningen Y:44108 tripunctaria 477 26/8/07 Pabstsendam X:17389 Gortyna flavago 0 Wageningen Y:44108 478 6/6/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 479 6/6/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 480 21/6/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 481 5/8/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 482 26/8/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 483 26/8/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 484 26/8/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 485 11/9/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 486 11/9/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 487 11/9/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 488 11/9/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 489 11/9/07 Pabstsendam X:17389 Habrosyne 0 Wageningen Y:44108 pyritoides 490 26/8/07 Pabstsendam X:17389 Hoplodrina 0 Wageningen Y:44108 ambigua 491 26/8/07 Pabstsendam X:17389 Hoplodrina female single 0 Wageningen Y:44108 blanda 492 21/6/07 Pabstsendam X:17389 Hoplodrina 0 Wageningen Y:44108 octogenaria 493 31/7/07 Pabstsendam X:17389 Hydraecia 0 Wageningen Y:44108 micacea 494 5/8/07 Pabstsendam X:17389 Hydraecia 0 Wageningen Y:44108 micacea 495 5/8/07 Pabstsendam X:17389 Hydraecia 0 Wageningen Y:44108 micacea 496 11/9/07 Pabstsendam X:17389 Hylaea fasciaria male 0 Wageningen Y:44108 497 6/6/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 498 6/6/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 499 31/7/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 500 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis

40 501 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 502 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 503 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 504 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 505 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 506 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 507 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 508 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 509 5/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 510 26/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 511 26/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 512 26/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 513 26/8/07 Pabstsendam X:17389 Hypena 0 Wageningen Y:44108 proboscidalis 514 6/6/07 Pabstsendam X:17389 Idaea aversata 0 Wageningen Y:44108 515 26/8/07 Pabstsendam X:17389 Idaea aversata 0 Wageningen Y:44108 516 5/8/07 Pabstsendam X:17389 Lacanobia 0 Wageningen Y:44108 oleracea 517 5/8/07 Pabstsendam X:17389 Lacanobia female 0 Wageningen Y:44108 oleracea 518 26/8/07 Pabstsendam X:17389 Lacanobia 0 Wageningen Y:44108 oleracea 519 26/8/07 Pabstsendam X:17389 Lacanobia female group 0 Wageningen Y:44108 oleracea 520 26/8/07 Pabstsendam X:17389 Lacanobia 0 Wageningen Y:44108 oleracea 521 11/9/07 Pabstsendam X:17389 Lacanobia 0 Wageningen Y:44108 oleracea 522 21/6/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 523 31/7/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 524 31/7/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 525 5/8/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 526 26/8/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 527 26/8/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 528 26/8/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 529 26/8/07 Pabstsendam X:17389 Lomaspilis 0 Wageningen Y:44108 marginata 530 6/6/07 Pabstsendam X:17389 Lomographa 0 Wageningen Y:44108 temerata 531 6/6/07 Pabstsendam X:17389 Lomographa 0 Wageningen Y:44108 temerata 532 5/8/07 Pabstsendam X:17389 Macaria 0 Wageningen Y:44108 alternata 533 5/8/07 Pabstsendam X:17389 Macaria notata 0 Wageningen Y:44108 534 26/8/07 Pabstsendam X:17389 Macdunoughia 0 Wageningen Y:44108 confusa 535 31/7/07 Pabstsendam X:17389 Macuria 0 Wageningen Y:44108 alternata 536 31/7/07 Pabstsendam X:17389 Macuria 0 Wageningen Y:44108 alternata

41 537 5/8/07 Pabstsendam X:17389 Mesapamea 0 Wageningen Y:44108 didyma 538 26/8/07 Pabstsendam X:17389 Mesapamea 0 Wageningen Y:44108 secalis 539 5/8/07 Pabstsendam X:17389 Mesoligia 0 Wageningen Y:44108 furuncula 540 5/8/07 Pabstsendam X:17389 Mesoligia 0 Wageningen Y:44108 furuncula 541 5/8/07 Pabstsendam X:17389 Mesoligia 0 Wageningen Y:44108 furuncula 542 5/8/07 Pabstsendam X:17389 Mesoligia 0 Wageningen Y:44108 furuncula 543 5/8/07 Pabstsendam X:17389 Mesoligia 0 Wageningen Y:44108 furuncula 544 5/8/07 Pabstsendam X:17389 Mythimna 0 Wageningen Y:44108 albipuncta 545 21/6/07 Pabstsendam X:17389 Mythimna 0 Wageningen Y:44108 impura 546 18/9/07 Pabstsendam X:17389 Mythimna l- 0 Wageningen Y:44108 album 547 21/6/07 Pabstsendam X:17389 Noctua comes female 0 Wageningen Y:44108 548 11/9/07 Pabstsendam X:17389 Noctua comes 0 Wageningen Y:44108 549 15/10/07 Pabstsendam X:17389 Noctua comes 0 Wageningen Y:44108 550 13/7/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 551 13/7/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 552 13/7/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 553 13/7/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 554 13/7/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 555 5/8/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 556 5/8/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 557 5/8/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 558 26/8/07 Pabstsendam X:17389 Noctua interjecta 0 Wageningen Y:44108 559 11/9/07 Pabstsendam X:17389 Noctua janthe 0 Wageningen Y:44108 560 11/9/07 Pabstsendam X:17389 Noctua janthe 0 Wageningen Y:44108 561 13/7/07 Pabstsendam X:17389 Noctua janthina 0 Wageningen Y:44108 562 26/8/07 Pabstsendam X:17389 Noctua janthina 0 Wageningen Y:44108 563 6/6/07 Pabstsendam X:17389 Noctua pronuba 0 Wageningen Y:44108 564 21/6/07 Pabstsendam X:17389 Noctua pronuba 0 Wageningen Y:44108 565 21/6/07 Pabstsendam X:17389 Noctua pronuba 0 Wageningen Y:44108 566 13/7/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 567 13/7/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 568 13/7/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 569 13/7/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 570 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 571 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 572 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108

42 573 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 574 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 575 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 576 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 577 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 578 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 579 13/7/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 580 5/8/07 Pabstsendam X:17389 Noctua pronuba female 0 Wageningen Y:44108 581 5/8/07 Pabstsendam X:17389 Noctua pronuba 0 Wageningen Y:44108 582 26/8/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 583 26/8/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 584 26/8/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 585 11/9/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 586 11/9/07 Pabstsendam X:17389 Noctua pronuba 0 Wageningen Y:44108 587 15/10/07 Pabstsendam X:17389 Noctua pronuba male 0 Wageningen Y:44108 588 31/7/07 Pabstsendam X:17389 Notodonta 0 Wageningen Y:44108 ziczac 589 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 590 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 591 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 592 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 593 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 594 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 595 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 596 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 597 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 598 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 599 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 600 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 601 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 602 13/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 603 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 604 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 605 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 606 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 607 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 608 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta

43 609 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 610 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 611 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 612 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 613 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 614 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 615 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 616 31/7/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 617 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 618 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 619 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 620 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 621 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 622 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 623 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 624 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 625 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 626 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 627 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 628 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 629 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 630 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 631 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 632 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 633 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 634 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 635 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 636 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 637 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 638 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 639 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 640 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 641 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 642 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 643 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 644 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta

44 645 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 646 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 647 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 648 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 649 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 650 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 651 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 652 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 653 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 654 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 655 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 656 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 657 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 658 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 659 5/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 660 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 661 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 662 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 663 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 664 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 665 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 666 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 667 26/8/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 668 11/9/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 669 11/9/07 Pabstsendam X:17389 Ochropleura 0 Wageningen Y:44108 plecta 670 31/7/07 Pabstsendam X:17389 Opisthograptis 0 Wageningen Y:44108 luteolata 671 5/8/07 Pabstsendam X:17389 Opisthograptis 0 Wageningen Y:44108 luteolata 672 6/6/07 Pabstsendam X:17389 Orthosia cerasi 0 Wageningen Y:44108 673 6/6/07 Pabstsendam X:17389 Orthosia gothica 0 Wageningen Y:44108 674 6/6/07 Pabstsendam X:17389 Orthosia gothica 0 Wageningen Y:44108 675 21/6/07 Pabstsendam X:17389 Orthosia incerta 0 Wageningen Y:44108 676 5/8/07 Pabstsendam X:17389 Peribatodes male 0 Wageningen Y:44108 rhomboidaria 677 26/8/07 Pabstsendam X:17389 Peribatodes male 0 Wageningen Y:44108 rhomboidaria 678 26/8/07 Pabstsendam X:17389 Peribatodes male 0 Wageningen Y:44108 rhomboidaria 679 11/9/07 Pabstsendam X:17389 Peribatodes male 0 Wageningen Y:44108 rhomboidaria 680 11/9/07 Pabstsendam X:17389 Peribatodes male 0 Wageningen Y:44108 rhomboidaria

45 681 11/9/07 Pabstsendam X:17389 Peribatodes male 0 Wageningen Y:44108 rhomboidaria 682 26/8/07 Pabstsendam X:17389 Phlogophora 0 Wageningen Y:44108 meticulosa 683 11/9/07 Pabstsendam X:17389 Phlogophora female single/ 0 Wageningen Y:44108 meticulosa group 684 11/9/07 Pabstsendam X:17389 Phlogophora 0 Wageningen Y:44108 meticulosa 685 13/7/07 Pabstsendam X:17389 Phragmatobia 0 Wageningen Y:44108 fuliginosa 686 13/7/07 Pabstsendam X:17389 Phragmatobia 0 Wageningen Y:44108 fuliginosa 687 13/7/07 Pabstsendam X:17389 Phragmatobia 0 Wageningen Y:44108 fuliginosa 688 13/7/07 Pabstsendam X:17389 Phragmatobia 0 Wageningen Y:44108 fuliginosa 689 26/8/07 Pabstsendam X:17389 Plusia festucae 0 Wageningen Y:44108 690 31/7/07 Pabstsendam X:17389 Polygogon 0 Wageningen Y:44108 strigilata 691 31/7/07 Pabstsendam X:17389 Pterostoma 0 Wageningen Y:44108 palpina 692 11/9/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 693 11/9/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 694 11/9/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 695 11/9/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 696 11/9/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 697 15/10/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 698 15/10/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 699 15/10/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 700 15/10/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 701 15/10/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 702 15/10/07 Pabstsendam X:17389 Rivula sericealis 0 Wageningen Y:44108 703 6/6/07 Pabstsendam X:17389 Spilosoma 0 Wageningen Y:44108 lubricipeda 704 5/8/07 Pabstsendam X:17389 Thaumetopoea 0 Wageningen Y:44108 processionea 705 11/9/07 Pabstsendam X:17389 Thera britannica 0 Wageningen Y:44108 706 6/6/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 707 6/6/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 708 6/6/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 709 6/6/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 710 6/6/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 711 21/6/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 712 5/8/07 Pabstsendam X:17389 Thyatira batis 0 Wageningen Y:44108 713 13/7/07 Pabstsendam X:17389 Timandra comae 0 Wageningen Y:44108 714 13/7/07 Pabstsendam X:17389 Timandra comae 0 Wageningen Y:44108 715 5/8/07 Pabstsendam X:17389 Timandra comae 0 Wageningen Y:44108 716 26/8/07 Pabstsendam X:17389 Timandra comae 0 Wageningen Y:44108

46 717 26/8/07 Pabstsendam X:17389 Timandra comae 0 Wageningen Y:44108 718 26/8/07 Pabstsendam X:17389 Triodia sylvina male 0 Wageningen Y:44108 719 26/8/07 Pabstsendam X:17389 Triodia sylvina female 0 Wageningen Y:44108 720 11/9/07 Pabstsendam X:17389 Xanthia icteritia 0 Wageningen Y:44108 721 15/10/07 Pabstsendam X:17389 Xanthia togata 0 Wageningen Y:44108 722 26/8/07 Pabstsendam X:17389 Xanthorhoe 0 Wageningen Y:44108 ferrugata 723 26/8/07 Pabstsendam X:17389 Xanthorhoe 0 Wageningen Y:44108 ferrugata 724 26/8/07 Pabstsendam X:17389 Xanthorhoe 0 Wageningen Y:44108 ferrugata 725 26/8/07 Pabstsendam X:17389 Xanthorhoe female single 0 Wageningen Y:44108 ferrugata 726 11/9/07 Pabstsendam X:17389 Xanthorhoe 0 Wageningen Y:44108 ferrugata 727 5/8/07 Pabstsendam X:17389 Xanthorhoe 0 Wageningen Y:44108 fluctuata 728 31/7/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 729 31/7/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 730 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 731 5/8/07 Pabstsendam X:17389 Xestia c-nigrum female 0 Wageningen Y:44108 732 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 733 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 734 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 735 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 736 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 737 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 738 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 739 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 740 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 741 5/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 742 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 743 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 744 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 745 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 746 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 747 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 748 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 749 26/8/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 750 11/9/07 Pabstsendam X:17389 Xestia c-nigrum 0 Wageningen Y:44108 751 26/8/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 752 26/8/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa

47 753 26/8/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 754 26/8/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 755 26/8/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 756 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 757 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 758 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 759 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 760 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 761 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 762 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 763 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 764 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 765 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 766 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 767 11/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 768 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 769 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 770 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 771 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 772 18/9/07 Pabstsendam X:17389 Xestia female single 0 Wageningen Y:44108 xanthographa 773 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 774 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 775 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 776 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa 777 18/9/07 Pabstsendam X:17389 Xestia 0 Wageningen Y:44108 xanthographa

48 Appendix 3: List of caught moths during day time per research location Reported are the research locations and their coordinates, caught moths, gender of caught moths, found eggs in the vials, number of caught Trichogramma wasps, which Trichogramma species was on the moth and where it was found in the vial.

No Date Research location Coordinates Moth species Gender Eggs N Trichogramma Body Tricho species part Entomology garden X:17570 1 1/8/07 Wageningen Y:44195 Autographa gamma 0 Entomology garden X:17570 2 13/8/07 Wageningen Y:44195 Autographa gamma 0 Entomology garden X:17570 3 28/8/07 Wageningen Y:44195 Cabera exanthemata 0 Entomology garden X:17570 4 28/8/07 Wageningen Y:44195 Camptogramma bilineata 0 Entomology garden X:17570 5 25/707 Wageningen Y:44195 Epirrhoe alternata 0 Entomology garden X:17570 6 21/8/07 Wageningen Y:44195 Epirrhoe alternata 0 Entomology garden X:17570 7 24/8/07 Wageningen Y:44195 Epirrhoe alternata 0 Entomology garden X:17570 8 28/8/07 Wageningen Y:44195 Epirrhoe alternata 0 Entomology garden X:17570 9 30/8/07 Wageningen Y:44195 Epirrhoe alternata female single 0 Entomology garden X:17570 10 30/8/07 Wageningen Y:44195 Epirrhoe alternata female 0 Entomology garden X:17570 11 31/8/07 Wageningen Y:44195 Epirrhoe alternata female 0 Entomology garden X:17570 12 31/8/07 Wageningen Y:44195 Epirrhoe alternata female 0 Entomology garden X:17570 13 15/5/07 Wageningen Y:44195 Euclidia glyphica female 0 Entomology garden X:17570 14 25/7/07 Wageningen Y:44195 Euclidia glyphica 0 Entomology garden X:17570 15 3/8/07 Wageningen Y:44195 Idaea dimidiata 0 Entomology garden X:17570 16 12/11/07 Wageningen Y:44195 Operophtera brumata male 0 Entomology garden X:17570 17 5/10/07 Wageningen Y:44195 Rivula sericealis 0 Entomology garden X:17570 18 30/8/07 Wageningen Y:44195 Timandra comae male 0 Entomology garden X:17570 19 22/6/07 Wageningen Y:44195 Tyria jacobaeae 0 Entomology garden X:17570 20 28/8/07 Wageningen Y:44195 Xanthorhoe spadicearia 0 Klein aboretum X:17497 21 6/9/07 Wageningen Y:44223 Eupithecia absinthiata 0 Marijkeweg X:17311 22 1/5/07 Wageningen Y:44108 Agrotis puta male 0 Marijkeweg X:17311 23 11/10/07 Wageningen Y:44108 Agrotis segetum male 0 Marijkeweg X:17311 24 22/10/07 Wageningen Y:44108 Epirrita dilutata male 0 Marijkeweg X:17311 25 22/10/07 Wageningen Y:44108 Epirrita dilutata male 0 Marijkeweg X:17311 26 18/9/07 Wageningen Y:44108 Noctua comes 0 Marijkeweg X:17311 27 8/10/07 Wageningen Y:44108 Noctua pronuba 0 Marijkeweg X:17311 28 7/11/07 Wageningen Y:44108 Operophtera brumata male 0 Marijkeweg X:17311 29 12/11/07 Wageningen Y:44108 Operophtera brumata male 0 Marijkeweg X:17311 30 12/11/07 Wageningen Y:44108 Operophtera brumata male 0 Marijkeweg X:17311 31 16/11/07 Wageningen Y:44108 Operophtera brumata male 0 Marijkeweg X:17311 32 21/11/07 Wageningen Y:44108 Operophtera brumata male 0 Marijkeweg X:17311 33 22/10/07 Wageningen Y:44108 Rhizedra lutosa 0 Marijkeweg X:17311 34 11/9/07 Wageningen Y:44108 Xestia xanthographa 0 Pabstsendam X:17389 35 3/5/07 Wageningen Y:44108 Macaria alternata 0

49