Hitch-Hiking Behavior of Trichogramma Wasps on Cabbage White Butterflies and Moths
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WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY Hitch-hiking behavior of Trichogramma wasps on cabbage white butterflies and moths ? 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).