Department of Zoology Laboratory of Entomology Institute of animal ecology Dr. Hans Smid Prof. Dr. Johannes Steidle University of Wageningen University of Hohenheim (220c) The Netherlands Stuttgart, Germany

Comparative study on memory phases in the parasitic wasps Cotesia glomerata and Cotesia rubecula

Diploma Thesis

This work was financially supported by the Herzog-Carl-Foundation, University of Hohenheim

Submitted by Silja Tribuhl (07.05)

Hohenheim, Stuttgart, March 2007 Faculty of Agricultural Sciences Table of contents I

Table of contents

Comparative study on memory phases in the parasitic wasps Cotesia glomerata and Cotesia rubecula Listof figures III Listof tables IV 1.Summary 1 2.Introduction 2 2.1 Insects and learning...... 2 2.2 Associative learning...... 2 2.3 Learning and memory formation...... 3 2.4 Cotesia glomerata and Cotesia rubecula...... 5 2.5 Memory phases in Cotesia glomerata and Cotesia rubecula...... 7 3.Material and Methods 11 3.1 Insects...... 11 3.2 Plants...... 11 3.3 Odour source...... 12 3.4 Wind tunnel...... 12 3.5 Experimental design...... 13 3.6 Wind tunnel assay...... 14 3.7 Statistical analysis...... 15 4.Results 15 4.1 Results of different learning trials in Cotesia glomerata...... 15 4.2 Results of single learning trials in C. glomerata and C. rubecula after 4 hours...... 17 5.Discussion 20 5.1 Cotesia glomerata...... 20 5.2 Cotesia rubecula...... 21 5.3 Variations in learning during the experiments...... 22 5.4 Costs and benefits of learning...... 24 5.5 Cotesia glomerata and Cotesia rubecula – why are they so different in learning?...25 6.References 28 Table of contents II

Appendix:

Transition from solitary to gregarious development in parasitic wasps

1.Introduction 32 1.1 Solitary and gregarious development...... 32 1.2 Transition from solitary to gregarious development...... 34 1.3 Cotesia glomerata and Cotesia rubecula...... 34

2.Material and Methods 35 2.1 Experimental setup...... 35 2.2 Statistical analysis...... 36

3.Results 36

4.Discussion 39

5.References 43 List of figures III

List of figures

Comparative study on memory phases in the parasitic wasps Cotesia glomerata and Cotesia rubecula

Fig.1: Putative pathway of memory formation. 4 Fig.2: Putative pathway of memory formation. 5 Fig.3: Life cycle of parasitic wasps. 6 Fig. 4: Memory retention in C. glomerata and C. rubecula. 8 Fig. 5a: Memory retention in C. rubecula afterthree spaced trials. 9 Fig. 5b: Memory retention in C. glomerata after three spaced trials. 9 Fig. 6: Memory retention in C. glomerata and C. rubecula 4 hours after single training. 10 Fig. 7: Wind tunnel set-up. 13 Fig.8: Wind tunnel. 14 Fig. 9: Cotesia glomerata: Choice for cabbage or nasturtium after no, one single or threespaced learning trials. 16 Fig. 10: C. glomerata and C. rubecula: Choice for nasturtium depending on the treatment. 18

Appendix: Transition from solitary to gregarious development in parasitic wasps

Fig. 1: Mean numbers of twisting and folding in mymariform larvae of the solitary

A. victus and the gregarious A. listronoti. 35

Fig. 2: Mean numbers of lateral bending of the solitary C. rubecula and the gregarious C. glomerata. 37 Fig. 3: Larvae of C. glomerata and C. rubecula 6 days after oviposition. 37 Fig. 4: Mouthparts of larvae of C. glomerata and C. rubecula 5 to 8 days after oviposition. 38 Fig.5: The evolution of gregariousness. 41 List of tables IV

List of tables

Comparative study on memory phases in the parasitic wasps Cotesia glomerata and Cotesia rubecula

Table 1: C. glomerata: Choice for nasturtium after different learning trials. 17 Table 2: C. glomerata: Choice for nasturtium 4 hours after one learning trial. 19 Table 3: C. rubecula: Choice for nasturtium 4 hours after one learning trial. 19

Appendix: Transition from solitary to gregarious development in parasitic wasps

Table 1: Mean numbers of bending in C. glomerata and C. rubecula. 37 1. Summary 1

Comparative study on memory phases in the parasitic wasps Cotesia glomerata and Cotesia rubecula

1. Summary

The parasitic wasps Cotesia glomerata and Cotesia rubecula are closely related and live in the same habitat in the Netherlands. The wasps lay their eggs in caterpillars of Pieris butterflies. To find their hosts the wasps are innately attracted by volatiles emitted by infested cabbage plants. If hosts are encountered on plants different than cabbage the wasps learn to associate the odour of this plant species with the presence of suitable hosts. Although the wasps are closely related, clear differences in learning and memory formation occur. C. glomerata forms long-term memory (LTM) already after one successful egg laying experience whereas C. rubecula needs three spaced experiences. Former experiments assumed that after three spaced conditioning trials LTM is consolidated directly out of anesthesia sensitive memory (ASM) in C. glomerata and no anesthesia resistant memory (ARM) is present. In C. rubecula two memory traces coexist after three spaced trials, ARM and LTM. To test if ARM is present in C. glomerata a classical conditioning setup is used, in combination with cooling to inhibit ASM and with a translation inhibitor (anisomycin) that blocks LTM. Both species were tested with one conditioning trial. In C. glomerata both, cooling and anisomycin inhibited 4 hour memory retention, which shows that ARM seems to be absent in C. glomerata after 4 hours. ASM is directly consolidated into LTM in this species suggesting that LTM induction prevents formation of ARM. In C. rubecula there was no effect of cooling on 4 hour memory retention, and previous results showed that 4 hour memory retention in this species cannot be inhibited by anisomycin. This shows that 4 hour memory retention is constituted exclusively by ARM. Looking at learning and memory formation in the context of the natural behaviour of the wasps, it becomes clearer why these differences in memory formation might occur. For C. glomerata finding a host on a special plant species is reliable information due to the egg laying performance of its host P. brassicae which chooses a stand of plants of the same species. If C. glomerata associates the odour of the new encountered plant with the presence of suitable hosts it can be sure to find enough hosts for its offspring. Therefore new memory is stored directly into LTM. For C. rubecula finding a host is not reliable information because 2. Introduction 2 its host P. rapae spreads its eggs widely on different host plants. For the wasp it would be maladaptive to change its innate behaviour and search for the new encountered plant species. Still associations are formed in C. rubecula but this memory is stored in ASM and ARM which wane within one day and are a form of “low cost” memories, demanding less energetic costs than the formation of LTM which requires new gene expression.

2. Introduction

2.1 Insects and learning When you think of insects you think of tiny little animals flying or crawling around maybe disturbing you. The idea that these little insects are able to learn and that they react in a flexible way to their environment would maybe never come to your mind. “Do insects have a brain?” is a frequently asked question when I tell people about my research. Of course the brains of insects look a little bit different than brains in higher animals or humans but the way they function is not very different. Insects are able to learn and this fact has been of great interest for a lot of researchers all over the world. Are there any advantages for the insects if they are able to learn? Do they all learn in the same way, with the same speed? How long are they able to remember learned information? Which genes are involved in memory formation? Especially the fruit fly Drosophila has been well-investigated concerning these questions but also parasitic wasps are used to know more about different forms of learning behaviour. Knowing more about learning in insects enables us to know more about our own learning behaviour.

2.2 Associative learning To know more about learning, experiments with all kinds of animals have been performed until now. Famous experiments have been carried out by PAVLOV (1927). In his experiments Pavlov always rang a bell directly before he fed a dog. After a while the dog started salivating when it heard the sound of the bell alone. This association between a neutral stimulus (called conditioned stimulus or CS) the bell, and a meaningful stimulus (called unconditioned stimulus or US) the food, is called pavlovian or classical conditioning and is a form of associative learning. Classical conditioning will only take place if the CS is directly followed by the US. Associative learning also plays an important role in memory formation in insects. 2. Introduction 3

Another form of associative learning is operant conditioning (THORNDICKE 1901, SKINNER 1938). In operant conditioning an association is formed between the response to a CS and the reinforcer (US). In one of his experiments Thorndicke put a cat in a puzzle box with a lockable door which could be opened from the inside. He observed the behaviour of the cat and noted the time the cat needed to escape from the box. After several trials the cat learned to unlock the door and escape from the box very quickly. The cat associated its specific behaviour (unlock the door) with the reinforcer (US), the freedom. This means that in operant conditioning the important stimulus (reinforcer) occurs after the response and not before it like in classical conditioning (LUTZ 2005). Operant learning is also found in parasitic wasps like Cotesia glomerata (L.) and Cotesia rubecula (Marshall) (Hymenoptera: Braconidae). C. glomerata and C. rubecula lay their eggs in caterpillars of Pieris butterflies. The larvae of the wasps develop in the caterpillars until they finally leave their dying host and pupate. To find their hosts the wasp smells a damaged plant with caterpillars feeding on it, flies towards the plant, lands on it and finds suitable hosts for oviposition. The odour of the damaged plant represents the CS, the wasp responds by landing on this plant where it is rewarded with suitable hosts (US) (SMID 2006). An association is formed between the flight response and the reward, which is represented by the host.

2.3 Learning and memory formation If insects learn, the information has to be stored somehow so memory is formed. Instead of one general memory form, different memory phases can be distinguished. JAMES (1890) first separated memory into primary and secondary memory, which are now known as short-term (STM) and long-term (LTM) memory. Memory phases can be classified by their duration but this is a criterion which differs considerably between animal species including humans. Another possible criterion to distinguish is the biochemical processes which are critically involved in forming these different memory phases (ROSENZWEIG et al. 1993). The biochemical pathways are not completely understood until now, most of all chemical substances or treatments are found which inhibit the different phases without really knowing why. STM is sensitive to amnestic treatments (e.g. cooling, narcosis with CO2 or N2), and is therefore also called anesthesia sensitive memory (ASM) (e.g. TULLY et al. 1994). LTM is sensitive to protein synthesis inhibitors showing that LTM requires new gene expression

(TULLY et al. 1994). 2. Introduction 4

The formation of memory phases is time and event dependent meaning that different learning experiences depending on their chronology cause different memory phases. After single learning experiences STM occurs but also another form of memory appears which is called anesthesia resistant memory (ARM). ARM is resistant to amnestic treatments (e.g. TULLY et al. 1994, MARGULIES et al. 2005, SMID 2006), and is also formed after massed (repeated experiences without a break) and spaced (several experiences with breaks in between) learning experiences. LTM normally occurs only after spaced learning. Spaced learning experiences ensure that only important information is stored in long-term memory. For animals it is important not to learn too fast to prevent the storage of wrong information (SMID 2006). A lot of research about memory and memory formation has been done in the fruit fly Drosophila. The flies are trained with classical conditioning with different learning experiences (single, massed, spaced). In Drosophila STM appears immediately after training and fades away within 60 minutes. ARM appears slowly, shows its highest level two hours after training and can last for three days after massed training. LTM appears 4-5 hours after spaced learning trials (10 sessions with a 15 minute rest interval in between) lasting at least one week (TULLY et al. 1994, MARGULIES et al. 2005). In literature you find contradictive opinions about the way these different memory phases occur, if they appear sequentially, meaning one phase consolidating into the other, or also in parallel. MARGULIES et al. (2005) state in their review that ARM and LTM are genetically distinct, formed independently of each other and function in an additive way (Fig. 1).

ARM

Acquisition ASM

LTM

Fig. 1: Putative pathway of memory formation: ARM and LTM are genetically distinct and

formed in parallel (adapted from TULLY et al. 1994).

These statements are based on studies of TULLY et al. (1994) and YIN et al. (1994) that showed that LTM but not ARM is protein synthesis dependent. This means that ARM is not affected by protein synthesis inhibitors and there has to be a different underlying mechanism. 2. Introduction 5

ISABEL et al. (2004) presented a different hypothesis; in their research ARM and LTM do not coexist in Drosophila but LTM replaces ARM (Fig. 2). Drosophila mutants in which LTM is deficient showed almost no memory retention 5 hours after spaced conditioning. Normally ARM should be present 5 hours after spaced training and last for several days. It is suggested that ARM is blocked during LTM-specific training. The assumption is that in wild-type flies LTM is formed after spaced conditioning and replaces ARM which is only formed after massed conditioning. This mechanism is thought to ensure that only information with a high predictive value is stored in LTM.

Acquisition ASM ARM LTM

Fig. 2: Putative pathway of memory formation: LTM replaces ARM during spaced learning experiences.

2.4 Cotesia glomerata and Cotesia rubecula C. glomerata and C. rubecula are two closely related parasitic wasps coexisting in the same habitat in the Netherlands and are indigenous to Europe. They belong to the endoparasitoids, they lay their eggs in young larvae (first to third larval instar) of Pieris butterflies. The larvae of the wasps leave the caterpillar approximately after 2 weeks by eating through its cuticle and thereafter the caterpillar dies. The larvae spin a cocoon and develop to the pupal stage

(Fig. 3) (SMID 2006). C. glomerata is a generalist, it can parasitize Pieris rapae (L.), Pieris brassicae (L.) and Pieris napi (L.) but in the Netherlands it parasitizes mainly the large cabbage white

P. brassicae (GEERVLIET et al. 2000a). C. glomerata is a gregarious species; approximately

20 eggs are laid in a single host (VOS & VET 2004). The solitary wasp C. rubecula is a specialist on the small cabbage white P. rapae; only one egg is laid in a single host

(GEERVLIET et al. 2000b). 2. Introduction 6

Fig. 3: Life cycle of parasitic wasps (TUMLINSON et al. 1993).

The wasps emerging from the cocoons have to find food, a mate and a host to lay their eggs. To find a suitable host is quite a difficult task, the environment of the wasps is full of different odours emitted by plants and non-hosts. Also the young caterpillars are very small; they are well camouflaged and do not emit attractants which are detectable from a distance (SMID 2006). So, how are the wasps able to find their hosts? Caterpillars of Pieris butterflies are restricted to plants containing glucosinolates like most of the cruciferous plants (CHEW 1980). The wasps could orientate itself from distance towards the plant odours but still this stimulus is not very reliable (VET & DICKE 1992) because the plant odours by themselves do not promise the presence of suitable hosts. GEERVLIET et al. (1994) showed that volatiles of cabbage plants on which caterpillars are feeding are highly attractive to the wasps. This means plants change their volatile composition when they are damaged by feeding caterpillars. The wasps orientate at first towards the volatiles which are emitted by the damaged plants and not towards the odour of the caterpillars. The wasps do not have to learn to recognize the plant odour of cabbage; naïve wasps already have this preference (GEERVLIET et al. 1996). After landing on a host plant with caterpillars, the wasps starts local searching behaviour by using host-derived stimuli like frass or silk. Also non-cruciferous plants containing glucosinolates, e.g. nasturtium (Tropaeolum major L.), are used as a host plant by Pieris butterflies (FELTWELL 1982, GEERVLIET 1997). If the wasp encounters a host e.g. on nasturtium it can learn to associate the odour of this different plant species with the presence of a suitable host. The wasp changes its innate 2. Introduction 7 behaviour and starts searching for hosts on nasturtium. C. glomerata changes its preference already after one oviposition experience on a plant different than cabbage and forms a long- lasting memory of at least 2-3 days. C. rubecula also learns to associate the odours of other plant species with the presence of a suitable host but this memory already wanes within one day (BLEEKER 2005, SMID submitted). Thus there is a difference in host-searching strategies between the generalist C. glomerata and the specialist C. rubecula (GEERVLIET et al. 1998,

SMID 2006). In the case of C. glomerata and C. rubecula the difference in preference behaviour might be due to their different host distributions (BLEEKER 2006). P. brassicae females lay their eggs in clusters of approximately 150 eggs per plant. The caterpillars are conditioned on the first species they feed on and need to find more plants of the same species. P. brassicae therefore selects a stand of host plants of the same species to ensure enough food for their offspring (ROTHSCHILD 1987, LE MASURIER 1994). C. glomerata will find enough hosts for its own offspring if it orientates itself towards the odour of the specific plant species. Therefore it is useful for the wasp to associate the odour of the newly encountered plant with the presence of its host. This change in behaviour ensures C. glomerata to find enough hosts to lay all its eggs. P. rapae usually lays only one egg per plant and spreads its eggs widely in their habitat on different host plant species (ROOT & KAREIVA 1984). For C. rubecula the presence of one caterpillar does not indicate the presence of other suitable hosts so it would be a disadvantage to change its preference towards the newly encountered plant (BLEEKER 2006). C. glomerata and C. rubecula are two closely related wasps; they live in the same habitat and show clear differences in learning behaviour. Therefore these two wasps are a good model for comparative investigations about different memory phases and duration of memory retention.

2.5 Memory phases in Cotesia glomerata and Cotesia rubecula In C. glomerata and C. rubecula 3 memory phases are distinguished until now. We find ASM, ARM and LTM but there seem to be differences in the two parasitic wasps.

SMID et al. (submitted) trained the wasps with classical conditioning on nasturtium plants (infested with Pieris caterpillars) and tested them for memory retention in a dual choice test in the wind tunnel. The wasps had a choice between infested cabbage and nasturtium plants. Memory retention could be seen on the basis of the preference shift from cabbage to nasturtium. If the wasps receive one learning trial both wasps show high memory retention after one and four hours (Fig. 4); around 70 % of the wasps chose for nasturtium in the wind 2. Introduction 8 tunnel. After 24 hours there is no memory left in C. rubecula whereas in C. glomerata there is still high memory retention. C. glomerata was also fed with the translation blocker anisomycin which inhibits LTM. After one conditioning trial 24 hour memory retention was completely inhibited showing that the wasp forms long-term memory already after one oviposition experience (SMID et al. submitted). This phenomenon has also been shown for e.g. appetitive conditioning in the snail

Lymnea (FULTON et al. 2005), fear conditioning in rats (IGAZ et al. 2002), and oviposition learning in parasitic wasps (COLLATZ et al. 2006). In C. rubecula no LTM is formed after one learning trial (SMID et al. submitted). Choicefornasturtium (%)

Time (h)

Fig. 4: Memory retention in C. glomerata and C. rubecula after 1, 4, and 24 hours after one

learning trial (SMID submitted).

Both wasp species also received three learning trials (spaced training with 10 minutes break in between), and were fed with sucrose solution alone or with anisomycin. Again there are differences between the two species. In C. rubecula, fed with sucrose, there are still high levels of memory retention after 5 days. Fed with anisomycin the wasp still shows memory retention after 24 hours (Fig. 5a). Memory is only partially inhibited by anisomycin showing that 24 hour retention is composed of a protein-synthesis independent component (ARM) and of a protein-synthesis dependent component (LTM). 2. Introduction 9 Choicefornasturtium (%)

Time (h)

Fig. 5a: Memory retention in C. rubecula after three spaced trials. Bright bars: Wasps were fed with 3 % sucrose solution. There is still memory retention after 5 days. Dark bars: Wasps were fed with anisomycin in 3 % sucrose solution. Although LTM is inhibited by anisomycin there is still memory retention left after 24 and 48 hours

(SMID submitted). Choicefornasturtium (%)

Time (h)

Fig. 5b: Memory retention in C. glomerata after three spaced trials. Bright bars: Wasps were fed with 3 % sucrose solution. There is still memory retention after 5 days. Dark bars: Wasps were fed with anisomycin in 3 % sucrose solution. After 4 hours there is

no memory retention left (SMID submitted).

Feeding C. glomerata with anisomycin the picture looks different (Fig. 5b). After 4 hours there is no memory retention left which shows that from 4 hours on memory retention is 2. Introduction 10 composed completely of a protein-synthesis dependent component (LTM). This result leads to the question if ARM might be absent in C. glomerata (SMID submitted). To get more information about ASM also experiments with cooling were carried out by

WANG (unpublished). Cooling inhibits ASM without blocking ARM and LTM. The wasps were trained with classical conditioning as explained before, received a single training and were cooled in ice for two minutes to inhibit ASM. Cooling was carried out directly, 20 min, 1, 2 and 3 hours after training (Fig. 6). The wasps were tested after 4 hours in the wind tunnel.

80 70 60 50 40 30 Cotesia glomerata 20 Cotesia rubecula 10 choice for nasturtium (%) 0 0 0,33 1 2 3 cooling time after oviposition (h)

Fig. 6: Memory retention in C. glomerata and C. rubecula 4 hours after single training. The

wasps were cooled directly, 20 min, 1, 2 and 3 hours after oviposition (WANG unpublished).

Direct cooling after training had no effect on memory retention but cooling after 20 min seems to have a major effect because in both species there is no memory retention left after 4 hours. The later the wasps were cooled the less effect is seen on memory retention showing that ASM is consolidating into ARM and/or LTM which are not sensitive to cooling. There seems to be a sensible phase 20 min after training concerning the consolidation of ARM and LTM. This could be explained if ARM and LTM are consolidated out of ASM because inhibiting ASM seems to influence the formation of ARM and LTM.

This thesis will focus on some remaining questions of the studies mentioned above. Comparing the memory phases of C. glomerata and C. rubecula you see that in C. rubecula

ARM seems to be present and formed in parallel with LTM (SMID submitted). This is comparable with the situation that MARGULIES et al. (2005) described for Drosophila (Fig. 1). 3. Materials and methods 11

In C. glomerata ARM seems to be absent and LTM might be directly consolidated out of

ASM. This would support the suggestions of ISABEL et al. (2004) that memory phases are mutually exclusive and one phase replaces the other. This research study will try to find out if ARM is present in C. glomerata 4 hours after one conditioning trial. 4 hours after one trial both species show high memory retention (Fig. 4). This memory does not seem to consist of ASM because cooling 3 hours after training had no effect on memory retention after 4 hours any more (Fig. 6) and ASM seems to be consolidated into ARM or LTM by that time. If C. glomerata is cooled after 3 hours (to inhibit ASM) and fed with anisomycin (to block LTM) there should be no memory retention left after 4 hours if ARM is missing. The same experiments will be carried out with C. rubecula where ARM seems to be present. Another question is if LTM consolidates out of ASM and at which point in time LTM starts in C. glomerata. Data of WANG (unpublished) show, that cooling seems to have an influence on the consolidation of LTM. Cooling should normally only inhibit ASM. In this study wasps will be trained and tested in the wind tunnel after different points in time and also cooled after different times to state more precisely when LTM starts and if it is formed out of ASM.

3. Materials and methods

3.1 Insects C. glomerata and C. rubecula originated from individuals collected on cabbage fields near Wageningen, The Netherlands. The parasitoids were maintained on P. brassicae and P. rapae larvae respectively, feeding on cabbage plants in a climatic room at 20-22 °C, 50-70 % r.h. and a L16:D8 regime as described by GEERVLIET et al. (1994). The cocoons were kept in cages under the same conditions and the emerging parasitoids were supplied with water and honey. P. brassicae and P. rapae larvae were maintained on cabbage plants under the conditions described above. For the experiments first instar larvae were used.

3.2 Plants Cabbage (Brassica oleracea gemmifera cv. Cyrus) and nasturtium plants (Tropaleum majus L. cv. Glorious Gleam) were reared in a greenhouse at 20-22 °C, 50-60 % r.h. and a L16:D8 regime. 3. Materials and methods 12

3.3 Odour source Cabbage and nasturtium plants of approximately the same size were chosen. On two leaves of each plant 20 early first instar larvae were transferred and were allowed to feed for 24 hours to induce feeding damage. For the oviposition experience nasturtium plants were treated the same way.

3.4 Wind tunnel The wind tunnel (Fig. 7) used in these experiments measures 200 x 60 x 60 cm. The flight- compartment is made out of polyacryl; both ends are covered with fine mesh copper gauze. At the observer’s side there are three little gates which can be opened to put in the odour source and the wasps. During the experiments outside air is filtered over glass wool and led over a humidity controlled air humidifier. The air is then pressurised, filtered over an active charcoal filter and passes through a temperature controlled heating system. Wind speed was constantly at 18 cm/s. The temperature varied between 21-27 °C, relative humidity fluctuated between 40 and 60 %. Above the flight-compartment there is one half of a concave metal cylinder with 8 fluorescent high-frequency tubes and 4 bulbs as a light source. The inner side of the cylinder is covered with aluminium plates for light dispersion. Light intensity during the tests was 1700 lux on the bottom, increasing to 2000 lux at the ceiling. The wasps were released in a horizontal glass cylinder (30 cm long, 15 cm diameter) 40 cm in distance of the odour source

(GEERVLIET et al. 1994). 3. Materials and methods 13

Fig. 7: Wind tunnel set-up: AI = air inlet, CF = charcoal filter, HS = heating system, RP = parasitoid release point, FT = high-frequency fluorescent tubes, B = bulbs, X =

position of odour source, HM = hygrometer, G = gate (GEERVLIET et al. 1994).

3.5 Experimental design 5-8 days old females of C. glomerata and C. rubecula were deprived from water and honey over night and fed the next day with 0,5 ȝl of a solution containing 5 mM anisomycin (2-p- methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine) in 3 % sucrose. The wasps were kept in vials for 2.5 hours until they consumed the entire solution. After this they were transferred for one hour to a glass cage with honey and water. The wasps were trained by giving them a single oviposition experience on first instar larvae of P. brassicae and P. rapae, which had been feeding on nasturtium plants for 24 hours. In these experiments the wasps are trained with classical conditioning to standardize the experiments. The wasps are placed directly on the leaf (odour = CS) and the taste/odour of the host or host-derived products represents the US. An association is formed between the neutral stimulus (plant odour) which is directly followed by a meaningful stimulus, the suitable host. The test for memory formation in the wind tunnel represents an operant context because the reinforcing stimulus (suitable host) occurs after the response (flight) to the conditioned stimulus (plant odour). The wasps are able to transfer the information from the classical conditioning to the operant context. 3. Materials and methods 14

Three hours after oviposition the wasps were cooled for 2 minutes in ice. Four hours after oviposition the wasps were tested in the wind tunnel for their preferences, infested cabbage or nasturtium. Forty individuals were tested per group. Control groups were given the same treatment as explained above.

Control group 1: training + anisomycin Control group 2: training + cooling Control group 3: cooling + anisomycin Control group 4: training

3.6 Wind tunnel assay

The wind tunnel as described in GEERVLIET et al. (1994) was used for all experiments. The wasps had the choice between odour sources of cabbage and nasturtium plants, both infested with P. brassicae or P. rapae larvae for 24 hours (Fig. 8). Each wasp was released 40 cm downwind from the plants. Every test lasted a maximum time of five minutes. Landing on one of the plants was recorded as a positive response. Wasps showing no preference or landing somewhere else within 5 min were recorded as a “no response”. After testing five wasps the position of the plants was switched. All wasps were used only once.

Fig. 8: Wind tunnel (REMCO VAN POECKE).

Learning is defined in this study as the preference shift from cabbage to nasturtium. If there is a significant difference between naïve and trained wasps in their preference for nasturtium, learning takes place. 4. Results 15

3.7 Statistical analysis Generalized linear modelling (GLM) procedures using procedure GENMOD in SAS version 8.02 (Proc GENMOD, SAS Inc, Chicago, IL, U.S.A.) were used for data with a binominal distribution of error variance and a logit-link function. When main effects or their interactions were found significantly different, further separation of the treatment levels was carried out by acquiring specific contrasts for particular comparisons, which are given in the text. Data collected on different experimental days were considered as replicates. We tested whether treatments (4 levels: training; training + anisomycin; training + cooling; training + anisomycin + cooling) and species (2 levels: C. glomerata and C. rubecula) had an effect on the response level to nasturtium plants (GLM treatment Ȥ²6 = 41.42, P < 0.0001). Choices between odour sources were analysed with a two-tailed binominal test with continuity correction.

4. Results

4.1 Results of different learning trials in C. glomerata

Experiments from SMID et al. (submitted) and WANG (unpublished) were carried out with a different population than the population that was used in this study. Every year a new population of Cotesia wasps is collected from the field and established to avoid inbreeding. Years of rearing showed that it is normal to observe a decrease in yield in the beginning of the new breed. Also this time low yields were obtained and high mortality rates. In addition, it has been found that the learning capability of the wasps was very low. Normally the wasps show a pronounced preference shift towards nasturtium hours after classical conditioning, 70-90 % of the wasps choose nasturtium instead of cabbage. Only 36 % of the wasps chose for nasturtium 4 hours after training (Fig. 9) in the beginning of this study. The wasps were tested after different times (1, 4, 24 hours) and with different learning protocols (single, spaced) in the wind tunnel to check if memory retention changed dependent on different learning experiences (Fig. 9). Data were not statistically analyzed because not enough replicates were taken. Still it can be seen that there was a low preference shift from cabbage to nasturtium also after three spaced trials which should normally lead to a high level of memory retention. In contrast, control experiments with C. rubecula showed normal memory retention in an identical setup, showing that the experimental setup was functioning well. Unfortunately also 4. Results 16 in the rearing of C. rubecula low yields and high mortality rates were obtained. Therefore only 30 individuals were tested in the “Training” and “Training + Cooling”- groups and around 10 individuals in the “Training + Anisomycin” and “Training + Anisomycin + Cooling” Groups. It was not possible to include a naïve control group due to the low availability of the wasps. C. glomerata was tested every week to see if the mortality rates became normal in successive generations and learning performance improved to values comparable to results obtained by previous studies. Meanwhile an alternative study was performed which is described in the appendix. After several generations, batches of C. glomerata came available that showed adequate levels of memory retention again so the experiments could be started. In this study single and spaced trials were planned but finally only single training trials could be tested due to lack of individuals with adequate learning capabilities. The wasps were only tested after 4 hours and not after 5 and 6 hours as planned before.

Nasturtium Cabbage

naïve after 4 hours

1 hour after single trial

4 hours after single trial

24 hours after three spaced trials 50 0 50 100 Choice (%)

Fig. 9: Cotesia glomerata: Choice for cabbage or nasturtium after no, one single or three spaced learning trials. Test in the wind tunnel 1, 4 and 24 hours after oviposition. 4. Results 17

Table 1: C. glomerata: Choice for nasturtium after different learning trials, 1, 4 and 24 hours after training.

Treatment Hours after Nasturtium Cabbage N N oviposition responding

naïve n.a. 0 24 29 24 single trial 1 9 22 38 31 single trial 4 13 23 59 36 spaced trial 24 4 4 9 8

4.2 Results of single learning trials in C. glomerata and C. rubecula after 4 hours A new batch of C. glomerata which showed adequate levels of memory retention was taken for the final experiments. Naïve wasps fed with anisomycin and cooled after 3 hours flew mostly to cabbage (94 %), showing their natural preference for this plant. The wasps behaved normally and showed low no-response levels (22 %) which indicates that feeding them with anisomycin and cooling has no detrimental effect on the behaviour of the wasps. In trained C. glomerata a significant preference shift from cabbage to nasturtium plants could be observed compared to naïve wasps (GLM treatment Ȥ²1 = 11.91, P = 0.0006) (Fig. 10). 61 % of the wasps chose for nasturtium in the wind tunnel which was a result not as high as observed in previous studies (SMID submitted), normally 70-90 % chooses for nasturtium. Still it shows that the wasps were able to learn and that they showed memory retention after 4 hours. Cooling trained wasps after 3 hours reduced memory retention significantly from trained wasps (61 %) to 30 % (GLM treatment Ȥ²1 = 4.40, P = 0.0360) but the memory retention level of cooled wasps was still significantly different from naïve control wasps (GLM treatment

Ȥ²1 = 5.36, P = 0.0206) (Fig. 10). Feeding wasps with anisomycin before training reduced memory retention from trained wasps (61 %) to 38 % but this reduction was not significant

(GLM treatment Ȥ²1 = 2.44, P = 0.1180) and the memory retention level of anisomycin fed wasps was significantly different from naïve control wasps (GLM treatment Ȥ²1 = 6.97, P = 0.0083) (Fig. 10). In trained wasps which were fed with anisomycin and were cooled after 3 hours, memory retention was reduced significantly compared to trained wasps (GLM treatment Ȥ²1 = 4.43, P = 0.0353) (Fig. 10). 4. Results 18

100 90 Cotesia glomerata 80 Cotesia rubecula 70 60 50 40 30 20

Choice for nasturtium (%)Choice for 10 0 T TAC TC TA AC

Treatments

Fig. 10: C. glomerata and C. rubecula: Choice for nasturtium depending on the treatment. T = Training; TC = Training + Cooling; TA = Training + Anisomycin; TAC = Training + Anisomycin + Cooling; AC = Anisomycin + Cooling.

For C. rubecula it was not possible to include a naïve control group due to the low availability of the wasps. Results from SMID et al. (submitted) show that naïve C. rubecula are attracted significantly more to cabbage than to nasturtium plants. The preference for cabbage can be compared with the preference observed in naïve C. glomerata, normally 70-90 % of the wasps choose for cabbage. Trained C. rubecula showed high memory retention after 4 hours; 79 % of the wasps flew to nasturtium (P = 0.0023). Trained wasps which were cooled after 3 hours showed no significant difference in preference for nasturtium compared to trained wasps

(GLM treatment Ȥ²1 = 0.92, P = 0.3376) (Fig. 10). After one conditioning trial there is no significant difference in memory retention between

C. glomerata and C. rubecula (GLM species Ȥ²1 = 2.30, P = 0.1290). Cooling after three hours led to a significant difference in memory retention between both species (GLM species

Ȥ²1 = 10.66, P = 0.0011) (Fig. 10). Only results of “Training” and “Training + Cooling” of C. rubecula were included into the statistical analysis. Not enough replicates of “Training + Anisomycin” and “Training + Anisomycin + Cooling” and could be carried out. Data of these groups are added to figure 10 to show the tendency. Also feeding anisomycin seems not to have an influence on memory retention in C. rubecula. 4. Results 19

Table 2: C. glomerata: Choice for nasturtium 4 hours after one learning trial. AC = Anisomycin + Cooling; T = Training; TAC = Training + Anisomycin + Cooling; TC = Training + Cooling; TA = Training + Anisomycin.

Treatment Hours after Nasturtium Cabbage N N training responding

AC 4 2 33 45 35 T 4 25 16 63 41 TAC 4 11 31 52 42 TC 4 14 32 63 46 TA 4 17 28 64 45

Table 3: C. rubecula: Choice for nasturtium 4 hours after one learning trial. T = Training; TC = Training + Cooling; TAC = Training + Anisomycin + Cooling; TA = Training + Anisomycin.

Treatment Hours after Nasturtium Cabbage N N training responding

T 4 23 3 51 29 TC 4 20 11 36 31 TAC 4 12 1 16 13 TA 4 6 2 13 8 5. Discussion 20

5. Discussion

5.1 Cotesia glomerata In this study C. glomerata was trained with one conditioning trial. Disrupting ASM with cooling, and feeding anisomycin before training (to block LTM), resulted in no memory retention after 4 hours; showing that no ARM is present like assumed in the hypothesis. If there is no ARM, ASM is directly consolidated into LTM in this species. This suggests that

LTM induction prevents formation of ARM. Data from SMID et al. (submitted) suggested the same results after three spaced trials. Wasps which were fed with anisomycin before training showed no memory retention after 4 hours. 4 hour memory retention after one or three spaced conditioning trials seems to consist exclusively out of protein-synthesis dependent LTM. Unexpectedly, cooling 3 hours after oviposition affected memory retention after 4 hours, showing that ASM is at least present for 3 hours. This result was unexpected and is contradicting to data from WANG (unpublished). WANG (unpublished) also cooled trained wasps after 3 hours but no effect was seen after 4 hours. These results show that in C. glomerata 4 hour memory retention most likely does not consist exclusively out of LTM after one conditioning trial because ASM is still present after 3 hours. Actually planned experiments with cooling after 4 and 5 hours and testing after 5 and 6 hours respectively could not be carried out because of problems with the rearing (see 4.3). These experiments could give more clarity about the duration of ASM. Feeding anisomycin before training also reduced memory retention after 4 hours compared with trained wasps but this reduction was not significant. This result suggests that 4 hour memory retention contains a protein synthesis dependent component, but that this component has not yet been completely consolidated. This suggests that at 4 hours, there is a consolidation process going on from ASM into LTM. The fact that the combination of cooling and anisomycin has an additive effect on 4 h memory retention further suggests that ASM is consolidated into LTM directly. LTM formed after one conditioning trial seems to be different compared to LTM formed after three spaced trials. This is apparent when you look at the duration of LTM after different conditioning trials. In C. glomerata LTM lasts 2-3 days after one trial (GEERVLIET 1997) whereas LTM after three spaced trials lasts at least 5 days (SMID et al. submitted). How these differences can be explained on the neuronal level is unknown so far. It may explain that the effect of anisomycin on 4 hour memory retention in C. glomerata after 3 spaced trials (SMID et al. submitted) is more pronounced than after 1 single trial. 5. Discussion 21

In C. glomerata, ASM is consolidated directly into LTM. There is no long lasting memory phase like ARM coexisting with LTM after one conditioning trial. The situation in

C. glomerata, where memory phases are mutually exclusive, is similar as described by ISABEL et al. (2004) for Drosophila mutants (ala mutants) in which LTM is deficient but with normal ASM and ARM. In wild-type Drosophila, single and massed conditioning trials induce ASM and ARM. A combined memory trace consisting from ASM, consolidating into ARM and

LTM is induced by spaced training (MARGULIES et al. 2005). In ala flies ARM is fully functional and formed after single and massed learning. Training the flies with spaced learning experiences resulted in no memory retention after 5 hours. Normally ARM should be present 5 hours after spaced training, lasting for several days. ISABEL et al. (2004) concluded that ARM and LTM do not coexist in wild-type flies but instead ARM is blocked during LTM-specific training. It is assumed that LTM is formed after spaced conditioning and replaces ARM which is only formed after massed conditioning. LTM is formed in C. glomerata after single, massed and spaced conditioning trials and no ARM seems to be present in the wasp. Therefore also in C. glomerata formation of ARM seems to be prevented by LTM induction.

5.2 Cotesia rubecula C. rubecula was also trained with one conditioning trial but the process of memory consolidation was totally different. Contrary to C. glomerata cooling after 3 hours has no effect on memory retention in C. rubecula after one conditioning trial, showing that ASM is already absent. LTM is not formed in C. rubecula after one trial (SMID submitted) but only after several spaced experiences (Fig. 5a). Therefore ARM has to be present 4 hours after single training. WANG (unpublished) showed that ARM is present at least 8 hours after one trial and wanes between 8 and 24 hours. If C. rubecula receives three spaced learning trials LTM and ARM are formed in parallel and coexist. This was shown by SMID et al. (submitted) by inhibiting LTM with anisomycin; ARM was still present 48 hours after three spaced trials. Coexisting memory phases are described by e.g. TULLY et al. (1994) for Drosophila. After spaced learning trials, ARM and LTM are consolidated out of ASM and appear in parallel. The two memory phases are genetically dissected and dependent on different biochemical pathways. Evidence for this statement came out of the discovery that ARM is not sensitive to protein-synthesis inhibitors like cycloheximide (CXM). In contrast LTM is sensitive to protein-synthesis inhibitors and therefore requires gene expression. 5. Discussion 22

In C. glomerata and C rubecula we see clear differences in memory formation and memory retention. My results suggest, according to my hypothesis, that there is no ARM in C. glomerata. If there is no ARM, ASM is directly consolidated into LTM in this species. This suggests that LTM induction prevents formation of ARM in C. glomerata. In C. rubecula LTM is not formed after one conditioning trial and ARM is present after 4 hours. Comparing memory retention of both species we see that in C. glomerata ASM is present at least for 3 hours because cooling reduced memory retention significantly. In C. rubecula ASM lasts shorter; 3 hours after training only ARM is present, cooling had no effect on memory retention. In C. glomerata also feeding trained wasps with anisomycin affected memory retention after 4 hours but this difference was not significant compared to trained wasps. 4 hour memory retention seems to contain a protein synthesis dependent component, but this component has not yet been completely consolidated. C. glomerata can be compared with ala mutants in Drosophila described by ISABEL (2004). In ala mutants ARM is blocked during LTM-specific training. With C. glomerata we found a wild-type species where ARM is also blocked during LTM-specific training (single, massed and spaced). The situation in C. rubecula where ARM is formed after LTM-specific training (spaced) was described by TULLY et al. (1994) for wild-type Drosophila. LTM induction does not prevent formation of ARM in these species. Both models about the pathway of memory formation (Fig. 1, 2) described by TULLY et al. (1994) and ISABEL (2004) seem to have their validity if we look at the results of C. glomerata and C. rubecula. C. glomerata and C rubecula show clear differences in memory formation and memory retention. To find out why these closely related wasps differ so much in preference learning, a closer look has to be taken at memory formation in the context of natural behaviour. During my study unexpected variations in learning were observed in C. glomerata. First I would like to offer some suggestions why these variations might have occurred.

5.3 Variations in learning during the experiments During the first months of my study the rearing of C. glomerata did not work very well and in addition we found that the learning capability of the wasps was very low (see 4.1). Reasons for differences in learning and memory retention are hard to specify. A lot of factors could influence the learning behaviour of the wasps.

TAUTZ et al. (2003) showed for honeybees that temperature during pupal development influences behavioural performances in adult bees. Temperature affects their dance communication and also their odour-learning abilities which are important for foraging. 5. Discussion 23

Several studies also focus on parasitic infections and its influence on behavioural traits in vertebrates and invertebrates. Parasitic infections often affect the function of the central nervous system (CNS); directly by destroying neurons or secreting neuroactive compounds or indirectly through substances which are produced by the host’s immune system as a response to the infection (ADAMO 1997, 2002; THOMAS et al. 2005). This leads to an impairment of cognitive abilities which might be important for learning, memory and decision-making in animals (GEGEAR et al. 2006). In vertebrates it is well known that there are complex interactions between the immune system and the nervous system. In insects little is known but it is becoming clear that the cost of an immune response affects the survival of the insect itself, and also every day behaviour and memory formation (MALLON et al. 2003).

GEGEAR et al. (2006) showed for the social bumble-bee Bombus impatiens that the gut parasite Crithidia bombi influences their ability to utilize floral information impairing foragers to monitor and acquire floral resources. The following food stress increases the mortality rate in infected bees and has therefore detrimental effects on the reproductive success of the colony. Although the parasite is found exclusively in the gut it can influence indirectly the CNS by the immune response of the bee. This study is one example how parasitic infections can disrupt ecologically important aspects of cognition in animals (e.g.

KAVALIERS et al. 1995).

MALLON et al. (2003) infected honeybees with a non-pathogenic immune elicitor (lipopolysaccharide) to simulate an immune response. The honeybees performed, after conditioning experiments, a reduced ability to associate an odour with a sugar reward, showing that the immune system seems to interfere with learning and/or memory formation. If both processes (immune response and memory formation) rely on the same intercellular communication system, using the same substances or messengers, there could be a trade-off in substrates. Specific substances like octopamine, which is important in immune response

(WIESNER et al. 1996), might be used for the immune response instead for associative learning and/or memory formation (MALLON et al. 2003). Studies in vertebrates showed a way of signalling between the nervous system and the immune system (ADER et al. 1991, PUGH et al. 2001). Substances released by the immune response affect the hippocampus, and reduce memory consolidation (PUGH et al. 2001). In insects a similar signalling pathway could be possible, substances like eicosanoids are known to be involved in immune responses but also in learning processes (PIOMELLI 1994). Generally it is thought that immune responses must have substantial energetic costs and are therefore traded off against other costly functions (SHELDON & VERHULST 1996). For 5. Discussion 24

C. glomerata no parasitic infections are known so far but the high mortality rates and the low memory performances support the assumption that the individuals have been infected by a parasite or a pathogen. The differences in memory formation and consolidation between the wasps used by WANG (unpublished) and my population leads to the conclusion that the condition of the individuals has a huge impact on memory formation and consolidation.

Learning is a costly process affecting animal fitness (DUKAS 1998), so maybe C. glomerata could not afford to spend energy on learning and memory formation. If the formation of LTM, which requires gene expression, is too costly, only ASM can be formed in C. glomerata because ARM seems to be absent. ASM and ARM do not require gene expression and are therefore also called a form of “low cost” memory (MERY & KAWECKI 2005). In C. glomerata the infection might have a higher impact on the wasp compared to other species because ARM, as a form of long lasting memory phase, is absent and only ASM can be formed. It would be interesting to know how long ASM lasts in individuals with a bad state of health.

In these experiments ASM is still present after 3 hours whereas in the population of WANG (unpublished) LTM was exclusively present after 3 hours. Future studies should try to find out if infections play a role in variations of learning behaviour in Cotesia species. As a first step the mortality rate of the individuals should be correlated to the learning performance of the wasps.

5.4 Costs and benefits of learning To find out why C. glomerata and C. rubecula, two closely related species, differ in their learning behaviour you have to look at several factors. Learning and storing memory is a costly process which affects animal fitness. Energetic costs are involved to form and maintain memory and there are also ecological costs to learning; learning takes time and is vulnerable to mistakes (DUKAS 1998). Therefore innate behaviour might be favourable in some cases. Why should animals learn at all if it affects their fitness and why did learning evolve? It was thought that learning has developed because animals live in unpredictable environments (e.g. THORPE 1963, MACKINTOSH 1983). If the environmental situation would be completely stable, learning would be useless and innate behaviour favourable. STEPHENS (1993) rephrased a compromise between these two extreme situations; relatively little change is required for learning to evolve. For STEPHENS (1993) learning makes sense if the environment changes unpredictably between generations but is predictable within generations. For example if there are several generations of an insect in one year, which live in different seasons and exploit different food sources. In this case the environment is predictable within 5. Discussion 25 the generation and learning is favoured above innate behaviour. If the environment is very variable within a generation innate behaviour is beneficial. Animals are then confronted with so many different decisions that they would spend too much energy and time for changing behaviour. During their lifetime animals encounter a lot of new stimuli which they have to assess for its importance. Information is only valuable if it has the potential to change behaviour considerably (STEPHENS 1993). How do animals know which information is useful and reliable? When should they adapt to changes in their environment by changing their innate behaviour? One way of assessing the value of information takes place when information is repeated several times. Learning and the subsequent storage of memory in short- or long-term memory is always dependent on the frequency of the stimuli but most of all on the time between the repetitions. Single experiences lead to the storage of memory in STM and ARM whereas only spaced experiences lead to LTM. Still this is not true for every species as we can see e.g. in C. glomerata. C. glomerata does not need confirmative experiences (spaced learning) to form LTM showing the value and the reliability of the information. Learning and memory formation is a process, which proceeds differently in various species. In some species a considerable amount of time is needed for consolidation of memory phases but this time is not required for the underlying neuronal processes; these can react and change very quickly (FREY & MORRIS 1997). Instead, memory formation is adapted to the specific demands and constraints of every single species in its natural environment. A more ecological view of learning contains that memory formation has to be seen in the context of natural behaviour and the specific requirements to which animals are exposed (MENZEL 1999). Learning always means to find a balance between costs and benefits. E.g. learning fast can be a disadvantage by storing wrong information but it might also be advantageous if the information is reliable. Referring to memory formation this means that new memories have to be evaluated on the basis of their relevance. Forming new memory is a costly process indicating that new memories should only be consolidated when they are reliable and of importance.

5.5 Cotesia glomerata and Cotesia rubecula – why are they so different in learning? Looking at the natural behaviour, differences in preference learning might be an adaptation to the specific differences in host-finding behaviour between the two wasp species. If we look at the generalist C. glomerata we see a gregarious species which lays in average 20 eggs mainly 5. Discussion 26 in the caterpillars of P. brassicae. P. brassicae is also gregarious and a generalist, it uses different host plants (cruciferous species) to lay its eggs on. For the wasp this causes a problem in its searching behaviour. On all cruciferous plants hosts could be present and therefore the host plants have a low predictive value for the wasps. If there are a lot of potential plants which might offer hosts, a lot of time and energy is wasted for host searching.

GEERVLIET et al. (1994) showed that C. glomerata is attracted innately by cabbage plants with caterpillars feeding on it but still the wasp encounters hosts on plants different than cabbage. If this happens associative learning takes place. The wasp associates the odour of the new encountered plant with the presence of suitable hosts and this process causes temporal specialization (DUKAS 1998). Specialization is thought to be a way out of inefficient searching behaviour. If C. glomerata encounters a host on a specific plant species this is reliable information for the wasp. It can be sure to find other hosts on the surrounding plants of the same species due to the egg laying performance of its host P. brassicae. P. brassicae uses different host plants but if it lays its egg clusters it chooses a stand of plants of the same species to ensure the food supply for its offspring. The caterpillars are conditioned on the first species they feed on and need to find more plants of the same species. Therefore the wasp will find enough hosts for its own offspring if it orientates itself towards the odour of that plant species. For this reason C. glomerata not only changes its behaviour after one experience but it also stores this memory directly in LTM. For the generalist C. glomerata, parasitizing a generalist host, learning is more valuable than retaining to its innate behaviour. Through learning it benefits from the advantages of specialization (SMID 2006). For the solitary specialist C. rubecula, finding a host on a certain plant species is not reliable information. Its host P. rapae lays only one egg per plant and spreads its eggs widely in its habitat on different plant species (ROOT & KAREIVA 1984). For C. rubecula the presence of one caterpillar does not indicate the presence of other suitable hosts on other plants of the same species. It would be maladaptive for the wasp to change its innate behaviour and start searching for this plant species. C. rubecula is able to associate the odour of a plant with a suitable host but this memory is stored in ASM and ARM and wanes within one day. The wasp does not change its innate behaviour (BLEEKER 2006, SMID submitted). In C. rubecula, memory formation works as a filter keeping out unreliable information. Only after several verifying experiences information is stored in LTM. The wasp is called a slow learner which might sound negative but in the case of C. rubecula learning slowly is the way to successful oviposition. 5. Discussion 27

Comparing the memory formation in C. glomerata and C. rubecula we see a “quick” and a “slow” learner but both ways of learning are successful in their natural environment. My results show that it seems not to be important for C. glomerata to have an additional long lasting memory phase like ARM. The information the wasp gets is so reliable that LTM is directly consolidated out of ASM. LTM requires new gene expression but although the formation is connected to high energetic costs, these costs are worth to spend. The benefits from forming LTM outweigh the disadvantage of spending energy for the formation. Maybe the wasp can also save some energy by leaving out ARM. In C. rubecula the situation is different, the predictability of its environment is very low and innate behaviour is favoured. Still associations are formed after one learning experience but these are stored in ASM and ARM. ASM and ARM are a form of “low cost” memories, demanding less energetic costs than the formation of LTM (MERY & KAWECKI 2005). Like this C. rubecula found a successful way to benefit on the one hand of innate behaviour but also from learning without wasting too much energy. Closely related parasitic wasps which differ in their learning behaviour are useful model organisms to study more about learning and memory formation. Results of my study offer some interesting findings concerning differences in memory consolidation in C. glomerata and C. rubecula. Still a lot of questions remain and future studies could focus on these. Together with a more ecological approach on learning with behavioural tests in the wind tunnel, biomolecular techniques can help to find out more about underlying mechanisms of memory formation. Working six months intensively with parasitic wasps was a fascinating experience for me. Although I read a lot about learning in insects it is still different to experience it by yourself. Wasps which were put for some seconds on a nasturtium leaf with suitable hosts on it remembered this odour 4 hours later! Watching them in the wind tunnel, seeing them move their antennae in agitation, and flying directly to the new learned plant odour fascinated me every time again. 6. References 28

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TULLY, T., T. PREAT, S.C. BOYNTON, M. DEL VECCHIO: Genetic Dissection of Consolidation Memory in Drosophila. Cell 79, 35-47, 1994.

TUMLINSON, J.H., W.J. LEWIS, L.E.M. VET: Parasitic wasps, chemically guided intelligent foragers. Scientific American 268, 100-106, 1993.

VET, L.E.M., M. DICKE: Ecology of infochemical use by natural enemies in a tritrophic context. Annual Review of Entomology 37, 141-172, 1992.

VOS, M., L.E.M. VET: Geographic variation in host acceptance by an insect parasitoid: genotype versus experience. Evolutionary Ecology Research 6, 1021-1035, 2004.

WIESNER, A., D. WITTWER, P. GOTZ: A small phagocytosis stimulating factor is released by and acts on phagocytosing Galleria mellonella haemocytes in vivo. Journal of Insect Physiology 24, 829-835, 1996.

YIN, J.C., J.S. WALLACH, M. DEL VECCHIO, E.L. WILDER, H. ZHOU, W.G. QUINN, T. TULLY: Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79, 49-59, 1994. 1. Introduction 32

Appendix:

The role of larval mobility in the transition from solitary to gregarious development in Cotesia glomerata and Cotesia rubecula

1. Introduction

1.1 Solitary and gregarious development In parasitic wasps two different ways of developing are known, solitary and gregarious. In solitary species only one egg can develop in one host. If more than one egg is laid into the host only one of the larvae survives (e.g. SALT 1961) because of physical attack, physiological suppression or resource competition (GODFRAY 1994). Despite the fact that adult females are able to destroy larvae or eggs of other females

(ovicide) with their ovipositor (GODFRAY 1994) or by injecting venoms (HÅGVAR 1988,

MACKAUER 1990), mostly the behaviour of the immature parasites determines their own survival. First instar solitary larvae have strong mandibles and they search actively for conspecific competitors and individuals of other species. The death of rivals is not necessarily caused by fighting itself but wounding makes sure that invaders are recognized by the host’s immune system and die (GODFRAY 1994). Older larvae are able to eliminate younger larvae by physiological suppression. They are thought to excrete chemicals that inhibit or kill competitors (TIMBERLAKE 1910, 1912; MACKAUER 1986). Also the competition for limited resources is a possibility to fight against opponents. The host can be already consumed before a competitor hatches (FISKE & THOMPSON 1909) or e.g. the oxygen content in the host’s haemolymph can be reduced (asphyxation) and younger more sensible larvae or eggs perish

(FISHER 1961, 1963). For the young larvae which have fighting capabilities mobility plays the key role for finding and killing competitors to exploit the whole resources of the host. Solitary development seems to be the ancestral condition in parasitoid Hymenoptera. Still gregariousness has evolved at least 43 times in 26 different families of Hymenoptera

(ROSENHEIM 1993, MAYHEW 1998) and is stable in these cases. In gregarious species several eggs are laid in one host and the larvae develop successfully together (ODE & ROSENHEIM 1. Introduction 33

1998). Gregarious development has the advantage that the adult insect can exploit its hosts better, the larger the host the more eggs are laid into it. In evolutionary biology the transition of solitary to gregarious development has been of interest for a long time (MOCK & PARKER 1997). Most research has been done on birds

(GODFRAY & HARPER 1990) and insect parasitoids (GODFRAY 1987, MAYHEW 1998). The question is how the transition from solitary and siblicidal to gregarious and nonsiblicidal individuals took place. If the individuals are siblicidal there should be a selection towards solitary individuals, laying only one egg in one host. How can nonsiblicidal behaviour enter under these conditions in siblicidal populations? HARVEY and PARTRIDGE (1987) called this situation an “evolutionary black hole” because this evolution seems to be irreversible. Normally nonsiblicidal brood mates would always be killed by their siblicidal mates (MAYHEW et al. 1998). In addition why should solitary species start to lay more than one egg in a host? Several hypotheses are proposed until now. Beside the suggestion, that this could happen by accident

(GODFRAY 1987), it is proposed that parasitoids may lay additional eggs to ensure the successful development of their offspring (PARKER & COURTNEY 1984, GODFRAY 1987). If the first egg is not viable, the second egg has a chance to develop and survive (GODFRAY 1994). Additional eggs are also an advantage when the risk of multiparasitism (interspecific superparasitism) is high. Two or more eggs of the same species enhance the chance to be the winner in competitive interactions between the developing larvae of other species (PARKER &

MOCK 1987, VAN ALPHEN & VISSER 1990). Also the discouragement of conspecifics is seen as a possibility. If conspecific adults are able to determine the number of eggs being present in a host they might be prevented from parasitizing if there are enough hosts in the patch. For the offspring the chance of surviving decreases with the number of eggs already present in the host. Therefore additional eggs are a good option to ensure the survival of the own offspring (VAN ALPHEN & VISSER 1990). Eggs of the parasitoid wasps also have to deal with the immune system of its hosts, haemocytes of the host can form multicellular capsules which surround the eggs and the larvae are not able to hatch any more (BAUER 1999, VAN ALPHEN & VISSER 1990). In this case superparasitism or self-superparasitism can be advantageous because the encapsulation of the first egg in the host might have exhausted the haemocyte supply of the host (VAN

ALPHEN & VISSER 1990). Normally, brood reduction by fighting larvae seems to be advantageous because there is a higher amount of host resources left over for the winner. Still the laying of additional eggs can 1. Introduction 34 be of advantage when the host can support the development of more than one individual and there is a fitness advantage for several individuals developing together. Improvement of host quality through host shift or change in host size might be a reason why gregarious development has been evolved in some cases (GODFRAY 1994). Still the question remains how the transition from solitary to gregarious development took place.

1.2 Transition from solitary to gregarious development One assumption of the successfully transition to gregarious development has been that the brood mates tolerate each other through a loss of aggression while the larvae retain mobile

(MAYHEW & VAN ALPHEN 1999). Still, the invasion of an aggressive individual would disturb the transition to tolerant behaviour. In Argochrysis armilla the spread of tolerant behaviour seems to be supported by single sex broods. If a tolerant gene enters a population it has a bigger chance to be spread if the brood is closely related to each other (PEXTON & MAYHEW 2001).

BOIVIN and BAAREN (2000) assume that instead of a loss of aggression (and retaining mobility), the larvae retain aggressive behaviour but have a reduced mobility. They compared the mobility of Anaphes listronoti , a gregarious egg parasitoid of Listronotus oregonensis, with the solitary A. victus (Fig. 1). Larvae of A. victus were more mobile (twisting and folding) than larvae of A. listronoti. Both first instar larvae have highly developed mandibles and are able to defend themselves against siblicidal brood mates. The assumption is that the brood mates of gregarious species don’t search actively for conspecific larvae and they develop beyond the aggressive first larval instar, without encountering each other. If they encounter a siblicideous larvae invading the population they have equal chances to survive because of their developed mandibles. To investigate if this phenomenon also occurs in other species the mobility of larvae of the gregarious wasp Cotesia glomerata (L.) and the solitary Cotesia rubecula (Marshall) (Hymenoptera: Braconidae) will be compared in Pieris caterpillars.

1.3 Cotesia glomerata and Cotesia rubecula The parasitic wasps C. glomerata and C. rubecula lay their eggs in young larvae of Pieris butterflies. C. rubecula is classified as solitary and prefers the larvae of the solitary small cabbage white Piries rapae (L.) (Lepidoptera: Piridae) as a host. Only one egg is laid in a single caterpillar (GEERVLIET et al. 2000, SMID 2006). If more than one egg is laid into a host only one larva will survive (HARVEY, unpublished). 2. Materials and methods 35

Fig. 1: Mean numbers (± SEM) of twisting (lateral torsion) and folding (vertical bending)

observed for 5 min in mymariform larvae of the solitary A. victus and the

gregarious A. listronoti (BOIVIN & BAAREN 2000).

In contrast, C. glomerata is classified as gregarious, i.e. the wasp lays up to 20 eggs in a single host. Although C. glomerata is regarded as a generalist, the wasp prefers to lay its eggs in the larvae of the gregarious large cabbage white Pieris brassicae (L.) (Lepidoptera:

Piridae) (VOS & VET 2004). The larvae develop successfully together without killing each other, i.e. they are regarded as nonsiblicidal (PEXTON et al. 2003). To see if the loss of mobility plays also an important role in these two species for the transition from solitary to gregarious development a similar approach as in the experiments of

BOIVIN and BAAREN (2000) will be done.

2. Materials and methods

2.1 Experimental setup Insects and plants were reared as explained before. Freshly hatched larvae of P. brassicae and P. rapae were transferred to cabbage plants and were allowed to feed for 24 hours. After 24 hours the larvae were parasitized by C. glomerata and C. rubecula respectively. The wasp larvae hatched after 5 days and the Pieris caterpillars were dissected after 5, 6, 7 and 8 days. Larvae of the wasps were observed with a microscope and filmed in physiological saline for a period of 5 min. 15 larvae of each species were analysed at the specific days. 3. Results 36

To compare the morphology of the mouthparts, pictures of the head of both species were taken with a JEOL JSM 6300F field emission scanning electron microscope (SEM). Pieris caterpillars were parasitized as described above and the wasp larvae were dissected out of the caterpillars after 5, 6, 7 and 8 days. The larvae were transferred to 100 % ethanol, critical point dried and sputtered with 10 nm platinum for observation in the microscope. Unfortunately larvae of C. glomerata at day 5 and 6 were so small that they were washed through the gauze during the drying process.

2.2 Statistical analysis Generalized linear modelling (GLM) procedures using procedure GENMOD in SAS version 8.02 (Proc GENMOD, SAS Inc, Chicago, IL, U.S.A.) were used for data with a binominal distribution of error variance and a logit-link function. When main effects or their interactions were found significantly different, further separation of the treatment levels was carried out by acquiring specific contrasts for particular comparisons, which are given in the text. Data collected on different experimental days were considered as replicates. We tested whether time (4 levels: 5, 6, 7 or 8 days) and species (2 levels: C. glomerata and C. rubecula) had an effect on twisting in Cotesia larvae (GLM treatment Ȥ²6 = 41.42, P < 0.0001).

3. Results

Observing the larvae of both species, the lateral bending were counted. Five days after oviposition early first instar larvae of C. rubecula twisted significantly more than larvae of C. glomerata (GLM, P = 0.0014) (Fig. 2). The larvae bended with quick and dynamic movements, enabling them to move through the body of the host. Movements of C. glomerata were more slowly and consistent with breaks in between. From day 6 on there was no difference in movements any more between both species. In C. rubecula a rapid decline of movements from 5 to 8 days after oviposition is shown (GLM, P < 0.0001) whereas in C. glomerata the twisting remain at the same level (GLM, P = 0.2771). 3. Results 37

50 Cotesia glomerata 45 40 Cotesia rubecula 35 30 25 20 15 10 twistings in 5 minutes 5 0 5 6 7 8 days after oviposition

Fig. 2: Mean numbers of lateral bending of the solitary C. rubecula and the gregarious C. glomerata within 5 minutes, 5 to 8 days after oviposition.

Table 1: Mean numbers of bending in C. glomerata and C. rubecula, 5, 6, 7 and 8 days after oviposition.

Species 5 days 6 days 7 days 8 days

C. glomerata 30 30 23 20 C. rubecula 47 31 21 9

Fig. 3: Larvae of C. glomerata and C. rubecula 6 days after oviposition. Bar = 300 ȝm. 3. Results 38

A B mp

C D

mp md lp

E F

Fig. 4: Larvae of C. glomerata (A, B) and C. rubecula (C – F). A, B: Mouthparts and ventral view of C. glomerata on day 7 and 8. C: Lateral view of C. rubecula on day 5. D: Ventral-frontal view of C. rubecula and mouthparts on day 5. E, F: Dorsal and ventral view of the head of C. rubecula on day 8. Note the absence of mandibles and labial palpes in C. glomerata. mp = maxillary palpus; md = mandible; lp = labial palpus. 4. Discussion 39

The mouthparts of C. glomerata and C. rubecula show clear differences in morphology. Unfortunately no pictures of C. glomerata on the first two days after eclosion could be made. The period directly after eclosion represents the most interesting point in time because the early first larval instar is suggested to be the aggressive instar, at least in solitary species. Also my results on the mobility indicate that of the first larval instar is the most interesting phase. Therefore a comparison between both larvae directly after eclosion would be of interest. In C. glomerata maxillary palpes are visible on day 7 and 8 (Fig. 4 A, B). In C. rubecula beside maxillary palpes also large mandibles and labial palpes are visible from day 5 to 8 (Fig. 1 C – F). Until now it is not known how many instar the larvae pass through before emergence (Fig. 3). More studies about the change in length, size and morphology of head and mouthparts and the morphology of the abdomen and anal vesicle would make it possible to find a system for precisely staging the larvae. The time until emergence is variable and dependent on the host; therefore the age of the larvae is not a useful parameter.

4. Discussion

My results show that also in the larvae of C. rubecula and C. glomerata a difference in mobility occurs. One day old larvae of the solitary C. rubecula twisted significantly more than larvae of the gregarious C. glomerata. These findings support the hypothesis of BOIVIN and

BAAREN (2000). BOIVIN and BAAREN (2000) suggested for A. victus and A. listronoti that one possible way for the transition from solitary to gregarious development could be a loss of mobility instead of a loss of aggression. Reduced mobility while retaining the capacity to fight seems to be one possible answer to the question how transition takes place and above all prevail. If the immobile individuals retain their capability to fight they are less vulnerable to invading solitary individuals. If a solitary species enters a host with more than one immobile, but aggressive species its own survival is dramatically reduced and the survival of the gregarious species can be successful. The difference in mobility in Cotesia larvae occurs only on the first day. After that the twistings of C. rubecula decline rapidly and are not different from C. glomerata any more. Why are the larvae of the solitary C. rubecula only mobile on the first day? What happens if a second larva is located in the host? The early first larval instar is suggested to be the aggressive period; the larvae have developed mandibles which give them fighting capabilities. 4. Discussion 40

Solitary larvae search actively for competitors; if they find another larva they will try to kill it

(GODFRAY 1994). If hosts are parasitized at the same time with more than one egg the hatched larvae fight and only one individual survives (e.g. SALT 1961). If eggs are laid in a different point in time the first larvae which hatches searches for competitors and will find the eggs of other individuals. MAYHEW & VAN ALPHEN (1999) showed that eggs are also attacked by first instar larvae of the solitary Aphaereta genevensis. In this case it would be sufficient for the solitary C. rubecula to be mobile only on the first day. The larvae search for competitors, try to kill other brood mates or eggs and after this the reduction of its mandibles could take place and the larvae stay more immobile than before. It might also be likely that other mechanisms take over the fight for the own survival and explain the rapid decline of mobility. MACKAUER (1959) and TREMBLAY (1966) assume that cytolytic enzymes are released by the first-hatching larvae to suppress younger competitors.

Also starvation (FISKE & THOMPSON 1909) or asphyxation (FISHER 1961, 1971) are seen as a possibility to inhibit the development of brood mates. Still the adult female may also play a role in suppressing the offspring of conspecifics by injecting substances which kill embryos already present in the host (HÅGVAR 1988). How could the transition from solitary to gregarious development be explained in Cotesia sp.? Models suggest that solitary species can become gregarious by a gain of tolerance and a subsequent increase in clutch size (Fig. 5, route 1) or the clutch size is increased and a gain of tolerance appears subsequently (Fig. 5, route 2) (PEXTON & MAYHEW 2001). As said before there are several factors influencing solitary species to lay more than one egg in a host such as laying of insurance eggs (PARKER & COURTNEY 1984, GODFRAY 1987) the presence of conspecifics (e.g. MICHAUD & MACKAUER 1995), low encounter rates with hosts

(e.g. ROSENHEIM & HONGKHAM 1996), high egg loads (MICHAUD & MACKAUER 1995) and increasing host size (CHAU AND MACKAUER 1999).

PEXTON and MAYHEW (2005) compared the clutch size of the solitary wasp A. genevensis with the gregarious wasp A. pallipes (both parasitize Drosophila virilis) under intraspecific competition. They showed that A. genevensis lays more eggs when there is a high risk of superparasitism. With this adaptive behaviour the wasps ensure that the surviving offspring is hers. They also showed that the clutch size under the risk of superparasitism seems to be dependent on the size of the adults. Small females increase the average clutch size noticeable due to their limited fat resources (PEXTON & MAYHEW 2005). Adults cannot increase their fat reserves during their life which means that small individuals have limited time to encounter 4. Discussion 41 enough hosts to deposit their eggs (ELLERS 1996, OLSEN et al. 2000, RIVERO & WEST 2002). Therefore the clutch size is increased. As this example shows there seem to be reasons why solitary species adapt to specific factors by laying more than one egg on or into their hosts which may lead to a gregarious development under certain circumstances.

Fig. 5: The evolution of gregariousness. Larvae (a) and (b) are tolerant or immobile; they can evolve towards gregarious development with increasing clutch size (a to b). Larvae (c) and (d) are mobile, intolerant and develop solitary. Gregariousness can develop by a gain of tolerance and a subsequent increase in clutch size (Route 1) or

by an increase in clutch size and the subsequent gain of tolerance (Route 2) (PEXTON

& MAYHEW 2001).

This study shows that there is a clear difference between the mobility of early first instar larvae of the two closely related wasps C. rubecula and C. glomerata. For C. glomerata a loss of mobility seems to be one factor being involved in the transition from solitary to gregarious development. Of course the question remains if the larvae of the gregarious C. glomerata are able to defend itself against siblicidal brood mates. Therefore pictures of the mouthparts of C. glomerata and C. rubecula were taken. Clear differences are visible between both species. C. rubecula has impressive mandibles at least up to 4 days after eclosion whereas these are absent in C. glomerata. This fact might be interpreted that also a loss of aggression plays a role in the transition from solitary to gregarious development. Models described by PEXTON & 4. Discussion 42

MAYHEW (2001) suggest that solitary species can become gregarious by a gain of tolerance and a subsequent increase in clutch size. Still, pictures of C. glomerata directly after eclosion are missing. Pictures of the early larval instar should be conducted to see if changes in morphology of the mouthparts are visible between day 5 and 8. It seems that more than one factor is involved as a route out of solitary development in C. glomerata. A loss of mobility but also a loss of aggressiveness might play role in the transition from solitary to gregarious development. 5. References 43

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VOS, M., L.E.M. VET: Geographic variation in host acceptance by an insect parasitoid: genotype versus experience. Evolutionary Ecology Research 6, 1021-1035, 2004. Erklärung

Hiermit versichere ich, dass ich die vorliegende Diplomarbeit selbständig verfasst und keine anderen Quellen und Hilfsmittel benutzt habe. Alle Stellen der Arbeit, die wörtlich oder sinngemäß aus Veröffentlichungen oder aus anderweitigen fremden Äußerungen entnommen wurden, sind also solche einzeln kenntlich gemacht.

Die Diplomarbeit habe ich noch nicht in einem anderen Studiengang als Prüfungsleistung verwendet.

Stuttgart-Hohenheim, den 5. März 2007