Host searching and host selection of Trichogramma cacoeciae:
Potential and limitations of an egg parasitoid to locate and parasitise eggs from Lobesia botrana and Eupoecilia ambiguella
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau
vorgelegt von Cornelia Rüdiger geboren am 11.09.1980 in Sigmaringen
1
Dekan der Fakultät: Prof. Dr. Gunther Neuhaus
Prodekan Biologie I: Prof. Dr. Wolfgang Driever
Promotionsvorsitzender: Prof. Dr. Samuel Rossel
Betreuer der Arbeit: PD Dr. Thomas Schmitt
Gutachter und Zweitprüfer: Prof. Dr. Josef K. Müller
Drittprüfer: Prof. Dr. Michael Schere-Lorenzen
Prüfung: 7.10.2011
2 Erklärungen:
1) Die vorliegende Dissertation wurde korrigiert und wird als Ersatz für die erste Version eingereicht. Somit wurde die vorliegende Arbeit in keiner Form anderweitig als Prüfungsarbeit verwendet oder einer anderen Fakultät als Dissertation vorgelegt.
2) Hiermit erkläre ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quellen gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die erste Version, sowie die hier vorliegende, korrigierte Arbeit wurden bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
3) Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion zur Führung des Doktortitels nicht berechtigt bin.
Freiburg, den
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Für meine Familie, die in den Jahren meines Studiums
immer für mich da war und der ich so viel verdanke.
Danksagung
Mein besonderer Dank gilt PD Dr. Thomas Schmitt, der mir ermöglichte das Thema meiner Dissertation selbst mitzuentwickeln und mich bei der Durchführung meiner Arbeit unterstützt hat. Ich danke ihm für die vielen Stunden anregender Diskussionen und die Möglichkeit der Betreuung zweier Diplomandinnen im Rahmen meines Dissertationsthemas.
Ebenso herzlich möchte ich mich bei Herrn Prof. Dr. Klaus Peschke bedanken, der mir die Arbeit in seinem Labor und den Zugang zu sämtlichen relevanten Materialien und Geräten ermöglichte.
Prof. Dr. Josef Müller danke ich für die konstruktiven Anregungen bei der Ausarbeitung der vorliegenden Arbeit und die vertieften Einblicke in die Welt der Statistik.
Dr. Michael Breuer danke ich für die gute Zusammenarbeit am Staatlichen Weinbauinstitut Freiburg und den Zugang zur Traubenwicklerzucht. Sämtliche Freilandprojekte waren nur durch seinen tatkräftigen Einsatz möglich und seine Erfahrung hat mich vor vielen Überraschungen bewahrt.
Dr. Olaf Zimmermann und Bernd Wührer von AMW-Nützlinge GmbH danke ich für die regelmäßige Übersendung der Trichogramma-Wespen, ohne die meine Arbeit nicht möglich gewesen wäre, und die vielen Tipps und Tricks zur Haltung der Winzlinge.
Für die Unterstützung bei allen kleineren und größeren Problemen, sowie für die stets schnelle, unkomplizierte und bereitwillige Hilfe, danke ich Stefan Heyl, Birgit Büsch, Daniela Noeske, Marc Spelleken, Dr. Martin Schaefer und besonders Renate Kendlinger.
Harald Noeske, Matthias Siegel und dem ganzen Werkstatt-Team danke ich recht herzlich für die Herstellung sämtlicher Sonderanfertigungen für meine Versuchsreihen, für die häufig recht kurzfristigen Reparaturen und die Beratung bei allen Materialfragen.
Bei meiner Arbeitsgruppe und der ganzen Abteilung bedanke ich mich für die vielen schönen und gemütlichen Stunden, sowie die wissenschaftlichen Diskussionen und Anregungen zur Durchführung der vorliegenden Arbeit. Ein besonderer Dank geht an Thomas Braun, Cordula Neumann, Nina Stobbe, Tamara Prokorny und an meine liebe Freundin Ragna Franz. Recht herzliche möchte ich mich außerdem bei Mareike Wurdack und Sandra Steiger bedanken, die diese Arbeit mit großem Zeitaufwand korrigiert haben und denen ich viele unvergessliche Stunden verdanke. Bei meinen Diplomandinnen Barbara Kagerer und Magdalena Daum bedanke ich mich für die erfolgreiche Zusammenarbeit und die tollen Ergebnisse, die in diese Dissertation mit eingegangen sind.
Ein großes Dankeschön geht außerdem an Martin Köcher, der während der gesamten Dissertation immer für mich da war und mich wärend der ganzen Zeit tatkräftig unterstützt hat.
Zuletzt möchte ich nochmals meiner Familie danken, ohne die mein Studium und diese Dissertation nicht möglich gewesen wären und der ich diese Arbeit widme.
Table of contents
1. Introduction ...... 10
1.1. Prospects to gain host access...... 10 1.1.1. Host habitat location...... 13 1.1.2. Host location and induced plant volatiles ...... 14 1.1.3. Host location and enhancing background odours ...... 15 1.1.4. Host location and host sex pheromones ...... 16 1.1.5. Host location and egg volatiles...... 17 1.1.6. Host location and moth wing scales ...... 18 1.1.7. Identification of involved chemicals ...... 18 1.1.8. Host location and host egg age...... 21 1.1.9. Host suitability and host egg age ...... 21 1.1.10. Host suitability and host species ...... 22 1.1.11. Parasitoids and their natural environment...... 23 1.2. Limitations on host access ...... 25 1.2.1. Masking plant volatiles ...... 26
2. Issues & Approaches ...... 27
3. Material & Methods...... 30
3.1. Model organisms...... 30 3.1.1. Trichogramma cacoeciae ...... 30 3.1.2. Lobesia botrana & Eupoecilia ambiguella ...... 31 3.2. Laboratory bioassays ...... 32 3.2.1. General setup: Y-tube olfactometer ...... 32 3.2.2. Host location and plant volatiles ...... 35 3.2.3. Host location and the source of infochemicals ...... 35 3.2.4. Identification of involved chemicals ...... 38 3.2.5. Host location and host egg age...... 40 3.2.6. Host suitability and host egg age ...... 40 3.2.7. Host suitability and host species ...... 41 3.3. Field bioassays...... 43 3.3.1. Natural occurring Trichogramma species...... 43 3.3.2. Influences of sulphur treatment and artificial application...... 45
-6- 4. Results...... 49
4.1. Statistikal declarations ...... 49 4.2. Laboratory bioassays ...... 49 4.2.1. Control trials: Y-tube olfactometer...... 49 4.2.2. Host location and plant volatiles ...... 50 4.2.3. Host location and the source of infochemicals ...... 54 4.2.4. Identification of involved chemicals ...... 57 4.2.5. Host location and host egg age...... 60 4.2.6. Host suitability and host egg age ...... 62 4.2.7. Host suitability and host species ...... 65 4.3. Field bioassays...... 66 4.3.1. Natural occurring Trichogramma species...... 66 4.3.2. Influence of sulphur treatment and artificial application ...... 68
5. Discussion ...... 70
5.1. Influence of plant volatiles on host location ...... 71 5.2. Source of infochemicals used for host location ...... 75 5.3. Involved chemicals ...... 76 5.4. Moth scales ...... 80 5.5. Influence of host egg age on host location and suitability ...... 80 5.6. Influence of host species and its suitability for T. cacoeciae...... 82 5.7. Host location and parasitisation...... 83 5.8. Performance of Trichogramma in natural environment ...... 85
6. Conclusions ...... 87
6.1. Potential of T. cacoeciae to gain host access ...... 87 6.2. Limitations ...... 88
7. Summary ...... 90
8. References ...... 92
9. Participation at conferences...... 110
9.1. Oral presentations...... 110 9.2. Poster presentations...... 110
-7- List of figures and tables
1. Introduction
Figure 1-1: Pathway of an egg parasitoid to gain host access...... 11
3. Material & Methods
Figure 3-1: T. cacoeciae on a L. botrana egg ...... 30 Figure 3-2: Abdominal tips of female grapevine moths ...... 32 Figure 3-3: General setup of the Y-tube olfactometer ...... 33 Figure 3-4: Graphical description of results gained from Y-tube olfactometer bioassays ...... 34 Figure 3-5: Aerial view of the sampling sites...... 43 Figure 3-6: Areal partition of the sampling site and area treatments...... 46
Table 3-1: List of samples used for choice experiments via Y-tube olfactometer and/or GC-MS analyses...... 37 Table 3-2: Composition of available egg associated substances...... 39 Table 3-3: Concept types of area treatments ...... 47
4. Results
Figure 4-1: Reaction of T. cacoeciae towards plant volatiles ...... 50 Figure 4-2: Reaction of T. cacoeciae towards infested grapes ...... 52 Figure 4-3: Reaction of T. cacoeciae towards infested inflorescences ...... 53 Figure 4-4: Reaction of T. cacoeciae towards egg mass extracts and fractions ...... 55 Figure 4-5: Reaction of T. cacoeciae towards egg associated substances ...... 56 Figure 4-6: Part of a chromatogram of an L. botrana egg mass extract...... 57 Figure 4-7: Details of polar fraction of L. botrana egg mass extract...... 59 Figure 4-8: Reaction of T. cacoeciae towards eggs of different ages ...... 61 Figure 4-9: Parasitisation rates on different aged eggs...... 63 Figure 4-10: Breeding success on different aged eggs...... 64 Figure 4-11: Parasitisation rates during flight cage experiments ...... 65 Figure 4-12: Parasitisation rates in the field due to different area treatments...... 69 Figure 4-13: Rating of larval infestation in differentially treated areas ...... 69
Table 4-1: Glossary of performed statistical analyses...... 49 Table 4-2: Identified substances from polar fraction of L. botrana egg mass extracts ...... 58 Table 4-3: Proportion of parasitised eggs ...... 66 Table 4-4: Species classification of baited parasitoids...... 67 Table 4-5: Results of Tukey-Kramer post hoc analysis...... 67 -8- Glossary
Semiochemicals: Chemicals involved in the chemical interaction between organisms. They include toxins and nutrient that themselves benefit or detriment the interacting organisms and information conveying chemicals (infochemicals) (senus Nordlund & Lewis 1976).
Infochemicals: Chemicals that are involved in conveying information in intra- and interspecific interactions (e.g. pheromones, kairomones, synomones) (sensu Dicke & Sabelis 1988).
Pheromones: Substances that is secreted by an animal or plant to the outside that cause a specific reaction in a receiving individual of the same species (senus Nordlund & Lewis 1976).
Kairomones: Substances, produced, acquired by, or released as a result of the activities of an organism that in contact with an individual of another species in the natural context, evokes a behavioural or physiological reaction adaptively favourable to the receiver but not to the emitter (sensu Dicke & Sabelis 1988).
Synomones: Substances produced or acquired by an organism that in contact with an individual of another species in the natural context, evokes a behavioural or physiological reaction adaptively favourable to both emitter and receiver (sensu Murata et al. 1986).
Enemy free space: EFR; Region or place where eggs and larvae of the herbivore can develop and crow up to adult stage without being disturbed and attacked (sensu Jeffries & Lawton 1984).
-9- 1. Introduction
Interactions between insect parasitoids and their hosts usually result in hosts’ death and are obligatory for the development of parasitic insects. This obviously led to a strong selection pressure on host species to avoid detection by parasitoids and on parasitoid species to improve their access to suitable hosts. Insect parasitism first was discovered and described in 1096 by Lu Dian for tachnid parasitoids in China (Cai et al. 2005). In Europe, 1678 Swammerdam and Marsilius were the first who observed and described the phenomenon of insect parasitism (Van Lenteren & Godfray 2005). Usage of parasitoids for augmentative biological control (= inundative releases of natural enemies) then attracted the attention of scientists since the early 18th and finally in 1911 Radeckij was the first who initiated experiments on rearing and introducing Trichogramma evanescens (Hymenoptera: Trichogrammatidae) to biologically control Cydia pomonella (Lepidoptera: Tortricidae) in apple orchards (Kot 1964). Since then, an extensive literature has been generated concerning the potential of parasitoids to biologically control phytophagous pest species. Besides this, several studies considered biological, ecological and evolutionary factors that influence interactions between several parasitoids and their host species (Hawkins 1994). However, comprehensive studies on the whole process of searching and selecting a host by a parasitoid are rare. Especially studies including the skills of all trophic levels - the hosts’ food plant, the host and the parasitoid. Aim of the present study was to provide an elaborated study on the potential of an egg parasitoid to locate and parasitise eggs of its two host species and on limitations due to adverse factors and conditions. Therefore, fundamental researches on host location and parasitisation of the egg parasitoid Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) for eggs of the two European grapevine moth species (EGM), Lobesia botrana and Eupoecilia ambiguella (Lepidoptera: Tortricidae), were performed. Furthermore, applied studies on the potential of natural populations and artificial induced T. cacoeciae under different environmental conditions, were conducted.
1.1. Prospects to gain host access
Female egg parasitoids are forced to gain access to suitable host eggs as they are the only food source for their offspring and therefore directly linked to their reproductive success (Van Alphen & Vet 1986, Castelo et al. 2006). In general, successful parasitisation is initiated by different steps of searching behaviour that lead the parasitoid females into closest vicinity of
-10- their potential host eggs (Vinson 1976). Although the intensity and inalienability of steps differ between different parasitoid species, in general parasitoids host searching persists of two phases - host habitat location (1) and host location (2). After reaching the hosts habitat and landing near a hosts egg or egg mass, the next step on the pathway for successful reproduction is host selection. In general, this part of gaining host access is divided into two phases: first host recognition (3) and host acceptance (4) followed by the last important host attribute - host suitability (5). (Figure 1-1; after Fatouros et al. 2008).
Host selection
Host constitution Host constitution
4. Host acceptance -> Parasitisation
5. Host suitability 3. Host recognition -> Eclosion/Adult Plant Plant cues Host searching cues Host
1. Host habitat 2. Host location location
Chemical cues e.g. OVIV, FIV
Host habitat Host on plant
Figure 1-1: Pathway of an egg parasitoid to gain host access. In arrows the associated chemical and physical stimuli are stated. Steps 1 and 2 comprise host searching from a greater distance, followed by host recognition and acceptance in much nearer vicinity (steps 3 and 4). Hosts’ suitability (step 5), is important for parasitoids breeding success and crucial for the appearance of a new generation (modified from Fatouros et al. 2008). OVIV = oviposition induced volatiles, FIV = feeding induced volatiles
-11- For several parasitoid species, the processes to reach, recognize and accept a host (1-4) were shown to be mediated by visual, physical and chemical stimuli (Schmidt 1991, Vinson 1998, Borges et al. 1999, Mansfield & Mills 2002). Thereby, for egg parasitoids, visual cues (if at all) only are involved in host habitat location, whereas physical stimuli play a role during host recognition and acceptance in direct contact with the host eggs (reviewed by Fatouros et al. 2008). Chemical stimuli are reported to be involved in all steps of host searching and host selection. However, to locate host habitats and get information about the hosts` presence within, informative chemical cues - called infochemicals - are the most prominent resources (Conti et al. 2003). There is a wide range of possible infochemicals that could facilitate parasitoids search. For example volatiles emitted by host food plants - infested as well as uninfested - were shown to act as kairomones during host habitat location for several parasitoid species (Reddy et al. 2002, Romeis et al. 2005, Fatouros et al. 2005a, 2005b, Lou et al. 2005, Hilker & Meiners 2006, reviewed by Fatouros et al. 2008). For host location, sex pheromones from calling host females (Zaki 1985, Noldus 1988, Noldus et al. 1990, 1991a, 1991b, Arakaki & Wakamura 2000, Boo & Yang 2000, Hilker et al. 2000, Schöller & Prozell 2002), wing scale volatiles from ovipositing host females (Lewis et al. 1971, 1972, Noldus & Van Lenteren 1985, Kainoh et al. 1990, DeLury et al. 1999, Yong et al. 2007) as well as semiochemicals emitted directly from the host (Frenoy et al. 1992, Renou et al. 1992, Bai et al. 2004) were shown to attract many parasitoids, including egg parasitoids. Thereby, stimuli directly emitted from the needed host stage provide most reliable information. However, such infochemicals may be hard to detect, especially for egg parasitoids due to the small biomass of insect eggs and the small amount of emitted infochemicals relative their surrounding environment (Greevliet et al. 1994). Phytochemical information, although obviously much more detectable, at first sight seem to be less reliable. However, the use of phytochemicals for host location was reported for several parasitoid species (e.g. Vet & Dicke 1992, Vinson 1998, Takabayashi et al. 2006). In this case, especially plant volatiles emitted due to oviposition or feeding of the herbivorous host species are suggested to serve as probable infochemicals (reviewed by Fatouros et al. 2008).
After successful host location, egg parasitoids are about to recognise and accept their host. In this context, a series of behaviour patterns - walking, tapping, drumming and drilling - was described for several species (Vinson 1976, Suzuki et al. 1984, Meiners et al. 1997). However, this aspect remains disregarded in the present study due to the fact that it was already described for Trichogramma egg parasitoids in detail (Schmidt 1994).
-12- The last step of successful reproduction is host suitability, which also may be influenced by several factors. The nutritional value of the host as well as its defence mechanisms - mechanical and/or molecular - may affect the parasitoids reproduction success (Vinson & Iwantsch 1980). In this context, for egg parasitoids, egg age was mentioned to play a major role in their reproductive success (e.g. reviewed by Pak 1986, Reznik et al. 1997, Monje et al. 1999, Saour 2004, Makee 2005).
1.1.1. Host habitat location
In order to locate the hosts’ habitat, and reside within, besides visual cues, specific plant volatiles may serve as long-range infochemicals. For example, Williams et al. (2007) showed that Platygaster subuliformis (Hymenoptera: Platygastridae) shows chemically mediated anemotaxis towards their hosts’ food plant, Brassica napus (Brassicaceae) in field studies. Additionally, there are a number of studies on the impact of host food plants on the host location of different Trichogramma species. They report positive responses to volatiles emitted from undamaged plants and plant extracts (e.g. Kaiser et al. 1989, Romeis et al. 1997, Boo & Yang 1998, Fatouros et al. 2005a, 2005b, reviewed by Romeis et al. 2005). However, it was shown that small parasitoids, like T. cacoeciae, have limited flying abilities in open field and mostly disperse passively by wind drifts rather than to fly actively to an odour source (reviewed by Keller et al. 1985). Therefore, it was assumed that the parasitoids more likely were arrested by plant volatiles rather than actively move towards the plants over far distances (Romeis et al. 1997). In field studies, only under calm conditions or in wind protected areas within vegetation, individuals of Trichogramma spp. were shown to be able to actively disperse in different directions (Fournier & Boivin 2000). Similar results were gained from a laboratory study. Volatiles emitted by uninfested cabbage to elicited a chemically mediated anemotaxis in Trichogramma chilonis, only when tested in a Y-tube olfactometer with low air flow of 30 ml/min (Reddy et al. 2002). However, there are no additional studies on the impact of volatiles from undamaged plant materials on a Trichogramma egg parasitoid. Therefore, this was examined in the present study, using a Y-tube olfactometer with low-wind conditions. Undamaged plant organs of Vitis vinifera (Vitaceae) – the food plant of L. botrana and E. ambiguella- were infestigated for its attractiveness towards T. cacoeciae.
-13- 1.1.2. Host location and induced plant volatiles
Host location, as already mentioned, can be mediated by several chemical substances emitted by a variety of sources. For instance, plant volatiles emitted from infested plants that are “calling for help” due to herbivorous are used as kairomones by several parasitoids and predators. Such induced volatiles that are about to recruit enemies of the plants` enemies - called synomones - are known from a variety of plant species. Obviously they provide reliable information for egg parasitoids about the presence of their hosts. Actually, synomones are used for host location by several parasitoid species (Kaiser et al. 1989, Turlings et al. 1990, Tumlinson et al. 1992, Dicke & Van Loon 2000, Turlings & Waeckers 2004, Fatouros et al. 2005a, 2005c, Hilker & Meiners 2006, reviewed by Fatouros et al. 2008).
As activators for production and release of induced phytochemicals larval frass and egg deposition by herbivorous insects are known. When infested, the plants produce so called feeding induced (FIV) or oviposition induced plant volatiles (OVIV) that serve as synomones (reviewed by Fatouros et al. 2008). For egg parasitoids, the use of FIV for host location is only adaptive if all developmental stages of the host co-occur on the same plant (Lou et al. 2005). Therefore, in general, more reliable information on the presence of the needed stage for an egg parasitoid can be gained from OVIV.
Using OVIV to locate insect eggs may be especially advantageous due to a better detectability of such infochemicals. Compared to the general small biomass of insect eggs, plants are able to emit much higher amounts of reliable infochmicals. Besides, many phytophagous insects lay their eggs scattered and concealed (Averill & Prokopy 1981, 1982, Gabel & Thièry 1992, Thièry et al. 1995, Li & Ishikawa 2005, Doak et al. 2006, Broad et al. 2008, Korosi et al. 2008, Liu et al. 2008), which additionally decreases the chance of host location by using egg emanated volatiles. Thus, again OVIV provide reliable and more detectable information to the parasitoid.
Several plants are known to produce OVIV that guide egg parasitoids to their hosts. For example the field elm Ulmus minor (Ulmaceae) (Meiners & Hilker 2000, Hilker et al. 2002) or bean plants, Vicia faba and Phaseolus vulgaris (Fabaceae) (Colazza et al. 2004) are known to produce such volatiles. U. minor was shown to emit a volatile blend from its leaves induced by egg deposition of elm leaf beetles, Xanthogaleruca luteola (Coleoptera: Chrysomelidae) that attracts females of the egg parasitoid Oomyzus gallerucae (Hymenoptera: Eulophidae) (Wegener et al. 2001). O. gallerucae was attracted only to OVIV, neither to feeding damaged plants, pure host eggs nor to uninfested plant materials (Meiners & Hilker 1997).
-14- In terms of the present study, it was hypothesised that inflorescences and grapes of Vitis vinifera produce OVIV due to egg deposition of the two EGM species that attract T. cacoeciae. The potential of V. vinifera to react to infestation was demonstrated by the finding that feeding of spider mites, Tetranychus urticae (Acari: Tetranychidae), elicits production of volatile ketones, some non-polar terpenoids and methyl salicylate ester (Van den Boom et al. 2004). Methyl salicylate is known to attract predatory mites that attac the spider mites (Dicke et al. 1990). Additionally, it is known to function as synomone in Ulmus minor due to oviposition of elm leaf beetles (Wegener at al. 2001). Refering to this it is feasible that V. vinifera emits volatiles due to EGM egg deposition that guide T. cacoeciae during host location.
Concerning insect induced volatiles, until now only leaves were shown to process defence responses (Hilker & Meiners 2006). Likewise, for V. vinifera emissions of induced phytochemicals so long only are known from leaves. However, we suggested other plant organs to also be capable to react to insect eggs by producing effective attractants. If this could be demonstrated for grapes and inflorescences of V. vinifera, this would be the first evidence of OVIV that attract an egg parasitoid in this plant species. Additionally, it would be the first study that demonstrates the production of synomones from plant organs other than leaves.
1.1.3. Host location and enhancing background odours
Searching parasitoids are exposed to a huge amount of additional volatiles in addition to odours that directly or indirectly indicate the presence of a host when searching in open field. Such accessory volatiles were defined as background odours and were shown to affect the response of some parasitoids to host indicating infochemicals (Schröder and Hilker 2008). For example, Chrysonotomia ruforum (Hymenoptera: Eulophidae) needs volatiles emitted by twigs of the Scots pine, Pinus sylvestris, as essential background odours for host location. The egg parasitoid is attracted to oviposition induced (E)-β-farnesene, only if presented in context with uninfested pine twigs (Mumm & Hilker 2005). (E)-β-farnesene is emitted by P. sylvestris in high quantities due to infestations with eggs of the herbivorous sawfly, Diprion pini (Hymenoptera: Diprionidae). However, C. ruforum never shows attraction to (E)-β- farnesene on its own. In this context, the authors suggest that background odours emanated from the host plant are crucial to recognize or identify (E)-β-farnesene as host indicating -15- signal. Therefore, even if odours of undamaged plant materials or host induced volatiles are inneffective on their own, they may be important for successful host location in addition to volatiles directly linked to the hosts´ presence.
For T. cacoeciae nothing is known about influences of plant volatiles on host location. Therefore, behavioural responses of the species to host eggs in the presence of undamaged inflorescences of V. vinifera were tested in a flight cage. We hypothesised that T. cacoeciae shows a different behavioural response to host eggs when offered in combination with the volatile blend of their host plant.
1.1.4. Host location and host sex pheromones
Besides herbivorous induced plant volatiles, sex pheromones from calling host females were reported frequently to be used for host location (Zaki 1985, Noldus 1988, Noldus et al. 1990, 1991a, 1991b, Arakaki & Wakamura 2000, Boo & Yang 2000, Schöller & Prozell 2002). At first view, the use of cues emitted by the adult host stage seems unfeasible for host location of an egg parasitoid. However, it is possible that sex pheromones represent a "bridge in time" for egg parasitoids as it was shown for Trichogramma evanescens (Noldus et al. 1991b). In this case, sex pheromones of calling Mamestra brassicae moths (Lepidoptera: Noctuidae) were adsorbed onto the surface of cabbage leaves, Brassica oleracea (Brassicaceae) to such extent that they elicited behavioural responses in female T. evanescens up to 24 hours after ‘treatment’. Besides this, the use of sex pheromones may be advantageous if host females oviposit close to their calling sites, as it was shown for Diprion pini (Hymenoptera: Diprionidae) (Hilker et al. 2000). Actually, Chrysonotomyia ruforum (Hymenoptera: Eulophidae), egg parasitoids of D. pini, were arrested in the chamber provided with major components of host sex pheromone when tested in a four-chamber olfactometer.
Sex pheromones of host females also may be attached to eggs. For example, traces of sex pheromone from Euproctis taiwana and E. pseudoconspersa (Lepidoptera: Lymantriidae) were found adsorbed to, retained on and released from scale hairs that cover their egg masses (Arakaki & Wakamura 2000). Telenomus euproctidis (Hymenoptera: Scelionidae), an egg parasitoid of E. taiwana and E. pseudoconspersa uses the sex pheromone as a kairomone to locate host egg masses (Arakaki et al. 1996).
-16- In sustainable agriculture, sex pheromones are used for mating disruption techniques to control several Lepidopteran pest species (reviewed by Cardé & Minks 1995, Moschos et al. 2004). Thereby, the components of female moths’ sex pheromone essential for mating are applied with dispensers in the field. The synthetic pheromones evaporate from dispensers and permeate the atmosphere to such extend that sexual communication of pest species is disrupted. However, if parasitoids use sex pheromones as kairomones, the treatment with pheromone dispensers also might inhibit their host location. Niwa and Daterman (1989) investigated effects of releasing pheromones for mating disruption purposes on a parasitoid. In this study, the abundance of the ichneumonid wasps Glypta zozanae (Hymenoptera: Ichneumonidae), that parasitises lettuce tortricid Eucosma conterminana (Lepidoptera: Tortricidae), was reduced significantly due to treatment with the mating disruption technique. So long it is not known if Trichogramma cacoeciae uses EGM sex pheromones for host location. However, if the wasps are affected by main components of Lobesia botrana or Eupoecilia ambiguella sex pheromones, it also might be affected by the pheromone disruption technique. The technique is used to control the two species in almost all vineyards of German viticulture (Kast 2001, Louis & Schirra 2001). To answer these questions laboratory and field experiments were conducted.
1.1.5. Host location and egg volatiles
Airborne chemicals, directly emitted by egg masses, were shown to stimulate an intensive searching behaviour in egg parasitoids (Frenoy et al. 1992, Renou et al. 1992, Hofstetter & Raffa 1998, DeLury et al. 1999, Bai et al. 2004, Yong et al. 2007). However, infochemicals present on the egg surface act as kairomones only in close vicinity or in contac with the eggs (e.g. Strand & Vinson 1982, Nordlund et al. 1987, Bin et al. 1993, Meiners & Hilker 1997, Meiners et al. 1997, Conti et al. 2003). Thus they are more likely to play a role in host recognition then in host location. Additionally, Fatouros et al. (2008) expect that host eggs do not elicit chemicals serving as long-range attractants for egg parasitoids. However, eggs of Ostrinia nubilalis and M. brassicae were shown to elicit activity over some distance in Trichogramma brassicae tested in a linear olfactometer (Renou et al. 1992) and Yong et al. (2007) found egg masse volatiles of O. nubilalis to be attractive to T. ostrinae in a Y-tube olfactometer. Therefore, in the present study we investigated if T. cacoeciae uses egg emanated volatiles for host location by offering eggs on plastic foil in a Y-tube olfactometer.
-17- 1.1.6. Host location and moth wing scales
During oviposition females of various Lepidopteran species leave wing scales, which are attached to egg masses or the surface of their food plant at oviposition sites. This was shown for naturally occurring eggs of Heliothis assulta (Lepidoptera: Noctuidae) (Boo & Yang 2000) and for oviposition sites of great white cabbage butterflies, Pieris brassicae (Lepidoptera: Pieridae) (Fatouros et al. 2005b). Lewis et al. (1971) were the first who reported wing scales left by ovipositing Heliothis zea (Lepidoptera: Noctuidae) or Plodia interpunctella (Lepidoptera: Pyralidae) to arrest egg parasitoids of the species Trichogramma evanescens. Concerning host location, usage of moth scale volatiles for host location was reported by Yong et al. (2007). They demonstrated an innate positive response of female Trichogramma ostriniae to egg mass volatiles and to scale volatiles of its host, the European corn borer, Ostrinia nubilalis, in a Y-tube olfactometer. However, the attraction to egg mases might also be due to volatiles emanated from attached scales that were not removed from egg masses. Therefore, in the present study, if T. cacoeciae positively responds to egg masses of L. botrana and/or E. ambiguella, it has to be investigated if the involved infochemicals are emitted by eggs or by moth scales attached to them. This can for example be realised by identification and comparison of attractive chemicals from egg masses with scales to identified chemicals present on pure scales.
1.1.7. Identification of involved chemicals
Infochemicals used for host location by egg parasitoids can belong to different chemical classes (e.g. aldehydes, alcohols, esters and terpenes) and can function as kairomones or synomones (reviewed by Afsheen et al. 2008, reviewed by Fatouros et al. 2008). In closer vicinities to the host, various alkanes, fatty acids and amino acids are known to be attractive to different parasitoid species (reviewed by Rutledge 1996, Boo & Yang 2000, Reddy et al. 2002). Yet, it is not known which source of infochemicals is used by the egg parasitoid T. cacoeciae to locate the eggs of its two host species, L. botrana and E. ambiguella. Consequently, also the chemical structure of infochemicals so long remained unknown. In order to not only detect the source of infochemicals used for host location by T. cacoeciae, but also to identify the chemical structure of the attractants for T. cacoeciae, chemical analyses and olfactometer bioassays were performed.
-18- Oviposition induced plant volatiles (OVIV). OVIV are phytochemical substances that are induced by oviposition of herbivores, so long known only from plant leaves. As already mentioned, OVIV are able to serve as synomones for some egg parasitoid species. In a tritrophic system of herbivore, plant and parasitoid the identification of chemical structures of plant produced volatiles contributes to the understanding of insect-plant interactions (reviewed by Arab & Bento 2006). Wegener et al. (2001) identified more than 40 compounds (almost all terpenoid hydrocarbons) from the OVIV of the field elm Ulmus minor that is induced by egg deposition of the elm leaf beetle and is used for host location of Oomyzus gallerucae (Hymenoptera: Eulophidae). In another study, bean plants (Vicia faba and Phaseolus vulgaris) were shown to produce increased amounts of terpenoids (Colazza et al 2004) due to infestation with egg masses of Nezara viridula (Heteroptera: Pentatomidae). In highly attractive to Trissolcus basalis (Hymenoptera: Scelionidae) when tested in a Y-tube olfactometer. So long, almost all identified OVIV belong to the class of terpenoid hydrocarbons. As largest class of natural produced substances they are known to defend many species of plants against predators and competitors. Additionally, they are involved in insect communication, conveying messages to conspecifics and mutualists regarding the presence of food, mates and enemies (reviewed by Gershenzon & Dudareva 2007). Thus, it would not be surprising that if T. cacoeciae reacts to OVIV, this would be due to terpenoids.
Sex pheromones. Female sex pheromones are reported to attract egg parasitoid species in many cases. However, there are only few studies which identified involved chemical compounds. From the few studies identifying chemical structures of sex pheromones it is known that they comprises of alkanes and esters. For example, the major component of Helicoverpa asaulta (Lepidoptera: Noctuidae) sex pheromones was identified as (Z)-11- hexadecenyl acetate and that of Ostrinia furnacalis was (E)-12-tetradecenyl acetate. Both substances were shown to be attractive to female wasps of Trichogramma chilonis (Boo & Yang 2000). Likewise, the (Z)-11-hexadecenyl acetate from sex pheromones of Plutella xylostella (Lepidoptera: Plutellidae) was shown to elicit positive chemotaxis in T. chilonis, when tested in a Y-tube olfactometer (Reddy et al. 2002).
For L. botrana the main component of sex pheromone, (E,Z)-7,9-dodecadienylacetate was already identified 1973 (Roelofs et al. 1973) and for E. ambiguella Z-9-dodecenylacetate and a number of related compounds was shown to make up its sex pheromone (Arn et al. 1986). However, although the chemical components were identified long time ago, since the present study nothing was known about their impact on host location of T. cacoeciae.
-19- Egg volatiles. Concerning infochemicals emitted by host eggs that elicit searching behaviour in egg parasitoids, there are only three studies identifying chemical structures of attractive substances. Renou et al. (1992) reported palmitic acid and a blend of five saturated hydrocarbons (heneicosane, tricosane, pentacosane, heptacosane, nonacosane.) from the surface of Ostrinia nubilalis eggs, to be attractive to females of T. brassicae when tested in a linear olfactometer. Additionally, from egg masses of O. furnacalis the airborne sex pheromone component (E)-12-tetradecenylacetate was shown to stimulate intensive searching behaviour in T. ostriniae (Bai et al. 2004) and T. chilonis females (Boo & Yang 2000), when tested in a four-arm olfactometer. From eggs of L. botrana some substances are already identified including saturated free fatty acids and unsaturated acids (Thièry et al. 1992a, Gabel & Thièry 1996). However, again, nothing is known about their impact on the egg parasitoid T. cacoeciae.
Moth wing scales. Moth wing scales, are often attached to egg masses or oviposition sites of herbivorous insects. Furthermore, they were reported to be used for host location by some egg parasitoid species. Identifications of the chemical substances showed alkanes (Jones et al. 1973, Shu et al. 1990), aldehydes, acetones, and esters (DeLury et al. 1999) to be present on moth scales. Attraction of egg parasitoids towards an alkane (13,17-dimethyl nonatriacontane), identified from moth wing scales of its host O. nubilalis, was reported for females of Trichogramma nubilale when tested in a walking arena (Shu et al. 1990). Alkanes isolated from wing scales were also found to be attractive for T. evanescens, which showed positive orientation towards a blend of docosane, tricosane and tetracosane (Jones et al. 1973).
As mentioned before, egg parasitoids attracted to egg masses may use volatiles emitted from attached moth scales instead of egg emanated volatiles. This was for example demonstrated by DeLury et al. (1999). They found that egg masses of Cydia pomonella (Lepidoptera: Tortricidae) were attractive to egg parasitoids, Ascogaster quadridentata (Hymenoptera: Braconidae), when tested in a Y-tube olfactometer. Additionally, they showed the parasitoids to be attracted to scale volatile extracts. After identifying numerous compounds from scale extracts it was shown that scale emitted substances elicit responses in A. quadridentata antennae, when tested via coupled gas chromatographic- electroantennographic detection analysis. Therefore, the finding that egg masses of C. pomonella were attractive to A. quadridentata was due to attached moth scales. This again demonstrates the importance of identification of substances emitted by the hosts’ eggs and the moths’ scales to precisely identify the source of infochemicals.
-20- 1.1.8. Host location and host egg age
As reported in detail, host location mainly is mediated by olfactory active, volatile substances. Thereby, compositions of involved infochemicals and their chemical structure not only play a major role on their olfactory perceptibility, but also on their persistence. In general, usage of volatile and non-permanent substances for host location, probably result in a restricting time frame. For example Arakaki & Wakamura (2000) found that attraction of the egg parasitoid Telenomus euproctidi (Hymenoptera: Scelionidae) to eggs from Euproctis taiwana (Lepidoptera: Lymantriidae) exists only for short time. In this species, compounds from host sex pheromones adsorbed on egg attached moth scales were used for host location. 24 hours after oviposition, T. euproctidi no longer was able to locate eggs of E. taiwana. The authors demonstrated that this was due to evaporation of sex pheromones that decrease to an unattractive amount 24 hours after oviposition. Similar results were obtained by Fatouros et al. (2007) who reported Trichogramma evanescens to positively respond to OVIV from cabbage plants only up to 24 hours after egg deposition. From another study it is known that the ability of T. evanescens to locate leaves treated with sex pheromones of Mamestra brassicae (Lepidoptera: Noctuidae) declines 4 hours after treatment (Noldus et al. 1991b). Similar findings were made for additional egg parasitoid species, with response times ranging from three to four days after egg deposition (Colazza et al. 2004, Fatouros et al. 2005a, 2005c). Concerning these studies, it could be concluded that egg parasitoids’ host location is restricted to the availability of volatile infochemicals. Fatouros et al. (2005a) hypothesised that the time frame during which parasitoids are attracted to their host is adapted to that when it is best suitable for parasitisation. However, there are no studies combining the effect of host egg age on host location and host suitability. Therefore, the present study was the first that investigates the synchrony of the host location and the suitability for successful parasitisation of an eg parasitoid.
1.1.9. Host suitability and host egg age
Egg age was shown to have a great bearing on parasitisation rates and fecundity of many egg parasitoid species (reviewed by Pak 1986, Hintz & Andow 1990, Reznik & Umarova 1990, Reznik et al. 1997, Monje et al. 1999, Saour 2004, Makee 2005). In order to examine the performance of egg parasitoids on eggs of different ages, choice and no-choice experiments
-21- were performed in the laboratory (Smith 1996). In general, from such studies it is known that younger eggs were accepted more often for parasitisation than older ones. Additionally, the fitness of the parasitoids’ offspring mainly decreases with increasing host egg age. However, for few parasitoids acceptance and reproductive success were shown to be independent from host age and some studies even showed older eggs to be more suitable for parasitisation (reviewed by Pak 1986, Reznik et al. 1997, Monje et al. 1999, Godin & Boivin 2000, Makee 2005). This indicates that age dependency for parasitisation is species specific and it is not possible to make a general statement about correlations between host egg age and parasitisation success. Moreover, this aspect has to be investigated separately for each host- parasitoid system as it is preformed for T. cacoeciae and its two host species L. botrana and E. ambiguella in the present study.
1.1.10. Host suitability and host species
Besides hosts’ egg age, also the host species might influence the suitability of eggs for parasitisation. Eggs of different species may for example differ in their direct and indirect resistances and defence mechanisms like volume, chorion thickness, nutritional contents or antibody load against invasive parasitoids (Barrett & Schmidt 1991, Bai et al. 1992, Mansfield & Mills 2004, Makee 2005, Roriz et al. 2006). Therefore, egg parasitoids might show differences in their efficacy on and suitability of different host species. In general, parasitisation rates and fecundity of an egg parasitoid found in laboratory behavioural assays is uses as evidence for its potential to control a certain pest species (e.g. Honda et al. 1999, Romeis et al. 1999, Murthy et al. 2003, Kalyebi et al. 2005). Therefore, there are different methods established to estimate the productivity and effectiveness of several species, such as choice and non-choice experiments (e.g. Bigler 1989, 1994, Leppla & Fisher 1989, Hassan & Guo 1991, Losey & Calvin 1995, Liu & Smith 2000).
Wasps of the genus Trichogramma predominantly parasitise eggs of various Lepidoptera species. In general, most Trichogramma species are known to be generalists (e.g. Brotodjojo & Walter 2006). However, there are many studies referring to differences in the suitability of different host species (Zhang & Cossentine 1995, Monje et al. 1999, Brotodjojo & Walter 2006, Roriz et al. 2006). For example, when offering eggs of Plutella xylostella and Pseudoplusia includens (Lepidoptera: Noctuidae) simultaneously, the egg parasitoid T. pretiosum parasitises significantly more eggs of P. xylostella than eggs of P. includens (Pluke
-22- & Leibee 2006). Additionally, a study on efficacy of T. brassicae on eggs of two different host species, Anagasta kuehniella and Plodia interpunctella (Lepidoptera: Pyralidae), revealed higher rates in reproduction, population increase and intrinsic birth on eggs of one (A. kuehniella) over the other (P. interpunctella) (Iranipour et al. 2009). Differences in number of parasitised eggs, preimaginal developmental time and the number of offspring per female on eggs from different host species were demonstrated for Trichogramma cordubensis by comparing suitabilities of eggs form six Lepidopteran pest species (Roriz et al. 2006). All these studies demonstrate that even for generalist egg parasitoids there may be host species that are more suitable for reproduction then others.
In the present study the suitability of eggs of the two species of EGM, L. botrana and E. ambiguella, was investigated by first recording the number of parasitised eggs separately for each species. Subsequently, breeding success of T. cacoeciae was recorded to investigate the species impact on parasitoids reproduction.
1.1.11. Parasitoids and their natural environment
In natural environment, there are several factors which may influence the host searching and selection of an egg parasitoid. Besides impacts of the host plants, in particular, temperature and humidity were shown to influence parasitisation performance, developmental time and adult longevity of different egg parasitoid species (Murthy et al. 2003, Chabi-Olaye et al. 2004, Kalyebi et al. 2005, 2006). Therefore, local parasitoid strains/species were reported to be more effective in parasitising pest species from their own regions than were parasitoid strains/species originating from habitats with different abiotic conditions (Browning & Melton 1987, Glenn et al. 1997, Herz & Hassan 2006). Hegazi et al. (2007) for example found that in an olive plantation in Egypt indigenous strains of Trichogramma accomplished higher egg parasitisation of eggs of olive moths, Prays oleae, and jasmine moths, Palpita unionalis, than did commercial strains. This result is encouraging for an efficient use of Trichogramma spp. that already occurs in the locality of exertion. Additionally, it reveals the importance of confirmation of natural occurring parasitoid species and strains prior to artificial parasitoids introduction. Thus, in the present study prior to subsequently application studies the natural occurrence of Trichogramma species in the sampling site was performed.
-23- Besides temperature and humidity, pesticides or other agricultural measures could affect the parasitoids performance in the field. There are several studies demonstrating an impact of different insecticides, herbicides and fungicides on lifespan, development and parasitisation rates of different egg parasitoid species (e.g. Reddy & Manjunatha 2000, Takada et al.2001, Youssef et al. 2004, Bueno et al. 2008, Giolo et al. 2008). Vianna et al. (2009) reported negative effects of seven insecticides, regularly used in tomato production, on parasitisation rate and viability of Trichogramma pretiosum wasps. Although pesticides are mainly used in chemical pest management, substances applied in ecological and sustainable agriculture as well were shown to be harmful to various egg parasitoid species (Consoli et al. 1998, Hassan et al. 1998, Thompson et al. 2000, Manzoni et al. 2006). Notably, most studies on the impact of insecticides on performances of parasitoids were carried out in laboratories, where standardised methods were used. Therefore, there might be distinct differences in effects of chemicals on parasitisation examined in laboratories compared to field studies. For instance, sulphur was shown to be highly toxic and causing high mortality rates in egg parasitoids, Anagrus erythroneurae (Hymenoptera:Mymaridae), when tested in laboratory assays (Martinson et al. 2001). However, Jepsen et al. (2007a) demonstrated that there is no effect of sulphur on parasitoid performance when comparing oviposition success and lifetime reproductive success of A. erythroneurae wasps collected from sulphur-treated versus untreated vineyards. Furthermore, in a following study, they also found no changes in parasitisation rates due to sulphur treatment in the field (Jepsen et al. 2007b). Thus, to confirm a negative impact of substances diagnosed in laboratory assays, field experiments have to be conducted.
Since sulphur is used as fungicide/acaricide to control powdery mildew and spider mites in sustainable viticulture, it is clearly important to investigate effects of sulphur on T. cacoeciae. From laboratory assays it is already known that sulphur is harmful to the egg parasitoids (Gruetzmacher et al. 2004). However, it is possible that the negative effects will disappear in open field as it was shown for A. erythroneurae. Therefore, the performance of natural as well as artificial applied T. cacoeciae in areas with and without sulphur treatment was investigated.
-24- 1.2. Limitations on host access
In order to minimize the risk of predation and parasitisation and to reduce subsequent larval competition many phytophagous insects lay their eggs scattered and concealed (Averill and Prokopy 1981, 1982, Gabel & Thiery 1992, Thiery et al. 1995, Li & Ishikawa 2005, Doak et al. 2006, Broad et al. 2008, Korosi et al. 2008, Liu et al. 2008). For example the cabbage butterfly Pieris rapae (Lepidoptera: Pieridae), oviposits single eggs and scatter them sparsely in diverse vegetations (Root & Kareiva 1984). L. botrana use an oviposition deterring pheromone to prevent other moth from oviposition on the same site to minimize competition of the larvae (Gabel & Thiery 1991). Thus, it is not surprising that the moths deposit their eggs scattered and discrete on the infructescence of Vitis vinifera (Gabel & Thièry 1992) and same was observed for E. ambiguella (Rüdiger, personal observations). The influence of this behaviour on the parasitisation of T. cacoeciae was examined and discussed in the present study.
Besides this simple method of risk spreading, another strategy to avoid predation and parasitisation could be selection of oviposition sites unfavourable for host searching enemies. Enemy free spaces (EFS) were found for several species (Fox & Eisenbach 1992, Meiners & Obermaier 2004, Moon & Stiling 2006, Randlkofer et al. 2007, Obermaier et al. 2008). In this context, reduced parasitisation rates are often due to unfavourable vegetation structures that mechanically deter parasitoids to reach the hosts oviposition site. Actually, rates of parasitisation and predation differ between different host food plant species (Haynes & Butcher 1962, Bombosch 1966, reviewed by Weseloh 1976) or different parts of the same food plant (Weseloh 1974, reviewed by Weseloh 1976).
An other strategy of herbivores to gain EFS may be to oviposit on plants that are surrounded by non-host plants emitting inhibiting volatiles or plants doing so themselves (reviewed by Sheehan 1986, Andow & Prokrym 1990, Randlkofer et al. 2007). Concerning inhibiting volatiles, a distinction is drawn between compounds that have a repelling effect and those that have only a masking effect without repellent activity (reviewed by Schröder & Hilker 2008). Volatiles of the first category are for example phytochemicals emitted by pigeon pea pods. They were shown to have a deterring effect on the egg parasitoid Trichogramma chilonis, when tested in a four-arm olfactometer (Romeis et al. 1998). However, this is the only study mentioning a plant repellent for an egg parasitoid. More frequently such volatiles play a role during herbivores search for oviposition sites, acting as defence mechanism of plants (reviewed by Schröder & Hilker 2008). However, volatiles of
-25- the second category, with only masking effects, are more likely to be involved into plant-host- parasitoid interactions and will be considered in more detail.
1.2.1. Masking plant volatiles
Masking effects of plant volatiles are defined as an impact that neutralizes the behavioural response of an insect to a resource-indicating cue (reviewed by Schröder & Hilker 2008). This means that odours of resources that are attractive in the absence of the masking odour become unattractive when masking background odours are added. For example, this was shown for the egg parasitoid Oomyzus galerucivorus (Randlkofer et al. 2007). O. galerucivorus responds positively to solely presented odours of its hosts´ food plant but no more when combined with odours from non-host plants. Likewise, Monteith (1960) demonstrated that olfactory stimuli from non-host plants inhibit the host location of parasitic tachinid flies, Drino bohemica (Diptera: Tachinidae). The non-host odours masked the attractive odour of larvae from the sawfly Diprion hercyniae (Hymenoptera: Diprionidae). For host species, the use of such plants as oviposition site and/or feeding site for their progeny probably creates EFS. Therefore, such plants should be favoured over plants that do not grow in the vicinity of “masking” non-host plants. However, up to now there are only few studies reporting negative effects of plant volatiles on host location of parasitoids. However, if masking effects of plant volatiles are widespread, the success of inundative releases for biological control may be affected not only by the host-parasitoid system, but also by particular planting of host and non-host plants. This might be important for example when choosing plants as understorey in sustainable agriculture. Therefore, this aspect has to be taken into account prior to the application of a parasitoid for biological control.
-26- 2. Issues & Approaches
The two species of European grapevine moth (EGM), Lobesia botrana and Eupoecilia ambiguella (Lepidoptera: Tortricidae), are among the most important pests in vineyards of Southern and Western Europe (Bovey 1966, Fermaud & Le Menn 1989, Gabel & Röhrich 1995). The moths use up to three different seasonal plant organs of their food plant Vitis vinifera (Vitaceae) to deposit their eggs on: inflorescences, grapes of early stages and grapes of late stages (Stavridis & Savopoulou-Soultani 1998). After hatching, the larvae start feeding and thereby cause severe damage on their food plant (Barney et al. 2001). Additionally, larval feeding facilitates infections with grey mould fungi, Botrytis cyneria (Schruft, 1983). Thus, it is important to prevent moths’ larvae from feeding on crops and therefore to fight them at an early stage of development. This is done for example with chemicals like Pyrhetroid Tombstone Helios®, which is toxic for adult moths or biological insecticides like Microbial insecticide Biobit® HP (see handout developed by Cooper & Varela 2010). Unfortunately, some of the chemicals are also toxic for other insects, like Pyrhetroid Tombstone Helios® that also kills honey bees. Therefore, the use of chemical pesticides should be supplemented if not replaced with more biocompatible methods. One such method is the pheromone disruption technique, which is used in scope of sustainable agriculture (Krieg et al 1987, Feldhege et al. 1993). In viticulture this method currently is used to control both species of EGM on about 4000 ha of German vineyards (Louis & Schirra 2001). However, in some cases this method lacks in function for example due to high moth densities of more than 4000 females (Feldhege et al. 1993) or at high temperatures (Cardé & Minks 1995, Moschos et al. 2004). Therefore, investigations on additional strategies are necessary. One alternative method for biological control is the use of natural antagonists. It was shown that species, which originate from regions with same or similar climatic conditions as in the sample area, are very efficient in controlling several pest species (Browning & Melton 1987, Glenn et al. 1997, Herz & Hassan 2006).
Egg parasitoids of the genus Trichogramma are used frequently to control Lepidopteran pest species in many agricultural domains (Li 1994). They are cosmopolitan and there are more than 140 described species (Pinto & Stouthammer 1994). All wasps of the family Trichogrammatidae are obligatory egg parasitoids (Strand 1986) exploiting eggs of more than 400 host species (Silva 1999). According to Hoffmann & Frodsham (1993) no other chalcid wasp genus has been used as extensively for biological control of Lepidopteran pests all over the world.
-27- In the present study, the performance of Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) on eggs of the two species of EGM was investigated. The tiny wasp (only 1 mm long) is widely distributed over Germany (Walter 1982, Ibrahim 2004, Zimmermann 2004) and is known to parasitise eggs of different Tortricidae and the two species of EGM, L. botrana and E. ambiguella (Castaneda-Samayoa et al. 1993, Zimmermann 1997, 2004). Since field application experiments of T. cacoeciae resulted in unpredictable damage reduction rates (Castaneda 1990, Wührer et al. 1995, Zimmermann et al. 1997, Hommay et al. 2002, Ibrahim 2004) it remains unprofitable to be used as biological control agent in commercial viticulture. However, although there are some assumptions why T. cacoeciae fluctuates in their parasitisation rates - like infestation rates with EGM and strain differences (Ibrahim 2004) - it was hardly examined which factors affect the performance of the species. However, general investigations on host location and parasitisation processes as well as on factors influencing the aptitude of T. cacoeciae to locate and successfully parasitise eggs of EGM would lead to a better understanding of the present host-parasitoid system. Therefore, in the present study laboratory bioassays and field experiments where conducted.
During host location female wasps of T. cacoeciae first are faced a reliability- detectability problem (Vet & Dicke 1992): The most reliable indicators for the hosts’ presence are infochemicals deriving from their herbivorous host itself, namely eggs. However, the detectability of such sources often is low due to their small biomass relative to that of the hosts’ food plant. In order to overcome this problem several strategies were realised by different egg parasitoid species. Since it is unknown which strategy is pursued by T. cacoeciae the first approach of the present study was to investigate which cues are used by T. cacoeciae to locate eggs of its two host species L. botrana and E. ambiguella. In a series of dual choice experiments a variety of possible sources for infochemicals were examined for their attraction to the egg parasitoids: undamaged and infested inflorescences and grapes of V. vinifera, the main sex pheromone components of adult hosts as well as odours of egg masses. Additionally, chemical analyses of egg mass extracts, moth wing scales, polar and non-polar fractions of egg mass extracts and synthetic blends of different chemical substances were combined with dual choice behavioural assays in the Y-tube olfactometer. This was done in order to identify volatile chemical substances involved into host location of T. cacoeciae.
The condition of the host egg greatly influences the performance of many egg parasitoids. Additionally, there are differences in the suitability of eggs from different host species. In order to examine if the performance of T. cacoeciae is influenced by these factors, experiments on host location, parasitisation rates and breeding success of T. cacoeciae on
-28- eggs of different ages, of both species of EGM were conducted. Therefore, choice experiments were performed, using different aged eggs. Experiments were completed with studies of parasitisation rates and breeding success on different aged eggs. Additionally, flight cage experiments were conducted that combine both host location and parasitisation in one experimental setup and make it possible to compare the performance of T. cacoeciae for eggs of L. botrana and E. ambiguella. This was never done before for any egg parasitoid species.
In order to examine the potential of natural populations and artificial introduced T. cacoeciae wasps to parasitise eggs of EGM a series of field studies were performed. Therefore, parasitisation rates were observed and larval infestation was investigated in a system of sustainable viticulture. Additionally, the impact of pheromone disruption techniques and sulphur on the parasitoids’ efficiency to locate and parasite eggs of the two host species was examined. The existence of such influences was reported for several other egg parasitoid species.
Effects of volatiles emitted from plant organs of V. vinifera on host location of T. cacoeciae was examined by performing choice experiments via Y-tube olfactometer and flight cage experiments. Although negative effects were seldom reported for egg parasitoids this might help explain the ineffectiveness of T. cacoeciae to control EGM in viticulture.
-29- 3. Material & Methods
3.1. Model organisms
3.1.1. Trichogramma cacoeciae
10 µm
Figure 3-1: T. cacoeciae on a L. botrana egg.
Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) is an obligate egg parasitoid that parasitises eggs of several Lepidopteran species. The small size, short generation period and easy access to rearing hosts (Hoffmann et al. 1995) make the wasp a capable model organism for research on parasitisation. Additionally, the thelytokous reproduction of the species - almost exclusively female offspring are produced from unfertilised but diploid eggs - make it convenient for host searching experiments where only female wasps are needed.
The wasps of strain D90, used in the present study, originated from a commercial breeding on flour moths eggs, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), conducted by AMW-Nützlinge GmbH (Pfungstadt, Germany). Eggs of E. kuehniella are pasted on paper strips and offered to T. cacoeciae wasps. After parasitisation, the stripes are stored in glass tubes (7.5 cm long X 1.0 cm ID) and closed up with cotton wool.
Per week, 21 glass tubes with successive parasitised eggs were sent to the University of Freiburg per mail. Here they were stored at 25 °C (16:8 light-dark cycle) and ten to 12 days later first wasps hatched. Offspring were fed with a mixture of honey, water and gelatine (2:1:0.03) on filter paper.
For all experimental setups, only wasps of T. cacoeciae without any ovipositing experience (naïve) were used. Additionally, only wasps hatched 12 to 36 hours prior to experiments were used.
-30- Prior to behavioural assays, the wasps were tested for their motivation to oviposit. Therefore, approximately 30 naïve wasps of one glass tube were transferred to some eggs of Lobesia botrana in a Petri dish. Motivation was scored when at least one wasp displayed the three phases of parasitisation - drumming, tapping and ovipositor inserting - within the first 30 seconds (Klomp et al. 1980). If not, all wasps of the particular glass tube were discarded. For each experimental setup a fresh batch of motivated wasps was used, with wasps used for motivation tests excluded from further experiments.
3.1.2. Lobesia botrana & Eupoecilia ambiguella
European grapevine moths (EGM), Lobesia botrana and Eupoecilia ambiguella (Lepidoptera: Tortricidae), are severe pests in vineyards of Southern and Western Europe (Bovey 1966, Fermaud & Le Menn 1989, Gabel & Röhrich 1995). In order to successfully control these moths using egg-parasitoids, comprehensive knowledge on host-parasitoid interactions is required.
Moths and eggs used in the present study were reared at Staatliches Weinbauinstitut Freiburg (WBI). All breeding cycles started with approximately 200 males and females kept within a “breeding box”. The “breeding box” consists of a paper bag (Swirl® 10 L Bio-Müll Papierbeutel, Melitta® Haushaltsprodukte GmbH & Co. KG, Minden, Germany) tightened to a plastic bucket and closed up with plastic foil (Toppits ® Gefrierbeutel, Melitta® Haushaltsprodukte GmbH & Co. KG, Minden, Germany). After moistening the boxes with water, they were stored at 25 °C (16:8 light-dark cycle) and 30 % humidity for L. botrana respectively 50 % humidity for E. ambiguella. After mating, female moths deposited their eggs on the plastic foil, which they prefer as substrate for oviposition compared to paper. Foils were replaced when fully covered with eggs or every three days at the latest. Egg loaded foils were cut into stripes and stored in smaller “feeding boxes” (Polypropylen, 7 cm high X 7 cm ID), provided with artificial food (private formula). The boxes were stored at same conditions as “breeding boxes”. After hatching, moths’ larvae fed on artificial food until they start crawling up inside the boxes. At this time, stripes of corrugated paper were added to “feeding boxes”, where larvae pupated. Paper stripes were refreshed every three days and stripes with pupae were transferred to new “breeding boxes” for next breeding cycle.
-31- Sexing. For some experimental setups only female moths were required. Therefore, moths were sexed after mating. Adult moth of both EGM species can easily be distinguished due to differences in their abdominal tips. Female moths have a tuft of hairs on their abdominal tip (Figure 3-2), which lacks in male moths.
Figure 3-2: Abdominal tips of female grapevine moths. The two sexes of adult grapevine moths can be distinguished on the basis of abdominal tips. Female moths have a hairy tuft, whereas males only have a smooth plate.
3.2. Laboratory bioassays
3.2.1. General setup: Y-tube olfactometer
For two sided choice experiments a transparent Y-tube olfactometer (Plexiglas, stem-length 7 cm, arm-length 9 cm, stem-arms angle 120°, ID 2.5 cm) was used (Figure 3-3). It consisted of a Y shaped baseplate, six wallplates, three endplates with valve tips and a Y shaped lid that could be fixed to the baseplate via 15 screws. Additionally, there were two perforated – permeable – plates (Plexiglas with holes ø 1 mm) 4 cm from both ends of arms that separate two sample chambers inside the olfactometer. Both chambers could be provided with samples through slots in the lid that could be sealed with fitting caps. Another closable slot in stem was used to transfer T. cacoeciae wasps into the Y-tube. A constant air-stream of 50 ml/min was created using an aquarium pump (ASF THOMAS, WISA, Wuppertal, Germany) flowing air through the valves from both arms to stem. The air was cleaned by flowing through activated charcoal (FLUVAL® Carbon, Hagen, Holm, Germany) and humidified by bubbling through a bottle with distilled water. Intensity of air stream and equality for both arms was controlled regularly after every 5th trial and, if necessary, readjusted to 50 ml/min using a digital gas flowmeter.
-32- Prior to each trial the olfactometer was disassembled and all components were washed with 90 % ethanol followed by distilled water. After reassembling, the olfactometer was affiliated to the air stream and dried for at least one hour.
Figure 3-3: General setup of the Y-tube olfactometer. Air flows from the aquarium pump through glass tubes with charcoal and water and enters the Y-tube at both arms next to sample chambers. A fresh batch of naïve T. cacoeciae was placed to the stem for each experimental trial.
For each trial, the sample and, if required, corresponding sample was placed into one sample chamber. The arm provided with sample was alternated every trial. After ten minutes of exposure, about 100 naïve T. cacoeciae wasps were transferred to the stem of the olfactometer. Wasps that walked around had to decide left or right when reaching the branching point of olfactometer. It was considered that wasps came to a decision for one direction when a line drawn 2 cm behind the branching point was passed. The number of decisions for each arm was listed until 40 wasps decided for any direction. Returning females were counted only for the first time when passing a line. For each behavioural assay performed with the Y-tube olfactometer ten replicates with a new sample set and fresh T. cacoeciae were conducted.
The possibility of interference among individuals as it was observed by Salt (1937) for T. semblidis was evaluated for T. cacoeciae performing control trials: Decisions of wasps were observed with both arms of Y-tube olfactometer empty. Five control trials were
-33- performed prior to each behavioural assay and after every 5th trial. If interference was absent, wasps were expected to show equal distribution over both arms and very low aberrations. In case of interactions between individuals, either a directed orientation of the majority of wasps to one direction or at least a high aberration would result. Thus, the decision of each individual can be regarded as unaffected and self-sufficient, if equal distribution and low aberration results.
Statistical analysis. All results of Y-tube olfactometer assays were statistically analysed via Wilcoxon signed rank test with continuity correction (WSR). Graphical description is given in Figure 3-4.
Control trials were analysed separately for equal distribution using Karl Pearsons Chi- square test (KP). Additionally, the results were pooled and analysed via Wilcoxon signed rank test with continuity correction (WSR).
significance niveau P Q75 & Q25 Q25 & Q75
air air ns
air sample **
left Q75 = right Q25 right Q75 = left Q25
100 50 0 50 100 median number of decisions (%)
Figure 3-4: Graphical description of results gained from Y-tube olfactometer bioassays. Median percentage of decisions for either sample or antagonist is opposed in the graph (boxes). Error bars for upper box represent 75 (Q75) and 25 quantiles (Q25). Q75 from the left box is equal to Q25 on the right and vice versa. For simplification, only Q75 are displayed for each side (box below).
-34- 3.2.2. Host location and plant volatiles
Plant material. Two types of plant material from Vitis vinifera (Vitaceae) were used for experiments: inflorescences (flower buds) and immature grapes. Both plant organs are highly affected by either the first or second generation of both species of EGM (Broumas et al. 1989). Inflorescences from the grape variety Müller-Thurgau and grapes from the variety Pinot noir were obtained from vineyards of the Staatliches Weinbauinstitut (Freiburg i.Br., Germany). For experiments with 24 hours old uninfested plant material, inflorescences and grapes were cut off their stalks one day before usage and stored in a climate chamber at 25 °C and 30 % humidity.
Infested plant organs. Plant material covered with EGM eggs was gained by placing inflorescences and grapes into a “breeding box” of either L. botrana or E. ambiguella for 24 hours. For assays plant organs with 25 eggs (± 5) were used immediately after removing from boxes.
Behavioural assays. Attractiveness of plant material of V. vinifera towards wasps of T. cacoeciae was examined via choice experiments in the Y-tube olfactometer. Freshly cut and 24 hour old uninfested inflorescences and grapes were tested for their attractiveness against clean air. Inflorescences and grapes covered with eggs were tested against clean air, against uninfested plant material and against 24 hour old eggs deposited on plastic foil.
3.2.3. Host location and the source of infochemicals
Main component of sex pheromones. Attraction of T. cacoeciae towards main components of sex pheromones from E. ambiguella ((Z)-9-dodecenylacetate) [226.36 g/mol] and L. botrana ((E,Z)-7,9-dodecadienylacetate) [224.34 g/mol] was investigated via choice experiments in the Y-tube olfactometer. Both substances, (Z)-9-dodecenylacetate and (E,Z)- 7,9-dodecadienylacetate, were of artificial origin. They were gained from dispensers (RAK® 1+2 SD) that are commercially available from BASF (Limburgerhof, Germany). Dispensers liquid contained both main components of sex pheromones. The liquid was extracted with an injection needle, transferred to 1.5 ml glass tubes and diluted with pentane. Pheromone concentrations were measured via GC-analyses (see 3.2.4. Identification of involved chemicals).
-35- For behavioural assays two different concentrations were adjusted. One extract contained compounds at concentrations of 0.0532 たg/µl of (Z)-9-dodecenylacetat and 0.0269 たg/µl of (E,Z)-7,9-dodecadienylacetat. The other extract contained much lower concentrations, with 0.004378 µg/µl of (Z)-9-dodecenylacetat and 0.001063 µg/µl of (E,Z)-7,9-dodecadienylacetat. Both extracts were tested for their attractiveness towards against pure pentane in the Y-tube olfactometer.
Host eggs of Lobesia botrana. Attractiveness of 24 hour old L. botrana egg masses was investigated via Y-tube olfactometer assays. For methods, materials and a detailed description of conducted bioassays see “3.2.5. Host location and host egg age”.
Egg mass extracts. In order to gain egg mass extracts, mated moths of L. botrana were anaesthetized using carbon dioxide. Afterwards, they were sexed and 25 females were captured within a sterile glass tube (9 cm long X 2 cm ID) closed up with cotton wool. In total 30 tubes were provided with female moths and were stored at room temperature for two days. Afterwards, tubes were stored at -20 °C to kill the moths and remove them from tubes. For each extract about 2000 (1800-2200) egg masses from up to seven glass tubes were extracted. Thereby, to not extract chemicals from the eggs inside, each glass tube was repeatedly rinsed with 1 ml of dichloromethane for only 30 seconds in total. All extracts were stored at -20 °C when not in use and were adjusted to room temperature before use. Attraction of T. cacoeciae towards extracts compared to pure dichloromethane was investigated via Y-tube olfactometer bioassays.
Fractions of egg mass extracts. Prior to fractionation the dichloromethane egg mass extracts were carefully dried to dry-point using a gentle stream of nitrogen. Instantly the extract was reconstituted with pure pentane. For fractionation, the reconstituted extract was transferred to a silica gel column (SiOH, CHROMABOND, 500 mg, Macherey-Nagel, Düren, Germany), which was previously conditioned with pentane. Instantly the column was first eluted with 400 µl of pentane (first fraction) and subsequently with 400 µl of dichloromethane (second fraction). Both fractions were examined for their attractiveness to T. cacoeciae compared to their corresponding solvent via Y-tube olfactometer bioassays.
Blends of egg associated substances. Attractiveness of substances, which have been identified from egg mass extracts of L. botrana was investigated using artificial blends as samples for Y-tube olfactometer assays. Only volatile substances with C-19 or less were used for examinations. However, not all substances could be tested due to their unavailability. Thereby, the attractiveness of some substances identified from extracts remained unclear. All
-36- available substances were diluted with dichloromethane and concentrations were adjusted to identified amounts via GC-analyses (see 3.2.4. Identification of involved chemicals). They were tested for their attractiveness compared to pure dichloromethane.
Behavioural assays: All samples tested for their attractiveness via Y-tube olfactometer are summarised in Table 3-1. In order to avoid chemical reactions between solvents and Plexiglas of the olfactometer, glass cover slips were placed into sample chambers. For each trial 10 µl of extract, blend or solvent was applied to a piece of filter paper and placed to the cover slip. After each trial a new glass cover slip and new pieces of filter paper were used. Prior to every behavioural assay solvent was allowed to evaporate for two minutes.
Table 3-1: List of samples used for choice experiments via Y-tube olfactometer and/or GC-MS analyses. The first and second fraction were of L. botrana egg mass extracts. Aldehydes, ketones, fatty acids and esters were constituent parts of the attractive polar fraction.
Sample Solvent sex pheromones high concentration pentane sex pheromones low concentration pentane eggs of L. botrana and E. ambiguella extracts of 24 hour old eggs (L. botrana) dichloromethane first fraction (polar) pentane second fraction (non- polar) dichloromethane aldehydes dichloromethane ketones dichloromethane fatty acids dichloromethane esters (methyl- and isopropylesters) dichloromethane three main aldehydes dichloromethane nonanal dichloromethane moth wing scales dichloromethane
-37- 3.2.4. Identification of involved chemicals
GC-MS-analysis. Identification and quantification of the different extracts, fractions and blends listed in Table 3-1 were performed using gas chromatography coupled with mass spectrometry (GC-MS).
For analyses a Hewlett Packard HP 6890 Series GC System directly coupled to a Hewlett Packard HP 5973 Mass Selective Detector (Agilent Technologies, Böblingen, Germany) was used. The system was controlled by a computer with HP Enhanced ChemStation G1701AA software (version A.03.00). The gas chromatograph was equipped with a fused silica capillary column coated with DB-5 (30m x 0.25 mm ID; 0.25 µm thickness; J & W Scientific, Folsom, CA, USA) using helium as carrier gas at a flow of 1 ml/min. Electron impact mass spectra (EI-MS) were recorded with an ionization voltage of 70 eV and a source temperature of 230°C. Samples were injected in splitless mode using an auto sampler. Solvent delay was set at 60 sec and injector port was set at 250°C. Oven temperature was programmed to increase from 60°C at 5°C/min to a final temperature of 300°C which was held for 10 min.
Prior to analyses all extracts and fractions were reduced to a volume of 30 µl using a gentle flow of nitrogen. The artificial blends were reduced to a volume of 1 ml to measure concentrations of substances prior to concentration adjustment.
Egg mass extracts. The substances comprised within extracts of 24 hours old L. botrana egg masses were tentatively identified by investigating their equivalent chain length. Moth wing scales. In order to differentiate between egg emanated infochemicals and those from moth scales, the chemical constitution of the latter was examined apart from eggs. Moth scales can not be removed from the inside of glass tubes without destroying or altering the eggs surface. Therefore, moth wing scales were extracted separately. Therefore, 20 freshly mated, CO2 anaesthetized females of L. botrana were transferred to sterile glass tubes (9 cm long X 2 cm ID) closed up with cotton wool. Immediately the tubes were stored for 15 min at -20 °C to kill the female moths. Tubes with dead moths were slowly shaken for one minute to get scales attached to glass. Afterwards, moths were removed and extraction was performed by repeatedly rinsing each glass tube with 1 ml of dichloromethane for 30 seconds in total. The extract was removed from the tubes and stored at -20 °C prior to use for GC-MS analyses. After identifying attractive substances from egg mass extracts, moth wing scale extracts were scanned for these substances.
-38- Fractionation of egg mass extracts. Since only the polar fraction of egg mass extracts was attractive to T. cacoeciae, volatile substances (up to C-19) of this fraction were identified more precisely. Their structures were determined by equivalent chain length and the use of MS-database Wiley275 software (John Wiley and Sons Inc. N.Y, USA). Substances comprised within the non-polar fraction were identified tentatively.
Blends of egg associated substances. From analyses of polar fractions of egg mass extract it arose that comprised chemicals belong to one of four substance classes: aldehydes, fatty acids, esters (methyl- & isopropylesters) or ketones. For behaviour assays, four synthetic blends consisting of available substances of these substance classes were produced (Table 3- 2). The relative abundance of substances within each blend was evaluated via GC analysis and was adjusted to the relative amount of the particular substance within the polar fraction. Besides the four blends a mixture of three aldehydes, heptanal, nonanal and dodecanal was produced. The three aldehydes were dissolved in dichloromethane and their concentrations were adjusted to concentrations measured in the polar fraction. Additionally, solely nonanal, a prominent aldehyde within the polar fraction was dissolved in dichloromethane for Y-tube olfactometer tests.
Table 3-2: Composition of available egg associated substances. For each artificial blend of substance classes four to six substances have been available.
Chain Esters lenght Aldehydes Ketones Fatty acids Methylesters Isopropylesters C5 3-Pentanone C6 3-Hexanone C7 Heptanal Heptanoic acid
C8 3-Octanone Caprylic acid C9 Nonanal Nonanoic acid C10 Capric acid C12 Dodecanal Lauric acid C13 Tridecanal 2-Tridecanone Tridecanoic acid C17 Methyl palmitate Isopropyl myristate C18 Methyl heptadecanoate
-39- 3.2.5. Host location and host egg age
T. cacoeciae wasps were tested for their attraction towards egg masses of different ages from both species of EGM, deposited on plastic foil via Y-tube olfactometer assays.
Egg masses were gained from breeding of L. botrana and E. ambiguella. For 24 hour old eggs, a “breeding box” was overlaid with fresh plastic foil where moths oviposit for one day. After 24 hours, foils were removed and either immediately used for behavioural assays or stored at room temperature for another 24 or 48 hours to receive 48 and 72 hours old eggs.
Behavioural assays. For each species (L. botrana and E. ambiguella) and egg age (24 hours, 48 hours, 72 hours), a piece of 1 cm² plastic foil covered with approximately 25 (± 7) eggs and scales was used as sample and tested for its attractiveness against empty plastic foil in the Y-tube olfactometer.
3.2.6. Host suitability and host egg age
Suitability of eggs of three different age groups - young, medium aged and old- for T. cacoeciae was tested. Eggs of both species of EGM ranging from 24 hours to 144 hours in age, with an interval of 24 hours, were used. Youngest eggs were up to 24 hours old and oldest eggs were 144 hours old. After that time larvae of EGM begin to hatch from eggs (personal observations).
Examined eggs were grouped into three categories: young (24 hours and 48 hours old), medium aged (72 hours and 96 hours old) and old eggs (120 hours and 144 hours old). This was made due to statistical requirements and to classify the type of relationship between host age and parasitisation rate as it was described by Pak (1986) and Godin & Boivin (2000).
Behavioural assays. For each trial a Petri dish (Ø 9.5 cm) was provided with a 1.5 cm² piece of plastic foil covered with on average 60 (32 to 91) eggs of either L. botrana or E. ambiguella. The plastic foil was fixed to the dish with water droplets. Prior to each trial the exact number of eggs was counted. 25 (± 5) wasps of T. cacoeciae were transferred to each Petri dish, which afterwards was closed up and sealed with adhesive tape (Scotch® Magic 19mm, Cergy Pontoise Cedex, France). The dishes were stored at 25 °C (light-dark cycle: 16:8 hours) for five days. Suitability of EGM eggs as good resource for T. cacoeciae depends
-40- on both, the potential of eggs to be parasitised and proportion of emerging wasps from parasitised eggs.
Parasitised host eggs turned black during third instar of parasitoids larvae (three to four days after parasitism) as a result of dark melanin granules deposited on the inner surface of egg chorion (reviewed by Vinson & Iwansch 1980). The number of blackened eggs for each species and egg age compared to the number of offered eggs was regarded as parasitisation rate.
After five days parasitoids were carefully removed and dishes with eggs were stored at 25 °C until offspring has hatched and died. The number of emerged wasps compared to the number of parasitised eggs was regarded as breeding success of T. cacoeciae. For each egg age and species ten replicates were performed.
Statistical analysis. Differences in parasitisation rates and breeding success comparing the three egg age categories were analysed using Kruskal-Wallis tests (KW) which were, if significant, followed by Tukey-Kramer post-hoc procedures (TKph).
3.2.7. Host suitability and host species
Host location and parasitisation performance of naïve T. cacoeciae wasps on egg masses of L. botrana in comparison to egg masses of E. Ambiguella were examined via flight cage experiments. Experiments were conducted in the presence and absence of inflorescences of V. vinifera.
Flight cages were cuboid shaped aluminium frames (30cm x 30cm x 60cm) covered with densely woven organza gaze (mesh size Ø 0.5 mm). The upper side of the cage was used as door that could be closed tightly using set screws on two sides of the door. At a height of 20 cm within the cage, a wire was bonded diagonal from one side to the other where samples could be fixed.
Samples. 24 hours old egg masses on plastic foil were gained from the breeding of the two EGM species at Staatliches Weinbauinstitut Freiburg (WBI) as described before. Inflorescences from V. vinifera of the grape variety Müller-Thurgau were cut from field plants right prior to conducted experiments.
-41- Behavioural assays. For trials without plant material ten pieces of plastic foil, each with a single 24 hour old egg and scales of either L. botrana or E. ambiguella, were adjusted to the wire within the flight cage using green and yellow miniature pegs. The colour of the pegs was alternated between the two EGM species after every trial.
For behavioural assays with plant material, six pieces of plastic foil with a single 24 hour old egg of L. botrana were adjusted to the wire within the flight cage as described above. Additionally, four inflorescences were added to the wire situated between every two pieces of plastic foil. As control in another cage, six pieces of plastic foil with a single egg of L. botrana were adjusted with positions with inflorescences in experimental setup remained empty.
After assembling the cage with eggs and respectively inflorescences, 400 to 500 naïve females of T. cacoeciae were released into the cages. After one day, the pieces of plastic foil were carefully removed and transferred to Petri dishes. Thereby, all wasps were removed from foils before closing up the dishes and store them at 25 °C for at least five days. The number of parasitised eggs compared to the total number of exposed eggs was taken as parasitisation rate. For each trial fresh pieces of plastic foil with single eggs on them and a fresh bunch of inflorescences were used. For comparison of parasitisation rates on eggs of the two EGM species, ten replicates were conducted. When analysing influences of the inflorescences presence, 16 replicates were conducted.
Statistical analysis. Potential impact of coloured pegs was tested by comparing the number of parasitised eggs for each of the two colours using Wilcoxon rank sum test (WRS). Differences in parasitisation rates of T. cacoeciae related to the two host species of grapevine moth and the presence respectively absence of inflorescences were analysed via two Wilcoxon rank sum tests (WRS).
-42- 3.3. Field bioassays
3.3.1. Natural occurring Trichogramma species
Sampling site. Investigations on parasitoids in their natural environment were carried out on vineyards of the Staatliches Weinbauinstitut Freiburg (WBI) (Figure 3-5). The sampling site is characterised by moderate temperatures and rainfall (mean annual temperature: +10.4°C; average annual rainfall: 930 mm). WBI vineyards are all cultivated as sustainable agriculture. All examined areas were generally treated with fungicides Stroby® WG, Polyram® WG and Folpan® WDG.
Possible influences of the mating disruption technique on host location of T. cacoeciae were examined. Therefore, investigations were performed in two areas with the grape variety Müller-Thurgau: WBI+ and WBI-. Both areas were treated similarly with pesticides. Additionally, area WBI+ was treated with the pheromone mating disruption technique, whereas area WBI- remained untreated.
47° 58' 41.05"N, N WBI 20 m 7° 50' 4.49"E S
Area WBI +
Area WBI -
Vinyard for sulphur treatment and artificial application experiments
Figure 3-5: Aerial view of the sampling sites. The areas were all situated near the Staatliches Weinbauinstitut (WBI) in Freiburg (47° 58' 45" N, 7° 49' 56" E). The two area types area WBI+ and area WBI- were used to examine natural occurrence of Trichogramma species. The area used for sulphur experiments is located next to the two other sampling areas.
-43- Baits. In order to lure natural occurring Trichogramma species and to investigate their natural parasitisation rates, stripes of binding wire that were covered with 24 hour old eggs of either L. botrana or E. ambiguella were used as baits. In order to obtain baits, 6 cm long stripes of green plastic coated binding wire (Westfalia ®) were exposed to moths within “breeding boxes” for 24 hours. To induce moths to oviposit on wire stripes boxes were covered with paper towels instead of plastic foil. After one day stripes were removed from boxes and the number of eggs on each bait was recorded. In total 1200 wires were used, with a mean number of 120 eggs (± 20).
Baiting. Within each of the two areas WBI+ and WBI-, ten stocks of grapevines with a maximum distance of 1 m to each other were chosen. For each species 20 baits were used, which means two baits with eggs of L. botrana and two baits with eggs of E. ambiguella for ten stocks. The baits were attached to the top of inflorescences in spring, later in the year to imatured grapes. After every seven days of exposure, baits were removed from the field and a new batch of baits was deployed. Baiting experiments were carried out for 15 weeks from end of May to end of August in 2006.
Parasitisation rates and species classification. The number of remaining eggs on removed baits was recorded. Subsequently, wires were separated in glass tubes (7.5 cm long X 1.0 cm ID) and stored at 25 °C and 30 % humidity until parasitisation became visible. The number of parasitised baits compared to the number of exposed baits was regarded as parasitisation rate. For species classification the baits with parasitised eggs were sent to Carlos Monje from the University of Hohenheim, Germany (Institute for phytomedicine).
Statistical analysis. In order to analyse differences in the natural parasitisation rates for eggs of the two EGM species and between treated (WBI+) - with pheromone mating disruption technique - and untreated areas (WBI-), Kruskal Wallis test (KW) was performed, followed by Tukey-Kramer post hoc procedure (TKph) if significant.
-44- 3.3.2. Influence of sulphur treatment and artificial application
Sampling site and timing of experiments. Investigations on the influence of sulphur treatment and artificial application were carried out within a Müller-Thurgau vineyard of 360 m², located between the two areas WBI+ and WBI- at the area of WBI Freiburg (Figure 3-5). The vinyard used for the present experimental setup was not treated with the pheromone mating disruption technique.
Beginning of experiments depended on the degree-day assessment. All day temperatures > 0 °C are summarised from January 1st on. Experiments started, when the sum of day temperatures reaches 1100 day degrees. This is the critical sum of temperatures for L. botrana to start to fly. In 2008 this sum was reached at begin of May. Examinations were continued until end of July.
Baits. Green binding wires covered with 24 hour old eggs of L. botrana were used as baits. They were gained as described above. Prior to every trial, the number of eggs on every bait was estimated to have baits with similar numbers for each trial.
Area treatments. Within the vineyard 32 examination areas were distributed over twelve rows (Figure 3-6). Between every row with examination areas and the next, one row remained untreated. Every second row was treated with sulphur (S+) whereas the others remained untreated (S-). For treatment with sulphur (Thiovit® Jet, Syngenta Agro GmbH, Maintal, Germany) a tunnel applicator (SCHACHTNER Fahrzeug- und Gerätetechnik, Ludwigsburg-Oßweil, Germany) was used. First application was done at May 20th and was repeated at six dates: June, 6th, June, 16th, June, 25th, July, 8th, July, 15th, July, 22th.
Within each examination row a maximum of three examination areas were distributed uniformly over the row. Each area consisted of five contiguous plants (No 1 to No 5) that were numbered from northern edge. Between one area and the next there were at least 10 m with several plants. Every second area was provided with artificial applicated T. cacoeciae populations (T+). This was done by adding one Trichocard® (AMW-Nützlinge GmbH, Pfungstadt, Germany ) to plant No 3 in the middle of each area. Trichocard®s consist of covered board containing 2000-3000 parasitised eggs of Ephestia kuehniella from which T. cacoeciae emerges successively during one week. Eggs on cards were covered with coated board protecting them against predation. The cards were replaced every seven days during entire experimental time.
-45-
Figure 3-6: Areal partition of the sampling site and area treatments. To investigate influences of sulphur (S) and artificial induced egg parasitoids (T) on the parasitisation rate of T. cacoeciae, a vineyard of 360 m² was partitioned into 32 areas. One untreated row separated two types of rows with sulphur treatment (S+) or without (S-). Within treated rows a maximum of three areas provided with Trichocards® (T+) or without (T-) were distributed. Each area consisted of five plants which were numbered from the northern edge of the vineyard (box).
-46- Finally, there were 16 areas treated with sulphur (S+) and 16 areas without sulphur treatment (S-). Additionally, eight of sulphur treated areas and eight untreated areas were provided with Trichocards® (T+). Hence, there were eight areas of each of the four concept types A (S+, T+), B (S+, T-), C (S-, T+) and D (S-,T-) (Table 3-3).
Parasitisation rates. Within each of the 32 areas, one bait (wire with eggs) was applied to each of the five plants No 1 to No 5. Baits were attached to grapevines stems (before blossom), inflorescences (bloom of V. vinifera) or verdant grapes (later in year) and were exchanged every two days by a fresh batch of baits. Removed wires were stored separately in glass tubes (7.5 cm long X 1.0 cm ID) at 25 °C and 30 % humidity until parasitisation became visible. The number of baits with parasitised eggs compared to the total number of applied baits was regarded as parasitisation rate.
Table 3-3: Concept types for area treatments. There where eight areas of each of the four concept types within the sampling area. Areas differed in their treatment with sulphur and Trichocards® which was either present (+) or absent (-).
Differences in larval infestation. In order to investigate influences of sulphur treatment and artificial application on the performance of T. cacoeciae, differences in infestation rates with L. botrana larvae were examined and compared between the four area types.
To create an artificially high rate of infestation, bags of gauze (10 cm x 15 cm) were provided with two mated females of L. botrana and were attached to an inflorescence of plant N° 3. This was done for all 32 areas at three different dates from late May to early June (27.05.08, 29.05.08, 03.06.08). After 24 hours, bags were removed from plants and the particular inflorescence was marked using a coloured binding wire. For each treatment, another inflorescence was used. After third treatment, a comparable number of eggs were deposited within each area.
-47- Two weeks after the last artificial infestation, plants N° 1 to N° 5 of each area were rated for infestation by searching for spun inflorescences. Infested inflorescences were cut of the plants and collected separately in plastic bags. In laboratory, the number of larvae from each area were counted and recorded. After five more days, rating was repeated. The median number of larvae recorded from each area type (A, B, C, D) was regarded as load of infestation.
Statistical analysis. Differences in parasitisation rates and in load of infestation between the four different area types (A, B, C and D), were statistically analysed using two- way analysis on ranks (Friedman-test (F)), followed by Wilcoxon-Wilcox posthoc test for within-sample comparison (WWph).
-48- 4. Results
4.1. Statistical declarations
For all statistical analyses (Table 4-1) P values < 0.05 were regarded as significant. All data analyses were performed using the free software R (version 2.4.0). For Wilcoxon signed rank tests with continuity correction R only incorporates results with differences. Therefore the sample size (N) reduces to the number of samples with differences (Nd). Additionally, for V the program R always gives only rank sums of the first column, which results in high and low V to be significant.
Table 4-1: Glossary of performed statistical analyses.
Bioassay Statistical test Shortcut Control trials for Y-tube olfactometer Karl Pearsons Chi-square test KP bioassays Y-tube olfactometer bioassays Wilcoxon signed rank test with continuity WSR correction Differences in parasitisation rates and Kruskal-Wallis test KW breeding success Post hoc for KW Tukey-Kramer post hoc procedure TKph
Flight cage experiments Wilcoxon rank sum test WRS
Comparison of parasitisation rates of the Friedman-test F four area types A,B,C,D Post hoc for F Wilcoxon-Wilcox test for within-sample WWph comparison
4.2. Laboratory bioassays
4.2.1. Control trials: Y-tube olfactometer
In order to evaluate possible external influences on decisions of Trichogramma cacoeciae wasps, control trials were performed.
All control trials resulted in equal distribution of T. cacoeciae wasps over the two arms of the Y-tube olfactometer (each KP: Chi² < 3.6, df = 1, P > 0.05). The number of aberrations of all control trials was low (Mean squared error = 1.52). The numbers of decisions for each arm ranged from 17 to 23 with a mean number of 20.5 wasps deciding for the right and 19.5 females deciding for the left arm. The median number of decisions during control trials and the statistically robustness is displayed graphically within each figure pooled for all control trials per assay.
-49- 4.2.2. Host location and plant volatiles
The attractiveness of uninfested and infested plant material of Vitis vinifera for wasps of T. cacoeciae was examined via choice experiments using a Y-tube olfactometer.
Pure plant material. Y-tube olfactometer bioassays showed that freshly cut grapes did not attract the parasitic wasps (WSR; V = 14.5, P = 0.67, Nd = 8) and same was true for 24 hours old pure grapes (V = 25.0, P = 0.84, Nd = 10). Similarly, fresh inflorescences did not attract T. cacoeciae wasps (V = 16.0, P = 0.83, Nd = 8) and also 24 hours old inflorescences did not lead to preference for one arm (V = 23.0, P = 0.15, Nd = 7) (Figure 4-1).
P
air air ns
fresh grapes air ns
24 hour old grapes air ns
fresh inflorescence air ns
24 hour old air inflorescence ns
100 50 0 50 100 median number of decisions (%)
Figure 4-1: Reactions of T. cacoeciae towards plant volatiles. Median percentage of decisions of naïve T. cacoeciae wasps in the Y-tube olfactometer for different reproductive plant organs of Vitis vinifera. Error bars show Q75 quantile. ns = not significant. (WSR; control trials – air/air: N = 60, all others: N = 10).
-50- Infested grapes. T. cacoeciae wasps were attracted by Lobesia botrana eggs deposited on grapes (Figure 4-2 a) when compared with clean air (WSR; V = 39.5, P < 0.05, Nd = 9). Same result was gained for comparisons of grapes with L. botrana eggs and grapes without eggs (V
= 53.5, P < 0.01, Nd = 10). However, wasps did not differentiate between L. botrana eggs deposited on grapes and L. botrana eggs deposited on plastic foil (V = 14.5, P = 0.67, Nd = 8).
In the olfactometer bioassays with eggs of Eupoecilia ambiguella deposited on grapes (Figure 4-2 b), female wasps were attracted to them when tested against clean air (V = 55.0, P
< 0.01, Nd = 10) as well as against uninfested grapes (V = 40.5, P < 0.05, Nd = 9). Again, comparisons of eggs on grapes to eggs on plastic foil revealed an equal distribution of T. cacoeciae wasps over the two arms of the olfactometer (V = 12.0, P = 0.83, Nd = 6).
Infested inflorescences. When analysing the results for L. botrana eggs deposited on inflorescences (Figure 4-3 a) T. cacoeciae wasps showed equal distribution when tested against clean air (WSR; V = 9.0, P = 0.83, Nd = 6). Similarly, when compared to uninfested inflorescences, wasps decision was not directed to one of the two sides (V = 22.0, P = 1.00,
Nd = 9). Additionally, when L. botrana eggs on plastic foil were tested against eggs deposited on inflorescences, T. cacoeciae did not decide more often for one of the two arms (V = 24.0, P
= 0.76, Nd = 10).
For inflorescences with eggs of E. ambiguella, again there was no attraction of T. cacoeciae to inflorescences with eggs (Figure 4-3 b); Neither when tested against air (V =
25.5, P = 0.76, Nd = 9) nor when tested against pure plant material (V = 41.5, P = 0.17, Nd = 10). Same was true when testing inflorescences with eggs of E. ambiguella against eggs on plastic foil (V = 20.5, P = 0.86, Nd = 9).
-51- a) P
air air ns
grape with eggs air *
grape with eggs grape **
grape with eggs foil with eggs ns
100 50 0 50 100 median number of decisions (%)
b) P
air air ns
grape with eggs air **
grape with eggs grape *
grape with eggs foil with eggs ns
100 50 0 50 100 median number of decisions (%)
Figure 4-2: Reactions of T. cacoeciae towards infested grapes. Median percentage of decisions of naïve T. cacoeciae wasps in the Y-tube olfactometer for grapes with eggs of (a) L. botrana and (b) E. ambiguella, compared to different samples. Error bars show Q75 quantiles. * = P < 0.05; ** = P < 0.01; ns = not significant (WSR; control trials – air/air: N = 45, for all others: N = 10). -52- a) P
air air ns
inflorescence with air eggs ns
inflorescence with inflorescence ns eggs
inflorescence with foil with eggs ns eggs
100 50 0 50 100 median number of decisions (%)
b) P
air air ns
inflorescence with air eggs ns
inflorescence with ns eggs inflorescence
inflorescence with ns eggs foil with eggs
100 50 0 50 100 median number of decisions (%)
Figure 4-3: Reactions of T. cacoeciae towards infested inflorescences. Median percentage of decisions of naïve T. cacoeciae wasps in the Y-tube olfactometer for inflorescences with eggs of (a) L. botrana and (b) E. ambiguella, compared to different samples. Error bars show Q75 quantiles. ns = not significant. (WSR; control trials: N = 45, for all others: N = 10).
-53- 4.2.3. Host location and the source of infochemicals
Main component of sex pheromones. Attraction of T. cacoeciae towards main components of E. ambiguella ((Z)-9-dodecenylacetate) and L. botrana ((E,Z)-7,9-dodecadienylacetate) sex pheromones was investigated via choice experiments in the Y-tube olfactometer. For 10 µl of high concentrated blend, with 0.0532 たg/µl of (Z)-9-dodecenylacetat [23 mM] and 0.0269 たg/µl of (E,Z)-7,9-dodecadienylacetat [1.2 mM], T. cacoeciae showed no positive chemotaxis (WSR; V = 11.0, P = 0.20, Nd = 9). The same was true for 10 µl of low concentrated blend with 0.004378 µg/µl of (Z)-9-dodecenylacetat [0.193 mM] and 0.001063 µg/µl of (E,Z)-7,9-dodecadienylacetat [47 µM], which did not elicit attraction in T. cacoeciae
(V = 12.5, P = 0.26, Nd = 9).
Egg mass extracts and fractionated extracts. Attractiveness of L. botrana egg mass extracts and their polar and non-polar fractions for T. cacoeciae was investigated via choice experiments with the Y-tube olfactometer.
T. cacoeciae preferred L. botrana egg mass extract over pure solvent (WSR; V = 0.0, P < 0.01, Nd = 10) (Figure 4-4). Similarly, the parasitoids preferred the polar fraction (V = 0.0,
P < 0.01, Nd = 10) over pure solvent. However, the non-polar fraction was not attractive to T. cacoeciae when tested against pure solvent (V = 21.5, P = 0.95, Nd = 9).
Egg associated substances. Attraction of T. cacoeciae towards synthetic semiochemicals comprised within the polar fraction of L. botrana egg mass extracts was investigated via choice experiments with the Y-tube olfactometer. Blends of compounds from four different substance classes were investigated for their attractiveness towards T. cacoeciae wasps (Figure 4-5 a). Blends of aldehydes, ketones, fatty acids and esters comprise substances identified from egg mass extracts that were available in laboratory (substances in bold Table 4-2).
Blends of components from substance classes, ketones (WSR; V = 21.0, P = 0.54, Nd =
10), fatty acids (V = 7.5, P = 0.15, Nd = 8) and esters (V = 8.5, P = 0.11, Nd = 9) were not attractive to T. cacoeciae. Only mixtures of aldehydes elicited positive chemotaxis in T. cacoeciae wasps ( V = 0.0, P < 0.01, Nd = 10).
-54- P
air air ns
pentane egg extract **
dichloro- polar fraction methane **
pentane non-polar fraction ns
100 50 0 50 100 median percentage of decisions (%)
Figure 4-4: Reactions of T. cacoeciae towards egg mass extracts and fractions. Median percentage of decisions of naïve T. cacoeciae wasps in the Y-tube olfactometer for L. botrana egg mass extract and its two fractions. Error bars show Q75 quantiles. ns = not significant; ** = P < 0.01 (WSR; control trials: N = 45, for all others: N = 10)
From GC-MS analyses of polar fractions it was known that two aldehydes, octanal and nonanal, occurred in remarkable high amounts. Octanal was not available for testing its attractiveness to T. cacoeciae wasps in the Y-tube olfactometer. Therefore, we tested a blend of the three next abundant aldehydes: heptanal, nonanal and dodecanal. T. cacoeciae wasps were attracted towards this blend of three aldehydes (WSR; V = 3.0, P < 0.05, Nd = 9) (Figure 4-5 b). However, when examined the attractiveness of the second prominent aldehyde, nonanal, on its own, T. cacoeciae did not show a positive chemotaxis (V = 16.0, P = 0.83, Nd = 8).
-55- a) P
air air ns
dichloromethane aldehydes **
dichloromethane fatty acids ns
dichloromethane ketones ns
dichloromethane esters ns
100 50 0 50 100 median percentage of decisions (%)
b) P
air air ns
heptanal, nonanal, dichloromethane dodecanal **
dichloromethane nonanal ns
100 50 0 50 100 median percentage of decisions (%)
Figure 4-5: Reaction of T. cacoeciae towards egg associated substances. Median percentage of decisions of naïve T. cacoeciae wasps in the Y-tube olfactometer to (a) blends of synthetic chemicals from four different substance classes and (b) three aldehydes and nonanal alone. All tested substances were identified from polar fractions of L. botrana egg mass extracts. Error bars show Q75 quantiles. ns = not significant; ** = P < 0.01 (WSR; control trials: (a) N = 60, (b) N = 30, for all others: N = 10).
-56- 4.2.4. Identification of involved chemicals
Egg mass extracts. GC-MS analyses of L. botrana egg mass extracts revealed several chemical substances. Tentative identifications of the volatile substances, from C1 to C19, showed different hydrocarbons, aldehydes, ketones, fatty acids and esters to be present within the extracts of L. botrana egg masses (Figure 4-6).
56 * Methyl hexanoate Methyl 4 Fatty acids: A Heptanoic acid B Caprylic acid C Capric acid Ester: ゴ undecanoate O Methyl IV C 3 III O 2 a Aldehydes: I Octanal II Nonanal Dodecanal III IV Tridecanal Ketones: a Undecanone B 1 A A A 10.00 15.00 20.00 25.00 10.00 15.00 20.00 25.00 II II II I Hexadecen Alcanes: 1 Dodecane 2 Tridecane 3 Tetradecane 4 Pentadecane 5 Hexadecane 6 Heptadecane Alcenes: * 0 500000 400000 300000 200000 100000 Time--> Time-->
Relative Abundance Relative
Figure 4-6: Part of a chromatogram of an L. botrana egg mass extract. Tentatively identified chemical substances are marked and listed next to the graph. Only volatile substances up to C19 were identified.
-57- Polar and non-polar fractions of egg mass extracts. In order to identify substances involved in host location of T. cacoeciae, possible candidates were narrowed down by fractionating the egg mass extracts. Fractionation with pentane and dichloromethane resulted in two fractions, separating the non-polar hydrocarbons from polar lipids.
Substance identification. Only polar fractions were attractive to T. cacoeciae wasps when tested in the Y-tube olfactometer. Tentative identification of substances comprising the attractive polar fraction (Figure 4-7) revealed several potential semiochemicals that could be partitioned into four substance classes (Table 4-2). The substances were tested for their attractiveness to T. cacoeciae wasps.
Moth wing scales. Moth wing scales were investigated via GC-MS for the presence of aldehydes. GC-MS data revealed that aldehydes do not occur in extracts of moth wing scales.
Table 4-2: Identified substances from polar fraction of L. botrana egg mass extracts. Substances in bold were available to mix blends for behavioural assays. Position of the keto-group in ketones was not elucidated.
Chain Esters lenght Aldehydes Ketones Fatty acids Methylesters Isopropylesters C5 Pentanone C6 Hexanone Methyl hexanoate C7 Heptanal Heptanoic acid C8 Octanal Octanone Caprylic acid C9 Nonanal Nonanoic acid C10 Decanal Capric acid C11 Undecanal Undecanone Methyl undecanoate C12 Dodecanal Lauric acid C13 Tridecanal Tridecanone Tridecanoic acid C16 Methyl hexadecanoate C17 Isopropyl myristate C19 Isopropyl palmitate
-58-
III Dodecanal
16.00
15.00
A Dodecanoic acid
O undecanoate Methyl
Undecanon Undecanon Undecanone Undecanone b b 2-Undecanon b b 2-Undecanon 14.00
a 3-
9.00 II Nonanal
8.00 Methyl hexanoate Methyl
ゴ
7.00 I Octanal
6.00
0 500000 400000 300000 200000 100000 Time-->
bundance
A
Figure 4-7: Details of polar fraction of L. botrana egg mass extract. The chromatogram show some identified substances from polar fractions that are listed in detail in Table 4-1.
-59- 4.2.5. Host location and host egg age
Eggs of different ages from L. botrana and E. ambiguella deposited on plastic foil were tested for their attractiveness to T. cacoeciae wasps via choice experiment in the Y-tube olfactometer.
Twenty-four hours old eggs of both species elicited attraction in T. cacoeciae wasps.
They react positively to eggs of L. botrana (WSR; V = 55.0, P < 0.01, Nd = 10) and eggs of E. ambiguella (V = 42.0, P < 0.05, Nd = 9). Likewise, for 48 hours old eggs T. cacoeciae showed a positive chemotaxis towards eggs of L. botrana (V = 54.0, P < 0.01, Nd = 10) and eggs of E. ambiguella (V = 49.0, P < 0.05, Nd = 10). However, 72 hours old eggs did not elicit a positive reaction in T. cacoeciae wasps. Neither eggs of L. botrana (V = 18.5, P = 0.67, Nd = 9) nor those from E. ambiguella (V = 27.0, P = 1.00, Nd = 10) were attractive (Figure 4-8).
-60- a) P
air air ns
72 hours old eggs air ns
48 hours old eggs air **
24 hours old eggs air **
100 50 0 50 100 median percentage of decisions (%)
b) P
air air ns
72 hours old eggs air ns
48 hours old eggs air *
24 hours old eggs air *
100 50 0 50 100 median percentage of decisions (%)
Figure 4-8: Reactions of T. cacoeciae towards eggs of different ages. Median percentage of decisions of naïve T. cacoeciae wasps in the Y-tube olfactometer for eggs of different ages of (a) L. botrana and (b) E. ambiguella when tested against clean air. Error bars show Q75 quantiles. ** = P < 0.01, * = P < 0.05, ns = not significant (WSR; control trials: N = 45, for all others: N = 10).
-61- 4.2.6. Host suitability and host egg age
Eggs of three different age groups (young, medium aged and old) from both species of EGM were tested for their suitability as host for T. cacoeciae.
Examining parasitisation rates for eggs of L. botrana a difference between eggs of the three age groups arose (KW; H = 20.19, df = 2, P < 0.001) (Figure 4-9 a). Differences occurred between young (24 to 48 hours old) and old eggs (120 to 144 hours old) (TKph; q = 5.42, K = 3, P < 0.001) as well as between medium aged (62 to 96 hours old) and old eggs (q = 5.58, K = 3, P < 0.001), but not between young and medium aged eggs (q = 0.16, K = 3, P > 0.05). Young eggs were parasitised to a percentage four times bigger than old ones. The average percentage of parasitised young eggs was 34 %, 40 % for medium aged eggs and 8 % for old eggs. There were two dishes with young eggs, one dish with medium aged eggs and seven dishes with old eggs where no eggs were parasitised.
The proportion of parasitised eggs from E. ambiguella did not differ between the three age categories (H = 5.59, df = 2, P = 0.06) (Figure 4-9 b). However, there was a trend for higher parasitisation rates on young compared to old eggs (q = 3.05, K = 3, P = 0.1). There were two dishes with young eggs, five with medium aged eggs and two with old eggs where no parasitisation took place.
There was a difference in breeding success of T. cacoeciae in regard to parasitised eggs of L. botrana between the three age categories (H = 7.30, df = 2, P < 0.05) (Figure 4-10 a). Higher proportion of offspring hatched from parasitised medium aged eggs (N = 19) compared to old ones (N = 13) (q = 3.81, K = 3, P < 0.05). However, there was no difference in the breeding success of T. cacoeciae between young eggs (N = 18) compared to old eggs (q = 0.50, K = 3, P > 0.05). And there was no difference in the breeding success between young and medium aged eggs (q = 0.50, K = 3, P > 0.05). On average offspring hatched from 42 % of young, 55 % of medium aged and 29 % of old parasitised eggs.
Breeding success of T. cacoeciae on parasitised eggs of E. ambiguella did not differ between eggs of the three age categories (KW; H = 0.42, df = 2, P > 0.05) (Figure 4-10 b). The mean percentage of hatching offspring from parasitised E. ambiguella eggs was 42 % for young, 40 % for medium aged and 40 % for old eggs.
-62- a) 100
90 ***
80 ns *** 70
60
50
40
30 % parasitised eggs
20
10
0 young medium aged old egg age categories
b) 100
90
80
70
60
50
40
aaiie eggs % parasitised 30
20
10
0 young medium aged old egg age categories
Figure 4-9: Parasitisation rates on different aged eggs. Median percentage of parasitisation rates of T. cacoeciae on eggs of (a) L. botrana and (b) E. ambiguella referring to young (24 to 48 hours old), medium aged (72 to 96 hours) and old (120 to 144 hours) eggs. Error bars show Q25 and Q75 quantiles. *** = P < 0.001; ns = not significant (KW, if significant followed by TKph; all N = 20).
-63- a) 100 ns 90 ns 80 * 70 N = 18 60
50 N = 13
40
% hatched% offspring 30 N = 19 20
10
0 young medium aged old egg age categories
b) 100
90
80
70 N = 18 N = 15 60 N = 18 50
40
% hatched offspring 30
20
10
0 young medium aged old egg age categories
Fig. 4-10: Breeding success on different aged eggs Median percentage of hatched offspring from parasitised eggs of (a) L. botrana and (b) E. ambiguella referring to young (24 to 48 hours old), medium aged (72 to 96 hours) and old eggs (120 to 144 hours). Error bars show Q25 and Q75 quantiles. * = P < 0.05; ns = not significant (KW, if significant followed by TKph).
-64- 4.2.7. Host suitability and host species
Eggs on plastic foil. Host location and parasitisation performance of naïve T. cacoeciae wasps on eggs of L. botrana in comparison to eggs of E. ambiguella was investigated using a flight cage. It resulted that T. cacoeciae parasitised more eggs of L. botrana than E. ambiguella (WRS; W = 76.5, P < 0.05, N1 = N2 = 10) (Figure 4-11 a). The color of pegs, used to attach foils with eggs to the flight cages, did not influence wasps parasitisation rates (W = 49.5, P > 0.999, N = 10).
Influence of background odours from inflorescences. Presence of inflorescences from V. vinifera in flight cage experiments did not influence parasitisation rates of T. cacoeciae (W = 91.5, P = 0.16, N = 16) (Figure 4-11 b).
a) b) 100 100
90 90 * ns 80 80
70 70
60 60
50 50
40 40 % parasitised eggs % % parasitised eggs 30 30
20 20
10 10
0 0 L. botrana E. ambiguella Inflorescences present Inflorescences absent
Figure 4-11: Parasitisation rates during flight cage experiments. Displayed are the median percentages of parasitised eggs when presented to T. cacoeciae in a flight cage. (a) Comparison of parasitisation rates for eggs of the two EGM species. * = P < 0.05 (WRS; N = 10) (b) Comparison of parasitised eggs from L. botrana in the presence or absence of inflorescences from V. vinifera. ns = not significant (WRS; N = 16). Error bars show Q25 and Q75 quantiles.
-65- 4.3. Field bioassays
4.3.1. Natural occurring Trichogramma species
Natural diversity of Trichogramma species. During the whole experimental time, 1200 baits with in total 144304 eggs were used. From these 51 % were lost after one week of exposure. 70709 eggs remained on binding wire stripes for analyses. Proportion of parasitised stripes and eggs from both area types WBI+ – treated with pheromone mating distribution technique – and WBI- are shown in Table 4-3. Parasitised baits were found in five of 15 weeks.
Table 4-3: Proportion of parasitised eggs. Total numbers of wires with parasitised eggs are given for the two EGM species within the two different area types WBI+ and WBI-.
Parasitised baits (eggs) total L. botrana E. ambiguella 64 (2192) 40 (1549) 24 (643) WBI+ WBI- WBI+ WBI- 23 (1044) 17 (505) 19 (591) 5 (52)
Species classification revealed that all baited individuals were of the genus Trichogramma, determined as T. cacoeciae and T. evanescence (Table 4-4).
Parasitisation rates. To investigate natural parasitisation rates and influences of pheromone disruption techniques on Trichogramma species the number of parasitised baits with eggs of L. botrana was compared to parasitised baits with eggs of E. ambiguella.
There was a significant difference in the number of parasitised baits between the two moths species and two area types (KW; H = 8.62, df = 2, P < 0.05). However, only the number of parasitised baits between E. ambiguella eggs in area WBI- compared to L. botrana eggs in are WBI+ differed (Table 4-5). Therefore, treatment with pheromone disruption technique alone did not affect the parasitisation rate of EGM eggs, due to the finding that there was no difference in the number of parasitised baits between the two area types.
-66- Table 4-4: Species classification of baited parasitoids. All parasitoids that hatched from offered eggs were of genus Trichogramma and were determined as T. evanescens or T. cacoeciae.
Area type WBI + WBI- Baited Baited Host species parasitoid Host species parasitoid Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. evanescens Lobesia botrana T. cacoeciae Lobesia botrana T. evanescens Lobesia botrana T. cacoeciae Eupoecilia ambiguella T. cacoeciae Lobesia botrana T. cacoeciae Eupoecilia ambiguella T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. cacoeciae Lobesia botrana T. evanescens Eupoecilia ambiguella T. cacoeciae Eupoecilia ambiguella T. evanescens
Table 4-5: Results of Tukey-Kramer post hoc analysis. Comparison of the number of parasitised baits with eggs of L. botrana (Lb) or E. ambiguella (Ea) in areas treated with pheromone distribution technique (WBI+) and without (WBI-). P gives the grade of significance. ns = not significant, * = P < 0.05.
Treatment 1 Treatment 2 P WBI+ Lb WBI+ Ea n.s. WBI+ Lb WBI- Lb n.s.
WBI+ Lb WBI- Ea * WBI- Lb WBI- Ea n.s. WBI- Lb WBI+ Ea n.s. WBI+ Ea WBI- Ea n.s.
-67- 4.3.2. Influence of sulphur treatment and artificial application
In order to examine influences of sulphur treatment and artificial application of T. cacoeciae on parasitisation rates in the field, eggs of L. botrana on binding wire were used as baits and analysed for parasitisation.
There were four different area types (Table 3-3, page 47) treated with either both, sulphur and artificial applicated T. cacoeciae on Trichocards® (A), with only one of these treatments (B with sulphur, C with Trichocards®) or with none (D). In total to area type 1040 baits were exposed. Baits with parasitised eggs were found in all four area types: A -> 31 (3.0 %), B -> 3 (0.3 %), C -> 44 (4.2 %) and D -> 4 (0.4 %) (Figure 4-12). The number of parasitised baits differed between the four area types (F; F-Chi² = 41.05, df = 3, P < 0.001). Differences were due to applications with Trichocards®, with higher parasitisation rates in area A (T+) and C (T+) compared to B (T-) and D (T-) (WWph ; both P < 0.05). Parasitisation rates did not differ due to sulphur treatment (WWph; P > 0.05).
Differences in larval infestation rates. In order to investigate damage reduction through artificial introduced T. cacoeciae wasps and sulphur influences, infestation loads in all four area types were compared to each other. The number of collected larvae displays the performance of parasitoids and predators that are about to destroy the exposed eggs.
From statistical analyses, a significant level of under 10 % resulted for differences between the different area types (F-Chi² = 7.30, df = 3, P = 0.06). In order to increase the statistical power a two-way ANOVA was performed (Figure 4-13). From these analyses it resulted that sulphur treatment did not lead to different infestations rates with L. botrana larvae (two-way ANOVA; F = 1.341, df = 1, P = 0.26). However, application of Trichocards® did (F = 7.724, df = 1, P < 0.05). Most larvae were collected from sulphur treated areas without Trichocard® application (B) and lowest number was found in areas with applicated Trichocards® (A, C) irrespective of sulphur treatment.
-68- 4 a
3 a
2 b
parasitised baits 1
b S- 0 T+ S+
T- Figure 4-12: Parasitisation rates in the field due to different area treatments. Plots display the median number (bars) of parasitised egg stripes and interquartile range (box) according to area treatment: S+ = with sulphur, S- without sulphur, T+ with applicated Trichocards®, T- without applicated Trichocards®. Different letters are consistent with significant differences (P < 0.05) (WWph; N = 8).
6
b a 5
4 a a 3
2
1 S-
0 S+ T+
T- Figure 4-13: Rating for larval infestation in differentialy treated areas. Mean number of detected larvae according to treatments of the sampling areas with sulphur (S+; A and B) or without (S-; C and D), respective with applicated Trichocards® (T+; A and C) or without (T-; B and D). Different letters are consistent with significant differences (P < 0.05) (two-way ANOVA, N = 8). Error bars show standard deviation.
-69- 5. Discussion
Egg parasitoids of the family Trichogrammatidae are used frequently to control Lepidopteran pest species (Li 1994). In European viticulture, two of the most important pest species are European grapevine moths (EGM), Lobesia botrana and Eupoecilia ambiguella (Bovey 1966, Fermaud & Le Menn 1989, Gabel & Röhrich 1995). Promising candidates for biological control of these pest species are chalcid wasps of the species Trichogramma cacoeciae. The egg parasitoids are known to parasitise eggs of different Tortricidae including eggs of the two species of EGM (Castaneda-Samayoa et al. 1993, Zimmermann 1997, 2004). However, since field application experiments resulted in unpredictable damage reduction rates (Castaneda 1990, Wührer et al. 1995, Zimmermann et al. 1997, Hommay et al. 2002, Ibrahim 2004) wasps of T. cacoeciae remain unprofitable to be used as biological control agent in commercial viticulture. As stated already, there are some speculations why T. cacoeciae fails in effectively controlling EGM (Ibrahim 2004). However, there are only few studies that investigated factors influencing the general ability of the species to locate and successfully parasitise eggs of EGM. Therefore, in the present study laboratory bioassays and field experiments were conducted to give information about host location of T. cacoeciae.
In a series of dual choice experiments influences of the host food plant, Vitis vinifera, and of main components of moth sex pheromones on host location of T. cacoeciae were examined. It resulted that neither volatiles from uninfested nor from infested grapes or inflorescences elicit positive chemotaxis in parasitoids. Likewise, main components of sex pheromones did not attract T. cacoeciae when tested in Y-tube olfactometer bioassays. However, odours from egg masses elicited positive chemotaxis in the parasitoids and this was found for eggs of both EGM species. From another series of choice experiments it resulted that egg mass extracts and the polar fraction of egg mass extracts were attractive to T. cacoeciae. Dual choice experiments with synthetic blends of chemicals, identified from polar fractions, revealed only aldehydes to be attractive. All other substance classes did not elicit positive reactions. Finally, performed chemical analyses of moth wing scales resulted that aldehydes were not present within their extracts. Therefore, attractive aldehydes were emitted from EGM eggs and can be detected by T. cacoeciae wasps over some distance.
Influences of egg age on host location, parasitisation rates and breeding success of T. cacoeciae were examined in a series of behavioural assays. Y-tube olfactometer experiments showed that eggs can be localised up to an age of 48 hours after oviposition. However, parasitisation was possible even for some 144 hours old eggs of L. botrana and E. ambiguella.
-70- Highest parasitisation rates were gained for young (24 hours to 48 hours old) and medium aged eggs (72 hours to 96 hours) of L. botrana. The experiments were completed with flight cage experiments that combined investigations on host location and parasitisation rates. It resulted that T. cacoeciae gains higher parasitisation rates on eggs of L. botrana compared to eggs of E. ambiguella. Together with the finding that eggs of L. botrana were parasitised the most this result might give a first hind on species preference of T. cacoeciae.
Natural parasitisation rates under different conditions were measured. Thereby, influences of the pheromone disruption technique, sulphur treatment and artificial applicated T. cacoeciae on parasitisation rates were investigated. It resulted, that sulphur treatment did not affect the wasps’ performance. However, sex pheromones influenced the parasitisation rates in one special case; in the area with pheromone treatment more baits of L. botrana were parasitised compared to baits of E. Ambiguella in the area without pheromone treatment. Applicated T. cacoeciae wasps resulted in higher parasitisation rates compared to areas without application. However, parasitisation rates were low, with a maximum of 4.2 % baits with parasitised eggs in one area.
5.1. Influence of plant volatiles on host location
The ability of parasitic Hymenoptera to locate hosts over long distances by using volatile chemicals, associated with their host, has been well documented (Vinson 1976, Price et al. 1980, Nordlund et al. 1983, Van Alphen & Vet 1986). Egg parasitoids have to detect the immobile stage of their host species that is very small and probably emit only small amounts of infochemicals compared to those of other sources within the environment. Therefore, egg parasitoids probably are faced a detectability problem when searching for host eggs. In order to solve the problem, usage of additional infochemicals, available besides egg odours, was ascertained for several parasitoid species, e.g. plant odours (reviewed by Tumlinson et al. 1992, reviewed by Fatouros et al. 2008). Volatiles from uninfested plants are assumed to be used for host habitat location or as cue leading to arrestment within a habitat. Actually, Reddy et al. (2002) found females of Trichogramma chilonis to be attracted to volatiles from their hosts’ food plant, Brassica oleracea (Brassicaceae): The parasitoids showed positive chemotaxis towards volatile synthetic blends of substances identified from uninfested cabbage leaves, tested in a Y-tube olfactometer. Some other Trichogramma species are known to be arrested by odours of uninfested plant materials (Romeis et al. 1997, Boo & Yang 1998,
-71- Romeis et al. 2005). Therefore, we suggested a possible effect of plant volatiles on T. cacoeciae as well. However, the present study revealed T. cacoeciae neither to be attracted to uninfested grapes nor to uninfested inflorescences. Given this result, we concluded that naïve T. cacoeciae wasps do not use volatiles from uninfested host plant material for host location.
Concerning plant semiochemicals, host induced volatiles seem to be a good source of infochemicals, being detectable and reliable. Such volatiles can be produced due to feeding (FIV) or oviposition (OVIV) of herbivores. However, for host location of T. cacoeciae, only OVIV were considered as possible induced infochemicals. First T. cacoeciae is an obligate egg parasitoid and second, both species of EGM have distinct generations with only one instar occurring on the host plant at one time (Zimmermann 1997, Barney et al. 1999). Therefore, FIV that displays larval presence remain useless for T. cacoeciae locating EGM eggs.
OVIV from a variety of plants were shown to attract several parasitoid species (Meiners & Hilker 2000, Hilker et al. 2002, Colazza et al. 2004, Fatouros et al. 2005a, 2005c, Hilker & Meiners 2006, reviewed by Fatouros et al. 2008). Experiments conducted in the present study referred to olfactometer bioassays on egg parasitoids like Oomyzus gallerucae (Hymenoptera: Eulophidae). This species was shown to respond to OVIV from elm leaves with present and removed eggs of elm leaf beetles, Pyrrhalta luteola (Coleoptera: Chrysomelidae) (Meiners & Hilker 1997). Likewise, Trichogramma brassicae was reported to be arrested to cabbage leaves with freshly laid and one day old eggs (Fatouros et al. 2005a). In the present study, dual choice experiments with a Y-tube olfactometer were conducted. Inflorescences and grapes of Vitis vinifera (Vitaceae) with eggs deposited one day prior to assays were offered to naïve T. cacoeciae in a Y-tube olfactometer. Parasitoids were not able to experience OVIV beforehand. It resulted that eggs deposited on grapes were highly attractive to T. cacoeciae when compared to clean air and uninfested grapes. However, grapes with eggs were not more attractive as was plastic foil with eggs. Therefore, we concluded that OVIV from eggs deposited on grapes up to 24 hours prior to experiments, if existent at all, are not involved in the host location of naïve T. cacoeciae.
However, there are few studies noting experience with host linked infochemicals to be necessary for successful host location of in some parasitoid species (Bjorksten & Hoffmann 1995, Nurindah & Gordh 1999, Peri et al., 2006). For example, Chrysonotomyia ruforum (Hymenoptera: Eulophidae), an egg parasitoid of Diprion pini (Hymenoptera: Diprionidae) on Pinus sylvestris (Penaceae), has to associate OVIV with host presence and food supply in order to learn and to respond to them (Schröder et al. 2008). Additionlly, the period of time
-72- since eggs were deposited on plants was mentioned to have an impact on host location of several species. For example, a positive response of C. ruforum was found only to OVIV from pine twigs with 3-days-old eggs (Schröder et al. 2008). OVIV from twigs with other induction times (one, two, four days) were not attractive. The authors suggested an unusual learning strategy of the parasitoid as reason for the limited period for host location in this species. However, another reason for the finding that twigs with one- and two-days-old eggs did not attract parasitoids might be that the plant needs some time to start OVIV production. Indeed, there are some studies demonstrating that attractiveness of odours from infested leaves persists only after three and up to five days after egg deposition (Colazza et al. 2004, Fatouros et al. 2005a, 2005c). This aspect remains disregarded in the present study. Therefore, it is possible that T. cacoeciae uses OVIV to locate eggs deposited for more than 24 hours. Additionally, after gaining experience with this source of infochemicals the species may be attracted more to eggs on grapes compared to eggs on plastic foil. This has to be investigated in further studies.
In contrast to results gained for grapes with eggs of EGM, naïve wasps of T. cacoeciae were never attracted to infested inflorescences when used as sample in the Y-tube olfactometer. Wasps were never able to locate eggs deposited on inflorescences, neither when tested against air nor when tested against uninfested inflorescences. Even eggs deposited on plastic foil did not elicit a positive chemotaxis in T. cacoeciae when presented simultaneously with the “unattractive” infested inflorescences. This led us to the conclusion that odours of inflorescences not only mask egg odours deposited on them, but also inhibits host location for surrounding eggs in this experimental setup. Such effect of inflorescences probably hinders T. cacoeciae to detect eggs of EGM during the first generation. This migth give a first hint why this parasitoid fails in controlling EGM in an economic way. In order to confirm this result, flight cage experiments were performed where T. cacoeciae wasps were about to locate and parasitise eggs of L. botrana in presence and absence of V. vinifera inflorescences. It resulted that there was no difference in the number of parasitised eggs in the presence or absence of inflorescences. However, fluctuations were great, ranging from 17 % to 67 % in both test arrangements. Probably, this might be due to the small number of offered eggs, which should be increased for further tests. Additionally, withering of the inflorescences during experiments could be a reason for the undetermined results. Thus, greenhouse or field studies on effects of inflorescences on T. cacoeciaes host location should be conducted. However, negative effects of plant volatiles on Trichogramma egg parasitoids were reported in one other study that mentions volatile compounds emitted by pods of pigeon peas, Cajanus sp. (Fabaceae) to repel
-73- T. chilonis. (Romeis et al. 1998). Interestingly, Cajanus and Vitis both belong to the class Rosidae. Therefore, volatile floral components of this plant class might comprise repellences that favour herbivores that deposit their eggs on them. A closer look on this topic might be important, in particular when using Trichogramma parasitoids to control pest species on Rosidae plant species, e.g. T. dendrolimi used to control Cydia pomonella on apple (Rosaceae) (Hassan et al. 1988).
In regard of host species, a masking effect of inflorescences probably hinders parasitoids to detect their eggs deposited on them and nearby. Therefore, selection of inflorescences as oviposition site might be a strategy to save their eggs from parasitisation. In general, selection of an oviposition site for herbivores is mediated by several factors: plant conditions, plant defence mechanisms, competition, predatory and parasitisation pressures (Thompson & Pellmyr 1991, Fox & Eisenbach 1992, Meiners & Obermaier 2004, Stiling & Moon 2005, Randlkofer et al. 2007, Obermaier et al. 2008). Since it is probably impossible to find an oviposition site with optimal conditions, herbivores are certainly faced a trade-off. For example, female herbivores might place their eggs to physically and/or chemically defended sites that however do not assure best feeding conditions for their progeny (Renwick & Chew 1994). This was demonstrated for salt marsh planthoppers, Pissonotus quadripustulatus (Homoptera: Delphacidae) (Moon & Stiling 2006). The planthoppers oviposit on both, woody and green stems of their host plant, Borrichia frutescens (Asteraceae). Green stems have much higher food qualities. However, P. quadripustulatus gains higher quantities of offspring and increased fitness from eggs deposited on woody stems, which represent a barrier for their parasitoid, Anagrus armatus (Hymenoptera: Mymaridae). Thus, lower parasitisation rates on woody stems facilitate planthoppers that oviposit on plant parts with reduced food qualities. This demonstrates the selective pressure of parasitisation on herbivores selection of an oviposition site. Therefore, inflorescences of V. vinifera as oviposition site of L. botrana and E. ambiguella might have resulted from parasitisation of T. cacoeciae. Since more than 95 % of all Lepidoptera species in Santa Rosa National Park eat green leaves (Janzen 1988) and most larvae of Tortricidae live as leaf rollers, leaf webbers or leaf miners (Van der Geest & Evenhuis 1991), the use of other plant organs than leaves represents an unusual behaviour. Besides this, leaves of V. vinifera are available during the whole season and therefore represent a possible substrate for larval feeding. However, EGM were never found to oviposit on leaves of this plant species. They exclusively oviposit on inflorescences and grapes. Usage of grapes may be explained due to higher food qualities of fruits over leaves, as it was reported for other Tortricidae (Geier 1963, Agnello et al. 1996, Trimble et al. 2001).
-74- However, barely anything is known about food quality of inflorescences compared to leaves. Concerning our results, usage of inflorescences as oviposition site might have evolved as strategy to avoid parasitisation. Therefore, finding larvae of EGM to only feed on inflorescences and grapes of V. vinifera (Thompson 1988, Jones 1991, Röhrich & Boller 1991) probably resulted from an evolutionary process including different selective pressures like food qualities and enemy pressures (Thompson & Pellmyr 1991, Stavridis & Savopoulou-Soultani 1998).
In summary, the performed experiments on influences of V. vinifera on host location of T. cacoeciae demonstrates that naïve wasps neither use plant volatiles of uninfested nor of infested grapes and inflorescences for host location. Therefore, in order to locate eggs of EGM, T. cacoeciae has to use another source of infochemicals. Furthermore, inflorescences are likely to inhibit the host location due to masking effects, making it unlikely for T. cacoeciae to detect eggs of first generation of EGM.
5.2. Source of infochemicals used for host location
There are a series of possible sources for infochemicals that may be used for host location of T. cacoeciae. For example, sex pheromones emitted by adult hosts were shown to act as effective kairomones for many egg parasitoids (Lewis et al. 1982, Nordlund et al. 1983, Noldus 1988, Reddy et al. 2002, Schöller & Prozell 2002). However, usage of sex pheromones is reported only for parasitoids whose hosts’ oviposit close to their mating site and for parasitoids that display phoresy. In contrast, both species of EGM oviposit far from their mating sites (Masante-Roca et al. 2007) and T. cacoeciae is not known to show phoresy. Thus we expected T. cacoeciae to not react to sex pheromone blends of the two EGM species when tested in the Y-tube olfactometer. Nevertheless, it was important to verify our succession due to the usage of pheromone mating disruption techniques in European vinyards, that may influence T. cacoeciaes performance. Actually, the predictions were confirmed by the performed experiments. Additionally, field studies on influences of the pheromone mating disruption technique revealed no difference in parasitisation rates of T. cacoeciae in treated (WBI+) compared to untreated areas (WBI-).
Since neither plant volatiles nor sex pheromones were attractive to T. cacoeciae wasps, host egg masses themselves were considered as source for infochemicals. Although it was not
-75- expected that egg sources elicit chemical long-range attraction in egg parasitoids by Fatorous et al. (reviewed 2008), from the present study it resulted that T. cacoeciae was highly attracted by egg masses of both EGM species when tested in Y-tube olfactometer bioassays. This for the first time demonstrates the potential of airborne chemicals of EGM egg masses to attract T. cacoeciae during host location over some distance.
Most studies concerning volatiles directly emitted from host egg masses depict increased mobility or stimulated searching behaviours rather than active orientation towards egg masses (Frenoy et al. 1992, Renou et al. 1992, Hofstetter & Raffa 1998, Bai et al. 2004, Yong et al. 2007). For example, Trichogramma brassicae was shown to increase its activity in the presence of volatile chemicals emanating from Ostrinia nubilalis eggs (Lepidoptera: Pyralidae) in a linear airflow olfactometer (Frenoy et al. 1992). However, T. brassicae was not attracted by host eggs. Similarly, airborne chemicals from Ostrinia furnacalis egg masses were shown to stimulate intensive search behaviour in Trichogramma ostriniae in a four-arm olfactometer. However, again the egg masses did not elicit positive chemotaxis in T. ostriniae egg parasitoids (Bai et al. 2004). Only Yong et al. (2007) demonstrated females of T. ostriniae to show innate positive responses to egg mass volatiles of O. nubilalis eggs, in a Y- olfactometer bioassay. This is the only study on Trichogramma egg parasitoids that corresponds to our findings for T. cacoeciae and eggs of L. botrana and E. ambiguella.
T. ostriniae also responds to moth sex pheromones that probably increase detectability of their hosts’ eggs. However, T. cacoeciae did not respond to any additional source of infochemicals tested in the present study. This finding might be another explaination for the low performance of this species to control EGM. Nevertheless, T. cacoeciae is able to locate some eggs. In this context, we assumed the parasitoids to overcome the detectability problem of insect egg masses by using highly volatile and easy to detect infochemicals from this source. Therefore, analyses of the chemical structures of infochemicals present on egg masses were conducted in the present study.
5.3. Involved chemicals
Since egg masses were known to be used as infochemicals for host location of T. cacoeciae, we were interested in the chemical structure of substances involved. Studies on identified chemical substances from insect egg surfaces are rare, due to the finding that most egg
-76- parasitoids use infochemical sources other than eggs. Besides, most of the few studies concerning chemical substances from Lepidopteran eggs are about oviposition-deterring pheromones (Thièry & Le Quere 1991, Thièry et al. 1992b, Li et al. 2001, Liu et al. 2008). Such studies exist for eggs of L. botrana. Several straight chain fatty acids and their esters are known to make up part of the oviposition-regulating pheromone (ORP) of this species (Thièry et al. 1992a, Thièry & Gabel 1993, Gabel & Thièry 1996). ORP are about to elicit intraspecific avoidance and therefore are volatile and easy to detect substances. Consequently, such substances might become eavesdropped by enemies to locate eggs. However, there are barely any investigations on chemical substances from insect eggs having interspecific activity so far. To my knowledge the only studies concerning chemical characterisation of egg emanated volatiles with kairomonal effects were performed by Renou et al. (1992) and Frenoy et al. (1992). In the first study five saturated hydrocarbons (heneicosane, tricosane, pentacosane, heptacosane and nonacosane) were identified from eggs of Ostrinia nubilalis (Lepidoptera: Pyralidae) and Mamestra brassicae (Lepidoptera: Noctuidae). They elicit increased upwind locomotion in Trichogramma brassicae females, when tested in a linear olfactometer. In the latter study, additionally oleic acid from eggs of O. nubilalis was shown to increase the mobility of T. brassicae.
In the present study extracts of L. botrana egg masses were shown to be as attractive as 24 hours old egg masses. Approximate identification of chemicals revealed that alkanes, alkenes, fatty acids, ketones, esters and aldehydes are present within the extracts. Thereby, short-chain hydrocarbons and fatty acids (nonanoic and octanoic acid) identified from the eggs of L. botrana are highly volatile and were already known to be part of the ORP (Thièry et al. 1992a, Gabel & Thièry 1996). Furthermore, different substances of almost all of these substance classes are known to elicit attraction in some Trichogramma species (reviewed by Rutledge 1996). As already mentioned alkanes and fatty acids were found to be attractive to T. brassicae (Renou et al. 1992, Frenoy et al. 1992). Additionally, Rani et al. (2007) showed long chain alkanes and alkenes from cuticular extracts of adult Scirpophaga incertulas (Lepidoptera: Pyralidae) to be attractive to T. japonicum. However, although aldehydes were found to elicit positive reactions in some egg parasitoid species (Lewis et al. 1982, DeLury et al. 1999, Laumann et al. 2009), there are no studies demonstrating Trichogramma egg parasitoids to be attracted to aldehydes so long.
In order to narrow down the possible infochemicals involved in host location of T. cacoeciae egg mass extracts were fractionated into polar and non-polar fractions. Based on the study of Renou et al. (1992) T. cacoeciae was supposed to be attracted to non-polar rather
-77- than polar substances. However, this was not the case. When tested in the Y-tube olfactometer wasps of T. cacoeciae showed positive reactions only towards polar fractions of L. botrana egg mass extracts. This fraction consisted of esters, fatty acids, ketones and aldehydes.
Subsequent investigations on the attraction of T. cacoeciae to synthetic blends of several identified substances revealed only aldehydes to elicit positive chemotaxis. This was the first time when aldehydes were shown to be used for host location of an egg parasitoid. Studies concerning effects of aldehydes on parasitoids, so long only reported increased neural activity, stimulated searching behaviour or increased parasitisation rates. An increase of neural activity was shown for the egg parasitoid Ascogaster quadridentata (Hymenoptera: Braconidae) examined via gas chromatographic-electroantennographic detection, in the presence of different aldehydes (including heptanal, octanal, nonanal, decanal, dodecanal, (Z)- 9-hexadecenal) (DeLury et al. 1999). Stimulation of searching behaviour was shown for Trisolcus basalis (Hymenoptera: Scelionidae) when exposed to two aldehydes of glandular secretions of their host Nezara viridula (Heteroptera: Pentatomidae) (Laumann et al. 2009). Finally, increased parasitisation rates were found for Trichogramma pretiosum in the presence of four synthesised stereoisomers of hexadecanal (Lewis et al. 1982). Nevertheless, since aldehydes were never shown to be used for active host location before, the present study is the first that describes such phenomenon.
T. cacoeciae only was attracted to aldehydes and to no other blend of substances from any other chemical class tested in the present study. Thus, there might be an advantage for the parasitoid using this chemical class. The examined aldehydes that elicited positive chemotaxis in T. cacoeciae were of short chain length (C-5, C-9, C-12) and therefore easy detectability was assumed due to high volatility. Studies on male sawflies, Pikonema alaskensis (Hymenoptera: Tenthredinidae), revealed that they are attracted to (Z)-10-nonadecenal (C-19) from the sex pheromone of female sawflies even at very low concentrations (1 µg/cm²) (Bartelt & Jones 1983). This aldehyde is generated by oxidation of (Z, Z)-9,19 hydrocarbon dienes and actually, synthesis of aldehydes as well might explain their usefulness as kairomone. Aldehydes are generated by oxidation of unsaturated hydrocarbons (Swedenborg & Jones 1992, reviewed by Tillman et al. 1999, Bartelt et al. 2002) which themselves are comprised within cuticular waxes of almost all insect species (reviewed by Howard & Blomquist 2005) and on the surface of Lepidopteran eggs (Renou et al. 1992). Oxidative conversion of hydrocarbons to aldehydes occurs slowly in air (Bartelt & Jones 1983). Thus, the advantage of using aldehydes rather than hydrocarbons lies within the finding that
-78- aldehydes are permeable for longer times (Gibbs 2002). In summary, aldehydes are easy to detect and their long availability might increase the chance for host location.
However, aldehydes represent a rather unspecific class of chemicals, occurring as compound of many floral scents (e.g. Gerlach & Schill 1991, Patt et al. 1995, Campeol et al. 2001). Thus, the use of such unspecific infochemicals for host location seems unreliable. Anyhow, it was demonstrated to be advantageous for generalist parasitoids. For example, Cephalonomia tarsalis (Hymenoptera: Bethylidae), a larval parasitoid of Oryzaephilus surinamensis (Coleoptera: Cucujidae) was shown to be attracted to odours from seed–host complexes of their host as well as of their alternative host O. mercator. Additionlly, the parasitoid was attracted to odours from non-host seed-larvae complexes of Sitophilus granarius (Collatz & Steidle 2008). Subsequently, attraction was found to be due to two aldehydes: nonanal and decanal (Collatz 2010). This demonstrates the general character of this chemical substance class that even led parasitoids to larvae of non-host species.
T. cacoeciae wasps are known to be generalists, parasitising eggs of L. botrana and E. ambiguella. Additionally, they were shown to parasitise eggs of Ephestia kuehniella (Lepidoptera: Pyralidae) and Prays oleae (Lepidoptera: Yponomeutidae) (Barney et al. 1999, Pereira et al. 2004, Hegazi et al. 2005). Thus, the use of unspecific infochemicals might be advantageous for T. cacoeciae when searching for eggs of different host species. Nevertheless, to differentiate between host eggs and non-host materials (such as flowers) the parasitoids probably have to use additional stimuli. It is possible that T. cacoeciae needs to experience host odours either during development inside the egg or during first parasitisation events. This was for example demonstrated for the larval parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae). It was shown that host recognition response increased, after giving the wasps opportunity to gain experience on their host (Steidle et al. 2001). However, L. disinguendus as well not only uses infochemicals generated directly from their host. It was shown that they learn specific cues from herbivore-damaged wheat, rice and cowpea seeds and use this information for host recognition (Collatz et al. 2006). Thus, the finding that T. cacoeciae only responded to one kind of tested infochemicals may give a further hint on the inefficiency of this species to locate and therefore parasitise eggs of EGM. However, further experiments on the impact of experience and learning have to be made.
-79- 5.4. Moth scales
From chemical analyses combined with Y-tube olfactometer assays it resulted that within extracts of L. botrana egg masses only aldehydes elicit positive chemotaxis in T. cacoeciae. However, concerning egg masses as source of infochemicals, the possible impact of moth scales have to be taken into account. Moth scales are attached to almost all moth egg masses (reviewed by Fatouros et al. 2008) and same was true for egg masses of L. botrana and E. ambiguella in the present study. In general, if egg masses elicit a positive reaction in a parasitoid, eggs and moth scales have to be examined separately in order to precisely identify the infochemicals source. For example, DeLury et al. (1999) demonstrated that the egg-larval parasitoid Ascogaster quadridentata (Hymenoptera: Braconidae) is attracted towards odours of both, eggs and body scales of Cydia pomonella (Lepidoptera: Tortricidae). However, when using “eggs” as sample moths scales were attached to them just as we observed it for eggs of EGM in our experiments. Therefore, attraction of A. quadridentata towards host “eggs” has to be regarded as attraction towards egg masses and may be due to adhered moth scales. This aspect remained uninvestigated in the addressed study.
In the present study, it was not possible to examine moth eggs exclusive of moth scales without destroying or modifying the eggs surface. Accordingly, it is impossible to perform behavioural studies on attractiveness of eggs without moth scales. Therefore, in the present study chemical analyses of both, egg masses with scales and separated scales were performed to precisely determine which source attracts T. cacoeciae during host location. Subsequent analyses of body and wing scales from L. botrana revealed that within extracts of moth scales no aldehydes were present. Together with the finding that only aldehydes from egg mass extracts were attractive to T. cacoeciae in the Y-tube olfactometer, we concluded that this is due to kairomones emanated directly from host eggs.
5.5. Influence of host egg age on host location and suitability
Time limitation for host location and host suitability was reported for several egg parasitoid species (e.g. reviewed by Vinson & Iwantsch 1980, Pak 1986, Godin & Boivin 2000). However, studies investigating time limitation of host location and suitability in the same study are rare (Colazza et al. 2004, Fatouros et al. 2005a, 2005c). In the present study both parameters were taken into account.
-80- Host location on basis of volatile infochemicals probably is restricted to the amount of essential components evaporating from the informative source. This was shown for example for host location of Telenomus euproctidi (Hymenoptera: Scelionidae). The egg parasitoid is attracted to butterfly sex pheromones attached to egg masses of Euproctis taiwana (Lepidoptera: Lymantriidae). However, the attraction decreases 24 hours after egg deposition. Thereby, the loss of attraction is congruent with the achievement of infochemical amounts no longer attractive to the parasitoid (Arakaki & Wakamura 2000).
Host location of T. cacoeciae was supposed to be limited to eggs of about 72 hours as it was shown for other egg parasitoids before (Colazza et al. 2004, Fatouros et al. 2005a, 2005c). However, T. cacoeciae only showed positive chemotaxis for 24 and 48 hours old eggs of EGM when tested in the Y-tube olfactometer. 72 hours old eggs of L. botrana and E. ambiguella did no longer elicit attraction in wasps. Therefore, time restriction for host location of T. cacoeciae is exceeded compared to that of Trissolcus basalis (Colazza et al. 2004) or Trichogramma brassicae (Fatouros et al. 2005a, 2005c).
Since egg age have an influence on parasitisation rates and breeding success of many egg parasitoid species, it was assumed to also affect T. cacoeciae wasps. For most egg parasitoids, young eggs were reported to be more suitable for parasitisation than older eggs (Pak et al. 1986, Reznik & Umarova 1990, Reznik et al. 1997, Monje et al. 1999, Colazza et al. 2004, Saour 2004, Makee 2005). Accordingly, two comparison studies on the influence of egg age on parasitisation rates and breeding success of different Trichogramma species revealed younger eggs to be more suitable then were old eggs (Pak 1986, Godin & Boivin 2000). Results of the present study for eggs of L. botrana confirm these findings. T. cacoeciae had higher parasitisation rates on young (24 to 48 hours old) and medium aged eggs (72 to 96 hours old) compared to old ones (120 to 144 hours old). After Pak (1986) and Godin & Boivin (2000) the relationship between egg age and parasitisation rate of this species can be classified as type II response (preference for young AND medium aged eggs), which is seldom found. For breeding success a type VI response can be found with medium aged eggs be most suitable. However, for eggs of E. ambiguella parasitisation rates and breeding success were independent of egg ages with all age classes parasitised equally and same breeding successes for all categories (type I responses). Age independency on parasitisation rates and reproduction is hardly known from other parasitoid species (reviewed by Pak 1986). However, the high fluctuations in both parasitisation rates and breeding success of T. cacoeciae on eggs of E. ambiguella may refer to an unpredictable suitability of such eggs.
-81- In general, timeframes for host location fitting that of best host suitability are predicted to be adaptive (Colazza et al. 2004, Fatouros et al. 2005a, 2005c). Actually, for T. cacoeciae highest parasitisation rates and breeding successes on EGM eggs were gained for eggs best detectable: 24 to 48 hour old eggs. However, eggs of 72 and 96 hours as well showed hight parasitisation rates and breeding successes, but were no longer attractive to T. cacoeciae. Additionally, 48 hours for host location is very short compared to other host-parasitoid systems. The impact of this finding is intensified due to the fact that EGM oviposit during first hours of night (Hurtrel & Thièry 1999), whereas T. cacoeciae is active during daytime (Pompanon et al. 1995). Therefore, there is only little time left for T. cacoeciae to locate eggs of L. botrana and E. ambiguella. This increases the chance for eggs of EGM, to remain undetected by T. cacoeciae. However, in our experimental setup we only used naïve T. cacoeciae wasps and the impact of possible induced plant volatiles of older eggs remained disregarded and have to be considered in further studies.
5.6. Influence of host species and its suitability for T. cacoeciae
Egg parasitoids of the genus Trichogramma are known to be generalists, with ability to parasitise eggs of a wide range of Lepidopteran species, but with preferences for some species (Brotodjojo & Walter 2006, Roriz et al. 2006, Monje et al. 1999, Zhang & Cossentine 1995). For T. cacoeciae it is known that eggs of both species of EGM, L. botrana and E. ambiguella, are preferably parasitised when compared to eggs of the mealy moth Ephestia kuehinella (Lepidoptera: Pyralidae) (Zimmermann 1997, Herz et al. 2005). When comparing parasitisation rates of T. cacoeciae between eggs of the two species of EGM in a direct choice experiment, it was reported that there were no differences (Castaneda-Samayoa et al. 1993). Likewise, in the present study, parasitisation rates of 24 hours old L. botrana eggs (ø 40 %) and E. ambiguella eggs (ø 57 %) did not differ significantly. However, from another study it is known that some strains of T. cacoeciae prefer eggs of L. botrana over eggs of E. ambiguella for oviposition, when offered simultaneously (Ibrahim 2004). The inconsistent results from different studies on host preferences and host suitability of EGM eggs for T. cacoeciae give a good hind on differences in the performance of different strains of this species. This point is discussed continuative in the following chapters.
-82- 5.7. Host location and parasitisation
In general, the potential of egg parasitoids to control a certain pest species is estimated on the basis of their parasitisation rates found in laboratory behavioural assays (e.g. Honda et al. 1999, Romeis et al. 1999, Murthy et al. 2003, Kalyebi et al. 2005). T. cacoeciae wasps from D-90 strain, used for the present study, reached maximum parasitisation rates of 57 % on young eggs of E. ambiguella and on average 40 % breeding success from these eggs. Most suitable eggs were medium aged eggs of L. botrana with a breeding success of 55 % on 40 % of parasitised eggs on average. In comparison with other Trichogramma egg parasitoid species, T. cacoeciae reaches an average level of parasitisation rates of EGM eggs in laboratory. For example one-day-old, mated and fed females of T. brassicae show parasitisation rates of up to 100 % (92 % on average) for eggs of Ephestia kuehinella (Cerutti & Bigler1995). For T. exiguum, parasitisation rates of 58 % are mentioned for eggs of Plutella xylostella (Lepidoptera: Plutellidae) (Polanczyk et al. 2007) and T. nubilale wasps reach 66 % parasitised eggs of Ostrinia nubilale (Lepidoptera: Pyralidae) (Losey & Calvin 1995). Finally, T. pretiosum parasitises 68 % of Sitotroga cerealella eggs (Lepidoptera: Gelechiidae) (Goncalves et al. 2003). However, high parasitisation rate is not the only criterion for successful parasitisation. Preliminary to parasitisation, successful host location is essential for the reproduction of the parasitoid. Unfortunately, the important criterion of successful host location is disregarded in most studies on the potential and efficacy of egg parasitoids (e.g. Bigler 1994, Leppla & Fisher 1989, Hassan & Guo 1991, Losey & Calvin 1995, Liu & Smith 2000). Therefore, in the present study, an experimental setup to investigate both, host location and parasitisation was generated.
Studies on the ability of parasitoids to parasitise hosts subsequently to host location are almost known from field studies. Therefore, parasitoids are inundative released within a certain area and parasitisation rates or reduction rates of the target pest species are measured (e.g. Babendreier et al. 2003, Kuhar et al. 2004, Herz & Hassan 2006, Agamy 2007, Hegazi et al. 2007). Remarkably, sometimes results from field application studies did not match the assumed parasitisation rates gained from laboratory bioassays (Bigler 1989, 1994, Cerutti & Bigler 1995, Dutton et al. 1996). This might be due to influences of the host plant or other environmental influences. Additionally, low potential of parasitoids to locate the hosts may result in lower parasitisation rates in the field compared to laboratory bioassays (Smith 1996). Therefore, an experimental setup combining host location and parasitisation is essential for better predictions on performances of parasitoid species.
-83- Until know, studies considering host location and parasitisation in laboratory are known only for parasitoids searching for their hosts by crawling over substrate in crop storage (Schöller et al. 1996, Steidle & Schöller 2002, Stolk et al. 2005, Grieshop et al. 2006, 2007). Therefore, in the present study an experimental setup was processed to examine the potential of an egg parasitoid that flies. Flight cage experiments under semi-natural conditions demonstrate the ability of parasitoids to first locate the host prior to parasitisation. For T. cacoeciae wasps it resulted that they parasitised 60 % of offered L. botrana eggs and 40 % of E. ambiguella eggs. Comparing these results to those performed with Petri dishes, a distinct difference in the parasitisation rates of T. cacoeciae for eggs of both EGM species becomes obvious. When offered directly, 40 % of young L. botrana eggs and 57 % of young E. ambiguella eggs have been parasitised. The impact of host location on the result becomes clear in regard of results gained from Y-tube olfactometer assays with an average percentage of decisions for 24 hours old L. botrana eggs (71 %) being higher than for E. ambiguella eggs (56 %). Therefore, intraspecific differences of parasitisation rates gained from the different experimental setups probably are due to different requirements on T. cacoeciae performance.
However, it is also important to always use parasitoids from the same strain for comparable results. This is e.g. due to the findings of different host preferences of different T. cacoeciae strains found in different studies, as described in the previous chapter. Additionally this is demonstrated by findings on different strains of Trichogramma brassicae. Wasps of different strains, reared under similar conditions, differed in emergence rates, sex ratios and fecundity on eggs of their natural host species Ostrinia nubilalis (Cerutti & Bigler 1995, Hassan & Zhang 2001). Furthermore, different strains of Trichogramma bourarachae greatly differ in their infestation efficiency on eggs of Ephestia kuehniella, with 60 eggs/five days/female for one strain and 25 eggs/five days/female for another (Girin & Bouletreau 1995). Such great differences in performance of individuals of “same” species could evolve due to several reasons. Microorganism load (Girin & Bouletreau 1995), artificial selection, breeding depressions (Geden et al. 1992) and natural selection (Colazza & Rosi 2001) are reported. Therefore, to precisely investigate performances of defined parasitoids, careful choice of individuals from one parasitoid strain is essential.
-84- 5.8. Performance of Trichogramma in natural environment
Several studies on species selection for inundative releases reported locally collected Trichogramma species to be more capable of parasitising host eggs in the field than artificially introduced species. This was true, even when locally collected species were compared to species that showed improved adaptation to pests’ eggs in laboratory (Browning & Melton 1987, Glenn et al. 1997, Herz & Hassan 2006). Therefore, in the present study prior to application experiments, baiting with eggs of L. botrana and E. ambiguella was conducted to investigate natural occurring Trichogramma species. It resulted that within the examined areas, two Trichogramma species were present: T. cacoeciae and T. evanescence. Unfortunately, it was not possible to use baited populations for application studies. Therefore, a laboratory strain of T. cacoeciae was used, that was ecologically responsive and of economical expedience.
In the present study, the potential of T. cacoeciae from D-90 strain to locate and parasitise eggs of L. botrana and E. ambiguella was examined in laboratory studies, as described by several authors (Van Lenteren et al. 1982, Hassan 1989, Vasquez et al. 1997). Petri dish experiments with directly offered EGM eggs were performed. Additionally, host location ability of T. cacoeciae was examined via Y-tube olfactometer and flight cage assays. Since T. cacoeciae was shown to reach an average level of parasitisation as was able to locate eggs of both species of EGM, field applications were conducted. Therefore, parasitisation rates and damage reduction rates of areas with artificially induced T. cacoeciae in comparison to untreated control areas have to be investigated (Naranjo et al. 1992, Hassan 1994, Andow et al. 1995). Studies on this subject so long resulted in inconsistent parasitisation rates of T. cacoeciae on eggs of L. botrana (Castaneda-Samayoa et al. 1993, Wührer et al. 1995, Zimmermann 1997, Hommay et al. 2002, Zimmermann 2004). Likewise, in the present study remarkably low parasitisation rates of little more than 4 % resulted for applicated T. cacoeciae. In general, parasitisation rates for successful biological control are stated to more than 80 % of parasitisation in order to reduce pest populations (Goodenough & Witz 1985, Knipling & McGuire 1968). More recent studies presumed rates of 75 % to be effective in biologically control (Bigler 1994). However, from the present study, applicated T. cacoeciae from strain D-90 never reached more than 11 % of parasitised eggs (two days eggs of L. botrana) making this strain insufficient as biological control agent.
As already mentioned, there are several reasons for parasitisation rates to be lower in field studies compared to laboratory bioassays. Besides, differences between strains of the
-85- “same” species also influences of host and non-host plants, limited searching capacities or hosts counter strategies may affect parasitoids performance. Certainly, different biotic and abiotic factors are involved (Smith 1996) as for example, negative impacts of different insecticides and fungicides (e.g. Reddy & Manjunatha 2000, Takada et al.2001, Youssef et al. 2004, Bueno et al. 2008, Giolo et al. 2008, Vianna et al. 2009).
As a result of insecticide use unpredictable parasitisation rates of applied biocontrol agents may occur. For example, Trichogramma pretiosum, parasitising eggs of the cotton bollworm, Heliothis armigera (Lepidoptera: Noctuidae), reached up to 89 % of parasitisation (Johnson 1985). However, from other studies parasitisation rates of 5 % to 35 % are known for T. pretiosum on eggs of H. armigera (Fletcher & Thomas 1943, Kring & Smith 1995). Remarkably, high parasitisation rates in the study of Johnson (1985) were found only during early season and sharply declined later in year, simultaneous to the start of insecticide treatments. Therefore, we assumed the low parasitisation rates for T. pretiosum gained from other studies probably are due to strain differences or insecticide treatments. In the present study, sulphur treatment was assumed as probable reason for low parasitisation rates of T. cacoeciae. Sulphur is used to fight powdery mildew in viticulture and applications are conducted during the whole vegetative phase of grapevine. Besides, sulphur was shown to be harmful to T. cacoeciae when tested in laboratory (Hassan et al. 1998, Gruetzmacher et al. 2004). Additionally, from inundative release programs it was reported that T. pretiosum perform better in fields without sulphur treatment compared to those with sulphur treatment (Simser 1994, Scholz et al. 1998). Therefore, T. cacoeciae was supposed to show higher parasitisation rates in areas without sulphur treatment compared to those with sulphur treatment. However, in the present study, parasitisation rates of T. cacoeciae did not differ between the two area types. This was true for natural and for artificially induced parasitoids. In order to see if other chemicals, used as pesticides in sustainable viticulture, affect the performance of T. cacoeciae, further studies have to be performed.
-86- 6. Conclusions
Field applications of Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) remain unprofitable for commercial use as biological control agent against the two pest species Lobesia botrana and Eupoecilia ambiguella (Lepidoptera: Tortricidae), due to unpredictable efficacy (Wührer et al. 1995, Zimmermann et al. 1997, Hommay et al. 2002, Zimmermann 2004). In fact, from the present study very low parasitisation rates resulted from both natural and artificial induced T. cacoeciae. Since so far it was hardly examined which factors affect the performance of T. cacoeciae the present study was conducted to answer this question. Therefore, basic researches and applied studies on the potential and limitations of the egg parasitoid were conducted. Although, reasons for low parasitisation rates of T. cacoeciae on eggs of L. botrana and E. ambiguella in the field remain not fully answered, the present study gives good information on different factors that probably inhibit the wasps during the whole process of gaining access to their host.
6.1. Potential of T. cacoeciae to gain host access
Naïve wasps of the thelytokous egg parasitoid species T. cacoeciae were shown to use olfactory active volatiles from the surface of host eggs as infochemicals for host location. This is rarely known from other parasitoids due to the small biomasses of insect eggs compared to that of surrounding vegetation. Nevertheless, it seems as if T. cacoeciae overcomes the problem of low detectability by using a volatile oxidative decomposition product of hydrocarbons: aldehydes. The high efficacy of aldehydes is demonstrated by male sawflies, Pikonema alaskensis (Hymenoptera: Tenthredinidae), that are attracted to (Z)-10-nonadecenal from the sex pheromone of female sawflies (Bartelt & Jones 1983). This aldehyde is generated by oxidation of (Z, Z)-9,19 hydrocarbon dienes and was shown to be sufficient even in very low concentrations (1 µg/cm²) to attract male sawflies in greenhouse bioassays. Additionally, aldehydes were shown to be advantageous for generalist parasitoids, that are about to locate different host species. This was demonstrated for Cephalonomia tarsalis (Hymenoptera: Bethylidae), a larval parasitoid that uses two aldehydes (nonanal and decanal) to locate larvae of their host Oryzaephilus surinamensis (Coleoptera: Cucujidae), their alternative host O. mercator (Collatz 2010) and was attracted even to larvae of a non-host species Sitophilus granarius (Coleoptera: Curculionidae) (Collatz & Steidle 2008). Therefore,
-87- finding T. cacoeciae to be attracted to a blend of three aldehydes (heptanal, nonanal and dodecanal) probably reveals an effective strategy to locate eggs of both species of European grapevine moths (EGM), L. botrana and E. ambiguella.
Host location bioassays via Y-tube olfactometer showed T. cacoeciae to be able to locate eggs of both EGM species 24 to 48 hours after deposition. Additionally, parasitisation rates achieved in laboratory studies for eggs of these ages were satisfying with on average 40 % (L. botrana) and 57 % (E. ambiguella) parasitised eggs. Therefore, eggs of ages that are parasitised the most fit that of detectable eggs, which probably benefits the use of T. cacoeciae as biological control agent against EGM. However, the results only represent parasitisation rates under laboratory conditions. Concerning field studies, another dimension appears.
6.2. Limitations
In the present study, parasitisation rates of natural occurring T. cacoeciae were low with a maximum of less than 2 %. Likewise, parasitisation rates of artificial induced T. cacoeciae were remarkably low (4 %) and far from effective values, postulated for successful biological control (Goodenough & Witz 1985, Knipling & McGuire 1968, Bigler 1994). As reason for this in the present study, several probable limitations on T. cacoeciae were identified, affecting different steps of the process of host searching and selection.
One limitation might be that naïve T. cacoeciae wasps were attracted only to egg emanated volatiles. In the present study, they were found to neither use plant volatiles, oviposition induced volatiles nor sex-pheromones from adult hosts for host location. Additionally, although many chemicals from the surface of L. botrana eggs were tested for their attractiveness, T. cacoeciae was attracted only to aldehydes. Although being highly volatile and the use of such unspecific infochemicals may be advantageous for a generalist, volatiles emanated from insect eggs probably remain hard to detect within a complex environment (reviewed by Fatorous et al. 2008).
Another limiting factor for successful parasitisation of L. botrana and E. ambiguella eggs may be the restricted time for host location. Maximum egg age for which host location is possible is 48 hours. Although, this matches the time when parasitisation rates are high, the most suitable eggs for T. cacoeciae with highest breeding success are medium aged L.
-88- botrana eggs (72 to 96 hours old). However, eggs of this age were not attractive to parasitoids anymore. Additionally, medium aged eggs of L. botrana were as suitable for parasitisation as were young eggs. Thus, finding the parasitoids to be unable to locate eggs older than 48 hours probably restricts their access to suitable eggs. Additionally, the discrepancy in periods of activity, which for EGM is in first hours of night (Hurtrel & Thièry 1999) and during the day for T. cacoeciae (Pompanon et al. 1995) reduces the time window for host location. However, the possibility of associative learning as well as a probable use of oviposition induced volatiles from grapes infested with eggs for more than 24 hours, remained disregarded in the present study. However, even if T. cacoeciae is able to locate eggs older than 48 hours, the overall low parasitisation rates found in field studies suggests that other limiting factors inhibit the parasitoids performance.
Besides probable low amounts of essential infochemicals and time limiting factors for host location a third limiting factor is the masking effect of inflorescences odours from Vitis vinifera (Vitaceae). They prevent T. cacoeciae from locating eggs of both EGM species in Y- tube olfactometer assays. However, flight cage experiments did not confirm this result. Nevertheless, if a negative effect of inflorescences on the host location of T. cacoeciae acts in field, this might be a further explanation for the remarkably low parasitisation rates of T. cacoeciae in the field.
From the present study we conclude that naïve wasps of the commercially available T. cacoeciae strain D-90 (AMW-Nuetzlinge GmbH, Pfungstadt, Germany) are insufficient as biological control agent against L. botrana and E. ambiguella in commercial viticulture, due to the identified limiting factors.
-89- 7. Summary
Successful reproduction of an egg parasitoid depends on its ability to locate and successful parasitise eggs of its host species. By contrast, in the life history of target pests, there is a strong selective pressure to develop strategies which reduces risks of parasitisation and predation. Purpose of the present study was to figure out potentials and limitations on the egg parasitoid Trichogramma cacoeciae to locate and parasitise eggs of its two host species, the European grapevine moths (EGM), Lobesia botrana and Eupoecilia ambiguella. We investigated how T. cacoeciae locates eggs of its two host species. Therefore, the source of infochemicals and identification of their chemical structures were determined. Additionally, influences of the hosts’ food plant and egg conditions on host location and parasitisation were investigated. Finally, field studies were performed to examine parasitisation rates of natural and artificial induced T. cacoeciae and the impact of the pheromone disruption technique and sulphur on the performance of T. cacoeciae.
In order to gain information about the origin of infochemicals used by T. cacoeciae for host location a series of dual choice experiments with a Y-tube olfactometer were performed. It resulted that neither odours of uninfested plant materials, oviposition induced plant volatiles nor sex-pheromones of EGM attracts the parasitoids. However, volatiles directly emitted from eggs were highly attractive and were found to also attract wasps to grapes with deposited eggs. However, inflorescences were found to have a masking effect preventing T. cacoeciae from locating eggs on them but also eggs in their nearer vicinity when tested in a Y-tube olfactometer. Subsequent flight cage experiments that were performed in order to examine both, host location and parasitisation in the presence and absence of inflorescences could not confirm this result. Overall, from investigations on the infochemicals source, it revealed that naïve T. cacoeciae only use egg emanated volatiles for host location.
In order to identify chemicals involved in host location of T. cacoeciae, GC-MS analyses and Y-tube behavioural assays were performed using egg mass extracts, their non- polar and polar fraction as well as artificial synthesised blends of identified substances. It resulted that egg mass extracts, polar fractions and aldehydes were attractive to T. cacoeciae wasps. Furthermore, a blend of three short chained aldehydes was sufficient to elicit a positive chemotaxis in the parasitoids. This was the first time when aldehydes were shown to be used for host location of an egg parasitoid over some distance.
Differences in detectability and suitability of eggs of the two EGM species and eggs of different ages were performed via Y-tube olfactometer assays, flight cage experiments and no
-90- choice experiments in Petri dishes. From choice experiments in a Y-tube olfactometer it resulted that eggs of both EGM species were successfully localised up to an age of 48 hours. From Petri dish behavioural assays it resulted that T. cacoeciae gained high parasitisation rates for young (24 to 48 hours old) E. ambiguella eggs and young and medium aged (72 to 96 hours old) L. botrana eggs and. Breeding success was highest on medium aged eggs of L. botrana. The flight cage behavioural assays, depicting semi-natural conditions, finally showed that T. cacoeciae located and parasitised significantly more eggs of L. botrana (60 %) compared to eggs of E. ambiguella (40 %). However, parasitisation rates for both species of EGM were satisfying compared to other egg parasitoid species used for biological control.
Field studies on natural parasitisation rates of EGM eggs revealed two species of Trichogramma, T. cacoeciae and T. evanescence, that reach parasitisation rates of less than 2 %. Thereby, the pheromone disruption technique did not affect natural parasitisation rates. Parasitisation rates of artificial induced T. cacoeciae as well were low with a maximum of little more than 4 %. Investigations of a possible impact of sulphur treatment on low parasitisation rates and on damage reduction was examined. It resulted that sulphur treatment did not affect the parasitoids performance.
The gained results were discussed in regard to the potential and limitations of T. cacoeciae to gain access to eggs of L. botrana and E. ambiguella and to successful parasitise them.
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-107- Van Lenteren, JC, Glass, PCG, Smits, PH, 1982. Evaluation of control capabilities of Trichogramma and results of laboratory and field research on Trichogramma in the Netherlands. In: Les Trichogrammes, Antibes (France). Les Colloques de l'INRA, No. 9, 257– 268. Van Lenteren, JC , Godfray, HCJ, 2005. European science in the Enlightenment and the discovery of the insect parasitoid life cycle in The Netherlands and Great Britain. Biological Control 32: 12-24. Vasquez, LA, Shelton, AM, Hoffmann, MP, Roush, RT, 1997. Laboratory evaulation of commercial Trichogrammatid products for potential use against Plutella xylostella (L.) (Lepidoptera: Plutellidae). Biological Control, 9: 143-148. Vet, L, Dicke, M, 1992. Ecology of infochemical use by natural enemies in a tritrophic context. Annual Review of Entomology, 37: 141-172. Vianna, UR, Pratissoli, D, Zanuncio, JC, Lima, ER, Brunner, J, Pereira, FF, Serrao, JE, 2009. Insecticide toxicity to Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) females and effect on descendant generation. Ecotoxicology, 18 (2): 180-186. Vinson, SB, 1976. Host selection by insect parasitoids. Annual Review of Entomology, 21: 109-133. Vinson, SB, Iwantsch, GF, 1980. Host suitability for insect parasitoids. Annual Review of Entomology, 25: 397-419. Vinson, SB, 1998. The general host selection behavior of parasitoid hymenoptera and a comparison of initial strategies utilized by larvaphagous and oophagous species. Biological Control, 11 (2): 79-96. Walter, S, 1982. The occurrence of species belonging to the genus Trichogramma in forest biocenoses in East Germany. Entomologische Nachrichten und Berichte, 26 (6): 255-259. Wegener, R, Schulz, S, Meiners, T, Hadwich, K, Hilker, M, 2001. Analyses of volatiles induced by oviposition of elm leaf beetle Xanthogaleruca luteola on Ulmus minor. Journal of Chemical Ecology, 27(3): 499-515. Weseloh, RM, 1974. Host-related microhabitat preferences of the gypsy moth larval parasitoid, Parasetigena agilis. Environmental Entomology, 3: 363-64. Weseloh, RM, 1976. Behavior of forest insect parasitoids. In: Perspectives in Forest Entomology. Anderson, JF, Kaya, HK. Academic Press, NY, 99-110. Williams, IH, Frearson, DJT, Barari, H, McCartney, A, 2007. First field evidence that parasitoids use upwind anemotaxis for host-habitat location. Entomologia Experimentalis et Applicata, 123(3): 299-307. Wührer, B, Hassan, SA, Holst, H, 1995. Die Anwendung von Eiparasiten der Gattung Trichogramma zur Bekämpfung der Taubenwickler Eupoecilia ambiguella und Lobesia botrana. Tagungsband: Entomologen-Tagung, Göttingen, 1995. Deutsche Gesellschaft für Allgemeine und Angewandte Entomologie, 239. Yong, T-H, Pitcher, S, Gardner, J, Hoffmann, MP, 2007. Odor specificity testing in the assessment of efficacy and non-target risk for Trichogramma ostriniae (Hymenoptera : Trichogrammatidae). Biocontrol Science and Technology, 17 (1-2): 135-153. Youssef, AI, Nasr, FN, Stefanos, SS, Elkhair, SSA, Shehata, WA, Agamy, E, Herz, A, Hassan, SA, 2004. The side-effects of plant protection products used in olive cultivation on the hymenopterous egg parasitoid Trichogramma cacoeciae Marchal. Journal of Applied Entomology, 128 (9-10): 593-599. -108- Zaki, FN, 1985. Reactions of the egg parasitoid Trichogramma evanescens Westwood to certain insect sex-pheromones. Zeitschrift für Angewandte Entomologie, 99 (5): 448-453. Zhang, Y, Cossentine, JE, 1995. Trichogramma platneri (Hym.: Trichogrammatidae): Host choices between viable and nonviable codling moth, Cydia pomonella, and three-lined leafroller, Pandemis limitata (Lep.: Tortricidae) eggs. Entomophaga, 40 (3-4): 457-466. Zimmermann, O, 1997. Biologische Bekämpfung der Traubenwickler Eupoecilia ambiguella Hb. und Lobesia botrana Schiff. mit Trichogramma cacoeciae. Mitteilungen der Deutschen Gesellschaft für Allgemeine und Angewandte Entomologie, 11: 363-366. Zimmermann, O, 2004. Der Einsatz von Trichogramma-Schlupfwespen in Deutschland. Gesunde Pflanzen, 56: 157-166.
-109- 9. Participation at conferences
9.1. Oral presentations
2007 Die Wirtsfindung bei Trichogramma cacoeciae Ma. einem Eiparasitoiden der Traubenwicklerarten Lobesia botrana Den. & Schiff. und Eupoecilia ambiguella Hb. 23rd regular meeting of the Deutsche Gesellschaft für allgemeine und angewandte Entomologie (DGaaE), Innsbruck, Austria.
2007 Host location and success of parasitisation of Trichogramma cacoeciae Ma. (Lep.: Trichogrammatidae) 100th annual meeting of the Deutsche Zoologische Gesellschaft (DZG), Köln, Germany.
2008 Abiotische und biotische Einflüsse auf den Parasitierungserfolg des Eiparasitoiden T. cacoeciae 1st meeting of chemical ecologists of Baden-Württemberg
2008 Host location behaviour of the egg parasitoid Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) towards its two host species Lobesia botrana and Eupoecilia ambiguella (Lepidoptera: Tortricidae) 101st annual meeting of the DZG, Jena, Germany.
2010 Genetic and morphometric differentiation of three distinct strains of L. distinguendus 15th PhD meeting of Evolutionary Biology of the DZG, Freiburg, Germany
2010 Genetic differentiation of multiple distinct strains of Lariophagus distinguendus (Hymenoptera: Pteromalidae) 103rd Annual meeting of the DZG, Hamburg, Germany
9.2. Poster presentations
2006 Host location and parasitisation by Trichogramma cacoeciae, an egg parasitoid of Lobesia botrana 9th annual meeting of the DZG, Münster, Germany.
2008 A different kind of Easter: Host location of Trichogramma cacoeciae 13rd PhD meeting of Evolutionary Biology of the DZG, Hamburg, Germany.
2009 Alternating relation between the egg parasitoid Trichogramma cacoeciae and its two host species Lobesia botrana and Eupoecilia ambiguella: A host-parasitoid armsrace? 102nd Annual meeting of the DZG, Regensburg, Germany.
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