Anja S. Rott

New insights in analysing parasitoid attracting synomones: early volatile emission and use of stir bar sorptive extraction

Summary.It is well known that feeding by Pieris brassicae caterpillars on cabbage leaves triggers the release of volatiles that attract natural antagonists such as the parasitoid Cotesia glomerata. The temporal dynamics in the emissions of parasitoid attracting volatiles has never been elucidated in this system. In a time course experiment, caterpillar infested leaves attracted the parasitoid within one hour after infestation. At such an early stage of infestation, as much as fifty percent of the parasitoids flew towards the infested plant in a wind tunnel bioassay, while only five percent flew towards the non-infested control plant. Three hours after infestation and later, the response to the volatiles from the infested plant reached its maximum and then continued at a constantly high level for the remaining 14 hours of the experiment. Chemical analyses of volatiles collected from infested leaves at short time intervals during the first 24 hours identified a total of ten compounds, comprising green leaf volatiles, terpenoids, and a nitrile. Significant increase of emission within the first 5 hours following initial herbivory was detected for (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate, cineole and benzylcyanide. Subsequently, a coupled bioassay-chemical analysis procedure was developed allowing for testing and analyzing the same sample for future identification of the bioactive compounds. This was achieved by using stir bar sorptive extraction for the analysis of solvent extracts of caterpillar-damaged leaves.

The effects of temperature, relative humidity and host plant on the behaviour of Stethorus punctillum as a predator of the two-spotted spider mite Tetranychus urticae

The predatory behaviour of Stethoruspunctillum larvae was studied on the two-spottedspider mite (Tetranychus urticae), in order toassess how it responded to temperatures and relativehumidities typical of glasshouse conditions on fouredible crop plant species. Locomotory activity(distance covered, time spent walking, walking speed,angular velocity, and turning rate) was recorded at20, 25 and 30 °C and relativehumidity levels of 33%, 65% and 90% RH on tomato,pepper, aubergine and cucumber and analysed usingvideo/computer techniques. The results show thatactivity of S. punctillum significantly increased athigher temperatures. Host plant species also stronglyinfluenced the performance of the predator, which wasmost active on pepper and tomato and least active onaubergine. Relative humidity had no significantinfluence.

The role of the plant in attracting parasitoids: response to progressive mechanical wounding

Based on the model system of Brussels sprouts [Brassica oleracea var. gemmifera (Brassicaceae)], the herbivore cabbage white caterpillar, Pieris brassicae (L.) (Lepidoptera: Pieridae), and the parasitoid wasp, Cotesia glomerata (L.) (Hymenoptera: Braconidae), the influence of plant damage type, and damage duration were assessed on plant volatile emission and subsequent recruitment of natural antagonists of the herbivore. Plants were damaged by three methods for a period of either 3 or 8 h: herbivore damage (HD), progressive mechanical damage, and final mechanical damage inflicted in a single event. Wind‐tunnel bioassays evaluated whether the mode of damage affected female parasitoid oriented flight. After both periods of damage, all treatments were highly significantly preferred by naïve C. glomerata to undamaged control plants. After 3 h, herbivore‐damaged plants were significantly preferred to plants with final damage (FD). Most remarkably, following 8‐h damage, the parasitoid preferred both herbivore‐damaged and progressively damaged plants to plants with FD and did not significantly discriminate between herbivore and progressively damaged plants, thus indicating a similarity in plant response to herbivore and progressive mechanical damage. In addition to wind‐tunnel bioassays, emitted plant volatiles were collected and analysed by thermal desorption gas chromatography/mass spectrometry, following 3 and 8 h of damage in order to correlate volatiles released from different damage types with the attraction of the parasitoid. Differences in volatile profiles from all damage types were similar following both 3 and 8 h of damage, with only (Z)‐3‐hexenyl acetate found to be emitted in significantly higher quantities by final mechanical damage compared with HD after 3 h. In conclusion, the plants response to progressive mechanical damage was more similar to HD than final mechanical damage deployed at a single point in time, irrespective of damage duration, and C. glomerata did not significantly discriminate between progressive damage and HD.

Comparison between volatile emissions from transgenic apples and from two representative classically bred apple cultivars

While most risk assessments contrast a transgenic resistant to its isogenic line, an additional comparison between the transgenic line and a classically bred cultivar with the same resistance gene would be highly desirable. Our approach was to compare headspace volatiles of transgenic scab resistant apple plants with two representative cultivars (the isogenic ‘Gala’ and the scab resistance gene-containing ‘Florina’). As modifications in volatile profiles have been shown to alter plant relationships with non-target insects, we analysed headspace volatiles from apple plants subjected to different infection types by gas chromatography-mass spectrometry. Marked differences were found between healthy and leafminer (Phyllonorycter blancardella) infested genotypes, where emissions between the transgenic scab resistant line and the two cultivars differed quantitatively in four terpenes and an aromatic compound. However, these modified odour emissions were in the range of variability of the emissions recorded for the two standard cultivars that proved to be crucial references.

Parasitoid exploitation of the seasonal variation in host plant volatile emission for herbivore location

Volatile compounds from the apple, Malus domestica Borkh. (Rosaceae), change considerably as the season progresses, and this is successfully exploited by the female codling moth Cydia pomonella L. (Lepidoptera, Tortricidae), as it searches for oviposition sites. In this study, we investigated the effect of seasonal emissions of apple fruit volatiles on the host location behaviour of a parasitoid of the codling moth larvae, Hyssopus pallidus (Askew) (Hymenoptera: Eulophidae). In dual choice olfactory bioassays, the behaviour of the parasitoid in response to apple cues was observed over the complete 2003 growing season. Our results show that codling moth infested apples evoked a strong response from the parasitoid at the beginning of the season, until July. Then, attraction dropped drastically, increasing again at the beginning of August. At the end of the growing season, just prior to harvest, infested apples hardly evoked any behavioural response. Interestingly enough, mid‐season emissions of healthy apples were per se attractive to the parasitoid, and even preferred over volatiles from infested apples. Simultaneous volatile collections from healthy apples on twigs in the field were analysed throughout the season, showing that the overall quantity of headspace volatiles peaks at the beginning of June and mid‐August. The seasonal volatile emission is correlated with the behaviour of the parasitoid during the fruit ripening stage. The results are discussed in relation to the use of H. pallidus as a potential biocontrol agent, in order to enhance current integrated pest management (IPM) programs.

13C-labelling patterns of green leaf volatiles indicating different dynamics of precursors in Brassica leaves.

To investigate the dynamics of precursor compounds of green leaf volatiles (GLV)s and other biogenic compounds released by mechanically damaged Brassica oleracea leaves, plants were exposed for two consecutive 16h light phases to highly enriched (13)CO(2). Analysis by GC-MS indicated (1) biogenic compounds released upon wounding, (2) a different labelling pattern between and (3) within compounds, and (4) evidence for spatial heterogeneity of the precursor pool extrapolated from points (1)-(3). First, GLVs comprised C(5) and C(6) molecules, with the GLV pentenyl acetate being reported here for the first time from higher plants. Second, the labelling pattern found in most GLVs indicates a low turnover of the precursor alpha-linolenic acid. Moderate labelling of dimethyldisulphide indicates a connection to an active plastidic methyl pool closely connected to CO(2) fixation, and very weak labelling of terpenes indicates a constitutive monoterpene pool. Third, not all GLVs exhibit similarly strong labelling patterns (hexenyl acetate vs. hexyl acetate), indicating different precursors. As the labelling patterns of alcohol and acetate moieties in the esters differ, with only the former being strongly labelled, the precursor of the acetate moiety, acetyl-CoA, is likely to derive from a different cellular pool to that used in chloroplastic fatty acid synthesis, or was rapidly synthesised after the end of labelling. Fourth, the exceptionally high relative abundance of labelled GLV and the low concentration of unlabelled molecules are likely to occur because recently synthesized alpha-linolenic acid is bound in lipids that are organised in distinct areas, or are chemically different from the older lipids. They must be preferentially used as precursors.

Terpene-mediated parasitoid host location behavior on transgenic and classically bred apple genotypes.

Terpene-mediated interactions between transgenic or classically bred apple genotypes and associated insects were investigated. Apple genotypes were either resistant or susceptible to Venturia inaequalis that causes apple scab. They were subjected to infestation by Phyllonorycter leafminers and/or inoculation with V. inaequalis. Apple leaf extracts were analyzed by gas chromatography-mass spectrometry to quantify squalene, a triterpene known to mediate host location by Pholetesor parasitoids that are specialized on leafminers. Squalene contents in leafminer-infested leaves differed between the transgenic apple scab resistant line and a classically bred cultivar sharing the same resistance gene. This resistant cultivar showed an increase in squalene contents from healthy to leafminer-infested leaves. This was not the case in the transgenic resistant line. However, there was also no increase in the susceptible isogenic cultivar. Behavioral bioassays with parasitoid females also reflected these findings. Hence, alterations in leaf chemistry and corresponding responses of the parasitoid are apparent among classically bred cultivars, rather than in the genetically modified resistant line.

How transgenic and classically bred apple genotypes affect non-target organisms on higher trophic levels

Current plant breeding comprises classical techniques as well as biotechnological methods suitable to insert target genes into the plant genome. Potential non‐target effects of these newly developed plants have to be evaluated in appropriate risk assessment studies. We investigated non‐target effects of four apple genotypes (Malus × domestica L. Borkh.; Rosaceae). They were either susceptible or resistant to the fungal phytopathogen causing apple scab, Venturia inaequalis (Cke.) Wint. (Ascomycotina: Pleosporales), and were either of transgenic origin or classically bred, differing in or sharing a single trait each. Experiments on insect performance were carried out with two non‐target species, namely the apple leafminer Phyllonorycter blancardella Fabricius (Lepidoptera: Gracillariidae) and its parasitoid Pholetesor circumscriptus Nees (Hymenoptera: Braconidae). Development time of the apple leafminer was not affected by the four apple genotypes. By contrast, the number of emerged adult moths significantly differed between the two classically bred cultivars, whereas no effect of the two transgenic apple genotypes was detected. Paralleling the observations of the apple leafminer development, no impact of the four apple genotypes on parasitoid development time and parasitism success could be detected. Although mean numbers of emerged parasitoids were lower on the cultivars with fewer emerged apple leafminers, these differences were not significant. Herbivore and parasitoid performance was not altered in the presence of pathogen inoculation. In conclusion, host plant suitability was found to be more determined by variability among classically bred apple cultivars than by the transgenic genotypes tested. To elucidate possible effects of transgenic plants within multitrophic interactions, an appropriate risk assessment incorporating the relevant controls is crucial.

Can interventions that aim to decrease Lyme disease hazard at non-domestic sites be effective without negatively affecting ecosystem health? A systematic review protocol

BackgroundLyme disease (LD) is the most commonly reported, broadly distributed vector-borne disease of the northern temperate zone. It is transmitted by ticks and, if untreated, can cause skin, cardiac, nervous system and musculoskeletal disease. The distribution and incidence of LD is increasing across much of North America and Western Europe. Interventions to decrease exposure to LD hazard by encouraging behavioural change have low acceptance in high risk groups, and a safe, effective human LD vaccine is not presently available. As a result, habitat level interventions to decrease LD hazard itself (i.e. levels of infected ticks) have been proposed. However, some interventions may potentially negatively affect ecosystem health, and consequentially be neither desirable, nor politically feasible. This systematic review will catalogue interventions that aim to reduce LD hazard at non-domestic sites, and examine the evidence supporting those which are unlikely to negatively affect ecosystem health.MethodsThe review will be carried out in two steps. First, a screening and cataloguing stage will be conducted to identify and characterise interventions to decrease LD hazard at non-domestic sites. Secondly, the subset of interventions identified during cataloguing as unlikely to negatively affect ecosystem health will be investigated. In the screening and cataloguing step literature will be collected through database searching using pre-chosen search strings, hand-searching key journals and reviewing the websites of public health bodies. Further references will be identified by contacting stakeholders and researchers. Article screening and assessment of the likely effects of interventions on ecosystem health will be carried out independently by two reviewers. A third reviewer will be consulted if disagreements arise. The cataloguing step results will be presented in tables. Study quality will then be assessed independently by two reviewers, using adapted versions of established tools developed in healthcare research. These results will be presented in a narrative synthesis alongside tables. Though a full meta-analysis is not expected to be possible, if sub-groups of studies are sufficiently similar to compare, a partial meta-analysis will be carried out.