Adaptation an Pflanzliche Pyrrolizidinalkaloide Bei Phytophagen Käfern (Coleoptera, Chrysomelidae)

Adaptation an pflanzliche Pyrrolizidinalkaloide bei phytophagen Käfern (Coleoptera, Chrysomelidae)

im Fachbereich Biologie der Universität Hamburg eingereichte Dissertation zur Erlangung des Doktorgrades

von Ingo Narberhaus aus Aachen

Hamburg 2004 Tag der Disputation: 30.04.2004

Gutachter: Prof. Dr. S. Dobler

G Inhalt

1 Einführung 7

2 Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles (Coleoptera: Chrysomelidae) adapted and non adapted to alkaloid containing host plants 12

2.1 Summary 12 2.2 Introduction 12 2.3 Material and Methods 14 2.4 Results 18 2.5 Discussion 24

3 Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles (Coleoptera, Chrysomelidae) 27

3.1 Summary 27 3.2 Introduction 27 3.3 Material and Methods 29 3.4 Results 31 3.5 Discussion 35

4 Direct evidence for membrane transport of host plant derived pyrrolizidine alkaloid N-oxides in two leaf beetle genera 38

4.1 Summary 38 4.2 Introduction 38 4.3 Material and Methods 41 4.4 Results 46 4.5 Discussion 54 5 Pyrrolizidine alkaloids on three trophic levels – evidence for toxic and deterrent effects on phytophages and predators 59

5.1 Summary 59 5.2 Introduction 59 5.3 Material and Methods 61 5.4 Results and Discussion 63

6 Abschliessende Diskussion 67

6.1 Speicherung und Metabolisierung von PAs bei Longitarsus 67 6.2 Membran-Carrier für PAs 69 6.3 Strategien der PA-Sequestration im Vergleich 70 6.4 Evolutionäre Aspekte der PA-Adaptation bei Longitarsus 71 6.5 Toxizität von PAs 73

Literatur 76 4

Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus fremden Arbeiten direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Die Ergebnisse der Arbeit wurden in den unten aufgeführten vier Beiträgen vorab veröffentlicht bzw. eingereicht; die Hauptkapitel der Dissertation sind in dieser Form, also auch in englischer Sprache, belassen. Teilergebnisse wurden außerdem in Tagungsbei- trägen veröffentlicht.

Publikationen

NARBERHAUS, I., THEURING, C., HARTMANN, T., DOBLER, S. 2003. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles (Coleoptera: Chrysomelidae) adapted and non adapted to alkaloid containing host plants. Journal of Comparative Physiology 173:483-491.

NARBERHAUS, I., THEURING, C., HARTMANN, T., DOBLER, S. 2004. Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles (Coleoptera, Chrysomelidae) Chemoecology 14:17-23.

NARBERHAUS, I., PAPKE, U., BEUERLE, T., THEURING, C., HARTMANN, T., DOBLER, S. submitted. Direct evidence for membrane transport of host plant derived pyrrolizidine alkaloid N-oxides in two leaf beetle genera. Journal of Chemical Ecology.

NARBERHAUS, I., ZINTGRAF, V., DOBLER, S. submitted. Pyrrolizidine alkaloids on three trophic levels – evidence for toxic and deterrent effects on phytophages and predators. Functional Ecology.

Tagungsbeiträge

Narberhaus I: Comparative physiology of evolutionary adaptations to host plant alkaloids in a phytophagous beetle genus. 7th Meeting of PhD students in Evolutionary Biology, Bernried 21.3.-24.3.01 (Vortrag) 5

Zintgraf V, I Narberhaus, S Dobler: Zur Evolution des schlechten Geschmacks - Varia- bilität und Funktion der Alkaloid-Sequestration bei Flohkäfern der Gattung Longitarsus (Coleoptera, Chrysomelidae). Graduiertentreffen der Studiengruppe Evolutionsbiologie der DZG, Regensburg, 8.-10.2.02 (Poster)

Narberhaus I, S Dobler: Physiologische Anpassungen an Wirtspflanzen-Alkaloide bei Flohkäfern der Gattung Longitarsus. Multitrophische Interaktionen, Göttingen, 11.- 12.4.02 (Vortrag)

Narberhaus I, T Hartmann, S Dobler: Physiological adaptations to host plant alkaloids in leaf beetles of the genus Longitarsus. 19th Annual Meeting of the International Society of Chemical Ecology, Hamburg, 3.-8.8.02 (Vortrag)

Narberhaus I, T Hartmann, S Dobler: Pyrrolizidine alkaloid sequestration by Longitarsus flea beetles (Coleoptera, Chrysomelidae). DZG-Tagung Berlin, 9.-13.6.03 (Poster) 6

1 Einführung

Die Interaktionen zwischen Insekten und Pflanzen sind auf vielfältige Weise von pflanzlichen Inhaltsstoffen abhängig, insbesondere von Sekundärstoffen. Sekundäre Pflanzenstoffe sind Substanzen, die nicht dem pflanzlichen Grundstoffwechsel, sondern eigenen metabolischen Synthesewegen entspringen und dabei keine physiologischen, sondern vorwiegend ökologische Funktionen haben. Sie dienen der Pflanze zum Anlocken von Bestäubern, wirken allelopathisch gegen Konkurrenten, schützen sie als Fungizide oder Bakterizide vor Pathogenen oder als Giftstoffe vor Herbivoren (Harborne, 1995; Schlee, 1992). Etwa ein Drittel der rund 100.000 bekannten pflanzlichen Sekundärstoffe sind Alkaloide. Eine chemisch vielfältige Gruppe darunter, die besondere Relevanz in Tier-Pflanze-Interaktionen hat, sind die Pyrrolizidin-Alkaloide (PAs) (Hartmann, 1991, 1995). Hierbei handelt es sich um Esteralkaloide, bestehend aus einer Necinbase (dem bizyklischen Pyrrolizidin-Kern), die mit einer oder zwei Necinsäuren verestert ist (Abb. 1.1). Sie können als Monoester, offenkettige Diester oder auch als makrocyclische Diester vorkommen. Bisher sind fast 400 verschiedene Strukturen aus ca. 600 Pflanzenarten isoliert und beschrieben worden. PAs sind häufige Inhaltsstoffe in einigen Gattungen der Asteraceae (Tribus Senecioneae und Eupatorieae), in den meisten Boraginaceae und sporadisch in weiteren Pflanzenfamilien (z.B. Fabaceae, Orchidaceae, Apocynaceae) (Hegnauer, 1962-2001; Roeder, 1995). Obgleich es an direkten Belegen mangelt, wird generell davon ausgegangen, dass Pflanzen PAs zur chemischen Verteidigung einsetzen (Boppré, 1990; Hartmann, 1995). Boppré (1986) berichtet von Fütterungstests, in denen Tiere verschiedenster Taxa PA-kontaminierte Nahrung ablehnten, darunter Säuger, Vögel, Reptilien, Amphi- bien und Insekten. Zu proximaten Merkmalen der Tiere wie geschmackliche Erkennung von PAs und Fraßhemmung kann als ultimater Faktor nur die Toxizität der Stoffe führen. Diese ist durch zahlreiche Studien gut belegt. So ist lange bekannt, dass PAs zu ernsthaften Erkrankungen bei Weidetieren oder Menschen führen können (z.B. Fowler, 1968; Kumana et al., 1985; Stillman et al., 1977). PAs wirken bei Wirbeltieren hepatotoxisch und pneumotoxisch (Mattocks, 1986) und, wie durch in vitro Untersuchungen gezeigt wurde, auch neurotoxisch (Schmeller, 1997). Einführung 7

Necinsäure

Necinbase

Abb. 1.1 Aufbau eines tertiären Pyrrolizidin-Alkaloids (Senecionin).

Die salzartigen PA-N-oxide, wie sie in den meisten Pflanzenarten vorkommen, sind jedoch nicht per se toxisch (Hartmann et al., 1989). Erst durch ihre chemische Reduktion werden harmlose N-Oxide zu protoxischen tertiären Alkaloiden („freien Basen“), ein Vorgang, der im reduzierenden Darmmilieu der meisten Herbivoren passiv abläuft. Tertiäre PAs sind lipophil und diffundieren leicht durch biologische Membranen wie die Darmwand. Wenn dies geschieht, können in der Leber eines herbivoren Vertebraten in Folge einer Bioaktivierung durch mikrosomale Cytochrom P-450 Enzyme PAs in instabile Pyrrolverbindungen umgewandelt werden. Dies sind hoch aktive, alkylierende Zwischenprodukte (Winter and Segall, 1989), die mit Proteinen und Nukleinsäuren reagieren und diese damit inaktivieren. Hiermit gehen Veränderungen der Zellfunktionen einher, die bis zum Zelltod oder zur Auslösung einer Karzinogenese führen können (Mattocks, 1986). Einige Säugetierarten besitzen die Möglichkeit einer Re-N-oxidierung von PAs als Entgiftungsmechanismus. Hierbei wird das potentiell toxische tertiäre Alkaloid in sein ungiftiges N-Oxid überführt, welches wiederum nicht in das pyrrolische Toxin umgewandelt werden kann (Cheeke, 1994). Meerschweinchen und Schafe z.B. verfügen über mikrosomale Multisubstrat-Flavin-Monooxygenasen, die eine schnelle Transforma- tion aufgenommener PAs in N-Oxide ermöglichen. Diese N-Oxide werden daraufhin rasch ausgeschieden, was die Resistenz dieser Tiere gegenüber PAs erklärt (Huan et al., 1998a; Huan et al., 1998b; Miranda et al., 1991). Auch bei Insekten werden toxische Effekte von PAs angenommen. Obgleich direkt schädigende Effekte auf Stoffwechsel Einführung 8 und Entwicklung des Individuums bisher sehr mangelhaft überprüft sind, werden hier P- 450-Enzyme mit schädlichen Effekten vermutet, vergleichbar mit denen der Vertebraten (Hodgson, 1985). Nachgewiesen wurde bisher lediglich, u.a. in „wing-spot-tests“ mit Drosophila, eine mutagene Wirkung von PAs (Frei et al., 1992; Zijlstra and Vogel, 1988). Hoch spezialisierte phytophage Insektenarten machen sich die Giftigkeit von PAs bisweilen zunutze. Substanzen, die von der Pflanze als Abwehrstoffe hergestellt werden, werden von diesen eng angepassten Arten nicht nur toleriert, sondern in ihren Körpern aktiv angereichert und dienen ihnen mitunter als Stimulanzien. In einer klassischen Untersuchung am Jakobskrautbär Tyria jacobaeae (Lepidoptera, Arctiidae) zeigten Aplin et al. (1968) zum ersten Mal, dass Insekten PAs in hohen Konzentrationen speichern und teilweise in neue Metaboliten umwandeln können. Später wurde deutlich, dass die Tiere Alkaloide auf diese Weise „recyceln“ und zur Verteidigung gegen ihre eigenen Raubfeinde benutzen (Boppré, 1986; Hartmann, 1999; Hartmann and Witte, 1995; Schneider, 1987). Dieser Vorgang wird als Sequestration bezeichnet. Inzwischen sind PA-sequestrierende Insekten aus diversen Insektentaxa bekannt: aus verschiedenen Gruppen der Lepidoptera, Coleoptera (v.a. Blattkäfern), Orthoptera (der Heuschrecke Zonocerus) und einigen Homoptera (einigen Blattläusen) (Hartmann, 1999). Ein eindrucksvolles Beispiel einer besonders hochgradigen Anpassung an PAs ist die Arctiide Utetheisa ornatrix (Eisner and Meinwald, 1987, 1995). Deren Larven sequestrieren hohe Konzentrationen von PAs aus Wirten der Gattung Crotalaria (Fabaceae) und transferieren sie durch die Metamorphose hindurch in die Imagines. Um seine Eier mit den schützenden PAs auszustatten, verwendet das Weibchen neben eigenen PAs auch solche, die es vom Männchen während der Samenübergabe als „Hochzeitsgeschenk“ erhalten hat (Dussourd et al., 1991; Dussourd et al., 1988). Die meisten PA sequestrierenden Insekten speichern diese Gifte in jener unschädlichen Form, in der sie auch in den Zellvakuolen der Wirtspflanzen vorliegen (Mattocks, 1971), als PA-N-oxide. Beispiele, für die dies bekannt ist, sind die Raupen einiger Schmetterlingsarten, darunter verschiedene Arctiiden (Ehmke et al., 1990; Hartmann et al., 1990; von Nickisch-Rosenegk et al., 1990; von Nickisch-Rosenegk and Wink, 1993) und Ithomiinen (Brückmann et al., 2000; Trigo et al., 1996), Blattkäfer der Gattung Oreina (Hartmann et al., 1997; Pasteels et al., 1988; Rowell-Rahier et al., 1991) oder die Heuschrecke Zonocerus variegatus (Bernays et al., 1977; Biller, 1994; Lindigkeit et al., 1997). Obgleich das Ziel der ungiftigen Speicherung in all diesen Fällen dasselbe ist, gibt es verschiedene Möglichkeiten, dieses zu erreichen. Eine davon ist die passive Aufnahme reduzierter, tertiärer PAs in den Körper und deren Re-N-oxidierung mittels eines Enzyms, wie es oben schon für die Meerschweinchen beschrieben ist. Einführung 9

Unter den Insekten sind einige Arctiiden und die Heuschrecke Zonocerus variegatus in ihrer Fähigkeit zur N-Oxidierung bekannt (Lindigkeit et al., 1997). Eine zweite Lösung ist die Unterdrückung der Reduktion aufgenommener PA-N-oxide im Darm und ein schneller, spezifischer Transfer dieser hydrophilen Moleküle durch die Darmmembran in den Körper. Dieser Mechanismus wurde zwar bereits bei der Blattkäfergattung Oreina angenommen, konnte bisher jedoch nicht eindeutig nachgewiesen werden (Hartmann et al., 1999; Rowell-Rahier et al., 1991). Auch für Blattkäfer der Gattung Longitarsus ist die Sequestration von Pyrrolizidin- Alkaloiden nachgewiesen worden (Dobler et al., 2000; Haberer and Dobler, 1999). Diese Käfer stellen mit weltweit über 500 beschriebenen Arten eine der artenreichsten Gattungen der Chrysomeliden dar. Allein für die paläarktische Region sind 150, für Mitteleuropa 77 Arten nachgewiesen worden (Döberl, 1994; Douget, 1994). Bei der Betrachtung einer auf DNA-Sequenzdaten basierenden Stammbaumhypothese der Gattung (Dobler, 2001) fällt die breite Streuung von Kladen auf, die Arten mit PA-haltigen Asteraceen und Boraginaceen als Wirtspflanzen enthalten. Diese Verteilung legt nahe, dass die Besiedlung von PA-haltigen Pflanzengruppen innerhalb der Gattung Longitarsus mehrfach erfolgt ist und wirft damit die folgende Frage auf: Sind die chemischen Hürden, die die Pflanzen gegen ihre Bedrohung durch Phytophage aufstellen, ein einziges Mal von einem gemeinsamen Vorfahren der heute PA-angepassten Longitarsus-Arten überwunden worden oder ist dies in der Evolution der Gattung mehrmals erfolgt? Die vorliegende Arbeit soll dazu beitragen, die evolutionären Pfade der Anpassung an PAs innerhalb der Gattung Longitarsus nachzuvollziehen. Durch die Charakterisierung und den zwischenartlichen Vergleich adaptiver Merkmale auf Verhaltens- und physiologischer Ebene sollen Aussagen ermöglicht werden, ob solche Anpassungen als „Wegweiser“ für Wirtswechsel zwischen Pflanzen mit ähnlichen Sekundärstoffkompositionen dienen können. Primäres Ziel der Arbeit war daher ein detailliertes Verständnis der Anpassungen von Longitarsus-Arten an Pyrrolizidin- Alkaloide als Inhaltsstoffe ihrer Wirtspflanzen. Die im Rahmen der vorliegenden Dissertation durchgeführten Experimente lassen sich in vier Teilprojekte aufteilen, gemäß derer die Ergebnisse in Kapiteln präsentiert werden. In einer ersten Studie wurde zunächst in einem größeren Artvergleich die Wirkung von PAs auf das Fraßverhalten der Käfer untersucht (Kapitel 2). In Wahlversuchen wurde untersucht, ob PAs auf Longitarsus-Arten ohne PA-haltige Wirte einen abschreckenden Effekt haben und ob sie auf solche mit PA-haltigen Wirten einen fraßstimulierenden Effekt haben. Weiterhin sollte in diesem Teil anhand exemplarischer Arten das Schicksal der Substanzen nach der Aufnahme durch die Käfer verfolgt Einführung 10 werden. Anhand von Fütterungs- und Injektionsexperimenten mit radioaktiv markierten Alkaloiden wurde geprüft, wie PAs in den Käfern metabolisiert und in welcher Form sie in ihren Körpern gespeichert werden. Das PA oxidierende Enzym, als im Wesentlichen sich herausstellende physiologische Schlüsselanpassung, wurde im Weiteren in seiner Spezifität gegenüber PAs getestet, indem noch weitere Tracer anderer Klassen von Pflanzenalkaloiden eingesetzt wurden. Außerdem wurde bei zwei Käferarten die Affinität ihrer Enzyme auf verschiedene PA-Typen, in denen sich die Wirte der beiden Arten unterscheiden, getestet. In einem unmittelbar daran anschließenden Teilprojekt wurde in längerfristigen Speicherexperimenten der Zeitverlauf der PA-Sequestration untersucht (Kapitel 3). Außerdem wurden hier vergleichende Fütterungsexperimente mit unterschiedlichen PA- Konzentrationen angesetzt, die einerseits einer Abschätzung der Effizienz des Enzyms und andererseits seiner Lokalisierung dienten sollten. Ein drittes Experiment in diesem Teil war die Sektion einiger Käfer, die mit einem Tracer-PA gefüttert waren, um die PA- Speicherorte in den Insektenkörpern zu lokalisieren. Neben dem N-oxidierenden Enzym wurde noch einer zweiten Schlüssel- anpassung nachgegangen (Kapitel 4). Um die Form der Aufnahme von PAs durch die Darmmembran zu identifizieren, wurde ein Fütterungsexperiment mit einem 18O- markierten Alkaloid durchgeführt, das eigens hierfür hergestellt worden war. Als Vergleichsarten dienten neben dem bereits näher bekannten Longitarsus jacobaeae zwei Blattkäferarten der Gattung Oreina. Diese alpin verbreiteten Käfer besitzen exokrine Wehrdrüsen, aus denen sie eine konzentrierte PA-Lösung abgeben. Das letzte Teilprojekt nahm sich der Frage nach dem Zweck der PA- Sequestration durch Insekten an (Kapitel 5). Im Mittelpunkt der manipulativen Experimente mit Carabiden stand die Hypothese, dass sequestrierte PAs in Longitarsus Blattkäfern eine abschreckende Wirkung gegenüber potentiellen Raubfeinden haben. Außerdem wurde in diesem Teil die Toxizität von PAs bei nicht angepassten Insekten überprüft, die wie oben erwähnt bisher nur schlecht belegt ist. Hierzu wurden über einen längeren Zeitraum Raupen des Seidenspinners Philosamia ricini (Lepidoptera, Saturniidae) mit künstlich PA-behandelten Wirtsblättern gefüttert und in ihrem Lebenszyklus verfolgt. 11

2 Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles (Coleoptera: Chrysomelidae) adapted and non adapted to alkaloid containing host plants

2.1 Summary

Several Longitarsus flea beetle species sequester pyrrolizidine alkaloids (PAs) acquired from their Asteraceae and Boraginaceae host plants. Here we carried out feeding and injection experiments using radioactively labeled PAs to investigate the physiological mechanisms of uptake, metabolism and storage of alkaloids in adult beetles. We examined six Longitarsus species belonging to different phylogenetic clades in a comparative approach. All species that accepted PAs in a preceding food choice study showed the ability both to store PA N-oxides and to metabolize tertiary PAs into their N- oxides. Regardless of whether the beetles' natural host plants contain PAs or not, these species were found to possess an oxidizing enzyme. This oxygenase appears to be specific to PAs: [3H]atropine and [14C]nicotine, two alkaloids not related to PAs, were neither stored nor N-oxidized by any of the tested species. One species, L. australis, that strictly avoids PAs behaviorally, exhibited a lack of adaptations to PAs on a physiological level as well. After injection of tertiary [14C]senecionine, beetles of this species neither N- oxidized nor stored the compounds, in contrast to L. jacobaeae, an adapted species that underwent the same treatment. L. jacobaeae demonstrated the same efficiency in N- oxidation and storage when fed or injected with tertiary [14C]senecionine.

2.2 Introduction

Various groups of phytophagous insects are known to sequester pyrrolizidine alkaloids (PAs) from their host plants (Brown and Trigo, 1995; Hartmann, 1999; Hartmann and Ober, 2000). Many of these animals have been shown to make use of the acquired substances to defend themselves against vertebrate and invertebrate predators (Brown, 1984; Gonzales et al., 1999; Orr et al., 1996; Rowell-Rahier et al., 1995; Trigo et al., 1993). Possibly, PAs occur so frequently as defensive chemicals because they exist in two transformable chemical forms (Hartmann, 1999): On the one hand, the protoxic, lipophilic tertiary alkaloid and on the other the non-toxic, hydrophilic N-oxide, as which PAs are stored in most plants and adapted insects. Studies with PA sequestering insects Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 12 brought to light different strategies to safely handle these substances. In any case they prevent exposure to detrimental concentrations of the protoxic tertiary form of PAs. Food plant records of Longitarsus flea beetles (Coleoptera, Chrysomelidae) (Döberl, 1994; Douget, 1994) indicate that many species of this genus use plants that are known to contain PAs as hosts (Hartmann and Witte, 1995; Roeder, 1995). These PA host plants of Longitarsus constitute two groups that can be distinguished by the PA types they produce (Figure 2.1). The first group is comprised of species of the Asteraceae tribe Senecioneae containing PAs of the senecionine type. The second group covers species of the Asteraceae tribe Eupatorieae and of the Boraginaceae containing PAs of the lycopsamine type (Hartmann and Witte 1995). Several Longitarsus species have recently been shown to sequester PAs in concentrations of up to 0.5% of their dry weight (Dobler et al., 2000; Haberer and Dobler, 1999). So far there is little evidence on the physiological mechanisms involved, whether there are parallels to the other PA sequestering leaf beetle taxa and whether they are identical for different Longitarsus species on different plants or belonging to different clades.

senecionine senecionine N-oxide

rinderine

Figure 2.1 Structures of the two pyrrolizidine alkaloid (PA) types in Longitarsus host plants, senecionine representing the senecionine type and rinderine the lycopsamine type. The structure of senecionine N-oxide is shown as an example for an alkaloid N-oxide. For tracer experiments both alkaloid types were [14C]-labeled at carbons 3, 5, 8 and 9. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 13

The purpose of the present study was to use a comparative approach to analyze the behavioral response of Longitarsus beetles to PAs and to follow their uptake, storage and metabolism. Our long term goal is to understand the evolution of beetle adaptations to PAs in their host plants by integrating the physiological data with a molecular phylogeny of the genus (Dobler, 2001). However, this goal still awaits a stable resolution of decisive nodes within the phylogenetic tree. Here, we want to address the following questions: 1) How do PAs influence the host choice behavior of Longitarsus species? 2) What are the physiological mechanisms of the beetles' adaptation to their host PAs? 3) How specific is the detoxification process? Is it only acting on PAs or does it merely reflect a more general unspecific detoxification process? 4) Do species discriminate between chemically different types of PAs and are there differences in the efficiency of PA handling between species? 5) What happens to PAs in non adapted species? The influence of PAs on the beetles' host choice behavior (1) was tested in different feeding experiments using host leaves that were artificially covered with an unlabeled PA. All investigations on the physiological level (2) were performed by feeding radioactively labeled PAs to the beetles. To test the PA specificity of the adaptation (3) other alkaloid groups, structurally not related to PAs, were tested with two species. The tropane alkaloid atropine and the tobacco alkaloid nicotine were fed to L. succineus and L. jacobaeae as tertiary [3H]atropine and [14C]nicotine. To examine a possible specialization of the sequestration mechanism to certain PA types (4), L. jacobaeae and L. aeruginosus were fed both with the PA type of their host plants and with a PA type that they are not confronted with in nature. [14C]Senecionine and its N-oxide were chosen as representatives of PAs of the senecionine type and [14C]rinderine plus its N-oxide for those of the lycopsamine type. With both species the efficiency to accumulate and metabolize the different PA types was studied. Finally, to compare the metabolism of adapted and non adapted species (5), injection experiments with tertiary [14C]senecionine were performed with L. jacobaeae and L. australis.

2.3 Material and Methods

Insects and host plants Adult beetles and their host plants were collected at various field sites in the Black Forest, the Swiss Alps and the Swiss Jura from April to September 2000/01 and kept at 20°C under long day conditions (16h/8h light/dark) until use (Table 2.1). Identification of species were performed according to Döberl (1994). Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 14

Table 2.1 Investigated Alticinae species, the host plants from which they were collected and collection sites. L. = Longitarsus, A. = Aphtona, BW = Baden-Württemberg, BE = Bern, CH = Switzerland, D = Germany, JU = Jura, VS = Wallis.

PAs in host Beetle species Host plant Host family Collection site plant L. aeruginosus Eupatorium cannabinum Asteraceae + Freiburg, BW, D L. anchusae Symphytum officinale Boraginaceae + Freiburg, BW, D L. atricillus Ranunculus repens Ranunculaceae - Ebringen, BW, D L. australis Scrophularia canina Scrophulariaceae - Grißheim, BW, D L. exoletus Echium vulgare Boraginaceae + Grißheim, BW, D L. luridus Plantago lanceolata Plantaginaceae - Ebringen, BW, D L. jacobaeae Senecio jacobaea Asteraceae + St. Imier, BE, CH L. melanocephalus Plantago lanceolata Plantaginaceae - Ebringen, BW, D L. membranaceus Teucrium scorodonia Lamiaceae - Freiburg, BW, D L. nigrofasciatus Verbascum thapsus Scrophulariaceae - Riegel, BW, D L. pellucidus Convolvulus arvensis Convolvulaceae - Freiburg, BW, D L. pratensis Plantago lanceolata Plantaginaceae - Ebringen, BW, D L. quadriguttatus Cynoglossum officinale Boraginaceae + Delemont, JU, CH L. rubiginosus Convolvulus arvensis Convolvulaceae - Waltershofen, BW, D L. succineus Achillea millefolium Asteraceae (-)* Wiler, VS, CH L. suturellus Petasites albus Asteraceae + Zastler, BW, D A. coerulea Iris pseudacorus Liliaceae - Opfingen, BW, D A. cyparissiae Euphorbia cyparissias Euphorbiaceae - Grißheim, BW, D * L. succineus has hosts with and without PAs, Achillea does not contain PAs.

Food choice experiments Seneciphylline was chosen as exemplary PA due to its frequent occurrence in the beetles’ host plants and its commercial availability. Seneciphylline N-oxide was produced by oxidation of tertiary seneciphylline (Roth) according to Craig and Purushothaman (1970). The purity of the resulting N-oxide was verified by TLC and detection according to Mattocks (1967). A total of 13 species were tested for feeding preference: Longitarsus atricillus, L. australis, L. exoletus, L. luridus, L. melanocephalus, L. membranaceus, L. nigrofasciatus, L. pratensis, L. quadriguttatus, L. rubiginosus, L. suturellus, Aphtona coerulea and A. cyparissiae. For each experiment 15 beetles were placed individually in Petri dishes (12 cm diameter) and kept at 20°C and 16h/8h light/dark regime. To every beetle two leaf discs (1 cm diameter) of their host plant were offered, one painted with 10 – 20 µl of a methanolic seneciphylline N-oxide solution and one with the same volume of pure methanol as a control. The total alkaloid concentration applied to the leaf discs was adjusted to 2% seneciphylline per leaf dry weight. After a 48h feeding period the leaf discs were scanned and the surface areas eaten measured. In some species the feeding period was extended to 72 h for all individuals if the ingested amounts were considered too small. The Wilcoxon paired sample test was used to test for differences in eaten leaf surfaces between treated and untreated leaves. Species that did not eat any of the PA- Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 15 treated leaf material were additionally tested for PA avoidance under no-choice conditions where beetles were only offered seneciphylline-N-oxide painted leaves.

Tracer feeding experiments [14C]Senecionine and [14C]rinderine (1.07-1.34 GBq⋅mmol-1) and their N-oxides were prepared biosynthetically from [1,4-14C]putrescine-dihydrochloride (4.1 GBq⋅mmol-1; Amersham Biosciences, Freiburg, Germany) using root cultures of Senecio vulgaris L. (for [14C]senecionine) and Cynoglossum officinale L. (for [14C]rinderine) according to Hartmann (1994). [3H]Atropine was supplied by DuPont NEN Products (Boston, MA) and [14C]nicotine by Movarek Biochemicals (Brea, CA). Respective N-oxides of labeled PAs were produced as described above. Six species of Longitarsus were fed with [14C]senecionine and [14C]senecionine N- oxide: three species that naturally encounter PAs in their host plants (L. aeruginosus, L. jacobaeae, L. anchusae), one species that occurs on both PA- and non PA hosts (L. succineus) and two species that accept artificial PA diet but do not naturally feed on PA hosts (L. luridus, L. pellucidus). Since single beetles are too small (1-2 mg fresh weight) to be analyzed individually, we carried out all tracer feeding experiments with groups of ten and all injection experiments with 20 beetles each. This approach evens out biological variability between individuals and yields a reliable mean of accumulated and metabolized amounts but does not provide information on variation. However, this information is not relevant to the interpretation of most experiments described here, since the aim was simply to decide whether the beetles have or do not have the capacity to take up or metabolize the alkaloids offered. An interpretation of species specific differences was only intended in additional comparative feeding experiments with L. jacobaeae and L. aeruginosus using different PA-types. These experiments were repeated in five replicates each which allowed us to measure variability and to quantify differences statistically. The beetles were placed in groups of ten in Petri dishes (12 cm diameter) lined with moist filter paper and kept at 20°C at 16 h light and 8 h dark. For every tracer experiment ca 30 µl of a methanolic solution of the labeled alkaloid (2.5 ⋅ 105 cpm) was painted on a 1 cm diameter disc of a fresh host plant leaf and offered to the beetles for 48 h. After the feeding period the remains of the leaf were removed and a piece of untreated host plant leaf was offered for another 48 h. At the end of every experiment, the beetles were killed by freezing and stored in methanol until extraction. The feces were washed from the Petri dish surfaces and stored in methanol together with the filter paper until extraction. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 16

Injection experiments To localize the N-oxidizing enzyme activity within the beetle, we injected labeled tertiary senecionine into the hemolymph of beetles of two species, one adapted to PAs, i.e. L. jacobaeae, and one not adapted, i.e. L. australis. Individuals were mechanically fixed in plasticine to immobilize them during injection on a cooled aluminum block. Under a stereo lens the intersegmental skin between the beetles' last abdominal sternites was perforated with a fine needle and a glass capillary (diameter < 1 µm) was introduced. Using a hydraulic microinjector device (Sutter, USA) ca 150 nl of a methanol-ringer solution (1:4, by volume) of labeled tertiary senecionine were injected into the hemolymph. Exuding body fluid was removed with a piece of filter paper. After the treatment the beetles were released into cages and allowed to feed on fresh leaves of their host plant for 48 h. The subsequent treatment of the animals was identical to that of the orally fed ones. For every species 20 individuals were injected and stored in methanol until extraction.

Extraction procedure The beetles were ground in a mortar and extracted with 500 µl methanol. After centrifugation the supernatant was recovered and the residual pellet extracted twice with 500 µl methanol. The combined solutions were dried under an air stream and the residue re-dissolved in 400 µl methanol. The feces fractions were extracted three times with methanol, dried and recovered in 2 ml methanol. Small aliquots of these crude extracts were analyzed for total radioactivity by scintillation counting.

Thin-Layer Chromatography (TLC) Crude methanol extracts from beetles and feces were separated by TLC and analyzed using a multichannel radioactivity detector (Rita-32a, Raytest). Separation was achieved on silica gel 60 plates (Merck) using the solvent system dichloromethane:MeOH:NH4OH (25%) (82:15:3, by volume) for senecionine and the solvent system ethylacetate:isopropanol:NH4OH (25%) (45:35:20, by volume) for rinderine, atropine and nicotine. References of tertiary and N-oxide PAs were used to identify metabolites. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 17

2.4 Results

Feeding behavior The response to seneciphylline of 13 flea beetle species, 11 Longitarsus species and two of the sister genus Aphthona for comparison, was analyzed (Table 2.2). Of the 13 species tested seven did not respond to PAs at all and preferred the untreated host plant leaf. Three of these species refused feeding even under no-choice conditions which means that they would rather starve than ingest seneciphylline. These were the two Scrophulariaceae feeding Longitarsus species, L. australis and L. nigrofasciatus, and the Iridaceae feeding Aphthona coerulea. Only one species, L. suturellus, showed a preference for the alkaloid treated leaf.

Table 2.2 Feeding experiments with several Longitarsus and Aphthona species with unlabelled seneciphylline (sen) under choice (FC) and no choice (NC) conditions. Rejections or preferences are given, if eaten leaf areas differed significantly (p≤0.05).

Preference for Rejection of sen Rejection of sen Beetle species Food plant used sen in FC in FC in NC L. atricillus Ranunculus repens - - - L. australis Scrophularia canina - + + L. exoletus Echium vulgare - - - L. luridus Ranunculus repens - - - L. melanocephalus Plantago lanceolata - + - L. membranaceus Teucrium scorodonia - - - L. nigrofasciatus Verbascum thapsus - + + L. pratensis Plantago lanceolata - + - L. quadriguttatus Cynoglossum officinale - + - L. rubiginosus Convolvulus arvensis - - - L. suturellus Petasites albus + - - A. coerulea Iris pseudacorus - + + A. cyparissiae Euphorbia cyparissias - + -

Uptake and storage of [14C]senecionine and its N-oxide In the tracer feeding experiments, the amounts of absorbed radioactivity varied strongly between species (Figure 2.2). However, the distribution of radioactivity recovered from beetles and from feces extracts showed that all species tested have the ability to store PAs in their bodies. Two days after uptake of the labeled molecules, beetles of the different species held between 2 and 60 percent of the ingested alkaloids in their bodies. The other proportion was eliminated with the feces. The amounts of recovered radioactivity from beetle extracts was higher in most species in the N-oxide fed beetles than in the ones fed with tertiary senecionine. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 18

70 Sen fed 60 SNox fed

10 Recovered radioactivity from beetles (%)

0 L. aeruginosus* L. anchusae* L. jacobaeae* L. luridus L. pellucidus L. succineus(*)

Figure 2.2 Total radioactivity recovered from Longitarsus species two days after ingestion of labeled senecionine (Sen) or senecionine N-oxide (SNox). Columns represent absorbed radioactivity in beetles out of total ingested radioactivity. Species marked with * are PA adapted, L. succineus has both hosts with and without PAs.

N-oxidation of [14C]senecionine The proportions of radioactively labeled metabolites recovered from the beetles' bodies are presented in Figure 2.3. The feeding experiments with senecionine demonstrate that all species are able to metabolize this alkaloid to senecionine N-oxide (Figure 2.3A). At least half of the tertiary alkaloid uptake had been N-oxidized over the two day period. In most experiments minor amounts of unidentified polar metabolites (Rf lower than the PA N-oxides) were detectable. The beetles are also able to reduce the alkaloids. The N-oxide feedings (Figure 2.3B) resulted in a similar distribution of metabolites as in the preceding experiment. Tertiary senecionine was found in all beetles and accounted for 10-35 % of the total radioactivity of the beetle extracts. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 19

Figure 2.3 Distribution of radioactively labeled metabolites recovered from Longitarsus species two days after ingestion when fed with labeled senecionine (A) and senecionine N-oxide (B). Species marked with * are PA adapted, L. succineus has both hosts with and without PAs.

Injection experiments The tracer injection experiment with L. jacobaeae can be directly compared to the oral tracer feeding experiment with this species. The two experiments yielded almost identical results (Figure 2.4A+B). In both cases the beetles converted the major proportion of senecionine into its N-oxide. After injection this transformation was even more pronounced, only traces of non metabolized senecionine were recovered after two days (Figure 2.4B). Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 20

Figure 2.4 Radioactively labeled metabolites identified by TLC separation of crude methanol extracts of beetles two days after treat-ment with tertiary [14C] senecionine. A = L. jacobaeae after oral feeding, B = L. jacobaeae after injection, C = L. australis after injection. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 21

The injection experiment with L. australis yielded completely different results. First, injection of [14C]senecionine showed that the beetles are unable to N-oxidize the tertiary alkaloid (Figure 2.4C). Secondly, the beetles excreted most of the injected alkaloid with the feces (93% of total radioactivity). Only 7% were recovered in the beetles compared to 73% in L. jacobaeae.

Specificity of alkaloid N-oxidation The result of the [3H]atropine and [14C]nicotine feeding experiment with L. succineus and L. jacobaeae was unequivocal (Figure 2.5). Neither of the two species was able to efficiently store the two alkaloids. Only traces of radioactivity could be recovered in the bodies of both species. Most of the ingested radioactivity had been excreted with the feces. In addition, neither of the two species N-oxidized the two alkaloids effectively. While no nicotine N-oxide could be detected in the feces of either species, only a small proportion of 15% N-oxide could be detected in the feces of atropine fed L. jacobaeae.

100 A

60 Beetles

40 Feces

Relative abundance (%) 0 L. succineus L. jacobaeae

100 B

60 Beetles

40 Feces

Relative abundance (%) 0 L. succineus L. jacobaeae

Figure 2.5 Storage abilities for atropine (A) and nicotine (B) in two Longitarsus species: Recovery of radioactivity in beetles and their feces. Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 22

Species-specific differences in uptake and metabolism of different PA types Two Longitarsus species were fed each with the labeled PA typical for the PA type found in its respective host plant. In a parallel experiment the two species received the other tracers to which they were not adapted. L. aeruginosus naturally feeds on Eupatorium cannabinum which contains lycopsamine type PAs. L. jacobaeae, in contrast, sequesters the senecionine type PAs that are present in its Senecio hosts. Both species were fed with [14C]rinderine (lycopsamine type) and [14C]senecionine (senecionine type) in separate experiments. The results show that the two species have the capacity to take up both types of PAs (Table 2.3). A difference in the efficiency of PA uptake between the two PA types could be discovered in L. jacobaeae. These beetles absorbed about 15% of the ingested labeled alkaloids in their bodies when fed with [14C]senecionine and only 2% when fed with [14C]rinderine (Mann-Whitney test, p<0.01). In contrast, L. aeruginosus took up similar amounts of the two alkaloids (p=0.12). In terms of their N-oxidation ability the two species did not show specificities to either PA type. However, L. jacobaeae proved to be generally more efficient than L. aeruginosus in converting tertiary PAs into their N-oxide regardless of type. In L. jacobaeae 80% of total recovered tertiary and N- oxide PAs in the beetles' bodies were identified as N-oxides (Table 2.3). In L. aeruginosus this percentage of recovered N-oxide was significantly lower and lay at around 50% in both PA type feeding experiments (Mann-Whitney test p<0.01 for both PA types).

Table 2.3 Affinity to host and non host PAs in two Longitarsus species. Radioactivity recovered from beetles two days after ingestion of a senecionine type ([14C]senecionine) or a lycopsamine type PA ([14C]rinderine). Values represent percentages ± standard error.

Beetle species PA type in Fed with senecionine Fed with rinderine host Total stored Recovered N-oxide Total stored Recovered N-oxide L. jacobaeae Senecionine 15.2 ± 2.4 78.5 ± 2,6 2.3 ± 0,7 79.3 ± 1,0 L. aeruginosus Lycopsamine 25.7 ± 3.4 50.3 ± 3,1 38.5 ± 5,5 49.2 ± 4,9 Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 23

2.5 Discussion

Insects that feed on plants containing pyrrolizidine alkaloids all have to cope with the potential toxicity of these compounds. Species that employ the strategy of sequestration must possess a mechanism to prevent self-poisoning and safely store these substances in their bodies. The two PA adapted taxa of Chrysomelidae that have been studied so far, the palaearctic genus Oreina and the neotropical genus Platyphora, have developed different adaptations to deal with these problems. Oreina leaf beetles ingest PA N-oxides of the senecionine type from their host plants, suppress their reduction in the gut, and take them up as N-oxides into the hemolymph, probably via a specific carrier in the gut. They build up a storage pool in the hemolymph and body tissues that serves to fill the exocrine defensive glands located in the elytra and pronotum. The PA N-oxide concentrations in the defensive secretions may reach levels up to 0.3 mol ⋅ 10-1 (Hartmann et al., 1997). When Oreina ingests the pro-toxic tertiary form of their host plant PAs, these are efficiently detoxified by glucosylation (Hartmann et al. 1999). Platyphora boucardi, in contrast, takes up the tertiary alkaloids as they are supplied by their host plant. The PAs are very efficiently transported into defensive glands where they are stored as tertiary alkaloids (Hartmann et al., 2001; Pasteels et al., 2001). In contrast to Oreina, P. boucardi does not build up a body storage pool but instead prevents an accumulation of detrimental concentrations of the pro-toxic tertiary PAs in the hemolymph and body tissues. Platyphora is totally unable to N-oxidize PAs. In Oreina sometimes traces of N-oxides are found upon feeding of labeled tertiary PAs (Hartmann et al, 1999) but N-oxidation of PAs is certainly not efficiently developed for PA detoxification in this genus. The present investigation provides evidence of a third mechanism of PA sequestration that has not previously been documented for the Chrysomelidae. Flea beetles of the genus Longitarsus with PA containing host plants also accumulate ingested PAs and mainly store them in their N-oxide form (see also Haberer and Dobler, 1999). Tertiary alkaloids ingested by these beetles are N-oxidized efficiently. In most species only minor amounts of tertiary alkaloids are stored in the body. This strategy resembles the one described for the cinnabar moth Tyria jacobaeae (Lepidoptera, Arctiidae), which stores PAs exclusively as N-oxides. T. jacobaeae takes up tertiary senecionine into the hemolymph where it is detoxified by N-oxidation catalyzed by a specific flavin-dependent monooxygenase (Lindigkeit et al., 1997). This enzyme must have been recruited in the course of evolutionary adaptation of arctiids to PA plants (Naumann et al., 2002). The results of our feeding experiments with PA adapted Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 24

Longitarsus species using various labeled alkaloids also indicate the presence of a specific enzyme. As demonstrated by the tracer feeding experiments with [3H]atropine and [14C]nicotine, alkaloids chemically different from PAs are neither taken up nor N- oxidized quantitatively by the beetles. This argues against an unspecific detoxification mechanism. Some understanding has been reached concerning the site of action of the oxygenase within the beetles' bodies. The comparison of PA application by injection and by oral feeding with L. jacobaeae, a species adapted to PAs, provided evidence for an efficient N-oxidation in the beetle’s body and not in the gut. Since N-oxidation of injected tertiary senecionine is as efficient as of orally ingested alkaloid we can assume that the enzyme is either located in the hemolymph as in the arctiid moths (Lindigkeit et al., 1997) or in associated tissue like fat body or integument. However it remains unclear in which form the alkaloids pass the gut membrane. If they are taken up as N-oxide a carrier of unknown specificity must be presumed. Current feeding experiments using [18O] labeled senecionine N-oxide will address this problem as it has been done for the Arctiidae (Lindigkeit et al., 1997). The tracer feeding experiments with the PA accepting Longitarsus species of different clades confirm that PA storage and N-oxidation ability is a widespread trait in this genus. Even species with hosts that do not contain PAs (L. luridus and L. pellucidus) and species that only occasionally feed on hosts with PAs (L. succineus) show similar sequestration abilities as beetle species that are confronted with PAs throughout their life history. This suggests that a once acquired mechanism has been retained throughout the subsequent evolutionary history of the genus. PA adaptations of species that do not feed on PA plants today consequently have to be regarded as a phylogenetic relic. Those species that do utilize PA plants, however, seem to differ in their efficiency to take up PAs. While in L. aeruginosus host and non host PAs were equally well taken up, in L. jacobaeae the uptake was selective to the type of PA that naturally occurs in their hosts. The physiological adaptation to a certain chemical structure of PA could imply selective benefits of a more efficient sequestration process. Most likely such a benefit consists in the importance of PAs as a defensive agent against predation. Preliminary experiments with potential predator species support this notion (V. Zintgraf & S. Dobler, unpublished data). When the results of the food choice experiments are considered in conjunction with a preliminary phylogenetic tree of the genus Longitarsus (Dobler, 2001), a conspicuous pattern arises. The two Longitarsus species that reject PAs on the behavioral level both belong to a well defined clade of Longitarsus species feeding on Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles 25

Scrophulariaceae. One of these PA avoiding species (L. australis) was exemplarily tested for its N-oxidation ability and proved not to convert injected tertiary senecionine into the non-toxic N-oxide. Instead it excreted most of it with the feces. A marked contrast was the outcome of the same experiment with L. jacobaeae, a species that efficiently takes up and N-oxidizes injected PAs. These results suggest that tolerance and sequestration ability of PAs have either evolved after the split of the Scrophulariaceae feeding clade or have been lost in its ancestor. More phylogenetic data as well as behavioral and physiological studies of other species are needed to decide between these alternatives. However, it can be stated that the N-oxidation of PAs, as it occurs in most Longitarsus species (discussed above), is unlikely to be based on a general xenobiotic mechanism or on a physiologically constrained side effect of unknown metabolic processes. Rather, N-oxidation is catalyzed specifically by an enzyme that has been recruited for PA detoxification during the evolution of the genus. 26

3 Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles (Coleoptera, Chrysomelidae)

3.1 Summary

Several species of Longitarsus take up, metabolize and store pyrrolizidine alkaloids (PAs) from their host plants. In feeding experiments using radioactively labeled PAs of different types we examined the time course of the sequestration process in L. jacobaeae and L. aeruginosus. We found that adapted species efficiently store PAs for at least two weeks without major losses. During that time, there is virtually no change in the ratio of tertiary alkaloids to stored non-toxic N-oxides, regardless of chemical form fed to the beetles. This implies a transient N-oxidation process where the alkaloids are only temporarily accessible to the enzyme. A dissection experiment with L. aeruginosus six days after uptake of labeled PAs showed that the tertiary alkaloids are not found in the hemolymph but are stored in the elytra and other body compartments. This conforms with earlier experiments that localized the enzyme’s site of action in the hemolymph. Furthermore we show that different total alkaloid doses in the diet of L. jacobaeae and the potentially less adapted L. succineus do not affect the ratio of recovered N-oxides to tertiary molecules. Thus, the efficiency of the N-oxidizing enzyme is not dependent on the concentration of alkaloids offered.

3.2 Introduction

Longitarsus flea beetles (Coleoptera, Chrysomelidae) are known to take up and store pyrrolizidine alkaloids (PAs) from their Asteraceae and Boraginaceae hosts (Dobler et al., 2000; Haberer and Dobler, 1999). PA sequestration occurs in many taxa of phytophagous insects (Brown and Trigo, 1995; Hartmann, 1999; Hartmann and Ober, 2000) and mainly serves the insects to defend themselves against predators (Brown, 1984; Gonzales et al., 1999; Orr et al., 1996; Rowell-Rahier et al., 1995; Trigo et al., 1993). The frequent occurrence of PAs as defensive agents is possibly due to its dual chemical nature: On the one hand, there is the protoxic, non-polar tertiary alkaloid (free base) and on the other the non-toxic, polar N-oxide, as it is synthesized and stored in most plants. The alkaloid is a threat to herbivores after ingestion as it is easily reduced into the toxic form under the anaerobic conditions of the gut. Therefore the strategies of Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 27 adaptation to PAs should either be a mechanism to prevent this reduction in the gut, as demonstrated for leaf beetles of the genus Oreina, (Hartmann et al., 1999), or a re-N- oxidation of the alkaloids once they have been reduced. The second strategy is realized in arctiid moths and the grasshopper Zonocerus (Lindigkeit et al., 1997). PA sequestration in Longitarsus species appears to resemble the process used by arctiid moths (Narberhaus et al., 2003). We suggest the existence of an N-oxygenase similar to the specific enzyme that had been characterized from arctiids (Lindigkeit et al., 1997; Naumann et al., 2002) However, the storage form of PAs in Longitarsus is not exclusively the N-oxide. In all species analyzed through pulse feeding experiments with radioactive tracers, tertiary PAs were also recovered from the beetles. In L. jacobaeae for example the proportion of tertiary PAs two days after feeding on a diet of radioactively labeled senecionine amounted to an average of 21,5% in five replicates (Narberhaus et al., 2003). The purpose of the present study was to investigate whether the proportion of labeled tertiary PAs is held constant or if it changes over time. If the N-oxidizing enzyme had permanent access to the alkaloids we would expect a continuous decrease of the proportion of tertiary PAs during the time course after uptake. However, if the proportion of tertiary to total labeled alkaloid remains stable over time, then we would have to assume that substrate and enzyme are separated by some kind of compartmentalization. Therefore, long term tracer storage experiments with two selected species of Longitarsus were performed. We applied two labeled alkaloids, each one as tertiary PA and its N- oxide. The alkaloids [14C]senecionine and its N-oxide as well as [14C]rinderine and its N- oxide represent two different types of PAs (Hartmann and Witte, 1995). The two beetles tested utilize host plants that exclusively contain one of these two alkaloid types so that an adaptation of the insects to their respective PA type might be expected. By performing these experiments, we also intended to gain insights into the storage half-life of PAs in the beetles. The evidence that part of the ingested PAs remain in the tertiary form led us to investigate the efficiency of N-oxidation. Tracer feeding of high PA concentrations should show whether the efficiency of N-oxidation is dose dependent. Finally, a dissection experiment with L. aeruginosus was aimed at localizing the sites of PA storage within the beetles. Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 28

3.3 Material and methods

Study Organisms Adult beetles and their host plants were collected at field sites in Switzerland and Germany (Table 3.1). They were kept in cages at 20°C and 16/8 h L/D and were fed with fresh leaves of their host plants until use. Identification of beetle species was performed according to Döberl (1994).

Table 3.1 Investigated Alticinae species, the host plants from which they were collected and collection sites. BW = Baden-Württemberg, BE = Bern, CH = Switzerland, D = Germany, VS = Wallis

Beetle species Collection site Host plant PA type in host plants

L. aeruginosus Freiburg, BW, D Eupatorium cannabinum lycopsamine

L. jacobaeae St. Imier, BE, CH Senecio jacobaea senecionine

L. succineus Lötschental, VS, CH Achillea none*

* Other populations of this beetle, however, feed on plant species that contain senecionine type PAs.

Tracer biosynthesis [14C]Senecionine and [14C]rinderine (1.07-1.34 GBq⋅mmol-1) and their N-oxides were prepared biosynthetically from [1,4-14C]putrescine-dihydrochloride (4.1 Gbq⋅mmol-1; Amersham Biosciences, Freiburg, Germany) using root cultures of Senecio vulgaris L. (for [14C]senecionine) and Cynoglossum officinale L. (for [14C]rinderine) according to Hartmann (1994).

Long term storage experiments For the long term experiments two Longitarsus species that feed on hosts with different types of PAs were chosen. L. jacobaeae (Waterhouse), that occurs on plants containing PAs of the senecionine type, was fed with [14C]senecionine or its N-oxide. L. aeruginosus (Foudras), that has hosts with lycopsamine type PAs, was fed with [14C]rinderine or its N- oxide. Every experiment consisted of a group of ten beetles that were placed in a Petri dish (∅12 cm) lined with moist filter paper and kept at 20°C at 16 h light and 8 h dark. About 30 µl of a methanolic solution of the labeled alkaloid were painted on a 1 cm ∅ disc of a fresh host plant leaf which was offered to the beetles for 48 h. The amount of alkaloids applied in each experiment of the long term study was ≈1.8 µg with a total Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 29 radioactivity of 2.5 ⋅ 105 cpm. After termination of the feeding period the leaf remains were removed, and untreated leaves were offered for a defined post feeding period. Four parallel replicates that differed in the length of this post feeding period (i.e. 2, 6, 10 , and 14 days) were carried out for each species. At the end of every experiment, the beetles were killed by freezing and stored in methanol until extraction. The feces were washed from the Petri dish surfaces and stored in methanol together with the filter paper until extraction.

Feeding experiments with high PA doses The concentration dependent uptake and metabolism of PAs was tested with L. jacobaeae and L. succineus (Foudras). The first was applied as a highly PA adapted species, the latter as a potentially less adapted. An experiment using a higher dose of 52 µg of PAs with the same total amount of radioactivity was applied in parallel to the usual feeding experiment described above. In the analyses only the labeled molecules were detected. They were representative of the total ingested alkaloids and served to indicate ratios of stored and metabolized molecules. The experimental procedure was as described above for the long term experiments. The post feeding period here was 48 h.

Dissection experiments To localize the sites of PA storage in L. aeruginosus 15 individuals were fed with [14C]rinderine N-oxide. After a 5 day post feeding period on untreated leaves the beetles were lightly shaken in a centrifuge tube to seize possible defensive secretions. The tube was then washed with methanol and the insects were killed by freezing. Feces and eggs were collected from the petri dishes in which the beetles were kept after tracer feeding. Hemolymph was drawn off the beetles using glass capillary tubes that were inserted through the intersegmental skin between abdominal sternites, while the elytra were detached from the pronota with foreceps. Further dissections were prohibitive due to the minute size of the animals.

Extraction procedure The beetles or body parts were ground in a mortar and extracted with 500 µl methanol. After centrifugation the supernatant was recovered and the residual pellet extracted twice with 500 µl methanol, each. The combined solutions were dried under an air stream and the residue dissolved in 400 µl methanol. The feces fractions were extracted three times with methanol, dried and recovered in 2 ml methanol. Using small aliquots of these crude extracts total radioactivity was analyzed by scintillation counting. Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 30

NH4OH (25%) (82:15:3, by volume) for senecionine and the solvent system ethylacetate: isopropanol:NH4OH (25%) (45:35:20, by volume) for rinderine.

3.4 Results

Time course of PA storage and metabolism A large amount of total PAs were taken up and stored by the two Longitarsus species and remained consistently high in the four different post feeding treatments (Figure 3.1). The efficiency and time course was almost identical for the tertiary PA and its respective N-oxide. Even two weeks after ingestion of the labeled PAs, high quantities of radioactive molecules could be recovered from the beetles. The amounts of radioactivity detected in these long term samples were 32 % (senecionine fed) and 51 % (senecionine N-oxide fed) of the initial dose ingested in L. jacobaeae and 48 % (rinderine fed) and 47 % (rinderine N-oxide fed) in L. aeruginosus. The ratio of tertiary PA to N-oxide was very similar in the four sets of post exposure trials (Figure 3.2). As can be seen from the analysis, there was no evidence for a decrease in tertiary PA between day 2 and day 14 after tracer feeding. However, the total amount of the toxic tertiary alkaloid did vary depending on the chemistry of the PAs fed to the beetles. The average amount of non-oxidized alkaloid in L. jacobaeae was ca 23% in the senecionine fed series and ca 7% in the senecionine N-oxide fed series. The results of the experiments with L. aeruginosus were similar. Here too, the amounts of N- oxide recovered from the rinderine N-oxide fed beetles were higher than in those fed with tertiary rinderine. Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 31

Figure 3.1 Time course of alkaloid storage in the two PA adapted species L. jacobaeae (A) and L. aeruginosus (B): Radioactivity in beetle extracts as percent of total ingested alkaloids (ingested = radioactivity recovered from beetles plus from feces). Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 32

Figure 3.2 Time course of PA N-oxidation in the two PA adapted species L. jacobaeae (A) and L. aeruginosus (B): Relative amounts of stored N-oxide (total equals tertiary plus N-oxide PAs).

Efficiency of PA N-oxidation When fed with leaf discs that were painted with a thirty fold concentrated alkaloid solution the beetles reacted similarly as when fed with low doses of alkaloids not exceeding the natural concentrations in their host plants. In L. succineus half of the radioactive tertiary alkaloids were N-oxidized and about 80 % in L. jacobaeae (Figure 3.3). These ratios between N-oxide and the respective tertiary form were independent of the alkaloid dose offered. The proportion of labeled tertiary PA to N-oxide recovered from the beetles was also equivalent for the two experiments where N-oxides were fed. In L. succineus two Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 33 thirds remained in the N-oxide state, in L. jacobaeae this metabolite accounted for more than 90 % no matter which concentration was fed to the beetles. At the same time the absolute amounts of PAs taken up in the high dose feeding experiments were between 3 and 40 fold greater than in the low dose trials. The reaction to the different doses fed demonstrates a high efficiency of the PA oxygenase in both species.

100 A

-oxide (%) normal

N 30fold concentrated 40

20 Alkaloid

0 Sen fed SNox fed

100 B

-oxide (%) normal

N 30fold concentrated 40

20 Alkaloid

0 Sen fed SNox fed

Figure 3.3 Efficiency of PA N-oxidation in L. succineus (A) and L. jacobaeae (B): Relative amounts of N-oxides (total equals tertiary plus N-oxide PAs) in beetles two days after ingestion of a normal and 30fold concentrated alkaloid diet.

Localization of the alkaloid storage sites Dissections of L. aeruginosus to localize the storage site of PAs recovered a quarter of the total radioactivity from the hemolymph and 8% from the elytra. The remaining amount, accounting for most of the ingested tracer, was recovered from the residual body parts (67%). Calculating PA concentrations per mg body tissue, the pattern becomes more obvious (Figure 3.4). The highest concentration was found in the hemolymph, amounting to 0,47 µg PAs per mg fresh weight. In contrast, lower PA concentrations were recovered from the elytra (0,02 µg/mg) and residual body parts (0,04 µg/mg). Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 34

The ratios of N-oxide to tertiary PA differed in the analyzed body parts (Figure 3.4). In the elytra ca 32 % of the recovered metabolites consisted of tertiary rinderine, however no tertiary PA was detected in the hemolymph. In the remaining body tissue an intermediate proportion of 10 % tertiary PA was found. A third of the labeled tracer had been excreted with the feces, in this case entirely as N-oxide. In the eggs a small fraction of 0,3 percent of the ingested radioactivity were found. The attempt to check for defensive secretions by shaking the beetles and washing the vial did not reveal any radioactivity in this fraction.

Figure 3.4 Ratio of tertiary PA (Rin) to N-oxide (RNox) in different body sections, eggs and feces of L. aeruginosus, six days after ingestion of [14C]rinderine N-oxide. Numbers on top of columns indicate the total radioactivity measured in the extract of the respective body section (in 103 cpm out of 3 500⋅10 cpm offered; top row) and total amounts of recovered PAs in µg per mg fresh body tissue (italics; weight of eggs and feces not measured).

3.5 Discussion

Most insects that sequester PAs from their host plants keep and utilize these substances through out their lives. The vast majority of arctiid moths for example, sequester PAs as larvae and transfer them through the pupa to the adult stage (Boppré, 1990; Hartmann and Witte, 1995). Well studied species include the moths Utetheisa ornatrix (Eisner, 1980; Eisner and Meinwald, 1987) and Tyria jacobaeae (Aplin et al., 1968; Ehmke et al., Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 35

1990; Rothschild et al., 1979). Some butterflies in contrast take up PAs only in the adult stage. Most Danainae and Ithomiinae extract PAs from dry plant material using a liquid produced by their proboscides (Edgar, 1982; Rothschild and Marsh, 1978). Also two leaf beetle species have been demonstrated to store alkaloids over long time spans. In Oreina cacaliae, labeled alkaloids could be recovered 25 days after ingestion (Pasteels et al., 1992) and in Platyphora boucardi PAs accumulated during the larval stage are stored life long. For the latter species a specific and efficient alkaloid transfer from larvae via pupae into the defensive secretions of adult beetles was revealed. Almost four months after uptake by the larvae, labeled alkaloids could still be detected in the adult beetles' secretions (Pasteels et al., 2003b). The present study revealed that Longitarsus beetles store ingested PAs efficiently over a period of at least 14 days. Within this time the beetles fed ad libitum on leaves of their natural host plants that contain substantial concentrations of the same PAs as the labeled alkaloids fed previously. In view of this continuous flow of alkaloids into the beetles' bodies, it is the more remarkable that the tracer was held in the body to such a large extent. Obviously, there was no steady loss of the stored labeled PAs. These data suggest that the beetles accumulate the substances to such high amounts that a percentage of every additional dose provided in the food is absorbed. Most PA sequestering insects store the alkaloids as N-oxides. Larvae of the arctiid Tyria jacobaeae (Lindigkeit et al., 1997), of ithomiine butterflies (Brückmann et al., 2000) and the grasshopper Zonocerus variegatus (Lindigkeit et al., 1997) reduce ingested PAs in their gut and re-N-oxidize them in their hemolymph. Oreina leaf beetles both take up and store the alkaloids unmodified in the N-oxide form, probably mediated by a specific carrier. PAs entering the hemolymph as tertiary alkaloid are detoxified by efficient glucosylation (Hartmann et al., 1999). So far, only species of leaf beetles from the neotropical genus Platyphora are known to store PAs exclusively as tertiary alkaloids (Pasteels et al., 2001; Pasteels et al., 2003a). Tracer studies with P. boucardi revealed that this beetle seems to efficiently transport the poisonous tertiary PAs directly into its defensive glands. Any accumulation of detrimental concentrations of tertiary alkaloids in the hemolymph and body tissues is thereby avoided (Hartmann et al., 2001). In Longitarsus PA sequestration follows yet another strategy. The investigated species stored PAs from their host plants both as N-oxides and, to a lesser extent, as tertiary alkaloids (Narberhaus et al., 2003). As the present study showed, two days after ingestion in L. jacobaeae beetles around 20% of the stored alkaloids were in the tertiary form, and in L. aeruginosus about 15%, respectively. Moreover, as the long term experiments have shown, this ratio of N-oxide to tertiary PA did not change when beetles Time course of pyrrolizidine alkaloid sequestration in Longitarsus flea beetles 36 were analyzed 6, 10 or 14 days after ingestion of the tracer. We therefore suppose that the enzyme responsible for the N-oxidation has only transiently access to the PAs. Once they are accumulated in storage tissues and are thereby out of reach of the enzyme, the non-oxidized PAs remain unaltered. As we have shown previously by injection experiments, the enzyme appears to be located in the hemolymph (Narberhaus et al., 2003). This would agree with the idea of a transient oxidation of PAs. The dissection of tracer fed L. aeruginosus demonstrated that PAs were present in the hemolymph in highest concentrations and were entirely in the non-toxic N-oxidized form. In comparison, in the beetles’ elytra around 30% and in the remaining body parts 10% of the PAs were recovered in the tertiary form. Hence it appears that tertiary PAs in the elytra and other tissues are not accessible for N-oxidation by the enzyme and may be harmless to the beetles since they are excluded from metabolism. The results of the long term storage experiments were similar whether the beetles had been fed with N-oxides or tertiary PAs. However, in both analyzed species we observed a tendency of slightly higher recovered N-oxide proportions in the N-oxide fed samples. This could suggest the existence of an active transport mechanism of PA N- oxides through the beetles’ guts, in addition to the N-oxidizing enzyme present in the hemolymph. Recent experiments with L. jacobaeae that we fed with oxygene labeled senecionine N-oxide confirm this interpretation (Narberhaus et al., unpublished results). Finally, the feeding experiments with a high PA concentration show no indication for a lower enzyme efficieny in the potentially less PA adapted L. succineus. On the other hand, the experiments show for both species that the ratio of stored N-oxides to tertiary alkaloids remains largely unaffected. The raison d'être for the remaining tertiary alkaloids in the beetles' bodies cannot lie in the enzyme's inefficiency but rather in a rapid transfer of PAs from the enzyme’s site of action into storage tissues. Potential specialized storage organs in the form of defensive glands are known in a number of leaf beetles including the Alticinae (Deroe and Pasteels, 1982) and we have microscopic evidence for their existence in Longitarsus as well (I. Narberhaus; S. Dobler & J. Pasteels, unpublished data). However, the large proportion of stored PAs in the body (without elytra) implies the existence of further so far unknown storage compartments. 37

4 Direct evidence for membrane transport of host plant derived pyrrolizidine alkaloid N-oxides in two leaf beetle genera

4.1 Summary

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae and O. speciosissima sequester pyrrolizidine alkaloids (PAs) from their asteracean host plants and store them as non-toxic N-oxides. Our previous analyses have shown that Longitarsus is able to N-oxidize pro-toxic tertiary PAs but did not resolve in which form N- oxides are taken up. For Oreina it has been indicated that the beetles are able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of the PA N-oxides in the gut yielding pro-toxic free bases. Here we confirm the predicted but never experimentally confirmed direct uptake of PAs as N-oxides by Oreina and elucidate the situation for Longitarsus by applying double labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC and submitted to ESI-MS, GC-MS and analysis of the specific radioactivity. The results revealed that the 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC purified senecionine N-oxide by a second compound subsequently identified as a dihydrosenecionine N-oxide by high resolution CID analysis. The dihydrosenecionine N-oxide, most probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle’s alkaloidal background. The recovered senecionine N- oxide retained 74% 18O-label. The remaining 25% are mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar non-toxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).

4.2 Introduction

Pyrrolizidine alkaloids (PAs) are plant secondary metabolites that are potentially toxic and produced by the plant to protect itself against herbivory. In the plant PAs are usually Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 38 present as N-oxide (Figure 4.1B), a form that is hydrophilic and a priori non toxic. When ingested by a herbivore, PA N-oxides are generally reduced to lipophilic tertiary alkaloids (free bases, Figure 4.1A) and passively absorbed into the herbivore's body (Hartmann, 1999). In vertebrates these tertiary PAs are then converted into pyrrolic metabolites that easily react with biological nucleophiles causing hepatotoxic and pneumotoxic effects (Cheeke, 1989; Mattocks, 1986; Winter and Segall, 1989). In phytophagous insects severe detrimental effects have similarly been described, reaching from mutagenesis (Frei et al., 1992) to lethal developmental failures (Narberhaus et al., submitted). However, a number of specialized insects from diverse taxonomic groups have developed adaptations that even allow the sequestration of PAs from plants, i.e. their accumulation and utilization for their own antipredator defense (Hartmann, 1999; Hartmann and Ober, 2000). Even though the hurdle that the PAs' toxicity imposes on insects is the same, the strategies evolved to overcome it differ greatly in various taxa: larvae of the arctiid moths Creatonotos transiens and Tyria jacobaeae and the grasshopper Zonocerus variegatus passively take up reduced PAs into their bodies and detoxify them by N-oxidation. In T. jacobaeae a specific flavin-dependent monooxygenase (FMO) in the hemolymph has been found to be responsible for this reaction (Lindigkeit et al., 1997; Naumann et al., 2002). Some vertebrates like guinea pigs (Cheeke, 1994) and sheep (Huan et al., 1998a) are also known to N-oxidize tertiary PAs through vertebrate-specific multisubstrate FMOs making these highly polyphagous grazers resistant to the toxicity of PA food-plants.

Figure 4.1 Structures of the two forms of senecionine: A) tertiary alkaloid (free base) B) PA N-oxide. Carbons 3,5,8 and 9 were14C-labeled. Labeled oxygene atom in [14C]senecionine-[18O]N-oxide is shaded in grey. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 39

A variety of different strategies to sequester PAs is found in the beetle family Chrysomelidae. The neotropical species Platyphora boucardi, for example, takes up tertiary PAs as they are supplied by its host plants (Pasteels et al., 2001; Pasteels et al., 2003a). They are absorbed and efficiently transported into defensive glands where they are stored unchanged as tertiary alkaloids. The efficient transport prevents accumulation of detrimental concentrations of tertiary PAs in the hemolymph and body tissues (Hartmann et al., 2001). The adapted alticine leaf beetles of the genus Longitarsus N- oxidize tertiary PAs and store them mainly in their N-oxide form (Narberhaus et al., 2003), most similarly to the way the arctiid moths are handling the alkaloids. Enzymatic observations also indicate the existence of a flavin-dependent monooxygenase (FMO) located in the beetles' hemolymph (Narberhaus et al, unpublished data), analogous to the one responsible for N-oxidation in Tyria jacobaeae. In contrast to the arctiid moths, Longitarsus also stores minor amounts of PAs in the tertiary form. This portion appears to be separated from the enzyme in so far unidentified storage tissues and remain there unchanged for prolonged time periods (Narberhaus et al., 2004). While in arctiids only tertiary PAs are able to permeate the gut membrane, the situation in Longitarsus is still unclear. The possibilities would be either a reduction in the gut followed by passive uptake of tertiary PAs across the gut membrane and re-oxidation, or, as demonstrated for leaf beetles of the alpine genus Oreina, a suppression of reduction in the gut combined with an active transport of N-oxides into the body. Oreina leaf beetles store PAs also in the N-oxide form. However, no N-oxidation could so far be detected in these beetles. After ingestion as N-oxides from their hosts, the alkaloids can be recovered in the same form from the beetles' defensive secretions. In the hemolymph and body tissues a storage pool of PA N-oxides serves to fill up exocrine defensive glands located in the elytra and pronotum. When Oreina cacaliae ingests tertiary PAs these are efficiently detoxified by glucosylation (Hartmann et al., 1999). It hence seems probable that Oreina possesses both a carrier in the gut membrane that transports the hydrophilic N-oxides unchanged into the hemolymph and a carrier that transports them from the body into the defensive glands. Even though biochemical evidence is strong, no direct proof for the state in which PAs cross membranes in Oreina has hitherto been brought forth. The goal of the current investigation was to elucidate the process of membrane transfer of PAs in Longitarsus and Oreina. To establish the molecular state in which PAs cross membranes only one experiment can provide direct evidence, the oral administration of a PA N-oxide with a labeled N-oxide oxygen atom. We therefore prepared [14C]senecionine [18O]N-oxide from tertiary [14C]senecionine using a senecionine-N- Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 40 oxygenase obtained from the hemolymph of Tyria jacobaeae caterpillars. As exemplary PA-sequestering species we fed Longitarsus jacobaeae, Oreina speciosissima and O. cacaliae with this tracer. All three species feed on PA containing plants. L. jacobaeae is specialized on the asteracean genus Senecio and has already been shown to efficiently N-oxidize PAs in tracer feeding and injection experiments (Narberhaus et al., 2003). This beetle species can be regarded as a highly adapted PA plant feeder, which is corroborated by the PA-specificity of the N-oxygenase. The two Oreina species have hosts in the asteracean genera Petasites and Adenostyles and have been well investigated for their PA sequestration mechanisms. They were here further analyzed to complete our knowledge on PA uptake mechanisms in these species and to serve as comparison to Longitarsus.

4.3 Material and Methods

Biosynthesis of [14C]senecionine [18O]N-oxide [14C]Senecionine (1.07-1.34 GBq⋅mmol-1) was prepared biosynthetically from [1,4- 14C]putrescine-dihydrochloride (4.1 GBq⋅mmol-1; Amersham Biosciences, Freiburg, Germany) using root cultures of Senecio vulgaris L. according to Hartmann (1994). The preparation of [14C]senecionine [18O]N-oxide was performed according to a slightly modified protocol of Lindigkeit et al. (1997). The senecionine N-oxygenase from hemolymph of Tyria jacobaeae was used to catalyze the N-oxidation. To isolate the enzyme, late instar caterpillars of this species were field caught in the Meijendahl dunes (Leiden, NL) from their hosts in June 2002. Around 280 larvae were used to collect 8.4 ml of hemolymph which was precipitated in a slowly stirring saturated ammonium sulfate solution at 4°C. After centrifugation the supernatant was discarded and the remaining protein pellet could be stored at -80°C without losing its activity. Before use, the enzyme was desalted via Sephadex G-25 (PD-10 columns, Amersham Biosciences, Freiburg, Germany). A gas-tight glass apparatus was used, consisting of a 100 ml round-bottomed 18 18 flask that was connected to an O2 gas flask (99% purity, 95% O excess) on one side and to a vacuum pump on the other side. Approx. 9 mg senecionine (Roth, Karlsruhe; 100% pure according to GC) was suspended in 5 ml 0.01 M phosphate buffer, pH 2; after dissolving the solution was adjusted to pH 7.0 with 0.01 M K2HPO4 containing 2 mM dithioerythritol. The final volume was adjusted to 25 ml and 30 µl [14C]senecionine (107 cpm) in methanol was added. Finally the desalted enzyme solution containing crude senecionine N-oxygenase in 0.01 M potassium phosphate buffer, pH 7.0 was added. All Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 41 buffers and solutions had been thoroughly degassed with helium before use. The top was sealed with a Teflon septum and flushed and evacuated 10 times with > 99.9% N2. 18 18 After one flush with O2 followed by evacuation, the round flask was filled with O2 and the reaction was started by injecting 1 ml degassed reaction buffer containing 50 mg NADPH through the septum. The apparatus was smoothly shaken in a 30°C water bath for 24 h before the reaction was stopped by injection of 3 ml 25 % HCl. Precipitating protein was removed by centrifugation. A small aliquot of the supernatant was subjected to thin-layer chromatography and analyzed using a multichannel radioactivity detector (Rita-32a, Raytest). The enzymatic N-oxidation turned out to be quantitative; the applied substrate senecionine was no longer detectable in the assay. The remaining solution was dried under an air stream, re-dissolved in 6 ml CHCl3, and applied on a 9 g Al2O3 column. After flushing the column several times with a total of 20 ml CHCl3, alkaloid N- 6 oxides were eluted with the solvent MeOH:CHCl3 (3:7 by volume). A total of 3 · 10 cpm (ca. 5.5 mg) was recovered. The 18O-content of the sample as well as of the beetle extracts was determined by mass spectrometry after the feeding trial, as described below. Specific radioactivity was measured by scintillation counting and quantitative gas chromatography of a Zn/H+-reduced aliquot. Using heliotrine as an internal standard the yield was measured to be 1.94 · 108 cpm/mmol (equal to 5.8 · 105 cpm/mg [14C]senecionine or 5.5 · 105 cpm/mg [14C]senecionine [18O]N-oxide)

Tracer feeding experiments Beetles of three species were caught in the field. Longitarsus jacobaeae was collected in St.Imier (Ct. Jura, CH) on Senecio jacobaea in August 2002, Oreina cacaliae on the Stöckalp (Ct. Obwalden, CH) on Adenostyles alliariae in July 2002 and Oreina speciosissima on the Gfelalp (Ct. Bern, CH) on A. alliariae and Petasites albus in August 2003. All insects were kept under long day conditions at 18°C in plastic cylinders on their host leaves until use. To reduce the risk of complications with background PAs in the insects, the individuals of both Oreina species were fed with Petasites albus, whose leaves are practically free of alkaloids, for two weeks before start of the feeding experiments. For the tracer feeding of every species, a methanolic solution of 1 mg of [14C]senecionine [18O]N-oxide was painted on leaves of Senecio jacobaeae (for L. jacobaeae) or Petasites albus (for O. cacaliae and O. speciosissima). The subsequent procedures differed between the species, depending on how well the insects ingested the leaves and on whether secretions were collected. With L. jacobaeae, four replicate experiments were performed with groups of 50 beetles each that were placed together in Petri dishes (day 0). Every group fed on leaf disks covered with 0.25 mg (1.4 · 105 cpm) Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 42 of the tracer. On day 2, the beetles had consumed the treated leaves completely and were placed on non-treated fresh leaves in clean containers. This step was repeated on day 4. On day 7 the insects were killed by freezing and stored at –20°C until extraction. Likewise, four replicate experiments were initiated with O. cacaliae. Each experiment consisted of a group of three individuals that were placed in a Petri dish (ø 9 cm) and fed with a 3x3 cm P. albus leaf covered with 0.25 mg (1.4 · 105 cpm) [14C]senecionine-[18O]N-oxide. The beetles were transferred to new Petri dishes with untreated leaves on days 3 and 5. After one week defensive secretions were collected by disturbing the animals with a pair of forceps and by wiping exuding liquid from elytra and pronota with a piece of filter paper. Secretions were stored in Methanol in a centrifuge tube at -20°C. Of O. speciosissima seven individuals were collected. They were placed together in a Petri dish and fed with 1 mg (5.5 · 105 cpm) [14C]senecionine [18O]N-oxide painted on a 5x5 cm leaf piece of P. albus. All had been consumed on day 3. On days 3 and 5 the beetles were transferred to a new Petri dish with a fresh untreated leaf. On day 8 the defensive secretions were collected from the insects, as described above.

Extraction of L. jacobaeae beetles Since no secretions could be collected from L. jacobaeae, the whole beetles were extracted. The frozen beetles were ground in a mortar and extracted with 2 ml MeOH. After centrifugation the supernatant was recovered and the residual pellet extracted twice again with 2 ml MeOH. The combined solutions were dried under an air stream and the residue dissolved in 2 ml MeOH. Small aliquots of these crude extracts were subjected to scintillation counting to evaluate total radioactivity. Extracts were then further purified through Al2O3 columns. After drying and re-dissolving of the residue in 500 µl CH2Cl2 the extracts of the four beetle groups were each applied on 300 mg Al2O3. To remove tertiary

PAs the columns were washed with 30 ml CH2Cl2 before N-oxides were successively eluted with four 500 µl-portions CH2Cl2:MeOH (7:3, by volume). The resulting fractions 1- 4 were collected separately. A TLC analysis revealed the necessity of an additional washing to remove persisting residual tertiary PAs. Therefore the fractions 1 and 2, that contained most of the radioactivity, were pooled, dried and dissolved in 40 µl H2O. Any remaining tertiary PAs were removed by extraction with 40 µl toluene. The aqueous phase containing the PA N-oxide was recovered, dried, and subjected to HPLC for separation of senecionine N-oxide from other contaminant PAs, such as seneciphylline N-oxide. HPLC was performed using an RP-18 column (Nucleosil 120-5 C18, 250 mm long, 4 mm ID, Macherey & Nagel). Samples were redissolved in 20 µl MeOH and Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 43 injected into a 20 µl loop. Separation was achieved using Helium-washed acetonitrile and

H2O/CF3COOH (pH2) as solvents (2:8, by volume) at a flow rate of 1 ml/min, UV- detection was by absorbance at 210 nm. The retention time (RT) for senecionine N-oxide is 9.2 min. The fractions with the highest radioactivity (RT 8-10 min) were pooled separately for each of the four replicates. They contained on average a radioactivity of 20,600 cpm, corresponding to 37.9 µg labeled senecionine N-oxide. The solvent was removed by evaporation and the samples subjected to MS analysis.

Purification of defensive secretions from O. cacaliae and O. speciosissima The filter papers with secretions were extracted three times each with 500 µl MeOH, evaporated and redissolved in 200 µl MeOH. Total radioactivity was measured by scintillation counting of an aliquot. Since the recovered radioactivity in secretions of O. cacaliae was low, the samples obtained from the four replicate groups were pooled. Purification of senecionine N-oxide was performed via HPLC following the procedure described above for L. jacobaeae. Fractions of retention time 9-10 min, containing 4,400 cpm (=7.9 µg senecionine N-oxide) in the O. cacaliae sample and 6,500 cpm (=11.7 µg senecionine N-oxide) in the O. speciosissima sample, were evaporated and subjected to MS analysis.

Determination of 18O abundance in senecionine N-oxide by electrospray mass spectrometry Mass spectrometric analysis was carried out on a MAT 95 XL Trap hybrid tandem mass spectrometer (Thermofinnigan MAT, Bremen, Germany) equipped with the standard electrospray interface supplied by the manufacturer. The instrument consists of a double focussing sectorfield mass spectrometer with high resolution and accurate mass measurement capabilities, coupled to a quadrupole ion trap mass analyzer. The second mass analyzer is used to investigate the fragmentation behavior of monoisotopically isolated precursor ions from the first mass analyzer by collision induced dissociation (CID). A reference sample of senecionine N-oxide, a reference sample of 18O-labeled senecionine N-oxide and the dry samples obtained by radio-HPLC as described above were reconstituted with 100 µl of HPLC-grade acetonitrile and directly analyzed by ESI MS. For all low resolution measurements a slightly modified version of the microspray Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 44 device supplied with the instrument was used for sample introduction1 and operated at a flow rate of approximately 0.8 µl/min and a spray voltage of 1.5 to 1.7 kV. The temperature of the heated capillary was set to 195°C. The remaining voltages of the electrospray interface were optimized for maximum signal intensity of the protonated molecular ion of senecionine N-oxide at m/z = 352. The mass spectrometer was operated at a resolution of 3000 (10% valley definition) and magnetically scanned from 50 to 1200 amu at a scan speed of 2 sec/dec. About 50 spectra for each sample were acquired in centroid mode and subsequently averaged. For high resolution and accurate mass measurements samples were introduced by a nanospray device using gold coated tapered nanospray emitters with approximately 2 µm ID tip openings (MasCom, Bremen, Germany) operating at a flow rate of approximately 50 nl/min and a spray voltage of 1.0 to 1.1 kV. The temperature of the heated capillary was set to 120°C. The remaining voltages of the electrospray interface were optimized as described above. For accurate mass measurements employing the peak match method the resolution was adjusted to 7,000 (10% valley definition). An appropriate amount of a polypropylene glycol standard solution serving as an internal mass calibrant was added and mixed with the sample in the nanospray emitter by means of a microliter syringe. In some cases interfering signals from isobaric impurities necessitated the application of far higher resolutions up to 22,000 (10% valley definition) for the correct assignment of target molecule peak intensities. In these cases, where due to the high resolution conditions only weak signal intensities can be obtained, the addition of a mass calibrant was omitted in order to avoid ion suppression effects leading to even more decreased sensitivity. Consequently, data acquisition was performed in profile mode scanning magnetically from 350 to 360 amu at a scan speed of 30 sec/dec. About 150 spectra for each sample were acquired and averaged. Recalibration of the mass scale was done manually by assigning the theoretical values to signals of known components that had been identified by their CID product ion spectra prior to the experiment. The conditions for the acquisition of CID product ion spectra were essentially the same for low and high resolution precursor ion selection. The transfer ion optics were optimized for maximum transmission using the protonated molecular ion of senecionine N-oxide (reference sample) at m/z = 352. The same signal was subsequently used for optimization of the relevant ion trap parameters, i.e. multipole 1 and multipole 2 offset,

1 The original fused silica sample transfer capillary was replaced by stainless steel tubing of similar dimensions (SMS Service für Massenspektrometrie GmbH, Idstein, Germany). And the non conductive ferrule that holds the capillary was replaced by a graphite ferrule of appropriate size. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 45 multipole lens, trap offset and mulitpole RF DAC by semiautomatic tune procedures of the instrument control software. CID spectra of the respective precursor ions were then recorded with a parent isolation width of 3.0 amu and a normalized collision energy of 35% in normal centroid scan mode between 95 and 400 amu. Depending on precursor ion intensity between 10 and 50 spectra had to be acquired and averaged subsequently with respect to the required signal to noise ratio of the resulting spectra. Total sample consumption was between 5 and 15 µl depending on the performed experiments.

Gas Chromatography – Mass Spectrometry (GC-MS) The GC-MS data were obtained with a Hewlett Packard 5890A gas chromatograph equipped with a 2 m fused silica guard column (deactivated, ID 0.32 mm) and a 30 m x 0.32 mm analytical column (ZB1 and ZB5, Phenomenex). The capillary column was directly coupled to a triple quadrupole mass spectrometer (TSQ 700, Finnigan). The analyses were under the following conditions: Injector and transfer line were set at -1 280°C; the temperature program used was: 100°C (3 min)-300°C at 6°C min for PA -1 separation and 70°C (6 min)-300°C at 10°C min for the separation of necic acid methyl esters, respectively. The injection volume was 1 µl. The split ratio was 1:20, the carrier gas flow was 1.6 ml min-1 He, and the mass spectra were recorded at 70 eV. Quantitative Gas Chromatography was achieved using a capillary column (15 m x 0.25 mm fused-silica; DB-1, J&W Scientific) (Witte et al., 1993). All other GC conditions were the same as given for GC-MS. Detectors were FID and PND. Quantitative analyses were performed via the FID signals using heliotrine as internal standard.

4.4 Results

Preparation of [14C]senecionine [18O]N-oxide The 18O-labeled senecionine N-oxide was prepared enzymatically using a crude preparation of senecionine N-oxygenase (Lindigkeit et al., 1997) obtained from the larval hemolymph of the arctiid Tyria jacobaeae. The senecionine applied as substrate contained approximately 107 cpm [14C]senecionine prepared biosynthetically using Senecio vulgaris root cultures (Hartmann, 1994). The substrate was essentially free of seneciphylline. This is important because seneciphylline [18O]N-oxide has the same molecular mass as unlabeled senecionine N-oxide and could thus interfere with MS analysis. The prepared approximately 5.5 mg double-labeled [14C]senecionine [18O]N- oxide contained 96.5% 18O in N-oxide oxygen as established by ESI MS and had a Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 46 specific activity of 1.94 · 108 cpm/mmol. The double labeled tracer was radiochemically and chemically pure. An accurate mass measurement confirmed the correct elemental + 18 composition ([M+H] : C18H26NO5 O; found 354.1812 Da S. D.: 0.4 mDa; calculated: 354.1797 Da). CID product ion spectra were recorded as a reference for latter identification by comparison. No contamination with other PAs could be detected by high resolution ESI MS and GC-MS of the Zn/H+ reduced sample.

Uptake and storage of [14C]senecionine [18O]N-oxide by Oreina cacaliae and O. speciosissima From feeding studies with radioactively labeled PAs it is well documented that the two PA sequestering leaf beetles O. cacaliae and O. speciosissima are able to absorb PA N- oxides and transfer them into their defensive secretions (Rowell-Rahier et al., 1991). Moreover, Oreina has been shown to be unable to N-oxidize tertiary PAs (Hartmann et al., 1999). However, the unaltered transfer of orally fed PA N-oxide was never directly demonstrated. Therefore, the two leaf beetles were fed with the tracer painted on their host plant leaves, and their defensive secretions were collected subsequently. The secretions were subjected to HPLC purification and the fraction containing senecionine N-oxide was recovered. The recovered material upon ESI-MS almost exclusively exhibited the expected signals for 18O-labeled and unlabeled senecionine-N-oxide (Figure 4.2). The CID product ion spectra of the signals at m/z = 352 ([M + H]+ senecionine [16O]N-oxide) and 354 ([M + H]+ senecionine [18O]N-oxide) in the samples of O. speciosissima and O. cacaliae proved to be identical with those obtained from the synthetic tracer. Thus the degree of 18O labeling could be calculated directly from the signal intensities (Table 4.1). In O. speciosissima, analysis of the senecionine N-oxide in the defensive secretion revealed a recovery of 99.1% of the 18O and 102% of specific activity. This clearly indicates that the orally fed senecionine N-oxide was unaltered taken up and transferred from the hemolymph via the exocrine glands into the defensive secretion. The same applies for O. cacaliae. In this case, however, the recovery of 18O was only 62.6%. Since, however, the specific radioactivity is reduced to almost the same extent (68.8%) the reason is not loss of 18O label from fed tracer but dilution by non- labeled background alkaloid already present in the beetle’s secretion. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 47

Figure 4.2 Electrospray mass spectra (ESI-MS) of synthetic senecionine [18O]N-oxide tracer (A), reisolated senecionine N-oxide from O. speciosissima defensive secretions (B) and from L. jacobaeae beetle extracts (C).

Table 4.1 Feeding of [14C]senecionine [18O]N-oxide to Oreina speciosissima and O. cacaliae. Analysis of the 16O/18O composition and specific radioactivity of senecionine N-oxide recovered from the defensive secretions.

Ratio 18O Specific radioactivity Sample 16O/18O recovered (cpm/mmol) Recovered (%) (%) (x108) (%)

Tracer fed 3.5 : 96.5 100 1.94 100

Oreina speciosissima 4.4 : 95.6 99.1 1.99 102.5

Oreina cacaliae 37.4 : 62.6 64.9 1.37 68.8 Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 48

Uptake and storage of [14C]senecionine [18O]N-oxide by Longitarsus jacobaeae The tracer feeding experiment with L. jacobaeae was performed in the same way as described above for the Oreina species. Since so far no defensive secretions could be recovered from Longitarsus the sequestered senecionine N-oxide had to be recovered from whole beetle extracts. Contamination of the extracted materials with unabsorbed applied tracer could be excluded since after consumption of the tracer the beetles were kept for five days on untreated food plant leaves that were changed twice before termination of the experiment. The crude alkaloid extracts were prepurified by Al2O3 chromatography and the fraction containing senecionine N-oxide was subsequently recovered by HPLC separation. In this case the determination of the degree of 18O labeling by a simple low resolution ESI measurement as described for the other samples was not feasible since the CID product ion spectra of the precursor ion at m/z = 354 showed minor but significant differences compared to those of the tracer standard. Additional peaks at m/z = 222, 254 and 292 not present in the spectra of the standard material were evident (Figure 4.3). An accurate mass measurement (354.1853 Da S. D.: 0.3 mDa) showed a deviation from the theoretical value (354.1797 Da) exceeding the error limits that are typically valid for the instrument in this mode of operation as can be seen from the value obtained for the tracer standard (354.1812 Da S. D.: 0.4 mDa). This suggested the presence of an isobaric impurity that coeluted in the preceding HPLC isolation. The appearance of a second component contributing to the signal at m/z = 354 could be confirmed in a subsequent experiment with drastically increased resolution of 22,000 (10% valley definition) revealing a partially resolved doublet of peaks with a ratio of roughly 3:2 (Figure 4.4). Due to the immanently low sensitivity in this mode a further increase of resolution and the addition of a mass calibrant in order to enable accurate mass measurement by peak matching were not feasible. The achieved resolution and signal intensities proved to be sufficient for the successful detection of distinct CID product ion spectra of both components (Figure 4.3). It could be shown that the additional signals at m/z = 222, 254 and 292 mentioned above originate exclusively from the minor component while the major component was unambiguously identified as senecionine [18O]N-oxide by comparison with the reference data. This in turn allows for the recalibration of the mass scale by assigning the correct mass value of 354.1797 Da 18 (C18H26NO5 O) to the apex of the major component. Linear extrapolation, which should be valid for small mass increments, furnishes an accurate mass of 354.1905 Da for the minor component. This strongly suggested the presence of a hydrogenated congener of senecionine N-oxide (C18H28NO6 354.1911 Da) which had not been separated by HPLC. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 49

Figure 4.3 CID product ion spectra of the senecionine N -oxide fraction from extracts of Longitarsus jacobaeae after feeding [14C]senecionine [18O]N-oxide: synthetic tracer (A), purified beetle extract (B), major component from beetle extract (C), minor component from beetle extract (D). A + B = low resolution precursor ion selection. C + D = high resolution precursor ion selection (see Fig 4).

Figure 4.4 High resolution (22,000) ESI-MS of the signal at m/z = 354 from L. jacobaeae beetle extract showing a partially resolved doublet: left peak = senecionine [18O]N-oxide, right peak = dihydrosenecionine [16O]N-oxide (see text). Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 50

A computer based simulation of the superimposed signals of the two compounds obtained by ESI ensured that the chosen resolution provided sufficient peak separation for the determination of the individual abundances from the peak maxima. Thus, the ratio of senecionine [18O]N-oxide and the hydrogenated congener assuming negligible differences in ionization efficiency was determined to be 61% : 39%. By comparing the peak top intensities of the signals at m/z = 352 (senecionine [16O]N- oxide; 35.0%) to the one at m/z = 354 (senecionine [18O]N-oxide; 100%) the degree of 18O labeling was determined to be 74% (Table 4.2).

Table 4.2 Feeding of [14C]senecionine [18O]N-oxide to Longitarsus jacobaeae. Analysis of the 16O/18O composition and specific radioactivity of the senecionine N-oxide fraction recovered from beetle extracts. The sample was found to consist of senecionine N-oxide (47%) and dihydrosenecionine N- oxide (53%) that were not separated by HPLC purification.

Ratio 18O Specific radioactivity Sample 16O/18O recovered (cpm/mmol) Recovered (%) (%) (x108) (%)

Tracer fed 3.5 : 96.5 100 1.94 100

Senecionine N-oxide 26 : 74 77 2.21 113.8

Dihydrosenecionine N- 92 : 8 < 8 oxide

Senecionine N-oxide + dihydrosenecionine N- 1.03 53.2 oxide Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 51

OH CH3 A O

CH3

CH3 O O O H

OH CH3 O B CH3

CH3 O O O H

Figure 4.5 EI mass spectra of the two main components recovered from L. jacobaeae beetle extracts after Zn/H+ reduction: senecionine (A) and dihydrosenecionine (B).

In order to obtain more information about the suspected hydrogenated congener the N- oxides were reduced and analyzed by GC-EI-MS. Two major components were observed in the corresponding chromatogram, one of which was unambiguously identified to be senecionine by its retention index (RI 2294) and its mass spectrum (Figure 4.5A). The other component (RI 2275) shows an almost identical pattern of signals in the lower mass range representing the fragments of a presumably unmodified necine base (m/z, 42, 53, 80, 120, 136, 138) (Figure 4.5B). In contrast many of the fragments in the upper mass range still containing parts of the necine acid moiety show an increase of two mass units (e.g., m/z 222 vs 220, 250 vs 248, 292 vs 290, 322 vs 320, 337 vs 335). This strongly suggests that the unknown compound represents a dihydrosenecionine tentatively assigned as 15,20-dihydrosenecionine (Figure 4.5B). The GC-MS analysis revealed a composition of 47% senecionine and 53% dihydrosenecionine (Figure 4.6). The differences in relative abundance of senecionine with respect to dihydrosenecionine in ESI MS vs. GC-MS may be explained by the different samples analyzed. In the case of ESI MS one of the four replicates was analyzed whereas for GC-MS an aliquot of the combined replicates was analyzed. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 52

A B

S S

Figure 4.6 Sections of GC profiles of (A) the free base obtained from reduction of [14C]senecionine [18O]N-oxide fed as substrate to Longitarsus jacobaeae and (B) the free bases obtained from reduction of the HPLC purified senecionine N-oxide fraction of beetle extract. S, heliotrine as internal standard; 1, senecionine (RI 2375); 2, dihydrosenecionine (RI 2348)

In summary the results of the L. jacobaeae experiment are: (1) The HPLC purified senecionine N-oxide fraction obtained from extracts of L. jacobaeae beetles previously fed with [14C]senecionine [18O]N-oxide revealed two compounds: senecionine N-oxide and dihydrosenecionine N-oxide. (2) 74% of the senecionine N-oxide consisted of the 18O-labeled N-oxide, whereas less than 8% of the dihydro derivative was labeled (Table 4.2). (3) Most likely the unlabeled dihydrosenecionine N-oxide originates from the alkaloidal background of the beetles; only a small proportion of ca. 8% may be derived by hydrogenation of the labeled senecionine N-oxide fed. (4) The recovery of 74% 18O in the senecionine N-oxide fraction but no obvious dilution of the specific radioactivity strongly indicates that the lowered recovery of 18O could not be due to dilution by 16O background N-oxide but must be caused by partial reduction of the fed [18O]N-oxide and re-N-oxidation of the respective radioactively labeled senecionine. (5) Nevertheless, the fact that 74% of the original 18O-label of the tracer fed to the beetles is retained clearly indicates that the intact N-oxide is absorbed and stored by the beetles. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 53

4.5 Discussion

Proof of a carrier-mediated transport of PA N-oxides The feeding experiments with senecionine N-oxide 18O-labeled in its N-oxide oxygen clearly prove that Oreina and Longitarsus leaf-beetles are able to absorb plant acquired PA-N-oxides without preceding reduction. For Oreina this result confirms previous biochemical studies showing that plant acquired PA-N-oxides build up a storage pool in the hemolymph and body-tissues that serve to fill the defensive glands (Hartmann et al., 1999; Hartmann et al., 1997) where the concentration of PA N-oxides may reach levels of up to 0.3 mol·l-1 (Rowell-Rahier et al., 1991). Oreina leaf beetles are not able to N- oxidize any absorbed tertiary PA (Ehmke et al., 1991), instead they convert tertiary PAs into glucosides, that are assumed to be detoxification products (Hartmann et al., 1999). With L. jacobaeae the situation is more complex. In the present study the main proportion (> 75%) of the orally applied 18O-labeled senecionine N-oxide accumulated unaltered in the beetle. The remaining portion of <25% accounted for 16O-N-oxide. Since the specific radioactivity indicates that no unlabeled background senecionine N-oxide was present in the beetles, this fraction must be derived from re-N-oxidation of radioactively labeled senecionine produced by reduction of the 18O-N-oxide. This corroborates previous studies where we showed that radioactively labeled senecionine is N-oxidized when fed orally or injected into the beetles’ hemolymph (Narberhaus et al., 2003). Thus, in comparison to Oreina, Longitarsus jacobaeae is not only able to directly sequester the PA N-oxides but also to detoxify any tertiary PA by N-oxidation. To be sequestered, plant derived PAs have to cross cellular barriers. While tertiary alkaloids passively diffuse through biological membranes, PA N-oxides are hydrophilic, salt-like substances that are unable to do so unless a specific membrane carrier is present. The present study unequivocally demonstrates the presence of such PA membrane carriers in the beetles investigated. In all three species the gut epithelium has to be crossed to enter the body. In the case of the Oreina species a second passage is necessary to enter the secretory gland cells (Figure 4.7B). Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 54

Figure 4.7 Deduced PA-pathways in Longitarsus jacobaeae (A) and Oreina cacaliae (B). Solid arrows: main pathways, dotted arrows: minor pathways. Thick white arrows: PA transport and specific metabolization. PA, tertiary pyrrolizidine alkaloid; PA-O, pyrrolizidine alkaloid N-oxide; PA-G, pyrrolizidine alkaloid glucoside. Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 55

In L. jacobaeae defensive secretions could not be detected so far. However, storage organs must be present that are inaccessible to the N-oxygenase according to our earlier long term storage experiments with this species. These analyses showed that a stable proportion of tertiary PAs was stored over prolonged time periods without being N- oxidized, thus suggesting a transient N-oxidation of PAs in the beetles' hemolymph (Narberhaus et al., 2004) (Figure 4.7A).

The different strategies of handling PAs in adapted insects We conclude that both Longitarsus and Oreina are equipped with specific but partly different key adaptations to PAs. Both may absorb plant derived PA-N-oxides directly via carrier-mediated membrane transport and store the N-oxides in their bodies (Figure 4.7). In the case of Oreina there is a second specific membrane transfer from the hemolymph via the cells of the exocrine glands into the defensive secretions (Figure 4.7B). Since the PA N-oxides are concentrated in the defensive secretions the transport into the glands must be an energy-dependent active transport counteracting a steep concentration gradient (Hartmann et al., 1999). Whether the carrier-mediated transfer of PA N-oxide from the gut into the hemolymph is active or passive is yet unknown. The major difference between Oreina and Longitarsus is how they handle tertiary PAs. It should be recalled that PA-N-oxides are easily reduced even in presence of weak reducing agents such as cysteine (Hartmann and Toppel, 1987). Orally ingested PA N- oxides are spontaneously reduced in the guts of most vertebrates and absorbed passively as lipophilic tertiary alkaloids (Mattocks, 1986). The same seems to be the general rule in insects, too. For instance, in Spodoptera littoralis, a noctuid moth that tolerates but does not sequester PAs, ingested PA N-oxides are reduced and resulting tertiary alkaloids absorbed. Nevertheless, S. littoralis is able to tolerate PAs in its food because it efficiently eliminates the absorbed pro-toxic tertiary PAs from the hemolymph back into the gut (Lindigkeit et al., 1997). PA adapted arctiids and the African grasshopper Zonocerus variegatus behave like S. littoralis but instead of eliminating the absorbed pro-toxic PAs they detoxify them by efficient and specific N-oxygenation, the resulting non-toxic N-oxides remain trapped in the body (Lindigkeit et al., 1997). N-oxide reduction and passive absorption thus appears to be the normal fate of ingested PA N- oxides in most animals. In contrast, as demonstrated, Oreina and Longitarsus are both able to suppress the N-oxide reduction in the gut and absorb the plant acquired PAs as non-toxic N-oxides. However, any pro-toxic tertiary PA formed in the gut or body is efficiently detoxified, either by N-oxidation, as confirmed for Longitarsus (Narberhaus et al., 2003) or by glucosylation as demonstrated for Oreina (Hartmann et al., 1999). Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 56

In addition to the different modes of detoxification of tertiary PAs there is another striking difference between the two beetles. Oreina is unable to utilize tertiary PAs (Ehmke et al., 1991), whereas Longitarsus sequesters tertiary PAs with almost the same efficiency as N-oxides (Narberhaus et al., 2003). In this case, most of the alkaloids are stored as N- oxides by autogenous oxidation, while small amounts are retained as tertiary PAs in still unknown compartments where they are no longer accessible to the N-oxygenase (see above, Figure 4.7A). Apparently, the suppression of reduction of ingested PA N-oxides is less efficient in Longitarsus than in Oreina. This is indicated by the substantial proportion of recovered senecionine N-oxide that has lost the 18O label in the double tracer feeding experiment (i.e., about 23%, see Table 4.2). Thus it appears that in Longitarsus reduced PAs are recycled by re-N-oxidation, whereas in Oreina the suppression of reduction appears to be so efficient that a “recycling” of tertiary PAs may be dispensable. The results presented here complete our vision about the strategies of PA adapted insects to absorb, maintain and store PAs and prevent self-poisoning. In any case an accumulation of detrimental concentrations of pro-toxic tertiary PAs in metabolically active tissues is avoided. Presently the following strategies can be documented. (i) arctiid moths: the passively absorbed tertiary PAs are specifically N- oxidized and always maintained in the state of the non-toxic N-oxides. (ii) Oreina leaf beetles: the reduction of ingested N-oxides is efficiently suppressed and the N-oxides are absorbed via specific carrier-mediated transport and build up a storage pool in the body that serves to fill the exocrine glands; Oreina is unable to N-oxidize tertiary PAs that are, in contrast, detoxified by glucosylation. (iii) Platyphora leaf beetles: the passively absorbed tertiary PAs are efficiently removed from the hemolymph and pumped into the exocrine glands; no PA storage is found in the body outside the glands; Platyphora is unable to N-oxidize tertiary PAs. (iv) Longitarsus leaf beetles: the reduction of ingested N-oxides is suppressed and the N-oxides are absorbed via specific carrier-mediated transport; passively absorbed tertiary PAs are partly recycled into the N-oxides by specific N-oxidation and partly stored as tertiary PAs in a compartment where they are apparently separated from the metabolically active tissue. Thus, L. jacobaeae combines the basic strategies of the other three PA adapted insect taxa: It is able to N-oxidize tertiary PAs, specifically absorb the N-oxides and safely store pro-toxic tertiary PAs.

Is dihydrosenecionine a specific metabolite of Longitarsus jacobaeae? A specific metabolic ability of L. jacobaeae was observed by chance: the presence of a dihydrosenecionine N-oxide, tentatively identified as the 15,20-dihydro derivative, that as an unexpected hydrogenated congener of the senecionine [18O]N-oxide caused some Direct evidence for membrane transport of plant acquired pyrrolizidine alkaloids in two leaf beetle genera 57 complications in the 18O-tracer experiment. The dihydrosenecionine is already known from the PA-profile of a L. jacobaeae field population (Dobler et al., 2000). It has, however, not been found in the leaves of the beetle’s food plant nor has it ever been reported to occur in Senecio jacobaea which is one of the best studied Senecio species (Witte et al., 1992). Thus, it is very likely that the beetles themselves catalyze the hydrogenation of plant acquired senecionine. In our feeding experiment of L. jacobaeae with senecionine [18O]N-oxide the direct MS analysis resulted in a proportion of up to 8 % double labeled molecules in the recovered dihydrosenecionine N-oxide (Table 4.2). The hydrogenation of a small proportion of labeled senecionine N-oxide is also evidenced by a comparison of the specific activities of the recovered reduced PAs (Table 4.2). However, the efficiency of senecionine hydrogenation in adult beetles appears to be very low although dihydrosenecionine is generally dominating in field collected control samples. Therefore, the substance might possibly originate in larval metabolism. Only tracer experiments with larvae might clarify this issue. 58

5 Pyrrolizidine alkaloids on three trophic levels – evidence for toxic and deterrent effects on phytophages and predators

5.1 Summary

We demonstrate the toxic effects of a pyrrolizidine alkaloid (PA) on growth and survival of the eri silk moth Philosamia ricini. In a feeding experiment, larvae of this generalist herbivore fed with an artificial PA diet gained weight significantly slower than control animals, and died as pupae. We suggest that derivatives of the ingested PA N-oxide damage developmental functions during metamorphosis. A tracer test with [14C]senecionine N-oxide revealed that the caterpillars lack adaptations that would prevent conversion of the chemical into the pro-toxic free base. In contrast, the PA adapted leaf beetle Longitarsus anchusae accumulates PAs as N-oxides. We tested the purpose of sequestration in this species as defence against predators. Through a series of prey choice experiments with three carabid predator species, chemically non-protected bark beetle pupae were chosen almost uniformly over L. anchusae pupae. In a following choice test with one of these predators, artificially PA-treated mealworm segments deterred the predator from feeding. Overall the study corroborates the immediate toxic effect of PAs on non-adapted herbivores and the protective effect that adapted insects may gain by sequestering them. It thereby underlines the potential for PAs to play a central role in multitrophic interactions between plants, phytophages and their predators.

5.2 Introduction

Pyrrolizidine alkaloids (PAs) are secondary plant compounds, commonly found in the plant families Asteraceae, Boraginaceae and Fabacae (Hartmann and Witte, 1995). It is widely accepted that their major function lies in the plant’s protection against herbivory (Boppré, 1986, 1990; Hartmann and Witte, 1995). Feeding experiments with several animal taxa demonstrate that generalist herbivore vertebrates and invertebrates reject PA contaminated food (Bernays and Chapman, 1977; Boppré, 1986; Van Dam et al., 1995). In addition, PA synthesis can be induced by herbivory (Van Dam et al., 1993), indirectly indicating that alkaloid production is a variable and highly adaptive defence character in plants. Pyrrolizidine alkaloids on three trophic levels 59

The idea of PAs functioning as defence agents in plants implies their deleteriousness to non-adapted herbivores. However, to date there is no direct empirical proof of a negative physiological impact of PAs on insects. Due to economical interest in human pharmacology and in animal livestock, existing toxicological studies of PAs are abundant for mammals (Cheeke, 1989; Mattocks, 1986; Prakash et al., 1999; Schmeller, 1997; Steenkamp et al., 2000; Stewart and Steenkamp, 2001), but for insects, this issue has been largely unexplored. PAs have been shown to cause mutagenic effects in a wing spot test of Drosophila melanogaster (Frei et al., 1992). In vivo, however, no relevant effects have hitherto been demonstrated. Such a proof is generally regarded as difficult because in standard non-adapted herbivores the strong deterrent effect of PAs precludes studies of their toxicity. To investigate direct consequences of alkaloid uptake to the individual insect we performed feeding experiments with an exemplary generalist herbivore. We found the eri silkworm Philosamia ricini (Boisduval, 1854) (Saturniidae) to be the ideal system for bioassays with an artificial diet since the larvae seem to lack sensual and/or behavioural discrimination of detrimental chemicals in their food (Kotaro Konno, personal communication). In our experiments, P. ricini caterpillars proved to feed indiscriminately on PA treated leaves. We were thus able to observe growth and survival in the animals during a complete developmental cycle. In an additional feeding experiment with radioactively labelled senecionine N-oxide we investigated the metabolic processing of PAs in P. ricini. In addition to examining the toxicity of PAs to a generalist herbivore, we investigated the role of PAs on the next trophic level, in the interaction of phytophages and their predators. A number of flea beetle species in the genus Longitarsus (Chrysomelidae, Alticinae) are specialist feeders on PA plants and have the ability to detoxify ingested alkaloids through N-oxidation by a specific enzyme (Narberhaus et al., 2003). Furthermore, several species with PA hosts analyzed even accumulate these substances (Dobler et al., 2000; Haberer and Dobler, 1999), a process referred to as sequestration. Such behaviour has been observed in a number of insect taxa and has often been shown to act as a type of chemical co-option: the phytophages reutilize the plant substances for their own antipredator protection (Bernays and Chapman, 1977; Brown, 1984; Eisner and Eisner, 1991; Eisner et al., 2000; Eisner and Meinwald, 1987; Gonzales et al., 1999; Orr et al., 1996). The evolution of chemical defence in herbivores has even become such a far reaching analogue to the one in plants that there are examples of secondary sequestration by predators storing the plant-derived PAs from their prey (Witte et al., 1990). Pyrrolizidine alkaloids on three trophic levels 60

In the present work we investigated the role of alkaloid storage in the PA adapted Longitarsus anchusae (Paykull, 1799) for the purpose of defence against predators. Adult Longitarsus flea beetles are well protected from many vertebrate and invertebrate predators by their pronounced jumping ability. We therefore suggest that chemical defensive compounds are particularly valuable during the insects’ relatively immotile, soil-living pupal or larval stages. To test the defensive properties of PAs sequestered by Longitarsus we performed prey choice experiments with carabid beetles as generalist predators that are a likely threat to the flea beetle larvae and pupae. In a first series, a choice between PA-containing Longitarsus anchusae pupae and pupae of a bark beetle species was offered. In a second series, to equalize prey properties, predators were given a choice between artificially PA-treated and non-treated baits.

5.3 Material and Methods

PA toxicity Eggs of Philosamia ricini (Saturniidae), the eri silk moth, were obtained from OPIE- insectes (http://www.inra.fr/Internet/Hebergement/OPIE-Insectes/pa.htm) and were kept in Petri dishes lined with moist filter paper until hatching. First instar larvae were individually put into plastic boxes and fed with fresh leaf discs of the tree-of-heaven Ailanthus altissima (Simaroubaceae), a plant that naturally does not contain PAs. A. altissima is a common garden tree in the town of Freiburg, where the experiments were carried out. Ten larvae were fed with leaves that were artificially covered with a methanolic seneciphylline N-oxide solution, another ten individuals were fed with leaf material covered with methanol only and served as control group. Seneciphylline N-oxide was chosen as exemplary PA due to its frequent occurrence in host plants of specialists in the Chrysomelidae, and due to its commercial availability. The N-oxide was produced by oxidation of tertiary seneciphylline (Roth) according to Craig and Purushothaman (1970). The purity of the resulting substance was verified by TLC and detection according to Mattocks (1967). The total alkaloid concentration applied to the leaf discs was adjusted to 2 % seneciphylline N-oxide per leaf dry weight to match that seen in typical chrysomelid host plants. All individuals were fed ad libitum and were weighed regularly. To restrict the usage of expensive seneciphylline the PA treatment was ceased after 15 days. After that period all caterpillars were equally fed with untreated Ailanthus leaves and were subsequently observed in their development. Pyrrolizidine alkaloids on three trophic levels 61

To examine the metabolic fate of PAs in P. ricini, three additional caterpillars were fed with a radioactively labelled PA, [14C]senecionine N-oxide. Insects and faeces were subsequently extracted and analyzed. The tracer method including extraction procedure, measurement of total radioacticity, and metabolite analysis via thin layer chromatography is described elsewhere (Narberhaus et al., 2003).

Predator experiments with Longitarsus pupae As predators, three carabid beetle species were chosen that naturally inhabit the upper soil layer and that could be potential enemies of Longitarsus in its pupal stage: adults of Pogonus persicus Chaudoir and Acupalpus elegans (Dejean) and larvae of Pterostichus oblongopunctatus (Fabricius). Adult beetles of all three species were field caught in Illmitz, Austria (A. elegans and P. oblongopunctatus) and Freiburg, Germany (P. oblongopunctatus) in June 2001. They were kept at 20°C under long day conditions (16 h/8 h light/dark) in 10 x 15 x 10 cm plastic boxes lined with moist filter paper. Eggs produced by P. oblongopunctatus were kept separately in small Petri dishes until hatching. Emerging larvae were transferred to turf filled Drosophila tubes. Before use in the experiments, predators were maintained on a diet of freshly cut mealworms. As prey choice objects, pupae of Longitarsus anchusae (Paykull) (Chrysomelidae) and pupae of Pityogenes chalcographus (L.) (Scolytidae) were chosen. Longitarsus pupae were obtained from field-caught adults collected from their host Symphytum officinale (Boraginaceae) early spring. Pityogenes pupae were collected from bark of freshly chopped spruce that accrued in the local forestry. The wood-boring species is not known to be chemically protected and its pupae do not contain alkaloids. Every choice experiment consisted of the two types of living prey and an individual predator in a 3 x 5 x 2 cm plastic box. For each predator, the first pupa eaten was recorded. Each individual predator was used only once.

Artificial prey treatment with a PA To test whether PAs are responsible for the predator’s avoidance of Longitarsus pupae in their diet, binary choice experiments with artificially PA-treated ‘prey’ were performed. Each experiment consisted of both a piece of PA-treated and a control piece of ‘prey’ that were offered simultaneously to a Pterostichus oblongopunctatus larva in 2 x 3 x 1 cm plastic boxes. As model ‘prey’ single abdominal segments of mealworms were used, of the approximate size of a Longitarsus pupa. For PA treated ‘prey’ 1 µl of an aqueous seneciphylline N-oxide solution (25 mg/ml) was applied onto the mealworm segments. The final alkaloid concentration in the ‘prey’ thus amounted to 1 % per dry weight. The Pyrrolizidine alkaloids on three trophic levels 62 control ‘prey’ was treated with 1 µl of water. The ‘prey’ choice of the predator was defined as the first ‘prey’ object on which the predator fed continuously for at least 30 seconds. Each Pterostichus larva was used in only one trial.

5.4 Results and Discussion

Toxicity of PAs The alkaloid-fed larvae of Philosamia ricini grew significantly slower than the control individuals (Figure 5.1). After ten days, the average weight difference amounted to 30%. Furthermore, the entire set of PA-fed insects died within or before the pupal stage. In contrast, nine out of ten control animals completed metamorphosis after ca. 40 days and lived another ca. 10 days.

Figure 5.1 Growth of Philosamia ricini fed with seneciphylline N-oxide treated leaves (Sen) and control. Measurements were taken until day 15. Control animals gained weight significantly faster than PA-fed animals (time vs. treatment interaction p<0.001, repeated measures ANOVA). After 40 days, control animals emerged from pupae, whereas all PA-fed individuals died as pupa or earlier. Pyrrolizidine alkaloids on three trophic levels 63

The tracer feeding experiment with [14C]senecionine N-oxide showed that the caterpillars were not able to efficiently detoxify this chemical. After ingestion by a herbivore, PA N- oxides, the storage form of PAs in plants, are easily converted to tertiary alkaloids through the mildly reducing conditions in the gut. Due to the lipophilic properties of tertiary PAs they can passively penetrate through gut membranes (Hartmann, 1999; Lindigkeit et al., 1997). For vertebrates it is known that tertiary PAs can then be converted into highly reactive pyrrolic metabolites by microsomal cytochrome P450 oxidases, with the consequence of detrimental cytotoxic and genotoxic effects (Winter & Segall 1989). Adapted PA plant feeding insects usually store PAs in their non-toxic N- oxide form in their bodies, either by preventing reduction or by re-N-oxidation of reduced PAs (e.g. (Hartmann and Ober, 2000). Longitarsus species for example are able to rapidly metabolize major amounts of ingested tertiary PAs into the safe storage form through N-oxidation by a specific oxygenase (Narberhaus et al., 2004). The non-adapted P. ricini proved to be unable to do so. In our tracer experiment, the caterpillars accumulated only traces of the substance (ca. 2 % of recovered radioactivity) and excreted major amounts with the faeces. Faeces that was subjected to further analysis by thin layer chromatography contained no significant amounts of N-oxides. Instead, 8 % of the detected metabolites consisted of tertiary [14C]senecionine and 92 % could be recovered as three polar substances that have not been identified yet. We assume that PA N- oxides in P. ricini are reduced to the tertiary form in the insects’ guts and diffuse into their bodies. The lack of a re-oxidizing enzyme in P. ricini possibly results in further processing of PAs into compounds that damage functions of metabolism and development. Apart from size and weight, no ectomorphological differences between the long-term PA-fed P. ricini and the control group were recognized. However, since death of the PA fed animals occurred chiefly during the pupal stage, the toxic effects of PAs must be particularly dramatic on developmental processes related to metamorphosis. To our knowledge, our results represent the first experimental evidence of PAs causing severe developmental dysfunctions in phytophagous insects. In the light of such a toxic effect it can be understood that the behavioural avoidance of PAs in the food by insect herbivores is under strong selection pressure. Besides, in view of the toxicity of PAs, it becomes obvious that such compounds impose a serious hurdle to the colonization of PA plants by a non-adapted phytophage. The ancestors of well adapted species like the arctiid Tyria jacobaeae (Lindigkeit et al., 1997) and chrysomelids of the genera Oreina (Hartmann et al., 1997) or Longitarsus (Narberhaus et al., 2003) must therefore have undergone a number of behavioural and physiological sophistications before they shifted to the present-day PA-containing hosts. Pyrrolizidine alkaloids on three trophic levels 64

PA sequestration in Longitarsus as protection against predators In the first prey choice series, all three carabid predator species preferred the Pityogenes pupa over the Longitarsus pupa (Figure 5.2). All tested Pogonus persicus and Pterostichus oblongopunctatus individuals chose the bark beetle, in the case of Aucupalpus elegans 17 out of 18 individuals did so. The results are in all cases highly significant. The bark beetles used as prey in the experiments do not contain alkaloids. L. anchusae in contrast, sequesters diverse lycopsamine-type PAs from its Symphytum officinale host plant. As shown earlier, L. anchusae accumulates alkaloids beginning with the larval stages, that feed on the fine hair roots of Symphytum. As pupae, they contain around 1.8 µg PA per mg dry weight. The compounds are then transferred into the adult stage that contain similar amounts as the pupae (Dobler et al., 2000; Haberer and Dobler, 1999).

bark beetle pupa p<0.001 p<0.001 Longitarsus pupa 100

p<0.01 80

20 Proportion of preferred prey (%) 0 Acupalpus elegans Pogonus persicus Pterostichus oblongopunctatus Predator species

Figure 5.2 Prey preference of three carabid predator species between PA sequestering Longitarsus anchusae and bark beetle pupae (Wilcoxon paired samples, n=18,19, and 8).

The deterrent effect of PAs in the prey was corroborated in the choice experiment with artificially PA laden mealworm prey. The predator species used, Pterostichus oblongopunctatus, significantly preferred the control prey over the PA treated prey (Figure 5.3). Since the only difference between the two prey objects in this case was their alkaloid content, it can be concluded that PAs have a deterrent effect on the Pyrrolizidine alkaloids on three trophic levels 65 predator. PAs have similarly been found to be deterrent to a number of other predator taxa. As examples, Nephila maculata spiders for example refuse to feed on PA sequestering Tellervo butterflies (Orr et al., 1996), the predatory ant Leptothorax longispinosus preys upon Utetheisa ornatrix (Arctiidae) eggs only if the eggs were produced by parents artificially reared on an alkaloid free diet (Hare and Eisner, 1993), and the pileated finch Coryphospingus pileatus avoids PAs by taste and learns to associate the bad taste to visual cues (Cardoso, 1997).

80 p<0.05 70

Proportion of preferred prey (%) 10

0 Sen - Sen + Prey treatment

Figure 5.3 Prey preference of Pterostichus oblongopunctatus larvae. Prey labelled as Sen+ were mealworm segments treated with seneciphylline N-oxide, Sen- were controls (Wilcoxon paired samples, n=15).

Compared to other sequestering insect taxa, PA concentrations in Longitarsus beetles are low. While for example PA sequestering ithomiine butterflies amount to 2 - 7 % of dry weight on average and reach up to 20 % of dry weight in extreme cases, pupae of Longitarsus anchusae only contain 0,2 % PAs. Nevertheless the carabid predators were deterred from L. anchusae pupae in our experiments. Storage of alkaloids in the juvenile stages of this species should therefore not be considered a within-body-disposal to prevent self poisoning but rather a useful defensive strategy. Our results point out that plant-derived PAs, even if present in low doses, may play an important role in the behavioural interaction of phytophages and their predators. 66

6 Abschließende Diskussion

6.1 Speicherung und Metabolisierung von PAs bei Longitarsus

Die in dieser Arbeit vorwiegend untersuchte Chrysomelidengattung Longitarsus erwies sich hinsichtlich ihrer Assoziationen gegenüber der Sekundärstoffklasse der Pyrrolizidin- Alkaloide als besonders vielschichtiges System. Bereits auf Verhaltensebene konnte festgestellt werden, dass Arten dieser Gattung trotz ihrer nahen Verwandtschaft auf PA- haltige Nahrung sehr verschieden reagieren. Nach den Futterwahl- und „No-choice“- Experimenten aus Kapitel 2 (Narberhaus et al., 2003) kristallisierten sich die folgenden PA-Reaktionstypen bei Longitarsus heraus: (i) strikte Ablehnung von PAs in der Nahrung (L. nigrofasciatus, L. australis), (ii) „neutrale Arten“, weder Ablehnung noch Präferenz (z.B. L. atricillus, L. rubiginosus), (iii) Präferenz PA-haltiger Nahrung (L. suturellus). Die einzigen als PAs strikt ablehnend aufgezeigten Arten leben oligophag auf Scrophulariaceen, einer Pflanzenfamilie ohne natürliche PA-Vorkommen. Unter den „neutral“ reagierenden Käfern finden sich sowohl Arten, die auf PA-haltigen Wirten leben (L. exoletus), als auch einige, die im Freiland mit PAs nicht in Kontakt kommen (L. membranaceus, L. pratensis). Die Tracerstudien (ebenfalls Kapitel 2) (Narberhaus et al., 2003) beweisen, dass alle PA-akzeptierenden Arten die Fähigkeit besitzen, PAs in ihrer ungiftigen Form, als PA-N-Oxide, zu speichern. Auch nach Aufnahme tertiärer PAs können diese Moleküle größtenteils als N-Oxide aus den Käfern zurück gewonnen werden. Die Tiere besitzen demnach die Fähigkeit zur N-Oxidation, ein Prozess, der der Katalyse durch ein spezifisches Enzym bedarf. Im Weiteren wurden zwei ausgewählten PA-angepassten Arten radioaktiv markierte Alkaloide gefüttert, die nicht zu den Pyrrolizidin-Alkaloiden gehören: [3H]Atropin und [14C]Nicotin. Diese beiden Substanzen wurden von dem Enzym der Käfer nicht N-oxidiert, was dessen Spezifität für PA- Substrate anzeigt. Die Tatsache, dass auch Longitarsus-Arten ohne alkaloidhaltige Wirtspflanzen diese Stoffe umwandeln können, stimmt demnach mit den Ergebnissen der Verhaltens- experimente überein. Ohne eine kausale bzw. evolutionäre Abfolge implizieren zu wollen, kann man konstatieren: Käferarten ohne PA-Vermeidungsmechanismen in ihrem Verhalten besitzen die Fähigkeit, aufgenommene PAs durch N-Oxidation unschädlich zu machen. Wie bereits erwähnt, erwiesen sich die auf Scrophulariaceen lebenden Arten bei den Verhaltenstests durch ihre strikte Ablehnung von PAs als Sonderfall. Um hier dennoch eine mögliche PA-Metabolisierung zu untersuchen, wurde Käfern einer dieser Arten, L. australis, radioaktiv markiertes tertiäres Senecionin in die Hämolymphe injiziert. Abschließende Diskussion 67

Das Ergebnis bewies eindeutig, dass diesen Käfern jegliche Fähigkeit zur N-Oxidation fehlt: Der Tracer wurde unverändert in der Tertiärform zurück gewonnen, während im Kontrollexperiment mit der angepassten Art L. jacobaeae eine vollständige Umwandlung festgestellt werden konnte. Auch diese Tatsache ist im Einklang mit den Verhaltenstests – dieses Mal umgekehrt formuliert: Eine Insektenart ohne einen physiologischen PA- Entgiftungsmechanismus benötigt entsprechende Verhaltensmechanismen, die eine Aufnahme von PAs mit der Nahrung verhindern. Bei einer Freilandpopulation erwarten wir allerdings das Entstehen eines Vermeidungsverhaltens nur dann, wenn durch dauerhafte Konfrontation mit PA-haltigen Pflanzen ein entsprechender Selektionsdruck verursacht wird. Die untersuchten PA-sequestrierenden Longitarsus-Arten speichern neben dem ungiftigen PA-N-Oxid immer auch einen geringeren Anteil an tertiärem PA. Trotz Anwesenheit eines N-oxidierenden Enzyms bleibt dieser Anteil auch zwei Wochen nach Aufnahme in den Käfern vorhanden, wie die Tracer-Experimente zur Langzeit- Speicherung von PAs zeigten (Narberhaus et al., 2004) (s. Kapitel 3). Dies ließ schlussfolgern, dass die PAs in den Käfern außerhalb der Reichweite des Enzyms gelagert werden. Ein weiteres Experiment, in dem einige Individuen der Art L. aeruginosus sechs Tage nach Aufnahme eines markierten PA in verschiedene Körpersektionen zerlegt wurden, unterstützte diese Hypothese und wies auf eine transiente N-oxidation von PAs während der Passage durch die Hämolymphe hin. In der Radio-Dünnschichtchromatographie der verschiedenen Körperextrakte zeigte sich, dass die tertiären PAs vor allem in den Elytren gespeichert werden, nicht jedoch in der Hämolymphe, in der sich ausschließlich PA-N-oxide fanden. Als wahrscheinlicher Wirkungsort des Enzyms kommt daher nur die Hämolymphe in Frage, wie es auch bereits für die Senecionin-N-oxygenase des Schmetterlings Tyria jacobaeae beschrieben wurde (Lindigkeit et al., 1997; Naumann et al., 2002). Obgleich die N-Oxygenase bei Longitarsus einen steten Anteil des zugeführten Substrats unverwandelt belässt, scheint sie ein hocheffizientes Enzym zu sein, da sich der Anteil umgewandelter tertiärer PAs unabhängig von der angebotenen Substratmenge erwies. In einem Tracer-Experiment mit 30-facher PA-Dosis wurden aus den Käfern die gleichen Anteile Tertiär-PAs zu N- Oxiden zurück gewonnen, wie im Kontrollexperiment mit einfacher Dosis (Narberhaus et al., 2004). Abschließende Diskussion 68

6.2 Membran-Carrier für PAs

Wie oben dargelegt, ist die Fähigkeit zur Entgiftung tertiärer PAs eine adaptive Einrichtung zur gefahrlosen Sequestration von Pyrrolizidin-Alkaloiden. Dies ist bei Longitarsus durch die Existenz einer PA-N-oxygenase als Entgiftungsenzym verwirklicht. Nun enthalten die Asteraceen- und Boraginaceen-Wirtspflanzen von Longitarsus jedoch ausschließlich PA-N-Oxide. Eine N-Oxidation von PAs ist daher nur notwendig, wenn die Stoffe vorher zur protoxischen „freien Base“ reduziert worden sind. Durch die bisher beschriebenen Experimente ist dies nicht ausreichend erkennbar. Um zu identifizieren, in welcher chemischen Form die PAs in die Körper der Käfer aufgenommen werden, d.h. in welcher Form sie die Darmmembran der Tiere passieren, stellten wir ein doppelt markiertes Traceralkaloid her, [14C]Senecionin [18O]N-Oxid, und fütterten es adulten Longitarsus jacobaeae. Im Falle einer Reduktion, der passiven Aufnahme tertiären Senecionins und einer anschließenden Re-N-oxidierung in der Hämolymphe würden wir den Verlust des markierten Sauerstoffs annehmen, im Falle eines N-Oxid-Transports würde die Doppelmarkierung des Senecionins erhalten bleiben. Einige Tage nach der Behandlung ließen sich 75% des Alkaloids unverändert mit markiertem Sauerstoffatom aus den Körpern der Tiere zurückgewinnen, ein eindeutiger Beleg für die Existenz eines Membrancarriers bei Longitarsus (s. Kapitel 4). Auch für die in die Experimente mit einbezogenen Vergleichsarten der Gattung Oreina belegte die Rückgewinnung der unverwandelten Tracersubstanz die Existenz von PA-Carriern. Da der Tracer bei diesen Arten aus den Wehrsekreten zurück gewonnen wurde, die die Tiere über ihre Elytren und Pronota ausscheiden, muss bei Oreina neben dem Membrancarrier im Darmepithel noch ein weiterer PA-Carrier in den sekretorischen Zellen der Wehrdrüsen angenommen werden. Da dieser letztere Transportvorgang in die Drüsen gegen ein starkes Konzentrationsgefälle (ca. 1:150) zwischen Hämolymphe und Drüsensekret erfolgt, muss es sich hier um einen energieabhängigen, aktiven Prozess handeln (Hartmann et al., 1999). Bei Oreina Blattkäfern wurde bereits aufgrund indirekter Hinweise der Membran- transfer von N-Oxiden vermutet (Hartmann et al., 1999; Rowell-Rahier et al., 1991). Die vorliegende Studie an Longitarsus und Oreina liefert nun den eindeutigen Beleg für die Existenz eines Carrier-vermittelten PA-Transports. Abschließende Diskussion 69

6.3 Strategien der PA-Sequestration im Vergleich

Den bisher bekannten Strategien zur Aufnahme und Speicherung von Pyrrolizidin- Alkaloiden bei Insekten ist ein wesentlicher Aspekt gemeinsam: Alle Arten vermeiden die Ansammlung protoxischer Tertiäralkaloide in metabolisch aktivem Gewebe. Schmetter- linge der Familie Arctiidae erreichen dies durch vollständige Reduktion der PA-N-oxide im Darm, die passive Aufnahme von tertiären PAs durch die Darmwand und deren effiziente Re-N-oxidierung in der Hämolymphe mittels einer spezifischen Flavomonooxygenase (Lindigkeit et al., 1997; Naumann et al., 2002). Nach der Re- Oxidierung werden N-Oxide in hoher Konzentration in der Hämolymphe der Tiere gespeichert und akkumuliert. Der neotropische Blattkäfer Platyphora boucardi dagegen speichert tertiäre PAs. Als solche sind diese Stoffe bereits in den Wirtspflanzen der Gattung enthalten, womit die Apocynacee Prestonia portobellensis eine seltene Ausnahme unter den PA-Pflanzen darstellt. Nach ebenfalls passiver Diffusion durch die Darmmembran werden die Stoffe in diesen Käfern nicht oxidiert, sondern schnell und effizient in die Wehrdrüsen weiter transportiert, wo sie fern von metabolisch aktivem Gewebe sicher gespeichert werden (Hartmann et al., 2001; Pasteels et al., 2001; Pasteels et al., 2003b). Käfer der alpinen Chrysomelidengattung Oreina sequestrieren wie die Arctiiden PA-N-oxide, sind jedoch nicht in der Lage, tertiäre PAs zu oxidieren. Wie oben beschrieben, werden in diesen Käfern aufgenommene N-Oxide nicht reduziert und mittels Membrancarrier durch Darmwand und Drüsenzellen bis in ihre Wehrsekrete transportiert. Dennoch in die Hämolymphe gelangende tertiäre PAs werden durch Glucosylierung entgiftet und ausgeschieden (Hartmann et al., 1999). Dieser Prozess ersetzt bei Oreina die entgiftende N-Oxidation der freien Basen, wie es bei verschiedenen Longitarsus-Arten der Fall ist. Oreina und Longitarsus besitzen also je zwei Schlüsselanpassungen, die ihnen die sichere Sequestration von Wirtspflanzenalkaloiden ermöglichen. Zum einen ist dies die Fähigkeit zur Absorption mit der Nahrung aufgenommener PA-N-oxide über den Carrier-vermittelten Membran- transport sowie deren Speicherung im Körper, zum anderen die N-Oxidation bzw. Glucosylierung zur Entgiftung in den Körper gelangter tertiärer PAs. Der Flohkäfer L. jacobaeae kann nach den Ergebnissen der vorliegenden Arbeit als der „vielseitigste“ unter den bekannten PA-Sequestrierern betrachtet werden. Seine Sequestrationsstrategie besteht im Wesentlichen aus einer Kombination der Mechanismen der anderen Taxa: Er nimmt spezifisch N-Oxide in den Körper auf wie Oreina, kann reduzierte PAs N-oxidieren wie die Arctiiden und speichert tertiäre PAs wie Platyphora. Abschließende Diskussion 70

6.4 Evolutionäre Aspekte der PA-Adaptation bei Longitarsus

Bei Betrachtung einer auf mtDNA-Sequenzdaten basierenden Phylogenie-Hypothese der Gattung Longitarsus (Dobler, 2001) wird deutlich, dass die Fähigkeit zur PA-N-oxidation bei Arten verschiedener Kladen, im gesamten Stammbaum verteilt auftaucht (Abb. 6.1). Sie scheint weit gehend unabhängig davon in den Käfern vorhanden zu sein, ob deren Wirtspflanzen Pyrrolizidin-Alkaloide enthalten. Neben den in Kapitel 2 vorgestellten Arten sind bei der Darstellung weitere Longitarsus-Arten aus anderen Verwandtschaftsgruppen berücksichtigt, die zusätzlich anhand von Tracer-Fütterungen hinsichtlich ihrer N-Oxidationsfähigkeit überprüft wurden (L. membranaceus, L. melanocephalus, L. pratensis, L. ballotae, L. suturellus). Auch einer Art der Schwestergruppe Aphthona wurde markiertes tertiäres Senecionin verabreicht. Hierfür wurde die in der Stammbaumanalyse nicht enthaltene A. cyparissiae herangezogen, die im „No-choice“ Fütterungstest PA-behandelte Nahrung angenommen hatte (Kapitel 2). Die fünf Longitarsus-Arten, von denen vier auf PA-freien Lamiaceen- und Plantaginaceen-Wirten fressen, erwiesen sich in der Lage zu N-oxidieren, nicht jedoch die Aphthona-Art, von der wie im Falle des Injektionsexperiments mit L. australis ausschließlich tertiäres Senecionin zurück gewonnen wurde. Insgesamt stellte sich also heraus, dass neben den auf PA-haltige Boraginaceen und Asteraceen spezialisierten Longitarsus-Arten auch eine beträchtliche Anzahl von Arten ohne PA-Wirte in der Lage sind, PAs zu N-oxidieren. Dies sind unter anderen L. pellucidus, L. ballotae, L. luridus, L. melanocephalus, L. membranaceus, Arten, die nach der Stammbaumhypothese von Dobler (2001) Angehörige verschiedener und zum Teil von PA-adaptierten Gruppen umgebenen Kladen sind. Da solchen Arten spezifische Anpassungen an Sekundärstoffe, mit denen sie in der Natur nie konfrontiert sind, augenscheinlich nicht von Nutzen sein können, scheint es sich hier um phylogenetische Relikte zu handeln, die die Käfer aus Zeiten der Assoziation mit PA-haltigen Wirtspflanzen beibehalten haben. Derzeit lässt es sich nicht entscheiden, ob die Enzyme in diesen Käfern noch dauerhaft produziert werden, oder ob sie erst durch Kontakt mit dem PA-Substrat induziert werden. Eine im laufenden Projekt unternommene molekulare Charakterisierung des entsprechenden Proteins wird hierzu weiteren Aufschluss geben. Abschließende Diskussion 71

L. australis – L. tabidus L. nigrofasciatus L. apicalis L. atricillus L. pellucidus + L. dorsalis L. languidus L. succineus + L. succineus 2 Asteraceae L. aeruginosus + L. brisouti L. suturellus + L. echii Boraginaceae L. rubiginosus L. brunneus L. ballotae + L. ballotae 2 L. minusculus L. luridus 1 + L. luridus 2 L. luridus L. parvulus L. jacobaeae + Asteraceae L. absynthii L. longiseta L. holsaticus L. melanocephalus + L. melanocephalus 2 L. anchusae + L. nasturtii Boraginaceae L. indigonaceus L. reichei L. pratensis + L. pratensis 2 L. lewisii L. lewisii 2 L. minimus L. membranaceus + L. exoletus L. lateripunctatus Boraginaceae L. pinguis L. ventricosus L. lycopi L. salviae L. obliteratoides L. obliteratus 2 L. obliteratus Aphtona lutescens (– ) Aphtona venustula

Abb. 6.1 Maximale Parsimonie-Analyse der Gattung Longitarsus, basierend auf mitochondrialen Cytochrom-Oxidase Gensequenzen. Zwei Arten der Schwestergattung Aphthona wurden zur Wurzelung des Baumes verwendet. Äste, die zu Arten mit PA-haltigen Wirten führen, sind hellgrau unterlegt, der Kladus der PA-ablehnenden Longitarsus-Arten ist schraffiert. Arten mit Fähigkeit zur PA- N-oxidation sind mit + gekennzeichnet, Arten ohne diese Fähigkeit mit –. Von Aphthona wurde A. cyparissiae getestet, die in diesem Sequenzvergleich nicht aufgenommen ist und ebenfalls nicht N- oxidiert. Verändert nach Dobler (2001). Abschließende Diskussion 72

In der Stammbaumhypothese von Longitarsus fällt noch eine zweite Beziehung auf. Die einzigen Arten, auf die PAs eine stark deterrente Wirkung haben (siehe die „No-choice“ Fütterungsexperimente in Kapitel 2), finden sich in den basalen Ästen des Stammbaums. Zum einen ist dies die Schwestergruppe Aphthona, von der A. cyparissiae getestet wurde. Diese Art ist zwar selbst nicht im vorliegenden Stammbaum enthalten, die Monophylie der Gattungen Longitarsus und Aphthona sowie ihre nahe Verwandtschaft ist jedoch morphologisch gut belegt. Zum anderen liegen in dieser Stammbaum- hypothese die bereits erwähnten Scrophulariaceen-Fresser L. australis und L. nigrofasciatus basal zur restlichen Gattung (oberer Ast in Abb. 6.1). Im Falle von L. australis und A. cyparissiae konnte deren mangelnde Anpassung an Pyrrolizidin- Alkaloide sowohl durch die Verhaltenstests als auch in den Tracer-Injektionen bzw. - Fütterungen belegt werden: Beiden Arten fehlt eine N-Oxygenase. Basierend auf meinen Daten und dem vorliegenden Stammbaum wäre es die sparsamste Erklärung, dass die Anpassung an Pyrrolizidin-Alkaloide erst n a c h der Abspaltung des Vorfahren des „Scrophulariaceae-Kladus“ entstanden ist. So ist denkbar, dass erst nach dieser Abspaltung die Besiedlung PA-haltiger Wirtstaxa, von Asteraceae oder Boraginaceae, stattgefunden hat, von wo aus wiederum andere, PA-freie Wirtsgruppen kolonisiert wurden. Es muss hier allerdings darauf hingewiesen werden, dass die Auflösung des betrachteten Baums in den basalen Verzweigungen noch nicht ausreichend gesichert ist und eine derzeitige neue Auswertung der Daten unter Umständen eine neue Position des Kladus um L. australis nahe legen könnte (Dobler, mündl. Mittlg.). Durch hohe Bootstrap-Werte stark unterstützt ist in jedem Falle jedoch die Monophylie dieser Gruppe, so dass man im Falle einer Positionierung innerhalb von PA-sequestrierenden Arten eine Reduktion des Merkmals „PA-N-oxidation“ in einem gemeinsamen Vorfahren der Scrophulariaceen-Käfer annehmen müsste. Mögliche Erklärungen wären dann z.B. Verlust oder Mutation des Enzym-kodierenden Gens, oder auch Verlust oder Veränderung des Regulationsapparates, mit der Folge, dass die kodierenden Sequen- zen nicht mehr ansteuerbar sind. Untersuchungen des Genprodukts auf mRNA oder DNA-Ebene könnten auch diese Problematik erhellen.

6.5 Toxizität von PAs

Es wird generell davon ausgegangen, dass PAs vor allem zum Schutz der Pflanze vor Herbivorie produziert werden (Boppré, 1986, 1990; Hartmann, 1995, 1999). Das setzt voraus, dass diese Stoffe auf nicht-angepasste phytophage Insekten toxisch wirken, eine Abschließende Diskussion 73

Annahme, die bisher kaum überprüft wurde. Wie eingangs erwähnt, gibt es zwar einen Hinweis auf eine mutagene Wirkung von PAs auf Insekten (Frei et al., 1992), jedoch keine Belege für direkt schädigende Effekte auf Stoffwechsel und Entwicklung des Individuums. Der Mangel an Daten zu diesem Problem ist vor allem darauf zurück zu führen, dass geeignete Versuchstiere fehlten: Insektenarten, die nicht an PA-Pflanzen adaptiert sind, können aufgrund der abschreckenden Wirkung von PAs nicht mit diesen Substanzen gefüttert werden, wogegen bei Arten, die an PA-haltige Wirte angepasst sind, keine negativen Effekte sichtbar sind. Mit dem Experiment, das ich in Kapitel 5 vorgestellt habe, gibt es nun einen solchen Beleg. Die nicht an PA-haltige Wirte angepasste Seidenspinnerraupe Philosamia ricini eignete sich hervorragend für ein Toxizitätsexperiment, da sie PA-behandelte Nahrung wahllos akzeptierte. Das Ergebnis des Experiments war, dass die künstlich PA-gefütterten Raupen deutlich langsamer wuchsen als die Kontrolltiere und vor oder während des Puppenstadiums starben. Da sich die Nahrung der beiden Gruppen ausschließlich in ihrem PA-Gehalt unterschied, ist der lethale Effekt der PAs eindeutig. Ein begleitendes Tracer-Experiment bewies zudem, dass P. ricini nicht in der Lage ist, PAs durch N-Oxidation zu entgiften. Stattdessen wurden in Tierkörpern und Kot eine Anzahl anderer polarer Metaboliten des Tracers gefunden, die bisher nicht weiter identifiziert wurden. PAs haben also auf nicht-angepasste Insekten potentiell wachstumshemmende Wirkung und können zur Mortalität während der Metamorphose führen. Sie scheinen folglich direkt oder indirekt in entwicklungsphysiologische Prozesse des Insekts einzugreifen. Das Ziel der Pflanze, sich vor Biomasseverlust durch Herbivorie zu schützen, wäre hiermit erreicht. Bei andauernder Konfrontation einer nicht PA- angepassten Phytophagenpopulation mit Pflanzen, die diese Toxine enthalten, entsteht für die Phytophagen ein Selektionsdruck auf Erkennung und Vermeidung dieser Pflanzen. So ist auch anzunehmen, dass die abschreckende Wirkung von PAs, wie sie oben für L. australis beschrieben wurde, Ergebnis eines solchen Prozesses ist. Für die Schädlichkeit von PAs auf Insekten habe ich, ebenfalls in Kapitel 5, einen weiteren Hinweis dargelegt, wenn auch indirekter Natur: Die Nutzung von PAs durch einen PA-sequestrierenden Phytophagen als Schutz gegen räuberische Insekten. In Wahlexperimenten mit drei Carabidenarten wurde gezeigt, dass diese Prädatoren chemisch nicht geschützte Borkenkäferpuppen vor PA-haltigen Longitarsus-Puppen klar bevorzugen. In einem weiteren Wahlexperiment mit PA-behandelten und Kontroll- Mehlwurmsegmenten, wurde deutlich, dass sich dieselben Prädatoren von PAs in Beuteobjekten abschrecken lassen. Mit großer Wahrscheinlichkeit sind PAs auch als Speicherstoffe von Longitarsus für dessen abschreckende Wirkung auf Prädatoren Abschließende Diskussion 74 verantwortlich. Zusammenfassend sind diese Experimente Zeugnis der komplexen ökologischen Vernetzung, die durch sekundäre Pflanzenstoffe in den multitrophischen Interaktionen zwischen Pflanzen, Herbivoren und deren Prädatoren erreicht werden. 75

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