biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid...

Journal of Insect Physiology 45 (1999) 1085–1095www.elsevier.com/locate/jinsphys

Biochemical strategy of sequestration of pyrrolizidine alkaloids byadults and larvae of chrysomelid leaf beetles

Thomas Hartmanna,*, Claudine Theuringa, Jurgen Schmidtb, Martine Rahierc, JacquesM. Pasteelsd

a Institut fur Pharmazeutische Biologie der Technischen Universita¨t Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germanyb Institut fur Pflanzenbiochemie, D-06018 Halle/Saale, Germany

c Ecologie Animale, Universite´ de Neuchaˆtel, CH-2007 Neuchaˆtel, Switzerlandd Laboratoire de Biologie Animale et Cellulaire, Universite´ Libre de Bruxelles, B-1050 Bruxelles, Belgium

Received 7 January 1999; accepted 16 February 1999

Abstract

Tracer feeding experiments with14C-labeled senecionine and senecionineN-oxide were carried out to identify the biochemicalmechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetleOreina cacaliae(Chrysomelidae). The taxo-nomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosenas a ‘biochemically naive’ control. InC. coerulansingestion of the two tracers resulted in a transient occurrence of low levels ofradioactivity in the hemolymph (1–5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from thehemolymph was senecionine. This indicates reduction of the alkaloidN-oxide in the gut. Adults and larvae ofO. cacaliaesequesteringested senecionineN-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiaryalkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulatesin the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionineO-glucoside. Thefollowing mechanism of alkaloid sequestration inO. cacaliaeis suggested to have developed during the evolutionary adaptationof O. cacaliaeto its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloidN-oxides, (ii) efficientuptake of the alkaloidN-oxides, and (iii) detoxification of the tertiary alkaloids byO-glucosylation. The biochemical mechanismsof sequestration of pyrrolizidine alkaloidN-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect totoxicity, safe storage and defensive role of the alkaloids. 1999 Elsevier Science Ltd. All rights reserved.

Keywords:Chrysomelidae; Pyrrolizidine alkaloidN-oxide; Pyrrolizidine alkaloid glycoside; Sequestration; Chemical defense

1. Introduction

Plant pyrrolizidine alkaloids with certain structuralfeatures such as the presence of a 1–2 double bond,esterification of the allylic hydroxyl group and a free oresterified second hydroxyl group (Fig. 1) are potentiallytoxic (Mattocks, 1986; Cheeke, 1989). If these alkaloidsare ingested by a herbivore they are passively absorbedas tertiary alkaloids and are converted into reactive pyr-rolic metabolites by multisubstrate cytochrome P450oxidases. These pyrrolic intermediates easily react with

* Corresponding author. Tel.:+49-531-391-5680; fax:+49-531-391-8104E-mail address:[email protected] (T. Hartmann)

0022-1910/99/$ - see front matter. 1999 Elsevier Science Ltd. All rights reserved.PII: S0022-1910 (99)00093-1

Fig. 1. Senecionine a potentially toxic tertiary pyrrolizidine alkaloidand itsN-oxide. The structural features of senecionine which are essen-tial for cytochrome P450 catalyzed bioactivation of pyrrolizidine alka-loids: (A) 1–2 double bond; (B) esterification of the allylic hydroxylgroup at C9; and (C) a free or esterified hydroxyl group at C7.

1086 T. Hartmann et al. / Journal of Insect Physiology 45 (1999) 1085–1095

biological nuclophiles, with the consequence of detri-mental cytotoxic and genotoxic effects. In vertebratesthis bioactivation is catalyzed by microsomal cyto-chrome P450 enzymes (EC 1.14.14.1) (Winter andSegall, 1989). These enzymes are mainly localized inliver and lung tissues. They are components of the xeno-biotic metabolism and transform absorbed foreign com-pounds (xenobiotics) into excretable metabolites. In thecase of pyrrolizidine alkaloids per se non-toxic com-pounds are converted into toxic metabolites which areresponsible for the hepatotoxicity and pneumotoxicity ofpyrrolizidine alkaloids in vertebrates (Mattocks, 1986;Cheeke, 1989). Insects which have a similar xenobioticmetabolism with microsomal cytochrome P450 enzymes(Hodgson, 1985; Brattsten, 1992) should be affected bypyrrolizidine alkaloids similarly as vertebrates. In fact,pyrrolizidine alkaloids were shown to be genotoxic inthe Drosophilawing spot test (Frei et al., 1992).

A number of insect species from unrelated taxa havedeveloped adaptations to sequester pyrrolizidine alka-loids from their host plants and utilize them against theirown predators. Pyrrolizidine alkaloid sequestering spec-ies are found among Lepidoptera (many moths or butter-flies of the Arctiidae, Danainae and Ithomiinae) (Boppre´,1986; Schneider, 1987; Hartmann, 1999; Hartmann andWitte, 1995) and Coleoptera such as leaf beetles(Chrysomelidae) of the genusOreina (Pasteels et al.1988b, 1994; Hartmann et al., 1997). In addition isolatedoccurrences of alkaloid sequestering species are knownfrom the Orthoptera, i.e. the African grasshopperZonoc-erus (Bernays et al., 1977; Fischer and Boppre´, 1997)and Homoptera, i.e.Aphis jacobaeae, a specialistphloem-feeder onSeneciospecies (Witte et al., 1990).

The question is, how pyrrolizidine alkaloid seques-tering insects protect themselves against alkaloid tox-icity. Arctiids (Ehmke et al., 1990; Hartmann et al.,1990) andZonocerus(Biller et al., 1994) store plant-derived pyrrolizidine alkaloids exclusively in theN-oxide state.N-oxidation converts the tertiary alkaloidinto a derivative which cannot be bioactivated. Somevertebrates detoxify potentially toxic tertiary alkaloidsby N-oxidation (Cheeke, 1994). For example, guineapigs possess a microsomal multisubstrate flavin monoox-ygenase (EC 1.14.13.8) which efficiently convertsingested pyrrolizidine alkaloids into theirN-oxides. Inguinea pig liver N-oxigenation by far exceeds cyto-chrome P450 mediated bioactivation (Miranda et al.,1991). This explains the high resistance of guinea pigsto the toxic effects of pyrrolizidine alkaloids. Similarly,Arctiids store sequestered pyrrolizidine alkaloids in thesafeN-oxide state (Lindigkeit et al., 1997).

Pyrrolizidine alkaloid sequestration in leaf beetles dif-fers from sequestration in lepidopterans. Pyrrolizidinealkaloid sequestering leaf beetles have two alkaloid stor-age sites: (i) exocrine defensive glands located in theelytre and the pronotum of adult beetles from which the

defensive secretion is actively released upon attack(Pasteels et al. 1988a, 1989); (ii) the whole body inwhich adults (Ehmke et al., 1991; Rowell-Rahier et al.,1991; Pasteels et al., 1992) and larvae (Dobler and Row-ell-Rahier, 1994; Ehmke et al., 1999) store alkaloidsacquired from the host plant. The major storage site inthe body is the hemolymph. Like lepidopterans,Oreinalarvae and adults store the alkaloids asN-oxides. How-ever, in contrast to lepidopterans they are not able toefficiently convert the tertiary alkaloids into the respect-ive N-oxides (Ehmke et al., 1991).

The purpose of this study was to elucidate the mech-anisms by which pyrrolizidine alkaloid sequestering leafbeetles, e.g.O. cacaliae, handle tertiary alkaloids andprotect themselves against pyrrolizidine alkaloid tox-icity. Chrysolina coerulanswas chosen as a non-adaptedcontrol. The results provide some general insight intothe mechanisms and strategies adopted by leaf beetlesduring their evolutionary adaptation to host plant-derived chemical defense. These mechanisms will becompared with the those adopted by lepidopteran speciesutilizing host-derived pyrrolizidine alkaloids as defens-ive compounds.

2. Material and methods

2.1. Insects

Adult beetles ofC. coerulans(Scriba) were collectedin May and June in the Medicinal Plant Garden of theInstitute of Pharmaceutical Biology, Technical Univer-sity Braunschweig. The beetles fed onMentha spicataL.(Lamiaceae). Adults ofOreina cacaliae(Schrank) werecollected in May in the Val Ferret (Valais, Switzerland)and kept on their food plantAdenostyles alliariae(Gouan) Kern. (Asteraceae) at room temperature or in acooling-chamber at 8°C until use. The offspring wasraised onA. alliariae; 2nd and 3rd instar larvae wereused in the experiments. All feeding experiments werecarried out at room temperature.

2.2. Tracer feeding experiments

[14C]Senecionine (1.07-1.34 GBq×mmol21) and itsN-oxide were prepared biosynthetically from [1,4-14C]pu-trescine (4.1 GBq×mmol21; Amersham Buchler,Braunschweig) using root cultures ofSenecio vulgarisL. according to Hartmann (1994).

2.2.1. C. coerulansTracer feeding of adult beetles was successfully achi-

eved using small rooted mint plants in pots. All leavesexcept one pair were removed. Tracers were applied asaqueous solutions (ca 10µl) on the lower surface of theleaves (1.5×105 cpm per plant). TheN-oxide was directly

1087T. Hartmann et al. / Journal of Insect Physiology 45 (1999) 1085–1095

dissolved in water, whereas the tertiary alkaloid was dis-solved in a small volume of methanol and then dilutedwith water; the final methanol concentration did notexceed 5%. After evaporation of the solvent one beetlewas placed on each plant. Host plant and beetle werecaged in a plastic tube (85×25 mm) with its bottompressed into the earth and its top covered with gauze.The beetles (two individuals per group) were allowed tofeed for 4, 24 or 45 hr. In pulse feeding experimentsthe beetles were transferred to untreated host plants aftertermination of the feeding-pulse and were allowed tofeed till the end of the experiment.

2.2.2. O. cacaliaeTracer feeding experiments with adult beetles and lar-

vae of O. cacaliaewere performed in Petri dishes asdescribed previously (Ehmke et al. 1991, 1999). Fivelarvae (2nd and 3rd instar; individual larval weightbetween 15 to 22 mg) were placed in a Petri dish andwere fed with 1.2×105 cpm of [14C]senecionine or1.2×105 cpm of [14C]senecionineN-oxide, respectively.Fifteen Petri dishes were prepared with each tracer. Tra-cers were painted as aqueous solutions on the upper sur-face of fresh leaf discs (10 mm[) of Adenostyles alliar-iae, one disc per dish. Larvae were allowed to feed for17 hr, then a total of 25 larvae from five dishes of eachtreatment were preserved in methanol and the larvaefrom the remaining 10 dishes of each set were trans-ferred to fresh untreated host plant leaves and preservedin methanol after an additional feeding period of 24 and48 hr, respectively.

Experiments with adult beetles were arranged in thesame way as described above for larvae. However, onlyone indvidual was placed in each Petri dish and the dur-ation of tracer feeding was 13 hr. After termination ofthe experiment and weighing of the beetles the hemo-lymph was recovered. Hemolymph and the carcasseswere preserved separately in methanol until analysis.

2.3. Sampling of hemolymph

2.3.1. Centrifugation methodThe legs of a beetle were cut off at the tibia and the

beetle was placed head-down on the top of a pre-weighed 200µl Eppendorff micro-vial. Hemolymph wasrecovered by centrifugation at 3500 rpm for 15 min(Sigma 101M microcentrifuge). The vial was re-weighedand the amount of hemolymph calculated. During centri-fugation the beetle remained stuck to the top of the coni-cal micro-vial and the body fluid was collected at thebottom of the vial. The hemolymph obtained by thismethod was a clear fluid.

2.3.2. Capillary methodOne leg or antenna of a beetle was cut off at the basal

segment and the extruding hemolymph was collectedwith a capillary and immediately preserved in methanol.

2.4. Extraction procedure

Individual beetles or 5 larvae were ground in a mortarwith ca 500µl methanol and washed with another 500µl methanol into a 2 ml Eppendorff vial. After centrifug-ation (Sigma 101 M microcentrifuge) at 15 000 rpm for10 min, the supernatant was recovered and the pelletextracted with another 300µl methanol. The super-natants were combined and directly used for analysis oftotal radioactivity by scintillation counting or chromato-graphic analysis. Isolated hemolymph (between 3 and 25mg) was mixed with 200µl methanol and the precipitateremoved by centrifugation. The supernatant was ana-lyzed in the same way as the extracts.

2.5. Chromatographic methods

2.5.1. Thin-layer chromatography (TLC)Crude methanol extracts of larvae, adult beetles, hem-

olymph or tracer-treated food plant remains were separ-ated by TLC and evaluated quantitatively by means ofa multichannel radioactivity detector (Rita-32a, Raytest).Separation was achieved on silica gel 60 (Merck) usingthe solvent system: dichloromethane:methanol:NH4OH(25%) (84:14:2, by volume).

2.5.2. High pressure liquid chromatography (HPLC)Crude methanol extracts were separated isocratically

on an RP-18 column (25 cm×0.4 cm i.d.; Nucleosil;Macherey and Nagel). Solvent: 15% acetonitrile: 85%0.01 M potassium phosphate, pH 2.2. Flow: 1 ml×min21.Rt (min): presumed senecionine glucoside, 7.53; sene-cionine, 11.00; senecionineN-oxide, 12.30. Compoundswere detected by (a) UV 209 nm; (b) radioactivity moni-tor LB-506D (Berthold) equipped with a 2 ml flow-celland a split-mixer LB-5034; Rialuma (Baker) was usedas liquid scintillator.

2.6. Alkaloid hydrolysis

To isolate the radioactively labeled necine base moi-ety (retronecine) of [14C]senecionine the labeled alkaloidwas subjected to alkaline hydrolysis in 5% NaOH at55°C for 3 hr. The necine base was recovered from thehydrolysis solution by extraction with diethyl ether.

2.7. Isolation and identification of pyrrolizidinealkaloid O-glycosides

2.7.1. Seneciphylline feedingAdults of C. cacaliae(99 specimens) were kept in

Petri dishes (5 beetles per dish) on fresh leaf discs (15mm Ø) of A. alliariae each painted with 20µl of anaqueous solution of 1 mM non-labeled seneciphylline,which previously had been isolated and purified fromyoung sprouts ofA. aliariae. The beetles were allowed


to feed ad libitum on seneciphylline for 48 hr; consumedalkaloid-treated leaf discs were immediately replaced bynew ones. After termination of the experiment thebeetles were kept frozen (218°C) until analysis.

2.7.2. ExtractionThe frozen beetles were ground in a mortar and

extracted twice with methanol. After centrifugation thesolvent was evaporated at room temperature, the residuedissolved in a small volume of methanol and subjectedto TLC on silica gel (PSC plates, 1 mm, Merck). Theassumed alkaloid glycoside was localized by co-chroma-tography in the same lane with a radioactively labeledsample of the assumed senecionine glycoside whichshould migrate with the sameRF-value as the respectiveseneciphylline derivative. The marked zone was scrapedoff the plate and extracted with methanol. The methanolextract was subjected to HPLC using the same procedureas described above, but using 15% acetonitrile: 85% tri-fluoroacetic acid as solvent. Promising fractionslocalized by co-chromatography with the radioactivelylabeled assumed glycoside were collected, the solventevaporated and the fractions directly applied to LC-MS analysis.

2.7.3. LC-MS analysisThe positive ion electrospray (ES) spectra and the ES-

MS/MS measurements were obtained with a FinniganMAT TSQ 7000 instrument (electrospray voltage, 4.5kV; heated capillary temperature, 220°C; sheath gas,nitrogen) coupled with a Micro-Tech Ultra-Plus Micro-LC system equipped with a RP18-column (4µm, 1×100mm, SEPSERV). Separation was achieved with the fol-lowing solvent gradient: water:acetonitrile (9:1), eachcontaining 0.2% acetic acid, to water:acetonitrile (1:1)within 10 min, then holding the 1:1 ratio for 10 min.The flow rate was 70µl min21. Collision-induced dis-sociation (CID) mass spectra were generated during theHPLC run under the following conditions: collisionenergy (collision cell)240 eV (seneciphylline) and250eV (seneciphyllineO-glucoside); collision gas, argon;collision pressure, 1.8×1023 Torr. All mass spectra areaveraged and corrected for background.

3. Results

3.1. Uptake and metabolism of [14C]senecionine andits N-oxide by Chrysolina coerulans

Preliminary experiments revealed that it is possible tofeed C. coerulanson mint leaves painted with smallquantities (,1 µg) of radioactively labeled pyrrolizidinealkaloids without refusal reactions of the beetles.

Adults ofO. coerulanswere fed with [14C]senecionineN-oxide and [14C]senecionine for 4 to 45 hours. At inter-

vals some beetles were sacrificed, their hemolymph reco-vered, and analyzed for total radioactivity and its metab-olite composition. The results are summarized in Table1. With both tracers a small proportion of radioactivity(1 to 5% of total radioactivity offered) was detected inthe hemolymph. Their presence there was temporary.Beetles which were transferred from the radioactivesource to a non-labeled food leaf lost almost all of theradioactivity from their bodies within 24 hours. TLCanalysis of the radioactivity recovered from the hemo-lymph showed that the two compounds are metabolizedin a characteristic manner. Upon feeding of [14C]sene-cionineN-oxide, 80–90% of the radioactivity recoveredfrom the hemolymph was tertiary senecionine. Inaddition, a small proportion of non-metabolized[14C]senecionineN-oxide (up to 12%) was found. Thelatter result was confirmed with two different methodsof hemolymph sampling (i.e. centrifugation and thecapillary collection). Thus, contamination of hemolymphsamples with external tracer can be excluded. The radio-activity recovered from the hemolymph upon feeding of[14C]senecionine was mainly (.60%) non-metabolizedsenecionine, the remainder of radioactivity was associa-ted with a polar metabolite (PM in Table 1). Becausethe amount present was small it was not possible to per-form HPLC and to compare this metabolite with thealkaloid glycoside found inO. cacaliae(see below). Innone of the feeding experiments with labeled senecion-ine, could labeledN-oxide be detected in the hemo-lymph.

3.2. Uptake and metabolism of [14C]senecionine andits N-oxide by Oreina cacaliae

The same types of experiment as described above forthe non-adaptedC. coerulanswere carried out withadults and larvae ofO. cacaliae.Tables 2 and 3 showthe total radioactivity found in the hemolymph, in thecarcasses of adults and in larval bodies, respectively,upon feeding of [14C]senecionine and itsN-oxide.Recovery of radioactivity (expressed as percentage oftotal tracer offered on the food-leaf) in adult beetles was16 to 34% (senecionineN-oxide feeding) and 15 to 22%(senecionine feeding). With larvae, the respective valueswere 13 to 18% (N-oxide) and 6 to 12% (tertiaryalkaloid). The percentages of accumulated radioactivitywere evaluated at the end of the tracer feeding period(13 and 17 hours, respectively) and 24 and 48 hours afterthe insects had been transferred from tracer containingto normal diet. No effort was made to correct accumu-lation for the non-consumed radioactivity on the food-leaf remains. This explains the result that total radioac-tivity appeared to increase with time after an initial pulsefeeding of 14C-labeled tracer (e.g. senecionine in thehemolymph; see Table 2). The data just indicate that the24/48 hour groups consumed more labeled leaf-mass


Table 1Uptake and metabolism of radioactively labeled senecionineN-oxide (Sen-Nox) and senecionine (Sen) by the non-adapted leaf beetleChrysolinacoerulansf

Tracer Time (hours) Total radioactivity Radioactivity (relative abundance, %)in hemolymph (%of applied)a

PMb Sen-Nox Sen

Exp. IAdults

Sen-Nox 45 2 18 ,3 81Sen 45 1 38 nd 62

Food-leafc

Sen-Nox 45 .85 9Sen 45 nd ,75d

Exp. IIAdults

Sen-Nox 4 1.7 nd 12 88Sen-Nox 24 5.1 10 ,3 89Sen-Nox 24 + 24e ,0.5

a A total of 1.5×105 cpm (100%) of the respective tracer were painted on two leaves of a small mint plant and offered to one individual; n=2 per group.

b Polar metabolite.c Analysis of the food leaf remains at the end of the feeding period (n= 4).d Ca 25% loss of sen by unspecific decay, i.e. several labeled spots of unknown identity on the TLC plate.e 24 hr pulse feeding of tracer, followed by 24 hr feeding on non-treated leaves.f Hemolymph extracts were separated by TLC and analyzed by radioactivity detection. nd= not detected.

Table 2Uptake of [14C]senecionineN-oxide and [14C]senecionine by adults ofOreina cacaliaea

Tracer Time Uptake cpm (×105)×g21 fresh weight(hours)

In the hemolymph In the remainingbody

SenecionineN- 0 19.73± 10.83 7.19± 3.16oxide 24 15.24± 4.02 5.35± 1.62

48 8.50± 3.23 2.13± 0.51Senecionine 0 4.42± 1.68 1.58± 0.65

24 7.32± 1.94 2.27± 0.4348 8.20± 1.78 2.75± 0.84

a n = 5 per group; means± standard error. Individuals were allowedto feed on host plant leaves treated with the respective tracer for 13hours, then one group was analyzed (hour 0). Two other groups weretransferred to untreated host plant leaves and fed for a further 24 hours(24) and 48 hours (48), respectively.

than the zero-group. Moreover, we know from previousstudies that during the period after an initial pulse feed-ing of labeled alkaloid there is no (adults) or only aminor loss (larvae) of total accumulated radioactivityfrom the bodies (Pasteels et al., 1992; Ehmke et al.,1999). The main goal of the following experiments wasa comparative analysis of the metabolite patterns of theaccumulated radioactivity and not the comparison of theabsorbed total amounts.

The results summarized in Tables 2 and 3 show that:

Table 3Uptake of radioactively labeled senecionineN-oxide and senecionineby larvae (2nd and 3rd instar) ofOreina cacaliaea

Tracer Time (hours) Uptakecpm×105×g21 freshweight

SenecionineN-oxide 0 14.69± 1.7024 2.61± 0.6048 2.84± 1.18

Senecionine 0 2.41± 0.5824 0.90± 0.2048 1.51± 0.45

a n = 5 per treatment; mean± standard error. Larvae were fed onhost plant leaves treated with the respective tracer for 17 hours, thenone group was analyzed (hour 0). The two other groups were trans-ferred to untreated host plant leaves and allowed to feed for a further24 hours (24) and 48 hours (48), respectively.

(i) the two tracers are taken up in comparable amounts;(ii) the sequestered radioactivity is retained by adultbeetles and by larvae over two days following the tracerfeeding-pulse; (iii) in adult beetles, hemolymph andremaining-body contain almost equal concentrations ofsequestered radioactivity.

All extracts were analyzed by TLC and HPLC toestablish the respective metabolite patterns. The resultsof the TLC analysis are illustrated in Fig. 2. When theN-oxide was fed as tracer more than 95% of the radioac-tivity found in the hemolymph of adult beetles was


Fig. 2. Fate of [14C]senecionineN-oxide (A, C, E) and [14C]senecion-ine (B, D, F) ingested by adults (A-D) and larvae (E, F) ofOreinacacaliae. Individuals were allowed to feed on pieces of host plantleaves treated with the respective tracer for 13 hours (adults) or 17hours (larvae). Samples of each group each were then analyzed (hour0). Others were transferred to untreated host plant leaf material andallowed to feed for a further 24 hours and 48 hours, respectively. Aand B = adults, methanol extracts of hemolymph; C and D= adults,methanol extracts of carcasses; E and F= methanol extracts of larvae.For uptake and tissue distribution of total radioactivity see Tables 2and 3.

unalteredN-oxide (Figs. 2A and 3A). Body extracts con-tained 65 to 80% unaltered senecionineN-oxide, but inaddition also [14C]senecionine and a very polar com-pound with a lowRF value (Fig. 3B). The polar com-pound was later identified as an alkaloid glycoside (mostlikely the glucoside). In the larval extracts the recoveryof senecionineN-oxide accounted for 80 to 95% of totalradioactivity; it was always accompanied by the glyco-side which appeared to increase slightly with time(Fig. 2E).A completely different result was obtained when adultsor larvae were fed with [14C]senecionine instead of theN-oxide (Fig. 2B, D and F). Hemolymph extracts fromadults (Figs. 2B and 3C) contained senecionine glyco-side (75 to 92% of total activity) as major metabolite,accompanied by small proportions of senecionineN-oxide (7 to 20%) and less than 15% of residual senecion-ine. Body extracts revealed a more complex comparison,but in any case the alkaloid glycoside was the predomin-ating metabolite (Figs. 2D and 3B). SenecionineN-oxidewas always present (8 to 24%). The relative amounts of

Fig. 3. [14C]SenecionineN-oxide and [14C]senecionine fed to adultsof O. cacaliae. TLC separation of crude methanol extracts. (A) hemo-lymph, tracer senecionineN-oxide (24 hours); (B) body extract, tracersenecionine (24 hours); (C) hemolymph, tracer senecionine (48 hours).S = Start, F= Front. See legend of Fig. 2 for details.

residual senecionine were higher in the carcass than inthe hemolymph and decreased with time. TLC separationof the larval extracts revealed a composition similar tothat of adult body extracts (Fig. 2F). Two points wereremarkable: in comparison to adults, residual senecion-ine appears to be lower or absent, whereas senecionineN-oxide was always present in adults and larvae.

3.3. Identification of pyrrolizidine alkaloid O-glycosides

HPLC analysis of hemolymph extracts from a feedingexperiment with [14C]senecionine confirmed that thepolar metabolite eluted as a single peak. Alkalinehydrolysis of this compound yielded one labeled productwith the same chromatographic behavior as authenticretronecine. This confirmed that the polar metabolite stillcontains the intact necine base moiety. To obtain suf-ficient amount of the polar metabolite for reliable analy-sis by liquid chromatography/mass spectrometry


(LC/MS), adults ofO. cacaliaewere fed with non-lab-eled seneciphylline. Seneciphylline was chosen insteadof the closely related senecionine because it is the majoralkaloid found in the host plantA. alliariae. Positive ionelectrospray (ES) mass spectrometry revealed a com-pound with a prominent [M+ H]+ ion at m/z 496 andusing skimmer CID a further ion showed up atm/z334as base peak (Fig. 4A). The loss of 162u formally corre-sponds to the elimination of a hexose unit at formationof the protonated alkaloid. This result was confirmed bya collision induced (CID) mass spectrum of compoundm/z496 (Fig. 4B) in comparison to the respective spec-trum of authentic seneciphylline ([M+ H]+, m/z 334)(Fig. 4C). The two mass spectra show almost identical

Fig. 4. Identification of presumed seneciphylline O-glucoside by LC-MS. (A) positive ion electrospray (ES) mass spectrum of a compoundobtained by LS-MS (Rt 7.35 min), showing a prominent [M+H]+ ionat m/z496 and using skimmer CID a ion atm/z334 as base peak. (B)collision induced dissociation (CID) mass spectrum of compoundm/z496. (C) CID mass spectrum of reference seneciphylline ([M+H]+,m/z334).

fragment ions (e.g. atm/z 306, 151, 138, 120, and 94)in the mass range betweenm/z 50 to 350. From thesedata we conclude, that the compound with a [M+ H ]+

at m/z 496 represents anO-glycoside composed of ahexose and seneciphylline. It is reasonable to assumethat the hexose is a glucose and thus the alkaloid glyco-side most likely is seneciphyllineO-glucoside (Fig. 4A).In the same extract an additional compound with a [M+ H]+ ion atm/z498 could be recorded by ES-MS. Usingskimmer CID a further ion atm/z336 (presumably rep-resenting senecionine) was detected as base peak.Although the concentrations were too low to record therespective CID spectra, the compound with a [M+ H ]+

at m/z 498 most likely represents senecionineO-gluco-side.

4. Discussion

The generaChrysolinaandOreina are taxonomicallyclosely related (Daccordi, 1994; Hsiao and Pasteels,1999).C. coerulans,which in his life history never facespyrrolizidine alkaloids, was chosen as a ‘biochemicallynaive’ control. It should help to detect any biochemicaladaptation ofO. cacaliaethat enables this leaf beetle tocope with and safely store host-plant-derived pyrrolizid-ine alkaloids. The following conclusions can be drawnfrom the results of the tracer-feeding experiments withC. coerulans: (i) as expected,C. coerulansdoes notsequester pyrrolizidine alkaloids; (ii) during feeding onboth labeled senecionine and labeled senecionineN-oxide, low levels of radioactivity are temporarilydetected in the hemolymph; (iii) [14C]senecionineN-oxide appears to be efficiently reduced in the gut, sinceupon feeding of either of the two alkaloidal forms, 60to 80% of the radioactivity recovered in the hemolymphis associated with tertiary senecionine; (iv) since afterfeeding of labeled senecionineN-oxide, a small pro-portion of senecionineN-oxide could be detected in thehemolymph, it appears thatC. coerulans is able toabsorb the alkaloidN-oxide; (v) As no N-oxide wasfound after feeding of senecionineC. coerulansis appar-ently unable toN-oxidize the alkaloid; (vi) althoughC.coerulans is not specifically adapted to pyrrolizidinealkaloids, it is able to excrete efficiently the absorbedalkaloids and their metabolites (Fig. 5A).

The reduction of ingested pyrrolizidine alkaloidN-oxides is in agreement with the general observation thatpyrrolizidine alkaloidN-oxides are easily reduced in thegut. All vertebrates so far studied show it includinghumans (Mattocks, 1986; Winter and Segall, 1989) aswell as various arctiids (Lepidoptera) and the grass-hopperZonocerus variegatus(Lindigkeit et al., 1997).Liver toxicity of pyrrolizidine alkaloids in humans anddomestic animals (Mattocks, 1986; Huxtable, 1992) isinitiated in the digestive tract by reduction of the


Fig. 5. Strategies of uptake, metabolism and maintenance of tertiary pyrrolizidine alkaloid (PA) and itsN-oxide (PA-O) in (A) Chrysolinacoerulans(non-adapted, no sequestration; ‘biochemical control’); (B)Oreina cacaliae(adapted, sequestration), (C)Spodoptera littoralis(toleratingalkaloids, no sequestration); (D)Tyria jacobaeae(adapted, sequestration). Solid arrows: main pathways; dotted arrows: minor pathways. PA, tertiarypyrrolizidine alkaloid; PA-O, pyrrolizidine alkaloidN-oxide; PA-C, pyrrolizidine alkaloid conjugate; PA-G, pyrrolizidine alkaloid glucoside.

ingested alkaloidN-oxide followed by nonspecific pass-ive absorption of the lipophilic tertiary alkaloid. ThealkaloidN-oxides are easily reduced even in presence ofweak reducing agents such as cysteine (Hartmann andToppel, 1987). It has been suggested that the intestinalflora plays a major role inN-oxide reduction (Mattocks,1971; Winter and Segall, 1989), detailed studies, how-ever, are still missing.

In comparison to the non-adaptedC. coerulans,absorption and metabolism of pyrrolizidine alkaloids iscompletely different in alkaloid sequestering larvae andadults ofO. cacaliae. The most striking difference is thatthe N-oxide is not reduced in the gut but taken up assuch and maintained unchanged in the body. By contrast,ingested tertiary alkaloid is efficiently converted into therespectiveO-glycoside, most likely theO-glucoside.Both adults and larvae are capable ofN-oxidizing sene-cionine. However, in comparison to glycosylation,N-oxidation seems less efficient, and it was rather variablein different experiments. It is not clear whether glycosyl-ation andN-oxidation take place in the gut or in the hem-olymph. The increase of the relative proportion of thealkaloid glycoside in the course of the pulse feedingexperiments (e.g.N-oxide feeding Figs. 2A–E) may sug-gest that the hemolymph is the site of glycosylation, butconclusive evidence is lacking. This is the first report onthe formation of alkaloidO-glycosides from pyrrolizid-ine alkaloids.

Assuming that the reduction of pyrrolizidine alkaloid

N-oxides generally takes place in the gut of non-adaptedleaf beetles such asC. coerulans, we infer that the alka-loid sequestering specialist in the course of its adaptationto plants containing pyrrolizidine alkaloids has lost theability to reduce the alkaloidN-oxides in the gut. Thisis a prerequisite for the efficient uptake of pyrrolizidinealkaloid N-oxides (Fig. 5B). The direct uptake of thealkaloidN-oxide and its storage in the body of larvae oradult beetles has been demonstrated in this and previousstudies (Ehmke et al. 1991, 1999; Rowell-Rahier et al.,1991; Hartmann et al., 1997). The uptake of the alkaloidN-oxide from the gut into the hemolymph is difficult toexplain without postulating a carrier-mediated process.In the case of pyrrolizidine alkaloid sequestering larvaeof Lepidoptera it has been convincingly demonstratedthat the polar, salt-like alkaloidN-oxides are unable topermeate the membrane barrier between gut and hemo-lymph (Lindigkeit et al., 1997), although it had beenclaimed previously that such a carrier forN-oxides doesexist (Wink and Schneider, 1988). In plants containingpyrrolizidine alkaloids a specific membrane carrierfacilitates the transfer of the alkaloidN-oxides throughthe tonoplast into the cell vacuole (Ehmke et al., 1988;Hartmann et al., 1989). We know that inO. cacaliaethetransport of pyrrolizidine alkaloidN-oxides through thegut epithelia into the hemolymph is a rather unspecificprocess. A number of host-plant derived pyrrolizidinealkaloid N-oxides as well as several other pyrrolizidinealkaloidN-oxides which do not occur in the beetle’s host


plants, are absorbed into the hemolymph (Hartmann etal., 1997). By contrast, the transfer of pyrrolizidine alka-loid N-oxides from the hemolymph into the defensiveglands (Fig. 5B), which takes place in adult beetles(Pasteels et al., 1992), is much more selective. Only cer-tain alkaloidN-oxides taken up from the host plants aretransported into the defensive glands (Rowell-Rahier etal., 1991; Ehmke et al., 1991; Hartmann et al., 1997).Selective alkaloid transport into the defensive glands andmaintenance of a high concentration gradient (.150:1)between the secretion of the gland and the hemolymphargues for a specific energy-driven transport. Withrespect to the less specific uptake of the pyrrolizidinealkaloid N-oxides inC. coerulanswe noted a weak butunequivocal absorption of pyrrolizidine alkaloidN-oxidefrom the gut into the hemolymph. It is unlikely thatC.coerulanspossesses a specific carrier for these alkaloids,but it is possible that a nonspecific carrier systeminvolved in xenobiotic transport and elimination isresponsible for the temporary presence of theN-oxidein the hemolymph. We hypothesize that the developmentof such a carrier system for the uptake of the pyrrolizid-ine alkaloidN-oxides was an important step in the evol-utionary adaptation ofO. cacaliaeresulting in a host-mediated defense mechanism. Moreover,N-oxide uptakein O. cacaliaeis favored by the suppression ofN-oxidereduction in the gut. InC. coerulans N-oxide uptake islow because it is counteracted by the efficientN-oxidereduction.

Small proportions of tertiary alkaloids are formedfrom theN-oxide in the gut ofOreina (see Fig. 2C andE) but a more efficient mechanism converts tertiary pyr-rolizidine alkaloids into hydrophilic glycosides. Theseconjugates seem to remain in the body and are notexcreted or transferred into the defensive glands ofadults (Ehmke et al., 1991). Most likely the alkaloid gly-coside is a detoxification product. In comparison to theefficient detoxification of tertiary pyrrolizidine alkaloidsby conjugation,N-oxidation seems to play only a minorrole, if any, in alkaloid inactivation.

By contrast to pyrrolizidine alkaloid sequestering leafbeetles, alkaloid sequestering arctiid larvae completelyreduce ingested pyrrolizidine alkaloidN-oxide in the gutand passively absorb the tertiary alkaloid into the hemo-lymph. Such a mechanism is also found in larvae of thelepidopteranSpodoptera littoralis(Noctuidae) which tol-erates pyrrolizidine alkaloids because excretion is veryefficient (Fig. 5C). In arctiids the toxic tertiary alkaloidsare efficientlyN-oxidized by a soluble mixed functionN-oxigenase (senecionineN-oxygenase) localized in thehemolymph (Lindigkeit et al., 1997). The enzyme is spe-cific for toxic pyrrolizidine alkaloids. Interestingly, anenzyme with almost the same properties as the insectenzyme has recently been isolated and characterizedfrom a plant synthesizing pyrrolizidine alkaloids (Changand Hartmann, 1998).

Sequestration of pyrrolizidine alkaloids shows com-mon elements in specialized arctiids and specialized leafbeetles although the two unrelated taxa use quite differ-ent biochemical strategies (Fig. 5B and D). Both of themstore pyrrolizidine alkaloids in the form ofN-oxides andboth specifically prevent the accumulation of potentiallytoxic tertiary alkaloids. The specific mechanism for alka-loid sequestration in arctiids isN-oxidation of the pass-ively absorbed tertiary alkaloid by senecionineN-oxy-genase in the hemolymph (Fig. 5D). The specificmechanism for alkaloid sequestration in leaf beetles issuppression ofN-oxide reduction in the gut and efficientuptake of theN-oxide. The specificity ofN-oxide uptakeneeds further clarification. In arctiids and specialized leafbeetles the ability to excrete the stored alkaloidN-oxideis greatly reduced. This is a further specific prerequisitefor sequestration.O. bifrons (Ehmke et al., 1991) andSpodoptera (Lindigkeit et al., 1997) which do notsequester pyrrolizidine alkaloids but excrete senecionineN-oxide injected into the hemolymph almost completelywithin 24 hours.O. cacaliaedetoxify tertiary alkaloidsmore efficiently by conjugation than byN-oxidation(Fig. 5B).

To maintain sequestered pyrrolizidine alkaloids in theN-oxide state seems to be a prerequisite for alkaloidsequestration in specialized insects. It is reasonable toassume that this mechanism has been selected during theadaptation of insects to plants containing pyrrolizidinealkaloids to keep the alkaloids in a nontoxic and meta-bolically safe state. The success of the pyrrolizidinealkaloids as defensive compounds in plants and alkaloidsequestering insects is, that these alkaloids can exist intwo easily interchangeable forms: the nontoxicN-oxideand the potentially toxic tertiary alkaloid (Lindigkeit etal., 1997; Hartmann, 1999). In herbivores feeding onplants containing pyrrolizidine alkaloids, or in predatorsof insects loaded with alkaloidN-oxides, theN-oxidesare easily reduced in the gut and absorbed passively astertiary alkaloids. The tertiary alkaloids arebiotransformed into toxic metabolites in organisms withaccessible microsomal multisubstrate cytochromes P450.The biochemical strategies of pyrrolizidine alkaloidsequestration developed by arctiids and chrysomelid leafbeetles are in accordance with this general idea.

Acknowledgements

This work was supported by grants of the DeutscheForschungsgemeinschaft and Fonds der ChemischenIndustrie to T.H., of the Belgium Fund for Joint BasicResearch and the Communaute´ Francaise de Belgique toJ.M.P, and of the Swiss National Science Foundationto M.R.


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