Time-Course of Pyrrolizidine Alkaloid Processing in the Alkaloid
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Time-Course of Pyrrolizidine Alkaloid Processing in the Alkaloid Exploiting Arctiid Moth, Creatonotos transiens Eva von Nickisch-Rosenegkab, Dietrich Schneider13, and Michael Winka a Universität Heidelberg, Institut für Pharmazeutische Biologie, Im Neuenheimer Feld 364, D-6900 Heidelberg, Bundesrepublik Deutschland b Max-Planck-Institut für Verhaltensphysiologie, D-8130 Seewiesen-Stain'oerg, Bundesrepublik Deutschland Z. Naturforsch. 45c, 881 -8 9 4 (1990); received March 21, 1990 Creatonotos transiens, Pyrrolizidine Alkaloid, Pyrrolizidine Alkaloid-N-Oxide, 7 /?-Heliotrine Conversion, Alkaloid Storage The processing of dietary pyrrolizidine alkaloids by larvae and adults of the arctiid moth Creatonotos transiens was studied in time-course experiments: In larvae, pyrrolizidine alkaloid uptake is quickly followed by the transformation of the alkaloids into their N-oxides. Further more, if 7 S-heliotrine is applied, a stereochemical inversion of the hydroxyl group at C 7 to 7 /?-heliotrine can be observed within 48 h of feeding. The rate of this biotransformation is substantially higher in males which use the 7 /?-form later as a precursor for the biosynthesis of 7 /?-hydroxydanaidal, a pheromone. The resorbed pyrrolizidine alkaloids are deposited in the integument within 48 h, where they remain stored during the larval, pupal and partly also the imaginal stages. Virtually no alkaloids are lost during ecdysis. Some pyrrolizidine alkaloids can be recovered from the meconium which is released at eclosion by the imagines especially when disturbed. In the adults pyrrolizidine alkaloids are processed in different ways by the two sexes: In females, about 50-80% of total alkaloids are transferred from the integument to the ovaries and the eggs within 2 -3 days after eclosion. If females mate with alkaloid-rich males they additionally receive with the spermatophore up to 290 |ig pyrrolizidine alkaloid, which are further translocated to the eggs. A biparental endowment of eggs with acquired defence alkaloids is thus achieved. In males, 30-50% of pyrrolizidine alkaloids remain in the integu ment; about 10—30% are transferred to the scent organ, the corema, where they are converted into 7 /?-hydroxydanaidal. Another part (about 40%) is passed to the spermatophore. In the laboratory experiments, the sizes of the coremata and their respective 7 /?-hydroxydanaidal contents are strongly dependent on the availability of dietary pyrrolizidine alkaloids during L 6 and especially L7 stages. In the L7 stage even short-term feeding (4-6 h) on Senecio jaco- baea is sufficient to induce large coremata. Introduction Among them are two spccies of the South Asian genus Creatonotos which have an elaborate way to Insect herbivores cope with the plants’ defence cope with pyrrolizidine alkaloids (PA) (for reviews chemistry in various, often species-specific fash [5-7]). Pyrrolizidine alkaloids were found to be ions: 1. Polyphagous species often recognize plants phagostimulants for larvae of Creatonotos tran which store noxious allelochemicals and avoid siens [2]. Dietary pyrrolizidine alkaloids are re them. 2. Polar compounds are often not resorbed sorbed from the gut lumen with the aid of a specif but eliminated with the faeces. 3. If resorption ic intestinal carrier system [ 8]. Pyrrolizidine alka does take place, most herbivores have efficient de loids are then transferred to the larval integument toxicating enzymes. 4. Some herbivores are adapt where they remain stored through the subsequent ed to a specific allelochemical and may even use it life stages [9]. When 7 5-heliotrine is applied, a as an acquired defence compound. 5. A few species stereochemical inversion of the hydroxyl group at even have more elaborate ways to exploit the C l into 7Ä-heliotrine takes place [10]. After eclo plants’ defence chemicals as pheromone precur sion, substantial amounts of pyrrolizidine alka sors or morphogens [1-4]. loids are transferred to the ovaries and eggs in A number of arctiid moths (Lepidoptera) are females, whereas in males a smaller part is convert known to exploit some dietary allelochemicals. ed into the pheromone, 7/?-hydroxydanaidal [ 11]. Reprint requests to Prof. Dr. M. Wink or Prof. Dr. D. In males, pyrrolizidine alkaloids serve still another Schneider. function (at least in laboratory cultures [ 11]) as a Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen morphogen which induces the full development of 0341-0382/90/0700-0881 $01.30/0 a large abdominal scent organ, the corema [ 12, 13], 882 E. von Nickisch-Rosenegk et al. • Pyrrolizidine Alkaloid Processing in Creatonotos In this communication we report on the time- sex-specific way: Females were killed by decapita course of pyrrolizidine alkaloid processing in lar tion and their ovaries were removed. Males were vae and newly hatched imagines of Creatonotos decapitated and their abdomina were subjected to transiens: Emphasis was laid on the question of air pressure so that the coremata became fully in the time dependency of 1. the biotransformation flated [13]. After measuring the respective size of of 7 S-heliotrine into 7 Ä-heliotrine and their the corema, the scent organ was cut off and N-oxides, 2. pyrrolizidine alkaloid storage in the weighed on an electronic balance. Animals/tissues integument, 3. the transfer of pyrrolizidine alka were stored at -20 °C. loids into the ovaries and eggs in females, 4. the formation of the pheromone 7 /^-hydroxydanaidal in the male corema, 5. the induction of the corema Alkaloid extraction and gas-liquid chromatography anlage, and 5. the possible endowment of females Animals/tissues were ground in a mortar in with pyrrolizidine alkaloids by copulating males. 10 ml 0.5 m HC1. Zinc powder (about 500 mg) was added to reduce pyrrolizidine alkaloid-N-oxides. The homogenates were left standing overnight at room temperature. Next day they were made alka Materials and Methods line with 6 m NaOH and poured onto Chem elut columns (Analytichem, ICT, Frankfurt) for solid- Feeding experiments phase extraction. CH 2C12 was used as an eluent. A. Larvae. C. transiens has 7 larval stages, The eluate was collected and evaporated in vacuo. termed LI to L7 in the following. A laboratory Crude pyrrolizidine alkaloid extracts were taken culture of Creatonotos transiens was started with up in MeOH and analyzed by capillary GLC, animals from the Philippines as described in using a Varian 3300 instrument which was equip [6- 11] and maintained continuously in captivity ped with a nitrogen-specific detector. Column: for over 14 months. L5 to L7 larvae were kept sin DB1, 30 m x 0.3 mm, film thickness 1 |im. GLC gly in Petri dishes and reared on a semiartificial conditions: Oven: 190 °C to 300 °C with 20 °C/ diet (without any antibiotics or preservatives), min, then 5 min isothermal. Injector: 250 °C, split onto which defined amounts of pyrrolizidine alka injection (1:20); detectors: 300 °C; carrier gas: loids (7 ^-heliotrine (from Chemasea, Manuf. helium 90 kpascal; make up gas: nitrogen. 1 S- PTY. Ltd., Sydney, Australia)), senecionine or Heliotrine, seneciphylline or senecionine were used seneciphylline (kindly provided by R. Molyneux, as external standards for quantification (Spectra USDA Albany) were given. For some experiments physics integrator SP 4270). Coremata were sus leaves of Senecio jacobaea were supplied alterna pended in 200 (il ethyl acetate; their extracts were tively. At the intervals mentioned in the tables and analyzed by capillary GLC without further purifi figures animals were harvested, dissected into gut cation. 7 ^-Heliotrine and its metabolites were (food residues included), haemolymph, fat body, identified by GLC-MS in previous studies sex organs, and integument, immediately frozen to [10- 12]; in this study these compounds were iden -20 °C and stored at this temperature until alka tified according to their specific retention indices. loid extraction. Faeces were processed according About 1600 insects were treated with alkaloids ly. In parallel experiments we injected defined and later analyzed by capillary GLC for the exper amounts (up to 30 (il) of aqueous pyrrolizidine iments described in this publication. All values giv alkaloid solutions (adjusted to pH 7) laterally into en in tables or figures are means of measurements the larval haemolymph. Larvae were slightly of 3- 14 animals which were processed individual anaesthesized with ether for about 15 sec prior to ly. Pyrrolizidine alkaloid metabolism {i.e. uptake, injection. Before and after the application of biotransformation, storage and degradation) pyrrolizidine alkaloids the animals had no period seems to differ quantitatively between animals. Al of starvation. though the larvae were kept under controlled con B. Imagines. The time of eclosion was monitored ditions, we could not totally eliminate this varia separately for each animal. At the time interval tion (which was not due to extraction or chroma given the moths were collected and processed in a tography), even in carefully designed experiments. E. von Nickisch-Rosenegk et al. ■ Pyrrolizidine Alkaloid Processing in Creatonotos 883 Results applied could be recovered (ranging from 21 to Time-course of pyrrolizidine alkaloid storage and 41% after injection, and 25-56% after oral appli pyrrolizidine alkaloid transformation in larvae cation in experiments with 3 mg pyrrolizidine alkaloid). We assume that the difference is due to Larvae of C. transiens (last instars, i.e. L7) were degradation of 7 R- and