1H NMR Spectroscopy of pyrrolizidine alkaloids

The ¹H NMR spectra of three pyrrolizidine alkaloids of the macrocyclic diester type, retrorsine, seneciphylline and senecionine, plus their three N-oxides have been assigned. Previous ¹H NMR studies of these pyrrolizidine alkaloids have stressed the difficulties of spectral intrepretation. The results reported here will provide a useful resource for analysis of tertiary structure in these and related compounds.     

Pyrrolizidine alkaloids of probable host-plant origin in the pronotal and elytral secretion of the leaf beetle Oreina cacaliae

Oreina cacaliae (Coleoptera, Chrysomelidae) produces in its elytral and pronotal defensive secretion seneciphylline N-oxide together with small amounts of another pyrrolizidine alkaloid tentatively identified as senecionine N-oxide. This is a strong departure from the chemical composition of the defensive secretions in related species, characterized by complex mixtures of cardenolides, synthesized by the beetles from cholesterol. It is suggested that O. cacaliae sequesters the alkaloids from its host-plant, Adenostyles leucophylla. Other specimens of O. cacaliae from far distant populations feeding on Senecio nemorensis, Petasites paradoxus or P. album also produced pyrrolizidine alkaloids, but not O. speciosissima feeding on the same food plants and producing cardenolides. In addition to pyrrolizidine alkaloids, O. cacaliae secretes ethanolamine, which is also found in all the cardenolide-producing species.     

Sites of synthesis,translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L.

¹⁴C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3–6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [¹⁴C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [¹⁴C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a steam girdle translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.Abbreviations FW fresh weight - sen senecionine - sen-Nox senecionine N-oxide     

Pyrrolizidine alkaloids from Senecio longilobus and Senecio glabellus

TLC, capillary GC, packed column and capillary GC-MS, and¹H NMR were used to characterize pyrrolizidine alkaloids fromSenecio longilobus andS.glabellus.S.glabellus contained senecionine and integerrimine, andS.longilobus contained senecionine, integerrimine, seneciphylline and retrorsine, all present predominantly asN-oxides. Alkaloid content varied greatly in collections ofS.longilobus. This is the first report of integerrimine in these plants.     

Site of synthesis,metabolism and translocation of senecionine N-oxide in cultured roots of Senecio erucifolius

Root cultures of Senecio erucifolius (Asteraceae) efficiently took up and incorporated [¹⁴C]putrescine and [¹⁴C]arginine into the pyrrolizidine alkaloid (PA) senecionine N-oxide. Pulse-chase experiments covering a growth period of 10 to 19 days revealed the absence of any significant alkaloid turnover. The only metabolic activity was a slow but progressive transformation of senecionine N-oxide into its dehydrogenation product, seneciphylline N-oxide. Tracer experiments with single roots showed that the sites of enhanced PA synthesis coincided with the sites of preferred protein synthesis, i.e. root apices, indicating a close correlation between growth activity and alkaloid synthesis. Long-term pulse-chase experiments (10 to 12 days) with ¹⁴C-labelled arginine, putrescine and senecionine fed to single roots indicated that in spite of its metabolic inertia, senecionine N-oxide is a mobile compound which is translocated into tissues newly grown during the chase.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

Biological control of <Emphasis Type="Italic">Lantana camara</Emphasis> in South Africa: targeting a different niche with a root-feeding agent, <Emphasis Type="Italic">Longitarsus</Emphasis> sp.

The root-feeding flea beetle, Longitarsus sp. (Coleoptera: Chrysomelidae: Alticinae), was studied as a potential biological control agent for Lantana camara L. (Verbenaceae) in South Africa. Host range tests were carried out on 52 plant species in 11 families. Although 11 plant species, all in the family Verbenaceae, supported complete development of Longitarsus sp. during no-choice tests, the beetles showed very strong preferences for L. camara during paired-choice and multi-choice tests. The results confirm that the beetles have a narrow host range, and that under natural conditions they are highly unlikely to utilise plants other than L. camara. In the unlikely event that some of the Lippia spp. are attacked in the field, they are not expected to sustain populations of the flea beetle over time. Attributes that should enhance the biocontrol potential of Longitarsus sp. include: the adults are long-lived and highly mobile; and, the larvae cause extensive direct damage to the roots of L. camara, which could in turn expose the plants to soil-born pathogens. All indications are that Longitarsus sp. could make a substantial contribution to the biological control of L. camara in many countries around the world because the beetles pose no threat to non-target plant species and they damage a part of the plant (i.e. roots) not yet affected by any other agent species.     

Tritrophic interactions between aphids (Aphis jacobaeae Schrank), ant species,Tyria jacobaeae L., and Senecio jacobaea L. lead to maintenance of genetic variation in pyrrolizidine alkaloid concentration

Summary We hypothesize that the tritrophic interaction between ants, the aphid Aphis jacobaeae, the moth Tyria jacobaeae, and the plant Senecio jacobaea can explain the genetic variation observed in pyrrolizidine alkaloid concentration in natural populations of S. jacobaea. The ant Lasius niger effectively defends S. jacobaea plants infested with A. jacobaeae against larvae of T. jacobaeae. S. jacobaea plants with A. jacobaeae which are defended by ants escape regular defoliation by T. jacobaeae. Plants with aphids and ants have a lower pyrrolizidine alkaloid concentration than plants without aphids and ants. When these data are fitted to an existing theoretical model for temporal variation in fitness it is shown that varying herbivore pressure by T. jacobaeae in interaction with ants defending aphid-infested plants with a low pyrrolizidine alkaloid concentration can lead to a stable polymorphism in pyrrolizidine alkaloid concentration. Costs of the production and maintenance of pyrrolizidine alkaloids are not accounted for in the model.Publication of the Meijendel-comité, new series no. 114     

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination

Introduction  – Two recent mass spectrometry‐based reports concerning Senecio scandens yielded remarkably dissimilar pyrrolizidine alkaloid constituents. In both studies, and in a related analysis of Senecio scandens and Tussilago farfara using micellar electrokinetic chromatography, the presence of hazardous N‐oxides of the alkaloids was either not considered or was inadequately considered. This raises concerns about the effectiveness of the methodologies used in these, and similar, studies in assessing the pyrrolizidine alkaloid content and the safety of food, food supplements and medicines for human use. Objective  – To highlight essential analytical requirements for confident assessment of pyrrolizidine alkaloid‐related safety of food and herbal products for human use. Methodology  – Direct infusion‐ESI MS and HPLC‐ESI MS were used to analyse samples derived from liquid–liquid partitioning experiments and from strong cation exchange, solid‐phase extraction of pyrrolizidine alkaloids and their N‐oxides. Results  – A simple solvent partitioning experiment using pure senecionine and senecionine‐N‐oxide, two constituents reported in one of the mass spectrometry‐based studies of S. scandens, clearly demonstrated the inadequacy of the reported method to detect and quantitate hazardous pyrrolizidine alkaloid N‐oxide components. A preliminary LCMS analysis of commercially‐prepared extracts of comfrey roots (Symphytum officinale and S. uplandicum s. l.) was used as a model to highlight the analytical importance of N‐oxides in the safety assessment of pyrrolizidine alkaloid‐containing medicinal herbs. Conclusions  – This study highlighted significant differences in the reported identification of pyrrolizidine alkaloids from the same plant species, and clearly demonstrated the inadequacy of some procedures to include N‐oxides in the assessment of pyrrolizidine alkaloid‐related safety of food and herbal products. Copyright © 2008 John Wiley & Sons, Ltd.     

Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

UPLC-MS based metabolomics study on Senecio scandens and S. vulgaris: an approach for the differentiation of two Senecio herbs with similar morphology but different toxicity

Pyrrolizidine alkaloids show significant hepatotoxicity as they can bind to DNA or proteins after being activated in liver. Senecio vulgaris L., like many Compositae herbs containing pyrrolizidine alkaloids, was reported to have great hepatotoxicity. However, Senicio scandens Buch.-Ham., from the same genus, which was also used as a herb and documented in China Pharmacopoeia published in 2010, hardly showed any side effects or relevant toxicity. In the present study, we conducted the metabolomics study using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to obtain the different metabolic profiles of the two Senecio herbs. In addition, principle component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) were introduced for the multivariate analysis, and MS/MS was applied to the identification of target alkaloid markers which contributed most to the established models. As a result, ten pyrrolizidine alkaloids, including adonifoline, senecionine, senecionine N-oxide, retrorsine, retrorsine N-oxide and seneciphylline, were selected and identified. Among them, adonifoline was found to be a specific marker for S. scandens while senecionine and its N-oxidative were characteristic markers for S. vulgaris. Furthermore, the hepatotoxicity studies in vivo and in vitro showed that senecionine had more potent toxicity (LD₅₀, 57.3?mg/kg; IC₅₀, 5.41???M) than that of adonifonine (LD₅₀, 163.3?mg/kg; IC₅₀, 49.91???M). Taken together, the present study provides not only better understanding of the different toxicity between the two Senecio herbs containing pyrrolizidine alkaloids but also a reference method, which can be applied to other genetically closed species with similar morphology but different toxicity.     

Sequestration, metabolism and partial synthesis of tertiary pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi

Platyphora boucardi leaf-beetles sequester tertiary pyrrolizidine alkaloids of the lycopsamine type acquired from their host-plant Prestonia portobellensis (Apocynaceae) and synthesize their own alkaloids from exogenous retronecine and aliphatic 2-hydroxy acids. Tracer studies with [14C]rinderine and its N-oxide revealed that P. boucardi sequesters both alkaloidal forms with the same efficiency, but accumulates exclusively tertiary alkaloids. There is no substantial alkaloid accumulation in the body outside the defensive glands. Feeding studies with [2H][14C]rinderine confirmed that P. boucardi specifically epimerizes rinderine to its stereoisomers intermedine and lycopsamine. Feeding studies with [2H][14C]retronecine proved the ability of P. boucardi to synthesize O7- and O9-(2-hydroxyisovaleryl)-retronecine and O7-lactyl-O9-(2-hydroxyisovaleryl)-retronecine. Both, alkaloids of the lycopsamine type and self-synthesized retronecine esters accumulate in the defensive secretions at concentrations up to 38 mM and 33 mM, respectively. The different biochemical strategies to maintain pro-toxic pyrrolizidine alkaloids and to prevent self-poisoning, developed by specialized insects, are compared. There are two major findings: (1) the chemical defense mediated by plant acquired pyrrolizidine alkaloids in the taxonomically related palaearctic Oreina and neotropical Platyphora leaf beetles have been evolved independently, since the biochemical mechanisms of storing and maintaining the alkaloids is completely different in the two genera; (2) unexpected parallels exist between taxonomically unrelated Coleoptera and Lepidoptera in their ability to synthesize the same retronecine esters and to catalyze the same site-specific epimerizations of the lycopsamine stereoisomers.     

Senecionine n-oxide,the primary product of pyrrolizidine alkaloid biosynthesis in root cultures of Senecio vulgaris

Root cultures of Senecio vulgaris synthesize pyrrolizidine alkaloids which are accumulated in the form of their N-oxides. The cultures incorporate biosynthetic precursors, such as arginine, ornithine, isoleucine, putrescine and spermidine, with high efficiency into the alkaloids. Senecionine N-oxide is found to be the primary product of biosynthesis. With putrescine and spermidine incorporation rates of 20–30% are obtained. The N-oxide synthesized does not appear to undergo significant turnover. Tertiary pyrrolizidine alkaloids, if found at all, occur in small amounts in old tissues only. They are derived from the corresponding oxides, and are easily formed spontaneously during alkaloid extraction. The suitability of N-oxides in alkaloid storage is discussed.     

Implication of tyramine in the biosynthesis of morphinan alkaloids in Papaver

Doubly-labeled [³H, ¹⁴C]tyrosines, [1-¹³C-]tyramine or [2-¹⁴C]tyramine, administered to the stems of intact Papaver somniferum L. plants, were found to be incorporated into the morphinan alkaloids of the plant with comparable efficiency. ³H/¹⁴C ratios of alkaloids from plants fed the tyrosines were consistent with an almost equal conversion of this amino acid into the tetrahydroisoquinoline (TIQ) and benzyl-derived segments. Nuclear magnetic resonance (NMR) analyses of morphine isolated after administration of [1-¹³C]tyramine demonstrated selective labeling of C-16 of the alkaloid, indicating the conversion of this amine primarily into the TIQ-derived moiety. Morphine and thebaine labeled by [2-¹⁴C]tyramine were degraded to phenanthridines and N,N-dimethyl ethylamines. Of the total radioactivity in the alkaloids 97% was found to be associated with the ethylamines, a distribution consistent with the NMR data. This preferential utilization of tyramine in the biosynthesis of morphinan alkaloids can be explained by the compartmentalization of intermediates and enzymes of the pathway.Abbreviations L-dopa L-3,4-dihydroxyphenylalanine - HPLC high-pressure liquid chromatography - NMR nuelear magnetic resonance - TIQ tetrahydroisoquinoline     

Occurrence of theobromine synthase genes in purine alkaloid-free species of <Emphasis Type="Italic">Camellia</Emphasis> plants

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-¹⁴C]adenine and [8-¹⁴C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession numbers AB297451 (CjCS1), AB362882 (CgCS1), AB362883 (CgCS2), AB362884 (CkCS1), AB362885 (ClCS1), and AB362886 (CcCS2).     

Selective uptake of pyrrolizidine N-oxides by cell suspension cultures from pyrrolizidine alkaloid producing plants

The N-oxides of pyrrolizidine alkaloids such as senecionine or monocrotaline are rapidly taken up and accumulated by cell suspension cultures obtained from plants known to produce pyrrolizidines, i.e. Senecio vernalis, vulgaris, viscosus (Asteraceae) and Symphytum officinale (Boraginaceae). The transport of the N-oxides into the cells is a specific and selective process. Other alkaloid N-oxides such as sparteine N-oxide are not taken up. Cell cultures from plant species which do not synthesize pyrrolizidine alkaloids are unable to accumulate pyrrolizidine N-oxides. The suitability of the pyrrolizidine N-oxides in alkaloid storage and accumulation is emphasized.     

Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species

Root cultures of Senecio vulgaris, S. vernalis, S. erucifolius and S. squalidus were established. The patterns of pyrrolizidine alkaloids found in these root cultures were analyzed by high-resolution GC and GC-MS and compared with the alkaloids present in the respective plants. In vitro cultured roots produce alkaloid patterns and accumulate quantities which are comparable to those found in soil grown plants. With the exception of the otonecine derivative senkirkine all pyrrolizidines accumulate as N-oxides. Only senkirkine is partially released into the medium. The cultures incorporate biosynthetic precursors, e.g. ¹⁴C-labelled putrescine or spermidine with high efficiency into the alkaloids. Senecionine N-oxide was found to be the main product of biosynthesis. Evidence is presented that senecionine N-oxide is directly transformed into senkirkine, the main alkaloid of S. vernalis root cultures.Abbreviations GC Gas chromatography - MS Mass spectroscopy - PND Phosphorous-Nitrogen-Detector - FID Flame Ionization Detector - fr.wt Fresh weight     

Life cycle and abundance oflongitarsus jacobaeae [Col.: Chrysomelidae], biocontrol agent ofSenecio jacobaea

The life cycle and abundance of the tansy ragwort flea beetle,Longitarsus jacobaeae (Waterhouse), were investigated in a dune area in the Netherlands. The beetle overwinters in the egg stage, which is parasitised by a Mymarid wasp. No larvae were found until spring. Three larval instars can be separated by head capsule size and coloration. Initial larval numbers are high (up to 214 larvae per plant), but drop to very low levels by late spring. Adults appear during June or July, the numbers are high until October, the adults can be found until the end of December. This life cycle differs remarkably from those described for the species in Switzerland, Italy and Britain. Possible causes for these differences are discussed, as well as implications for the use ofLongitarsus in biological control.