Chemotaxonomy of Portuguese Ulex: quinolizidine alkaloids as taxonomical markers

Six species of Portuguese Ulex L. in a total of nineteen populations were studied by GC-EIMS as to their content in quinolizidine alkaloids. Sparteine, beta-isosparteine, jussiaeiine A, N-methylcytisine, cytisine, 5,6-dehydrolupanine, rhombifoline, lupanine, jussiaeiine B, N-formylcytisine, N-acetylcytisine, anagyrine, jussiaeiine C, jussiaeiine D, pohakuline, baptifoline, and epibaptifoline were detected. Analysis of the chromatograms showed that the chemical profile of all species was mainly composed of N-methylcytisine, cytisine, anagyrine, and jussiaeiines A, B, C and D. Therefore a quantification study of these alkaloids in all the populations studied was done by GC. These data were then submitted to cluster analysis and principal component analysis, which allowed the definition of five chemotypes and the recognition of hybrids. N-methylcytisine, cytisine, and jussiaeiines A, C and D are recognized as markers of this genus in Portugal.     

Biosynthese und Stoffwechsel der chinolizidinalkaloide von Sophora alopecuroides

Administration of matrine-U-³H and sophocarpine-U-³H to Sophora alopecuroides seedlings shows that these compounds were incorporated into quinolizidine alkaloids such as matrine, sophacarpine, and their N-oxides, but not into sophoridine. It is suggested that there is no stereochemical conversion of alkaloids of matrine configuration into sophoridine by the plant. The incorporation of cadaverine-1,5-¹⁴C was so low that it cannot be regarded with certainty as a physiological precursor of the alkaloids. The N-oxides of matrine and sophocarpine were isolated and identified by their chromatographic and chemical properties.     

Quinolizidine alkaloids from the curare adjuvant Clathrotropis glaucophylla

The bark of Clathrotropis glaucophylla (Fabaceae) is used as admixture of curare arrow poison by the Yanomami; Amerindians in Venezuela. A new quinolizidine alkaloid (QA), (-)-13alpha-hydroxy-15alpha-(1-hydroxyethyl)-anagyrine [(-)-clathrotropine], was isolated from the alkaloid extract of C. glaucophylla bark, together with eleven known QAs: (-)-anagyrine, (-)-thermopsine, (-)-baptifoline, (-)-epibaptifoline, (-)-rhombifoline, (-)-tinctorine, (-)-cytisine, (-)-N-methylcytisine, (-)-lupanine, (-)-6alpha-hydroxylupanine and (+)-5,6-dehydrolupanine. The isolation and structure elucidation were performed with the aid of chromatographic (TLC, HPLC and CC) and spectroscopic (UV and 1D/2D NMR) methods, and mass spectrometry. To our knowledge, this is the first time quinolizidine alkaloids have been isolated from an arrow poison ingredient. It is also the first report on Clathrotropis species being used for preparation of arrow poison.     

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents, are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetrahydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculeata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous 1H and 13C NMR data are presented for these alkaloids.     

Comparative study of the alkaloids in tribe Datureae and their chemosystematic significance

Sixty-six tropane alkaloids from crude leaf and root alkaloid mixtures of 12 different species and their varieties and subspecies from tribe Datureae were determined by GC–MS. The alkaloids 3β-hydroxy-6β-acetoxytropane, 3-propionyloxy-6-hyroxytropane, 3β-hydroxy-6β-tigloyloxytropane, 3β-tigloyloxy-6β-acetoxytropane and 3-tigloyloxy-7-isobutyryloxytropane are new reported tropane alkaloids.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Alkaloids in the venom of Messor ants

Tobacco alkaloids of the anabasine type have been found or confirmed in the venom of five species of arid-dwelling Messor ants. They are frequently accompanied by alkylpyrazines. Messor mediorubra contains four alkaloids, with anabasine the major component and also minor pyrazines. Anabasine was found alone in the venom of Messor semirufus and confirmed in Messor ebeninus. Messor rugosus from Tel Aviv contained a mixture of alkaloids and pyrazines, but those from Ein Yahav contained 3-ethyl-2,5-dimethylpyrazine, some 2-phenylethylamine, and N-ethylidene 2-phenylethylamine. Messor arenarius is confirmed as having a complex but variable mixture of alkaloids and pyrazines.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

Isoquinoline and isoindole alkaloids from Menispermum dauricum

Three isoquinoline alkaloids and an isoindole alkaloid, along with eight known compounds, were isolated from the roots of Menispermum dauricum (Menispermacese). The alkaloids were characterized as 7-hydroxy-6-methoxy-1(2H)-isoquinolinone, 6,7-dimethoxy-N-methyl-3,4-dioxo-1(2H)-isoquinolinone, 1-(4-hydroxybenzoyl)-7-hydroxy-6-methoxy-isoquinoline and 6-hydroxy-5-methoxy-N-methylphthalimide, on the basis of spectral evidence including 1D- and 2D-NMR and MS analyses.     

E/Z-Thesinine-O-4'-alpha-rhamnoside, pyrrolizidine conjugates produced by grasses (Poaceae)

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4'-alpha-rhamnoside (1) and Z-thesinine-O-4'-alpha-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).     

Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.     

Repeated plasticization and recovery of cuticular stiffness in the blood-sucking bug Triatoma infestans in the feeding context

Triatominae bugs experience changes in the mechanical properties of their cuticle prior to feeding. This process-plasticization-allows a rapid stretching of the unsclerotized abdominal cuticle of triatominae larvae and it is evoked by sensory inputs related to feeding. We tested: (a) whether the cuticle recovers its original mechanical properties after plasticization, (b) whether repeated stimulation would be able to evoke recurrent plasticization along the same larval instar, (c) the temporal course of recovering cuticular stiffness. We injected Ringer solution into the body cavity of the bugs at constant pressure, using the injection rate (ml/min) as a measure of the cuticle extensibility. To trigger plasticization, individuals were allowed to feed on blood from an artificial feeder at 32+/-2 degrees C. After plasticization occurred, the abdominal cuticle gradually recovered its original mechanical properties. Bugs were capable of plasticizing for a second time when repeatedly stimulated. The effects of plasticization vanished between 1 and 2 h after stimulation. Although one full meal could suffice to accomplish moult in other Triatomine species, Triatoma infestans is able to feed repeatedly during a single larval instar. Accordingly to this, their cuticle recovers stiffness in some hours and becomes able to respond repeatedly to sensory inputs associated with feeding.     

Dopamine-iridoid alkaloids in Carapichea affinis (= Psychotria borucana) confirm close relationship to the vomiting root Ipecac

Broad-based HPLC-UV comparison of 57 methanolic crude extracts of the Psychotria complex (Rubiaceae), representing about 20 different species collected in Costa Rica, exhibited a clear chemical segregation of Psychotria borucana. In contrast to the more widespread tryptamine-iridoid alkaloids it deviated by an accumulation of dopamine-iridoid alkaloids. Similar to the South American vomiting root Ipecac, Psychotria ipecacuanha (= Cephaelis ipecacuanha), cephaeline and emetine were found together with four related glycosides characterized by various N-acylations. Two glycosides were identified as the known N-acetates ipecoside and 6-O-methylipecoside, whereas the two others represent corresponding N-O-methylcaffeate derivatives from which borucoside was described as a new compound. Structure elucidation of all compounds was carried out by NMR- and MS-analyses supported by CSEARCH-NMR-database system. In addition to morphological characters and analyses of nucleotide sequence variation the chemical profile of P. borucana supported a recent taxonomic rearrangement where it has been grouped together with P. ipecacuanha in a separate genus Carapichea.     

Cuticular lipids and silverleaf whitefly stage affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus

Beauveria bassiana and Paecilomyces fumosoroseus are generalist entomopathogenic fungi that infect the silverleaf whitefly (Bemisia argentifolii). We found second and third instar whiteflies to be the most susceptible larval stage to both fungi. Conidia of B. bassiana germinated most readily on the cuticle of second instars (54% germinated) and P. fumosoroseus germination was highest on third instar cuticle (45%). Fourth instars (the ultimate instar) had low susceptibility to these pathogens, and spore germination on the cuticle of fourth instars was very low for B. bassiana (7%) and intermediate for P. fumosoroseus (33%). Cuticular lipids were found to have toxic or inhibitory effects on conidia of B. bassiana and P. fumosoroseus when the spores were germinated on nutrient agar in the presence of the lipids. In the absence of added nutrients, P. fumosoroseus conidial germination increased in the presence of the lipids. To test if the inhibitory effects of the lipids were due solely to hydrophobicity (preventing water from coming into contact with the conidia) we tested the effects of synthetic long-chain wax esters. The synthetic wax esters inhibited germination of P. fumosoroseus to a degree that was similar to the effect of the cuticular lipid extracts, but the synthetic lipids did not have a significant effect on B. bassiana. Thus, the thick coating of long-chain wax esters produced by whitefly nymphs affect spore germination of fungal pathogens, but whether they play a significant role in defense against disease is not clear.     

Cuticle Formation in Hemicycliophora arenaria,Aphelenchus avenae and HirschmannIella gracilis

The onset of molting in all stages of Hemicycliophora arenaria was preceded by the appearance of numerous, discrete globular structures which were termed "molting bodies" because they were present in the hypodermis only during the production of the new cuticle. In all parasitic stages the molt commenced with the separation of the cuticle from the hypodermis from which the new sheath and cuticle were differentiated. Following completion of the new sheath and cuticle most of the old outer covering was apparently absorbed before ecdysis. Electronmicrographs of body wall cross sections in molting L4 male specimens revealed the final molt to be a double molt in which an additional sixth cuticle was produced. Since both a new sheath and cuticle were produced during the molt of each stage, the sheath must be considered as an integral part of the cuticle and not as a residual cuticle or the result of an incomplete additional molt. Molting in Aphelenchus avenae and Hirschmanniella gracilis was less complex and "molting bodies" were not observed. After cuticle separation the hypodermis gave rise to a new trilaminate zone, the future cortex, and (later) the matrix and striated basal layers.     

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae,Nematomorpha): observations by TEM,SEM and AFM

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.     

Anisakis physeteris: Amino acids in body tissues

The whole cuticle, perienteric fluid, and reproductive organs of Anisakis physeteris Baylis, 1923 from the sperm whale were analyzed for amino acid composition. Amino acid nitrogen per total nitrogen accounted for 96.2% in the cuticle. Corresponding values of TCA supernatant and precipitated fractions of perienteric fluid and reproductive organ were obtained. Proline, glycine, and arginine occurred abundantly in cuticle hydrolyzate. Lysine, glutamic acid, glycine, valine, leucine and histidine occounted for approximately 62% of total nitrogen in reproductive organ fractions. Differences were observed in the amounts of certain amino acids present in corresponding tissue of males and females as well as different tissue fractions from nematodes of the same sex, e.g., more serine in male cuticle than female, more proline in the protein of female reproductive organs than male; methionine present in cuticular protein and reproductive organ protein and nonprotein fractions but absent in perienteric fluid, and cystine in cuticular protein but not the other fractions. Untreated Anisakis hemolymph showed at least a 2-fold increase in ammonia in analyses made 2 and 10 days after bleeding. The relatively large ammonia content could have been in part the result of functional ornithine cycle and amino acid oxidase activity in vivo.     

Circadian clock genes period and cycle regulate photoperiodic diapause in the bean bug Riptortus pedestris males

The photoperiodic response is crucial for many insects to adapt to seasonal changes in temperate regions. It was recently shown that the circadian clock genes period (per) and cycle (cyc) are involved in the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris females. Here, we investigated the involvement of per and cyc both in the circadian rhythm of cuticle deposition and in the photoperiodic diapause of R. pedestris males using RNA interference (RNAi). RNAi of per and cyc disrupted the cuticle deposition rhythm and resulted in distinct cuticle layers. RNAi of per induced development of the male reproductive organs even under diapause-inducing short-day conditions, whereas RNAi of cyc suppressed development of the reproductive organs even under diapause-averting long-day conditions. Thus, the present study suggests that the circadian clock operated by per and cyc governs photoperiodism of males as that of females.     

Chemical constituents of the seeds of Ammopiptanthus (Leguminosae) and their systematic and ecological significance

Eleven flavonoids including a new flavone glycoside, 7,3′-dihydroxy-4′-methoxyflavone-7-O-β-glucopyranoside, and a quinolizidine alkaloid, lupanine, were isolated from the seeds of two Ammopiptanthus (Leguminosae) species, Ammopiptanthus mongolicus and Ammopiptanthus nanus. 7,3′-Dihydroxy-4′-methoxyflavone and lupanine were the major constituents in the seeds of both species. The patterns of seed flavonoids presented a clear systematic relationship between the two species. The simple seed alkaloid composition was presumed to be responsible for the weak defense capability of the seeds to insect pests in both species.