Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus

Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.     

Uptake of linamarin and lotaustralin from their foodplant by larvae of Zygaena trifolii

Experiments in which unlabelled and [aglycone ¹⁴C-labelled cyanogenic glycosides, linamarin and lotaustralin, were fed to larvae of the moth Zygaena trifolii on leaves of an acyanogenic strain of their food plant, Lotus corniculatus, showed that the larvae retained about 20–45% of the glucosides consumed. The larvae in nature usually feed on plants of L. corniculatus which themselves contain linamarin and lotaustralin. Earlier experiments had shown that the larvae of Zygaena spp. are able to synthesize these glucosides from valine and isoleucine and so both sequestration and biosynthesis of the same compounds can occur. This is the only such occurrence yet known in the relationships between plants and insects.     

Evidence for site of biosynthesis and transport of the cyanoglucosides linamarin and lotaustralin in larvae of Zygaena trifolii (Insecta: Lepidoptera)

Zygaena larvae store relatively large amounts of cyanoglucosides within the haemolymph and cuticular cavities. Feeding experiments with ¹⁴C-labelled precursors demonstrate that valine and isoleucine are incorporated at different rates into the cyanoglucosides of the haemolymph and the defensive secretions. In conclusion from these different kinetics, we suggest that biosynthesis of cyanoglucosides takes place within the larval organs, such as fat body, gut, and/or haemolymph and that the epidermis is mainly involved in transporting and accumulating the cyanoglucosides within the integument.     

Intimate roles for cyanogenic glucosides in the life cycle of Zygaena filipendulae (Lepidoptera, Zygaenidae)

Zygaena larvae sequester the cyanogenic glucosides (CNglcs) linamarin and lotaustralin from their food plants (Fabaceae) and also de novo biosynthesize these compounds. In Zygaenidae, CNglcs serve as defence compounds during the entire life cycle, and their content and ratio are tightly regulated. We demonstrate that Z. filipendulae males transfer a nuptial gift of CNglcs to females during mating, and that females prefer males with a higher content of CNglcs for mating. Average HCN emission from female imagines is 19 times higher than from males, suggesting that plumes of HCN emitted from the perching female may serve to attract flying males. Analysis of the linamarin and lotaustralin content and ratio within different tissues in Z. filipendulae larvae shows that integument and haemolymph constitute the main sites of CNglc deposition. The data suggest that CNglcs may serve an additional role as storage compounds of reduced nitrogen that is mobilized during the transition of the last instar larva to imago, most likely to provide nitrogen for chitin synthesis. At least one of the enzymes responsible for de novo biosynthesis of CNglcs in Z. filipendulae is located in the integument. In conclusion, CNglcs play many important and different roles during the entire life cycle of Z. filipendulae in addition to defence.     

Little-known accessory glands in female Zygaena moths (Lepidoptera, Zygaenidae)

In the female genital system of Zygaena moths, an additional pair of accessory glands is present besides the Y-shaped sebaceous gland. The term 'Petersen's glands' is proposed for these organs. Anatomy, histology, histochemistry and cytology of Petersen's glands of Zygaena trifolii are described. The sac-like glands, situated in the extreme dorsocaudal part of the abdomen, can be divided into a purely secretory part consisting of acini with large pear-shaped gland cells and a reservoir part with combined secretory and storage function. The secretory cells of the acini are penetrated by long curved ductules or secretory end apparatuses having feltwork consisting of very fine filaments. The cytoplasm is characterized by abundance of smooth tubular endoplasmic reticulum (ATER) and the presence of peroxisomes. This cytoplasmic organization is in accordance with the chemical composition of the sticky secretion, which evidently consists completely of lipids. The ultrastructure of the epithelium lining the reservoir of the glands has both traits of secretory and of transporting epithelia. Besides contributing to the secretion, it may be involved in absorption of residual aqueous phase from the contents of the reservoir.     

Molecular evolution of a long wavelength-sensitive opsin in mimetic Heliconius butterflies (Lepidoptera: Nymphalidae)

This study examines the pattern of opsin nucleotide and amino acid substitution among mimetic species 'rings' of Heliconius butterflies that are characterized by divergent wing colour patterns. A long wavelength opsin gene, OPS1 , was sequenced from each of seven species of Heliconius and one species of Dryas (Lepidoptera: Nymphalidae). A parsimony analysis of OPS1 nucleotide and amino acid sequences resulted in a phylogeny that was consistent with that presented by Brower & Egan in 1997, which was based on mitochondrial cytochrome oxidase I and II as well as nuclear wingless genes. Nodes in the OPS1 phylogeny were well supported by bootstrap analysis and decay indices. An analysis of specific sites within the gene indicates that the accumulation of amino acid substitutions has occurred independently of the morphological diversification of Heliconius wing colour patterns. Amino acid substitutions were examined with respect to their location within the opsin protein and their possible interactions with the chromophore and the G-protein. Of the 15 amino acid substitutions identified among the eight species, one nonconservative replacement (A226Q) was identified in a position that may be involved in binding with the G-protein.     

The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles

Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.     

The evolution of ecologically important characters in Heliconius butterflies (Lepidoptera: Nymphalidae): a cladistic review

The biology of Heliconius butterflies has provided a rich source of data to test theories of ecological genetics, coevolution and community ecology. Many putatively adaptive characters have been discussed with reference to a phylogenetic hypothesis based on a variety of morphological and life-history traits interpreted from an evolutionary taxonomic perspective. Here, alternate interpretations of characters on the traditional tree and a more recent mitochondrial DNA cladogram with a substantially different topology are compared and contrasted. It is shown that many characters ostensibly providing support for the traditional phylogenetic hypothesis are almost equally parsimoniously distributed and in some cases more parsimoniously distributed on the mtDNA tree than on the tree inferred from those characters. Discussion of alternate evolutionary scenarios based on the mDNA-based topology is presented for pupal mating, pollen feeding, foodplant coevolution, and other ecologically significant features.     

Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera)

We examined chromosomal distribution of major ribosomal DNAs (rDNAs), clustered in the nucleolar organizer regions (NORs), in 18 species of moths and butterflies using fluorescence in situ hybridization with a codling moth (Cydia pomonella) 18S rDNA probe. Most species showed one or two rDNA clusters in their haploid karyotype but exceptions with 4–11 clusters also occurred. Our results in a compilation with previous data revealed dynamic evolution of rDNA distribution in Lepidoptera except Noctuoidea, which showed a highly uniform rDNA pattern. In karyotypes with one NOR, interstitial location of rDNA prevailed, whereas two-NOR karyotypes showed mostly terminally located rDNA clusters. A possible origin of the single interstitial NOR by fusion between two NOR-chromosomes with terminal rDNA clusters lacks support in available data. In some species, spreading of rDNA to new, mostly terminal chromosome regions was found. The multiplication of rDNA clusters without alteration of chromosome numbers rules out chromosome fissions as a major mechanism of rDNA expansion. Based on rDNA dynamics in Lepidoptera and considering the role of ordered nuclear architecture in karyotype evolution, we propose ectopic recombination, i.e., homologous recombination between repetitive sequences of non-homologous chromosomes, as a primary motive force in rDNA repatterning.     

Colonization of Northern Europe by Zygaena filipendulae (Lepidoptera)

Northern and mountainous ice sheets have expanded and contracted many times due to ice ages. Consequently, temperate species have been confined to refugia during the glacial periods wherefrom they have recolonized warming northern habitats between ice ages. In this study, we compare the gene CYP405A2 between different populations of the common burnet moth Zygaena filipendulae from across the Western Palearctic region to illuminate the colonization history of this species. These data show two major clusters of Z. filipendulae populations possibly reflecting two different refugial populations during the last ice age. The two types of Z. filipendulae only co‐occur in Denmark, Sweden, and Scotland indicating that Northern Europe comprise the hybridization zone where individuals from two different refugia met after the last ice age. Bayesian phylogeographic and ecological clustering analyses show that one cluster probably derives from an Alpe Maritime refugium in Southern France with ancestral expansive tendencies to the British Isles in the west, touching Northern Europe up to Denmark and Sweden, and extending throughout Central Europe into the Balkans, the Peleponnes, and South East Europe. The second cluster encompasses East Anatolia as the source area, from where multiple independent dispersal events to Armenia, to the Alborz mountains in north‐western Iran, and to the Zagros mountains in western Iran are suggested. Consequently, the classical theory of refugia for European temperate species in the Iberian, Italian, and Balkan peninsulas does not fit with the data from Z. filipendulae populations, which instead support more Northerly, mountainous refugia.     

Biosynthesis of cyanogenic glucosides: in vitro analysis of the glucosylation step

The last step in the biosynthesis of cyanogenic glucosides, the glucosylation of the cyanohydrin intermediate, has been investigated in detail using Triglochin maritima seedlings. The glucosyltransferase activity is not associated with membranes and appears to be a "soluble" enzyme. The cyanohydrin intermediate, which is formed by hydroxylation of 4-hydroxyphenylacetonitrile by a membrane-bound enzyme, is free to equilibrate in the presence of the glucosyltransferase and UDPG, because it can be trapped very efficiently. This indicates that this intermediate is not channeled (unlike some of the other intermediates), although it is probably the most labile of all of them. The glucosyltransferase of T. maritima responsible for the glucosylation of the cyanohydrin was separated from another glucosyltransferase, which used 4-hydroxybenzylalcohol as a substrate, and purified over 200-fold. It catalyzed the glucose transfer from UDPG to only 4-hydroxymandelonitrile and 3,4-dihydroxymandelonitrile, giving rise to the respective cyanogenic glucosides. Although the activities with these two substrates behaved differently in certain respects (e.g., extent of inactivation during purification and difference in activation by higher salt concentrations), most of the data acquired favor the view that only one enzyme in T. maritima is responsible for the glucosylation of both substrates.     

Biometrical evidence for adaptations of the salivary glands to pollen feeding in Heliconius butterflies (Lepidoptera: Nymphalidae)

The Neotropical genus Heliconius (Nymphalidae) is unique among butterflies for its pollen-feeding behaviour. With the application of saliva, they extract amino acids from pollen grains on the outside of the proboscis. We predicted that the salivary glands of pollen-feeding Heliconiinae would show adaptations to this derived feeding behaviour. A biometrical analysis of the salivary glands revealed that pollen-feeding butterflies of the genus Heliconius have disproportionately longer and more voluminous salivary glands than nonpollen-feeding Nymphalidae. The first two components in the principal component analysis explained approximately 95% of the total variance. The size-dependent factor score coefficients of body length and salivary gland parameters were predominately represented on axis 1. They significantly discriminated pollen-feeding from nonpollen-feeding heliconiines on that axis. Factor score coefficients for the volume of the secretory region of the salivary glands separated heliconiines from the outgroup species. The detailed biometrical analysis of salivary glands features thus provides strong evidence that the secretory regions of the salivary glands are larger in pollen-feeding butterflies. We concluded that pollen feeding is associated with a high production of salivary fluid.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 604–612.     

Phylogenetic studies of ribosomal RNA variation in higher moths and butterflies (Lepidoptera: Ditrysia).

The selection of exemplars has been shown both theoretically and empirically to affect tree topology, but the importance of the number and nature of taxa used to represent higher taxonomic lineages in molecular studies is rarely stressed. In our rRNA study of higher moths and butterflies (Lepidoptera: Ditrysia), the selection of different exemplars and outgroups caused major tree rearrangements. We also examined the effectiveness with which conserved rRNA regions track the diversification of Lepidoptera. Homoplasy is as prevalent at the few variable sites of conserved regions (18E, 18J, 28F) as at the many variable sites of a more rapidly evolving region (28B). Finally, 28B sequence variation differs qualitatively among lepidopteran superfamilies of presumed comparable age, the Papilionoidea (true butterflies) and Noctuoidea (cutworm moths and relatives).     

Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study

Background

In the mega-diverse insect order Lepidoptera (butterflies and moths; 165,000 described species), deeper relationships are little understood within the clade Ditrysia, to which 98% of the species belong. To begin addressing this problem, we tested the ability of five protein-coding nuclear genes (6.7 kb total), and character subsets therein, to resolve relationships among 123 species representing 27 (of 33) superfamilies and 55 (of 100) families of Ditrysia under maximum likelihood analysis.

Results

Our trees show broad concordance with previous morphological hypotheses of ditrysian phylogeny, although most relationships among superfamilies are weakly supported. There are also notable surprises, such as a consistently closer relationship of Pyraloidea than of butterflies to most Macrolepidoptera. Monophyly is significantly rejected by one or more character sets for the putative clades Macrolepidoptera as currently defined (P < 0.05) and Macrolepidoptera excluding Noctuoidea and Bombycoidea sensu lato (P ≤ 0.005), and nearly so for the superfamily Drepanoidea as currently defined (P < 0.08). Superfamilies are typically recovered or nearly so, but usually without strong support. Relationships within superfamilies and families, however, are often robustly resolved. We provide some of the first strong molecular evidence on deeper splits within Pyraloidea, Tortricoidea, Geometroidea, Noctuoidea and others. Separate analyses of mostly synonymous versus non-synonymous character sets revealed notable differences (though not strong conflict), including a marked influence of compositional heterogeneity on apparent signal in the third codon position (nt3). As available model partitioning methods cannot correct for this variation, we assessed overall phylogeny resolution through separate examination of trees from each character set. Exploration of "tree space" with GARLI, using grid computing, showed that hundreds of searches are typically needed to find the best-feasible phylogeny estimate for these data.

Conclusion

Our results (a) corroborate the broad outlines of the current working phylogenetic hypothesis for Ditrysia, (b) demonstrate that some prominent features of that hypothesis, including the position of the butterflies, need revision, and (c) resolve the majority of family and subfamily relationships within superfamilies as thus far sampled. Much further gene and taxon sampling will be needed, however, to strongly resolve individual deeper nodes.     

Genital and body allometry in two species of noctuid moths (Lepidoptera: Noctuidae)

One‐size‐fits‐all and related hypotheses predict that static allometry slopes for male genitalia will be consistently lower than 1.0 and lower than the slopes for most other body parts (somatic traits). We examined the allometry of genitalic and somatic morphological traits in males and females of two species of noctuid moths, Spodoptera exigua (Hübner, [1808]) and Helicoverpa armigera (Hübner, [1808]). The relationship between genitalic traits and body size was generally strongly negative‐allometric in males but with no significant differences from 1.00 in females of the two species examined. However, in females, the slope of genital traits was also lower than the slopes for somatic traits. The relationship between somatic traits and the body size indicator was approximately isometric in most cases in males, except in four traits in S. exigua, in which the slopes showed slight negative allometry, and the hind tibia in H. armigera, in which the slope had positive allometry. However, in females, some somatic traits showed isometric and some other showed negative allometry in both species. The coefficients of variation (CV) for all structures in the males were low, not exceeding 10%. Genitalic traits showed significantly lower CV than somatic traits in males. In females, somatic traits showed lower CV than genitalic traits but with no significant difference in the H. armigera. Our observations of strongly negative allometry for genitalic traits in males are consistent with stabilizing selection on genital size and we suggest that male performance in interactions with females is the source of selection on male genital allometry. The difference in the degree of phenotypic variation between genitalic and somatic traits in the two studied species is attributed to the different developmental‐genetic architectures of these traits. Female genitalia showed a similar trend to the males, although the difference between genital and somatic traits was not significant in females. This finding suggests that selection is acting differently on male and female genitalia. Positive allometry of hind tibia in H. armigera may be a result of secondary sexual function.     

DNA barcoding and species delimitation of butterflies (Lepidoptera) from Nigeria

Molecular Biology Reports - Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require...     

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.