Edmund Maliński

The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection.

Epicuticular lipids in many terrestrial arthropods consist of vast numbers of polar and non-polar aliphatic compounds, which are mainly responsible for the water balance in these animals but can also affect conidia germination of entomopathogenic fungi. In this work the qualitative and quantitative profiles of cuticular fatty acids from three insect species differing in their susceptibility to fungal infection were studied. In an innovative approach, laser light scattering detection was coupled with HPLC in order to identify the non-chromophoric chemicals usually present in cuticular extracts. The acids identified contained from 5 to 20 carbon atoms in the alkyl chain and included unsaturated entities such as C(16:1), C(18:1), C(18:2), C(18:3) and C(20:1). There was a marked dominance of acids containing 16-18 carbon atoms. The relative contents of fatty acids in the extracted waxes varied from trace amounts to 44%. Cuticular fatty acids profile of Calliphora vicina (species resistant to fungal infection) significantly differs from profiles of Dendrolimus pini and Galleria mellonella (both species highly susceptible to fungal infection). The major difference is the presence of C(14:0), C(16:1) and C(20:0) in the cuticle of C. vicina. These three fatty acids are absent in the cuticle of D. pini while G. mellonella cuticle contains their traces. The concentrations of four fatty acids dominating in the G. mellonella larval cuticle (C(16:0), C(18:0), C(18:1) and C(18:2)) were found to fluctuate during the final larval instar and correlate with fluctuations in the susceptibility of larvae to fungal infection. The possible role of cuticular fatty acids in preventing fungal infection is discussed.

ESTABLISHMENT OF HAIRY ROOT CULTURES OF AMMI MAJUS

Axenically grown Ammi majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains. Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402. The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures. Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains. The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A. majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography-mass spectrometry (GC-MS). However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures. The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A. majus seeds (19 µg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 µg/g DW, respectively).

Elicitation of secondary metabolites in in vitro cultures of Ammi majus L.

Abstract The present study was concentrated on the production of secondary metabolites in callus, cell suspension and hairy roots of Ammi majus L. by exposing them to elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION®) and autoclaved lysate of cell suspension of bacteria—Enterobacter sakazaki. GC and GC–MS analysis of chloroform and methanol extracts indicated a higher accumulation of umbelliferone in the elicited tissues than in the control ones. Using GC–MS, two compounds not earlier found in A. majus tissues were identified in callus cultures: scopoletin (7-hydroxy-6-metoxy-2H-1-benzopyron-2-one) and dehydrogeijerin (7-methoxy-6-(3-methyl-1-oxo-2-butenyl)-2H-1-benzopyran-2-one).

Application of chitin and chitosan as elicitors of coumarins and furoquinolone alkaloids in Ruta graveolens L. (common rue)

Common rue (Ruta gra veolens L.) accumulates various types of secondary metabolites, such as coumarins furanocoumarins, acridone and quinolone alkaloids and flavonoids. Elicitation is a tool extensively used for enhancing secondary‐metabolite yields. Chitin and chitosan are examples of elicitors inducing phytoalexin accumulation in plant tissue. The present paper describes the application of chitin and chitosan as potential elicitors of secondary‐metabolite accumulation in R. graveolens shoots cultivated in vitro . The simple coumarins, linear furanocoumarins, dihydrofuranocoumarins and furoquinolone alkaloids biosynthesized in the presence of chitin and chitosan were isolated, separated and identified. There was a significant increase in the growth rate of R. graveolens shoots in the presence of either chitin or chitosan. Moreover, the results of the elicitation of coumarins and alkaloids accumulated by R. graveolens shoots in the presence of chitin and chitosan show that both compounds induced a significant increase in the concentrations of nearly all the metabolites. Adding 0.01% chitin caused the increase in the quantity (μg/g dry weight) of coumarins (pinnarin up to 116.7, rutacultin up to 287.0, bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5 and rutamarin up to 133.7). The higher concentration of chitosan (0.1%) induced production of simple coumarins (pinnarin up to 116.7 and rutacultin up to 287.0), furanocoumarins (bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5) and dihydrofuranocoumarins (chalepin up to 18 and rutamarin up to 133.7). Such a dramatic increase in the production of nearly all metabolites suggests that these compounds may be participating in the natural resistance mechanisms of R. graveolens . The application of chitin‐ and chitosan‐containing media may be considered a promising prospect in the biotechnological production of xanthotoxin, isopimpinelin, psoralen, chalepin or methoxylated dictamnine derivatives.

Sex-dependent composition of cuticular hydrocarbons of the colorado beetle, Leptinotarsa decemlineata say

Abstract 1. 1. Qualitative and quantitative investigation of epicuticular and internal hydrocarbons of Colorado beetles of winter generation were performed. 2. 2. Quantitative compositional differences for sexes were found. 3. 3. Females contain more branched methylalkanes whose tentative structure was identified as either tetramethylhentriacontane or trimethyltritriacontane or a mixture of both. 4. 4. Compositional studies of potato leaves were also done. A prominent component of leaf waxes, 2-methyltricontane, was also found in cuticular hydrocarbons of both sexes as one of the most abundant compounds.

Cuticular hydrocarbons of the german cockroach, Blattella germanica L.

1. 1. The cuticular hydrocarbons from the German cockroach consist of n-alkanes, 3-methylalkanes, internally branched monomethylalkanes and dimethylalkanes. 2. 2. The composition of the hydrocarbon fraction differs markedly from the other species of cockroaches. n-Nonacosane and n-heptacosane as well as their monomethyl and dimethylderivatives are the major constituents of the hydrocarbon fraction from Blattella germanica. 3. 3. Lesser amounts of n-octacosane and two of its monomethylisomers have also been identified. 4. 4. The dimethylalkanes are nearly all 3,X-dimethylalkanes. There are 3, 5 and 7 methylene groups between the 3 and X in the various chains from carbon numbers 27–29.

Kováts retention indices of high-molecular-weight monomethyl-, cyclopentyl-, cyclohexyl- and phenylalkanes

Kovats retention indices of monomethyl-, cyclopentyl-, cyclohexyl- and phenylalkanes containing 17–24 carbon atoms were measured on Apiezon L, OV-1, Dexsil 300, SE-52 and Carbowax 20M stationary phases. Good agreement between the calculated and measured retention indices was found. The calculations were made by summing the contributions of atoms, bonds and interactions to the retention index. The elution order of 3- and 2-methylalkanes was correlated only with the Van der Waals volumes of the molecules. It was found that cycloalkanes and phenylalkanes have a high ΔI/ΔT coefficient. Therefore, to interpret the retention indices of these compounds we used fi(T) coefficients, which determine the influence of temperature on the contribution of interactions to the retention indices. A study of changes in the retention indices on individual stationary phases demonstrated that Dexsil 300 was the most suitable for the analysis of mixtures of isoalkanes, cycloalkanes and phenylalkanes.

Application of mass spectrometry to the study of prototropic equilibria in 5-substituted tetrazoles in the gas phase; experimental evidence and theoretical considerations

The tautomerism of tetrazole, 5-methyltetrazole, and its isotopically substituted derivatives is discussed on the basis of their fragmentation patterns, and of quantum chemical calculations by the LCAO MO method in the CNDO/2 approximation. The equilibrium of these compounds in the gas phase is displaced towards the 2H-tautomer. Analysis of the mass spectra of 5-methyl[2H3]tetrazole revealed randomization of hydrogen between the methyl and the NH groups.

Identification of Ruta graveolens L. Metabolites Accumulated in the Presence of Abiotic Elicitors

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC‐MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, γ‐fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.

Cuticular Hydrocarbons of the Colorado Beetle Leptinotarsa decemlineata say

Abstract The cuticular hydrocarbons of the Colorado beetle Leptinotarsa decemlineata say consist of 2-methylalkanes, internally branched monomethylalkanes, dimethyl-, trimethyl-and tetramethyl-alkanes. The hydrocarbons differ from those of most of the other insects so far investigated. The major components were found to be the dim ethyl branched alkanes, 2,6-and/or 7,22-dimethyl-octacosane and the tetram ethylalkane, 3,10,16,23-tetram ethyltritriacontane. The hydrocarbons identified are members of homologous series which differ by two m ethylene groups. The identification of hydrocarbons was achieved by mass spectrom etry and gas chromatographic determination of Kovats retention indices on two high efficiency capillary columns coated with Dexsil 300 and OV-1.