Molecular cloning, mRNA expression and biological activity of the pheromone biosynthesis activating neuropeptide (PBAN) from the European corn borer, Ostrinia nubilalis.

Pheromone biosynthesis activating neuropeptide (PBAN) is a member of the pyrokinin (FXPRLamide) insect neuropeptides. Here, we report the cloning of the gene Ostnu-PBAN from the E and Z pheromone strains of the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae), a major pest of maize. The Ostnu-PBAN genomic sequence is > 5 kb in length and consists of six exons. The deduced amino acid sequence revealed a 200-residue precursor protein including a signal peptide, a 24-amino acid (aa) diapause hormone, a 37-aa PBAN and three other FXPRLamide neuropeptides. Our in vivo assays suggest that the 37-aa synthetic Ostnu-PBAN is hormonally active in the pheromone gland. It restores sex pheromone production to normal levels in mated females and decapitated virgins of both E and Z cultures. The results of a real-time PCR analysis indicated that Ostnu-PBAN mRNA levels reached a plateau in the brain-suboesophageal ganglion complexes 1 day after eclosion, and mating did not affect the mRNA expression. Three size classes of Ostnu-PBAN mRNA (1.9, 2.0 and 2.1 kb) were obtained, differing only in the length of the 3' untranslated region. However, there was no correlation between sequence divergence and the pheromone composition, voltinism or geographical origin (Hungary, Slovenia, Sweden, Turkey) of ECB moths.

Optimization of blends of synthetic pheromone components for trapping male limabean pod borers (Etiella zinckenella Tr.) (Lepidoptera: Phycitidae): preliminary evidence on geographical differences.

From the five previously identified pheromone components of the limabean pod borer (Etiella zinckenella Tr.) (Lepidoptera: Phycitidae), the 100:3 mixture of (Z)-11-tetradecenyl acetate and (Z)-9-tetradecenyl acetate was necessary for maximal attraction of males into traps in tests performed in Hungary. The addition of (E)-11-tetradecenyl acetate in percentages higher than 10-30% generally had an adverse effect on catches, while the addition of the other two compounds present in pheromone extracts had no influence on catches. In contrast to the results in Hungary, none of the traps baited with combinations of the above compounds captured any moths in tests performed in Taiwan, suggesting possible geographical differences in pheromonal response of European and Eastern Asian populations of E. zinckenella.

Novel type of sex pheromone structure identified fromStigmella malella (Stainton) (Lepidoptera: Nepticulidae).

Short-chain unsaturated chiral methyl carbinols are identified as a new class of lepidopteran pheromone components. The natural female-produced pheromone of the banded apple pigmyStigmella malella (=Nepticula malella) (Stainton) (Lepidoptera: Nepticulidae) was identified to be a mixture of (S)-(E)-6,8-nonadien-2-ol and (S)-(Z)-6,8-nonadien-2-ol. For monitoring traps, a 10:3E:Z blend at 100-1000 µg is recommended. It is suggested that pheromones with similar structures may be specific to Nepticulidae and other related microlepidopteran families.

Pheromone differences between sibling taxaDiachrysia chrysitis (linnaeus, 1758) andD. tutti (Kostrowicki, 1961) (Lepidoptera: Noctuidae).

The noctuid sibling taxaDiachrysia chrysitis s. str. andD. tutti, of yet uncertain taxonomic status, have previously been shown to possess differences in morphology and to be attracted to different mixtures of the two presumed pheromone components (Z)-5-decenyl acetate and (Z)-7-decenyl acetate. TypicalD. tutti males (clearly broken forewing marking) are known to respond to a 2: 100 mixture of the two isomers, whereasD. chrysitis males (unbroken marking) are attracted to a 100: 10 mixture. We investigated female pheromone production and male electroantennographic (EAG) response inDiachrysia families raised in the laboratory from field-collected gravid females. Extracts of individual females from typicalD. tutti andD. chrysitis families were subjected to gas chromatography with simultaneous flame ionization and electroantennographic detection. All females produced mixtures of Z5- and Z7-10:OAc, but femaleD. chrysitis produced predominantly Z5-10:OAc and the antennae of their brothers responded more strongly to the Z5 peak than to the Z7-10:OAc peak, whereas the opposite was true forD. tutti families. The pheromone components were shown to be biosynthesized from hexadecanoic and tetradecanoic acid, respectively by Z11-desaturation followed by chain shortening, reduction, and acetylation. The EAG responses of males trapped with the typicalD. tutti andD. chrysitis blends, as well as with an intermediate blend, were investigated. Males trapped with theD. tutti mixture almost exclusively had a clearly broken wing marking and showed strongest EAG response to Z7-10:OAc. The intermediate blend and theD. chrysitis mixture gave more mixed catches, but with a prevalence of males with an unbroken (or almost unbroken) wing marking and with a higher mean response to Z5-10:OAc. Some males with typicalD. tutti EAG responses were attracted in the field to theD. chrysitis pheromone. In the flight tunnel someD. chrysitis males were attracted also to theD. tutti mixture. This indicates that cross attraction may take place between the two taxa under natural conditions.

2-(E)-nonen-1-ol: Male attractant for chafersAnomala vitis Fabr. andA. dubia Scop. (Coleoptera: Scarabaeidae).

Traps baited with 2-(E)-nonen-1-ol alone or in combination with other compounds caught large numbers of males of both the vine chafer,Anomala vitis Fabr. and the margined vine chafer,A. dubia Scop. (Coleoptera: Scarabaeidae), vineyard and orchard pests. In a dosage test, the largest numbers were caught by traps baited with 10 mg of 2-(E)-nonen-1-ol, which was the highest dosage tested. This is the first report on male attractants for chafer species occurring in Europe.

Species discrimination in five species of winter-flying geometrids (Lepidoptera) based on chirality of semiochemicals and flight season.

Enantiomer separation of (6Z,9Z)-cis-3,4-epoxynonadecadiene and (3Z,9Z)-cis-6,7-epoxynonadecadiene could be achieved using chiral high-resolution gas chromatography and a cyclodextrin-bond column. (3Z,9Z)-(6R,7S)-Epoxynonadecadiene was identified from ovipositor extracts ofColotois pen-Naria, while inErannis defoliaria the 6S,7R-enantiomer was found. In field trapping tests pure synthetic enantiomers caught only conspecific males of these species. (3Z,6Z,9Z)-Nonadecatriene was found in both species, while the presence of (3Z,6Z,9Z)-heneicosatriene was indicated inC. Pennaria only. A 10∶10∶3 blend of (3Z,9Z)-(6R,7S)-epoxynonadecadiene, (3Z,6Z,9Z)-heneicosatriene, and (3Z,6Z,9Z)-nonadecatriene was found to be optimal for catchingC. Pennaria, whileE. Defoliaria males were optimally caught by a 1∶1 mixture of (3Z,9Z)-(6S,7R)-epoxynonadecadiene and (3Z,6Z,9Z)-nona-decatriene. (6Z,9Z)-(3S,4R)-Epoxynonadecadiene was identified from ovipositor extracts ofAgriopis (Erannis) aurantiaria. In field tests the pure enantiomer proved to be a highly specific sex attractant for both the late autumn/early winter flyingA. Aurantiaria and the late winter/early spring flyingA. Leucophearia. Males ofAgriopis marginaria, which fly in late winter/early spring, were attracted to (3Z,9Z)-(6S,7R)-epoxynonadecadiene. The addition of (3Z,6Z,9Z)-nonadecatriene to theS,R-enantiomer increased captures. Optimal catches were recorded with a 10∶3 epoxide-hydrocarbon blend. Enantiomer specificity in all species was confirmed in EAG measurements.

Attraction of male turnip mothsAgrotis segetum (Lepidoptera: Noctuidae) to sex pheromone components and their mixtures at 11 sites in Europe, Asia, and Africa.

Selected combinations of (Z)-5-decenyl, (Z)-7-dodecenyl, and (Z)-9-tetradecenyl acetates, the pheromone components of the turnip mothAgrotis Segetum were tested for field attractancy at six, two, and three sites in Europe, Asia, and Africa, respectively. At all of the sites in Eurasia and in northern Africa the ternary mixture of the acetates captured most males, while at the sites south of the Sahara in Africa, (Z)-5-decenyl acetate alone was responsible for attraction. Differences in male attraction among the populations studied confirm the existence of significant population variation in the pheromone ofA. segetum. Interpretation of the present results together with earlier studies suggests that this variation is more or less continuous in Eurasia and north Africa, while a clearly distinct pheromone type is present in the areas south of the Sahara desert.

Abraxas grossulariata L. (Lepidoptera: Geometridae): Identification of (3Z,6Z,9Z)-3,6,9-heptadecatriene and (6Z,9Z)-6,9-cis-3,4-epoxyheptadecadiene in the female sex pheromone.

(3Z,6Z,9Z)-3,6,9-Heptadecatriene and (6Z,9Z)-6,9-cis-3,4-epoxyheptadecadiene have been identified in female sex pheromone gland extracts ofAbraxas grossulariata L. (Lepidoptera: Geometridae). The compounds were detected by gas chromatography with electroantennographic detection (GC-EAD) and identified by GC-mass spectrometry (GC-MS). The amounts of hydrocarbon and epoxide were 0.13 and 0.42 ng/female, respectively. These identifications were confirmed by synthesis; the absolute configuration of the epoxide remains to be determined. Another isomeric epoxide, (3Z,6Z)-3,6-cis-9,10-epoxyheptadecadiene, was tentatively identified in small amounts (0.04 ng/female). Racemic (6Z,9Z)-6,9-cis-3,4-epoxyheptadecadiene elicited the largest electroantennogram response within a series of heptadecadiene and nonadecadiene monoepoxides and the corresponding trienic hydrocarbons and, in field tests, attracted maleA. grossulariata into traps. Addition of 10% (3Z,6Z,9Z)-3,6,9-heptadecatriene to this epoxide enhanced attractiveness; addition of (3Z,6Z)-3,6-cis-9,10-epoxyheptadecadiene had no effect at low proportions while higher proportions caused a reduction in the numbers of males caught.

Study of the endotoxin sensitivity of pregnant rats and their fetuses.

The endotoxin sensitivity of pregnant rats and their fetuses was studied and the following conclusions were drawn: 1. In the third trimester the fetus-damaging effect of endotoxin also involves considerable damage to the mother which often ends with the mother's death (in this experiment in 40% of the cases). 2. The endotoxin sensitivity of the fetuses of one mother is relatively the same. 3. Within the race-specific range the endotoxin sensitivity of a given population of pregnant rats largely varies according to the individual sensitivity. 4. There is no "threshold dose" which would kill each fetus and would not kill any of the mothers. Based also on literary data, it is concluded that the effect of endotoxin causing fetal death is mediated by humoral factors released from the mother's organism, and fetal death is primarily due to anoxia.

Effect of bacterial endotoxin on placentation of rats.

The effect of bacterial endotoxin on placentation in rats was studied on 160 CFY pregnant rats. Based on this experiment, it was concluded that (i) the endotoxin (1 mg/animal i.p.) inhibited placentation (in 90% of animal). (ii) The endotoxin-induced fetopathy almost exclusively resulted in abortion. (iii) The fetuses reacted to endotoxin with relatively the same degrees of susceptibility. (iv) The growth of surviving fetuses seemed to be undisturbed. (v) Endotoxin-induced damages in mothers first of all depend on the individual susceptibility of these pregnant animals and (vi) the endotoxin tolerance induced by radio-detoxified endotoxin (TOLERIN) significantly protects both the mothers and the fetuses against endotoxin challenge.

Compounds modifying male responsiveness to main female sex pheromone component of the currant borer,Synanthedon tipuliformis clerk (Lepidoptera: Sesiidae) under field conditions.

Various blends of (E,Z)-2,13-octadecadien-1-ol acetate (E,Z2,13-18: OAc), (Z)-13-octadecen-1-ol acetate (ZOAc) (two previously identified pheromone components of the currant borer,Synanthedon tipuliformis females), (E,Z)-, (Z,Z)-3,13-octadecadien-1-ol acetates (E,Z- andZ,Z3,13-18:OAc), andZ,Z-3,13-octadecadien-1-ol (Z,Z3,13-18:OH) were evaluated in field traps in three geographic regions. MaleS. tipuliformis were attracted toE,Z2,13-18:OAc in Tasmania, New Zealand, and Hungary. Captures were not influenced by the addition ofZ13-18: OAc, tested in Tasmania and Hungary. In Hungary and New Zealand, the addition ofE,Z3,13-18: OAc toE,Z2,13-18:OAc in a ratio of 100:3 was strongly synergistic; however, in Tasmania captures were reduced. The addition ofZ,Z3,13-18: OAc toE,Z2,13-18:OAc in a ratio of 10∶1 resulted in strong inhibition of trap captures in Hungary. WhenZ,Z3,13-18: OAc was added in different ratios to a 100:3 binary mixture ofE,Z2,13-18:OAc/E,Z3,13-18:OAc in Hungary, it strongly reduced captures at, or above a ratio of 100∶3∶3 but no decrease was recorded at a ratio of 100∶3∶1. In New Zealand and Tasmania it reduced captures at a ratio of 100∶3∶1. Observations of behavioral responses of maleS. tipuliformis in Hungary to synthetic baits in the field showed thatE,Z2,13-18:OAcby itself evoked close-range approaches to the source from only 20% of males, whereas the addition ofE,Z3,13-18:OAc in a ratio of 100∶3 raised that value to 65 %. Landing on the source was significant only at sources with a 100∶3∶0.1∶3∶10 blend ofE,Z2,13-18:OAc/E,Z3,13-18: OAc/Z,Z3,13-18: OAc/Z,Z3,13-18: OH/Z13-18: OAc. A 100∶3 binary mixture ofE,Z2,13-18:OAc/E,Z3,13-18:OAc in a dose range of 10-1000 µg can be recommended for more effective field monitoring ofS. tipuliformis populations in Hungary and in New Zealand. In Tasmania, at present,E,Z2,13-18: OAc by itself is the most potent sex attractant of the species.

Pheromone variation among eastern European and a western Asian population of the turnip mothAgrotis segetum.

The female sex pheromone composition and the male electro-physiological response with respect to the three main sex pheromone components, (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate, and (Z)-9-tetradecenyl acetate, were investigated in populations ofAgrotis segetum from Armenia and Bulgaria. The percentage composition of the female-produced pheromone was 1∶52∶47 and 1∶42∶57 for the respective populations. Corresponding male receptor frequencies were 9∶90∶1 and 6∶92∶2. EAG response profiles of the male antennae were similar for the two populations. The populations from Armenia and Bulgaria differed from the earlier investigated French and Swedish populations, which have larger amounts of (Z)-5-decenyl acetate in gland extracts and have a majority of (Z)-5-decenyl acetate-sensitive receptors. Investigation of receptor frequencies on antennae of male Hungarian moths showed that individuals could be classified as either Swedish or Armenian/Bulgarian type. Males of the Swedish type were preferentially attracted to the three-component pheromone blend, whereas blends of (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetate, and (Z)-7-dodecenol [pure or in mixture with (Z)-5-decenol] attracted the Armenian/Bulgarian type. The nature of pheromone variation among European and Asian populations of the turnip moth and possible mechanisms maintaining the variation are discussed.

Electrophysiological and chemical analysis of sex pheromone communication system of the mottled umber,Erannis defoliaria (Lepidoptera: Geometridae).

(Z,Z,Z)-3,6,9-nonadecatriene (Z3,Z6,Z9-19Hy) and (Z,Z)-3,9-cis-6,7-epoxy-nonadecadiene (Z3,Z9-cis-6,7-epo-19Hy) were identified in pheromone gland extracts from femaleErannis defoliaria. The two components were found in a 1∶3 ratio, with the main component,Z3,Z9-cis-6,7-epo-19Hy present at an amount of about 1.5 ng per female. The components were identified by means of gas chromatography-mass spectrometry, gas chromatography-electroantennography and gas chromatography-single sensillum recordings. Single sensillum measurements on the male antenna showed two physiological types of sensilla. One type was characterized by a large spike amplitude cell responding toZ3,Z9-cis-6,7-epoxy-19Hy and a small spike amplitude cell responding toZ3,Z6,Z9-19Hy. A second type responded only with a large spike amplitude cell to the epoxide, and this cell was inhibited by the triene. Of the two pheromone components, the epoxide gave the higher response in the EAG tests. Preliminary field tests support the identification of the pheromone components. The epoxide was also found to be present in the extract of the pheromone gland ofColotois pennaria, and males ofC. pennaria andAgriopis marginaria were trapped by the mixture of the identified compounds.

Behavioral activity of optical isomers of 5,9-dimethylheptadecane, the sex pheromone ofLeucoptera scitella L. (Lepidoptera: Lyonetidae).

Among the pure stereoisomers of 5,9-dimethylheptadecane, a previously identified sex pheromone component ofLeucoptera scitella L., only theS,S isomer yielded trap captures in the field. The addition of the other stereoisomers had no effect on cathes. The addition of low percentages of racemic 5,9-dimethylhexadecane, a previously identified minor component in the sex pheromone, did not influence trap catches or alter behavior of males approaching an attractant source in the field.

Age-associated pharmacokinetic changes of metronidazole.

Serum concentration and urinary excretion of metronidazole were determined after p.o. administration of 500 mg to 15 young (20-25 years old) and 20 elderly (over 70) subjects. Serum levels were consistently higher and the AUCs were almost doubled in the aged group. One of the effects underlying the decrease in total clearance is the diminished renal excretion; however, the role of the reduction of distribution volume seems to be more important. Red cell binding of metronidazole significantly decreased in the aged group; this may also contribute to the reduction of distribution volume. Reduction of the standard dose of metronidazole by 30-40% is recommended in elderly patients.