Defensive chemistry of an aposematic bug, Pachycoris stallii Uhler and volatile compounds of its host plant Croton californicus Muell.-Arg.

Volatile components of Pachycoris stallii scent gland secretions and the bug's host plant, Croton californicus, were identified by gas chromatography and mass spectroscopy. The predominant compounds isolated from C. californicus fruit and leaves were beta-myrcene and beta-caryophyllene. Metathoracic gland secretions of P. stallii contained mostly (E)-2-hexenal, (E)-4-oxo-2-hexenal, (E)-2-hexenyl acetate, and n-tridecane. In males, n-tridecane was present throughout the metathoracic gland, but in females this compound was found only in the median reservoir/accessory gland. (E)-2-Hexenal was present throughout the gland of female bugs, but in males was primarily present in the median reservoir/accessory gland. (E)-4-Oxo-2-hexenal and n-dodecane were isolated from the median reservoir/accessory gland of male and female bugs. Metathoracic glands were sexually monomorphic. Data from chemical analyses and anatomical observations suggest that dorsal abdominal glands of adults were apparently obsolescent. In nymphs, dorsal abdominal glands produced (E)-2-hexenal, (E )-4-oxo-2-hexenal, n-dodecane, n-tridecane, and tetradecanal. The proportion of the predominant constituent, (E)-4-oxo-2-hexenal, decreased from 72% in the first instar to 47% in the fourth instar. Proportions of tetradecanal and n-tridecane were greater in the fourth instar than in the first instar. Observations of dissected glands indicated that median and posterior dorsal abdominal glands of all nymphal instars were more developed than anterior dorsal abdominal glands. Scanning electron micrography revealed the presence of polygonal microsculpturing on the integument surrounding the ostioles of metathoracic and dorsal abdominal glands. Chemical, anatomical, and behavioral data indicated that P. stallii has a chemical defense system based on short-chain carbonyl compounds and that this system is directed against arthropods. The abundance of arthropod natural enemies apparently has forced P. stallii to maintain this defense system despite feeding on a toxic host plant.

A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis.

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This omega-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.

Biomorphological alterations induced by an anti-juvenile hormonal compound, 2-(2-ethoxyethoxy)ethyl furfuryl ether, on three species of triatominae larvae (Hemiptera, Reduviidae).

Applied topically to larvae of Rhodnius prolixus Stal, Triatoma infestans (Klug) and Panstrongylus herreri Wygodzinsky, vectors of Trypanosoma cruzi, the causative agent of Chagas' disease, a synthetic, furan-containing anti-juvenile hormonal compound, 2-(2-ethoxyethoxy)ethyl furfuryl ether induced a variety of biomorphological alterations, including precocious metamorphosis into small adultoids with adult abdominal cuticle, ocelli, as well as rudimentary adultoid wings. Some adultoids died during ecdysis and were confined within the old cuticle. The extension of these biomorphological responses is discussed in terms of the complexity of the action of anti-juvenile hormonal compounds during the development of triatomines.

Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1.

Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize alpha-pinene, limonene, of the insect growth regulators hydroprene and methoprene. The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also the 6,7-epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers. These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site.

Feeding and oviposition deterrent activities of flower buds of globemallow,Sphaeralcea emoryi torrey, against boll weevil,Anthonomus grandis Boheman (Coleoptera: Curculionidae).

The globemallow,Sphaeralcea emoryi Torrey, a plant native to Arizona was evaluated as a source of feeding or oviposition deterrents to the boll weevil,Anthonomus grandis Boheman. Feeding and oviposition responses of reproductive weevils to the flower buds and artificial diets spiked with dry powder or extracts of the globemallow buds were determined. Boll weevils were deterred from feeding and ovipositing in the flower buds unless the calyxes were removed. Male and virgin female weevils were discouraged from feeding as much as gravid weevils. Secondary chemicals in the flower buds served primarily as feeding deterrents but also prevented oviposition. The concentration of these chemicals was highest in the calyxes of the buds, and potent deterrent activity could be extracted from the calyxes with methanol. Boll weevils were able to perceive the deterrents by contact chemosensory organs on the antennae, maxillary palps and labial palps.

Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very-high-density lipoproteins.

Facilitated diacylglycerol exchange between Manduca sexta [3H]diacylglycerol-labeled high-density lipophorin and Heliothis zea very-high-density chromolipoprotein was studied. M. sexta lipid transfer particle was employed in assays which measured exchange of [3H]diacylglycerol. Following incubations with lipid transfer particle, donor and acceptor lipoproteins were reisolated by density-gradient ultracentrifugation to determine facilitated exchange. The reaction was limited to diacylglycerol exchange, while donor or acceptor particle apoprotein exchange did not occur. Lipid analysis of donor and acceptor lipoproteins after the lipid-exchange reaction revealed that the labeled diacylglycerol remained unchanged. Lipid transfer particle-catalyzed diacylglycerol exchange was linear up to 0.3 micrograms lipid transfer particle protein in a standard assay and exchange occurred at a rate of 2.5 micrograms diacylglycerol min-1.micrograms-1 lipid transfer particle protein. The assay method was used to show that the hemolymph concentration of lipid transfer particle increased during development.

Purification of very high density lipoproteins by differential density gradient ultracentrifugation.

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.

Thallphytic alleopathy: isolation and identification of laetisaric Acid.

Laetisaria arvalis, a soil-dwelling basidiomycete fungus, secretes an allelopathic agent that induces rapid hyphal lysis in several phytopathogenic fungi. The active compound was isolated from chloroform:methanol extracts of L. arvalis mycelia and characterized as a previously unknown hydroxy fatty acid, (Z,Z-9,12-8-hydroxyoctadecadienoic acid.

A larval specific lipoprotein: purification and characterization of a blue chromoprotein from Heliothis zea.

The green color of the hemolymph of last instar larvae of the corn earworm, Heliothis zea (Boddie), is caused by the appearance of a blue chromoprotein. This protein (Mr approximately 560,000) is composed of 4 subunits (Mr approximately 150,000). It contains 8.4% lipid and has an equilibrium density of 1.26 g/ml. This protein is absent during all early larval stages, in pupae and in adult animals, but is a major hemolymph protein in 5th instar larvae. Although its physiological function remains unclear, this blue chromoprotein represents the first member of a new class of larval-specific insect lipoproteins.

Insect-plant interactions: endocrine defences.

It is the inevitable consequence of evolution that competitive species living together in a restricted space must try to exclude each other. Plants and insects are prime examples of this eternal competition, and although neither of these is in danger of extinction, their mutual defensive strategies are of compelling interest to the human race. Plant defences based on the insecticidal activity of certain of their secondary chemicals are readily apparent. Only through research into the fundamentals of insect physiology and biochemistry are more subtle defensive mechanisms revealed, linked to the disruption of the insect endocrine system. A diverse number of chemical structures are found in plants, which interfere with hormone-mediated processes in insects. Examples include: mimics of the insect's juvenile hormones such as juvabione from the balsam fir and the juvocimenes from sweet basil, which lethally disrupt insect development, and the precocenes found in Ageratum species, which act as anti-juvenile hormonal agents. The latter appear to serve as 'suicide substrates', undergoing activation into cytotoxins when acted on by specialized enzymes resident in the insect endocrine gland (corpus allatum) that is responsible for juvenile hormone biosynthesis and secretion. Consideration of these plant defensive strategies, which have been reached through aeons of evolutionary experimentation, may assist the human race in its defences against its principal competitors for food, fibre and health.

Synthesis of highly active juvenile hormone analogs, juvocimene I and II, from the oil of sweet basil,Ocimum basilicum L.

Juvocimene I and II are potent juvenile hormone mimics isolated from the essential oil of sweet basil,Ocimum basilicum L. The structures given by the formula I and II have been confirmed by synthesis withtrans-ß-ocimene andp-methoxycinnamyl chloride. Biological activity of the natural and synthetic juvocimenes was found to be identical.

Natural and synthetic allatotoxins: suicide substrates for juvenile hormone biosynthesis.

Cytotoxic agents with antijuvenile hormone activity in insects have been discovered. Their mechanism of action may involve an oxidative bioactivation into a reactive quinone methide.

The effects of precocene II on reproduction and development of triatomine bugs (Reduviidae: Triatominae).

Precocene II is a botanically derived chemical that inhibits the production of juvenile hormone (JH) in insects. The effects of this anti-JH compound on molting and growth by Rhodnius prolixus and Triatoma dimidiata and reproduction in R. prolixus were tested and the efficacy of precocene II as a fumigant was assessed. Precocene II induced precocious metamorphosis in both species when applied by either contact exposure or fumigation, and this effect could be prevented by juvenile hormone replacement therapy. The dosage effective in inducing precocious metamorphosis in T. dimidiata was similar to the EC50 previously reported for R. prolixus. The morphology of precocious adultoid T. dimidiata of different instars was similar to Rhodnius adultoids of corresponding instars. However, T. dimidiata was more sensitive than Rhodnius to the molt-inhibiting effects of precocene II. Rhodnius nymphs were fully susceptible to the anti-JH action of precocene when exposed for 24 hours one week before feeding. Precocene II was highly toxic to adult female Rhodnius and treatment of newly emerged females prevented oogenesis.

Juvocimenes: potent juvenile hormone mimics from sweet basil.

Two compounds with highly potent juvenile hormone activity were isolated and identified from the oil of sweet basil, Ocimum basilicum L., Labiatae.

Insect juvenile hormones and pheromones of isopentenoid biogenesis.

In their diversity, speciation, and sheer numerical superiority, few should question that insects are the dominant life-form on earth. Their utilization of the multifunctional isopentenoids to regulate their life processes is equally diverse. To catalog or even summarize the contribution of isopentenoids in the regulatory chemistry of insect feeding, development, reproduction, diaproduction, diapause, and behavior is beyond the scope of this review. However, a topical treatment of the chemistry of insect juvenile hormones and pheromones provides an insight into the dependence of insects upon isopentenoids.

Antiallatotropins: inhibition of corpus allatum development.

Treatment of newly eclosed adult milkweed bug (Oncopeltus fasciatus) females with precocene 2 prevents secretion of juvenile hormone by inhibition of postimaginal development of the corpus allatum. Ovarian development which is dependent upon juvenile hormone is prevented or reversed, depending upon the timing of precocene treatment. Juvenile hormone secretion is shown to be related to the development of the corpus allatum.