Palm Weevil Pheromones - Discovery and Use.

Male-produced aggregation pheromones of seven major pest species of weevils in the subfamily Rhynchophorinae have been identified as a closely related set of methyl-branched secondary alcohols. Although the weevils produce only one stereoisomer of these alcohols, no instances of isomeric inhibition have been observed, enabling stereoisomeric mixtures to be used in traps. Addition of fermenting plant material to traps synergizes attraction of weevils to the pheromones. The weevils are large, have long life cycles, and are strong fliers. These characteristics make mass trapping a suitable tactic to add to existing management strategies. When coupled with good phytosanitary practices, mass trapping of Rhynchophorus palmarum at 1 trap/5-ha significantly lowered the incidence of red ring nematode infection vectored by the weevil in commercial oil palm plantations in the Americas. Similarly, trap densities of 1-10 traps/ha have significantly lowered R. ferrugineus infestation of date palm throughout the Middle East. Although management of R. ferrugineus in urban areas is more problematic, trapping is an integral part of most programs aimed at protection of ornamental Canary palms in Europe. Overall, semiochemically-based management of these large weevils is now a mature and usually economically feasible control technology.

Management of Cosmopolites sordidus and Metamasius hemipterus in banana by pheromone-based mass trapping.

Mass trapping Cosmopolites sordidus (Coleoptera, Curculionidae) using a pheromone-baited pitfall trap and Metamasius hemipterus (Coleoptera, Curculionidae) using a pheromone-sugarcane-baited open gallon trap was conducted in commercial banana. Four traps for each insect per hectare were placed in each of two 5-hectare plots of banana. Two additional 5-hectare plots were designated as controls and treated according to the plantation protocol. Capture rates of C. sordidus and M. hemipterus declined by >75 % over 10-12 months. In the banana growing region studied, corm damage was due primarily to C. sordidus, while only a minor amount of damage was attributable to M. hemipterus. Corm damage reduction in trapping plots was, thus, attributed primarily to C. sordidus trapping. In trapping plots, corm damage decreased by 61-64 % during the experiment. Banana bunch weights increased 23 % relative to control plots after 11-12 months of trapping. Fruit diameter did not vary between bunches harvested from trapping plots vs. control plots. Plant vigor, however, as determined by stem circumference at one meter above ground increased in plots with traps compared to control plots. Trapping for C. sordidus in two plantations of over 200 hectares each, reduced corm damage 62-86 % relative to pre-trapping levels. Insecticide control measures in place when the experiment commenced resulted in about 20-30 % corm damage, while use of pheromone trapping to manage C. sordidus lowered corm damage to 10 % or less. It is estimated that the increase in value of increased yield obtained in this trial (23 %) is about $4,240 USD per year per hectare, while the cost of pheromone trapping is approximately $185 USD per year per hectare. The trapping program becomes revenue neutral if bunch weights increase by an average of 1 % per year of trapping. Approximately 10 % of all plantation area in Costa Rica use the pheromone trapping system described here. The system also is used in Martinique, Guadeloupe, and the Canary Islands.

Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.

Synthesis and inhibition studies of sulfur-substituted squalene oxide analogues as mechanism-based inhibitors of 2,3-oxidosqualene-lanosterol cyclase.

The synthesis and biological evaluation of three new sulfur-substituted oxidosqualene (OS) analogues (1-3) are presented. In these analogues, C-11, C-15, or C-18 in the OS skeleton was replaced by sulfur. The sulfur position in the OS skeleton was chosen to disrupt one or more key processes involved in cyclization: (a) the folding of the B-ring into a boat conformation, (b) the anti-Markovnikov cyclization leading to the C-ring, or (c) the formation of the D-ring during the lanosterol biosynthesis. Enzyme inhibition kinetics using homogeneous mammalian oxidosqualene cyclases (OSC) were also examined for the previously reported S-19 analogue 4. The four analogues were potent inhibitors of mammalian OSCs (IC50 = 0.05-2.3 microM for pig and rat liver OSC) and fungal cell-free Candida albicans OSC (submicromolar IC50 values). In particular, the S-18 analogue 3 showed the most potent inhibition toward the rat liver enzyme (IC50 = 50 nM) and showed potent, selective inhibition against the fungal enzyme (IC50 = 0.22 nM, 10-fold more potent than the S-19 analogue 4). Thus, 3 is the most potent OSC inhibitor known to date. The Ki values ranged from 0.5 to 4.5 microM for pig OSC, with 3 and 4 showing about 10-fold higher potency for rat liver OSC. Interestingly, the S-18 analogue 3 showed time-dependent irreversible inhibition with homogeneous pig liver OSC (kinact = 0.06 min-1) but not with rat OSC.

Identification of ergosta-8,24(28)-dien-3 beta,6 alpha-diol in A delta 8 goes to delta 7 sterol isomerase-blocked yeast mutant.

In addition to the monohydroxysterols found in the delta 8 goes to delta 7 isomerase-blocked Saccharomyces cerevisiae mutant erg 2, a noval dihydroxysterol, ergosta-8,24(38)-dien-3 beta,6 alpha-diol, was isolated. This sterol accumulated to the extent of 2.1% of the total sterol fraction when this mutant was treated with 23-azacholesterol, a known inhibitor of the 24-methylene-sterol-24(28)-reductase.

Sterol composition ofNeurospora crassa.

The sterols extracted from freeze-dried, log-phase cultures ofNeurospora crassa were separated by thin layer and analyzed by capillary gas liquid chromatography and mass spectrometry. Ergosterol, episterol, and ergosta-7,22 dienol were the major sterols of the free sterol fraction. The major esterified sterols, which constituted 4.95% of the total sterol fraction, were ergosterol, episterol, and ergosta- 7,22,24(28)-trienol, with lesser quantities of 4α-methyl-ergosta-8,24(28)-dienol. With the exception of lanosterol, all sterols were alkylated at the C-24 position.

Accumulation of ergosta-8,14-dien-3beta-ol by Saccharomyces cerevisiae cultured with an azasterol antimycotic agent.

15-Aza-25-methylene-D-homocholesta-8,14-dien-3beta-ol, an antimycotic agent, at a concentration of 75 ng/ml inhibited ergosterol biosynthetis in Saccharomyces cerevisiae strain 3701B resulting in the accumulation of an unusual sterol. Experimental data presented indicate that this sterol is ergosta-8,14-dien-3beta-ol. The accumulation of the compound is supportive of current models of biosynthetic pathways for sterols in yeast and is consistent with inhibition by the azasterol of the delta14 sterol reductase.

Optimization of a Trap for Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) and Trials to Determine the Effectiveness of Mass Trapping.

Management of the South American tomato leafminer, Tuta absoluta Meyrick, with insecticides has led to the widespread development of insect resistance. Mass trapping using traps baited with the female-produced sex pheromone is an attractive alternative for the management of this pest. The current study evaluated several commercial trap designs for capture of T. absoluta. Based on its small size and ease of handling, the most effective trap is a small plastic container with entry windows cut on the sides filled with motor oil over water. These traps are most effective when placed near ground level. Tests of septa containing 0.1 or 0.2 mg of the pheromone (95:5) E4, Z8-14Ac/E4,Z8,Z11-14Ac were slightly more attractive than septa loaded with 0.5, 1.0, or 2 mg during the first week of use, but the latter three loadings were slightly more attractive than the first two loadings after 9 weeks. Ideal trap baits were loaded with 0.5 mg of pheromone. Higher numbers of T. absoluta were captured near upwind borders of tomato fields suggesting that treatments against T. absoluta should be concentrated near upwind parts of fields. Comparisons of conventional insecticide treatment versus mass trapping to manage T. absoluta damage in three different test sites showed that even when initial captures in monitoring traps were high (>35 males trap(-1) day(-1)), mass trapping at 48 traps/ha reduced leaf damage more efficiently than conventional insecticide treatment. Based on the typical insecticide recommendations against T. absoluta, mass trapping is an economically viable alternative.

Nitroxide spin-probe study of amphotericin B-ergosterol interaction in egg phosphatidylcholine multilayers.

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B : ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity. Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (approximately 15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.

Investigation of polyene macrolide antibiotic-induced permeability changes in vesicles by 31P nuclear magnetic resonance.

The permeability of egg yolk lecithin (EYL) vesicles to Pr3+ has been measured by 31P nuclear magnetic resonance (nmr) spectroscopy. Measurable Pr3+ leakage into the internal aqueous compartment of EYL vesicles at ambient (21 degrees C) temperature required the presence of small (7--10 mol%) amounts of dicetyl phosphate (DCP). The permeability of DCP-containing vesicles is decreased by incorporation of sterol (cholesterol greater than ergosterol approximately 5.6-dihydroergosterol greater than zymosterol) into the lipid bilayer. Addition of the polyene macrolide antibiotic, nystatin, to DCP-containing EYL vesicles with and without sterol resulted in increased Pr3+ permeability at the three temperatures studied (21--37.5 degrees C). Permeability changes observed upon addition of nystatin to sterol-impregnated, DCP-containing vesicles varied with sterol structure: ergosterol approximately 5,6-dihydroergosterol greater than cholesterol approximately zymosterol. These results are compared with other polyene macrolide induced permeability changes on model and natural membrane systems. Permeability changes induced by nystatin in sterol-free EYL vesicles were generally greater than for comparable sterol-containing vesicles. This is attributed to a nonspecific interaction of the antibiotic with the latter vesicles.

Suppression of oviposition inOryzaephilus surinamensis (L.) (Coleoptera: Cucujidae) following prolonged retention in high-density cultures or short-term exposure to larval volatiles.

After 20 days in a high-density culture containing many larvae, female sawtoothed grain beetles,Oryzaephilus surinamensis (L.), laid half as many eggs in a 24-hr oviposition bioassay as females held for six days in the same culture, or for six or 20 days in a low-density culture. Oviposition by females held for six days in a high-density culture was reduced to a similar extent when they were exposed in the oviposition bioassay to an oat flake treated with an extract of Porapak Q-captured larval volatiles (equivalent to 5000 larval hours). A retained suppression of oviposition rate after prolonged exposure to larvae or an induced reduction caused by short-term exposure to larval volatiles both could be of adaptive advantage in reducing the risk of oviposition in an already densely populated habitat.

1-Methylcyclohex-2-en-1-ol as an aggregation pheromone ofDendroctonus pseudotsugae.

1-Methylcyclohex-2-en-1-ol (1,2-MCH-ol) was synthesized, identified as a compound found in volatiles of the female Douglas-fir beetle, and shown by bioassays to be an aggregation pheromone. 1,2-MCH-ol matches in both GC retention index and mass spectrum a compound released by the female after feeding. 3,3-MCH-ol was also synthesized as a candidate compound; its mass spectrum is presented because published mass spectra are incorrect for this compound. Synthetic 1,2-MCH-ol increased arrestment and stridulation of males in olfactory walkways and increased trap catches of flying beetles. Males were more responsive to 1,2-MCH-ol than females.

The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs.

The effect of low concentrations of a specifically designed sterol-24-transmethylase inhibitor, 25-aza-24, 25-dihydrozymosterol (10) on sterol production in Saccharomyces cerevisiae was examined. The synthesis of cholesta-5,7,22,24-tetraen-3beta-ol (4), its 7,22,24 analog (15) and the 7,24 analog (5) coupled with the availability of zymosterol (6) and cholesta-5,7,24-3beta-ol (3) derivatives facilitated a search for these sterols in cultures treated with this inhibitor. When S. cerevisiae was grown in the presence of 1.3 and 5 muM 10, it produced no ergosterol but accumulated zymosterol (6), cholesta-5,7,22,24-tetraen-3beta-ol (4) and related C27 sterols (3 and 5). These results indicate blockage of the side chain methylation that normally occurs during the biosynthesis of ergosterol in yeast by compound 10 is efficient. The cholesta-5,7,22,24-tetraen-3beta-ol is a close structural analog of provitamin D3 (7-dehydrocholesterol). The inhibited yeast thus provides a source of a potentially new provitamin D3 substitute.

Monoterpene metabolism in female mountain pine beetles,Dendroctonus ponderosae Hopkins, attacking ponderosa pine.

Abdominal volatiles of female mountain pine beetles,Dendroctonus ponderosae Hopkins, fed in ponderosa pine,Pinus ponderosa Dougl. ex Laws, and in lodgepole pine,P. contorta var.latifolia Engelmann, were analyzed by gas chromatography and coupled gas chromatography-mass spectrometry and were found to comprise host oleoresin components and beetle-produced alliylic alcohols, aldehydes, and ketones derived from host monoterpenes. Neitherexo- andendo-brevicomin nor frontalin were detected. Three metabolic pathways are proposed to account for the distribution of beetle-produced monoterpene alcohols. The first pathway involves hydroxylation of monoterpene substrates on allylic methyl groups which areE to a methylene or vinyl group. This oxidation pathway is indiscriminate with respect to substrate and probably functions to detoxify monoterpenes. A second pathway, which hydroxylates theendo-cyclic methyleneE to a vinyl methyl group of bicyclic monoterpenes to give almost exclusively thetrans alcohol, is hypothesized to be involved in pheromone production. A third detoxification pathway involves anti-Markovnikov addition of water to theexo-cyclic double bond of ß-phellandrene to give predominantlytrans-2-p-menthen-7-ol.

Influence of pheromone chirality on response byOryzaephilus surinamensis andOryzaephilus mercator (Coleoptera: Cucujidae).

The response of the sawtoothed grain beetle,Oryzaephilus surinamensis (L.), and the merchant grain beetle,O. mercator (Fauvel), to synthetic racemic and chiral macrolide aggregation pheromones was assessed in pitfall olfactometers.O.mercator utilizes theR enantiomers of (Z)-3-dodecen-11-olide and (Z,Z)-3,6-dodecadien-11-olide.O. surinamensis utilizes theR enantiomers of (Z,Z)-3,6-dodecadien-11-olide and the Synergist (Z,Z)-5,8-tetradecadien-13-olide in combination with achiral (Z,Z)-3,6-dodecadienolide. For both species, the racemates of the respective chiral pheromones were effective attractants. The respectiveS enantiomers were inactive for both species and had no effect on the biological activity of the active antipodes. No diel periodicity in responsiveness to pheromones was detected inOryzaephilus spp. reared either on a 12∶12 light-dark photoperiod or in darkness. Nonpheromone macrolides, naturally released in trace amounts byOryzaephilus spp., did not affect the aggregation response of either species to its pheromones when these additional macrolides were combined with the pheromone mixtures.

Chemical communication in cucujid grain beetles.

Males of five sympatric species of economically damaging cucujid grain beetles,Cryptolestes ferrugineus (Stephens),C. pusillus (Schönhen),C. turcicus (Grouvelle),Oryzaephilus mercator (Fauvel), andO. surinamensis (L.), produce macrolide aggregation pheromones especially in the presence of food. Work leading to the isolation, identification, and establishment of biological activity of these semiochemicals is reviewed. The trivial name "cucujolide" is proposed and used to identify these compounds that are characteristic of the Cucujidae. The twoOryzaephilus share species share a common cucujolide pheromone, whileCryptolestes species use cucujolides that are either enantiomeric, unique to the genus, or released in trace quantities byOryzaephilus spp. and not used as pheromones by the latter species. The major mechanisms for species specificity in chemical communication are: (1) presence of a unique pheromone (C. ferrugineus andC. pusillus); (2) use of pheromones that are inactive alone but synergize response to cucujolides unique to a species (C. pusillus, C. turcicus, andO. surinamensis); (3) response to only one enantiomer of a pheromone (C. ferrugineus, O. surinamensis, andO. mercator); and (4) synergism between enantiomers of a pheromone (C. turcicus). The only species for which cross-attraction was evident wasO. mercator toO. surinamensis. Both sexes ofOryzaephilus spp. produce (R)-1-octen-3-ol, which highly synergizes response to the cucujolide pheromones. Similar synergism occurs between hexanal, octanal, and nonanal and the cucujolide pheromones ofOryzaephilus spp. The males of a sixth cucujid species,Cathartus quadricollis (Guér) produce a different aggregation pheromone, (3R,6E)-7-methyl-6-nonen-3-yl acetate. Trapping ofCryptolestes andOryzaephilus spp. in cardboard traps baited with pheromones is efficient in environments mimicking food-storage areas. Pheromone-baited plastic probe traps are the most efficient at capturing these species in infested grain.

Azasterol inhibition of delta 24-sterol methyltransferase in Saccharomyces cerevisiae.

The inhibition of the delta 24-sterol methyltransferase (24-SMT) of Saccharomyces cerevisiae by side-chain azasterols is related to their nuclear skeleton and side chain nitrogen position. Inhibitory power [I50 (microM)] was found to be in the order of 25-azacholesterol hydrochloride salt (0.05) greater than 25-aza-24,25-dihydrozymosterol (0.08) greater than 25-azacholesterol approximately equal to 25-azacholestanol (0.14) greater than (20R)- and (20S)-22,25-diazacholesterol (0.18) greater than 24-azacholesterol (0.22) greater than 25-aza-24,25-dihydrolanosterol (1.14) greater than 23-azacholesterol (4.8). In the presence of azasterols, S. cerevisiae produces increased amounts of zymosterol, decreased amounts of ergosterol and ergostatetraenol, and the new metabolites cholesta-7,24-dienol, cholesta-5,7,24-trienol, and cholesta-5,7,22,24-tetraenol. Kinetic inhibition studies with partially purified 24-SMT and several azasterols suggest the azasterols act uncompetitively with respect to zymosterol and are competitive inhibitors with respect to S-adenosyl-L-methionine (SAM). These results are consistent with at least two kinetic mechanisms. One excludes competition of azasterol and zymosterol for the same site, whereas a second could involve a ping-pong mechanism in which 24-SMT is methylated by SAM and the methylated enzyme reacts with sterol substrate.

Pheromone chirality of african palm weevil,Rhynchophorus phoenicis (F.) and palmetto weevil,Rhynchophorus cruentatus (F.) (Coleoptera: Curculionidae).

There are four stereoisomers of both 3-methyl-octan-4-ol, the aggregation pheromone of the African palm weevil,Rhynchophorus phoenicis (F.) and 5-methyl-octan-4-ol, the aggregation pheromone of the palmetto weevil,Rhynchophorus cruentatus (F.). Synthetic stereoisomers of 3-methyl-octan-4-ol and 5-methyl-octan-4-ol were baseline-separated on a Cyclodex-B fused silica column. Use of this column in gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometric (GC-MS) analyses revealed that only one stereoisomer, (3S,4S)-3-methyl-octan-4-ol and (4S,5S)-5-methyl-octan-4-ol, is produced by maleR. phoenicis and maleR. cruentatus, respectively, and elicits good antennal responses by conspecific male and female weevils. In field trapping experiments, withR. phoenicis in Côte d'Ivoire andR. cruentatus in Florida, (3S,4S)-3-methyl-octan-4-ol and (4S,5S)-5-methyl-octan-4-ol strongly enhanced attraction of fresh palm tissue, whereas other stereoisomers were behaviorally benign. Stereoisomeric 3-methyl-octan-4-ol and 5-methyl-octan-4-ol may be utilized to monitor and/or manage populations of these two palm weevils.

Isolation of apigeninidin from leaf sheaths ofSorghum caudatum.

A stable 3-deoxyanthocyanidin, apigeninidin chloride, a potential fungal growth inhibitor and a useful dye, has been isolated with a high yield (10% in dried material) as the major pigment in the sheaths ofSorghum caudatum.