Selective Enrichment of Conjugated Linoleic Acid Isomers in Their Mixtures Using Combined Chemical and Enzymatic Methods

The aim of this study was to selectively enrich t10,c12-conjugated linoleic acid (t10,c12-CLA) and c9,t11-CLA in commercial CLA mixtures using a combination of urea crystallization and lipase-catalyzed esterification. The objective of the urea fractionation is to remove saturated and monounsaturated fatty acids (FA) from the CLA mixtures. CLA-enriched free FA (FFA) mixtures containing 53.8 wt% t10,c12-CLA and 39.1 wt% c9,t11-CLA were produced from the CLA mixtures containing ~34 wt% each of the two CLA isomers by a urea crystallization using methanol and the urea-to-FA weight ratio of 2.5:1. The CLA-enriched FFA mixtures were partially esterified with dodecan-1-ol in a recirculating packed-bed reactor using an immobilized lipase from Candida rugosa to further enrich the t10,c12-CLA and c9,t11-CLA in an FFA fraction and an FA dodecyl ester fraction, respectively, under the optimal conditions, i.e., temperature, 20 °C; FA-to-dodecan-1-ol molar ratio, 1:1; water content, 2 wt% of total substrates; residence time, 5 min; and reaction time, 24 h (for t10,c12-CLA enrichment) and 12 h (for c9,t11-CLA enrichment). After the reaction, an FFA fraction with 72.6 wt% t10,c12-CLA was obtained. Another FFA fraction with 62.0 wt% c9,t11-CLA was recovered after the saponification of the FA dodecyl ester fraction. The yields of t10,c12-CLA and c9,t11-CLA in the FFA fractions were 43.6 and 21.5 wt%, respectively, based on their initial weights in the CLA mixtures.     

Oxidizable Phenolic Concentrations Do Not Affect Development and Survival of <Emphasis Type="Italic">Paropsis Atomaria</Emphasis> Larvae Eating <Emphasis Type="Italic">Eucalyptus</Emphasis> Foliage

Insect folivores can cause extensive damage to plants. However, different plant species, and even individuals within species, can differ in their susceptibility to insect attack. Polyphenols that readily oxidize have recently gained attention as potential defenses against insect folivores. We tested the hypothesis that variation in oxidizable phenolic concentrations in Eucalyptus foliage influences feeding and survival of Paropsis atomaria (Eucalyptus leaf beetle) larvae. First we demonstrated that oxidizable phenolic concentrations vary both within and between Eucalyptus species, ranging from 0 to 61 mg.g^?1 DM (0 to 81% of total phenolics), in 175 samples representing 13 Eucalyptus species. Foliage from six individuals from each of ten species of Eucalyptus were then offered to batches of newly hatched P. atomaria larvae, and feeding, instar progression and mortality of the first and second instar larvae were recorded. Although feeding and survival parameters differed dramatically between individual plants, they were not influenced by the oxidizable phenolic concentration of leaves, suggesting that P. atomaria larvae may have effective mechanisms to deal with oxidizable phenolics. Larvae feeding on plants with higher nitrogen (N) concentrations had higher survival rates and reached third instar earlier, but N concentrations did not explain most of the variation in feeding and survival. The cause of variation in eucalypt herbivory by P. atomaria larvae is therefore still unknown, although oxidizable phenolics could potentially defend eucalypt foliage against other insect herbivores.     

Dietary fatty acids influence the growth and fatty acid composition of the yellow mealworm <Emphasis Type="Italic">Tenebrio molitor</Emphasis> (Coleoptera: Tenebrionidae)

Fat is the second most abundant component of the nutrient composition of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) that represents also an interesting source of PUFA, especially n-6 and n-3 fatty acids, involved in prevention of cardiovascular diseases. This study investigated the possibility of modifying the fat content and the FA composition of yellow mealworms through feeding and how this would be influenced by developmental stages, pupal sex, and generation with the future aim of applying this coleopteran as a diet supplement for human health. Growth rate and cumulative mortality percentage on the different feeding substrates were also evaluated to select the optimal conditions for a mass-raising of this insect species. Despite the different fat content in the six different breeding substrates used, T. molitor larvae and pupae contained a constant fat percentage (>34% in larvae and >30% in pupae). A similar total fat content was found comparing larvae and male and female pupae of the second generation to those of the first generation. On the contrary, FA composition differed both in larvae and pupae reared on the different feeding substrates. However, the exemplars reared on the diets based on 100% bread and 100% oat flour showed SFA, PUFA percentages, and an n-6/n-3 ratio more suitable for human consumption; the diet based on beer yeast, wheat flour, and oat flour resulted in a contemporary diet that most satisfied the balance between a fat composition of high quality and favorable growth conditions.     

Stingless Bees: Chemical Differences and Potential Functions in <Emphasis Type="Italic">Nannotrigona testaceicornis</Emphasis> and <Emphasis Type="Italic">Plebeia droryana</Emphasis> Males and Workers

Cuticular wax, abdominal and cephalic extracts of foraging workers and males of Nannotrigona testaceicornis and Plebeia droryana, from the “Aretuzina” farm in São Simão, SP, Brazil, were analyzed by GC-MS. The principal constituents were hydrocarbons, terpenes, aldehydes, esters, steroids, alcohols, and fatty acids. Interspecific differences for both cuticular wax and cephalic extracts were found. The composition of cuticular wax and cephalic extracts was similar at the intraspecific level, with minor component differences between males and workers. Abdominal extracts differentiated sexes (male and worker) at the intraspecific and interspecific levels. The main chemical components in abdominal extracts of N. testaceicornis workers and males were geranylgeranyl acetate and (Z)-9-nonacosene, respectively. The principal components of abdominal extracts from P. droryana workers and males were tetradecanal and unsaturated fatty acids (linoleic and linolenic acids), respectively. A secondary alcohol, (S)-2-nonanol, was detected in Plebeia droryana males only, but not in workers. Preliminary field experiments showed that (S)-(+)-2-heptanol and (S)-(+)-2-heptanol/ (S)-(+)-2-nonanol (1:1) attracted workers of P. droryana, N. testaceicornis, and Frieseomelitta silvestrii. However, males did not respond suggesting that these compounds do not function as alarm or recruitment pheromones . In addition, racemic mixtures were inactive.     

Volatiles of <Emphasis Type="Italic">Solena amplexicaulis</Emphasis> (Lam.) Gandhi Leaves Influencing Attraction of Two Generalist Insect Herbivores

Epilachna vigintioctopunctata Fabr. (Coleoptera: Coccinellidae) and Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae) are important pests of Solena amplexicaulis (Lam.) Gandhi (Cucurbitaceae), commonly known as creeping cucumber. The profiles of volatile organic compounds from undamaged plants, plants after 48 hr continuous feeding of adult females of either E. vigintioctopunctata or A. foveicollis, by adults of both species, and after mechanical damaging were identified and quantified by GC-MS and GC-FID analyses. Thirty two compounds were detected in volatiles of all treatments. In all plants, methyl jasmonate was the major compound. In Y-shaped glass tube olfactometer bioassays under laboratory conditions, both insect species showed a significant preference for complete volatile blends from insect damaged plants, compared to those of undamaged plants. Neither E. vigintioctopunctata nor A. foveicollis showed any preference for volatiles released by heterospecifically damaged plants vs. conspecifically damaged plants or plants attacked by both species. Epilachna vigintioctopunctata and A. foveicollis showed attraction to three different synthetic compounds, linalool oxide, nonanal, and E-2-nonenal in proportions present in volatiles of insect damaged plants. Both species were attracted by a synthetic blend of 1.64 μg linalool oxide?+?3.86 μg nonanal?+?2.23 μg E-2-nonenal, dissolved in 20 μl methylene chloride. This combination might be used as trapping tools in pest management strategies.     

Cuticular Hydrocarbon Cues Are Used for Host Acceptance by <Emphasis Type="Italic">Pseudacteon</Emphasis> spp. Phorid Flies that Attack <Emphasis Type="Italic">Azteca sericeasur</Emphasis> Ants

Parasitoids often use complex cues to identify suitable hosts in their environment. Phorid fly parasitoids that develop on one or a few host species often use multiple cues, ranging from general to highly specific, to home in on an appropriate host. Here, we describe the hierarchy of cues that Pseudacteon phorid flies use to identify Azteca ant hosts. We show, through behavioral observations in the field, that phorid flies are attracted to two cryptic Azteca species, but only attack Azteca sericeasur (Hymenoptera: Formicidae: Dolichoderinae). To test whether the phorid flies use cuticular hydrocarbons (CHCs) to distinguish between the two Azteca taxa, we first documented and compared cuticular hydrocarbons of the two Azteca taxa using gas chromatography/mass spectrometry. Then, using cuticular hydrocarbon-transfer experiments with live ants, we characterized the cuticular hydrocarbons of A. sericeasur as a short-range, host location cue used by P. lasciniosus (Diptera: Phoridae) to locate the ants.     

Beyond Cuticular Hydrocarbons: Chemically Mediated Mate Recognition in the Subsocial Burying Beetle <Emphasis Type="Italic">Nicrophorus vespilloides</Emphasis>

Burying beetles have fascinated scientists for centuries due to their elaborate form of biparental care that includes the burial and defense of a vertebrate carcass, as well as the subsequent feeding of the larvae. However, besides extensive research on burying beetles, one fundamental question has yet to be answered: what cues do males use to discriminate between the sexes? Here, we show in the burying beetle Nicrophorus vespilloides that cuticular lipids trigger male mating behavior. Previous chemical analyses have revealed sex differences in cuticular hydrocarbon (CHC) composition; however, in the current study, fractionated-guided bioassay showed that cuticular lipids, other than CHCs, elicit copulation. Chemical analyses of the behaviorally active fraction revealed 17 compounds, mainly aldehydes and fatty acid esters, with small quantitative but no qualitative differences between the sexes. Supplementation of males with hexadecanal, the compound contributing most to the statistical separation of the chemical profiles of males and females, did not trigger copulation attempts by males. Therefore, a possible explanation is that the whole profile of polar lipids mediates sex recognition in N. vespilloides.     

Differential Response of a Local Population of Entomopathogenic Nematodes to Non-Native Herbivore Induced Plant Volatiles (HIPV) in the Laboratory and Field

Recent work has shown the potential for enhanced efficacy of entomopathogenic nematodes (EPN) through their attraction to herbivore induced plant volatiles. However, there has been little investigation into the utilization of these attractants in systems other than in those in which they were identified. We compared (E)-β-caryophyllene and pregeijerene in the highbush blueberry (Vaccinium corymbosum) agroecosystem in their ability to enhance the attraction of EPN to and efficacy against the system’s herbivore, oriental beetle (Anomala orientalis). The relative attractiveness of (E)-β-caryophyllene and pregeijerene to a local isolate of the EPN species Steinernema glaseri was tested in a six-arm olfactometer in the laboratory to gather baseline values of attraction to the chemicals alone in sand substrate before field tests. A similar arrangement was used in a V. corymbosum field by placing six cages with assigned treatments and insect larvae with and without compound into the soil around the base of 10 plants. The cages were removed after 72 h, and insect baits were retrieved and assessed for EPN infection. The lab results indicate that in sand alone (E)-β-caryophyllene is significantly more attractive than pregeijerene to the local S. glaseri isolate Conversely, there was no difference in attractiveness in the field study, but rather, native S. glaseri were more attracted to cages with G. mellonella larvae, no larvae, and cages with the blank control and G. mellonella larvae.     

<Emphasis Type="Italic">n</Emphasis>-Pentacosane Acts as both Contact and Volatile Pheromone in the tea Weevil, <Emphasis Type="Italic">Myllocerinus aurolineatus</Emphasis>

Insect cuticular hydrocarbons (CHCs) play important roles in chemical communication, as well as having ecological and physiological roles. The use of CHCs for mate recognition has been shown in many insect genera, but little is known about their use in the tea weevil Myllocerinus aurolineatus. Here, we provide evidence that CHCs on the surface of sexually mature M. aurolineatus females act as contact sex pheromones, facilitating mate recognition and eliciting copulatory behavior in male weevils. Using gas chromatography-mass spectrometry, we identify n-pentacosane and n-heptacosane as two potential contact pheromone components. Results from arena bioassays showed that n-pentacosane is a component of a contact pheromone of M. aurolineatus. Further results from the Y-tube olfactometer bioassays showed that n-pentacosane also acts as a volatile attractant. Our results greatly improve our understanding of the chemical ecology of M. aurolineatus.     

Feminization of Male Brown Treesnake Methyl Ketone Expression via Steroid Hormone Manipulation

Pheromones are useful tools for the management of invasive invertebrates, but have proven less successful in field applications for invasive vertebrates. The brown treesnake, Boiga irregularis, is an invasive predator that has fundamentally altered the ecology of Guam. The development of control tools to manage Boiga remains ongoing. Skin-based, lipophilic pheromone components facilitate mating in brown treesnakes, with females producing the same long-chain, saturated and monounsaturated (ketomonoene) methyl ketones known to function as pheromones in garter snakes, Thamnophis sirtalis. Boiga also express novel, diunsaturated methyl ketones (ketodienes) with a purported function as a sex pheromone. In our study, we implanted 17 β-estradiol in adult male brown treesnakes in order to manipulate methyl ketone expression as sex attractants, an effect that would mirror findings with garter snakes. Specifically, estrogen promoted production of two ketomonoenes, pentatriaconten-2-one and hexatriaconten-2-one, and suppressed production of one ketodiene, heptatriacontadien-2-one. In bioassays, estrogen-implanted males elicited tongue-flicking and chin rubbing behavior from unmanipulated males, though the responses were weaker than those elicited by females. On Guam, wild males exhibited greatest responses to whole female skin lipid extracts and only weak responses to the methyl ketone fractions from females and implanted males. Our results suggest that sex identity in brown treesnakes may be conferred by the ratio of ketomonoenes (female) to ketodienes (male) from skin lipids and may be augmented by a sex-specific endocrine signal (estradiol). However, a blend of long-chain methyl ketones alone is not sufficient to elicit maximal reproductive behaviors in male Boiga.     

<Emphasis Type="Italic">cis</Emphasis>-, <Emphasis Type="Italic">trans</Emphasis>- and Saturated Fatty Acids in Selected Hydrogenated and Refined Vegetable Oils in the Indian Market

The fatty acid composition of 27 samples of commercial hydrogenated vegetable oils and 23 samples of refined oils such as sunflower oil, rice bran oil, soybean oil and RBD palmolein marketed in India were analyzed. Total cis, trans unsaturated fatty acids (TFA) and saturated fatty acids (SFA) were determined. Out of the 27 hydrogenated fats, 11 % had TFA about 1 % where as 11 % had more than 5 % TFA with an average value of about 13.1 %. The 18:1 trans isomers, elaidic acid was the major trans contributor found to have an average value of about 10.8 % among the fats. The unsaturated fatty acids like cis-oleic acid, linoleic acid and α-linolenic acid were in the range of 21.8–40.2, 1.9–12.2, 0.0–0.7 % respectively. Out of the samples, eight fats had fatty acid profiles of low TFA (less than 10 %) and high polyunsaturated fatty acids (PUFA) such as linoleic and α-linolenic acid. They had a maximum TFA content of 7.3 % and PUFA of 11.7 %. Among the samples of refined oils, rice bran oil (5.8 %) and sunflower oil (4.4 %) had the maximum TFA content. RBD palmolein and rice bran oils had maximum saturated fatty acids content of 45.1 and 24.4 % respectively. RBD palmolein had a high monounsaturated fatty acids (MUFA) content of about 43.4 %, sunflower oil had a high linoleic acid content of about 56.1 % and soybean oil had a high α-linolenic acid content of about 5.3 %.     

Host Plant Suitability in a Specialist Herbivore, <Emphasis Type="Italic">Euphydryas anicia</Emphasis> (Nymphalidae): Preference,Performance and Sequestration

The checkerspot butterfly, Euphydryas anicia (Nymphalidae), specializes on plants containing iridoid glycosides and has the ability to sequester these compounds from its host plants. This study investigated larval preference, performance, and sequestration of iridoid glycosides in a population of E. anicia at Crescent Meadows, Colorado, USA. Although previous studies showed that other populations in Colorado use the host plant, Castilleja integra (Orobanchaceae), we found no evidence for E. anicia ovipositing or feeding on C. integra at Crescent Meadows. Though C. integra and another host plant, Penstemon glaber (Plantaginaceae), occur at Crescent Meadows, the primary host plant used was P. glaber. To determine why C. integra was not being used at the Crescent Meadows site, we first examined the host plant preference of naïve larvae between P. glaber and C. integra. Then we assessed the growth and survivorship of larvae reared on each plant species. Finally, we quantified the iridoid glycoside concentrations of the two plant species and diapausing caterpillars reared on each host plant. Our results showed that E. anicia larvae prefer P. glaber. Also, larvae survive and grow better when reared on P. glaber than on C. integra. Castilleja integra was found to contain two primary iridoid glycosides, macfadienoside and catalpol, and larvae reared on this plant sequestered both compounds; whereas P. glaber contained only catalpol and larvae reared on this species sequestered catalpol. Thus, although larvae are able to use C. integra in the laboratory, the drivers behind the lack of use at the Crescent Meadows site remain unclear.     

The Role of Leaf Volatiles of <Emphasis Type="Italic">Ludwigia octovalvis</Emphasis> (Jacq.) Raven in the Attraction of <Emphasis Type="Italic">Altica cyanea</Emphasis> (Weber) (Coleoptera: Chrysomelidae)

Larvae and adults of Altica cyanea (Weber) (Coleoptera: Chrysomelidae) feed on the rice-field weed Ludwigia octovalvis (Jacq.) Raven (Onagraceae), commonly known as willow primrose, which is considered a biocontrol agent of the weed. Volatile organic compounds from undamaged plants, plants after 4, 12, and 36 h of continuous feeding by A. cyanea larvae or adult females and after mechanical damaging were identified by GC-MS and GC-FID analyses. Twenty nine compounds were identified from undamaged plants. 2Z–Penten-1-ol, geraniol, and 1-tridecanol were present in all plants damaged by larvae. In contrast, feeding by adults caused the release of 2Z–penten-1-ol only after 12 and 36 h; whereas geraniol and 1-tridecanol appeared only after 36 h. Farnesyl acetone was detected after 12 and 36 h of feeding by larvae and after 36 h of feeding by adults. Farnesene was detected after 36 h of feeding by larvae and adults. Linalool was unique after 36 h of feeding by larvae. In Y-shaped glass tube olfactometer bioassays, A. cyanea females were attracted to volatiles after 36 h of feeding by larvae or adults compared to volatiles released by undamaged plants. The insects were attracted to five synthetic compounds: 3-hexanol, α-pinene, linalool oxide, geraniol, and phytol. Synthetic blends were more attractive than individual compounds. Compared to undamaged plants, volatiles released by plants, damaged by conspecific individuals, were more attractive to A. cyanea females, due to elevated emissions of 3-hexanol, α-pinene, linalool oxide, geraniol, and phytol.     

Cuticular Hydrocarbons of <Emphasis Type="Italic">Tribolium confusum</Emphasis> Larvae Mediate Trail Following and Host Recognition in the Ectoparasitoid <Emphasis Type="Italic">Holepyris sylvanidis</Emphasis>

Parasitic wasps which attack insects infesting processed stored food need to locate their hosts hidden inside these products. Their host search is well-known to be guided by host kairomones, perceived via olfaction or contact. Among contact kairomones, host cuticular hydrocarbons (CHCs) may provide reliable information for a parasitoid. However, the chemistry of CHC profiles of hosts living in processed stored food products is largely unknown. Here we showed that the ectoparasitoid Holepyris sylvanidis uses CHCs of its host Tribolium confusum, a worldwide stored product pest, as kairomones for host location and recognition at short range. Chemical analysis of T. confusum larval extracts by gas chromatography coupled with mass spectrometry revealed a rich blend of long-chain (C25-C30) hydrocarbons, including n-alkanes, mono-, and dimethylalkanes. We further studied whether host larvae leave sufficient CHCs on a substrate where they walk along, thus allowing parasitoids to perceive a CHC trail and follow it to their host larvae. We detected 18 CHCs on a substrate that had been exposed to host larvae. These compounds were also found in crude extracts of host larvae and made up about a fifth of the CHC amount extracted. Behavioral assays showed that trails of host CHCs were followed by the parasitoids and reduced their searching time until successful host recognition. Host CHC trails deposited on different substrates were persistent for about a day. Hence, the parasitoid H. sylvanidis exploits CHCs of T. confusum larvae for host finding by following host CHC trails and for host recognition by direct contact with host larvae.     

Attraction of Three Mirid Predators to Tomato Infested by Both the Tomato Leaf Mining Moth <Emphasis Type="Italic">Tuta absoluta</Emphasis> and the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Plants emit volatile compounds in response to insect herbivory, which may play multiple roles as defensive compounds and mediators of interactions with other plants, microorganisms and animals. Herbivore-induced plant volatiles (HIPVs) may act as indirect plant defenses by attracting natural enemies of the attacking herbivore. We report here the first evidence of the attraction of three Neotropical mirid predators (Macrolophus basicornis, Engytatus varians and Campyloneuropsis infumatus) toward plants emitting volatiles induced upon feeding by two tomato pests, the leaf miner Tuta absoluta and the phloem feeder Bemisia tabaci, in olfactometer bioassays. Subsequently, we compared the composition of volatile blends emitted by insect-infested tomato plants by collecting headspace samples and analyzing them with GC-FID and GC-MS. Egg deposition by T. absoluta did not make tomato plants more attractive to the mirid predators than uninfested tomato plants. Macrolophus basicornis is attracted to tomato plants infested with either T. absoluta larvae or by a mixture of B. tabaci eggs, nymphs and adults. Engytatus varians and C. infumatus responded to volatile blends released by tomato plants infested with T. absoluta larvae over uninfested plants. Also, multiple herbivory by T. absoluta and B. tabaci did not increase the attraction of the mirids compared to infestation with T. absoluta alone. Terpenoids represented the most important class of compounds in the volatile blends and there were significant differences between the volatile blends emitted by tomato plants in response to attack by T. absoluta, B. tabaci, or by both insects. We, therefore, conclude that all three mirids use tomato plant volatiles to find T. absoluta larvae. Multiple herbivory did neither increase, nor decrease attraction of C. infumatus, E. varians and M. basicornis. By breeding for higher rates of emission of selected terpenes, increased attractiveness of tomato plants to natural enemies may improve the effectiveness of biological control.     

Volatile and Contact Chemical Cues Associated with Host and Mate Recognition Behavior of <Emphasis Type="Italic">Sphenophorus venatus</Emphasis> and <Emphasis Type="Italic">Sphenophorus parvulus</Emphasis> (Coleoptera: Dryophthoridae)

Beetles in the genus Sphenophorus Schönherr, or billbugs, potentially utilize both volatile and non-volatile behavior-modifying chemical signals. These insects are widely distributed across North America, often occurring in multi-species assemblages in grasses. However, details about their host- and mate-finding behavior are poorly understood. This study tested the hypothesis that volatile organic compounds from host-plants and conspecifics direct the dispersal behavior of hunting billbug S. venatus Say. Further, we characterized the cuticular hydrocarbon profiles of two widespread pest species, S. venatus and bluegrass billbug S. parvulus Gyllenhaal, to assess the potential role of contact pheromones in mate-recognition. In Y-tube olfactometer bioassays, S. venatus males were attracted to a combination of conspecifics and Cynodon dactylon host-plant material, as well as C. dactylon plant material alone. S. venatus females were attracted to a combination of male conspecifics and host-plants but were also attracted to male conspecifics alone. Field evaluation of a putative male-produced aggregation pheromone, 2-methyl-4-octanol, identified from two congeners, S. levis Vaurie and S. incurrens Gyllenhaal, did not support the hypothesis that S. venatus and S. parvulus were also attracted to this compound. Gas chromatography-mass spectrometry analysis of S. venatus and S. parvulus whole-body cuticular extracts indicated a series of hydrocarbons with qualitative and quantitative interspecific variation in addition to intraspecific quantitative variation between males and females. This study provides the first evidence that S. venatus orients toward host- and insect-derived volatile organic compounds and substantiates the presence of species-specific cuticular hydrocarbons that could serve as contact pheromones for sympatric Sphenophorus species.     

Herbivore-Induced Defenses in Tomato Plants Enhance the Lethality of the Entomopathogenic Bacterium, <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">kurstaki</Emphasis>

Plants can influence the effectiveness of microbial insecticides through numerous mechanisms. One of these mechanisms is the oxidation of plant phenolics by plant enzymes, such as polyphenol oxidases (PPO) and peroxidases (POD). These reactions generate a variety of products and intermediates that play important roles in resistance against herbivores. Oxidation of the catecholic phenolic compound chlorogenic acid by PPO enhances the lethality of the insect-killing bacterial pathogen, Bacillus thuringiensis var. kurstaki (Bt) to the polyphagous caterpillar, Helicoverpa zea. Since herbivore feeding damage often triggers the induction of higher activities of oxidative enzymes in plant tissues, here we hypothesized that the induction of plant defenses would enhance the lethality of Bt on those plants. We found that the lethality of a commercial formulation of Bt (Dipel® PRO DF) on tomato plants was higher if it was applied to plants that were induced by H. zea feeding or induced by the phytohormone jasmonic acid. Higher proportions of H. zea larvae killed by Bt were strongly correlated with higher levels of PPO activity in the leaflet tissue. Higher POD activity was only weakly associated with higher levels of Bt-induced mortality. While plant-mediated variation in entomopathogen lethality is well known, our findings demonstrate that plants can induce defensive responses that work in concert with a microbial insecticide/entomopathogen to protect against insect herbivores.     

The Extended Community-Level Effects of Genetic Variation in Foliar Wax Chemistry in the Forest Tree <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

Genetic variation in foundation trees can influence dependent communities, but little is known about the mechanisms driving these extended genetic effects. We studied the potential chemical drivers of genetic variation in the dependent foliar community of the focal tree Eucalyptus globulus. We focus on the role of cuticular waxes and compare the effects to that of the terpenes, a well-studied group of secondary compounds known to be bioactive in eucalypts. The canopy community was quantified based on the abundance of thirty-nine distinctive arthropod and fungal symptoms on foliar samples collected from canopies of 246 progeny from 13 E. globulus sub-races grown in a common garden trial. Cuticular waxes and foliar terpenes were quantified using gas chromatography - mass spectrometry (GC-MC). A total of 4 of the 13 quantified waxes and 7 of the 16 quantified terpenes were significantly associated with the dependent foliar community. Variation in waxes explained 22.9% of the community variation among sub-races, which was equivalent to that explained by terpenes. In combination, waxes and terpenes explained 35% of the genetic variation among sub-races. Only a small proportion of wax and terpene compounds showing statistically significant differences among sub-races were implicated in community level effects. The few significant waxes have previously shown evidence of divergent selection in E. globulus, which signals that adaptive variation in phenotypic traits may have extended effects. While highlighting the role of the understudied cuticular waxes, this study demonstrates the complexity of factors likely to lead to community genetic effects in foundation trees.     

Sexual Isolation and Cuticular Hydrocarbon Differences between <Emphasis Type="Italic">Drosophila santomea</Emphasis> and <Emphasis Type="Italic">Drosophila yakuba</Emphasis>

Drosophila santomea and Drosophila yakuba are two sister species inhabiting Saõ Tomé island. Previous studies showed that both species display strong reproductive isolation, although they can produce a few viable hybrids. Our study tried to understand the mechanism of this ethological isolation between two allopatric strains. A strong sexual isolation was confirmed, with a marked asymmetry. Comparisons of latency times to either courtship or copulation suggest that males do not discriminate females, whereas D. yakuba females, but not D. santomea females, accept their homospecifics more quickly. Cuticular hydrocarbon compositions of both species and sexes were also established with gas chromatography (GC) and GC/mass spectrometry analysis. All have (Z)-7-tricosene as their major compound. There are several quantitative differences between species for few minor compounds. The largest difference concerns n-heneicosane, which is more abundant in D. santomea than in D. yakuba flies (up to seven times more between males). A similar quantitative difference was also found in a pair of sympatric strains. Furthermore, D. yakuba males artificially perfumed with n-heneicosane were discriminated negatively by D. yakuba females, suggesting a role for this compound in the sexual isolation between these two species.     

Microbial Conversion of Arachidonic Acid to Arachidonyl Alcohol by a New <Emphasis Type="Italic">Acinetobacter</Emphasis> Species

Arachidonyl alcohol rarely occurs in natural oils. It can be used as a substrate for production of several ether lipids possessing beneficial functions. Although arachidonyl alcohol has been produced on a laboratory scale by the chemical reduction of arachidonic acid, it will be difficult to scale up this process for industrial application. The aim of this study was to develop a new bioprocess for converting arachidonic acid to arachidonyl alcohol. Screening was conducted using 11 wax ester- (esters of fatty acids and fatty alcohols) producing strains reported in our previous study, and a single-cell oil containing arachidonic acid. A new strain, Acinetobacter species N-476-2, most effectively converted arachidonic acid to arachidonyl alcohol, which accumulated inside the cells as a wax ester. GC–MS, FT–IR, and NMR analyses showed that this strain reduced the carboxyl group of 5-cis,8-cis,11-cis,14-cis-arachidonic acid to a hydroxyl group without altering the position or configuration of the double bonds; the product was identified as 5-cis,8-cis,11-cis,14-cis-arachidonyl alcohol. A time-course study of cultivation showed that the amount of arachidonyl alcohol produced by the strain after 4 days was 2.2 mg/mL culture. The bioprocess using Acinetobacter sp. N-476-2 can be applied to the large-scale production of arachidonyl alcohol.