Heritable variation of sex pheromone composition and the potential for evolution of resistance to pheromone-based control of the Indian meal moth,Plodia interpunctella

The short-term evolutionary effect of pheromone-based mating disruption on the mating ability of the Indian meal moth, Plodia interpunctella, was investigated. Three independent selection lines were established, and the mating ability of moths in plastic tents treated with high doses of pheromone and in control tents was compared for two consecutive generations. In addition, the heritability of the sex pheromone blend, measured as the ratio of two major pheromone components (Z,E)-9,12-tetradecadienyl acetate and (Z,E)-9,12-tetradecadienol, was estimated. Based on a mother–daughter regression analysis including 21 families, the heritability of the pheromone blend was 0.65 ± 0.14, indicating a potential for evolutionary change of the character. However, no increase in mating ability of females in pheromone-treated tents or alteration of the pheromone blend was observed in any selection line when compared with control lines, indicating no or weak selection on the pheromone blend as well as other traits influencing mating ability of this species under the created mating disruption conditions. Factors contributing to the lack of selection effects are discussed.     

Variables Affecting Pheromone Concentration in Vineyards Treated for Mating Disruption of Grape Vine Moth Lobesia botrana

Airborne pheromone concentration in a field is one of the most important variables for the successful application of mating disruption in pest control. In the present paper, we estimated the pheromone concentration with field EAG recordings in vineyards and investigated parameters affecting concentration. Pheromone concentration showed a positive correlation with number of dispensers per hectare (= number of point sources). A twofold increase in the absolute number of dispensers per hectare with a constant number of point sources (two dispensers at the same location) did not significantly affect relative pheromone concentration. Measurements carried out in plots where dispensers had been applied at different heights showed highest relative pheromone concentrations in plots with dispensers at 0.1 m and 1.4 m above the ground. Those concentrations were not significantly different from each other, but were significantly higher than in plots where dispensers had been placed at a height of 2 m. Foliage of grape vines substantially affected the development of high pheromone concentrations. In summer, in vineyards with fully developed leaf canopy, significantly higher pheromone concentrations could be measured shortly after application of the dispensers compared to vineyards in spring with sparse vegetation. The decline of pheromone concentrations after removal of dispensers is significantly prolonged in full vegetation, showing the impact of plant canopy on pheromone concentrations. In contrast, ground cover between the grapevine rows did not significantly affect mean pheromone concentrations.     

Anemotactic response threshold of the Indian meal moth,Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), to its sex pheromone

The effect of different concentrations of the sex pheromone (Z,E)-9,12-tetradecadien-1-ol acetate on the upwind anemotactic behavior of the malePlodia interpunctella (Hübner) was measured at 23 ± 1 ° C and 34 ± 1 ° C. The stimulus-response regression lines were analyzed by a new procedure that accounts both for control responses in the absence of pheromone and also for peak responses below 100% in the presence of concentrations considerably above the normal physiological levels. From the regression line for each temperature, the upwind anemotactic thresholds were calculated to be 1.34 × 10⁶ molecules/cm³ at 23 ° C and 1.65 × 10⁴ molecules/cm³ at 34 ° C, similar to other thresholds reported in the literature. Since departures from the two lines occurred at the highest concentrations tested, near 10⁸ molecules/cm³, the upwind anemotactic behavior may change qualitatively above an altered-behavior threshold that is about two orders of magnitude higher than the upwind anemotactic threshold. The lower response at 23 ° C suggests that cool temperatures inhibit flight in response to pheromonal stimulation.Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA.     

In vitro maintenance of the sex pheromone gland of the female Indian meal mothPlodia interpunctella (Hübner)

Sex pheromone glands ofPlodia interpunctella were isolated from surface-sterilized donors of different ages, freed of most of the attached gut, oviduct, and other tissues; rinsed in sterile medium; and cultured in 1 ml of culture medium. The sex pheromone gland cells that were cultured for 10 days in either chemically defined Grace's medium or modified Grace's medium appeared normal in histological examinations. Bioassays of extracted medium in which pheromone glands from mature females had been incubated showed that a greater percentage of the available pheromone was recovered from modified Grace's medium than from chemically defined Grace's medium.Lepidoptera: Pyralidae.This research was supported in part by NIH Grant 1-F34 GM 06251.     

Potential for Use of Synthetic Sex Pheromone for Mating Disruption of the Olive Pyralid Moth, Euzophera pinguis

The potential for pheromone-based mating disruption of the olive pyralid moth (OPM), Euzophera pinguis, in olive groves was investigated during the second flight period in small-plot trials in 2002. The female of this species emits a blend of (9Z,12E)-tetradecadien-1-ol and (9Z,12E)-tetradecadienyl acetate, which were synthesized for field tests. Mating disruption efficacy in 0.8-ha trials was evaluated using two parameters: reduction of male capture in pheromone traps and reduction of infestation in infestation-prone sites. White rubber septa containing 10 mg of pheromone blend as disruptant were applied at a density of 50 septa/ha for each treatment. Mean catches of E. pinguis males in pheromone traps were greatly reduced (> 95%) in pheromone-treated plots relative to similar traps placed in control plots. In addition, significant reductions were recorded (35-40%) in the oviposition and infestation levels during pheromone treatment. The total amount of pheromone blend released from disruption dispensers during the field trials was estimated to average 5.4 mg/ha/day, over 56 days.     

Seasonal Variation of Pheromone Concentration in Mating Disruption Trials Against European Grape Vine Moth Lobesia botrana (Lepidoptera: Tortricidae) Measured by EAG

Spatial and temporal distributions and relative concentrations of the pheromone of the European grape vine moth Lobesia botrana (Lepidoptera: Tortricidae) were measured in mating disruption trials with electroantennograms. Measurements were carried out over several years during the flight season of the two generations of this pest insect. In three consecutive years significantly higher mean relative pheromone concentrations were measured in summer during the flight of the second generation of L. botrana than in spring during the flight of the first generation (P < 0.001). The relative pheromone concentrations in 1989 reached a maximum between late July and early August, when the highest mean daily temperatures were registered. In addition to the differences in mean relative pheromone concentrations, the spatial and temporal distributions of the pheromone differed significantly between spring and summer. EAG recordings taken in summer showed high, uniform pheromone concentrations in the treated plots, whereas in spring strong temporal and spatial fluctuations were recorded. In a vineyard defoliated by a hailstorm, the mean relative pheromone concentrations measured in summer were not significantly different from those measured in spring (P > 0.05), but were significantly lower than those of a nearby intact vineyard (P < 0.001). The results provided additional evidence that foliage is an important parameter determining mean pheromone concentrations and temporal and spatial distribution of pheromone in mating disruption trials.     

Population-density-dependent mating frequency amongPlodia interpunctella (Lepidoptera: Phycitidae) in the presence of synthetic sex pheromone with behavioral observations

Mating frequency amongPlodia interpunctella (Hübner) held in enclosed environments was reduced in the presence of the synthetic sex pheromone, (Z,E)-9,12-tetradecadien-l-ol acetate. The effectiveness of any dose of pheromone was markedly increased as population densities were decreased from 10 to 0.1 pairs/m² of wall and ceiling surface. Mating behaviors of the insect are described.Mention of a commercial or proprietary product in this paper does not constitute an endorsement of the product by the U.S. Department of Agriculture.Florida employed under a cooperative agreement with the USDA.     

Production and release of (Z,E)-9,12-tetradecadienal by sex pheromone glands of females ofPlodia interpunctella (lepidoptera: pyralidae)

Extracts of sex pheromone glands obtained from females ofPloida interpunctella contained detectable amounts of (Z,E,)-9,12-tetradecadien-1-ol acetate (Z9,E12–14:Ac) and (Z,E.)-9,12-tetradecadien-1-ol (Z9,E12–14:OH) 4 hr prior to the first scotophase after adult emergence. The amount of pheromone increased during the first 4 hr of the scotophase and then declined to low levels during the subsequent photophase. Decapitation of females immediately after emergence, prior to expansion of the wings, inhibited production of pheromone during the subsequent 48 hr. Injection of extracts of the heads of 1-day-old females ofP. interpunctella of partially purified extracts of the cephalic ganglia of females of the corn earworm moth into decapitated females stimulated production of bothZ9,E12–14:Ac andZ9,E12–14:OH as well as production of (Z,E)-9,12-tetradecadienal (Z9,E12–14:Al). This aldehyde was subsequently identified from extracts of pheromone glands obtained from naturally calling females as well as from volatiles emitted by calling females. Studies on the terminal steps in biosynthesis of the pheromone showed thatZ9,E12–14:OH was produced from the corresponding acetate and thatZ9,E12–14:Al was produced from the alcohol via the action of an oxidase(s).     

Models for dispersal of vapors in open and confined spaces: Applications to sex pheromone trapping in a warehouse

Methods are presented for mathematically determining dispersal of a vapor in still air, in turbulent air of zero average velocity, and in turbulent or laminar air currents of constant, nonzero average velocity. The methods are combined with several assumptions about insect behavior to derive an insect attraction model that predicts: (1) In a warehouse a searching insect is likely to be attracted to a calling insect if it comes within an attraction sphere, 0.4–2.5 m in radius. (2) The attraction spaces of typical sex pheromone-baited traps that emit pheromone at rates greater than 0.01 ng/sec extend beyond the boundaries of a 10 × 10 × 10-m warehouse. (3) The searching behavior of an attracted insect is likely to be altered from an extensive to an intensive pattern if it comes within an altered-behavior sphere, 6–60 cm from a calling insect or within 10 m of a trap emitting 0.76 ng/sec. (4) Pheromone does not sink unless it is emitted along with a large amount of a high-vapor pressure solvent. The model is used in support of several hypotheses, including: (1) The effect of an adsorptive surface on the vapor concentration after an extended period of emission is negligible except at positions near the surface. (2) Sex pheromone-baited traps with sources of small dimensions have greater trapping efficiency than otherwise identical traps with sources of large dimensions.     

Mating Disruption of Citrus Leafminer Mediated by a Noncompetitive Mechanism at a Remarkably Low Pheromone Release Rate

The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a worldwide pest of citrus. A season-long investigation was conducted that evaluated mating disruption for this pest. Effective disruption of the male P. citrella orientation to pheromone traps (98%) and reduced flush infestation by larvae was achieved for 221 d with two deployments of a 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal/(Z,Z)-7,11-hexadecadienal at a remarkably low rate of 1.5 g active ingredient (AI)/ha per deployment. To gain insight into the mechanism that mediates the disruption of P. citrella, male moth catch was quantified in replicated plots of citrus treated with varying densities of pheromone dispensers. The densities of septum dispensers compared were: 0 (0/ha, 0.0 g AI/ha), 0.2 (one every fifth tree or 35/ha, 0.05 g AI/ha), 1 (215/ha, 0.29 g AI/ha), and 5 per tree (1,100/ha, 1.5 g AI/ha). Profile analysis by previously published mathematical methods matched predictions of noncompetitive mating disruption. Behavioral observations of male P. citrella in the field revealed that males did not approach mating disruption dispensers in any of the dispenser density treatments. The current report presents the first set of profile analyses combined with direct behavioral observations consistent with previously published theoretical predictions for a noncompetitive mechanism of mating disruption. The results suggest that disruption of P. citrella should be effective even at high population densities given the density-independent nature of disruption for this species and the remarkably low rate of pheromone per hectare required for efficacy.     

Simultaneous Disruption of Pheromone Communication in Choristoneura rosaceana and Pandemis limitata with Pheromone and Antagonist Blends

In British Columbia, trapping and wind-tunnel studies demonstrated that (Z)-9-tetradecenyl acetate (Z9–14:OAc), a minor component of the sex pheromone for Pandemis limitata, acted as a pheromone antagonist to a sympatric species, Choristoneura rosaceana. Addition of >1% Z9–14:OAc to the four-component C. rosaceana pheromone in a wind tunnel resulted in significant reductions in the proportion of male C. rosaceana that wing fanned, locked on to the plume in flight, oriented upwind, and made source contact, compared to the responses to the pheromone alone. Disruption of pheromone communication was tested in 33.3 × 33.3-m plots, at a release rate of 10 mg/ha/hr using Conrel fiber dispensers. Z9–14:OAc applied alone did not disrupt orientation to virgin-female-baited traps for either C. rosaceana or P. limitata. A 1:1 mixture of Z9–14:OAc and the four-component C. rosaceana pheromone was as effective as the pheromone alone at disrupting orientation of C. rosaceana males to virgin-female-baited traps, demonstrating that disruption apparently did not occur through false-trail following. The 1:1 mixture of Z9–14:OAc and the C. rosaceana pheromone also reduced catches of P. limitata males in virgin-female-baited traps, but not significantly more than the 83% disruption caused by the pheromone alone. Therefore, the C. rosaceana pheromone could be used alone or with Z9–14:OAc to disrupt communication and, presumably, mating in both leafrollers simultaneously.     

Prolonged Adaptation as Possible Mating Disruption Mechanism in Oriental Fruit MothCydia (=Grapholita) molesta

The effects of pheromone preexposure on flight behavior of male Oriental fruit mothCydia molestawere investigated in laboratory experiments using a wind-tunnel and in an orchard through insect releases at the center of a grid of pheromone traps. In the wind-tunnel experiments, the proportion of insects performing various behavioral phases was found to depend on the intensity and duration of the adapting stimulus and the recovery time allowed between exposure and measurement, indicating adaptation of at least some elements of the sensory system. The orchard experiments exhibited similar adaptation effects at similar exposure levels, causing the entry of treated insects into the traps to be delayed compared to controls and a reduction in insects caught for the highest preexposure concentration. The levels of pheromone exposure required to produce significant adaptation were, however, found to be much higher than could be expected under conditions of release of synthetic pheromone for insect control through mating disruption.     

Disruption of male spruce budworm orientation to calling females in a wind tunnel by synthetic pheromone

Male spruce budworm [Chorisloneura fumiferana (Clem.)] moths were held for 3 hr in a wind tunnel and subjected to various concentrations of background synthetic pheromone. They were then exposed to calling females and their response was recorded. The background pheromone was presented either as discrete turbulent plumes or as a uniform permeation throughout the tunnel. The numbers of males wing-fanning and flying in response to the calling females decreased as the concentration of background pheromone increased. Of the males which flew, a higher proportion progressed upwind in the discrete plumes than in the uniform permeation, an indication that structure in the pheromone cloud is necessary for upwind progression. In both discrete plumes and uniform permeation fewer males were able to locate the females (i.e., disruption was greater) as the concentration of synthetic pheromone increased, but for the same total release rates, disruption was greater when the synthetic pheromone was released in discrete plumes rather than in a uniform permeation. This implies that disruption which involves luring males to sources of synthetic pheromone is more effective than masking female plumes by uniform permeation and suggests that it is more efficient to release pheromone from a few potent sources than from numerous low-potency sources.     

Quantitative comparison of behavioral and neurophysiological responses of insects to odorants

A consistent pattern of relationships emerges from comparisons of insect electroantennograms, peripheral olfactory receptor neuron responses, and behavioral responses to quantified concentrations of odorants. One consistency is that all of the different response measurements can be described by stimulus-response curves of the same form. Another is that the responses have characteristic groupings when they are plotted against odorant concentration. The pattern of relationships is exemplified in the responses ofTrichoplusia ni (Hübner),Heliothis zea (Boddie), andPlodia interpunctella (Hübner) to several pheromone components and analogs. To quantify the relevant stimulus parameters for the response comparisons, the emission rates of the stimulus delivery system were calibrated for several 12 to 17-carbon pheromone components. The stimulus-response relationships determined forT. ni, H. zea, andP. interpunctella are combined with relationships reported for other insects in the literature, and applications are discussed for the interpretation of pheromone trapping and laboratory bioassays.Postdoctoral fellow employed through a cooperative agreement between the Insect Attractants, Behavior, and Basic Biology Research Laboratory     

Bidirectional Selection for Novel Pheromone Blend Ratios in the Almond Moth, <Emphasis Type="Italic">Cadra cautella</Emphasis>

The sex pheromone of the almond moth, Cadra cautella, is a blend of (Z,E)-9,12-tetradecadienyl acetate (Z9,E12–14:Ac, the major component capable of inducing attraction alone) and (Z)-9-tetradecenyl acetate (Z9–14:Ac, the minor component, which is unattractive alone but augments attraction of the major component). In this study, the ratio of the two components responded to artificial directional selection in five of six selected lines, whereas no change was observed in the three control lines. The mean ratio (±SE) of Z9,E12–14:Ac to Z9–14:Ac went from 13.72 ± 1.02:1 to 20.13 ± 0.68:1 in high line 1, an increase of 47%. In the second high-selected line, the mean ratio (±SE) increased from 9.87 ± 0.54:1 to 15.89 ± 0.85:1, an increase of 61%. In low line 1, the mean ratio (±SE) in the parental generation was 10.74 ± 0.78:1 and 7.35 ± 0.41:1 in the last selected generation, a decrease of 32%. The response to selection was greater in low line 2, as the mean ratio (±SE) decreased from 10.11 ± 0.66:1 to 5.65 ± 0.55:1 in the last generation, a decrease of 44%. In low line 3, the mean ratio (±SE) in the parental generation was 13.63 ± 0.82:1 and 6.47 ± 0.26:1 in the last generation, a decrease of 53%. The response to selection was approximately symmetrical with a mean increase of 54% and a mean decrease of 43%. The increases in ratio observed in the high lines were caused by an increase in the titer of the Z9,E12–14:Ac component with no concurrent change in the titer of the component Z9–14:Ac. Among the low selected lines, the titers of both components increased; however, there was a greater relative increase in the titer of the component Z9–14:Ac. The absolute and relative titers of the sex pheromone components had decreased significantly in the F10 generation in some of the selected lines, five generations after the discontinuation of selection.     

Identification of Female Sex Pheromone of the Legume Pod Borer, Maruca vitrata and Antagonistic Effects of Geometrical Isomers

An EAD-active component in a female abdominal tip extract of the legume pod borer, Maruca vitrata (Fabricius) (= M. testulalis), was identified as (E,E)-10,12-hexadecadienal (E10,E12–16:Ald). In laboratory bioassays, the purified synthetic E10,E12–16:Ald with 99% isomeric purity had attractancy to male moths equal to the crude extract. However, the unpurified chemical with 92% isomeric purity did not show any attractancy. Addition of 0.1 ng EZ isomer or 1 ng of EZ, ZE, or ZZ isomers of 10,12–16: Ald to 1 ng of the purified synthetic pheromone antagonized its attractancy as did addition of each isomer to 1 female equivalent of the crude extract. These results suggest that it is necessary to develop formulation and release techniques that maintain extremely high isomeric purity of the synthetic pheromone for male trapping in the field.     

Isolation, Identification, Synthesis, and Field Evaluation of the Sex Pheromone of the Brazilian Population of Spodoptera frugiperda

Several studies have shown intraspecific geographical variation in the composition of sex pheromones. Pheromone lures from North America and Europe were not effective against the fall armyworm Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) in Brazil, so we examined the composition of the sex pheromone produced by females from Brazilian populations. Virgin female gland extracts contained (Z)-7-dodecenyl acetate (Z7-12:Ac), (E)-7-dodecenyl acetate (E7-12:Ac), dodecyl acetate, (Z)-9-dodecenyl acetate, (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-10-tetradecenyl acetate, tetradecyl acetate/(Z)-11-tetradecenyl acetate (Z11-16:Ac), and (Z)-11-hexadecenyl acetate. The relative proportions of each acetate were 0.8:1.2:0.6:traces:82.8:0.3:1.5:12.9, respectively. This is the first time that E7-12:Ac has been reported from the pheromone gland of S. frugiperda. Only three compounds, Z9-14:Ac, Z7-12:Ac, and E7-12:Ac, elicited antennal responses, and there were no differences in catch between traps baited with either Z7-12:Ac + Z9-14:Ac or Z7-12:Ac + Z9-14:Ac + Z11-16:Ac blends. However, the Z7-12:Ac + Z9-14:Ac + E7-12:Ac blend was significantly better than Z7-12:Ac + Z9-14:Ac, indicating that E7-12:Ac is an active component in the sex pheromone of the Brazilian populations of S. frugiperda.     

Mating disruption of pea mothCydia nigricana F. (lepidoptera: Tortricidae) by a repellent blend of sex pheromone and attraction inhibitors

Synthetic sex pheromone of the pea mothCydia nigricana, (E,E)-8,10-dodecadien-1-yl acetate (E8,E10–12: Ac), was applied in polyethylene dispensers at a rate of 30 g/ha and 600 dispensers/ha in a 0.6-ha pea field. The release rate ofE8,E10–12: Ac was 140 mg/ha/day after six days, and 82 mg/ha/day after 20 days. Aerial concentrations ofE8,E10–12: Ac, as measured by a portable EAG apparatus, ranged from 2 ± 2 to 7 ± 3 ng/m³. The antennal signal was high and rather constant within pea canopy, but was lower and fluctuated strongly above canopy. Initially, >99% isomerically pureE8,E10–12: Ac was released, and male moths were attracted to dispensers. After nine days, isomeric blend composition had equilibrated to approx. 92%E8,E10–12: Ac and 8% of the inhibitory isomersE,Z-,Z,E-, andZ8,Z10–12: Ac. Males were then repelled from the pheromone-permeated field. Traps baited with 100 µgE8,E10–12: Ac caught 258 ± 133C. nigricana males/trap in the control, but no males at all in the disruption field.     

Characterization of Pheromone Blend for Grapevine Moth, Lobesia botrana by Using Flight Track Recording

The behavioral responses of Lobesia botrana males to calling females, pheromone gland extracts, and synthetic sex pheromones were recorded in a wind tunnel. Gland extracts and synthetic pheromones were released from a pheromone evaporator. The numbers of males reaching the source and their flight tracks in response to calling females and pheromone gland extracts were compared to those of synthetic blends. Upwind flights to natural sex pheromone were straighter and faster than to a three-component blend of (E)-7,(Z)-9-dodecadienyl acetate (E7,Z9–12:Ac), (E)-7,(Z)-9-dodecadien-1-ol (E7,Z9–12:OH), and (Z)-9-docecenyl acetate (Z9–12:Ac) (100:20:5). The optimum ratio of E7,Z9–12:OH and Z9–12:Ac to E7,Z9–12:Ac was found to be 5% and 1%, respectively. An additional seven compounds identified in the sex pheromone gland were investigated for their biological activity. Two unsaturated acetates, i.e., (E)-9-dodecenyl acetate (E9–12:Ac) and 11-dodecenyl acetate (11–12:Ac), increased the number of males reaching the source as well as straightness, linear velocity, and decreased the track angle of upwind flight. Optimum response was obtained by releasing 10 pg/min E7,Z9–12:Ac in a mixture with 0.5 pg/min E7,Z9–12:OH, 0.1 pg/min Z9–12:Ac, 0.1 pg/min E9– 12:Ac and 1 pg/min 11-12–Ac. The saturated acetates previously identified in the female glands were biologically inactive.     

Female Sex Pheromone of Brinjal Fruit and Shoot Borer, Leucinodes orbonalis Blend Optimization

The brinjal fruit and shoot borer, Leucinodes orbonalis is the major pest of eggplant in South Asia. Analysis of female pheromone gland extracts prepared from insects of Indian and Taiwanese origin confirmed (E)-11-hexadecenyl acetate (E11-16:Ac) as the major pheromone component with 0.8 to 2.8% of the related (E)-11-hexadecen-1-ol (E11-16:OH), as previously reported from Sri Lanka. The average quantity of E11-16:Ac extracted per female was estimated to be 33 ng, with a range of 18.9 to 46.4 ng when collected 2 to 3 hr into the scotophase. In field trials conducted in India, blends containing between 1 and 10% E11-16:OH caught more male L. orbonalis than E11-16:Ac alone. At the 1000 g dose, on white rubber septa, addition of 1% E11-16:OH to E11-16:Ac was found to be more attractive to male L. orbonalis than either 0.1 or 10% E11-16:OH. Trap catch was found to be positively correlated with pheromone release rate, with the highest dose tested, 3000 g, on white rubber septa catching more male moths than lower doses. Field and wind tunnel release rate studies confirmed that E11-16:OH released from white rubber septa and polyethylene vials at approximately twice the rate of E11-16:Ac and that the release rate of both compounds was doubled in polyethylene vials compared to white rubber septa. This difference in release rate was reflected in field trials conducted in Bangladesh where polyethylene vial dispensers caught more male moths than either black or white rubber septa, each loaded with the same 100 : 1 blend of E11-16:Ac and E11-16:OH in a 3000 g loading.