Introduction

Glandular trichomes may serve many functions in plants, but one of the more important is protecting them from herbivorous insects (Werker, 2000). The glandular trichomes of solanaceous plants contain or secrete a number of phytochemicals that are either toxic or deterrent to a wide variety of herbivorous insects (Dimock and Kennedy, 1983; Duffey, 1986; Goffreda et al., 1989; Neal et al., 1990; Buta et al., 1993; Severson et al., 1994; Yencho et al., 1994; Liedl et al., 1995; Eigenbrode et al., 1996; Wilkens et al., 1996; Liu et al., 1996). One class of compounds produced by glandular trichomes is acylsugars, or esters of simple sugars and one or more fatty acids. These compounds increase the mortality of whiteflies, aphids, mites, and psyllids, and inhibit the growth and survival of phytophagous Lepidoptera (Buta et al., 1993; Juvik et al., 1994; Neal et al., 1994; Puterka and Severson, 1995; van Dam and Hare, 1998a, b). However, the effects of acylsugars vary widely among insect species. They caused no mortality or inhibition of feeding of larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Neal et al., 1994), and at least one insect species utilizes these compounds as feeding and oviposition stimulants (van Dam and Hare, 1998a). Despite their potential role in insect pest management (e.g., Puterka et al., 2003), relatively little is known of the role of acylsugar production in plant defense against native insect herbivores.

Datura wrightii Regel, a native perennial plant, is dimorphic for leaf trichome morphology. Within populations, leaves of some plants are covered with relatively long, multicellular trichomes with a secretory gland at their tip and feel sticky when touched. Other plants are covered with a higher density of shorter, nonglandular trichomes, and feel velvety when touched. Trichome morphology is governed by a single gene, and the sticky allele is dominant (van Dam et al., 1999). Glandular trichomes of sticky plants produce several esters of glucose and straight-chain organic acids (van Dam and Hare, 1998b). In laboratory studies, these esters conferred resistance to whiteflies (van Dam and Hare, 1998a) and reduced the growth rate of Manduca sexta larvae (van Dam and Hare, 1998b). In the same laboratory studies, however, van Dam and Hare (1998a) showed that addition of acylsugars to velvety D. wrightii leaves stimulated feeding and oviposition by Tupiocoris notatus (Distant), a mirid bug with specific morphological adaptations to cope with glandular trichomes (Southwood, 1986).

The concentration of acylsugars on sticky D. wrightii leaves is strongly influenced by environmental conditions (Forkner and Hare, 2000). Acylsugar concentration per unit leaf area was approximately one-third less for plants that received twice-weekly irrigations than for plants that remained unirrigated for the whole growing season, despite the fact that irrigation did not affect the density of glandular trichomes (Forkner and Hare, 2000). Acylsugar concentration also differed between years, although this variation could have been the result of sampling plants at different times during the growing season in each of 2 yr (Forkner and Hare, 2000). Environmental variation in acylsugar concentration suggests that the level of herbivore resistance that glandular trichomes confer may vary both among populations growing under different environmental conditions and seasonally within populations.

To better understand the role of natural concentrations of acylsugars as a plant defense mechanism in D. wrightii, the following two objectives were addressed. First, the acylsugars produced by sticky plants were quantified monthly from field-grown plants in two irrigation treatments to determine the range of variation in concentration over one growing season. Second, the role of acylsugars as a defense against three common Coleopteran herbivores of D. wrightii was evaluated through a series of experiments in which insects were offered sticky and velvety leaves or leaf disks in a series of “choice” and “no choice” experiments. Treatments included unaltered disks from sticky and velvety plants, disks from sticky plants where the acylsugars were removed, anddisks or leaves from velvety plants to which esters were added in measured amounts.

The three herbivores were adults of Lema daturaphila Kogan and Goeden (formerly Lema trilineata daturaphila, see Riley et al., 2003), adults of the tobacco flea beetle, Epitrix hirtipennis (Melsheimer), and adults of a weevil, Trichobaris compacta (Casey). All are common native insect herbivores of wild and cultivated solanaceous plants. The first species damages sticky and velvety D. wrightii similarly in the field, whereas feeding damage by the latter two species is largely confined to velvety D. wrightii (Elle and Hare, 2000; Hare and Elle, 2002). Based upon the field observations, I predicted that E. hirtipennis and T. compacta would be inhibited more than L. daturaphila by the acylsugars of sticky D. wrightii.

Methods and Materials

Variation in Acylsugar Concentration in the Field

Because trichome morphology is a Mendelian trait with the sticky condition being dominant (van Dam et al., 1999), the distribution of phenotypes of backcrossing a heterozygous F1 individual to its homozygous recessive parent is expected to be a 1:1 mixture of velvety (homozygous recessive) and sticky (heterozygous) progeny. This experiment utilized progeny from backcross families # 1, 2, and 4, described in van Dam et al. (1999), and grown in the field in 2000. Although there were initially a total of 216 plants arranged in four blocks for other experiments, data on acylsugar concentration were taken from 48 plants that were sprayed weekly with acephate to protect them from insect attack. Plants in alternate blocks were assigned to two irrigation treatments. The two “irrigated” blocks were furrow-irrigated for 8 hr once a week from April through November, while the two remaining “unirrigated” blocks were left dry.

The youngest, fully expanded leaf was collected from all surviving plants on July 28, 2000 to determine trichome densities under a microscope using standard procedures (van Dam et al., 1999). Plants were flowering and producing seed capsules at this time.

Acylsugar concentration was determined for protected sticky plants every 4wk starting on June 21 and ending October 11, 2000 using methods described by Forkner and Hare (2000) and briefly outlined here. The youngest, fully expanded leaf on each plant was collected, placed into a plastic bag, and brought back to the laboratory. The use of plastic bags to transport the leaves may have introduced an error into the experimental design because some exudate might have been transferred from the leaves to the bags. This error is likely conservative, because such potential losses would actually decrease the probability of finding statistical differences in acylsugar concentration between treatments or among sampling dates.

Acylsugars were extracted three times with CHCl3, and leaf areas were measured with a leaf area meter (Li-Cor Model 3000, Li-Cor, Inc., Lincoln, NE, USA). CHCl3 was filtered and removed by evaporation, then the acylsugars were redissolved in MeOH and saponified with 0.04 N NaOH in water. The liberated glucose was quantified spectrophotometrically using a hexokinase-based glucose assay reagent [Sigma Glucose (HK) reagent; Sigma Diagnostics, St. Louis, MO, USA] prepared according to the manufacturer’s instructions. The molar concentration of glucose was calculated per leaf from the absorbance, then expressed as μmol of glucose esters per cm2 of leaf area.

Concentrations were analyzed statistically with a repeated-measures multiway analysis of variance. The repeated factor was the different sampling dates and the fixed treatment effect was the irrigation treatment. Potential random sources of variation included blocks within irrigation treatments, families, and the interactions of families with irrigation treatment and sampling date.

The data were analyzed using the PROC MIXED procedure of SAS because the design contained both fixed and random factors. PROC MIXED calculates significance tests using a restricted maximum likelihood (REML) method and is more robust for the analysis of mixed models with uneven sample sizes than PROC GLM (SAS Institute, 2000). The logarithmic transformation was applied to ensure normality of residuals.

Ester Removal Experiments

As in other solanaceous species (e.g., Neal et al., 1990), the acylsugars of D. wrightii can be removed from sticky foliage simply by washing the leaf for 15 sec in 95% ethanol, followed by a 15-sec rinse in deionized water, without damaging the leaves. In the following bioassays, the feeding activity of L. daturaphila, T. compacta, and E. hirtipennis was compared on: (1) sticky vs. velvety leaves, (2) sticky leaves vs. sticky leaves with their esters removed (hereafter “washed sticky leaves”), and (3) washed sticky leaves vs. velvety leaves. Both choice and no-choice experiments were performed. Adult insects of undetermined age and sex were collected from the field on the morning of the trial. For the no-choice experiment, there were 10 trials of 10 replicates, with each replicate comprising a pair of 1.5-cm leafdisks of one of the three leaf treatments above, and a single field-collected adult of L. daturaphila or T. compacta, or 10 adults of E. hirtipennis. Different numbers of adults of each species were used because of the differences in sizeand feeding mode of each species. Only five trials could be set up for E.hirtipennis because of a shortage of insects. After 48 hr, the quantity of foliage consumed by each L. daturaphila adult was determined by placing an acetate overlay marked with a grid (1-mm spacing) over the leaf and counting the number of 1×1 mm squares of leaf area removed. For both T. compacta and E.hirtipennis, the feeding pits per leaf were counted and recorded.

For the choice tests, there were five trials of 15 replicates, where each replicate comprised a pair of leaf disks from each of two treatments and individual adults of L. daturaphila or T. compacta, or 10 adults of E. hirtipennis. Feeding was scored after 48 hr as described above. In some cases, the leaf disks shriveled and the replicate was discarded. Additionally, a few T. compacta refused to feed on any leaf disks, and these replicates were also discarded. Both factors led to unequal sample sizes within trials. All bioassays were performed during the months of July and August to try to maximize the difference between sticky and velvety types (see Results), contingent upon the availability of insects in the field.

The “sticky vs. velvety” comparison provides a baseline difference between the leaf types, whereas the “washed sticky vs. sticky” comparison tests the effect of acylsugars on insect feeding on a background of long trichomes, and the “washed sticky vs. velvety” comparison tests the effect of variation in trichome morphology in the absence of acylsugars. In the comparison of sticky vs. washed sticky leaves, the appropriate control was sticky leaves washed for 15 sec in deionized water; in the comparison of washed sticky leaves vs. velvety leaves, the appropriate control was velvety leaves washed identically to sticky leaves, i.e., in both 95% ethanol and deionized water for 15 sec each.

Ester Addition Experiments

Acylsugars were extracted in bulk from various wild-grown sticky D. wrightii plants as described elsewhere (van Dam and Hare, 1998b). Numerous extractions were performed in order to obtain the large quantities of acylsugars required for study. Branches were clipped from plants and brought to the laboratory. All leaves were removed from their branches and soaked for 1 hr in 3 l of CHCl3. The extract was filtered through Whatman # 1 filter paper under vacuum to remove solid debris, then the volume of CHCl3 was reduced by rotary evaporation. The CHCl3 extract was filtered over anhydrous Na2SO4 to remove water then further concentrated, transferred to a preweighed vial, and evaporated to dryness under an air stream. The mass of the residue was determined and dissolved in CH3CN at a rate of 100 ml/g. The material was placed into a sonicating bath (10 min) to ensure that all material was dissolved or suspended in CH3CN. This material was partitioned three times against equal volumes of hexane to remove leaf waxes and other nonpolar compounds. Then CH3CN was taken to dryness by rotary evaporation. The residue was dissolved in 50 ml/g of CH2Cl2 and partitioned two times against 1 N tartaric acid and three times against water to remove alkaloids and other water-soluble compounds. The CH2Cl2 fraction was concentrated by rotary evaporation and transferred into a preweighed vial. Remaining traces of solvent were removed in vacuo. The mass of the residue, the “alkaloid-free acylsugars,” was determined, and yields ranged from 0.1% to 0.2% of the fresh mass of the leaves.

Acylsugars were applied to velvety leaves using methods derived from Puterka and Severson (1995). On the day that a feeding trial was to be established, a group of 12 young but fully expanded leaves from field-grown velvety D. wrightii were collected and brought to the laboratory. Leaves were surface-sterilized by soaking them for 3 min in a solution of 1% commercial bleach (equivalent to 0.0525% NaHClO3). Leaves were rinsed thrice in deionized water and kept turgid by placing their petioles in water reservoirs.

A suspension of acylsugars in water (10 mg/ml) was prepared, and a sonicating bath was used to keep the acylsugars in suspension. This was the highest concentration of acylsugars in water that would remain in suspension during the application process. Acylsugars were applied to velvety leaves by using an ultralow volume spray device modified from that described in Puterka and Severson (1995). A Nalgene aerosol spray bottle (# 2430-200, Nalgene, Rochester, NY, USA) was modified by affixing a commercial 3/8-in. pipe-to-hose adapter to the side of the spray bottle so that a hose from a cylinder of nitrogen or compressed air could be attached. For each application, 800 μl of the acylsugar suspension were transferred into a 12 × 75 mm test tube. This tube was placed into the spray bottle, and the pick-up tube of the sprayer was placed into the test tube.

To ensure uniform applications, a spraying stand was constructed from ring stands and clamps that consistently held the sprayer at the appropriate distance and angle from the leaf to be sprayed. The concept of the fixed spray platform was taken from Puterka and Severson (1995), but the design was modified in order to accommodate the larger leaves used in this study. The sprayer was clamped into a fixed position, and a 15-cm-diam plastic Petri dish was clamped 50 cm away from the nozzle of the sprayer in which the test leaf was placed. After the leaf and sprayer were set in proper position, the bottle was pressurized to 103 kPa, and all of the acylsugar suspension was sprayed onto the leaf and Petri dish.

In each trial, two of the 12 leaves were treated with 10 mg/ml of acylsugars per leaf surface (top and bottom) to serve as a check on the uniformity of application. Prior to treatment, the areas of these two leaves were determined with a leaf area meter. After treatment, the acylsugars were extracted from the leaves and quantified using methods described above. From this, the quantities of acylsugars actually applied to leaves per unit of leaf area were calculated.

Each trial consisted of five treatments. Control leaves were sprayed top and bottom with 800 μl of water. Leaves were treated with 20, 40, 80, or 120 mg/ml of acylsugars (1, 2, 4, or 6 applications per leaf surface of 800 μl of a 10 mg/ml mixture of acylsugars). These rates were chosen to span the range of acylsugars that occur on field-grown, sticky plants (see Results). Leaves sprayed top and bottom with 800 μl of a 10 mg/ml mixture of acylsugars received an average concentration of 0.097 (±0.003 SE, N = 40) μmol acylsugars/cm2 of leaf. Thus, the quantities of acylsugars applied to leaves were approximately equivalent to 0.097, 0.194, 0.388, and 0.582 μmol/cm2 of leaf. Leaves were returned to their water reservoirs to maintain leaf turgor while the leaf surfaces dried between applications.

For the no-choice bioassays, a piece of filter paper (Whatman #2, 12.5 cm) was placed into the bottom of a 15-cm-diam Petri dish and moistened with deionized water, then a whole treated leaf (above) was placed into the dish. One adult L. daturaphila was placed onto each treated leaf, whereas two T. compacta adults were used per leaf, and 10 adult E. hirtipennis were used per leaf. The dish was sealed with Parafilm® (American National Can, Menuasa, WI, USA) and the dish was placed into a growth room (constant 28°C, 16L:8D photoperiod). Insect feeding was scored after 48 hr as above.

For choice bioassays, only leaves from the control (water only) and 120 mg/ml treatments were utilized. Leaves were treated as described above and allowed to dry, then a cork borer was used to punch out 1.5-cm-diam disks from each leaf, avoiding the midrib. Pairs of disks were placed equidistant from the center of a 9.0-cm Petri dish on a disk of moistened filter paper (Whatman # 2, 7.0 cm) and insects added as above. For these tests, the number of T. compacta was reduced from two per dish to one. These experiments were repeated five times for L. daturaphila and T. compacta, but only three times for E. hirtipennis due to a shortage of insects. Additional procedures and data collection were as in the no-choice experiments above.

Data Analysis

Data from both the ester removal and ester addition experiments were analyzed by two-way mixed-model ANOVA using the PROC MIXED procedure of SAS with doses (no-choice) or treatments (choice) being fixed effects, and trials and the interaction between trials and treatments or doses being random sources of variation. For the choice trials, the potential variation among replicates within trials was also included in the model as a random source of variation to control for systematic differences in consumption rates among individual replicate insects (L. daturaphila and T. compacta) or groups of insects (E. hirtipennis) across treatments. The interaction between treatment and trials was pooled with the error term when not significant at P ≤ 0.25 prior to testing for significance of the fixed effects, following rules for pooling (Sokal and Rohlf, 1995). The square root (X + 0.5) transformation was applied to the feeding data for L. daturaphila and T. compacta, to ensure that the residuals were normally distributed, but the data for E. hirtipennis required no transformation.

Results

Variation in Acylsugar Concentration in the Field

Overall, acylsugar concentration varied from 0.03 to 0.84 μmol/cm2 of leaf. Acylsugar concentration differed significantly due to irrigation treatment, but not between blocks within treatments (irrigation F1,86 = 4.41, P = 0.039, pooled over blocks). Acylsugar concentration was 27% higher in unirrigated plants (Figure 1). The magnitude of variation over time was statistically significant (F4,86 = 34.26, P < 0.001), and mean concentrations in August were 2.6 times the concentrations in October (Figure 1). The interaction between irrigation treatment and month was not statistically significant (F4,86 = 1.41, P = 0.24). Families did not differ in their response either to irrigation or to season, as there was no significant effect of family or interactions of family with either months or irrigation (all P ≥ 0.15).

Fig. 1
figure 1figure 1

Least squares means (±SE) quantity of glucose ester produced per cm2 of leaf surface for watered and unwatered sticky plants sampled monthly. Data are pooled over blocks and families.

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The densities of glandular trichomes on sticky plants averaged 94.6 (±3.0 SE, N = 82) per mm2 and did not differ among families, irrigation treatments, or their interaction (all P ≥ 0.24, data not shown). Similarly, the density of nonglandular trichomes on velvety plants averaged 242.0 (±4.9 SE, N = 119) per mm2, and also did not differ among families, irrigation treatments or their interaction (all P ≥ 0.23, data not shown).

Ester Removal Experiments

When given a choice between sticky and velvety disks, both T. compacta and E. hirtipennis consumed more velvety foliage (F1,63 = 28.12, P< 0.001 for T. compacta and F1,59 = 9.26, P = 0.0035 for E. hirtipennis, Figure 2). There were more than 2.5 times the number of T. compacta feeding pits on velvety as sticky foliage (back transformed means) and 25% more E. hirtipennis feeding pits on velvety than sticky foliage (Figure 2). When given a choice between sticky foliage and washed sticky foliage, both species preferred washed sticky foliage (F1,44 = 8.78, P = 0.005 for T. compacta and F1,66 = 19.26, P < 0.001 for E. hirtipennis). There were more than 2.3 times the number of T. compacta feeding pits, and 46% more E. hirtipennis feeding pits on washed sticky foliage than on sticky foliage with acylsugars (Figure 2). When given a choice between washed sticky foliage and velvety foliage, the number of feeding pits from either species did not differ significantly (F1,36 = 2.63, P = 0.11 for T. compacta and F1,8 = 0.46, P = 0.52 for E. hirtipennis, Figure 2).

Fig. 2
figure 2figure 2

Least squares means (±SE) feeding by L. daturaphila, T. compacta, and E. hirtipennis adults on disks of sticky leaves with their acylsugars (S), disks of sticky leaves with the acylsugars removed by washing (WS), and disks of velvety leaves (V) in choice and no-choice bioassays. Top row: sticky vs. velvety choice; second row: washed sticky vs. sticky choice; third row: velvety vs. washed sticky choice; fourth row: no choice. The probability of a statistically significant difference in means is shown. n.s.: Not statistically significant.

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L. daturaphila consumed 1.6 times more sticky foliage (back transformed means) when given a choice between sticky and velvety foliage, (F1, 8 = 7.65, P = 0.025), but exhibited no significant choice for washed sticky foliage vs. sticky foliage with acylsugars (F1,8 = 1.56, P = 0.25), or washed sticky foliage vs. velvety foliage (F1,8 = 0.03, P = 0.87). In contrast to the bioassays with T. compacta and E. hirtipennis, the trial × type interactions were sufficiently large in the L. daturaphila bioassays that pooling of this potential source of variation with the error was not justified.

In no choice experiments, consumption of the three foliage types by T. compacta and E. hirtipennis differed significantly (F2,280 = 13.05, P < 0.001 for the former and F2,128 = 3.76, P = 0.026 for the latter.) Both species consumed more sticky foliage with the acylsugars removed than velvety foliage, followed by sticky foliage. Consumption of washed sticky foliage was 30% and 8% greater than velvety foliage, and 161% and 17% greater than sticky foliage for T. compacta and E. hirtipennis, respectively (Figure 2). L. daturaphila consumed similar quantities of all three types of foliage in no-choice experiments (F2,27 = 1.56, P = 0.23, Figure 2). Mortality of all three insect species was negligible in these experiments and was not recorded.

Ester Addition Experiments

In the choice experiments, only feeding by E. hirtipennis was significantly reduced by adding 0.582 μmol acylsugars/cm2 leaf (F1,61 = 43.81, P < 0.001), and the number of feeding pits was nearly cut in half by adding acylsugars to velvety foliage (Figure 3). Similarly, in the no-choice experiments, only feeding by E. hirtipennis was reduced by adding acylsugars to velvety leaves (F4,41 = 4.54, P = 0.004), and the highest quantity was no more effective in inhibiting feeding than the lowest (Figure 4). Neither T. compacta nor L. daturaphila showed any preference for treated or untreated velvety leaves in choice tests (F1,4 = 0.001, P = 0.97 for T. compacta and F1,4 = 0.06, P = 0.82 for L. daturaphila, Figure 3) nor feeding inhibition by acylsugars in no-choice tests (F4,41 = 0.92, P = 0.46 and F4,40 = 1.65, P = 0.18, respectively, Figure 4).

Fig. 3
figure 3figure 3

Least squares means (±SE) feeding by E. hirtipennis, T. compacta, and L. daturaphila adults on disks of velvety leaves either sprayed with water (Control) or with 0.582 μmol/cm2 of acylsugars in choice experiments. The probability of a statistically significant difference in means is shown. n.s.: Not statistically significant.

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Fig. 4
figure 4figure 4

Least squares means (±SE) feeding by E. hirtipennis, T. compacta, and L. daturaphila adults on velvety leaves either sprayed with water or with different concentrations of acylsugars in no-choice experiments. The probability of a statistically significant difference among means is shown. n.s.: Not statistically significant.

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Survival of L. daturaphila adults in the no-choice experiments averaged 98.0 ± 2.0%, whereas survival of T. compacta adults averaged 95.0 ± 2.0%, and survival of E. hirtipennis adults averaged 85.4 ± 2.4%. In no case was the variation in survival associated with acylsugar dose (all P ≥ 0.32, data not shown). Insect mortality in the choice experiments was similarly trivial and not analyzed.

Discussion

All three beetle species tolerate the acylsugars of D. wrightii, but the acylsugars moderately deter feeding by E. hirtipennis and T. compacta. The acylsugars were not toxic to any species. This is not surprising because all beetles commonly feed on D. wrightii in nature and might be expected to be less affected by the acylsugars of D. wrightii than herbivores that encounter D. wrightii less frequently, if at all, under natural conditions. The absence of any effect of acylsugars on feeding by L. daturaphila was not unexpected because feeding damage by this insect was shown to be independent of trichome type in detailed field studies (Elle and Hare, 2000; Hare and Elle, 2002). Observations of feeding behavior under the microscope show that both adults and larvae of L. daturaphila cope with glandular trichomes simply by feeding upon them and ingesting both the gland and the stalk until the trichome layer is short enough for the insects to begin feeding on leaf lamellar tissue. Such behavior implies that L. daturaphila has an effective physiological adaptation to acylsugars, although the mechanism of such an adaptation is not yet known.

T. compacta was more sensitive to acylsugars in the ester removal experiment than in the ester addition experiments. Feeding on sticky foliage was significantly reduced in all comparisons in the ester removal experiments (Figure 2), but not in the ester addition experiments (Figures 3 and 4). The application of acylsugars to velvety leaves does not completely mimic the presence of acylsugars on sticky leaves. Nonglandular trichomes of velvety leaves are less than 30% the length of glandular trichomes of sticky leaves (48 vs. 168 μm, van Dam et al., 1999), but occur at nearly 2.6 times the density. Application of acylsugars to velvety leaves replicates only the overall concentration per unit of leaf area, but not the location of the acylsugars at the tip of a long trichome. For weevils, the present results suggest that the location of the acylsugars on the tips of relatively long trichomes may be more important for deterrence than simply being present on the surface of leaves in a uniform layer. Future experiments to better understand the role of acylsugars on T. compacta, and perhaps other insect herbivores, might best be carried out by manipulating and exploiting natural variation in acylsugar production by glandular trichomes rather than by experimentally applying acylsugars to nonglandular foliage.

It is difficult to relate the relatively low biological activity of the acylsugars of D. wrightii reported here with the higher levels of other acylsugars against different insect species. High levels of mortality at relatively low doses (e.g., 1 mg/l or 1000 ppm or lower) are commonly reported against aphids, whiteflies, mites, and psyllids (Neal et al., 1994; Puterka and Severson, 1995; Liu et al., 1996), whereas only moderate feeding deterrence and negligible mortality occurred even after multiple applications of acylsugars totaling 120 mg/ml in the current study. It is unclear at this point to what extent the low mortality is the result of specific adaptations of all three coleopterans to D. wrightii’s acylsugars, or low overall toxicity of D. wrightii’s acylsugars in general. Different insect species may vary widely in their sensitivity to acylsugars (e.g., Neal et al., 1994; Puterka et al., 2003), and changes in either the sugars or the acid components of acylsugars often lead to unpredictable changes in biological activity (Puterka et al., 2003).

The range of acylsugar concentrations found in D. wrightii are similar to those found in other solanaceous species. For example, the concentration of total acylsugars in Lycopersicon pennellii lines ranged from 0.015 to 0.416 μmol/cm2 in one study (Goffreda et al., 1990) and from 0.035 to 1.265 μmol/cm2 in another (Mutschler et al., 1996). In L. pennellii, nearly a sevenfold range in concentration also occurred in different populations of the species when grown in a common garden (Shapiro et al., 1994). Although these acylsugars are responsible for resistance of L. pennellii to many pests of cultivated tomato, little is known about the biological significance of variation in the total quantities or composition of acylsugars in natural L. pennellii populations (Shapiro et al., 1994).

The prediction that E. hirtipennis and T. compacta would be more sensitive to the acylsugars of D. wrightii than L. daturaphila was supported by the ester removal experiments. Additional support for the hypothesis was provided by the ester addition experiments for E. hirtipennis, but the ester addition experiments provide more equivocal support of the hypothesis for T. compacta. In both the ester removal and ester addition experiments, biological activity appeared to be via inhibition of feeding rather than toxicity. In addition to providing absolute protection against whiteflies (van Dam and Hare, 1998a) and reducing feeding by Manduca sexta larvae (van Dam and Hare, 1998b), the acylsugars of D. wrightii also reduce feeding by E. hirtipennis and T. compacta. The more specialized L. daturaphila (Kogan and Goeden, 1970) was completely unaffected by D. wrightii’s acylsugars.