Effect of simulated and actual herbivory on alligator weed, Alternanthera philoxeroides, growth and reproduction

Biological Control 36 (2006) 74–79 www.elsevier.com/locate/ybcon

EVect of simulated and actual herbivory on alligator weed, Alternanthera philoxeroides, growth and reproduction Shon Schooler ¤, Zoë Baron, Mic Julien CSIRO Entomology and Cooperative Research Centre for Australian Weed Management, 120 Meiers Road, Indooroopilly, Qld 4068, Australia Received 30 March 2005; accepted 24 June 2005 Available online 24 August 2005

Abstract Simulated herbivory has often been used as a substitute for actual herbivory when assessing the eVects of herbivores on plants. However, mechanical damage does not always produce the same response as herbivore feeding. Similarity of plant response should be determined before mechanical damage is used as a surrogate for actual herbivory. This study compared two types of mechanical leaf tissue removal with actual herbivory on alligator weed [Alternanthera philoxeroides (Martius) Grisebach (Amaranthaceae)], a signiWcant wetland weed in Australia. We contrasted the eVects of simulated herbivory with those of actual herbivory by the alligator weed Xea beetle, Agasicles hygrophila Selman and Vogt (Chrysomelidae: Alticinae), on plant growth and reproduction. There was no signiWcant diVerence in the response of plant biomass to the three herbivory treatments. However, stem length and the number of stem nodes were reduced more by beetle damage than by the two simulated treatments. This was likely caused by preferential feeding on young leaf tissue and/or feeding damage to stem tissue as leaf tissue quality decreased at greater defoliation levels. To more closely mimic alligator weed response to A. hygrophila adult feeding for variables other than biomass, simulated damage methods should consider damage to new growth and stem tissue.  2005 Elsevier Inc. All rights reserved. Keywords: Agasicles hygrophila; Alternanthera philoxeroides; Alligator weed; Biological control of weeds; Predict eVectiveness; Simulate herbivory; Plant response; Insect–plant interactions

1. Introduction The chance of signiWcant non-target eVects from organisms used as biological control agents (Louda et al., 2003) places an increased onus on the part of biological control practitioners to demonstrate the eYcacy of candidate agents prior to their release (Colpetzer et al., 2004). One approach to understanding agent eVectiveness is to simulate herbivore damage and examine plant response (Raghu and Dhileepan, 2005). Simulated herbivory is a technique that oVers appreciable beneWts to researchers studying insect–plant interactions. It allows more precise control of the amount and *

Corresponding author. Fax: +617 3214 2885. E-mail address: [email protected] (S. Schooler).

1049-9644/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2005.06.012

timing of plant damage (Baldwin, 1990; Hjältén, 2004), as well as removing the logistical diYculties associated with manipulating herbivorous insects (Broughton, 2003). For practitioners of weed biological control, simulated herbivory can be used to determine plant response to diVerent types of damage (Colpetzer et al., 2004). Field experiments that simulate damage are a useful means of assessing eVectiveness of potential agents prior to expending resources on testing host speciWcity. As an example, a recent study using simulated herbivory on Lantana camara suggested that future directions in management of this weed should focus less on defoliation and more on reducing stored reserves (soluble carbohydrates in stems and roots) (Broughton, 2003). There are concerns surrounding the accuracy of simulated herbivory because it does not take into account the

S. Schooler et al. / Biological Control 36 (2006) 74–79

complexity of interactions between plant, herbivore, predator, and parasitoids that occur under natural conditions (Baldwin, 1990). A recent paper reviewed prior studies that compared simulated and actual herbivory (Lehtilä and Boalt, 2004). The authors found a mix of plant responses to the damage types, with some plants responding similarly to both and others responded diVerently. Even seemingly straightforward simulations, such as mimicking grasshopper damage to young wheat plants, can show signiWcant diVerences to actual herbivory (Capinera and Roltsch, 1980). However, simulated herbivory is a useful tool to study the eVects of herbivory when the diVerences are properly assessed (Baldwin, 1990; Hjältén, 2004; Welter, 1991). Two considerations can reduce the diVerences between actual and simulated damage. The Wrst is the choice of plant response variables. Simple response variables that are easy to measure (e.g., biomass and reproduction) are generally more similar in their responses to actual and simulated herbivory than those of complex plant responses (e.g., physiochemical responses of plant resistance) (Lehtilä and Boalt, 2004). Second, preliminary experiments that compare plant responses to diVerent types of mechanical damage with actual damage can lead to simulation techniques that more accurately mimic actual herbivory (Baldwin, 1990; Lehtilä and Boalt, 2004). Alligator weed, [Alternanthera philoxeroides (Martius) Grisebach (Amaranthaceae)], is a serious economic and environmental weed in Australia (Julien, 1995). It infests both aquatic and moist terrestrial habitats. A. philoxeroides does not produce viable seed in Australia, but reproduces asexually and spreads primarily through stem nodes containing axillary buds (Julien, 1995). Biological control is considered the best method of managing this weed due to its method of spread, which is exacerbated by mechanical control. In addition, its frequent proximity to potable water supplies often makes chemical control inappropriate (Sainty et al., 1998). A. philoxeroides has been successfully controlled in warm temperate aquatic habitats through the introduction of the alligator weed Xea beetle, Agasicles hygrophila Selman and Vogt (Chrysomelidae: Alticinae) (Julien, 1981; Sainty et al., 1998). However, there is a need for additional biological agents to control the weed in cooler climates and terrestrial habitats (Julien et al., 1995; Sainty et al., 1998). Simulated herbivory may be a useful method to determine whether selected agents will be eVective prior to introduction. In this study, we investigate the response of A. philoxeroides to defoliation by actual herbivory from the alligator weed Xea beetle, A. hygrophila, and two types of mechanical defoliation. If real and simulated damage are directly comparable for this agent, then simulated herbivory might also be useful for assessing the eVectiveness of potential leaf chewing biological control agents for

75

A. philoxeroides prior to release. We measured three response variables: two growth variables, biomass and stem length; and one reproductive variable, the number of stem nodes produced. We expected plant response to herbivory and simulated damage would be similar for all three variables.

2. Materials and methods 2.1. Experimental set-up Two hundred A. philoxeroides plants were propagated from tip cuttings approximately 1 month before the start of the experiment. The cuttings were 8 cm long (consisting of two nodes) and were planted vertically into 7 cm diameter pots. Ten days prior to the Wrst defoliation event, a homogeneous subset of 70 plants were selected and transplanted into 15 cm diameter pots. The 70 plants were randomly assigned to either caged treatments (60) or uncaged controls (10). The 60 plants used in the growth trial were placed in mesh cages (60 £ 60 £ 90 cm) and randomly arranged on benches in a temperature controlled glasshouse (25– 35 °C). The treatments were applied at Wve defoliation levels (0, 25, 50, 75, and 100%) and each level was replicated four times. Plants were randomly assigned to treatments and level of defoliation. Defoliation treatments were repeated three times (0, 7, and 21 days). The three treatments consisted of actual herbivory by A. hygrophila adults and two diVerent types of simulated herbivory; simple and complex. A defoliation event was initially scheduled for 14 days, however, an infestation of broad mite [Polyphagotarsonemus latus (Banks)] was discovered on some plants and consequently all plants in the trial were sprayed with a 0.15 mL/L water solution of the miticide Vertimec (Syngenta, Australia). Since the beetles may have been adversely aVected by the treatment, we didn’t damage the plants during that week. The mites were found on plants of all treatments as well as on the uncaged control plants and did not appear to particularly aVect any one treatment. 2.2. Actual herbivory Adult A. hygrophila beetles were used to damage leaves for the actual defoliation treatment. Approximately 400 beetles were collected from an A. philoxeroides infestation at Raymond Terrace (NSW) and maintained as a colony in an air-conditioned greenhouse (20–30 °C) for the duration of the experiment. Beetle colonies were maintained in a separate greenhouse to reduce chance of beetles feeding on uncaged control plants and to provide lower and less variable temperatures. During the defoliation events the beetles were moved to the experimental greenhouse and placed in

76

S. Schooler et al. / Biological Control 36 (2006) 74–79

cages with the plants. They were left to feed until the desired level of damage was reached. The number of beetles varied from 5 to 40 per cage and the period of time that they were allowed to feed varied from 2 to 4 days in most cases. In two cases the beetles were left on for 6 days before the required defoliation level had been achieved. To increase speed of defoliation, the beetles were starved for 24 h before putting them into the cages. When defoliation reached the target level the beetles were removed with an aspirator and returned to the colony. The study plants were then checked for eggs that had been oviposited while the beetles were feeding on the plant. Any eggs (that are laid in batches on the under side of leaves) were wiped oV with a damp cloth to avoid the experiment being complicated by larval damage. 2.3. Simulated herbivory The two types of simulated herbivory were a simple defoliation method and a complex method. The simple method involved cutting the leaf perpendicular to the mid vein. Each plant had the same percentage of each leaf removed. The complex method was designed to more closely mimic the patterns of beetle damage using a standard size hole punch (6 mm diameter). The hole size that adult beetles make tended to be smaller (2–6 mm diameter), but we used the maximum size to speed damage allocation. The punch removed a portion of the leaf while leaving the mid-vein intact. As with the simple simulated damage, each leaf of the plant had the same percentage of the leaf tissue removed. During the second and third defoliations it was necessary to re-clip some of the leaves that had increased in size since the previous defoliation. This was particularly the case with the terminal leaves. The timing of the manual defoliation coincided with the actual herbivory for each defoliation event, however, the duration of damage inXiction was diVerent. The simulated damage was done over several minutes for each plant, while the beetle damage was done over a number of hours or days. 2.4. Estimating the damage levels To maintain a consistent level of defoliation based on the treatment to which plants were allocated, damage levels were non-destructively estimated during the experiment using a series of damage charts that were prepared prior to the experiment. These charts exempliWed varying levels of damage for each of the treatments. In a preliminary experiment on a separate set of plants, leaves were subjected to the three defoliation methods, then removed from plants and pressed. The percent leaf damage for these charts was determined using graph paper (1 mm2 grid) and a light table. Area damaged was divided by total leaf area and multiplied by 100 to calculate percent damage. These charts were then used during the experiment to estimate plant damage. The defoliation

assessment was undertaken by one person throughout the experiment to minimise bias. The Wnal measurement of defoliation was calculated as the mean percent leaf damage from Wrst defoliation event to harvest. At the conclusion of the experiment, Wve leaves from each plant were randomly selected and the mean percent leaf area damaged was determined using Scion Imaging software (version 4.02, http://www.scioncorp.com/). Direct measurements were then compared to the visual estimates to assess observer bias. 2.5. Measuring the eVects of the treatments The eVect of the damage was assessed 1 week after the Wnal defoliation event. We measured three response variables: two growth variables, biomass and stem length; and one reproductive variable, number of stem nodes produced. After measuring stem length and the number of nodes, all plants were harvested and separated into roots, stem, and leaves. The roots were washed to remove soil. All plant parts were dried to constant weight at 60 °C and then weighed to determine total biomass. In addition, non-destructive measurements were taken each week and at the end of the experiment (number of leaves, stems, and nodes and length of stems). 2.6. Statistical analysis The eVect of the cages was determined through a Student’s t test. Linear regression analysis was used to examine whether the visual estimates of damage were similar to measurements using scanned leaves. Biomass, number of nodes, and stem length were regressed on damage level to determine signiWcance of the eVect of each type of defoliation. The similarity of treatment eVect for each plant response variable was determined by comparing regression coeYcients using F tests. SigniWcant diVerence was Wrst assessed for all three treatments. Where a diVerence was found, pair-wise comparisons were made to determine which treatments diVered from each other. Procedure follows Snedecor and Cochran (1967). Statistical analyses were done in Excel (2002, Microsoft) and S-plus (ver. 6.1, Insightful Corp.). One of the plants from the 50% defoliation level in the simple treatment was snapped in half while being handled and consequently its data were excluded from the analyses.

3. Results A comparison of the biomass between caged and uncaged control plants indicated that there was no eVect of cage (t D 1.287, dfs D 20, P > 0.05). In addition, visual damage estimates did not diVer from measurements using scanned leaves (beetle, n D 20; simple, n D 19; complex n D 20; P > 0.05 for all treatments).

S. Schooler et al. / Biological Control 36 (2006) 74–79

3.1. Biomass

70

3.2. Number of stem nodes Damage decreased node production for all treatments (beetle—F D 42.2; dfs D 1,26; P < 0.001: simple—F D 12.5; dfs D 1,25; P D 0.002: complex—F D 5.2; dfs D 1,26; P D 0.03) (Fig. 2). Beetle damage reduced the number of

simple

complex

beetle

simple

complex

50

40

30

beetle y = -0.32x + 41.4 R2 = 0.62 F = 42.2, d.f.s. = 1,26 P < 0.001

20

10

The biomass of the plants at the end of the experiment showed similar responses to the diVerent defoliation types (Fig. 1). Regression analysis indicated that all treatments resulted in lower biomass at the higher defoliation levels (beetle—F D 49.6; dfs D 1,26; P < 0.001: simple—F D 42.2; dfs D 1,25; P < 0.001: complex—F D 23.2; dfs D 1,26; P < 0.001). An analysis of the regression coeYcients revealed that there was no signiWcant diVerence between the treatments (F D 2.70; dfs D 2,77; P D 0.074).

beetle

60

Number of nodes

During the experiment we observed two diVerences between the simulated and actual damage treatments. First, beetles tended to concentrate damage on young tissues near the apical meristem. Second, beetles occasionally fed on stem tissues. The beetles caused minor damage to the stems of some plants at all defoliation levels but this was under 5% of total stem area for all but plants in the highest defoliation category (cf. 100%). In this category, the stem damage was as high as 40% and resulted in some of the stem tips falling oV. These plants were included in the analysis as it was a direct eVect of the herbivores.

77

simple y = -0.20x + 42.3 R2 = 0.34 F = 12.5, d.f.s. = 1,25 P = 0.002

complex y = -0.12x + 44.2 R2 = 0.17 F = 5.2, d.f.s. = 1,26 P = 0.03

0 0

20

40

60

80

100

Defoliation (%)

Fig. 2. Greater defoliation levels decreased node production for all three treatments (P < 0.05). Plants subjected to beetle defoliation produced fewer stem nodes than those of the simple (F D 6.33; dfs D 1,51; P D 0.015) and complex (F D 20.04; dfs D 1,52; P < 0.001) defoliation treatments. Manual damage treatments were not diVerent from each other in their node production (F D 3.30; dfs D 1,51; P D 0.075).

nodes by approximately double of that which occurred by manual defoliation and the diVerence was signiWcant for both simple (F D 6.33; dfs D 1,51; P D 0.015) and complex (F D 20.4; dfs D 1,52; P < 0.001) manual damage treatments. The two types of manual defoliation were not diVerent from each other (F D 3.30; dfs D 1,51; P D 0.075). 3.3. Stem length

2.5

beetle simple complex beetle

2.0

simple

Ln plant biomass (g)

complex

1.5

1.0 beetle y = -0.01x + 1.42 R2 = 0.66 F = 49.6, d.f.s. = 1,26 P < 0.001

0.5

simple y = -0.01x + 1.50 R2 = 0.63 F = 42.2, d.f.s. = 1,25 P < 0.001

complex y = -0.007x + 1.48 R2 = 0.47 F = 23.2, d.f.s. = 1,26 P < 0.001

0.0 0

20

40

60

80

100

Stem length decreased with increasing damage for the beetle treatment (F D 25.1; dfs D 1,26; P < 0.001), but not for the simple (F D 4.2; dfs D 1,25; P D 0.052) or complex (F D 1.9; dfs D 1,26; P D 0.176) manual damage treatments (Fig. 3). The plants that had been subjected to beetle defoliation had shorter stems than those that were exposed to simple (F D 11.80; dfs D 1,51; P D 0.001) or complex (F D 21.28; dfs D 1,52; P < 0.001) artiWcial damage, whereas stem length did not diVer between the two manual damage treatments (F D 0.74; dfs D 1,51; P D 0.39). However, the Wt of the regression line for the simple and complex simulated treatments was poor (R2 D 0.14 and 0.07, respectively).

4. Discussion

Defoliation (%)

Fig. 1. Increased defoliation resulted in lower plant biomass for all treatments (P < 0.001). Response of biomass did not diVer between the three defoliation treatments (F D 2.70; dfs D 2,77; P D 0.074). Biomass was natural log transformed to achieve constant variance.

The three diVerent treatments all showed a similar response in biomass production at the Wnal harvest. This suggests that manual defoliation is an appropriate method to use when investigating the eVects of potential

78

S. Schooler et al. / Biological Control 36 (2006) 74–79 1200

beetle

simple

complex

beetle

simple

complex

Stem length (mm)

1000

800

600

beetle y = -3.18x + 803 2 R = 0.49 F = 25.1, d.f.s. = 1,26 P < 0.001

400

simple y = -1.11x + 804 R2 = 0.14 F = 4.2, d.f.s. = 1,25 P = 0.052

200

complex y = -0.57x + 795 R2 = 0.07 F = 1.9, d.f.s. = 1,26 P = 0.176

0 0

20

40

60

80

100

Defoliation (%)

Fig. 3. Stem length decreased with increasing damage for beetle damaged plants (P < 0.001), but not for simple or complex damage treatments (P > 0.05). Plants subjected to beetle damage were more greatly aVected than those subjected to simple (F D 11.80; dfs D 1,51; P D 0.001) or complex (F D 21.28; dfs D 1,52; P < 0.001) manual defoliation treatments. Manual damage treatments were not diVerent from each other in stem length (F D 0.74; dfs D 1,51; P D 0.39).

biological control agents on biomass. Additionally, as both types of manual defoliation showed similar responses it would be acceptable to use the simple defoliation technique in such studies. An advantage of this method is that less time is taken to implement the damage (the complex damage took about three times as long). Stem length and node production diVered between the actual and simulated damage treatments. This indicates that the reproductive potential of the plant (node production) at diVering damage levels cannot be inferred from this type of simulated damage. A possible reason for this result is that during the trial the beetles preferentially fed on the young leaves of the plant and, in doing so, they may also have been destroying the axillary buds at the nodes. This may have resulted in reduced stem length and fewer nodes being produced. Alternatively, the diVerence in stem length and node number may be the result of stem damage by the beetles at the higher defoliation levels. Since biomass is directly related to stem length, the result of no diVerence in biomass response and diVerence in node and stem length response is counter-intuitive. However, we found that when stem tips were damaged, plants tended to produce new shoots from axillary meristems. This resulted in shorter plants that maintained equal biomass with taller plants. We did not detect a cage eVect. This indicates that the results we found were not due to a cage by treatment interaction. In addition, visual estimates of damage did not diVer from measured damage levels. This suggests that visual estimates were a reliable measurement of per-

cent leaf damage. However, even the method using scanned images involved some estimation to reconstruct leaf edges. This was primarily an issue at the higher levels of defoliation. The uniformity in shape of A. philoxeroides leaves aided in this estimate, however, the method might not be suitable for plants with irregular leaves or complex leaf margins. The review of prior comparisons of simulated damage with actual feeding indicated that the responses of simple variables (i.e., biomass and reproduction) were more likely to be similar than those of complex variables (i.e., inducing plant resistance compounds) (Lehtilä and Boalt, 2004). Therefore, we expected that eVects of herbivory on plant biomass, stem length, and node production would be relatively easy to simulate. However, we found that even these “simple” variables, such as stem length and node production, were not similar in their response. This result indicates that it is important to consider position of damage (i.e., feeding preference) particularly when investigating the response of plant morphological traits to herbivore damage. This study compared the eVect of adult beetle feeding with simulated damage methods. Observations of adult and larval feeding suggest that larval beetles inXict similar damage on leaf tissues and we expect that the eVect of larval herbivory on plant biomass, stem length, and node production will be similar to that of adult feeding. Both adults and larvae tend to preferentially feed on younger tissues and therefore the damage simulation techniques of this study will likely also underestimate the impact of larval feeding on plant height and node production. Studying the response of invasive plant populations to herbivore damage can improve our prediction of the eVectiveness of biological control agents (Colpetzer et al., 2004; Raghu and Dhileepan, 2005). As A. philoxeroides is not eVective at reducing A. philoxeroides abundance in terrestrial habitats or in cool temperate climates (Julien et al., 1995; Sainty et al., 1998), currently CSIRO Entomology is studying several additional agents. The most promising is the Xea beetle Disonycha argentinensis Jacoby (Chrysomelidae: Alticinae). This beetle is closely related to A. hygrophila and does similar damage to leaf tissue (Schooler, personal observation). The results of this study suggest that simulated damage in Weld plots will allow researchers to predict the eVectiveness of this agent on reducing A. philoxeroides biomass prior to its release. However, care must be taken when assessing competition with other plants species as plant height may be underestimated using simulated damage, which may lead to an underestimation of real herbivore impact. Improved simulation techniques, such as clipping apical tips, may remedy this problem. The ability to accurately simulate herbivory has a number of important advantages over natural herbivory. The amount of biomass removed can be directly measured, the magnitude of damage can be easily and accu-

S. Schooler et al. / Biological Control 36 (2006) 74–79

rately manipulated, and confounding biotic and abiotic eVects can be controlled (Hjältén, 2004). Another important advantage of simulated herbivory is that it gives researchers the ability to study the eVect of herbivory on invasive plants in their introduced habitats prior to the deployment of any specialist herbivores as biological control agents (Raghu and Dhileepan, 2005). The success of classical weed biological programs suggests that the populations of some invasive plants are primarily regulated by herbivores (Keane and Crawley, 2002; Raghu and Dhileepan, 2005). However, availability of resources and presence of disturbances speciWc to the invaded environment also aVects plant competition and these factors should be studied in concert with herbivory to identify integrated strategies for invasive plant management (McEvoy and Coombs, 1999; Paynter and Flanagan, 2004). Studies are currently underway that will use simulated herbivory to address the relative importance of bottom-up eVects of resource availability and the top-down eVects of herbivory on the competitive ability of invasive plants in Australia. In conclusion, plant biomass responded similarly to manual defoliation and beetle damage. Therefore, manual defoliation is an accurate means of simulating the eVects of beetle damage when biomass is the principal variable of interest. Furthermore, the simple damage method did not diVer from either the complex method or actual damage. This is beneWcial to future studies as the simple damage was both easier to apply and easier to accurately estimate. Although simulated damage is useful when considering biomass, it will need to be reWned when measuring the eVect of defoliation on other variables. This may include concentrating damage on young tissue and damaging stem tissues at higher defoliation levels. Acknowledgments Thanks to S. Raghu and Rose DeClerck-Floate for providing constructive comments on project design and for reviewing the manuscript. Gio Fichera helped with watering and provided advice on pest treatment. Dalio Mira, Areli Mira, and JeV Mackinson helped with the maintenance of the plants. Rose DeClerck-Floate and Mary Gellender helped with plant processing. Thanks to the editor, Andy Shepard, and two anonymous reviewers for their constructive comments. Funding for Z. Baron was provided by the CRC for Australian Weed Management through its Summer Student Program.

79

References Baldwin, I.T., 1990. Herbivory simulations in ecological research. Trends Ecol. Evol. 5, 91–93. Broughton, S., 2003. EVect of artiWcial defoliation on growth and biomass of Lantana camara L. (Verbenaceae). Plant Protect. Quart. 18, 110–115. Capinera, J.L., Roltsch, W.J., 1980. Response of wheat seedlings to actual and simulated migratory grasshopper defoliation. J. Econ. Entomol. 73, 258–261. Colpetzer, K., Hough-Goldstein, J., Harkins, K.R., Smith, M.T., 2004. Feeding and oviposition behaviour of Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae) and its predicted eVectiveness as a biological control agent for Polygonum perfoliatum L. (Polygonales: Polygonaceae). Environ. Entomol. 33, 990–996. Hjältén, J., 2004. Simulating herbivory: problems and possibilities. In: Weisser, W.W., Siemann, E. (Eds.), Ecological Studies, Vol. 173: Insects and Ecosystem Function Springer-Verlag, Berlin, pp. 243– 255. Julien, M.H., 1981. Control of aquatic Alternanthera philoxeroides in Australia: another success for Agasicles hygrophila. In: Delfosse, E.S. (Ed.), Proceedings of the Fifth International Symposium on Biological Control of Weeds. CSIRO, Melbourne, pp. 583–588. Julien, M.H., 1995. Alternanthera philoxeroides (Mart.) Griseb. In: Groves, R.H., Shepherd, R.C.H., Richardson, R.G. (Eds.), The Biology of Australian Weeds. R.G. and F.J. Richardson, Frankston, pp. 1–12. Julien, M.H., Skarratt, B., Maywald, G.F., 1995. Potential geographical distribution of alligator weed and its biological control by Agasicles hygrophila. J. Aquat. Plant Manage. 33, 55–60. Keane, R.M., Crawley, M.J., 2002. Exotic plant invasions and the enemy release hypothesis. Trends Ecol. Evol. 17, 164–170. Lehtilä, K., Boalt, E., 2004. The use and usefulness of artiWcial herbivory in plant-herbivore studies. In: Weisser, W.W., Siemann, E. (Eds.), Ecological Studies, Vol. 173: Insects and Ecosystem Function Springer-Verlag, Berlin, pp. 257–275. Louda, S.M., Pemberton, R.W., Johnson, M.T., Follett, P.A., 2003. Nontarget eVects—the Achilles’ heel of biological control? Retrospective analyses to reduce risk associated with biocontrol introductions. Annu. Rev. Entomol. 48, 365–396. McEvoy, P.B., Coombs, E.M., 1999. Biological control of plant invaders: regional patterns, Weld experiments and structured population models. Ecol. Appl. 9, 387–401. Raghu, S., Dhileepan, K., 2005. The value of simulating herbivory in selecting eVective weed biological control agents. Biol. Control 35, in press. Paynter, Q., Flanagan, G.J., 2004. Integrating herbicide and mechanical control treatments with Wre and biological control to manage an invasive wetland shrub, Mimosa pigra. J. Appl. Ecol. 41, 615–629. Sainty, G., McCorkelle, G., Julien, M., 1998. Control and spread of Alligator Weed Alternanthera philoxeroides (Mart.) Griseb., in Australia: lessons for other regions. Wetlands Ecol. Manage. 5, 195– 201. Snedecor, G.W., Cochran, W.G., 1967. Statistical Methods. Iowa State University Press, Ames, Iowa. Welter, S.C., 1991. Responses of tomato to simulated and real herbivory by tobacco hornworm (Lepidoptera: Sphingidae). Environ. Entomol. 20, 1537–1541.