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Confused by domestication: incongruent behavioral responses of the sunflower moth, Homoeosoma electellum (Lepidoptera: Pyralidae) and its parasitoid, Dolichogenidea homoeosomae (Hymenoptera: Braconidae), towards wild and domesticated sunflowers
Biological Control 28 (2003) 180–190 www.elsevier.com/locate/ybcon
Confused by domestication: incongruent behavioral responses of the sunflower moth, Homoeosoma electellum (Lepidoptera: Pyralidae) and its parasitoid, Dolichogenidea homoeosomae (Hymenoptera: Braconidae), towards wild and domesticated sunflowers Yolanda H. Chen* and Stephen C. Welter Division of Insect Biology, Wellman Hall, University of California, Berkeley, CA 94720-3112, USA Received 7 December 2001; accepted 2 April 2003
Abstract Due to domestication, agricultural plants and their wild ancestors encompass a wide range of variation, which is useful for identifying promising plant attributes that promote parasitoid activity. We examined the behavioral responses of the sunflower moth, Homoeosoma electellum Hulst and its major parasitoid, Dolichogenidea homoeosomae Muesebeck to agricultural and wild sunflowers. We found that female moths preferred to land on, and lay more eggs on agricultural plants. While female parasitoids preferred to land on infested agricultural sunflowers, they foraged poorly on them. Parasitoids spent 74% more time foraging on wild flowers than on agricultural flowers, and they probed for a significantly longer period of time for host larvae on wild flowers. As a result, each individual female parasitized 25.7% 0.06 more larvae on wild flowers than on agricultural flowers with the same larval density. Also, parasitoids probed for hosts four times longer on wild flowers than on agricultural flowers, while resulting parasitism levels were 19 times higher, indicating that parasitoids were more efficient on wild flowers. Agricultural flowers are about four times larger than wild flowers, and the increase in flower size correlated with higher moth oviposition but lower parasitoid efficiency. Our results show that changes to plant attributes through domestication can disrupt host–parasitoid interactions and limit the ability of parasitoids to control insect pests effectively. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Homoeosoma electellum; Dolichogenidea homoeosomae; Behavior; Sunflowers; Domestication; Agricultural; Wild; Foraging behavior; Sunflower moth; Parasitoid behavior
1. Introduction It is widely known that plant attributes can mediate interactions between insect herbivores and their natural enemies (Agrawal et al., 2002; Andow and Prokrym, 1990; Benrey et al., 1998; Benrey and Denno, 1997; Bottrell et al., 1998; Damman, 1987; Kalule and Wright, 2002; Lovinger et al., 2000; Price, 1986; Price et al., 1980; Turlings et al., 1995; Udayagiri and Welter, 2000). Parasitoid–herbivore interactions can be influenced by physical, nutritional, or chemical plant attributes * Corresponding author. Fax: +510-642-0477. E-mail address: [email protected] (Y.H. Chen).
(Cortesero et al., 2000; Price, 1986; Price et al., 1980). In order to manipulate these interactions, it is necessary to determine how plant attributes can enhance parasitoid activity. By understanding the relative behavioral responses of parasitoids and their herbivore hosts, plant breeders may select for particular plant attributes that promote biological control (Cortesero et al., 2000). Agricultural plants and their wild ancestors encompass a wide range of phenotypic variability in physical and chemical attributes as a result of domestication and selection (Evans, 1993; Gouinguene et al., 2001; Massei and Hartley, 2000; Rosenthal and Dirzo, 1997). Given that physical and chemical plant attributes play an essential role in host location and acceptance for
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parasitoids and hosts, it is likely that parasitoid–herbivore interactions may differ on agricultural plants and their wild ancestors (Evans, 1993; Geervliet et al., 1996; Gouinguene et al., 2001; Renwick and Chew, 1994; Thompson and Pellmyr, 1991). However, it is not clear how changes in parasitoid–herbivore interactions on agricultural plants may affect biological control. For example, agricultural plants have larger plant structures for economically important attributes and reduced architectural complexity, which may alter herbivore distribution and influence parasitoid foraging success (Andow and Prokrym, 1990; Grevstad and Klepetka, 1992). Also quantitative and qualitative differences in semiochemical production may differ between agricultural crops and their relatives (Gouinguene et al., 2001), which may alter herbivore or parasitoid host location and acceptance (Benrey et al., 1998). Understanding how herbivores and parasitoids respond to agricultural and wild plants will help identify promising plant attributes that may be manipulated to enhance biological control. Given that the majority of modern crops grown in North America originated from other continents (Evans, 1993; Harlan, 1971), assessing how domestication has altered parasitoid–herbivore interactions is limited within the United States. However, the domesticated sunflower, Helianthus annuus var. macrocarpus L., and its wild ancestor, H. annuus var. annuus L., represent a rare exception to this constraint, as wild and agricultural sunflower populations are sympatric, and the wild H. annuus is native throughout North America. Furthermore, molecular and isozyme data indicate that the domesticated sunflower was derived from a wild form of H. annuus (Arias and Rieseberg, 1995; Rieseberg and Seiler, 1990). The sunflower system is also particularly amenable to this study because it is attacked by a large assemblage of native herbivores (Rogers, 1988a,b). Among them, the sunflower moth, Homoeosoma electellum Hulst (Lepidoptera: Pyralidae) is considered the most important pest of sunflowers in North America (Charlet et al., 1997; Schultz, 1978). H. electellum is attacked by a large assemblage of parasitoids, and Dolichogenidea homoeosomae Muesebeck (Hymenoptera: Braconidae) is the most important parasitoid in both native and agricultural habitats in the San Joaquin Valley in California (Chen and Welter, 2002). We found previously that H. electellum was more abundant and less parasitized on agricultural plants, even when wild sunflowers were grown alongside them in a common garden setting (Chen and Welter, 2002). Our previous field results suggest that the differences in herbivory and parasitism may be due to differences in moth and parasitoid behavioral responses to agricultural and wild sunflowers. If D. homoeosomae prefer wild sunflowers, they may have less impact on agricultural plants. Clearly, the observed differences in
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parasitism levels between agricultural and wild sunflowers suggest that opportunities exist for manipulating plant attributes to enhance biological control in agricultural sunflowers. Our previous work indicated that nitrogen-enrichment increased plant phenotypic differences between wild and agricultural sunflowers, which further increased the differences in moth larval abundance and parasitism (Chen and Welter, unpublished results). Therefore, we designed this study to test for the most extreme differences in behavioral responses to agricultural and wild sunflowers using nitrogen-enriched plants. We recognize that behavioral responses studied here may be representative of interactions found in the field, but do not represent the entire spectrum of possibilities. One exception was made to test the effect of nitrogen-enrichment on moth ovipositional preferences to wild and agricultural sunflowers, because the field patterns suggested that sunflower moth ovipositional responses may be in response to flower size, which is influenced by nitrogen content (Chen and Welter, 2002). To determine whether there are differences in behavioral responses of the sunflower moth and its parasitoid, D. homoeosomae, to wild and agricultural sunflowers, we asked the following questions: (1) Do na€ıve female moths prefer to land on agricultural vs. wild sunflowers? (2) Does nitrogen-enrichment interact with plant genotype to influence na€ıve female moth ovipositional preference? (3) Do na€ıve parasitoids differ in their landing preferences on infested agricultural vs. infested wild sunflowers? (4) Does parasitoid foraging behavior differ by plant genotype and larval density? (5) Does previous experience on wild sunflowers influences parasitoid foraging behavior on agricultural sunflowers?
2. Materials and methods 2.1. Insect and plant cultures We used an agricultural sunflower genotype that is grown in the northern San Joaquin Valley and was previously studied in the field (Chen and Welter, 2002). The wild sunflower plants were grown from seeds collected in the Stone Lake Wildlife Refuge, Sacramento, CA and Yolo Bypass Wildlife Area, Yolo, CA. Although wild sunflowers represent multiple genotypes (Rieseberg and Seiler, 1990), they will be collectively identified as the wild genotype in this study. Wild sunflower seeds were germinated in a moistened paper towel held in a plastic bag. Agricultural sunflower seeds and germinated wild seedlings were planted into 6 cm diameter plastic cells on the same date to synchronize flowering. After two weeks of growth in the greenhouse, plants were transplanted into 11.4 liter pots containing a
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peat moss and sand soil mixture. In the fourth week, plants in the high nitrogen treatments received: 6.5 g 42–0–0 (NH4 NO3 with 6% S), 3.4 g 0–48–0 (P2 O5 with 15% Ca), and 7.3 g 0–0–51 (K2 O with 17% S), and non-enriched treatments received only phosphorous and potassium. Low-nitrogen treatments did not receive any fertilizer. These fertilizer formulations were used so that each macronutrient could be added individually, and therefore S and Ca levels could have been raised. Seed set was promoted by hand pollinating the flowers, and excess pollen was removed by tapping the flower heads. We established a colony of H. electellum using larvae collected from agricultural and wild sunflower fields in Yolo and Sacramento counties. Moths were reared on an agar-based diet (Wilson, 1990) at 22 °C under a 16L:8D light cycle for several generations before the study began to ensure adequate numbers for the study and remove maternal effects. Females observed to mate 1–3 days after emergence were used in the behavioral assay. Mating couples were isolated in the cage and then, females were held overnight under artificial light in a plastic cup with a cotton ball moistened with 5% sugar water solution. Female moths were used in the behavioral assays the next day. For the parasitism studies, we transferred first instar larvae equally spaced onto agricultural and wild flowers in bloom. Larvae were allowed to develop for two to four days at 25 °C in a greenhouse and exposed to the parasitoids as a second or third instar in the behavioral assays because parasitism in the field is highest in these stages (Chen, unpublished results). On the morning of the behavioral observations, artificially infested flowers were cut and the stems were placed into containers filled with water. Using calipers, we measured flower diameter, which was the distance across the face of the flower, and flower depth, which was the thickness of the flower from the base to the top of the florets. Na€ıve parasitoids were obtained by collecting wild parasitoids and allowing them to oviposit on infested sunflowers. Moth larvae were then reared out on artificial diet. We also obtained na€ıve parasitoids by rearing larvae collected from the field on artificial diet. We monitored parasitoid emergence by checking the cups three times a week. Experienced parasitoids were collected weekly from the field. All parasitoids were kept for 2–3 days in 36 ml vials streaked with honey before behavioral assays and between trials. 2.2. Moth landing preference Female moth landing preference was assessed in a rectangular wind tunnel (261 85:5 86:5 cm) with a laminar flow of 30 cm/s. The room was lit with a 60 W light bulb that was dimmed over an hour at a rate of 10% every 7 min to simulate dusk. Six 60 W red incan-
descent lights lined one side of the wind tunnel arena and were dimmed to 30% of full intensity. A 60 W incandescent light bulb was positioned outside of the wind tunnel to shine on the experimental flowers. This approach was used to stimulate a higher proportion of moths to fly upwind towards the part of the tunnel with the target plants, but did not influence landing preference. An agricultural and a wild flower were placed into test tubes filled with water and attached to a ring stand with test tube clamps. The position of the flowers within the wind tunnel was randomly determined on each day of the experimental assay. To reduce quantitative differences in volatile production due to the difference in flower head size, we standardized flower size by using the smaller lateral flowers on agricultural plants. The flowers were changed each day of the study or if moths landed on them. The area of the face of the flower, excluding the ray petals, was determined by measuring the longest diameter across the flower (r1 ) and the perpendicular diameter (r2 ), and calculating the area of an ellipse (A ¼ pðr1 r2 )). We determined that each pair of treatment flowers were equivalent in size using a paired t test. Each replicate consisted of the release of a single female moth onto a cloth-covered circular platform positioned at the same height as the target flowers (N ¼ 71). If the moth did not take off from the platform after 5 min or did not land on the flowers after 15 min, she was removed from the wind tunnel. Each female was allowed three trials, after which failure to land on a flower resulted in permanent removal from the study. For a moth that landed on a flower, its behavior was observed on the target flower in two ways: by recording the total time on the flower and by diagramming its route to calculate the proportion of the flower head covered. Female landing preference was analyzed using a binomial test with an expected probability of 0.5. Total time spent on the flower was analyzed using a WelchÕs t test for unequal variances in S-Plus 4.5 (Mathsoft Inc., 1998). The proportion of the flower head covered by the moth was analyzed with a StudentÕs t test. 2.3. Moth ovipositional preference We assessed female ovipositional response to plant genotype and nitrogen-enrichment. The four treatments were: (1) low nitrogen agricultural sunflowers, (2) high nitrogen agricultural sunflowers, (3) low nitrogen wild sunflowers, (4) high nitrogen wild sunflowers. Two types of assays were conducted: no-choice assays in cages where only a single treatment was present, and choice assays, in cages where all four treatment flowers were present. Flower size was not standardized in the moth oviposition studies.
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2.3.1. No-choice assay Mated females were held at room temperature until dusk and then introduced individually into a 0:03m3 metal cage with cloth sides. Each replicate consisted of a single treatment flower that was placed into the cage and exposed to a single moth for 24 h. A minimum of five replicate cages were used for each treatment. The cages were lit on a 16:8 light:dark schedule and room temperature was 22 °C. A fan, positioned 1.5 m away from the cages, blew air towards the cages at a low speed to provide air circulation. Flowers were processed under a dissecting microscope to count the total number of eggs and number of egg clusters. We tested whether the total number of eggs laid differed by plant genotype and nitrogen-enrichment in separate two-way ANOVA tests in JMP 3.2.2 (SAS Institute, 1997). For moths that laid eggs on the flowers, we tested whether the number of clutches and average clutch size differed using a two-way ANOVA test.
T 2 > ðK 1Þðn 1ÞðFK1;nKþ1 Þ=ðn K 1Þ:
2.3.2. Choice assay Female moth ovipositional preference to nitrogenenriched and non-enriched agricultural and wild flowers was experimentally assessed in a Plexiglas cage (61 61 30:5 cm) with cloth sides to allow airflow. The study was conducted in a greenhouse room at 27.1 °C 0.3, with two box fans to circulate the air within the greenhouse room. Each replicate consisted of a flower from each of the four treatment groups. Flowers were randomly assigned to the four 8.5 cm circular holes in the floor of the cage. Circular sponges, fit around the stem of the flower, were flush with the bottom of the floor and the stems were placed in 160 ml vials filled with water. Each replicate consisted of a single gravid female introduced into a cage for 24 h (N ¼ 62). Flowers were examined for the total number of eggs laid, the number of clutches laid, and clutch size. Moths that did not lay eggs on any plants were removed from the study. Because the treatments were enclosed together in a cage, the number of eggs laid on each treatment was not independent between treatments and thus violated the assumptions of v2 , nonparametric, and parametric tests (Roa, 1992). Therefore, we used an alternative multivariate test based on the Hotelling T 2 test, which has been used for the analysis of similar multiple choice preference tests (Roa, 1992). Also, the analysis used the proportion of eggs laid on each treatment rather than the total number of eggs, in order to prevent results from being biased by variation in total eggs laid by individual females (Lockwood, 1998). The T 2 statistic was significant if the following equation was true; where n is the number of replicates, K is the number of treatments, and F ¼ 95% quantile of F distribution for K 1 and n K 1 degrees of freedom:
2.4. Parasitoid landing preference
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Ninety-five percent confidence intervals were constructed for the differences between the means of each treatment in the manner described by Lockwood (1998). Treatments were considered to be significantly different if the confidence interval did not include zero. Calculations were performed in Microsoft Excel (Microsoft Inc., 1998). Although this method was useful in determining preference among treatments, there were limitations to the test, namely the interaction between plant genotype and nitrogen-enrichment could not be explicitly tested. We also tested whether flower size differed by plant genotype and nitrogen input using a two-way ANOVA test followed by a Tukey test in JMP 3.2.2 (SAS Institute, 1997). We used separate Wilcoxon ranksum tests to evaluate the effect of plant genotype and nitrogen-enrichment on clutch size, the number of clutches laid, and egg density per flower.
Parasitoid landing preference was assessed in a variable wind speed Plexiglas wind tunnel (114.5 cm length 41.2 cm width 41.7 cm height). Five first-instar larvae were placed on each treatment flower, and flower size was not standardized. An infested agricultural and infested wild flower were randomly assigned to two test tubes filled with water that were suspended from a ring stand. To encourage a higher proportion of parasitoids to fly upwind towards the target flowers, a 20 W light bulb dimmed to 50% was suspended outside of the wind tunnel to shine on the experimental plants. Each replicate consisted of placing an open vial containing a single female parasitoid onto a 15 cm high platform in the wind tunnel (N ¼ 33). The parasitoid was allowed 5 min to leave the vial, after which she was removed from the wind tunnel. After leaving the vial, each female was allowed 20 min to land on a flower before it was removed. Each parasitoid was allowed a total of three trials to reach a flower, after which they were permanently removed from the study. Treatment flowers were changed daily and after each successful landing. We recorded which flower genotype parasitoids first landed on and tested whether the proportion of first landings on each genotype differed from an expected probability of 0.5 using a binomial test. 2.5. Parasitoid foraging behavior 2.5.1. Experienced parasitoid foraging behavior We studied the foraging behavior of experienced female parasitoids on wild and agricultural infested flowers. We also manipulated larval density because the larger size of agricultural flowers could lower the frequency of contact with host larvae. On both agricultural
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and wild flowers, we placed 10 first-instar larvae on high density treatment flowers and five first-instar larvae on low density treatment flowers. Behavioral assays were conducted in the laboratory at 23 °C in a rectangular Plexiglas sleeve-cage (31 cm length 26 width 16 cm height). Each replicate consisted of a female parasitoid exposed to a wild and an agricultural flower with the same larval density, and the order of introduction was decided randomly. The female was introduced onto the flower using a pipette tip fitted onto an aspirator. If she failed to walk onto the flower from the pipette tip, she was gently blown onto the flower. Both methods were successful at stimulating the parasitoid to forage. If the female left the flower before 2 min had elapsed, the observation was discarded. Individual females were allowed three foraging attempts before being permanently removed from the study. Females were allowed to rest in vials with honey for at least an hour between behavioral assays. Thirty-one females were observed on low density flowers, and 18 females were observed on high density flowers. Behavioral observations were recorded in real time using event recording software (The Observer, Noldus Information Tech, 1995) until females left the flower. Flower area was measured in the same manner as described previously. The foraging route was diagrammed, allowing estimation of the flower area covered by the parasitoid. During the behavioral assay, the total foraging time and the amount of time a female spent walking, probing, cleaning, and resting was recorded. After the observation, larvae were put onto artificial diet in individual diet cups and reared at 23 °C until the emergence of either a parasitoid or an adult moth. Percent parasitism was calculated by (P =ðA þ P ÞÞ 100, where P is the number of parasitoids and A is the number of adult moths. Dead larvae were not included in the calculation because their fate could not be determined. The data were analyzed to determine whether the order of exposure was important in modifying behavior, because the same female was introduced onto both a wild and an agricultural flower. In all statistical tests, the order of exposure was not significant, and observations on each flower type could be considered independent, allowing eight additional unpaired observations to be included in the analysis. We tested for the influence of plant genotype and larval density on the total time spent on the flower, latency to first probing, and the percentage of total time spent walking, probing, cleaning, and resting with separate two-way ANOVA tests in S-Plus 4.5 (Mathsoft Inc., 1998). We analyzed both the percentage of total flower area covered and the total area covered, because the flower genotypes differed in size. Percent parasitism was analyzed by transforming the data into ranks and performing the Sheirer–Ray–Hare extension of the Kruskal–Wallis test for two-way
ANOVA design for ranked data (Sokal and Rohlf, 1997), because the data did not conform to the assumptions of normality. In addition, we measured the difference in individual behavioral responses on wild and agricultural flowers by testing whether the mean difference was equal to zero using a paired t test in JMP 3.2.2 (SAS Institute, 1997). 2.5.2. Na€ıve vs. experienced parasitoid foraging behavior and parasitism We compared the foraging behavior of na€ıve and experienced parasitoids on low density flowers (five larvae per flower) to assess whether previous experience foraging on wild sunflowers influenced parasitoid foraging behavior. Although both flower genotypes were not used in preconditioning, this comparison would indicate whether previous parasitoid experience on wild flowers can influence searching on agricultural flowers. Because almost all na€ıve parasitoids landed on agricultural flowers, we only compared behavioral responses for experienced and na€ıve parasitoids on agricultural flowers. For na€ıve parasitoids that successfully landed on a flower in the parasitoid landing preference study, we recorded the amount of time spent walking, resting, cleaning, and probing. We diagrammed the foraging path of each parasitoid to estimate the total area covered on the flower. Larvae were placed on artificial diet, and reared to determine the percent parasitized. Foraging behavior of na€ıve and experienced females on agricultural flowers with low larval density was compared using a StudentÕs t tests.
3. Results 3.1. Moth landing preference More females landed on agricultural flowers than on wild flowers (17 vs. 6 landings; v2 ¼ 5:26; df ¼ 1; P < 0:05). Females spent more time on agricultural flowers at 1308 195 s compared to 735 146 s on wild flowers (t ¼ 2:21; df ¼ 15:7; P < 0:05). The proportion of the flower covered by the moths did not differ between genotypes, 0:42 0:09 on agricultural flowers and 0:34 0:15 on wild flowers. Moth landing preference and retention time was not related to flower size in this study, because flower size was standardized. 3.2. Moth ovipositional preference 3.2.1. No-choice assay When female moths were exposed to flowers under no-choice conditions, total eggs laid on the four treatment groups were not significantly different. Moths laid an average of 9:14 1:95 eggs per flower and the total number of eggs did not differ by plant genotype or
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nitrogen-enrichment. For moths that oviposited on the flower, they laid an average of 7:94 1:54 clutches and 2:49 0:12 eggs per clutch, and average number of clutches and clutch size did not differ by genotype or nitrogen-enrichment. 3.2.2. Choice assay In total, 34 moths oviposited on at least one plant in the cage. Female moths preferred to lay their eggs on agricultural flowers (Fig. 1; T 2 ¼ 48:49; df ¼ 3; 34; P < 0:0001). They allocated 87% of their egg laying effort to agricultural flowers; 51:7% 6:46 of the eggs were laid on high nitrogen flowers and 35:2% 6:48 on low nitrogen agricultural flowers (Fig. 1). Females did not discriminate by nitrogen status, exclusive of the effect of plant genotype (Fig. 1). Also, more eggs were laid on agricultural flowers more frequently than on wild flowers (Fig. 2). Although average clutch size did not differ between plant genotype or nitrogen-enrichment, there were more clutches of eggs laid on agricultural flowers (6:21 0:64) than on wild flowers (2:56 0:50; v2 ¼ 11:47; df ¼ 1; P < 0:001). Nitrogen-enrichment did not influence the number of clutches laid. Females may prefer the larger size of agricultural flowers. Flower surface area for agricultural flowers was about four times larger than wild flowers (F ¼ 113:61; df ¼ 1; 132; P < 0:0001). Nitrogen-enrichment also increased flower size (Fig. 3; F ¼ 26:54; df ¼ 1; 132; P < 0:0001); however, nitrogen-enrichment doubled the size of the agricultural flowers but did not affect wild flowers, as shown by an interaction between flower genotype and nitrogen-enrichment (Fig. 3; F ¼ 23:81; df ¼ 1; 132; P < 0:0001). On agricultural flowers, females laid more eggs on larger flowers (Fig. 4A, R2 ¼ 0:15; F ¼ 15:96; P < 0:001), but this trend was not significant on wild flowers (Fig. 4B). However, it should be emphasized that only 15% of the variation
Fig. 2. Frequency distribution of moth eggs per flower on (A) agricultural and (B) wild flowers for ovipositional choice assays.
Fig. 3. Flower size for agricultural and wild flowers grown with and without nitrogen in choice assays. Letters represent significantly different treatment means using a Tukey test (P < 0:05).
Fig. 1. Female moth ovipositional preference by plant genotype and nitrogen status in choice assays ðT 2 ¼ 48:49; df ¼ 3; 34; P < 0:0001Þ. Different letters over bars indicate that treatment means (SE) were different (P < 0:05) using Lockwood (1998).
in total number of eggs laid per flower could be explained by flower size. Another limitation in comparing the regressions is that agricultural flowers were four times the size of wild flowers, and no wild flowers of equivalent size exist in nature (Figs. 4A and B). Furthermore, moths laid different densities of eggs on
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Fig. 5. Female ovipositional response per unit area in oviposition choice assays. Egg density differed by plant genotype (v2 ¼ 3:83; df ¼ 1; P ¼ 0:05), but did not differ by nitrogen status.
Fig. 4. Moth ovipositional response to (A) agricultural and (B) wild flower size (cm2 ) in choice assays. Females laid more eggs on larger agricultural flowers (R2 ¼ 0:15; F ¼ 15:96; P < 0:001), but not on wild flowers.
agricultural and wild flowers, because egg density was almost twice as high on wild flowers than on agricultural flowers (Fig. 5; v2 ¼ 3:83; df ¼ 1; P < 0:05). Moths did not vary the density of eggs laid in response to nitrogenenrichment.
behavioral assay was considered an independent event. Females spent on average, 1385:14 322:55 s (23.08 min) more, or 74% longer, foraging on wild flowers than on an agricultural flower with the equivalent host density (Fig. 6; F ¼ 15:14; df ¼ 1; 101; P < 0:001). Although parasitoids searched over 200 s longer on high density flowers than on low density flowers, host density did not significantly alter parasitoid residence time (Fig. 6). In addition to differences in total time budgets, parasitoid time allocation differed by plant genotype and host density. Flower genotype was more important than larval density in modifying parasitoid behavior (Table 1). Females allocated approximately 24.5% less time to walking on flowers with high host density, and there was an interaction between genotype and density (Table 1). Time allocation to cleaning did not differ by plant genotype or host density. Parasitoids rested twice as long
3.3. Parasitoid landing preference Eleven of the 33 na€ıve female parasitoids assayed landed on a target flower. More female parasitoids preferred to land on agricultural flowers than on wild flowers (10 vs. 1 landings; v2 ¼ 7:36; df ¼ 1; P < 0:01). Only a single female landed on a wild infested flower, preventing meaningful comparisons of foraging behavior on agricultural vs. wild sunflowers. Na€ıve females spent on average 2253:4 418:5 s foraging on agricultural flowers, while the single female that landed on the wild flower spent 1314 s foraging. 3.4. Parasitoid foraging behavior 3.4.1. Experienced parasitoid behavior Since the order of exposure to a plant genotype did not influence parasitoid behavioral responses, each
Fig. 6. Total time spent foraging by experienced parasitoid females on agricultural and wild flowers with low and high densities of larvae. Flower genotype influenced parasitoid residence time (F ¼ 15:14; df ¼ 1; 101; P < 0:001).
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Table 1 Summary of parasitoid foraging behavior in response to plant genotype, host density, and their interaction. Only significant results are reported in this table Behavior
Agricultural
Wild
Factor
F
df
P
High density
Low density
High density
Low density
Percent of total time walking
29:1 3:8
53:6 3:0
34:9 2:5
44:3 2:7
Density (D)
28.66
1
<0.0001
Percent of total time probing Percent of total time resting
16:8 3:8
13:5 2:4
31:8 2:5
27:7 2:5
GD Genotype (G)
5.19 27.55
1 1
<0.05 <0.0001
37:6 6:3
10:5 2:0
14:1 2:4
5:1 0:8
Genotype (G)
15.62
1
<0.001
37.28 8.44 6.9
1 1 1
<0.0001 <0.01 <0.01
Latency to first probing Frequency of probing Total area covered (cm2 ) Percent of total area covered
170:8 58:8
179:3 47:2
61:8 22:9
93:4 31:1
Density (D) GD Genotype (G)
28:5 6:4
17:7 4:3
43:1 7:9
39:8 6:5
Genotype (G)
7.22
1
<0.01
32:9 1:29
27:76 2:19
6:21 0:58
6:82 0:62
Genotype (G)
17.76
1
<0.0001
0:41 0:07
0:38 0:06
0:90 0:06
0:83 0:05
Genotype (G)
65.24
1
<0.0001
on agricultural flowers and almost three times longer on high density flowers (Table 1). Parasitoids appeared to be more stimulated by host cues on wild flowers, because they probed 2–3 times sooner after landing on wild flowers, probed more frequently, allocated twice as much time to probing, and spent more absolute time probing on wild flowers (Table 1). Although the area searched by parasitoids was 2–3 times larger on agricultural flowers, parasitoids covered twice as much of the total flower area on wild flowers due to their smaller size (Table 1). Parasitoid foraging efficacy may have been reduced on agricultural flowers with low host density due to a decreased frequency of parasitoid contact with host cues. Larval density was much lower on agricultural flowers (0:15 0:02 larvae= cm2 ) than on wild flowers (0:64 0:08 larvae=cm2 ; F ¼ 45:92; df ¼ 1; 79; P < 0:0001) due to the larger size of the agricultural flowers, and did not significantly differ between the high and low density treatments. Parasitism on wild flowers were double the levels found on agricultural flowers (Fig. 7, v2 ¼ 16:8; df ¼ 1; P < 0:0001), and host density did not influence parasitism rates. Each individual female parasitized 25.7% 0.06 more larvae on wild flowers than on the paired agricultural flower (t ¼ 4:41; df ¼ 44; P < 0:0001). While parasitoids probed for hosts on wild flowers four times longer than on agricultural flowers, the resulting parasitism levels were 19 times higher, indicating that parasitoids were much more successful per unit effort on wild flowers. 3.4.2. Na€ıve vs. experienced parasitoid foraging behavior Na€ıve and experienced female parasitoids foraged in a similar manner on agricultural flowers. Na€ıve parasi-
Fig. 7. Percent parasitism by plant genotype and larval density from parasitoid foraging behavior studies. Plant genotype significantly influenced parasitism (v2 ¼ 16:8; df ¼ 1; P < 0:0001), but host density did not influence parasitism rates.
toids allocated most of their time to walking (50.65% 3.29) and probing for hosts (28.54% 4.07), and they spent less time cleaning (16.32% 1.97) and resting (3.69% 1.02) on agricultural flowers. Previous experience did not influence female allocation to walking, probing, cleaning, resting, or the total time spent foraging on the flower. Na€ıve parasitoids covered a similar proportion of the total flower compared with experienced females. Even though both groups of parasitoids spent a large percentage of their total time probing for hosts, percent parasitism on agricultural flowers did not significantly differ between na€ıve (3.3% 3.3) and experienced (7.0% 3.6) parasitoids. Low parasitism rates on agricultural flowers were not
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due to inadequate coverage of the flower head, because na€ıve females covered 71% 7.0 of the flower head area. Therefore, previous foraging experience on wild flowers did not influence female foraging behavior on agricultural flowers.
4. Discussion Our data show that moths and parasitoids respond differently to agricultural and wild plants. Both H. electellum and D. homoeosomae preferred to land on agricultural flowers; however, they differed in their behavioral responses on them. While moths responded to the agricultural flowers by laying more eggs on them, parasitoids responded to agricultural flowers by spending less time probing and by parasitizing fewer larvae. Therefore, domestication has increased H. electellum abundance while reducing D. homoeosomae foraging efficiency and has thus reduced biological control. The most visible difference between agricultural and wild sunflowers is flower head size. Our data suggest that flower enlargement may be an important factor in altering parasitoid–herbivore interactions, because H. electellum laid more eggs on larger agricultural flowers. Although H. electellum is nocturnal, it may use visual cues, because other nocturnal Lepidopteran species have been found to prefer larger visual targets (Rojas et al., 2000). Similarly, enlargement of the flower can alter patterns of host plant use by altering semiochemical plant cues (De Moraes et al., 2001; del Campo et al., 2001; Renwick and Chew, 1994; Udayagiri and Mason, 1997). Agricultural flowers have larger individual florets and therefore have a higher pollen density than wild flowers (Chen, pers. obs.). As pollen is considered a stimulant for H. electellum oviposition (Delisle et al., 1989), the larger quantity of pollen correlated with larger agricultural flowers may cause H. electellum females to lay more eggs. Given that both moths and parasitoids were able to discriminate between agricultural and wild sunflowers in the landing preference tests suggest that there are differences in either the quantity or quality of volatiles emitted. The larger quantity of pollen on agricultural sunflowers may correlate with higher volatile emissions and provide a stronger signal for both moths and parasitoids. Also, H. electellum larvae appear to develop more quickly on agricultural sunflowers, so parasitoids may prefer agricultural flowers because of the larger total output of both host and plant-related volatiles. Both quantitative and qualitative variations in volatile production can be related to parasitoid preference (Geervliet et al., 1998; Hoballah et al., 2002). In other systems, parasitoids can respond to differences in semiochemicals between cultivars (Geervliet et al., 1996), and even finer scale differences between cultivar
lines (Elzen et al., 1986). Further research into the variability in volatile emissions within and between agricultural and wild plants will help determine whether they emit distinct profiles and which volatile compounds are important in eliciting favorable parasitoid responses (Gouinguene et al., 2001). Although parasitoids preferred to land on agricultural plants, they foraged poorly on them. There are several potential causes of the lower success in finding hosts for D. homoeosomae on agricultural flowers. First, female D. homoeosomae probed for host larvae on agricultural flowers, but they allocated far smaller percentage of their time to probing and probed for a shorter time. Second, larval density was much lower on agricultural flowers, and the lowered rate of contact with host products may have caused parasitoids to leave the patches sooner. While the number of larvae was equal on agricultural and wild flowers, larval density was much higher on wild flowers. If larval density is standardized between agricultural and wild genotypes, it would be possible to measure the direct effect of plant genotype on parasitoid behavior, independent of host density. Finally, the floral column is deeper on agricultural flowers than on wild flowers, and may provide a refuge from parasitism (Brown et al., 1995; Feder, 1995; Udayagiri and Welter, 2000). Although host frass and silk were present on agricultural flowers, repeated contact with host products without a successful oviposition has been shown to deter further searching and cause parasitoids to leave patches sooner (Papaj et al., 1994). We propose that the poor foraging efficacy of D. homoeosomae on individual agricultural flowers in this laboratory study contributes to their low efficacy in agricultural fields. It is unlikely that D. homoeosomae foraging behavior of leaving patches early and parasitizing few larvae is an adaptive strategy to ‘‘spread’’ its eggs over more patches like Anagrus delicatus, a parasitoid of a saltmarsh planthopper (Cronin and Strong, 1993). Parasitism levels within agricultural sunflower fields are uniformly low across all patches (1%) throughout the season (Chen and Welter, 2002). Coupled with the our previous field data, these results indicate that D. homoeosomae have not adapted to forage effectively on agricultural flower heads (Chen and Welter, 2002). Our results show that the greater abundance of H. electellum in agricultural sunflowers is in part due to the role of domestication in promoting herbivore activity and disrupting parasitoid efficacy. Developing a successful biological control program for H. electellum may be a challenge, because the remainder of the H. electellum parasitoid complex also appears to be ineffective at controlling H. electellum (Charlet, 1999; Chen and Welter, 2002). As this pattern may be repeated in other agricultural systems, our data suggest that parasitoid adaptation to domesticated crops may be an important
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barrier to overcome in order to develop successful biological control programs for insect pests in agricultural systems. On the other hand, many opportunities exist to work with plant breeders to promote biological control of H. electellum. The higher parasitism levels on wild sunflowers indicate that parasitoid activity can be enhanced considerably. Crop domestication may not always disrupt trophic interactions, because herbivore and parasitoid performance can be equally increased on agricultural crops (Benrey et al., 1998). The challenge for crop breeders is to develop plant attributes that promote the activity of parasitoids while minimizing the attributes favored by herbivores. Determining how herbivore and parasitoid responses have been altered by crop domestication and selection in this system and in other systems will help resolve the ecological factors limiting the successful biological control of insect herbivores by native parasitoids in agricultural habitats.
Acknowledgments We thank Tony Turkovich, US Fish and Wildlife Service, and California Department of Fish and Game for granting us field access to collect insects. We also greatly appreciate the help from John Andrews, Frances Cave, Jill Fortuna, Doug Light, Nick Mills, and Kathy Reynolds on this project. This manuscript has benefited from the comments of Gregory Fanslow, Nick Mills, Wayne Sousa, and several anonymous reviewers. This research was supported by a SAREP SAGA Grant, Van den Bosch Award, and a NSF Predoctoral Fellowship to Y.H. Chen, and also funds from the Division of Agriculture and Natural Resources to S.C. Welter.
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Confused by domestication: incongruent behavioral responses of the sunflower moth, Homoeosoma electellum (Lepidoptera: Pyralidae) and its parasitoid, Dolichogenidea homoeosomae (Hymenoptera: Braconidae), towards wild and domesticated sunflowers Yolanda H. Chen* and Stephen C. Welter Division of Insect Biology, Wellman Hall, University of California, Berkeley, CA 94720-3112, USA Received 7 December 2001; accepted 2 April 2003
Abstract Due to domestication, agricultural plants and their wild ancestors encompass a wide range of variation, which is useful for identifying promising plant attributes that promote parasitoid activity. We examined the behavioral responses of the sunflower moth, Homoeosoma electellum Hulst and its major parasitoid, Dolichogenidea homoeosomae Muesebeck to agricultural and wild sunflowers. We found that female moths preferred to land on, and lay more eggs on agricultural plants. While female parasitoids preferred to land on infested agricultural sunflowers, they foraged poorly on them. Parasitoids spent 74% more time foraging on wild flowers than on agricultural flowers, and they probed for a significantly longer period of time for host larvae on wild flowers. As a result, each individual female parasitized 25.7% 0.06 more larvae on wild flowers than on agricultural flowers with the same larval density. Also, parasitoids probed for hosts four times longer on wild flowers than on agricultural flowers, while resulting parasitism levels were 19 times higher, indicating that parasitoids were more efficient on wild flowers. Agricultural flowers are about four times larger than wild flowers, and the increase in flower size correlated with higher moth oviposition but lower parasitoid efficiency. Our results show that changes to plant attributes through domestication can disrupt host–parasitoid interactions and limit the ability of parasitoids to control insect pests effectively. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Homoeosoma electellum; Dolichogenidea homoeosomae; Behavior; Sunflowers; Domestication; Agricultural; Wild; Foraging behavior; Sunflower moth; Parasitoid behavior
1. Introduction It is widely known that plant attributes can mediate interactions between insect herbivores and their natural enemies (Agrawal et al., 2002; Andow and Prokrym, 1990; Benrey et al., 1998; Benrey and Denno, 1997; Bottrell et al., 1998; Damman, 1987; Kalule and Wright, 2002; Lovinger et al., 2000; Price, 1986; Price et al., 1980; Turlings et al., 1995; Udayagiri and Welter, 2000). Parasitoid–herbivore interactions can be influenced by physical, nutritional, or chemical plant attributes * Corresponding author. Fax: +510-642-0477. E-mail address: [email protected] (Y.H. Chen).
(Cortesero et al., 2000; Price, 1986; Price et al., 1980). In order to manipulate these interactions, it is necessary to determine how plant attributes can enhance parasitoid activity. By understanding the relative behavioral responses of parasitoids and their herbivore hosts, plant breeders may select for particular plant attributes that promote biological control (Cortesero et al., 2000). Agricultural plants and their wild ancestors encompass a wide range of phenotypic variability in physical and chemical attributes as a result of domestication and selection (Evans, 1993; Gouinguene et al., 2001; Massei and Hartley, 2000; Rosenthal and Dirzo, 1997). Given that physical and chemical plant attributes play an essential role in host location and acceptance for
1049-9644/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1049-9644(03)00084-7
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parasitoids and hosts, it is likely that parasitoid–herbivore interactions may differ on agricultural plants and their wild ancestors (Evans, 1993; Geervliet et al., 1996; Gouinguene et al., 2001; Renwick and Chew, 1994; Thompson and Pellmyr, 1991). However, it is not clear how changes in parasitoid–herbivore interactions on agricultural plants may affect biological control. For example, agricultural plants have larger plant structures for economically important attributes and reduced architectural complexity, which may alter herbivore distribution and influence parasitoid foraging success (Andow and Prokrym, 1990; Grevstad and Klepetka, 1992). Also quantitative and qualitative differences in semiochemical production may differ between agricultural crops and their relatives (Gouinguene et al., 2001), which may alter herbivore or parasitoid host location and acceptance (Benrey et al., 1998). Understanding how herbivores and parasitoids respond to agricultural and wild plants will help identify promising plant attributes that may be manipulated to enhance biological control. Given that the majority of modern crops grown in North America originated from other continents (Evans, 1993; Harlan, 1971), assessing how domestication has altered parasitoid–herbivore interactions is limited within the United States. However, the domesticated sunflower, Helianthus annuus var. macrocarpus L., and its wild ancestor, H. annuus var. annuus L., represent a rare exception to this constraint, as wild and agricultural sunflower populations are sympatric, and the wild H. annuus is native throughout North America. Furthermore, molecular and isozyme data indicate that the domesticated sunflower was derived from a wild form of H. annuus (Arias and Rieseberg, 1995; Rieseberg and Seiler, 1990). The sunflower system is also particularly amenable to this study because it is attacked by a large assemblage of native herbivores (Rogers, 1988a,b). Among them, the sunflower moth, Homoeosoma electellum Hulst (Lepidoptera: Pyralidae) is considered the most important pest of sunflowers in North America (Charlet et al., 1997; Schultz, 1978). H. electellum is attacked by a large assemblage of parasitoids, and Dolichogenidea homoeosomae Muesebeck (Hymenoptera: Braconidae) is the most important parasitoid in both native and agricultural habitats in the San Joaquin Valley in California (Chen and Welter, 2002). We found previously that H. electellum was more abundant and less parasitized on agricultural plants, even when wild sunflowers were grown alongside them in a common garden setting (Chen and Welter, 2002). Our previous field results suggest that the differences in herbivory and parasitism may be due to differences in moth and parasitoid behavioral responses to agricultural and wild sunflowers. If D. homoeosomae prefer wild sunflowers, they may have less impact on agricultural plants. Clearly, the observed differences in
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parasitism levels between agricultural and wild sunflowers suggest that opportunities exist for manipulating plant attributes to enhance biological control in agricultural sunflowers. Our previous work indicated that nitrogen-enrichment increased plant phenotypic differences between wild and agricultural sunflowers, which further increased the differences in moth larval abundance and parasitism (Chen and Welter, unpublished results). Therefore, we designed this study to test for the most extreme differences in behavioral responses to agricultural and wild sunflowers using nitrogen-enriched plants. We recognize that behavioral responses studied here may be representative of interactions found in the field, but do not represent the entire spectrum of possibilities. One exception was made to test the effect of nitrogen-enrichment on moth ovipositional preferences to wild and agricultural sunflowers, because the field patterns suggested that sunflower moth ovipositional responses may be in response to flower size, which is influenced by nitrogen content (Chen and Welter, 2002). To determine whether there are differences in behavioral responses of the sunflower moth and its parasitoid, D. homoeosomae, to wild and agricultural sunflowers, we asked the following questions: (1) Do na€ıve female moths prefer to land on agricultural vs. wild sunflowers? (2) Does nitrogen-enrichment interact with plant genotype to influence na€ıve female moth ovipositional preference? (3) Do na€ıve parasitoids differ in their landing preferences on infested agricultural vs. infested wild sunflowers? (4) Does parasitoid foraging behavior differ by plant genotype and larval density? (5) Does previous experience on wild sunflowers influences parasitoid foraging behavior on agricultural sunflowers?
2. Materials and methods 2.1. Insect and plant cultures We used an agricultural sunflower genotype that is grown in the northern San Joaquin Valley and was previously studied in the field (Chen and Welter, 2002). The wild sunflower plants were grown from seeds collected in the Stone Lake Wildlife Refuge, Sacramento, CA and Yolo Bypass Wildlife Area, Yolo, CA. Although wild sunflowers represent multiple genotypes (Rieseberg and Seiler, 1990), they will be collectively identified as the wild genotype in this study. Wild sunflower seeds were germinated in a moistened paper towel held in a plastic bag. Agricultural sunflower seeds and germinated wild seedlings were planted into 6 cm diameter plastic cells on the same date to synchronize flowering. After two weeks of growth in the greenhouse, plants were transplanted into 11.4 liter pots containing a
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peat moss and sand soil mixture. In the fourth week, plants in the high nitrogen treatments received: 6.5 g 42–0–0 (NH4 NO3 with 6% S), 3.4 g 0–48–0 (P2 O5 with 15% Ca), and 7.3 g 0–0–51 (K2 O with 17% S), and non-enriched treatments received only phosphorous and potassium. Low-nitrogen treatments did not receive any fertilizer. These fertilizer formulations were used so that each macronutrient could be added individually, and therefore S and Ca levels could have been raised. Seed set was promoted by hand pollinating the flowers, and excess pollen was removed by tapping the flower heads. We established a colony of H. electellum using larvae collected from agricultural and wild sunflower fields in Yolo and Sacramento counties. Moths were reared on an agar-based diet (Wilson, 1990) at 22 °C under a 16L:8D light cycle for several generations before the study began to ensure adequate numbers for the study and remove maternal effects. Females observed to mate 1–3 days after emergence were used in the behavioral assay. Mating couples were isolated in the cage and then, females were held overnight under artificial light in a plastic cup with a cotton ball moistened with 5% sugar water solution. Female moths were used in the behavioral assays the next day. For the parasitism studies, we transferred first instar larvae equally spaced onto agricultural and wild flowers in bloom. Larvae were allowed to develop for two to four days at 25 °C in a greenhouse and exposed to the parasitoids as a second or third instar in the behavioral assays because parasitism in the field is highest in these stages (Chen, unpublished results). On the morning of the behavioral observations, artificially infested flowers were cut and the stems were placed into containers filled with water. Using calipers, we measured flower diameter, which was the distance across the face of the flower, and flower depth, which was the thickness of the flower from the base to the top of the florets. Na€ıve parasitoids were obtained by collecting wild parasitoids and allowing them to oviposit on infested sunflowers. Moth larvae were then reared out on artificial diet. We also obtained na€ıve parasitoids by rearing larvae collected from the field on artificial diet. We monitored parasitoid emergence by checking the cups three times a week. Experienced parasitoids were collected weekly from the field. All parasitoids were kept for 2–3 days in 36 ml vials streaked with honey before behavioral assays and between trials. 2.2. Moth landing preference Female moth landing preference was assessed in a rectangular wind tunnel (261 85:5 86:5 cm) with a laminar flow of 30 cm/s. The room was lit with a 60 W light bulb that was dimmed over an hour at a rate of 10% every 7 min to simulate dusk. Six 60 W red incan-
descent lights lined one side of the wind tunnel arena and were dimmed to 30% of full intensity. A 60 W incandescent light bulb was positioned outside of the wind tunnel to shine on the experimental flowers. This approach was used to stimulate a higher proportion of moths to fly upwind towards the part of the tunnel with the target plants, but did not influence landing preference. An agricultural and a wild flower were placed into test tubes filled with water and attached to a ring stand with test tube clamps. The position of the flowers within the wind tunnel was randomly determined on each day of the experimental assay. To reduce quantitative differences in volatile production due to the difference in flower head size, we standardized flower size by using the smaller lateral flowers on agricultural plants. The flowers were changed each day of the study or if moths landed on them. The area of the face of the flower, excluding the ray petals, was determined by measuring the longest diameter across the flower (r1 ) and the perpendicular diameter (r2 ), and calculating the area of an ellipse (A ¼ pðr1 r2 )). We determined that each pair of treatment flowers were equivalent in size using a paired t test. Each replicate consisted of the release of a single female moth onto a cloth-covered circular platform positioned at the same height as the target flowers (N ¼ 71). If the moth did not take off from the platform after 5 min or did not land on the flowers after 15 min, she was removed from the wind tunnel. Each female was allowed three trials, after which failure to land on a flower resulted in permanent removal from the study. For a moth that landed on a flower, its behavior was observed on the target flower in two ways: by recording the total time on the flower and by diagramming its route to calculate the proportion of the flower head covered. Female landing preference was analyzed using a binomial test with an expected probability of 0.5. Total time spent on the flower was analyzed using a WelchÕs t test for unequal variances in S-Plus 4.5 (Mathsoft Inc., 1998). The proportion of the flower head covered by the moth was analyzed with a StudentÕs t test. 2.3. Moth ovipositional preference We assessed female ovipositional response to plant genotype and nitrogen-enrichment. The four treatments were: (1) low nitrogen agricultural sunflowers, (2) high nitrogen agricultural sunflowers, (3) low nitrogen wild sunflowers, (4) high nitrogen wild sunflowers. Two types of assays were conducted: no-choice assays in cages where only a single treatment was present, and choice assays, in cages where all four treatment flowers were present. Flower size was not standardized in the moth oviposition studies.
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2.3.1. No-choice assay Mated females were held at room temperature until dusk and then introduced individually into a 0:03m3 metal cage with cloth sides. Each replicate consisted of a single treatment flower that was placed into the cage and exposed to a single moth for 24 h. A minimum of five replicate cages were used for each treatment. The cages were lit on a 16:8 light:dark schedule and room temperature was 22 °C. A fan, positioned 1.5 m away from the cages, blew air towards the cages at a low speed to provide air circulation. Flowers were processed under a dissecting microscope to count the total number of eggs and number of egg clusters. We tested whether the total number of eggs laid differed by plant genotype and nitrogen-enrichment in separate two-way ANOVA tests in JMP 3.2.2 (SAS Institute, 1997). For moths that laid eggs on the flowers, we tested whether the number of clutches and average clutch size differed using a two-way ANOVA test.
T 2 > ðK 1Þðn 1ÞðFK1;nKþ1 Þ=ðn K 1Þ:
2.3.2. Choice assay Female moth ovipositional preference to nitrogenenriched and non-enriched agricultural and wild flowers was experimentally assessed in a Plexiglas cage (61 61 30:5 cm) with cloth sides to allow airflow. The study was conducted in a greenhouse room at 27.1 °C 0.3, with two box fans to circulate the air within the greenhouse room. Each replicate consisted of a flower from each of the four treatment groups. Flowers were randomly assigned to the four 8.5 cm circular holes in the floor of the cage. Circular sponges, fit around the stem of the flower, were flush with the bottom of the floor and the stems were placed in 160 ml vials filled with water. Each replicate consisted of a single gravid female introduced into a cage for 24 h (N ¼ 62). Flowers were examined for the total number of eggs laid, the number of clutches laid, and clutch size. Moths that did not lay eggs on any plants were removed from the study. Because the treatments were enclosed together in a cage, the number of eggs laid on each treatment was not independent between treatments and thus violated the assumptions of v2 , nonparametric, and parametric tests (Roa, 1992). Therefore, we used an alternative multivariate test based on the Hotelling T 2 test, which has been used for the analysis of similar multiple choice preference tests (Roa, 1992). Also, the analysis used the proportion of eggs laid on each treatment rather than the total number of eggs, in order to prevent results from being biased by variation in total eggs laid by individual females (Lockwood, 1998). The T 2 statistic was significant if the following equation was true; where n is the number of replicates, K is the number of treatments, and F ¼ 95% quantile of F distribution for K 1 and n K 1 degrees of freedom:
2.4. Parasitoid landing preference
183
Ninety-five percent confidence intervals were constructed for the differences between the means of each treatment in the manner described by Lockwood (1998). Treatments were considered to be significantly different if the confidence interval did not include zero. Calculations were performed in Microsoft Excel (Microsoft Inc., 1998). Although this method was useful in determining preference among treatments, there were limitations to the test, namely the interaction between plant genotype and nitrogen-enrichment could not be explicitly tested. We also tested whether flower size differed by plant genotype and nitrogen input using a two-way ANOVA test followed by a Tukey test in JMP 3.2.2 (SAS Institute, 1997). We used separate Wilcoxon ranksum tests to evaluate the effect of plant genotype and nitrogen-enrichment on clutch size, the number of clutches laid, and egg density per flower.
Parasitoid landing preference was assessed in a variable wind speed Plexiglas wind tunnel (114.5 cm length 41.2 cm width 41.7 cm height). Five first-instar larvae were placed on each treatment flower, and flower size was not standardized. An infested agricultural and infested wild flower were randomly assigned to two test tubes filled with water that were suspended from a ring stand. To encourage a higher proportion of parasitoids to fly upwind towards the target flowers, a 20 W light bulb dimmed to 50% was suspended outside of the wind tunnel to shine on the experimental plants. Each replicate consisted of placing an open vial containing a single female parasitoid onto a 15 cm high platform in the wind tunnel (N ¼ 33). The parasitoid was allowed 5 min to leave the vial, after which she was removed from the wind tunnel. After leaving the vial, each female was allowed 20 min to land on a flower before it was removed. Each parasitoid was allowed a total of three trials to reach a flower, after which they were permanently removed from the study. Treatment flowers were changed daily and after each successful landing. We recorded which flower genotype parasitoids first landed on and tested whether the proportion of first landings on each genotype differed from an expected probability of 0.5 using a binomial test. 2.5. Parasitoid foraging behavior 2.5.1. Experienced parasitoid foraging behavior We studied the foraging behavior of experienced female parasitoids on wild and agricultural infested flowers. We also manipulated larval density because the larger size of agricultural flowers could lower the frequency of contact with host larvae. On both agricultural
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and wild flowers, we placed 10 first-instar larvae on high density treatment flowers and five first-instar larvae on low density treatment flowers. Behavioral assays were conducted in the laboratory at 23 °C in a rectangular Plexiglas sleeve-cage (31 cm length 26 width 16 cm height). Each replicate consisted of a female parasitoid exposed to a wild and an agricultural flower with the same larval density, and the order of introduction was decided randomly. The female was introduced onto the flower using a pipette tip fitted onto an aspirator. If she failed to walk onto the flower from the pipette tip, she was gently blown onto the flower. Both methods were successful at stimulating the parasitoid to forage. If the female left the flower before 2 min had elapsed, the observation was discarded. Individual females were allowed three foraging attempts before being permanently removed from the study. Females were allowed to rest in vials with honey for at least an hour between behavioral assays. Thirty-one females were observed on low density flowers, and 18 females were observed on high density flowers. Behavioral observations were recorded in real time using event recording software (The Observer, Noldus Information Tech, 1995) until females left the flower. Flower area was measured in the same manner as described previously. The foraging route was diagrammed, allowing estimation of the flower area covered by the parasitoid. During the behavioral assay, the total foraging time and the amount of time a female spent walking, probing, cleaning, and resting was recorded. After the observation, larvae were put onto artificial diet in individual diet cups and reared at 23 °C until the emergence of either a parasitoid or an adult moth. Percent parasitism was calculated by (P =ðA þ P ÞÞ 100, where P is the number of parasitoids and A is the number of adult moths. Dead larvae were not included in the calculation because their fate could not be determined. The data were analyzed to determine whether the order of exposure was important in modifying behavior, because the same female was introduced onto both a wild and an agricultural flower. In all statistical tests, the order of exposure was not significant, and observations on each flower type could be considered independent, allowing eight additional unpaired observations to be included in the analysis. We tested for the influence of plant genotype and larval density on the total time spent on the flower, latency to first probing, and the percentage of total time spent walking, probing, cleaning, and resting with separate two-way ANOVA tests in S-Plus 4.5 (Mathsoft Inc., 1998). We analyzed both the percentage of total flower area covered and the total area covered, because the flower genotypes differed in size. Percent parasitism was analyzed by transforming the data into ranks and performing the Sheirer–Ray–Hare extension of the Kruskal–Wallis test for two-way
ANOVA design for ranked data (Sokal and Rohlf, 1997), because the data did not conform to the assumptions of normality. In addition, we measured the difference in individual behavioral responses on wild and agricultural flowers by testing whether the mean difference was equal to zero using a paired t test in JMP 3.2.2 (SAS Institute, 1997). 2.5.2. Na€ıve vs. experienced parasitoid foraging behavior and parasitism We compared the foraging behavior of na€ıve and experienced parasitoids on low density flowers (five larvae per flower) to assess whether previous experience foraging on wild sunflowers influenced parasitoid foraging behavior. Although both flower genotypes were not used in preconditioning, this comparison would indicate whether previous parasitoid experience on wild flowers can influence searching on agricultural flowers. Because almost all na€ıve parasitoids landed on agricultural flowers, we only compared behavioral responses for experienced and na€ıve parasitoids on agricultural flowers. For na€ıve parasitoids that successfully landed on a flower in the parasitoid landing preference study, we recorded the amount of time spent walking, resting, cleaning, and probing. We diagrammed the foraging path of each parasitoid to estimate the total area covered on the flower. Larvae were placed on artificial diet, and reared to determine the percent parasitized. Foraging behavior of na€ıve and experienced females on agricultural flowers with low larval density was compared using a StudentÕs t tests.
3. Results 3.1. Moth landing preference More females landed on agricultural flowers than on wild flowers (17 vs. 6 landings; v2 ¼ 5:26; df ¼ 1; P < 0:05). Females spent more time on agricultural flowers at 1308 195 s compared to 735 146 s on wild flowers (t ¼ 2:21; df ¼ 15:7; P < 0:05). The proportion of the flower covered by the moths did not differ between genotypes, 0:42 0:09 on agricultural flowers and 0:34 0:15 on wild flowers. Moth landing preference and retention time was not related to flower size in this study, because flower size was standardized. 3.2. Moth ovipositional preference 3.2.1. No-choice assay When female moths were exposed to flowers under no-choice conditions, total eggs laid on the four treatment groups were not significantly different. Moths laid an average of 9:14 1:95 eggs per flower and the total number of eggs did not differ by plant genotype or
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185
nitrogen-enrichment. For moths that oviposited on the flower, they laid an average of 7:94 1:54 clutches and 2:49 0:12 eggs per clutch, and average number of clutches and clutch size did not differ by genotype or nitrogen-enrichment. 3.2.2. Choice assay In total, 34 moths oviposited on at least one plant in the cage. Female moths preferred to lay their eggs on agricultural flowers (Fig. 1; T 2 ¼ 48:49; df ¼ 3; 34; P < 0:0001). They allocated 87% of their egg laying effort to agricultural flowers; 51:7% 6:46 of the eggs were laid on high nitrogen flowers and 35:2% 6:48 on low nitrogen agricultural flowers (Fig. 1). Females did not discriminate by nitrogen status, exclusive of the effect of plant genotype (Fig. 1). Also, more eggs were laid on agricultural flowers more frequently than on wild flowers (Fig. 2). Although average clutch size did not differ between plant genotype or nitrogen-enrichment, there were more clutches of eggs laid on agricultural flowers (6:21 0:64) than on wild flowers (2:56 0:50; v2 ¼ 11:47; df ¼ 1; P < 0:001). Nitrogen-enrichment did not influence the number of clutches laid. Females may prefer the larger size of agricultural flowers. Flower surface area for agricultural flowers was about four times larger than wild flowers (F ¼ 113:61; df ¼ 1; 132; P < 0:0001). Nitrogen-enrichment also increased flower size (Fig. 3; F ¼ 26:54; df ¼ 1; 132; P < 0:0001); however, nitrogen-enrichment doubled the size of the agricultural flowers but did not affect wild flowers, as shown by an interaction between flower genotype and nitrogen-enrichment (Fig. 3; F ¼ 23:81; df ¼ 1; 132; P < 0:0001). On agricultural flowers, females laid more eggs on larger flowers (Fig. 4A, R2 ¼ 0:15; F ¼ 15:96; P < 0:001), but this trend was not significant on wild flowers (Fig. 4B). However, it should be emphasized that only 15% of the variation
Fig. 2. Frequency distribution of moth eggs per flower on (A) agricultural and (B) wild flowers for ovipositional choice assays.
Fig. 3. Flower size for agricultural and wild flowers grown with and without nitrogen in choice assays. Letters represent significantly different treatment means using a Tukey test (P < 0:05).
Fig. 1. Female moth ovipositional preference by plant genotype and nitrogen status in choice assays ðT 2 ¼ 48:49; df ¼ 3; 34; P < 0:0001Þ. Different letters over bars indicate that treatment means (SE) were different (P < 0:05) using Lockwood (1998).
in total number of eggs laid per flower could be explained by flower size. Another limitation in comparing the regressions is that agricultural flowers were four times the size of wild flowers, and no wild flowers of equivalent size exist in nature (Figs. 4A and B). Furthermore, moths laid different densities of eggs on
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Fig. 5. Female ovipositional response per unit area in oviposition choice assays. Egg density differed by plant genotype (v2 ¼ 3:83; df ¼ 1; P ¼ 0:05), but did not differ by nitrogen status.
Fig. 4. Moth ovipositional response to (A) agricultural and (B) wild flower size (cm2 ) in choice assays. Females laid more eggs on larger agricultural flowers (R2 ¼ 0:15; F ¼ 15:96; P < 0:001), but not on wild flowers.
agricultural and wild flowers, because egg density was almost twice as high on wild flowers than on agricultural flowers (Fig. 5; v2 ¼ 3:83; df ¼ 1; P < 0:05). Moths did not vary the density of eggs laid in response to nitrogenenrichment.
behavioral assay was considered an independent event. Females spent on average, 1385:14 322:55 s (23.08 min) more, or 74% longer, foraging on wild flowers than on an agricultural flower with the equivalent host density (Fig. 6; F ¼ 15:14; df ¼ 1; 101; P < 0:001). Although parasitoids searched over 200 s longer on high density flowers than on low density flowers, host density did not significantly alter parasitoid residence time (Fig. 6). In addition to differences in total time budgets, parasitoid time allocation differed by plant genotype and host density. Flower genotype was more important than larval density in modifying parasitoid behavior (Table 1). Females allocated approximately 24.5% less time to walking on flowers with high host density, and there was an interaction between genotype and density (Table 1). Time allocation to cleaning did not differ by plant genotype or host density. Parasitoids rested twice as long
3.3. Parasitoid landing preference Eleven of the 33 na€ıve female parasitoids assayed landed on a target flower. More female parasitoids preferred to land on agricultural flowers than on wild flowers (10 vs. 1 landings; v2 ¼ 7:36; df ¼ 1; P < 0:01). Only a single female landed on a wild infested flower, preventing meaningful comparisons of foraging behavior on agricultural vs. wild sunflowers. Na€ıve females spent on average 2253:4 418:5 s foraging on agricultural flowers, while the single female that landed on the wild flower spent 1314 s foraging. 3.4. Parasitoid foraging behavior 3.4.1. Experienced parasitoid behavior Since the order of exposure to a plant genotype did not influence parasitoid behavioral responses, each
Fig. 6. Total time spent foraging by experienced parasitoid females on agricultural and wild flowers with low and high densities of larvae. Flower genotype influenced parasitoid residence time (F ¼ 15:14; df ¼ 1; 101; P < 0:001).
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Table 1 Summary of parasitoid foraging behavior in response to plant genotype, host density, and their interaction. Only significant results are reported in this table Behavior
Agricultural
Wild
Factor
F
df
P
High density
Low density
High density
Low density
Percent of total time walking
29:1 3:8
53:6 3:0
34:9 2:5
44:3 2:7
Density (D)
28.66
1
<0.0001
Percent of total time probing Percent of total time resting
16:8 3:8
13:5 2:4
31:8 2:5
27:7 2:5
GD Genotype (G)
5.19 27.55
1 1
<0.05 <0.0001
37:6 6:3
10:5 2:0
14:1 2:4
5:1 0:8
Genotype (G)
15.62
1
<0.001
37.28 8.44 6.9
1 1 1
<0.0001 <0.01 <0.01
Latency to first probing Frequency of probing Total area covered (cm2 ) Percent of total area covered
170:8 58:8
179:3 47:2
61:8 22:9
93:4 31:1
Density (D) GD Genotype (G)
28:5 6:4
17:7 4:3
43:1 7:9
39:8 6:5
Genotype (G)
7.22
1
<0.01
32:9 1:29
27:76 2:19
6:21 0:58
6:82 0:62
Genotype (G)
17.76
1
<0.0001
0:41 0:07
0:38 0:06
0:90 0:06
0:83 0:05
Genotype (G)
65.24
1
<0.0001
on agricultural flowers and almost three times longer on high density flowers (Table 1). Parasitoids appeared to be more stimulated by host cues on wild flowers, because they probed 2–3 times sooner after landing on wild flowers, probed more frequently, allocated twice as much time to probing, and spent more absolute time probing on wild flowers (Table 1). Although the area searched by parasitoids was 2–3 times larger on agricultural flowers, parasitoids covered twice as much of the total flower area on wild flowers due to their smaller size (Table 1). Parasitoid foraging efficacy may have been reduced on agricultural flowers with low host density due to a decreased frequency of parasitoid contact with host cues. Larval density was much lower on agricultural flowers (0:15 0:02 larvae= cm2 ) than on wild flowers (0:64 0:08 larvae=cm2 ; F ¼ 45:92; df ¼ 1; 79; P < 0:0001) due to the larger size of the agricultural flowers, and did not significantly differ between the high and low density treatments. Parasitism on wild flowers were double the levels found on agricultural flowers (Fig. 7, v2 ¼ 16:8; df ¼ 1; P < 0:0001), and host density did not influence parasitism rates. Each individual female parasitized 25.7% 0.06 more larvae on wild flowers than on the paired agricultural flower (t ¼ 4:41; df ¼ 44; P < 0:0001). While parasitoids probed for hosts on wild flowers four times longer than on agricultural flowers, the resulting parasitism levels were 19 times higher, indicating that parasitoids were much more successful per unit effort on wild flowers. 3.4.2. Na€ıve vs. experienced parasitoid foraging behavior Na€ıve and experienced female parasitoids foraged in a similar manner on agricultural flowers. Na€ıve parasi-
Fig. 7. Percent parasitism by plant genotype and larval density from parasitoid foraging behavior studies. Plant genotype significantly influenced parasitism (v2 ¼ 16:8; df ¼ 1; P < 0:0001), but host density did not influence parasitism rates.
toids allocated most of their time to walking (50.65% 3.29) and probing for hosts (28.54% 4.07), and they spent less time cleaning (16.32% 1.97) and resting (3.69% 1.02) on agricultural flowers. Previous experience did not influence female allocation to walking, probing, cleaning, resting, or the total time spent foraging on the flower. Na€ıve parasitoids covered a similar proportion of the total flower compared with experienced females. Even though both groups of parasitoids spent a large percentage of their total time probing for hosts, percent parasitism on agricultural flowers did not significantly differ between na€ıve (3.3% 3.3) and experienced (7.0% 3.6) parasitoids. Low parasitism rates on agricultural flowers were not
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due to inadequate coverage of the flower head, because na€ıve females covered 71% 7.0 of the flower head area. Therefore, previous foraging experience on wild flowers did not influence female foraging behavior on agricultural flowers.
4. Discussion Our data show that moths and parasitoids respond differently to agricultural and wild plants. Both H. electellum and D. homoeosomae preferred to land on agricultural flowers; however, they differed in their behavioral responses on them. While moths responded to the agricultural flowers by laying more eggs on them, parasitoids responded to agricultural flowers by spending less time probing and by parasitizing fewer larvae. Therefore, domestication has increased H. electellum abundance while reducing D. homoeosomae foraging efficiency and has thus reduced biological control. The most visible difference between agricultural and wild sunflowers is flower head size. Our data suggest that flower enlargement may be an important factor in altering parasitoid–herbivore interactions, because H. electellum laid more eggs on larger agricultural flowers. Although H. electellum is nocturnal, it may use visual cues, because other nocturnal Lepidopteran species have been found to prefer larger visual targets (Rojas et al., 2000). Similarly, enlargement of the flower can alter patterns of host plant use by altering semiochemical plant cues (De Moraes et al., 2001; del Campo et al., 2001; Renwick and Chew, 1994; Udayagiri and Mason, 1997). Agricultural flowers have larger individual florets and therefore have a higher pollen density than wild flowers (Chen, pers. obs.). As pollen is considered a stimulant for H. electellum oviposition (Delisle et al., 1989), the larger quantity of pollen correlated with larger agricultural flowers may cause H. electellum females to lay more eggs. Given that both moths and parasitoids were able to discriminate between agricultural and wild sunflowers in the landing preference tests suggest that there are differences in either the quantity or quality of volatiles emitted. The larger quantity of pollen on agricultural sunflowers may correlate with higher volatile emissions and provide a stronger signal for both moths and parasitoids. Also, H. electellum larvae appear to develop more quickly on agricultural sunflowers, so parasitoids may prefer agricultural flowers because of the larger total output of both host and plant-related volatiles. Both quantitative and qualitative variations in volatile production can be related to parasitoid preference (Geervliet et al., 1998; Hoballah et al., 2002). In other systems, parasitoids can respond to differences in semiochemicals between cultivars (Geervliet et al., 1996), and even finer scale differences between cultivar
lines (Elzen et al., 1986). Further research into the variability in volatile emissions within and between agricultural and wild plants will help determine whether they emit distinct profiles and which volatile compounds are important in eliciting favorable parasitoid responses (Gouinguene et al., 2001). Although parasitoids preferred to land on agricultural plants, they foraged poorly on them. There are several potential causes of the lower success in finding hosts for D. homoeosomae on agricultural flowers. First, female D. homoeosomae probed for host larvae on agricultural flowers, but they allocated far smaller percentage of their time to probing and probed for a shorter time. Second, larval density was much lower on agricultural flowers, and the lowered rate of contact with host products may have caused parasitoids to leave the patches sooner. While the number of larvae was equal on agricultural and wild flowers, larval density was much higher on wild flowers. If larval density is standardized between agricultural and wild genotypes, it would be possible to measure the direct effect of plant genotype on parasitoid behavior, independent of host density. Finally, the floral column is deeper on agricultural flowers than on wild flowers, and may provide a refuge from parasitism (Brown et al., 1995; Feder, 1995; Udayagiri and Welter, 2000). Although host frass and silk were present on agricultural flowers, repeated contact with host products without a successful oviposition has been shown to deter further searching and cause parasitoids to leave patches sooner (Papaj et al., 1994). We propose that the poor foraging efficacy of D. homoeosomae on individual agricultural flowers in this laboratory study contributes to their low efficacy in agricultural fields. It is unlikely that D. homoeosomae foraging behavior of leaving patches early and parasitizing few larvae is an adaptive strategy to ‘‘spread’’ its eggs over more patches like Anagrus delicatus, a parasitoid of a saltmarsh planthopper (Cronin and Strong, 1993). Parasitism levels within agricultural sunflower fields are uniformly low across all patches (1%) throughout the season (Chen and Welter, 2002). Coupled with the our previous field data, these results indicate that D. homoeosomae have not adapted to forage effectively on agricultural flower heads (Chen and Welter, 2002). Our results show that the greater abundance of H. electellum in agricultural sunflowers is in part due to the role of domestication in promoting herbivore activity and disrupting parasitoid efficacy. Developing a successful biological control program for H. electellum may be a challenge, because the remainder of the H. electellum parasitoid complex also appears to be ineffective at controlling H. electellum (Charlet, 1999; Chen and Welter, 2002). As this pattern may be repeated in other agricultural systems, our data suggest that parasitoid adaptation to domesticated crops may be an important
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barrier to overcome in order to develop successful biological control programs for insect pests in agricultural systems. On the other hand, many opportunities exist to work with plant breeders to promote biological control of H. electellum. The higher parasitism levels on wild sunflowers indicate that parasitoid activity can be enhanced considerably. Crop domestication may not always disrupt trophic interactions, because herbivore and parasitoid performance can be equally increased on agricultural crops (Benrey et al., 1998). The challenge for crop breeders is to develop plant attributes that promote the activity of parasitoids while minimizing the attributes favored by herbivores. Determining how herbivore and parasitoid responses have been altered by crop domestication and selection in this system and in other systems will help resolve the ecological factors limiting the successful biological control of insect herbivores by native parasitoids in agricultural habitats.
Acknowledgments We thank Tony Turkovich, US Fish and Wildlife Service, and California Department of Fish and Game for granting us field access to collect insects. We also greatly appreciate the help from John Andrews, Frances Cave, Jill Fortuna, Doug Light, Nick Mills, and Kathy Reynolds on this project. This manuscript has benefited from the comments of Gregory Fanslow, Nick Mills, Wayne Sousa, and several anonymous reviewers. This research was supported by a SAREP SAGA Grant, Van den Bosch Award, and a NSF Predoctoral Fellowship to Y.H. Chen, and also funds from the Division of Agriculture and Natural Resources to S.C. Welter.
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