Marc Rhainds 1,2. Institut de recherche en biologie végétale, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2

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1 728 Toward management guidelines for the soybean aphid in Quebec. I. Feeding damage in relationship to seasonality of infestation and incidence of native predators Marc Rhainds 1,2 Institut de recherche en biologie végétale, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2 Michèle Roy Pêcheries et Alimentation Québec, Direction de l innovation scientifique et technologique, Complexe scientifique, 2700 rue Einstein, Québec, Québec, Canada G1P 3W8 Gaétan Daigle Département de mathématiques et de statistique, Université Laval, Québec, Québec, Canada G1K 7P4 Jacques Brodeur Institut de recherche en biologie végétale, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec, Canada H1X 2B2 Abstract A study was conducted in 2004 and 2005 to test the hypotheses that the severity of damage caused by the soybean aphid, Aphis glycines (Hemiptera: Aphididae), is minimized by the activity of predators and declines with the maturity of soybeans, Glycine max (L.) Merr. (Fabaceae), at the time of infestation. In caged subplots where predators were excluded, aphids attained a high density following experimental infestation of soybeans, resulting in severe reductions of yield, particularly when plants were infested early in the season. A guild of generalist predators consisting predominantly of ladybird beetles colonized plants in uncaged subplots, resulting in a low rate of population growth following infestation of soybeans with aphids and a relatively weak impact on the soybean yield. The soybean yield declined as the density of aphids (number per plant), and the maturity of soybeans at the time of infestation, increased. Our results suggest that A. glycines represents an occasional pest of soybean in Quebec, because of (i) temporal asynchrony between the late-season infestation by aphids and the most susceptible phenological stage of soybeans (vegetative or. flowering) and (ii) biological control by natural enemies. Résumé Une étude réalisée en 2004 et 2005 a testé les hypothèses que l intensité des dommages causés par Aphis glycines (Hemiptera : Aphididae) est minimizée par l action des prédateurs généralistes, et qu elle diminue avec la maturité du soya, Glycine max (L.) Merr. (Fabaceae), au moment de l infestation. Dans des parcelles sous cage où les prédateurs étaient exclus, les pucerons ont atteint des densités élevées suite aux infestations expérimentales, ce qui s est traduit par des pertes considérables de rendement, surtout lorsque les plants avaient été infestés tôt en saison. Une communauté de prédateurs généralistes, essentiellement des coccinelles, a colonizé les plants de soya dans les parcelles sans cage, ce qui s est traduit par un faible taux de croissance des populations de puceron suivant les infestations expérimentales, ainsi que par un impact relativement faible du puceron sur le rendement du soya. Le rendement du soya a diminué avec des densités accrues de pucerons, et les réductions de rendement étaient plus importantes lorsque les infestations avaient lieu tôt en saison. Nos résultats suggèrent qu en dépit de son abondance dans les champs de soya, A. glycines représente un ravageur occasionnel au Québec, dû à (i) une asynchronie temporelle entre la colonization tardive par les pucerons et les stades phénologiques du Received 16 November Accepted 4 April Corresponding author ( mrhainds@purdue.edu). 2 Present address: Department of Entomology, Purdue University, Smith Hall, 901 W State Street, West Lafayette, Indiana , United States of America Can. Entomol. 139: (2007)

2 Rhainds et al. 729 soya les plus sensibles au dommage (stade végétatif et début de formation des gousses) et (ii) un contrôle biologique exercé par les ennemis naturels. Introduction Biological invasions are becoming increasingly frequent because of the rapid growth in international trade (Perrings et al. 2002). Fortunately, only a small fraction of exotic species accidentally introduced into a novel habitat become established, and even fewer reach the status of severe pest (Williamson and Fitter 1996). Incumbent generalist predators in particular may prevent the establishment and spread of introduced organisms or restrict their detrimental impact (Ehler 1998, 2004; Symondson et al. 2002). This has been particularly well documented for aphids (Hemiptera: Aphididae) (Stary and Gonzalez 1992; Briese and Jupp 1995; Michels et al. 2001; Cardinale et al. 2003), to the extent that local predators may be considered more effective than introduced enemies for managing populations of invasive aphids (Michaud 2002). The recent introduction and establishment of the soybean aphid, Aphis glycines Matsumura, from Asia into North America represents a spectacular example of biological invasion. The aphid was first noticed in Wisconsin in 2000, and 3 years later had spread to 21 states in the United States of America and three provinces in Canada (Ragsdale et al. 2004). Aphis glycines is a holocyclic and heteroecious species, alternating between primary (buckthorns, Rhamnus (L.) spp. (Rhamnaceae)) and secondary (soybean, Glycine max (L.) Merr. (Fabaceae)) hosts. Unless it undergoes an (unlikely) episode of continental mass extinction, A. glycines is now established in North America (Venette and Ragsdale 2004). Aphis glycines poses a serious threat to the commercial production of soybeans in North America. Soybean has traditionally been cultivated without insecticides (Heimpel and Shelly 2004), but the A. glycines invasion has already led to the widespread use of insecticides in the United States of America (North Central Soybean Research Program (NCSRP) 2004). Aphis glycines has the potential to reduce the soybean yield (Macedo et al. 2003; Wu et al. 2004; Myers et al. 2005a, 2005b) and has rapidly acquired pest status in the Midwestern United States and Ontario (NCSRP 2004; Baute et al. 2006). However, it may remain a relatively benign (occasional) pest in other regions of North America (Mignault 2005), as is reported in Asia (van den Berg et al. 1997; Heimpel et al. 2004; Liu et al. 2004; Ragsdale et al. 2004). In the province of Quebec, for example, an extensive and ongoing survey initiated in 2002 indicates that whereas A. glycines may be abundant on seedpod-bearing plants late in the season, population densities are typically low early in the season (Mignault 2005), and rarely reach the thresholds of economic damage postulated for China and North America ( aphids per plant at the early flowering stage; Liu et al. 2004; NCSRP 2004). Two main factors may limit the economic impact of A. glycines: (1) severe mortality caused by generalist predators (Fox et al. 2004; Rutledge et al. 2004; Costamagna and Landis 2006; Desneux et al. 2006; Mignault et al. 2006); and (2) colonization of soybean fields late in the season, when the mature plant is least sensitive to feeding injury (Mignault 2005), as has been shown for other plant herbivore associations (McPherson et al. 1993; Wiedenmann and Smith 1997; Cisneros and Godfrey 2001; Rhainds and English-Loeb 2003). The present study investigates the pest status of A. glycines following its establishment in the soybean agroecosystem in Quebec. Using manipulative experiments we quantified the severity of plant damage in relation to the timing of infestation by A. glycines and predation by native natural enemies. Seasonal effects were manipulated by infesting soybeans at different phenological stages with aphids, either by repeatedly infesting even-staged soybeans at different time periods or by infesting unevenstaged soybeans during one time period only. The effect of predation on aphid populations and soybean attributes (trophic cascade) was evaluated using exclusion cages. We tested the specific hypotheses that damage caused by A. glycines declines with the maturity of soybeans at the time of infestation, and is significantly reduced by the activity of aphidophagous native predators. Materials and methods Study site The 2 year study was conducted at a 30 ha experimental farm located in Pintendre, Quebec

3 730 Can. Entomol. Vol. 139, 2007 (46.77 N, W), at the northern end of the distribution of A. glycines in North America. Experiments were conducted in plots measuring approximately 30 m 20 m and located >50 m apart. Plots were fertilized in the spring with 250 kg/ha of (N P K) (three plots in 2004), 300 kg/ha of (N P K B) (one plot in 2004), or 150 kg/ha of (N P K) (four plots in 2005). The different fertilization regimes were determined following soil tests, as recommended in soybeanproduction guidelines for Quebec (Centre de Recherche en Agriculture et Agroalimentaire du Québec 2001). The plots were sown with determinate-growth soybean (<2550 UTM) at a density of one seed every 8 cm within rows and a distance of 18 cm between rows. Weeds were controlled by applying glyphosate (2.5 L/ha) days following planting. Asynchronous aphid infestations of evenstaged soybean plants The timing of infestation in 2004 was manipulated by infesting even-staged soybeans (four plots uniformly planted on 1 June) with aphids repeatedly over time (nine infestation periods). Twenty-four 65 cm 65 cm subplots were established in each plot on 30 June (four rows with each six subplots, with a distance of 4.5 m between rows and subplots), when soybean plants were at the two-foliole stage. Each subplot was isolated from the surrounding vegetation by excavating all plants within 20 cm from its periphery; the distance of 20 cm was selected as a compromise between isolating experimental plants from the remaining crop for several weeks and maintaining a continuous plant canopy in the study area. Subplots were randomly assigned to 1 of 20 treatments (10 infestation treatments 2 cage treatments, i.e., nine periods of infestation and three control subplots for each cage treatment and plot), with half the subplots enclosed in large 0.8 m high screen cages ( µm mesh) and the other subplots remaining uncovered. Cages were used to ensure that aphids used to infest soybeans reached high densities, as preliminary observations indicated substantial levels of predation (see also Fox et al. 2004; Rutledge et al. 2004). Although exclusion cages have the potential to influence some parameters of soybean growth, they seem to have only a moderate overall effect on yield (Simmons and Yeargan 1990). Exclusion cages are an established tool for analyzing interactions in arthropod communities (Messing et al. 2006) and are commonly used to estimate damage caused by arthropod pests on soybeans (Smith and Bass 1972; Ogunlana and Pedigo 1974; McPherson et al. 1979; Rowan et al. 1993; Yeargan et al. 1994; Killen and Lambert 1998), including A. glycines (Costamagna et al. 2007). Temperatures recorded for a period of 7 days (12 18 August) in 2004 below the soybean canopy in one caged subplot and one uncaged subplot (using a min max memory thermometer; Oakton Instruments, Vernon Hills, Illinois) indicated that conditions were similar for caged plants (19.9 ± 0.7 C) and uncaged plants (20.1 ± 0.9 C). This suggests that statistically significant differences between caged and uncaged plants cannot be primarily attributed to the influence of variation in temperature on either the abundance of aphids or the attributes of soybean. In a study using cages similar to the ones described herein, Desneux et al. (2006) observed no significant differences in temperature between caged and uncaged soybeans. Commencing on 1 July, when soybeans were at stage V1 (one trifoliate leaf), 18 randomly selected subplots were infested with aphids per plant every fifth day for a period of 40 days (1 caged and 1 uncaged subplot per plot on each infestation date for each of 9 infestations). The remaining 6 subplots in each plot remained uninfested and served as control, which corresponds to a total of 10 infestation treatments per plot. Visual inspection of one plant per subplot showed that no A. glycines were present at the beginning of the experiment. Infestations were conducted by attaching folioles with aphids per plant (all life stages combined) to soybean leaves; the aphids were collected from a colony maintained on potted soybeans in a growth chamber (24 C, 16L:8D photoperiod, and 60% RH). Soybeans were at the vegetative stage for the period corresponding to the first 4 infestations (1 16 July), at the flowering and early seedpod-bearing stages during the next 3 infestations (21 31 July), and at the late seedpod-bearing stage during the last 2 infestations (5 10 August). Commencing on 16 July, before soybeans initiated flowering, three to five plants were collected in each subplot every 15 days until 31 August in order to record the numbers of aphids (apterous and winged) and aphidophagous predators. On 14 September, three to five plants were sampled in each subplot to evaluate the fresh biomass (±0.1 g)

4 Rhainds et al. 731 of seedpods; arthropod densities were not evaluated at that time because tropical depression Jeanne had killed most aphids shortly before. Synchronous aphid infestations of unevenstaged soybean plants The timing of infestation relative to plant phenology was manipulated in 2005 by infesting plants at various developmental stages with aphids at one point in time. Four plots, each divided into three 6.5 m 30mtreatments, were sowed with soybeans on 19 May, 1 June, or 20 June. For each plot and planting date, five 65 cm 65 cm subplots were established on 3 July in the center of each row, with a distance of 4 m between subplots; each subplot was isolated from the surrounding vegetation by excavating all plants within 20 cm from its periphery. Soybeans were experimentally infested with aphids per plant on 18 July, when plants were either at the early seedpodbearing stage (R3; early-season planting), at the early flowering stage (R1 R2; midseason planting), or had three or four trifoliate leaves (V3 or V4; late-season planting). For each plot and planting date, subplots were randomly assigned to one of five treatments: (1) caged control plants not infested with aphids; (2) caged plants infested with aphids; (3) caged plants infested with aphids and 2 days later with six 1- to 3-week-old adult coccinellids, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) (four females and two males per cage); (4) uncaged control plants not infested with aphids; and (5) uncaged plants infested with aphids. Shortly before soybeans were experimentally infested with aphids, three plants per subplot were sampled for aphids. The density of aphids was low at the beginning of the experiment (an average of 0.3 and 1.2 aphids on caged and uncaged plants, respectively); two caged subplots planted early in the season and heavily infested with aphids were excluded from the experiment. Commencing on 2 August, up to five plants (depending upon availability) were sampled in each subplot to determine the number of aphids (apterous and winged individuals) and predators. Sampling was repeated six times between 2 August and 3 September at intervals of 7 15 days; arthropods were not sampled after 3 September because shortly before, tropical depression Katrina had killed most aphids. The fresh biomass of seedpods from three to five plants in each subplot was recorded on 11 September. Because we were interested in comparing variation in soybean yield as a function of planting date, the experiment had to be conducted for a longer physiological time for soybeans planted early in the season in 2005 than for any other treatment. Therefore, cages had to be removed from all experimental subplots after sampling assessments were made on 17 August because the soybean foliage occupied a large volume of the cage in some subplots, and this may have prevented further plant growth and favoured the development of fungal disease. It seems unlikely that the removal of cages late in the season strongly affected the expression of damage caused by aphids: insecticide applications when soybeans are at the full-pod stage (R4) reduce the abundance of A. glycines, yet have no discernible impact on yield (Myers et al. 2005b), which suggests that the feeding pressure exerted by aphids after the formation of seedpods results in minor damage. Impact of aphids on soybean yield The timing of aphid infestation was quantified for different planting dates in 2004 and 2005 using the cumulative day-degrees above 10 C between the sowing date and the infestation date; average daily temperatures were obtained from the nearest meteorological station in Québec City (46.47 N, W), approximately 30 km southwest of the experimental site. Yield of different subplots was estimated using the fresh biomass of seedpods per plant. For both caged and uncaged plants, an index of yield reduction (Y) was calculated for each subplot, i, as a function of infestation time using the equation Y i =1 (X ti /x i ), where X ti is the fresh biomass of seedpods per plant in subplot i infested with aphids at phenological stage t (cumulative day-degrees above 10 C since planting) and x i is the fresh biomass of seedpods per plant in control subplot i. Intervals of t values ranged between 158 and 528 day-degrees in 2004 (nine infestations) and between 320 and 463 day-degrees in 2005 (three infestations). Statistical analyses Statistical analyses were conducted using the SAS statistical package (SAS Institute Inc. 2002). For each subplot, the overall abundance of aphids and predators was measured throughout the season using cumulative indices of

5 732 Can. Entomol. Vol. 139, 2007 aphid-day and predator-day as defined by Ruppel (1983); cumulative values were computed up to the moment when the cage cover was removed in 2005, unless otherwise specified. Parameters (mean value per plant per subplot) recorded in the experiment (cumulative aphid-days, cumulative predator-days, fresh biomass of seedpods) were analyzed with ANOVA using the mixed procedure of SAS. For all analyses, the assumption of normality was verified with the Shapiro Wilk test, while homogeneity of variance was examined visually using graphs of residuals plotted against predicted values. Heterogeneity of variance was reduced by subjecting data to square-root (density of aphids and predators) and logarithmic (fresh biomass of seedpods) transformations. In 2004 the experimental design consisted of a two-way treatment structure, cage cover (2 levels) infestation (10 levels), in a generalized randomized complete block design with 4 blocks each containing 20 experimental treatments (Table 1). The design is generalized because the control treatment (plants not infested with aphids) was repeated in three subplots of each block for both caged and uncaged plants. This resulted in a number of experimental treatments (20) that differed from the number of subplots (24) in each block. Data in 2005 were analyzed using a split-plot ANOVA. The whole-plot experimental design consisted of a one-way treatment structure, planting date (3 levels), in a randomized complete block design structure with four blocks each containing three whole-plot experimental units. Each whole plot was split into five subplots and treatments were randomly assigned to one subplot within each whole plot. The subplot experimental design consisted of a one-way treatment structure, overall treatment effect (5 levels), in a randomized complete block design with 12 blocks (4 replicates 3 planting dates) each containing five subplot experimental units. The overall treatment effect included five treatments: (1) caged control plants not infested with aphids; (2) caged plants infested with aphids; (3) caged plants infested with aphids and predators; (4) uncaged control plants not infested with aphids; and (5) uncaged plants infested with aphids (Table 2). This treatment effect corresponds to an incomplete three-way factorial structure with three level factors: presence of a cage cover, infestation with aphids, and predator access. This treatment structure comprises eight treatment combinations, but only the five listed above were tested. 3 Following a significant overall treatment effect, a posteriori contrasts were computed on the basis of the type IV estimable functions to isolate the effects of cage cover, infestation with aphids, and predation (Table 2). Because of the pattern of treatment combinations, specific hypotheses couldnot be tested for the two-way and higher order interaction effects without making additional assumptions about the parameters in the model (Milliken and Johnson 1984). The contrasts were tabulated as a linear combination of averages for different treatments using the coefficients listed in Table 3. In 2004, linear regression was used to evaluate the relationship between cumulative aphiddays and biomass of seedpods in different subplots. In 2005, the effect of planting date and cumulative aphid-days throughout the season (including the period following removal of the cage cover) on the fresh biomass of seedpods was evaluated using analysis of covariance (ANCOVA), treating planting date as a class variable and abundance of aphids as a covariate. The relationship between the index of yield reduction in different subplots and the timing of aphid infestation (cumulative daydegrees in terms of soybean development) was 3 A statistically balanced three-way factorial structure would have included eight treatments: (1) caged control plants not infested with aphids; (2) caged control plants not infested with aphids and inoculated with adult H. axyridis; (3) caged plants infested with aphids; (4) caged plants infested with aphids and inoculated with adult H. axyridis; (5) uncaged control plants not infested with aphids; (6) uncaged control plants not infested with aphids and inoculated with adult H. axyridis; (7) uncaged plants infested with aphids; and (8) uncaged plants infested with aphids and inoculated with adult H. axyridis. Owing to a lack of adult H. axyridis of known age and sex, treatments 2, 6, and 8 were not included in the experimental design. Although this situation created an unbalanced factorial design from a statistical perspective, the omission of these treatments was sound from a biological point of view. Adult H. axyridis are predacious, and releasing them on caged plants without aphids (treatment 2) was not expected to have a measurable effect on the biomass of seedpods. Moreover, adult H. axyridis are highly mobile and would most likely have redistributed themselves on surrounding plants following their release on uncaged subplots (treatments 6 and 8).

6 Rhainds et al. 733 Table 1. ANOVA of parameters characterizing the tritrophic interaction between seedpod biomass of soybeans, cumulative abundance of soybean aphids (A. glycines), and cumulative abundance of generalist predators as a function of timing of infestation with aphids (I) and presence of a cage cover (C) in Aphid-days Predator-days Seedpod biomass Source df F P F P F P Block < < C < < < I < < C I < < Error 73 Note: Data were analyzed using a generalized randomized complete block design with all treatments randomized within blocks. Heterogeneity of variance was reduced by subjecting values (mean per plant per subplot) to square-root (densities of aphids and predators) and logarithmic (fresh biomass of seedpods) transformations. Table 2. ANOVA of parameters characterizing the tritrophic interaction between seedpod biomass of soybeans, cumulative abundance of A. glycines, and cumulative abundance of generalist predators in 2005 as a function of infestation with aphids (I), presence of a cage cover (C), planting date (D), and predator exclusion (P). Aphid-days Predator-days Seedpod biomass Source df F P F P F P Block D Main plot error 6 Treatment < < < Contrasts C I < < P < < D treatment Subplot error 34 Note: Data were analyzed using a split-plot ANOVA with planting date in the main plot and treatment effect in the subplot. Following the ANOVA with significant overall treatment effect, a posteriori contrasts were computed to isolate the effect of C, I, and P. The coefficients used to calculate the linear combination of averages for different contrasts are listed in Table 3. Heterogeneity of variance was reduced by subjecting data (mean values per plant per subplot) to squareroot (densities of aphids and predators) and logarithmic (fresh biomass of seedpods) transformations. The experimental design and ANOVA are detailed in the Statistical analysis section of Materials and methods. Table 3. Coefficients used to assess the linear combination of average values for different treatments in a posteriori contrasts to evaluate the effect of the presence of a cage cover (C), infestation with aphids (I), and predator access (P) in Independent variable Contrast Treatment C I P C I P 1 Yes No No Yes Yes No Yes Yes Yes No No Yes No Yes Yes Note: A detailed description of the ANOVA is provided in Table 2.

7 734 Can. Entomol. Vol. 139, 2007 evaluated in 2004 and 2005 using ANCOVA, treating year as a class variable and day-degree as a covariate; separate analyses were conducted for caged and uncaged plants. Results 2004 Overall, the abundance of aphids (expressed as aphid-days) was higher on soybean plants covered with a cage and infested early in the season (P < for both effects) (Fig. 1A), although the significant cage cover infestation interaction (P < ) indicates that these effects were not consistent for all experimental treatments (Table 1). Separate contrasts conducted across infestation periods revealed that a cage cover reduced the abundance of aphids on control plants but had the opposite effect on plants experimentally infested with aphids (Fig. 1A). Separate contrasts conducted across cage-cover treatments indicated that the cumulative aphid-days attained through the season was influenced by the timing of infestation in the presence of a cage cover (F 9,73 = 39.64, P < ) but not in its absence (F 9,73 = 0.88, P = 0.549). The significant variation in aphid abundance among plots (P < ; Table 1) may have been due to the differences in fertilization regime (Myers et al. 2005a). The fauna of natural enemies consisted mostly of generalist predators, including ladybird beetles (Coleoptera: Coccinellidae), midges (Diptera: Chironomidae), hoverflies (Diptera: Syrphidae), pirate bugs (Hemiptera: Anthocoridae), and lacewings (Neuroptera: Chrysopidae). Ladybird beetles represented the most common predators (53.4% of 319 individuals), Coccinella septempunctata L. being the most abundant species during both years, followed by H. axyridis. The cumulative abundance of predators was strongly suppressed by a cage cover (P < ) (Fig. 1B, Table 1), although the effect was not consistent for all infestation periods, as indicated by a significant cage cover infestation interaction (P = 0.014) (Fig. 1B, Table 1). The presence of a cage cover (P < ), the timing of infestation with aphids (P < ), and their interaction (P < ) all had a significant impact on the fresh biomass of seedpods (Table 1). Separate contrasts conducted across infestation periods revealed differences in biomass between caged and uncaged plants only for soybeans that had been infested early in the season when they were at the vegetative stage (the first four infestations of the season) (Fig. 1C). Separate contrasts conducted across cage-cover treatments indicated that the fresh biomass of seedpods was influenced by the timing of infestation in the presence of a cage cover (F 9,73 = 13.11, P < ) but not in its absence (F 9,73 = 0.90, P = 0.531) The date of soybean planting had no significant effect on any dependent variables recorded in this study, either by itself or in an interaction with overall treatment effect (Table 2). The overall treatment effect, in contrast, had a highly significant (P < ) influence on the cumulative abundance of aphids and of predators, as well as on the biomass of seedpods (Table 2). To assess the relative impact of a cage cover, infestation with aphids, and predator exclusion on the different dependent variables, a posteriori contrasts were computed from the overall treatment effect to isolate their separate effect (Table 2). Table 3 is a useful summary of the specific comparisons involved in these contrasts. The cumulative abundance of aphids throughout the season was considerably higher on plants experimentally infested with aphids than on control plants (P < ); the difference appeared more pronounced in caged subplots than in uncaged subplots (Fig. 2A), but the effect of the infestation cage cover interaction could not be tested statistically because the experimental design was unbalanced (see the Statistical analysis section in Materials and methods). In caged subplots, the inoculation of plants experimentally infested with aphids with H. axyridis greatly reduced the density of aphids compared with infested plants not inoculated with predators (P < ). The density of aphids was influenced by the presence of a cage cover to a significant but relatively small extent (P < ), which was due to the higher density of aphids on plants naturally colonized by local predators in uncaged subplots than on plants inoculated with H. axyridis in caged subplots; this effect of cage cover on the abundance of aphids was indirect, because the predator fauna differed in the two treatments (Table 2, Fig. 2A). Ladybird beetles were by far the most common predators in 2005 (92.6% of 359 individuals, excluding plants in caged subplots

8 Rhainds et al. 735 Fig. 1. Parameters characterizing the tritrophic interaction between seedpod biomass of soybeans, cumulative abundance of soybean aphids (A. glycines), and cumulative abundance of generalist predators for soybeans covered or not covered with exclusion cages and infested with aphids at different periods in Soybeans were infested at the vegetative stage for the first four infestations (60 75 days since infestation), at the flowering stage to early seedpod-bearing stage for the next three infestations (45 55 days since infestation), and at the late seedpod-bearing stage for the last two infestations (35 40 days since infestation); control soybeans were not experimentally infested with aphids (0 days since infestation). The significant cage cover time of infestation interaction for all dependent variables (Table 1) justified conducting separate contrasts across infestation periods. For each infestation period, an asterisk above a value indicates a significant difference between caged and uncaged plants (P < 0.05). experimentally infested with H. axyridis). As predicted, predators were considerably more abundant in caged subplots where plants had been inoculated than in caged subplots where they had not (P = ). Experimental infestation of soybeans with aphids did not significantly affect the cumulative abundance of predators (P = ). The lack of a significant cage-cover effect (P = ) was due to the fact that the overall abundance of predators in caged subplots on plants infested with aphids and inoculated with H. axyridis was similar to that on plants in uncaged subplots that were colonized by local predators (Table 2, Fig. 2B). The fresh biomass of seedpods was considerably lower on plants experimentally infested with aphids than on control plants (P < ); the difference appeared more pronounced in caged subplots than in uncaged subplots (Fig. 2C), but the effect of the infestation cage cover interaction could not be tested statistically because the experimental design was unbalanced (see the Statistical analysis section in Materials and methods). In caged subplots, the inoculation of plants experimentally infested with aphids with H. axyridis enhanced the biomass of seedpods compared with that of infested plants not inoculated with predators (P < ). The soybean yield was influenced by the presence of a cage cover to a significant but relatively small extent (P < ), which was due to the slightly smaller seedpod biomass on plants in uncaged subplots naturally colonized by local predators than on plants in caged subplots inoculated with H. axyridis (Fig. 2C); this effect of cage cover on the soybean yield was probably indirect, because the higher density of aphids in uncaged plots than in caged plots (Fig. 2A) may have contributed to depressing the biomass of seedpods (Table 2, Fig. 2C). Impact of aphids on the soybean yield in 2004 and 2005 The biomass of seedpods per plant was negatively correlated with the cumulative aphid-days in 2004 (Table 4). The significant aphid density planting date interaction in 2005 (F 2,52 = 3.71, P = ) indicates that the feeding impact varied among plants infested at different phenological stages. The relationship between the cumulative abundance of aphids and the biomass of seedpods was highly significant for soybeans planted late in the season (i.e., infested at the vegetative stage), whereas the relationship was not significant for soybeans planted early in the season (i.e., infested with aphids at the beginning of seedpod formation) (Table 4). The declining steepness of the slopes of the relationship with the date of planting (Table 4) further

9 736 Can. Entomol. Vol. 139, 2007 Fig. 2. Parameters characterizing the tritrophic interaction between seedpod biomass of soybeans, cumulative abundance of aphids, and cumulative abundance of generalist predators for soybeans planted on different dates in 2005 and assigned to one of five treatments: (1) caged control plants not infested with aphids; (2) caged plants infested with aphids; (3) caged plants infested with aphids and 2 days later with six 1- to 3-week-old adult coccinellids (Harmonia axyridis) (four females and two males per cage); (4) uncaged control plants not infested with aphids; and (5) uncaged plants infested with aphids. Soybean plants were infested with aphids on 18 July, when those planted on 20 June were at the vegetative stage, those planted on 1 June were at the early flowering stage, and those planted on 19 May were at the early seedpod-bearing stage. A summary of statistical analyses is provided in Table 2. Number of aphid-days ( 1000) Number of predator-days Biomass of seedpods (g) Planting date Uncaged plots A B C 19 May 1 June 20 June Caged plots 0 Infestation No Yes No Yes Yes Predators Yes Yes No No Yes revealed that soybeans planted late in the season were most susceptible to aphid infestation. In caged subplots, ANCOVA indicated a significant reduction of seedpod biomass with increasing maturity of soybeans at the time of infestation (expressed as cumulative day-degrees above 10 C) (F 1,43 = 14.90, P < 0.001); the lack of a significant effect of either year (F 1,43 = 0.18, P = 0.668) or day-degree year (F 1,43 = 0.80, P = 0.376) indicated that the relationship was similar for the two years (Fig. 3). In uncaged subplots, neither day-degree (F 1,44 = 0.00, P = 0.978), year (F 1,44 = 0.18, P = 0.673) nor the interaction (F 1,44 = 1.29, P = 0.263) was statistically significant (Fig. 3). Discussion The introduction of A. glycines into North America has had serious consequences, both in economic terms by reducing the soybean yield (Macedo et al. 2003; Wu et al. 2004; Myers et al. 2005a, 2005b; this study) and in environmental terms by necessitating insecticide applications over a vast acreage of agricultural land that was previously untreated (Heimpel and Shelly 2004; Mignault 2005). A formal economic threshold for A. glycines remains to be established and validated across North America, a threshold that should take environmental costs into consideration (Higley and Wintersteen 1996). Results from our manipulative experiments highlight two

10 Rhainds et al. 737 Table 4. Parameters of regression models analyzing the relationships between the cumulative density of A. glycines (sum of aphids sampled per plant per day) and the fresh biomass of seedpods (g). Date of planting r 2 β 0 β 1 1 June ** 3.21± ± May ± ± June * 3.38± ± June ** 3.63± ± Note: ANCOVA indicated that the biomass of seedpods in 2005 was significantly affected by the density of aphids (F 1,52 = 30.84, P < ) and the aphid density planting date interaction (F 2,52 = 3.71, P = ), thereby justifying separate analyses for the different planting dates. Heterogeneity of variance was reduced by subjecting data to square-root (density of aphids) and logarithmic (biomass of soybeans) transformations. (*, P < 0.05; **, P < ). Fig. 3. Reduction in fresh biomass of seedpods (mean ± SE) on caged and uncaged soybeans infested with A. glycines at different phenological stages (cumulative day-degrees above 10 C between the date of planting and the date when plants were infested with aphids) in 2004 ( ) and 2005 ( ). The phenological stages of soybeans at the time of infestation are shown in the lower section of the graphs. parameters that influence the feeding impact of A. glycines: the seasonality of infestation and the beneficial activity of aphidophagous predators. The development of a holistic approach, taking into account variables across multiple trophic levels (phenological stage of soybeans, abundance of aphids, incidence of predators) may lead to useful management guidelines. Seasonality of aphid infestation We investigated the seasonal impact of A. glycines by infesting soybeans at various developmental stages. In the experimental approach used in 2004, the length of infestation was manipulated by inoculating even-staged plants repeatedly over time, whereas in 2005, plants at various developmental stages were infested with aphids at one point in time. These complementary approaches are both valid and commonly used to assess the effect of seasonality of infestation on the feeding impact of herbivores (McPherson et al. 1993; Cisneros and Godfrey 2001; Wold and Hutchison 2003). Repeated experimental infestation of evenstaged soybeans with aphids in 2004 revealed that infestation of soybeans at the vegetative stage significantly reduced the fresh biomass of seedpods on plants in exclusion cages, whereas infestation of soybeans with aphids at subsequent developmental stages did not (Fig. 1C); interestingly, the former trend was not significant in uncaged subplots on plants naturally colonized by predators (see below). Experimental infestation of uneven-staged soybeans with aphids at one point in time in 2005, in contrast, resulted in a nonsignificant planting date overall treatment interaction effect (Table 2), which suggests that infestation with aphids

11 738 Can. Entomol. Vol. 139, 2007 reduces the soybean yield independently of the phenological stage of the soybeans (Fig. 2C). This conclusion ought to be interpreted cautiously, however, because (i) the yield loss caused by aphids in caged subplots declined with increasing maturity of soybeans at the time of infestation in the same way in 2004 and 2005, as revealed by a lack of significant effect of either year or year timing of infestation (Fig. 3); and (ii) the negative impact of aphids on seedbpod biomass was influenced by planting date in 2005, as revealed by a significant aphid density planting date interaction, and it was more pronounced for soybeans planted late in the season (i.e., infested at the vegetative stage) (Table 4). The latter two observations, combined with the data obtained in 2004, provide consistent support for the hypothesis that soybean is particularly susceptible to aphid injury when it is infested at an early growth stage. These results are consistent with previous findings that feeding by A. glycines restricts the growth and yield of soybean, especially when plants are infested with numerous individuals for a prolonged period (Macedo et al. 2003; Wu et al. 2004). The lack of significant effect of insecticide applications on yield of soybean treated at the full-pod stage (R4) (Myers et al. 2005b) provides further support for the hypothesis that the feeding pressure exerted by aphids late in the season results in only minor damage (see also Rutledge and O Neil 2006). Agronomic practices related to the phenology of soybean (date of planting, early-season cultivar) may influence the status of A. glycines as an economic pest. Suitable climatic conditions combined with a vast acreage of soybean have resulted in a rapid expansion of A. glycines in the province of Quebec. Despite its ubiquity, however, A. glycines may remain a minor (occasional) pest because plants are typically colonized late in the season (Mignault 2005), so aphid densities on flowering plants are consistently below the provisional economic threshold of individuals per plant (NCSRP 2004). In fact, not a single commercial soybean field out of more than 50 sampled each year between 2002 and 2006 has even approached a density of 250 aphids per plant when soybeans were at reproductive stage R2 (M. Rhainds, unpublished data). Impact of predators on the population dynamics of aphids Densities of A. glycines only occasionally reach economically damaging levels in some regions of Asia, which may be due to high levels of mortality caused by predators, parasitoids, and entomopathogens (van den Berg et al. 1997; Heimpel et al. 2004; Liu et al. 2004; Wu et al. 2004). As previously recorded in a survey conducted across the province of Quebec (Mignault et al. 2006) and direct observations conducted in soybean fields in Michigan (Costamagna and Landis 2007), a guild of generalist predators consisting predominantly of coccinellids responded to A. glycines populations in our experimental subplots. The collective impact of predators contributed to the regulation of aphid populations, as indicated by the relatively low abundance of aphids on uncaged plants experimentally infested with aphids in comparison with control plants (Figs. 1A, 1B, 2A, and 2B; see also Fox et al. 2004; Rutledge et al. 2004). The beneficial impact of generalist predators appears to be consistent across a wide range of soybean-management systems (Costamagna and Landis 2006), although the effectiveness of predators may be influenced by the relative abundance of aphids (numerical and functional responses; Costamagna and Landis 2007) and by the spatial distribution of aphid populations (Desneux et al. 2006). The higher population densities attained by aphids following infestation of soybeans in caged subplots than in uncaged subplots are indicative of natural biological control by predators (Figs. 1A, 1B, 2A, and 2B). Also, the confinement of aphids on caged plants may have been responsible in part for the high density of aphids. However, it seems unlikely that reproduction by the artificially confined winged adults was the main factor responsible for the massive buildup of aphid populations in cages. The following observations were made: (i) The proportion of winged individuals on caged plants remained low throughout the season, never exceeding 1.2% on any individual plant (see also Costamagna et al. 2007). (ii) Relatively large numbers of winged aphids were occasionally observed on the surface of the cage (rather than on the plants), but this occurred only after the aphid populations had become large (approximately1000 individuals per plant; see also Fox et al. 2004); the manual removal of winged cereal aphids from caged wheat plants did not affect the population dynamics of

12 Rhainds et al. 739 the aphids in a cage environment (Chambers et al. 1983). (iii) Winged aphids on the inner surface of the cages had an opportunity to escape when the cages were opened during sampling every 7 15 days. (iv) The hypothesis that the difference in aphid densities in caged subplots and uncaged subplots is due to the beneficial activity of predators is directly supported by the low abundance of aphids on caged plants infested with aphids in the presence of H. axyridis (Figs. 2A and 2B). The influence of predators in reducing the abundance of A. glycines cascaded across trophic levels and enhanced the soybean yield; this hypothesis is supported by the differential impact of aphids on seedpod biomass in the presence and absence of predators (Figs. 1C and 2C). Our results are consistent with those of Costamagna et al. (2007), which also demonstrate that the strong suppression of A. glycines by generalist predators cascades down to enhance the soybean yield. Timely colonization by generalist predators may therefore increase the abundance and mitigate the feeding impact of A. glycines, even when plants are infested early in the season at a highly susceptible developmental stage. The suppression of incipient populations of A. glycines by predators (Fox et al. 2004, 2005; Rutledge et al. 2004) may alleviate the negative impact of the aphids by delaying a massive population buildup beyond the phenological stage at which soybean is most vulnerable to aphid injury (see Fig. 1C in Wiedenmann and Smith 1997). Acknowledgments We are grateful to G. Boivin, S. Boudreault, É. Gagnon, M.E. Leclerc, A. Robichaud, J. Savard, and A. Vaillancourt for technical assistance. D. Marois and M. Lacroix helped to establish and maintain the experimental subplots. D. Gray and two anonymous reviewers provided useful comments on earlier versions of the manuscript. Funding was provided by the Fonds Québécois de la Recherche sur la Nature et les Technologies and by the program Prime- Vert of the Ministère de l Agriculture, des Pêcheries et l Alimentation (Québec). References Baute, T., Bohner, H., and Quesnel, G Economics of spraying foliar insecticides for the control of soybean aphid in on-farm strip trials in Available at _sba_onfarm_strip_trial_final_report.pdf [accessed 4 July 2007]. Briese, D.T., and Jupp, P.W Establishment, spread and initial impact of Aphis chloris Koch (Hemiptera: Aphididae), introduced into Australia for the biological control of St-Johns wort. Biocontrol Science and Technology, 5: Cardinale, B.J., Harvey, C.T., Gross, K., and Ives, A.R Biodiversity and biocontrol: emergent impacts of a multi-enemy assemblage on pest suppression and crop yield in an agroecosystem. Ecology Letters, 6: Centre de Recherche en Agriculture et Agroalimentaire du Québec Guide soya. Centre de Recherche en Agriculture et Agroalimentaire du Québec. pp Chambers, R.J., Sunderland, K.D., Wyatt, I.J., and Vickerman, G.P The effects of predator exclusion and caging on cereal aphids in winter wheat. Journal of Applied Ecology, 20: Cisneros, J.J., and Godfrey, L.D Midseason pest status of the cotton aphid (Homoptera: Aphididae) in California cotton: is nitrogen a key factor? Environmental Entomology, 30: Costamagna, A.C., and Landis, D.A Predators exert top-down control of soybean aphid across a gradient of agricultural systems. Ecological Applications, 16: Costamagna, A.C., and Landis, D.A Quantifying predation on soybean aphid through direct field observations. Biological Control, 42: Costamagna, A.C., Landis, D.A., and Di Fonzo, C.D Suppression of soybean aphid by generalist predators results in a trophic cascade in soybeans. Ecological Applications, 17: Desneux, N., O Neil, R., and Yoo, H.J.S Suppression of population growth of the soybean aphid, Aphis glycines Matsumura, by predators: the identification of a key predator and the effects of prey dispersion, predator abundance, and temperature. Environmental Entomology, 35: Ehler, L.E Invasion biology and biological control. Biological Control, 13: Ehler, L.E An evaluation of some natural enemies of Spodoptera exigua on sugarbeet in northern California. BioControl, 49: Fox, T.B., Landis, D.A., Cardoso, F.F., and DiFonzo, C Predators suppress Aphis glycines Matsumura population growth in soybean. Environmental Entomology, 33: Fox, T.B., Landis, D.A., Cardoso, F.F., and Di Fonzo, C.D Impact of predation on establishment of the soybean aphid, Aphis glycines in soybean, Glycine max. BioControl, 50: Heimpel, G.E., and Shelly, T.E The soybean aphid: a review of its biology and management. Annals of the Entomological Society of America, 97: 203.

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