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Assessment of three techniques for measuring natural enemy inflicted mortality of leafroller larvae in commercial orchards
Biological Control 41 (2007) 312–320 www.elsevier.com/locate/ybcon
Assessment of three techniques for measuring natural enemy inflicted mortality of leafroller larvae in commercial orchards Mark A. Sarvary *, Jan Nyrop, Harvey Reissig Department of Entomology, New York State Agricultural Experimental Station, Cornell University, Geneva, NY 14456, USA Received 7 June 2006; accepted 28 February 2007 Available online 12 March 2007
Abstract Measuring natural enemy (NE) inflicted mortality presents a serious challenge in managed habitats, such as apple orchards, where the crop-plant is frequently treated with a wide range of insecticides. Toxic residues keep natural populations of pests at too low levels to sample and prevent the use of techniques in which laboratory reared insects are released directly on foliage and used to measure NE activity. Three methods to implant obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae) larvae were tested: (1) larvae placed on potted apple trees, (2) larvae placed on excised leaves inserted in florists’ waterpicks and (3) larvae placed on branches placed into water filled Polyvinyl chloride (PVC) pipes. Use of potted trees allowed accurate estimation of NE inflicted mortality; however, this method was very labor intensive. The waterpick method required less labor, but it underestimated larval mortality caused by NEs. The use of PVC pipes to hold infested branches combined the benefits of the other two methods tested. This technique was simple to use and provided accurate estimates of NE inflicted mortality. 2007 Elsevier Inc. All rights reserved. Keywords: Choristoneura rosaceana; Outsourcing; Implantation; Apple orchard; Oncophanes americanus; Actia interrupta; Colpoclypeus florus; Parasitism; Predation
1. Introduction Measuring natural enemy (NE) inflicted pest mortality is required in many biological control studies. Mortality by NEs can be measured by either sampling the natural pest population directly (Kiritani and Dempster, 1973; Sunderland, 1988; Southwood and Henderson, 2000) or by implanting laboratory raised sentinel larvae (Jervis and Kidd, 1996). Laboratory raised sentinel larvae are ‘‘outsourced’’ or ‘‘implanted‘‘ into habitats where pest numbers are very low or direct pest sampling is precluded (Jervis and Kidd, 1996; Sarvary et al., 2007). Implantation of eggs (Burn, 1982; Fowler et al., 1991; Lawson et al., 1997; Merfield et al., 2004), larvae (Suckling et al., 2001; Cusson et al.,
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2002) or pupae (Speight and Lawton, 1976) has been used to collect parasitoids for laboratory trials or to compare relative treatment effects on NEs without the attempt to estimate actual parasitism and predation levels. This ‘‘trap host’’ method has been applied to many NE-host systems (Van Driesche, 1988; Van Driesche and Bellows, 1988; Lopez and Van Driesche, 1989; Van Driesche et al., 1991; Gould et al., 1992a,b; Sarvary et al., 2007) with known flaws such as abnormal pest density or the unnatural dispersal and behavior of the sentinel prey (Kiritani and Dempster, 1973; Van Driesche et al., 1991; Jervis and Kidd, 1996). Measuring mortality imposed by NEs presents a challenge when it is not possible to directly infest host plants or to sample natural pest populations. Implanting techniques using ‘‘mobile media’’, such as infested potted plants to outsource larvae and eggs have been used only in a few parasitism estimation studies (Fowler et al., 1991; Kruess and Tscharntke, 2000; Suckling et al.,
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2001), but the accuracy of these methods has rarely been evaluated. The use of ‘‘mobile media’’ to outsource hostinsects in parasitism and predation assessment studies is especially important in managed agro-ecosystems, such as apple orchards, where the crop-plant is frequently treated with a wide range of insecticides, resulting in toxic residues persisting on fruit and foliage. These toxic residues keep populations of pests at too low levels to sample and prevent the release of laboratory reared insects directly on crop foliage. The objective of our study was to assess methods to measure parasitism and predation rates in apple orchards where pesticide use makes direct collection or direct implantation of hosts impossible. Three mobile media: (1) potted trees aggregated into groups of nine, (2) two apple leaves placed into florists’ waterpicks and (3) apple branches with foliage held in pipes attached to trees, were tested as possible tools to carry implanted sentinel larvae. We hypothesized that these methods would provide as accurate an estimate of parasitism and predation as direct implantation of larvae onto field grown trees. These methods were tested in apple orchards using the Obliquebanded leafroller , Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). This bivoltine, polyphagous, North American totricid is a pest in apple (Chapman and Lienk, 1971; Reissig, 1978) and the larvae establish feeding sites by rolling leaves and feeding on the fruit or on surrounding foliage (Reissig, 1978). Because it is a native herbivore, C. rosaceana is a host for a wide range of natural enemies (Hagley and Barber, 1991; Brunner, 1996; Li et al., 1999; Cossentine et al., 2004a,b; Wilkinson et al., 2004; Sarvary et al., 2007). 2. Materials and methods 2.1. Experimental sites and laboratory colonies of C. rosaceana Three methods to assess predation and parasitism were evaluated by implanting sentinel larvae in ca. 5 hectare large apple orchards on the property of the New York State Agricultural Experimental Station in Geneva, NY (NYSAES). The tests were set up in ‘Macintosh’ and ‘Cortland’ apple trees that were at least 5 years old (referred to as field grown trees). The trees received normal treatments of fungicides and herbicides were used to control weed; however, trees were not sprayed with insecticides. The objective of these studies was to compare estimates of NE inflicted mortality on C. rosaceana larvae implanted using mobile media with larvae directly placed on foliage of field grown trees. Choristoneura rosaceana larvae were allowed to establish feeding sites on apple leaves in the tested media and on growing tips of field grown apple trees and then were exposed to NEs. Sentinel C. rosaceana larvae were selected from laboratory colonies, founded by larvae collected from commercial apple orchards and wild habitats in 1994 and
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2001 and maintained on fava bean plants, Vicia faba L. in a controlled environment (23 C, 16L:8D, 60%–80% RH). Leafroller neonates were removed from the colonies and 1–3 neonates were raised in 30 ml plastic cups (SOLO cup company, Canada) containing pinto bean, Phaseolus vulgaris L. diet (Shorey and Hale, 1965). Sentinel larvae were reared so that when they were used in experiments, their phenology coincided with the natural C. rosaceana populations in the field. After field exposure to NEs, larvae were retrieved and individually placed onto fresh pinto bean diet in 30 ml plastic cups and kept in a controlled environment at 24 C, 16 L:8D, 85% RH. Emerging parasitoid adults were identified. 2.2. Testing potted apple trees as a tool for measuring natural enemy activity One-year-old ‘Cortland’ apple trees were planted into potting soil (Cornell Mix-NYSAES) in 10 gallon plastic pots in 2001. The potted apple trees were exposed to natural conditions during spring, summer and fall, and kept in cold storage during the winter to allow trees to complete dormancy. The water content of the potted soil was constantly monitored and full water capacity of the soil was maintained throughout the experiment. Eighteen potted trees were used in each orchard in which the experiment was conducted. These 18 trees were aggregated into two groups of nine trees, each of which was considered a replicate. The nine trees were approximately one meter apart and were arranged in a square (Fig. 1A). Two groups of nine branches were chosen from surrounding field grown trees and were also considered a replicate. Groups of foliage were used as replicates because we wished to create a patch of host plant foliage when using the potted apple trees and then needed to maintain this spatial pattern when using field-grown plants. The total foliage size and the height of the nine potted trees were always smaller than of the field grown trees. Three C. rosaceana larvae were placed on each tree at the beginning of each trial. The larvae on the plants were protected by 100 · 30 cm mash sleeve cages for 48 h in order to exclude natural enemies while larvae established feeding sites. The cages were removed and the larvae in established feeding-sites were exposed to natural enemies for 96 h. The experiment was repeated six times in 2001 in two orchards and six times in 2002 in three orchards (2001: 7/2;7/12;7/18;7/31;8/7;8/21; 2002: 5/25;6/14;7/15;7/ 29;8/12;8/19). The proportions of larvae that were parasitized or that were lost following establishment of feeding sites and hence assumed to have been preyed upon were determined for each group of nine potted trees and for the nine surrounding field grown trees in each orchard. Establishment rate and loss due to predation were not estimated during the first trial in 2001. Proportions were transformed using arcsine square root transformation. Restricted Maximum Likelihood Models (Littell et al., 1996) were used to com-
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Fig. 1. Methods used to implant sentinel C. rosaceana larvae in apple orchards to measure parasitism and predation rates. (A) Caged 1-year-old potted apple trees surrounded by field grown trees and infested with C. rosaceana larvae; (B) florists’ waterpick with two leaves providing a C. rosaceana feeding site; (C) water filled 30 cm long PVC pipes holding larva infested branches.
pare the estimates on potted apple trees and on field grown apple trees (PROC MIXED, SAS Institute, 2000). Autoregressive covariance matrices were used for each model and were selected based on the Aikaike’s and Swartz Information Criterion (Littell et al., 1996). Proportions were compared in each temporal repeat separately using t-tests (Littell et al., 1996) on the least squares means (LSMEANS, SAS Institute, 2000). Least square means and the estimated proportions in each orchard in each trial were graphed to provide a complete visualization of the data (Fig. 2). Since the arcsine transformation is non-linear, the back transformed confidence limits provided in the text are asymmetrical (Zar, 1984; Steel et al., 1997). 2.3. Testing waterpicks as a tool for measuring natural enemy activity Waterpicks were tested as a mobile media for natural enemy assessment studies in 2003. Apple leaves were collected from insecticide-free orchards and two leaves were placed into a florists’ 6–8 cm tall waterpick (FloralSupply.com, Champaign, IL.) by pushing their petioles
through the top opening of the plastic cap. Waterpicks held enough water to prevent the leaves from wilting and the plastic cup prevented water evaporation. A single larva was allowed to establish a feeding site on each pair of apple leaves for 24 h in a screenhouse that excluded NEs, but allowed air movement. On the same day, larvae were placed onto growing tips of field grown trees (3 larvae/tree) protected by 100 · 30 cm mash sleeve cages to exclude NEs during larval establishment. Twenty-four hours later, the cages were removed from the field-grown trees and three waterpicks with one larva in each feeding site were attached to the same field-grown apple trees using plastic ties (Fig. 1B). Larvae were exposed to natural enemies for 48 h. All trees were located in the same apple orchard and the trials were carried out using a blocked design where each tree received both waterpick and direct larval infestation and each tree was treated as a block. The experiment was carried out three times during the development of the summer C. rosaceana generation using 111 larvae in waterpicks and 121 on branches in mid-July (7/15/2003), 139 larvae in waterpicks and 97 on branches in late-July (7/29/2003) and 183 larvae in waterpicks and 129 on branches in earlyAugust (8/12/2003). The proportions of larvae that dispersed within 48 h from foliage in waterpicks were estimated to allow for more accurate estimation of predation. To exclude predators and parasitoids, the dispersal from feeding sites was measured in a 30 m2 screenhouse, which provided seminatural environmental conditions. While wind speed in the screenhouse and the experimental orchards may have been different, it is reasonable to assume that wind was not an important factor causing ejection of larvae from already established, webbed-up feeding sites. Apple foliage was collected from insecticide-free orchards and leaves were placed into waterpicks. Larvae were placed on the leaves in waterpicks (1 larva/waterpick) and allowed to establish feeding sites for 24 h. A known number of these feeding sites were left in the screenhouse for 48 h and subsequently examined to determine the proportion of larvae that had dispersed away from the feeding sites. The experiment was repeated seven times (n = 583 larvae) and the average dispersal rate was 7 ± 3.2%. To estimate predation in the field experiment, the 7% estimated dispersal rate obtained from screenhouse studies was subtracted from the percentage of larvae lost in the field from the foliage in waterpicks. The design of the waterpick assessment experiment differed from the one used in the potted-apple test and logistic regression models were used to compare parasitism and predation of C. rosaceana larvae residing on foliage in waterpicks and on field grown trees (Proc Genmod, SAS Institute, 2000). Logistic regression was estimated as a generalized linear model with a binomial response probability distribution function and logit link function (Allison, 2001; Agresti, 2002). Separate models were created for Hymenoptera and Diptera parasitism rates. Unstructured covariance matrices were selected for all the models based on the
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Fig. 2. Comparison of C. rosaceana larval loss due to parasitism and predation in 2001 and in 2002 on potted and field grown apple trees. The mean and 95% confidence limits are plotted after back transformation. Data points marked by a ‘‘*’’ are significantly different within the same trial.
Aikaike’s and Swartz Information Criterion (Littell et al., 1996). Least square means and the 95% confidence limits were plotted in each trial after back transformation (Fig. 4). Since the logit transformation is non-linear, the confidence limits back transformed to the original scale were asymmetrical (Zar, 1984; Steel et al., 1997). 2.4. Testing PVC pipes as a tool for measuring natural enemy activity Polyvinyl chloride (PVC) pipes were tested as mobile media for assessment of NEs in 2004 and in 2005. At the beginning of the field season, 30 cm long and 2.54 cm diameter PVC pipes with a sealed cap on the bottom were painted green and attached to field-grown apple trees using plastic zip-ties (Fig. 1C). For each trial, apple branches were collected from insecticide-free apple orchards, trimmed to a length of one meter, with at last 6–8 healthy leaves and were placed into water-filled containers, so that the individual branches were separated. Two or three larvae were allowed to develop feeding sites on each branch for 24 h in the laboratory. On the same day, larvae were placed onto field grown trees as previously described. Twenty-four hours later the cages were removed from the field-grown trees and single branches infested in the laboratory were placed into the PVC pipes, one branch per pipe. Each pipe held sufficient amount of water to prevent the leaves from wilting. The pipes were kept at the same location throughout the field season, while the branches and the water were replaced before each trial.
Each tree harbored larvae residing on a branch in a PVCpipe and larvae that were directly infested. The experiment was carried out three times in 2004 using 64 larvae in pipes and 66 on branches in mid-June (6/13/2004), 67 larvae in pipes and 66 on branches in late-June (6/25/2004) and 55 larvae in pipes and 48 on branches in early-July (7/10/2004). Because parasitism levels were very low in June 2004, additional tests were conducted in 2005 using 87 larvae in pipes and 84 on branches in mid-July (7/15/2005) and 72 larvae in pipes and 72 on branches in late-July (7/27/2005). As with the waterpick method, larval dispersal was estimated in the absence of NEs to improve the estimate of predation loss. Three larvae were placed on each branch and allowed to develop feeding sites. Larval loss in the screenhouse was determined after 48 h. The experiment was repeated four times (n = 276 larvae) and 8.5 ± 4.1% of the larvae dispersed from feeding sites on branches. The 8.5% estimated dispersal level was subtracted from the percentage of larvae lost from feeding sites on the field to calculate more accurate predation rates. Because the caged branches of field grown trees had similar lengths as the branches placed in the PVC pipes, the dispersal loss from field grown trees in the orchard was also assumed to be 8.5% for both the waterpick and the PVC-pipe assessment experiments. Similarly to the waterpick assessment trials, logistic regression models with a binomial response probability distribution function and logit link function (Allison, 2001; Agresti, 2002) were fit using Proc Genmod procedure (SAS Institute, 2000) to compare C. rosaceana mortality rates inflicted by NEs on PVC pipes and field grown trees.
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Separate models were created for Hymenoptera and Diptera parasitism. Compound symmetry covariance matrices were selected for all the models based on the Aikaike’s and Swartz Information Criterion (Littell et al., 1996). Least square means and their 95% confidence limits were graphed in each trial after back transformation (Fig. 6). Since the logit transformation is non-linear, the confidence limits back transformed to the original scale were asymmetrical (Zar, 1984; Steel et al., 1997). 3. Results 3.1. Testing potted apple trees as a tool for measuring natural enemy activity Overall, the proportion of larvae that established feeding sites on potted and field-grown trees was not significantly different, although there was one out of the 11 trials where more larvae established feeding sites on field grown trees (p = 0.0255) (Fig. 2). A total of 68.30% (57.33; 78.33) of the larvae settled on field-grown trees and 65.21% (54.07; 75.56) on potted trees in 2001 (l% (95% confidence interval limits)). In the next year 68.28% (63.49; 72.88) of the released larvae established feeding sites on field grown trees and 62.86% (57.91; 67.67) on potted ones. During the two year study 715 larvae were recollected from field grown trees and 581 larvae were retrieved from potted apple trees. The overall parasitism levels (l% (95% confidence interval limits)) on potted trees in 2001 (40.45% (28.13; 53.42)) and in 2002 (9.35% (5.09; 14.74)) were not significantly different from those on the infested orchard trees in 2001 (48.01% (35.2; 60.94)) and in 2002 (11.10% (6.43; 16.84)) since during only one of the 11 trials was the parasitism inflicted larval mortality on potted apple trees lower (p = 0.0015) (Fig. 2). Larval mortality due to predation (= loss of larvae from feeding sites) on potted apple trees in 2001 (32.93% (2.39; 76.80)) and in 2002 (46.01% (29.09; 63.42)) were not significantly different from the estimated predation rates on field grown trees in 2001 (26.52% (0.70; 70.62)) and in 2002 (34.27% (18.88; 51.57)). A total of 275 parasitoids reached the adult stage and were identified. The eulophid Colpoclypeus florus (Walker) and the braconid Oncophanes americanus (Weed) parasitised the largest proportion of the larvae on both potted and field grown trees (Fig. 3). The tachinid Actia interrupta (Curran) was the dominant Diptera parasitoid. Incidental parasitism was observed by the tachinid, Nilea erecta (Coquillett), the braconids Mateorus trachynotus (Viereck) and Macrocentrus iridescens French, the bethylid Goniozus platynotae Ashmead and the ichneumonids Habronyx asclerivora Rohwer and Glypta spp (Fig. 3).
with larvae were placed in the field. The percentage of larvae that successfully established feeding sites in the sleeve-cages on the branches of field-grown trees was 76.35%. A total of 195 larvae were retrieved from direct infestation of field grown trees of which 36.39% (29.08; 44.39) were parasitised (l% (95% confidence interval limits)), a significantly higher proportion than the 16.90% (12.90; 21.83) of the 348 recollected from waterpicks (v2 = 19.10, df=1, p < 0.0001). Significant differences in parasitism were observed in all three trials (Fig. 4). Overall parasitism by Hymenoptera was not affected by the implanting method. Parasitism by Diptera was, however, underestimated by the waterpick method (v2 = 30.80, df = 1, p < 0.0001) due to the significant difference seen in the first (p < 0.0001) and second (p = 0.0010) trials (Fig. 4). Hymenoptera (14.94% (10.06; 21.62)) and Diptera (17.28% (12.02; 24.20)) parasitoids attacked similar proportions of the larvae placed on branches; however on waterpicks Hymenoptera (12.54% (8.66; 17.82)) parasitized larger proportion of the larvae than Diptera (2.85% (1.56; 5.17)). Larval mortality due to predation was also underestimated by the waterpick method (v2 = 43.41, df = 1 p < 0.0001) because of differences in the second (p < 0.0001) and third (p < 0.0001) trials (Fig. 4). The average predation inflicted mortality of larvae on branches of field grown trees and on leaves in waterpicks was 41.08% (35.84; 46.52) and 17.35% (13.81; 21.55), respectively. A total of six parasitoid species were reared from C. rosaceana larvae using the two implanting methods. The same four parasitoid species, A. interrupta, O. americanus, C. florus and M. trachynotus parasitized the largest proportions of the larvae with both methods while the ichneumonid wasp Exochus albifrons Cresson and G. platynotae were incidental parasitoids (Fig. 5).
3.2. Testing waterpicks as a tool for measuring natural enemy activity
3.3. Testing PVC pipes as a tool in measuring natural enemy activity
Choristoneura rosaceana larvae readily established feeding sites on waterpicks in the laboratory and only media
The proportion of larvae that successfully established feeding sites in the sleeve-cages on field-grown trees was
Fig. 3. Proportions of identified parasitoids of each species that attacked C. rosaceana larvae implanted on potted apple trees and field grown apple trees.
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Fig. 4. Comparison of mortality due to parasitism and predation of C. rosaceana larvae feeding on field grown apple trees and on leaves held in waterpicks. The mean and 95% confidence limits are plotted after back transformation. Mortality rates marked by a ‘‘*’’ are significantly different within the same trial.
78.89% in 2004 and 84.28% in 2005. A total of 195 larvae were retrieved from field grown trees and 250 from PVC pipes during the five trials. Parasitoid inflicted larval mortality was 13.26% (7.10; 23.42) and 37.97% (27.52; 49.68) on infested trees in 2004 and 2005 respectively (l% (95% confidence intervals)). These rates were not significantly different from those on PVC pipes in 2004 (10.12% (6.11; 16.33)) or 2005 (26.08% (17.31; 37.29)) (Fig. 6). Both parasitoid orders (Hymenoptera and Diptera) parasitized similar proportions of C. rosaceana larvae with the two outsourcing methods. Wasp attack rates were 8.58% (3.72; 18.57) on field grown trees and 6.47% (3.62; 11.29)
on PVC pipes in 2004, while dipterans parasitized only 3.18% (1.11; 8.73) on trees and 3.17% (0.98; 9.73) on PVC pipes. In 2005 Diptera attack rates increased to 34.04% (23.85; 45.96) on trees and 19.47% (11.58; 30.86) on PVC pipes, but the Hymenoptera parasitism rate stayed as low as 4.1% (1.21; 12.93) on trees and 6.73% (3.02; 14.35) on PVC pipes. Mortality due to predation was 25.48% (18.80; 33.57) and 26.78% (19.17; 36.05) on trees and 20.81% (14.68; 28.65) and 34.83% (27.08; 43.48) on PVC pipes in 2004 and in 2005, respectively. Overall predation rates were not significantly different, although there was a change in predation overtime (Fig. 6). Loss caused by predation from PVC pipes during the first (v2 = 7.07, df = 1, p = 0.007) and second (v2 = 4.03, df = 1, p = 0.045) trials in 2004 was significantly lower than loss of larvae from field grown trees, but these differences were not sustained in the later trials and we therefore inferred that there was no overall effect. The three dominant parasitoid species were the A. interrupta, C. florus and O. americanus, while E. albifrons, M. iridescens and the ichneumonid Itoplectis conquisitor (Say) were incidental parasitoid species (Fig. 7). 4. Discussion
Fig. 5. Proportions of identified parasitoids of each species that attacked C. rosaceana larvae on leaves held in waterpicks and field grown apple trees in 2003.
Abnormal pest density or the unnatural behavior of the sentinel prey is known to bias the results of studies using implanted larvae (Kiritani and Dempster, 1973; Van Driesche et al., 1991; Jervis and Kidd, 1996). In all of our studies larvae were allowed to establish natural feeding sites and densities of outsourced larvae were kept relatively
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Fig. 6. Comparison of mortality due to parasitism and predation of C. rosaceana larvae feeding on field grown apple trees and on leaves held in PVC pipes. The mean and 95% confidence limits are plotted after back transformation. Mortality rates marked by a ‘‘*’’ are significantly different within the same trial.
Fig. 7. Proportions of identified parasitoids of each species that attacked C. rosaceana larvae on branches held in PVC pipes and field grown apple trees in 2001 and in 2002.
low for each trial; therefore we did not expect any effects of artificially increased densities or abnormal behavior. 4.1. Testing potted apple trees as a tool for measuring natural enemy activity Parasitism and predation rates on potted trees were very similar to those measured on directly infested orchard trees; differences in size between the fairly large canopy of field-grown trees and the smaller canopy simulated by grouped potted trees did not affect natural enemy activity. Nine trees were clustered to increase foliage volume; therefore the results from experiments using only a single potted
tree may be different. This technique can provide realistic results for estimating the effects of natural enemies in a range of tree canopy sizes. The same parasitoid species were able to locate the larvae on both field-grown and potted apple trees and both Hymenoptera and Diptera parasitoids attacked similar proportions of C. rosaceana larvae on both tree types. Despite the increasing interest in using various potted ‘‘trap-plant’’ species (Fowler et al., 1991; Kruess and Tscharntke, 2000; Suckling et al., 2001), and that even though these methods facilitate accurate estimates of NE inflicted mortality, potted trees may be not the ideal assessment method in larger field experiments. Maintenance of these plants requires more labor and resources than the other two methods used in this study. Potted trees are surrounded by insecticide treated field-grown-trees and need to be moved or covered during spraying to maintain insecticide-free foliage for the outsourced larvae. Waterpicks and PVC pipes are more closely intertwined within the treated foliage canopy, but the insecticide-free leaves and branches held in them are replaced between trials. Potted trees also need to be frequently watered. Furthermore, a large proportion of larvae were lost prior to establishment of feeding sites; the loss due to unsuccessful settlement was as high as 69% in some trials. Due to the unpredictable rate of larval establishment, the number of larvae exposed to natural enemies varied from trial to trial. If cages are not used in the field to exclude NEs while larvae are rolling feeding sites, then it is likely that many of the larvae will become parasitized or fed upon before they establish their protective leaf-roll. In this study C. rosaceana larvae were allowed to establish feeding sites for 48 h under the sleeve-cages; however later laboratory observations
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showed that these larvae require less than 24 h to settle. Potted trees are adequate tools in NE comparison studies if resources and labor are plentiful for tree maintenance and movement. When treatments being compared are distant or the lack of plant maintenance may jeopardize the results, other outsourcing methods need to be selected. 4.2. Testing waterpicks as a tool for measuring natural enemy activity With the waterpick method larvae established feeding sites in the laboratory, therefore, the number of larvae used to measure NE inflicted mortality was kept constant. This was an advantage compared to the potted tree method. The untreated leaves may have to be obtained from unsprayed research orchards, feral apples, or potted trees established in the greenhouse or field away from commercial orchards so the foliage does not get contaminated with spray drift. Assembling the waterpicks and placing the larvae on the leaves was less time consuming than direct infestation on caged, potted trees. The use of waterpicks resulted in a ca. 50% underestimation of parasitism by the dipteran parasitoid, A. interrupta. In contrast, attacks by hymenopteran parasitoids were similar on larvae residing on foliage of field-grown trees and on leaves in waterpicks. However, one of the dominant species, the C. florus, seemed to be less successful in locating larvae on waterpicks. The activity of predators was also underestimated by ca. 50%, maybe because the plastic waterpick obstructed crawling predators. Winged predators, such as members of the family Vespidae, were observed taking larvae from both infested branches and foliage in waterpicks. The greatest benefit of this technique is its simplicity and it can be recommended in studies where relative effects of different treatments are compared without attempting to estimate an actual mortality rate from NEs (Sarvary et al., 2007). 4.3. Testing PVC pipes as a tool in measuring natural enemy activity Using PVC pipes to hold infested branches combined the benefits of the other two methods tested. Assembly and maintenance of the PVC pipes was simple, requiring only a one-time attachment to the trunk or branch of apple trees. Pipes were long enough to keep an adequate supply of water for at least 48 h, and refilling them was simpler than replenishing the waterpicks. Estimates of parasitism and predation obtained with this method were similar to those obtained using potted trees. In contrast to the potted trees, the number of larvae exposed to NEs can be kept constant. Infesting excised branches with three larvae was easier than placing one larva on foliage in each waterpick, and buckets used to transport excised branches to the permanently attached PVC pipes in the orchards consumed less space than the unstackable foam trays holding waterpicks. This method uses long cut branches placed into pipes thereby providing a more ‘‘natural’’ environment for forag-
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ing predators and parasitoids than leaves in waterpicks. Because of its simplicity, mobility and accuracy, this method can be recommended for comparative studies, estimating mortality inflicted by NEs (Sarvary et al., 2007). Acknowledgments The authors gratefully acknowledge the help of Dr. Biddinger (Pennsylvania State University, University Park, PA), Dr. Gates (National Museum of Natural History, Washington, DC), Dr. Sharkey (Kentucky State University, Lexington, KY) and Dr. Wahl (American Entomological Institute, Gainesville, FL) with Hymenoptera parasitoid identification and Dr. O’Hara (Agriculture and Agri-Food Canada, Ottawa, ON) with Diptera parasitoid identification. We thank Cynthia Smith, Karen Wentworth and David Combs for technical assistance on the field and in the laboratory. We also thank Francoise Vermeylen (Cornell University, Ithaca, NY) for assistance with the statistical analysis and the anonymous reviewers for their comments on the final draft of the manuscript. References Agresti, A., 2002. Categorical Data Analysis. Wiley–Interscience, New York. Allison, P.D., 2001. Logistic Regression Using the SAS System: Theory and Application. Wiley, Cary, NC. Brunner, J.F., 1996. Discovery of Colpoclypeus florus (Walker) (Hymenoptera: Eulophidae) in apple orchards of Washington. Pan Pacific Entomologist 72, 5–12. Burn, A.J., 1982. Role of predator searching efficiency in carrot fly Psila rosae egg loss. Annals of Applied Biology 101, 154–159. Chapman, P.J., Lienk, S.E., New York State Agricultural Experiment Station, 1971. Tortricid fauna of apple in New York (Lepidoptera: Tortricidae): including an account of apples’ occurrence in the state, especially as a naturalized plant. New York State Agricultural Experiment Station, Geneva, NY. Cossentine, J., Jensen, L., Deglow, E., Bennett, A., Goulet, H., Huber, J., O’Hara, J., 2004a. The parasitoid complex affecting Choristoneura rosaceana and Pandemis limitata in organically managed apple orchards. BioControl 49, 359–372. Cossentine, J.E., Deglow, E.K., Jensen, L.B.M., Bennett, A.M.R., 2004b. A biological assessment of Apophua simplicipes (Hymenoptera: Ichneumonidae) as a parasitoid of the oblique banded leafroller, Choristoneura rosaceana (Lepidoptera: Tortricidae). Biocontrol Science and Technology 14, 691–699. Cusson, M., Laforge, M., Regniere, J., Beliveau, C., Trudel, D., Thireau, J.C., Bellemare, G., Keirstead, N., Stolz, D., 2002. Multiparasitism of Choristoneura fumiferana by the ichneumonid Tranosema rostrale and the tachinid Actia interrupta: occurrence in the field and outcome of competition under laboratory conditions. Entomologia Experimentalis et Applicata 102, 125–133. Fowler, S.V., Claridge, M.F., Morgan, J.C., Peries, I.D.R., Nugaliyadde, L., 1991. Egg Mortality of the brown planthopper Nilaparvata lugens Homoptera Delphacidae and green leafhoppers Nephotettix spp Homoptera Cicadellidae on rice in Sri Lanka. Bulletin of Entomological Research 81, 161–168. Gould, J.R., Elkinton, J.S., Van Driesche, R.G., 1992a. Assessment of potential methods of measuring parasitism by Brachymeria intermedia Nees Hymenoptera Chalcididae of pupae of the gypsy moth. Environmental Entomology 21, 394–400.
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Gould, J.R., Elkinton, J.S., Van Driesche, R.G., 1992b. Suitability of approaches for measuring parasitoid impact on Lymantria dispar (Lepidoptera: Lymantriidae) populations. Environmental Entomology 21, 1035–1045. Hagley, E.A.C., Barber, D.R., 1991. Foliage-feeding lepidoptera and their parasites recovered from unmanaged apple orchards in Southern Ontario. Proceedings of the Entomological Society of Ontario 122, 1– 7. Jervis, M., Kidd, N., 1996. Population dynamics: demonstrating and quantifying predation and parasitism. In: Jervis, M., Kidd, N. (Eds.), Insect Natural Enemies. Chapman and Hall, London, pp. 293–315. Kiritani, K., Dempster, J.P., 1973. Different approaches to the quantitative evaluation of natural enemies. Journal of Applied Ecology 10, 323–330. Kruess, A., Tscharntke, T., 2000. Species richness and parasitism in a fragmented landscape: Experiments and field studies with insects on Vicia sepium. Oecologia 122, 129–137. Lawson, D., S, Nyrop, J.P., Reissig, W.H., 1997. Assays with commercially produced Trichogramma (Hymenoptera: Trichogrammatidae) to determine suitability for obliquebanded leafroller (Lepidoptera: Tortricidae) control. Environmental Entomology 26 (3), 684–693. Li, S., Y, Fitzpatrick, S.M., Troubridge, J.T., Sharkey, M.J., Barron, J.R., O’ Hara, J.E., 1999. Parasitoids reared from the obliquebanded leafroller (Lepidoptera: Tortricidae) infesting raspberries. Canadian Entomologist 131 (3), 399–404. Littell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D., 1996. SAS System for Mixed Models. SAS Institute, Inc., Cary, NC. Lopez, E.R., Van Driesche, R.G., 1989. Direct Measurement of host and parasitoid recruitment for assessment of total losses due to parasitism in a continuously breeding species the cabbage aphid Brevicoryne brassicae L. Hemiptera Aphididae. Bulletin of Entomological Research 79, 47–60. Merfield, C.N., Wratten, S.D., Navntoft, S., 2004. Video analysis of predation by polyphagous invertebrate predators in the laboratory and field. Biological Control 29, 5–13. Reissig, W.H., 1978. Biology and control of the oblique-banded leaf-roller on apples. Journal of Economic Entomology 71 (5), 804 809. Sarvary, M.A., Nyrop, J., Reissig, H., Gifford, K.M., 2007. Potential for conservation biological control of the obliquebanded leafroller
(OBLR) Choristoneura rosaceana (Harris) in orchard systems managed with reduced-risk insecticides. Biological Control 40 (1), 37–47. SAS Institute, 2000. SAS/SAT User’s Guide, release v8.00. SAS Institute, Cary, NC, USA. Shorey, H., Hale, R.L., 1965. Mass rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58 (3), 522–524. Southwood, T.R.E., Henderson, P.A., 2000. Observational and experimental methods for the estimation of natality, mortality, and dispersal. In: Southwood, T.R.E., Henderson, P.A. (Eds.), Ecological Methods, third ed. Blackwell Science, Oxford. Speight, M.R., Lawton, J.H., 1976. The Influence of weed cover on the mortality imposed on artificial prey by predatory ground beetles in cereal fields. Oecologia (Berlin) 23, 211–223. Steel, R.G.D., Torrie, J.H., Dickey, D.A., 1997. Principles and Procedures of Statistics. McGraw-Hill. Suckling, D.M., Burnip, G.M., Gibb, A.R., Daly, J.M., Armstrong, K.F., 2001. Plant and host effects on the leafroller parasitoid Dolichogenidia tasmanica. Entomologia Experimentalis et Applicata 100, 253–260. Sunderland, K.D., 1988. Quantitative methods for detecting invertebrate predation occurring in the field. Annals of Applied Biology 112, 201–224. Van Driesche, R.G., 1988. Field measurement of population recruitment of Apanteles glomeratus L. Hymenoptera Braconidae a parasitoid of Pieris rapae L. Lepidoptera Pieridae and factors influencing adult parasitoid foraging success in kale. Bulletin of Entomological Research 78, 199–208. Van Driesche, R.G., Bellows, T.S.J., 1988. Host and parasitoid recruitment for quantifying losses from parasitism with reference to Pieris rapae and Cotesia glomerata.. Ecological Entomology 13, 215–222. Van Driesche, R.G., Bellows, T.S.J., Elkinton, J.S., Gould, J.R., Ferro, D.N., 1991. The meaning of percentage parasitism revisited solutions to the problem of accurately estimating total losses from parasitism. Environmental Entomology 20, 1–7. Wilkinson, T.K., Landis, D.A., Gut, L.J., 2004. Parasitism of obliquebanded leafroller (Lepidoptera: Tortricidae) in commercially managed Michigan apple orchards. Journal of Economic Entomology 97, 1524– 1530. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall Inc., Englewood Cliffs, NJ.
Assessment of three techniques for measuring natural enemy inflicted mortality of leafroller larvae in commercial orchards Mark A. Sarvary *, Jan Nyrop, Harvey Reissig Department of Entomology, New York State Agricultural Experimental Station, Cornell University, Geneva, NY 14456, USA Received 7 June 2006; accepted 28 February 2007 Available online 12 March 2007
Abstract Measuring natural enemy (NE) inflicted mortality presents a serious challenge in managed habitats, such as apple orchards, where the crop-plant is frequently treated with a wide range of insecticides. Toxic residues keep natural populations of pests at too low levels to sample and prevent the use of techniques in which laboratory reared insects are released directly on foliage and used to measure NE activity. Three methods to implant obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae) larvae were tested: (1) larvae placed on potted apple trees, (2) larvae placed on excised leaves inserted in florists’ waterpicks and (3) larvae placed on branches placed into water filled Polyvinyl chloride (PVC) pipes. Use of potted trees allowed accurate estimation of NE inflicted mortality; however, this method was very labor intensive. The waterpick method required less labor, but it underestimated larval mortality caused by NEs. The use of PVC pipes to hold infested branches combined the benefits of the other two methods tested. This technique was simple to use and provided accurate estimates of NE inflicted mortality. 2007 Elsevier Inc. All rights reserved. Keywords: Choristoneura rosaceana; Outsourcing; Implantation; Apple orchard; Oncophanes americanus; Actia interrupta; Colpoclypeus florus; Parasitism; Predation
1. Introduction Measuring natural enemy (NE) inflicted pest mortality is required in many biological control studies. Mortality by NEs can be measured by either sampling the natural pest population directly (Kiritani and Dempster, 1973; Sunderland, 1988; Southwood and Henderson, 2000) or by implanting laboratory raised sentinel larvae (Jervis and Kidd, 1996). Laboratory raised sentinel larvae are ‘‘outsourced’’ or ‘‘implanted‘‘ into habitats where pest numbers are very low or direct pest sampling is precluded (Jervis and Kidd, 1996; Sarvary et al., 2007). Implantation of eggs (Burn, 1982; Fowler et al., 1991; Lawson et al., 1997; Merfield et al., 2004), larvae (Suckling et al., 2001; Cusson et al.,
*
Corresponding author. Fax: +1 607 255 0939. E-mail address: [email protected] (M.A. Sarvary).
1049-9644/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.02.014
2002) or pupae (Speight and Lawton, 1976) has been used to collect parasitoids for laboratory trials or to compare relative treatment effects on NEs without the attempt to estimate actual parasitism and predation levels. This ‘‘trap host’’ method has been applied to many NE-host systems (Van Driesche, 1988; Van Driesche and Bellows, 1988; Lopez and Van Driesche, 1989; Van Driesche et al., 1991; Gould et al., 1992a,b; Sarvary et al., 2007) with known flaws such as abnormal pest density or the unnatural dispersal and behavior of the sentinel prey (Kiritani and Dempster, 1973; Van Driesche et al., 1991; Jervis and Kidd, 1996). Measuring mortality imposed by NEs presents a challenge when it is not possible to directly infest host plants or to sample natural pest populations. Implanting techniques using ‘‘mobile media’’, such as infested potted plants to outsource larvae and eggs have been used only in a few parasitism estimation studies (Fowler et al., 1991; Kruess and Tscharntke, 2000; Suckling et al.,
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2001), but the accuracy of these methods has rarely been evaluated. The use of ‘‘mobile media’’ to outsource hostinsects in parasitism and predation assessment studies is especially important in managed agro-ecosystems, such as apple orchards, where the crop-plant is frequently treated with a wide range of insecticides, resulting in toxic residues persisting on fruit and foliage. These toxic residues keep populations of pests at too low levels to sample and prevent the release of laboratory reared insects directly on crop foliage. The objective of our study was to assess methods to measure parasitism and predation rates in apple orchards where pesticide use makes direct collection or direct implantation of hosts impossible. Three mobile media: (1) potted trees aggregated into groups of nine, (2) two apple leaves placed into florists’ waterpicks and (3) apple branches with foliage held in pipes attached to trees, were tested as possible tools to carry implanted sentinel larvae. We hypothesized that these methods would provide as accurate an estimate of parasitism and predation as direct implantation of larvae onto field grown trees. These methods were tested in apple orchards using the Obliquebanded leafroller , Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). This bivoltine, polyphagous, North American totricid is a pest in apple (Chapman and Lienk, 1971; Reissig, 1978) and the larvae establish feeding sites by rolling leaves and feeding on the fruit or on surrounding foliage (Reissig, 1978). Because it is a native herbivore, C. rosaceana is a host for a wide range of natural enemies (Hagley and Barber, 1991; Brunner, 1996; Li et al., 1999; Cossentine et al., 2004a,b; Wilkinson et al., 2004; Sarvary et al., 2007). 2. Materials and methods 2.1. Experimental sites and laboratory colonies of C. rosaceana Three methods to assess predation and parasitism were evaluated by implanting sentinel larvae in ca. 5 hectare large apple orchards on the property of the New York State Agricultural Experimental Station in Geneva, NY (NYSAES). The tests were set up in ‘Macintosh’ and ‘Cortland’ apple trees that were at least 5 years old (referred to as field grown trees). The trees received normal treatments of fungicides and herbicides were used to control weed; however, trees were not sprayed with insecticides. The objective of these studies was to compare estimates of NE inflicted mortality on C. rosaceana larvae implanted using mobile media with larvae directly placed on foliage of field grown trees. Choristoneura rosaceana larvae were allowed to establish feeding sites on apple leaves in the tested media and on growing tips of field grown apple trees and then were exposed to NEs. Sentinel C. rosaceana larvae were selected from laboratory colonies, founded by larvae collected from commercial apple orchards and wild habitats in 1994 and
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2001 and maintained on fava bean plants, Vicia faba L. in a controlled environment (23 C, 16L:8D, 60%–80% RH). Leafroller neonates were removed from the colonies and 1–3 neonates were raised in 30 ml plastic cups (SOLO cup company, Canada) containing pinto bean, Phaseolus vulgaris L. diet (Shorey and Hale, 1965). Sentinel larvae were reared so that when they were used in experiments, their phenology coincided with the natural C. rosaceana populations in the field. After field exposure to NEs, larvae were retrieved and individually placed onto fresh pinto bean diet in 30 ml plastic cups and kept in a controlled environment at 24 C, 16 L:8D, 85% RH. Emerging parasitoid adults were identified. 2.2. Testing potted apple trees as a tool for measuring natural enemy activity One-year-old ‘Cortland’ apple trees were planted into potting soil (Cornell Mix-NYSAES) in 10 gallon plastic pots in 2001. The potted apple trees were exposed to natural conditions during spring, summer and fall, and kept in cold storage during the winter to allow trees to complete dormancy. The water content of the potted soil was constantly monitored and full water capacity of the soil was maintained throughout the experiment. Eighteen potted trees were used in each orchard in which the experiment was conducted. These 18 trees were aggregated into two groups of nine trees, each of which was considered a replicate. The nine trees were approximately one meter apart and were arranged in a square (Fig. 1A). Two groups of nine branches were chosen from surrounding field grown trees and were also considered a replicate. Groups of foliage were used as replicates because we wished to create a patch of host plant foliage when using the potted apple trees and then needed to maintain this spatial pattern when using field-grown plants. The total foliage size and the height of the nine potted trees were always smaller than of the field grown trees. Three C. rosaceana larvae were placed on each tree at the beginning of each trial. The larvae on the plants were protected by 100 · 30 cm mash sleeve cages for 48 h in order to exclude natural enemies while larvae established feeding sites. The cages were removed and the larvae in established feeding-sites were exposed to natural enemies for 96 h. The experiment was repeated six times in 2001 in two orchards and six times in 2002 in three orchards (2001: 7/2;7/12;7/18;7/31;8/7;8/21; 2002: 5/25;6/14;7/15;7/ 29;8/12;8/19). The proportions of larvae that were parasitized or that were lost following establishment of feeding sites and hence assumed to have been preyed upon were determined for each group of nine potted trees and for the nine surrounding field grown trees in each orchard. Establishment rate and loss due to predation were not estimated during the first trial in 2001. Proportions were transformed using arcsine square root transformation. Restricted Maximum Likelihood Models (Littell et al., 1996) were used to com-
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Fig. 1. Methods used to implant sentinel C. rosaceana larvae in apple orchards to measure parasitism and predation rates. (A) Caged 1-year-old potted apple trees surrounded by field grown trees and infested with C. rosaceana larvae; (B) florists’ waterpick with two leaves providing a C. rosaceana feeding site; (C) water filled 30 cm long PVC pipes holding larva infested branches.
pare the estimates on potted apple trees and on field grown apple trees (PROC MIXED, SAS Institute, 2000). Autoregressive covariance matrices were used for each model and were selected based on the Aikaike’s and Swartz Information Criterion (Littell et al., 1996). Proportions were compared in each temporal repeat separately using t-tests (Littell et al., 1996) on the least squares means (LSMEANS, SAS Institute, 2000). Least square means and the estimated proportions in each orchard in each trial were graphed to provide a complete visualization of the data (Fig. 2). Since the arcsine transformation is non-linear, the back transformed confidence limits provided in the text are asymmetrical (Zar, 1984; Steel et al., 1997). 2.3. Testing waterpicks as a tool for measuring natural enemy activity Waterpicks were tested as a mobile media for natural enemy assessment studies in 2003. Apple leaves were collected from insecticide-free orchards and two leaves were placed into a florists’ 6–8 cm tall waterpick (FloralSupply.com, Champaign, IL.) by pushing their petioles
through the top opening of the plastic cap. Waterpicks held enough water to prevent the leaves from wilting and the plastic cup prevented water evaporation. A single larva was allowed to establish a feeding site on each pair of apple leaves for 24 h in a screenhouse that excluded NEs, but allowed air movement. On the same day, larvae were placed onto growing tips of field grown trees (3 larvae/tree) protected by 100 · 30 cm mash sleeve cages to exclude NEs during larval establishment. Twenty-four hours later, the cages were removed from the field-grown trees and three waterpicks with one larva in each feeding site were attached to the same field-grown apple trees using plastic ties (Fig. 1B). Larvae were exposed to natural enemies for 48 h. All trees were located in the same apple orchard and the trials were carried out using a blocked design where each tree received both waterpick and direct larval infestation and each tree was treated as a block. The experiment was carried out three times during the development of the summer C. rosaceana generation using 111 larvae in waterpicks and 121 on branches in mid-July (7/15/2003), 139 larvae in waterpicks and 97 on branches in late-July (7/29/2003) and 183 larvae in waterpicks and 129 on branches in earlyAugust (8/12/2003). The proportions of larvae that dispersed within 48 h from foliage in waterpicks were estimated to allow for more accurate estimation of predation. To exclude predators and parasitoids, the dispersal from feeding sites was measured in a 30 m2 screenhouse, which provided seminatural environmental conditions. While wind speed in the screenhouse and the experimental orchards may have been different, it is reasonable to assume that wind was not an important factor causing ejection of larvae from already established, webbed-up feeding sites. Apple foliage was collected from insecticide-free orchards and leaves were placed into waterpicks. Larvae were placed on the leaves in waterpicks (1 larva/waterpick) and allowed to establish feeding sites for 24 h. A known number of these feeding sites were left in the screenhouse for 48 h and subsequently examined to determine the proportion of larvae that had dispersed away from the feeding sites. The experiment was repeated seven times (n = 583 larvae) and the average dispersal rate was 7 ± 3.2%. To estimate predation in the field experiment, the 7% estimated dispersal rate obtained from screenhouse studies was subtracted from the percentage of larvae lost in the field from the foliage in waterpicks. The design of the waterpick assessment experiment differed from the one used in the potted-apple test and logistic regression models were used to compare parasitism and predation of C. rosaceana larvae residing on foliage in waterpicks and on field grown trees (Proc Genmod, SAS Institute, 2000). Logistic regression was estimated as a generalized linear model with a binomial response probability distribution function and logit link function (Allison, 2001; Agresti, 2002). Separate models were created for Hymenoptera and Diptera parasitism rates. Unstructured covariance matrices were selected for all the models based on the
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Fig. 2. Comparison of C. rosaceana larval loss due to parasitism and predation in 2001 and in 2002 on potted and field grown apple trees. The mean and 95% confidence limits are plotted after back transformation. Data points marked by a ‘‘*’’ are significantly different within the same trial.
Aikaike’s and Swartz Information Criterion (Littell et al., 1996). Least square means and the 95% confidence limits were plotted in each trial after back transformation (Fig. 4). Since the logit transformation is non-linear, the confidence limits back transformed to the original scale were asymmetrical (Zar, 1984; Steel et al., 1997). 2.4. Testing PVC pipes as a tool for measuring natural enemy activity Polyvinyl chloride (PVC) pipes were tested as mobile media for assessment of NEs in 2004 and in 2005. At the beginning of the field season, 30 cm long and 2.54 cm diameter PVC pipes with a sealed cap on the bottom were painted green and attached to field-grown apple trees using plastic zip-ties (Fig. 1C). For each trial, apple branches were collected from insecticide-free apple orchards, trimmed to a length of one meter, with at last 6–8 healthy leaves and were placed into water-filled containers, so that the individual branches were separated. Two or three larvae were allowed to develop feeding sites on each branch for 24 h in the laboratory. On the same day, larvae were placed onto field grown trees as previously described. Twenty-four hours later the cages were removed from the field-grown trees and single branches infested in the laboratory were placed into the PVC pipes, one branch per pipe. Each pipe held sufficient amount of water to prevent the leaves from wilting. The pipes were kept at the same location throughout the field season, while the branches and the water were replaced before each trial.
Each tree harbored larvae residing on a branch in a PVCpipe and larvae that were directly infested. The experiment was carried out three times in 2004 using 64 larvae in pipes and 66 on branches in mid-June (6/13/2004), 67 larvae in pipes and 66 on branches in late-June (6/25/2004) and 55 larvae in pipes and 48 on branches in early-July (7/10/2004). Because parasitism levels were very low in June 2004, additional tests were conducted in 2005 using 87 larvae in pipes and 84 on branches in mid-July (7/15/2005) and 72 larvae in pipes and 72 on branches in late-July (7/27/2005). As with the waterpick method, larval dispersal was estimated in the absence of NEs to improve the estimate of predation loss. Three larvae were placed on each branch and allowed to develop feeding sites. Larval loss in the screenhouse was determined after 48 h. The experiment was repeated four times (n = 276 larvae) and 8.5 ± 4.1% of the larvae dispersed from feeding sites on branches. The 8.5% estimated dispersal level was subtracted from the percentage of larvae lost from feeding sites on the field to calculate more accurate predation rates. Because the caged branches of field grown trees had similar lengths as the branches placed in the PVC pipes, the dispersal loss from field grown trees in the orchard was also assumed to be 8.5% for both the waterpick and the PVC-pipe assessment experiments. Similarly to the waterpick assessment trials, logistic regression models with a binomial response probability distribution function and logit link function (Allison, 2001; Agresti, 2002) were fit using Proc Genmod procedure (SAS Institute, 2000) to compare C. rosaceana mortality rates inflicted by NEs on PVC pipes and field grown trees.
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Separate models were created for Hymenoptera and Diptera parasitism. Compound symmetry covariance matrices were selected for all the models based on the Aikaike’s and Swartz Information Criterion (Littell et al., 1996). Least square means and their 95% confidence limits were graphed in each trial after back transformation (Fig. 6). Since the logit transformation is non-linear, the confidence limits back transformed to the original scale were asymmetrical (Zar, 1984; Steel et al., 1997). 3. Results 3.1. Testing potted apple trees as a tool for measuring natural enemy activity Overall, the proportion of larvae that established feeding sites on potted and field-grown trees was not significantly different, although there was one out of the 11 trials where more larvae established feeding sites on field grown trees (p = 0.0255) (Fig. 2). A total of 68.30% (57.33; 78.33) of the larvae settled on field-grown trees and 65.21% (54.07; 75.56) on potted trees in 2001 (l% (95% confidence interval limits)). In the next year 68.28% (63.49; 72.88) of the released larvae established feeding sites on field grown trees and 62.86% (57.91; 67.67) on potted ones. During the two year study 715 larvae were recollected from field grown trees and 581 larvae were retrieved from potted apple trees. The overall parasitism levels (l% (95% confidence interval limits)) on potted trees in 2001 (40.45% (28.13; 53.42)) and in 2002 (9.35% (5.09; 14.74)) were not significantly different from those on the infested orchard trees in 2001 (48.01% (35.2; 60.94)) and in 2002 (11.10% (6.43; 16.84)) since during only one of the 11 trials was the parasitism inflicted larval mortality on potted apple trees lower (p = 0.0015) (Fig. 2). Larval mortality due to predation (= loss of larvae from feeding sites) on potted apple trees in 2001 (32.93% (2.39; 76.80)) and in 2002 (46.01% (29.09; 63.42)) were not significantly different from the estimated predation rates on field grown trees in 2001 (26.52% (0.70; 70.62)) and in 2002 (34.27% (18.88; 51.57)). A total of 275 parasitoids reached the adult stage and were identified. The eulophid Colpoclypeus florus (Walker) and the braconid Oncophanes americanus (Weed) parasitised the largest proportion of the larvae on both potted and field grown trees (Fig. 3). The tachinid Actia interrupta (Curran) was the dominant Diptera parasitoid. Incidental parasitism was observed by the tachinid, Nilea erecta (Coquillett), the braconids Mateorus trachynotus (Viereck) and Macrocentrus iridescens French, the bethylid Goniozus platynotae Ashmead and the ichneumonids Habronyx asclerivora Rohwer and Glypta spp (Fig. 3).
with larvae were placed in the field. The percentage of larvae that successfully established feeding sites in the sleeve-cages on the branches of field-grown trees was 76.35%. A total of 195 larvae were retrieved from direct infestation of field grown trees of which 36.39% (29.08; 44.39) were parasitised (l% (95% confidence interval limits)), a significantly higher proportion than the 16.90% (12.90; 21.83) of the 348 recollected from waterpicks (v2 = 19.10, df=1, p < 0.0001). Significant differences in parasitism were observed in all three trials (Fig. 4). Overall parasitism by Hymenoptera was not affected by the implanting method. Parasitism by Diptera was, however, underestimated by the waterpick method (v2 = 30.80, df = 1, p < 0.0001) due to the significant difference seen in the first (p < 0.0001) and second (p = 0.0010) trials (Fig. 4). Hymenoptera (14.94% (10.06; 21.62)) and Diptera (17.28% (12.02; 24.20)) parasitoids attacked similar proportions of the larvae placed on branches; however on waterpicks Hymenoptera (12.54% (8.66; 17.82)) parasitized larger proportion of the larvae than Diptera (2.85% (1.56; 5.17)). Larval mortality due to predation was also underestimated by the waterpick method (v2 = 43.41, df = 1 p < 0.0001) because of differences in the second (p < 0.0001) and third (p < 0.0001) trials (Fig. 4). The average predation inflicted mortality of larvae on branches of field grown trees and on leaves in waterpicks was 41.08% (35.84; 46.52) and 17.35% (13.81; 21.55), respectively. A total of six parasitoid species were reared from C. rosaceana larvae using the two implanting methods. The same four parasitoid species, A. interrupta, O. americanus, C. florus and M. trachynotus parasitized the largest proportions of the larvae with both methods while the ichneumonid wasp Exochus albifrons Cresson and G. platynotae were incidental parasitoids (Fig. 5).
3.2. Testing waterpicks as a tool for measuring natural enemy activity
3.3. Testing PVC pipes as a tool in measuring natural enemy activity
Choristoneura rosaceana larvae readily established feeding sites on waterpicks in the laboratory and only media
The proportion of larvae that successfully established feeding sites in the sleeve-cages on field-grown trees was
Fig. 3. Proportions of identified parasitoids of each species that attacked C. rosaceana larvae implanted on potted apple trees and field grown apple trees.
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Fig. 4. Comparison of mortality due to parasitism and predation of C. rosaceana larvae feeding on field grown apple trees and on leaves held in waterpicks. The mean and 95% confidence limits are plotted after back transformation. Mortality rates marked by a ‘‘*’’ are significantly different within the same trial.
78.89% in 2004 and 84.28% in 2005. A total of 195 larvae were retrieved from field grown trees and 250 from PVC pipes during the five trials. Parasitoid inflicted larval mortality was 13.26% (7.10; 23.42) and 37.97% (27.52; 49.68) on infested trees in 2004 and 2005 respectively (l% (95% confidence intervals)). These rates were not significantly different from those on PVC pipes in 2004 (10.12% (6.11; 16.33)) or 2005 (26.08% (17.31; 37.29)) (Fig. 6). Both parasitoid orders (Hymenoptera and Diptera) parasitized similar proportions of C. rosaceana larvae with the two outsourcing methods. Wasp attack rates were 8.58% (3.72; 18.57) on field grown trees and 6.47% (3.62; 11.29)
on PVC pipes in 2004, while dipterans parasitized only 3.18% (1.11; 8.73) on trees and 3.17% (0.98; 9.73) on PVC pipes. In 2005 Diptera attack rates increased to 34.04% (23.85; 45.96) on trees and 19.47% (11.58; 30.86) on PVC pipes, but the Hymenoptera parasitism rate stayed as low as 4.1% (1.21; 12.93) on trees and 6.73% (3.02; 14.35) on PVC pipes. Mortality due to predation was 25.48% (18.80; 33.57) and 26.78% (19.17; 36.05) on trees and 20.81% (14.68; 28.65) and 34.83% (27.08; 43.48) on PVC pipes in 2004 and in 2005, respectively. Overall predation rates were not significantly different, although there was a change in predation overtime (Fig. 6). Loss caused by predation from PVC pipes during the first (v2 = 7.07, df = 1, p = 0.007) and second (v2 = 4.03, df = 1, p = 0.045) trials in 2004 was significantly lower than loss of larvae from field grown trees, but these differences were not sustained in the later trials and we therefore inferred that there was no overall effect. The three dominant parasitoid species were the A. interrupta, C. florus and O. americanus, while E. albifrons, M. iridescens and the ichneumonid Itoplectis conquisitor (Say) were incidental parasitoid species (Fig. 7). 4. Discussion
Fig. 5. Proportions of identified parasitoids of each species that attacked C. rosaceana larvae on leaves held in waterpicks and field grown apple trees in 2003.
Abnormal pest density or the unnatural behavior of the sentinel prey is known to bias the results of studies using implanted larvae (Kiritani and Dempster, 1973; Van Driesche et al., 1991; Jervis and Kidd, 1996). In all of our studies larvae were allowed to establish natural feeding sites and densities of outsourced larvae were kept relatively
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Fig. 6. Comparison of mortality due to parasitism and predation of C. rosaceana larvae feeding on field grown apple trees and on leaves held in PVC pipes. The mean and 95% confidence limits are plotted after back transformation. Mortality rates marked by a ‘‘*’’ are significantly different within the same trial.
Fig. 7. Proportions of identified parasitoids of each species that attacked C. rosaceana larvae on branches held in PVC pipes and field grown apple trees in 2001 and in 2002.
low for each trial; therefore we did not expect any effects of artificially increased densities or abnormal behavior. 4.1. Testing potted apple trees as a tool for measuring natural enemy activity Parasitism and predation rates on potted trees were very similar to those measured on directly infested orchard trees; differences in size between the fairly large canopy of field-grown trees and the smaller canopy simulated by grouped potted trees did not affect natural enemy activity. Nine trees were clustered to increase foliage volume; therefore the results from experiments using only a single potted
tree may be different. This technique can provide realistic results for estimating the effects of natural enemies in a range of tree canopy sizes. The same parasitoid species were able to locate the larvae on both field-grown and potted apple trees and both Hymenoptera and Diptera parasitoids attacked similar proportions of C. rosaceana larvae on both tree types. Despite the increasing interest in using various potted ‘‘trap-plant’’ species (Fowler et al., 1991; Kruess and Tscharntke, 2000; Suckling et al., 2001), and that even though these methods facilitate accurate estimates of NE inflicted mortality, potted trees may be not the ideal assessment method in larger field experiments. Maintenance of these plants requires more labor and resources than the other two methods used in this study. Potted trees are surrounded by insecticide treated field-grown-trees and need to be moved or covered during spraying to maintain insecticide-free foliage for the outsourced larvae. Waterpicks and PVC pipes are more closely intertwined within the treated foliage canopy, but the insecticide-free leaves and branches held in them are replaced between trials. Potted trees also need to be frequently watered. Furthermore, a large proportion of larvae were lost prior to establishment of feeding sites; the loss due to unsuccessful settlement was as high as 69% in some trials. Due to the unpredictable rate of larval establishment, the number of larvae exposed to natural enemies varied from trial to trial. If cages are not used in the field to exclude NEs while larvae are rolling feeding sites, then it is likely that many of the larvae will become parasitized or fed upon before they establish their protective leaf-roll. In this study C. rosaceana larvae were allowed to establish feeding sites for 48 h under the sleeve-cages; however later laboratory observations
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showed that these larvae require less than 24 h to settle. Potted trees are adequate tools in NE comparison studies if resources and labor are plentiful for tree maintenance and movement. When treatments being compared are distant or the lack of plant maintenance may jeopardize the results, other outsourcing methods need to be selected. 4.2. Testing waterpicks as a tool for measuring natural enemy activity With the waterpick method larvae established feeding sites in the laboratory, therefore, the number of larvae used to measure NE inflicted mortality was kept constant. This was an advantage compared to the potted tree method. The untreated leaves may have to be obtained from unsprayed research orchards, feral apples, or potted trees established in the greenhouse or field away from commercial orchards so the foliage does not get contaminated with spray drift. Assembling the waterpicks and placing the larvae on the leaves was less time consuming than direct infestation on caged, potted trees. The use of waterpicks resulted in a ca. 50% underestimation of parasitism by the dipteran parasitoid, A. interrupta. In contrast, attacks by hymenopteran parasitoids were similar on larvae residing on foliage of field-grown trees and on leaves in waterpicks. However, one of the dominant species, the C. florus, seemed to be less successful in locating larvae on waterpicks. The activity of predators was also underestimated by ca. 50%, maybe because the plastic waterpick obstructed crawling predators. Winged predators, such as members of the family Vespidae, were observed taking larvae from both infested branches and foliage in waterpicks. The greatest benefit of this technique is its simplicity and it can be recommended in studies where relative effects of different treatments are compared without attempting to estimate an actual mortality rate from NEs (Sarvary et al., 2007). 4.3. Testing PVC pipes as a tool in measuring natural enemy activity Using PVC pipes to hold infested branches combined the benefits of the other two methods tested. Assembly and maintenance of the PVC pipes was simple, requiring only a one-time attachment to the trunk or branch of apple trees. Pipes were long enough to keep an adequate supply of water for at least 48 h, and refilling them was simpler than replenishing the waterpicks. Estimates of parasitism and predation obtained with this method were similar to those obtained using potted trees. In contrast to the potted trees, the number of larvae exposed to NEs can be kept constant. Infesting excised branches with three larvae was easier than placing one larva on foliage in each waterpick, and buckets used to transport excised branches to the permanently attached PVC pipes in the orchards consumed less space than the unstackable foam trays holding waterpicks. This method uses long cut branches placed into pipes thereby providing a more ‘‘natural’’ environment for forag-
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