Final Report for ONE03-003
An attracticide formulation, LastCall™OFM, was tested against Grapholita molesta (Busck) (Lepidoptera: Tortricidae) in replicated small plot field trials in orchards of farmer cooperators in South Eastern Pennsylvania. LastCall™OFM treatments were applied using a calibrated hand pump and compared to similar non- treated plots. Male moth activity was monitored using virgin-female baited traps and the potential for reduction in mating activity was assessed using sentinel virgin females. A comparison of formulation application rates showed that a half rate of 1500 droplets per ha (600 per acre) was equally as effective as the recommended release rate of 3000 droplets per ha (1200 per acre) and both rates reduced capture in synthetic pheromone-baited traps for prolonged periods. Experimentation in standard tree plantings revealed an important effect of droplet placement within the tree canopy. Droplets placed either at high or low positions within the canopy significantly reduced trap capture and mating with sentinel females. However, only sentinel females located in the untreated portion of the tree canopy mated, as was reflected by a significant treatment*female interaction effect. Mate finding and mating behavior was equally disrupted by LastCall™OFM formulations with and without insecticide. Therefore, the main mechanism by which LastCall™OFM works in small plots is by disruption of male orientation and not removal of males due to insecticide poisoning. Two field cage experiments tested the impact of population density on the competitiveness of LastCall™OFM. A significant proportion of recaptured males flew to females at the highest female: droplet ratio tested. Equal proportions of recaptured males flew to the LastCall™OFM-baited traps at male moth densities of 10, 20, 40, and 80 males per cage, suggesting that more males flew to females at higher male population densities. These results clarify some of the factors influencing the effectiveness and the possible mechanisms of an attracticide management tactic against the Oriental fruit moth. This information has been disseminated to farmer cooperators verbally and in written form (receipt of final report). In addition, the information has been disseminated to a scientific audience on two occasions (Entomological Society of Alberta, October 2003; Entomological Society of America, October 2003). The data have already been submitted for publication in a peer-reviewed journal (Entomologia Experimentalis et Applicata) read widely by professional entomologists and pest managers alike.
Several moths in the family Tortricidae are considered key pests in apple pest management programs in the northeast. The Oriental fruit moth (OFM), Grapholita molesta (Busck) has traditionally been a major pest of stone fruit in the northeastern US, but infestation in apple has been on the increase since 1996. The significance of OFM to fruit production has also increased due to the development of resistance to organophosphate insecticides (OPs) in some populations (Usmani and Shearer 2001). Between 1998 and 2000, 68-464 loads of processing apples were rejected in the Biglerville, PA area due to OFM infestation (Hull, Pers. Comm.).
Insecticide application is ubiquitous in Pennsylvania apple orchards as 97% of all acreage received insecticidal inputs which totaled 2 086 000 lbs state-wide in 1999 (NASS 2000). The most commonly used OP in Pennsylvania orchards is azinphos-methyl that was applied to 89% of the total apple acreage in 1999 (NASS 2000). Azinphos-methyl is under scrutiny by the Food Quality Protection Act (1996) and is currently under a time-limited registration on apple for another four years (Krawczyck and Hull 2001). Therefore, alternative control measures for OFM on apple need to be established.
Attract and Kill formulations combine an attractant (sex pheromone) with a killing agent (pyrethroid insecticide) to control insect populations with reduced insecticidal input. A recent attracticide formulation consists of a viscous paste that incorporates insecticide (6% permethrin) and attractant material (sex pheromone) in a UV sensitive carrier material (Hofer & Brassel 1992). This formulation has been registered for use against both the Oriental fruit moth and another tree fruit pest, the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in the US under the trade names LastCall™OFM and LastCall™CM, respectively. Success of LastCall™CM in field trials (Charmillot et al. 2000; Krupke et al. 2002) and preliminary lab and field experimentation of this formulation against the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) in New Zealand (Brockerhoff & Suckling 19999; Suckling & Brockerhoff 1999) suggests it is a technology that could be effective in the control the Oriental fruit moth.
Our initial research (Evenden & McLaughlin 2004) demonstrated that individual droplets of the registered LastCall™OFM formulation attract wild and laboratory-reared moths and expose them to insecticide via source contact. Here we test LastCall™OFM in field settings to determine the most effective application rate and position, the impact of population density on the efficacy of the formulation, and the mechanism(s) invoked by the formulation.
The objective of this project, as outlined in the original NESARE Partnership grant proposal, was to determine the most effective treatment rate (droplets/acre) and position of the LastCall™OFM formulation in small field plots.
In one summer of field study, we met and exceeded the objectives outlined in our proposal. In addition to testing application rate and position of the LastCall™OFM formulation in small field plots, we also determined the mechanism by which LastCall™OFM works to control Oriental fruit moth populations in a small plot setting. Two field cage studies were also completed to examine the effect of pest population density on the attractiveness of LastCall™OFM.
LastCall™OFM formulations were formulated by D. Czokajlo (IPM Tech Inc., Portland, OR, USA). Formulations consisted of a clear viscous paste with base proprietary product plus other inert ingredients (93.8% of the formulation). Oriental fruit moth pheromone incorporated into formulations at a rate of 0.16% was a three-component blend consisting of 87.3 % (Z)-8-dodecenyl acetate, 3.6 % (E)-8 dodecenyl acetate, and 9.1 % (Z)-8-dodecenyl alcohol. In formulations with insecticide, permethrin was added at 6.0% of the formulation.
Small Plot Experiments.
Small plot experiments were established following a Randomized Block Design in commercial apple orchards in SE Pennsylvania during the 2003 field season. Three 0.1 ha (33.3 m x 33.3 m) plots, separated by a minimum of 40 m, were established at each site. Sites were established in three different orchards in Experiment 1 and these same plots were used in Experiment 2. Three different experimental sites were used in both Experiments 3 and 4 and in each case were located within an individual orchard. In these cases, sites were separated by 100 m and plots within sites were separated by 40 m. Individual treatments were assigned randomly to plots in each of the three sites per experiment. Treatments were applied as droplets (50 µl) using a calibrated hand pump to the central leader and branches of apple trees.
Treatments were assessed with sentinel virgin females and virgin female-baited traps (Figure 1A and 1B). Female moths were obtained from a laboratory colony maintained on a lima bean-based diet at L16:D8 and 24°C. Pupae were separated by sex and individual female pupae were held in 30 ml cups and monitored daily. Moths were provided with a water source until use at 1-5 days post eclosion. Sentinel virgin females were placed individually in small mating cages made from cone-orifice traps (6 cm tall by 2.5 cm diameter). Cages were modified to provide the moth with a water source and glued to the surface of a petri plate (10 cm diameter) lid that was fitted with a wire hanger for suspension within the tree. Virgin female-baited traps consisted of Intercept A traps (IPM Tech Inc, Portland, OR, USA) fitted with a removable sticky liner and baited with a small mesh bag (9 x 6.5 cm) containing one virgin female moth. Both mating cages and mesh bags containing virgin females were transported to field sites in a refrigerated container. During each of three, four-day assessment periods per experiment, six mating cages and six virgin female-baited traps were suspended at both low (head height) and high (upper third of the canopy) positions within the tree canopy of three trees at plot center. The mating status of sentinel females recovered from experimental plots was determined by dissection to reveal the presence (mated) or absence (virgin) of a spermatophore in the bursa copulatrix.
Virgin male moths were released in equal numbers in each plot during each assessment period (except in Experiment 1) to augment natural populations. Male moths were obtained from the laboratory colony and housed in the same manner as outlined for females until use 1-6 days post eclosion. Males were chilled at 5°C for 30 min and then transferred in groups of 10-25 to petri plates (5cm diameter) that were fitted with a wire hanger for suspension within the tree canopy. Petri plates containing male moths were transported to field sites in refrigerated containers. The lids of the petri plates were removed and two petri plates were suspended within the mid-canopy in the center row of each plot 7 m on either side of plot center.
Experiment 1 compared two treatment rates of LastCall™OFM to non-treated control plots. Treatments consisted of droplet application of 3000, 1500 droplets per ha (1200 and 600 droplets per acre, respectively). Droplets were applied in the low to mid-canopy on the center leader and branches of the trees. The three assessment periods occurred 16-53 days post treatment. During the first assessment period no laboratory-reared virgin males were released in the treated plots. Due to low male capture and mating rates of sentinel females in the first assessment period, we decided to supplement populations during the second and third assessment periods. In each of the second and third assessment periods, thirty virgin males were released in each plot at the time of female placement within plots. During the third assessment period, plots in only one site were assessed due to a shortage of reared moths at that time for a total N = 7 in Experiment 1. The proportion of sentinel females that mated were arcsin-square root transformed and the number of males captured in virgin female-baited traps were log (x+1) transformed to satisfy requirements for normality and homoscedasticity. Both data sets were then analyzed using a Randomized Block Design ANOVA with orchard specified as a random variable and assessment period treated as a repeated measure. Analysis of Variance was followed by Least Square Means tests to compare individual treatments.
Experiment 2 compared the capture of male Oriental fruit moths over a 16-week period in synthetic pheromone-baited traps to determine the longevity of LastCall™OFM formulations assessed in Experiment 1. One Intercept A trap was baited at field sites with a red rubber septa monitoring lure of the same Oriental fruit moth pheromone blend used in the LastCall™OFM formulations (IPM Tech Inc, Portland, OR, USA). Traps were placed 1.5 m off the ground in the center tree of each plot immediately following plot treatment with LastCall™OFM formulations. Trap captures were assessed at weekly intervals but traps were not present in plots during treatment assessment with sentinel virgin females and virgin female-baited traps (Experiment 1). Moths were removed from sticky liners weekly and liners were replaced as needed. Pheromone lures were replaced at 4-week intervals. Male moth catches in synthetic-baited traps were log (x+1) transformed and compared using a 2-way ANOVA (assessment period and treatment) with orchard specified as a random factor and assessment period treated as a repeated measure. Analysis of Variance was followed by Least Square Means tests to compare individual treatment * assessment period interactions.
Experiment 3 tested the hypothesis that droplet position would influence the effectiveness of LastCall™OFM. For this experiment, sites within the same orchard with similar standard tree plantings were chosen so there was a consistent difference between the high and low level droplet position. Treatments consisted of LastCall™OFM applied at 1500 droplets per ha (600 per acre) at low (head height) and high (upper third of canopy) positions and a non-treated control. The three assessment periods occurred 1-20 days post treatment. Forty-five male moths were released in each plot during each of the three assessment periods. All three sites were assessed during each period for a total of N = 9 for Experiment 3. Data sets were transformed as in Experiment 1 and analyzed using a 2-way ANOVA (treatment and female position) with site specified as a random factor and assessment period treated as a repeated measure. Analysis of Variance was followed by Least Square Means tests to compare individual treatments.
Experiment 4 tested the hypothesis that the principal mechanism of population control using LastCall™OFM is the removal of males from the population through permethrin exposure. LastCall™OFM formulations with and without the 6% permethrin component were applied to plots at a treatment rate of 3000 droplets per ha (1200 per acre) with droplets applied at both low (2/3 of droplets) and high (1/3 of droplets) positions within the canopy and compared to non-treated control plots. The three assessment periods occurred 3-15 days post treatment. Twenty, thirty-five and thirty males were released in each plot during each assessment period, respectively. All three sites were assessed during each period for a total of N = 9 for Experiment 4. The effect of treatments on the proportion of sentinel females that mated and number of males captured in female-baited traps were analyzed as in Experiment 1.
Field Cage Experiments.
Two field cage experiments were conducted to determine the impact of population density on the attractiveness of LastCall™OFM. Four field cages (1.83 m x 1.83 m x 1.83 m) were erected >25 m apart on grassy locations on the West Chester University campus (39º 58’ N 75º 38’ W) (Figure 2). Cages were placed close to buildings so that there was no direct airflow between cages. Within each cage, seven 1.9 l buckets fitted with lids and filled with water were placed in an “H” configuration with each bucket separated by 60 cm. The lid of each bucket was punctured and freshly cut apple branches were placed in each bucket to create a small (~60cm-high) canopy. Branches remained in cages for one replicate and were replaced at the beginning of each new replicate in both experiments. Each experiment consisted of four, one-night replicates. Treatments were assigned to cages randomly and were re-randomized over time following a Latin Square Randomization so that each treatment occupied each cage at one time. During each replicate, 1 to 5-day old virgin male moths were released at cage center from a 5cm- diameter petri plate placed within the branch canopy. Males were released in the late afternoon and given 1 h to acclimate to cage conditions. Males were reared and handled as outlined for the field experiments except that males were marked with one of four colors of Day-Glo UV fluorescent powder (Switzer, Cleveland, OH, USA) to ensure individuals were enumerated in only one replicate. Traps were introduced to cages 1 h after males were released and remained in the cages overnight. LastCall™OFM-baited traps consisted of Intercept A traps fitted with a sticky liner and baited with a 50 µl droplet weighed to between 42.5 and 52.5 mg, positioned in the center of a 1 x 2 cm piece of aluminum foil and suspended vertically by a short length of wire from the top of the trap. Virgin female-baited traps consisted of the same trap type but baited with an individual 2-4 day-old virgin female in a mesh bag suspended from the top of the trap. Females were reared and handled as outlined in the field experiments. Sticky traps were positioned on bamboo stakes placed adjacent to buckets within the canopy at 60 cm off the ground and separated by 60 cm. The LastCall™OFM-baited trap was always positioned in the center of the trap array and trap locations were maintained between replicates in both experiments. Traps were removed from cages the following morning and males captured in each trap were counted and recorded. Males that were not captured were removed from the cage with a vacuum.
Experiment 5 tested the hypothesis that the density of calling virgin females would influence the proportion of males within a population that were attracted to LastCall™OFM droplets. During each replicate, 25 virgin male moths were released within the canopy at cage center. Treatments consisted of: i) 1 LastCall™OFM-baited trap (control); ii) 1 LastCall™OFM-baited trap and 2 virgin-female baited traps; iii) 1 LastCall™OFM-baited trap and 4 virgin female-baited traps; and iv) 1 LastCall™OFM-baited trap and 8 virgin female-baited traps.
Experiment 6 tested the hypothesis that the density of virgin male moths could influence the relative attractiveness of LastCall™OFM droplets. During each replicate, 1 LastCall™OFM-baited trap and 8 virgin female-baited traps were positioned in each cage. Treatments consisted of the release of : i) 10 marked, virgin males; ii) 20 marked, virgin males; iii) 40 marked, virgin males; and iv) 80 marked, virgin males.
Field cage data were collected as the proportion of male moths that responded within each replicate. Proportions were arcsin-square root transformed and treatments were compared using a Latin Square Design ANOVA. Analysis of Variance was followed by Least Square Means tests to compare individual treatments.
Small Plot Experiments.
In Experiment 1, treatment of small plots with LastCall™OFM at application rates of 1500 and 3000 droplets per ha significantly and equally reduced the number of males captured in virgin female-baited traps and the proportion of sentinel females that mated (Figure 3A, 3B) as compared to the non-treated control plots. In Experiment 2, these same formulations shut down trap capture in synthetic pheromone-baited traps for seven weeks post treatment. However, due to the low population pressure at our sites, captures in control plots were only significantly greater than both treated plots for two weeks post treatment (Figure 4).
Experiment 3 revealed that treatment with LastCall™OFM at 1500 droplets per ha positioned either high or low in the canopy significantly reduced the number of males captured in virgin female-baited traps as compared to non-treated control plots (P<0.0001). However, trap capture was not influenced by the position of the female-baited trap within the canopy (P=0.3829) and there was no treatment * trap position interaction effect (P=0.1916) (Figure 5A). The proportion of females that mated was significantly greater in the control plots than in either of the treated plots (P=0.0003). All of the females that mated in plots treated with droplets in the low position were located high in the canopy. Similarly, all of the females that mated in plots treated with droplets at the high position were located low in the canopy. This resulted in a significant treatment*female position interaction effect (P=0.0282) on the proportion of sentinel females that mated (Figure 5B).
In Experiment 4, mate-finding and mating behaviors were equally disrupted by the LastCall™OFM formulations with and without the insecticide component. Treatment of small plots with LastCall™OFM with and without permethrin at 3000 droplets per ha positioned throughout the canopy significantly and equally reduced the number of males captured in female-baited traps and the proportion of females that mated (Figure 6A, 6B). Although no sentinel females mated in the plots treated with LastCall™OFM without insecticide, this was not significantly different from the proportion of females that mated in LastCall™OFM with insecticide plots which represents 2 mated females out of 43 tested.
Field Cage Experiments.
LastCall™OFM-baited traps in Experiment 5 captured the majority of recovered moths at all of the attracticide : virgin female ratios tested. However, when eight females were present in the cages, the proportion of responders captured in the LastCall™OFM trap was reduced significantly (Figure 7A), implying that a significant proportion of responders were captured in female-baited traps. In Experiment 6, with a constant female density of eight per cage, the LastCall™OFM-baited traps captured a similar proportion of responding males regardless of the male moth density within each cage (Figure 7B).
The results of these studies far exceed what we originally proposed in our NESARE partnership grant application. LastCall™OFM is now registered and available for use in the US. The data provided here will allow producers to use this technology in the most economic and effective manner. These small plot field trials and field cage experiments have demonstrated several important factors that could influence commercial application of LastCall™OFM. A reduction in the recommended release rate is unlikely to alter efficacy of this formulation and may enhance insecticide exposure of males through increased false-trail following to droplets at lower densities. Droplets need to be distributed throughout the tree canopy for complete disruption of mate-finding and mating behaviours. The LastCall™OFM formulation will also be most effective at low pest population densities.
Education & Outreach Activities and Participation Summary
The outcomes of this work were presented orally and in written format to the participating farmer cooperators. Because the Project Leader has relocated to the University of Alberta as of September 2003, she was unable to make local presentations to the grower community in the Fall 2003. However, the company (IPM Tech Inc.) collaborator, Dr. J. McLaughlin continues to disseminate the results of these studies to pest manager and producer organizations in the North East. In addition, this work was presented to an audience of scientists and pest managers at two Entomological Society meetings (Entomological Societies of America and Alberta).
This work has already been submitted for publication to an international entomology journal: Entomologia Experimentalis et Applicata. As the Oriental fruit moth is a world-wide pest found in most tree fruit growing areas of the world, this work has the potential to impact IPM systems in the US and internationally.
The multi-species pest pressure experienced in apple orchards in the North East makes it difficult for growers to adopt species-specific measures for pest control. That being said, LastCall™OFM is now registered for use in the US and will be used in 2 of the 3 participating farms in the next field season.
Areas needing additional study
This study tested the efficacy of LastCall™ to interfere with the mate-finding and mating ability of male Oriental fruit moths. Further work is needed to determine whether suppression of mating will result in reduced larval populations and fruit damage in a commercial setting. In addition, many of the participating farmers and other farmers as well, suggested that a combined attracticide formulation for the Oriental fruit moth and the codling moth would be very useful in their integrated pest management programs. We conducted trapping experiments and wind tunnel bioassays to assess the attractiveness of a combined formulation to these two species. Field trials assessing the suitability of a combined formulation against both species should be conducted in the future.