An initial field trapping experiment (Field Experiment 1: Effects of Blend Ratios and their Amounts on Male Captures) was conducted to determine whether pheromone blend ratio and amount affected trap captures in the field. This experiment was followed by laboratory evaluation (Laboratory Bioassay: Evaluation of Sex Pheromone Volatile Emissions) of the pheromone volatiles released by groups of laboratory reared females. Two subsequent field trapping experiments were conducted comparing the efficacy of newly formulated lures (Field Experiment 2: Evaluation of New Pheromone Blends with and without E11-14:Ac and Field Experiment 3: Comparison of Male Captures between New Lures and Commercial lures).
Insect Colonies. C. rosaceana adults were obtained from a laboratory colony originated from unsprayed orchards in Fennville, Michigan and maintained at the Michigan State University (East Lansing, MI, USA). Larvae were reared on a pinto bean-based diet (Shorey and Hale 1965) at 24 oC under a 16:8 light:dark photoperiod and 50% RH in an environmental chamber. To prevent mating by emerging adults, pupae were sorted by sex and kept separately in Bugdorm-1 cages (30 × 30 × 30 cm, Megaview Science Education Services Co., Taiwan) with 5% sucrose solution for adult emergence.
Pheromones. Pheromone compounds, Z11-14:Ac (98% purity), E11-14:Ac (95% purity), Z11-14:OH (95% purity), Z11-14:Al (95% purity) were purchased from Bedoukian Research, Inc. (Danbury, CT). Pheromone solutions were prepared using HPLC grade hexane (Aldrich, Milwaukee, WI).
Field Study Plots. Field studies were conducted in 2012 and 2014 in unmanaged apple plantings at the Michigan State University Clarksville Research Center (42.8423°N, 85.2425°W). Plots consisted of eight 0.21-ha apple orchards with 12 rows spaced at 4.5 m intervals and 26 apple trees (ca. 3 m height) within each row spaced at 1.5 m apart. Each plot was at least 100 m apart and planted with three apple cultivars (Idared, Empire, and Liberty) with four rows per cultivar (Gut et al. 2005). Among these eight plots, four were surrounded by a hedgerow barrier along its perimeter, consisting of three rows of hybrid popular, one row of Italian alder, and one row of white pine (Gut et al. 2005), the other four plots had no such hedgerow-barrier.
Field Experiment 1: Effects of Blend Ratios and their Dosages on Male Captures. The relative attractiveness of lures containing three- or four-component blends at different dosages was evaluated in the field between May 26 to July 5, 2012. The three-component blend at five doses (0.01, 0.1, 1, 10, and 20 mg) was randomized deployed in a plot surrounded by a hedgerow barrier, whereas the four-component blend at five doses was conducted in a plot without the barrier.
The four-component blend of Z11-14:Ac, E11-14:Ac, Z11-14:OH, and Z11-14Al was prepared in a ratio of 96.5:1.8:1.4:0.2 based on the ratio reported from female pheromone gland effluvium from a colony established from the same origin maintained the same way at Michigan State University as described above (El-Sayed et al. 2003). The 3-component blend of Z11-14:Ac, E11-14:Ac, and Z11-14:OH in a ratio of 96.5:1.8:1.4 was prepared similarly but without Z11-14Al. Either three- or four-component blends (200 µl) containing 0.01, 0.1, 1, 10, and 20 mg were applied to the sleeves of gray rubber septa (6 mm ID, 19 mm height, ISCA Technology, CA) in a fume hood. Pheromone impregnated septa were stored in a freezer at -20oC after hexane was evaporated.
Lures were deployed in Pherocon® VI orange delta traps (Trécé Incorporated, Adair, OK) with a sticky liner (18.5 × 18.5 cm) at the bottom by pinned centrally to the inside ceiling of the trap. Traps baited with one of 5 three-component lures were randomly placed at least 10 m apart in the 2nd and 11th rows in a plot with the hedgerow barrier and repeated three more times in the rest of the plots with the barrier. Traps were hung in the upper third of the tree canopy. Four-component lures were deployed in the same way but in four plots with no barrier. All the traps in each plot were checked weekly and sticky liners replaced as needed. Treatments within a plot were rotated weekly in a clockwise pattern to eliminate trap position bias.
Both weekly and total male captures over a 6-week trapping period were analyzed and compared among varying dosages within each blend. Since two blends were deployed in separate plots with or without hedgerow barriers and each plot had different levels of moth populations, the effect of blend type on male captures was not able to be directly compared. Instead, the proportion of male capture by each trap was generated for both blends by dividing its total capture over the 6-week trapping period by the grand total male capture from all the traps within each plot (i.e., sum of male captures by traps baited with 0.01, 0.1, 1, 10, and 20 mg dose). A linear regression analysis was performed on the newly generated data set for each blend; and their constants and slope coefficients were compared between the blends.
Laboratory Bioassay: Evaluation of Sex Pheromone Volatile Emissions from Live Females. Volatile collections were performed on groups of 42 1-2d old virgin females, males, a commercial lure, or an empty vessel (blank control) under dark conditions over a 16h period corresponding to the scotophase experienced by the source colony. Multiple moths were used to ensure adequate volatile collection, because each female pheromone gland contains a tiny fraction of pheromone (<10ng.gland-1) and not every female will call on a given night. The volatile collection was repeated 3 more times with additional groups of 42, 45, and 65 virgin females and males along with commercial lures.
Volatile collection chambers were constructed from 1-liter Teflon containers equipped with two 0.64-cm ports in their lids (Jensen, Coral Springs, Florida). The lid was completely sealed using Teflon tape to ensure that 100% of the air was directed through the trap. Air was passed through the collection chambers at a rate of 958 ml.min-1 after it passed through a moisture trap and a charcoal column. Volatiles were trapped using a trap containing 25 mg of Super Q adsorbent (Alltech Assoc., Deerfield, IL). Following collections, the chambers were cleaned with acetone three times and baked in an oven 120 oC for at least 2 h.
Volatile chemicals were eluted from individual traps with 150 μl hexane three times. The elute was concentrated under a nitrogen stream to approximately 20 μl of each sample, of which 1 μl was analyzed by capillary gas chromatography (Hewlett-Packard HP6890 equipped with a Hewlett Packard 7863 autosampler) and an HP-Innowax polyethylene glycol column (30 m × 250 μm i.d., 0.25 μm film thickness) with a splitless injector at 250 oC and flame ionization detector at 300 oC. Following injection, column temperature was held at 50oC for 5min, increased at 25 oC.min-1 to 155 oC and held for 5 min; then increased at 0.5 oC.min-1 to 165 oC and held for 3 min; finally increased at 30 oC.min-1 to 225 and held for 2 min. Helium was used as a carrier gas at a flow rate of 1.1 ml.min-1. Pheromone peaks released by the females were identified based on their retention times compared to those of synthetic compounds and further confirmed by their mass spectrums with those of synthetic ones by GC-MS using HP-5 5% phenyl methyl siloxane column. Data were collected with Hewlett- Packard ChemStation® software. Due to not knowing exactly how many females released pheromones during the collection period, the actual amount of each pheromone compound released was not calculated, instead, the percentage of each compound released was reported and calculated by 100 × its peak area divided by the total peak areas from all the pheromones combined.
Field Experiment 2: Evaluation of New Pheromone Blends with and without E11-14:Ac. This experiment was conducted to test how the addition of E11-14:Ac to a newly formulated 2-component pheromone lure (NL-2) would affect male capture. The 2-component lure contained 10 mg of Z11-14Ac and Z11-14OH in a ratio of 37:64 and the three-component lure contained 10 mg of Z11-14Ac, Z11-14OH, and E11-14Ac in a ratio of 37:64:0.68 (NL-3). Otherwise, lures were manufactured using the methods described above. Traps baited with either NL-2 or NL-3 were deployed on two adjacent trees in the middle 2nd row of one of four plots with the hedgerow barrier from June 23 to September 26, 2014. The numbers of males in each trap were counted and removed weekly. Moth captures from the week of 7/2 to the week of 7/23 were summed together to represent the total capture during the first flight, and data from 8/6 to 9/24 was added together to represent the total capture during the second flight. Both weekly and total male captures in the 1st and 2nd generations between the lures were compared.
Field Experiment 3: Comparison of Male Captures between New Lures and Commercial lures. This experiment was conducted to compare the relative attractiveness of the 3-component new blend lure (NL-3) with a commercial lure (3 mg, Trécé Incorporated, Adair, OK). Based on the results from Exp. 1 indicating that blend dosage significantly affected moth captures, 3 mg NL-3 was used to match the quantity of pheromones that was loaded in the commercial lure. Two traps, each baited with one of the two lures, were deployed on two adjacent trees in the center row of one of four plots without the hedgerow barrier and serviced the same way as described above during the second flight from August 15 to September 26, 2014. Both weekly and total male captures over the 6-week trapping period between the new and commercial lures were compared.
Statistical Analysis. In Field Experiment 1, weekly moth captures over time among traps baited with varying dosages of three-component or four-component blends were analyzed by repeated measures ANOVA with the dosage as the subject factor and time (week) as the within subject factor after data were log(X+1) transformed to meet their normality and homoscedasticity assumption (SAS Institute, 2020). Total numbers of moths captured over a 6-week period by traps baited with three- or four-component blends at various dosages were both analyzed by a RCBD design with study plots as blocks since original data met normality and homoscedasticity assumption tests (SAS Institute, 2020). Mean separations were performed via a post hoc Tukey’s HSD test (α=0.05). The proportion of male captured for both blends at varying dosages was analyzed using a linear regression model using Proc Reg after data was arcsine of square root transformed to meet normality and homoscedasticity assumption test (SAS Institute, 2020).
In Field Experiment 2, weekly moth captures over time between traps baited with NL-2 or NL-3, and moth captures between traps baited by NL-3 or the commercial lure in Field Experiment 3 were analyzed by repeated measures ANOVA with the lure type as the subject factor and time (week) as the within subject factor after data were log(X+1) transformed to meet normality and homoscedasticity assumptions (SAS Institute, 2020). Total numbers of moths captured during the first and second flight in Field Experiment 2 and total moth captures after log(X) transformed in Field Experiment 3 were analyzed by ANOVA with a randomized complete block design where the study plots served as blocks.