Journal of Science, (2018) 18(4): 15 1–10 doi: 10.1093/jisesa/iey082 Research

Spinosad Versus Spinetoram Effects on Kill and

2018 Oviposition of indifferens (Diptera: ) at Differing Ages and Temperatures

Wee L. Ye e

United States Department of Agriculture, Temperate Tree Fruit & Vegetable Research Unit, 5230 Konnowac Pass Road, Wapato, WA 98951 ([email protected])

Subject Editor: Cesar Rodriguez-Saona

Received 24 June 2018; Editorial decision 4 August 2018

Abstract Western fruit fly,Rhagoletis indifferens Curran (Diptera: Tephritidae), is a major quarantine pest of (Prunus spp.) in western North America that is often managed using the organic insecticide spinosad, but there is a question of whether its semisynthetic relative spinetoram is more toxic and better to use for controlling the fly. Here, spinosad and spinetoram effects on R. indifferens kill and oviposition were determined by exposing 3–4, 7–10, or 14–18 d old to dry spinosad and spinetoram (0.21 or 0.33 mg active ingredient [a.i.] per dish) and untreated cherries or to insecticide-treated cherries at 15.6, 22.5, and 29.4°C. Kill was not affected by fly age. Spinetoram killed more female flies by day 1 than spinosad at all temperatures. In both treatments, kill was lower at 15.6°C than 22.5 and 29.4°C, although a difference between 22.5 and 29.4°C was detected more often in spinosad treatments. Both insecticides killed 3–4 d old flies quickly enough to prevent oviposition, but neither prevented oviposition by 7–10 and 14–18 d old flies. Significantly, oviposition by flies exposed to spinosad and spinetoram did not differ at any temperature. Results indicate spinetoram is more toxic to R. indifferens than spinosad. However, this higher toxicity is not needed to prevent oviposition by younger flies. Furthermore, spinetoram residues are not sufficiently toxic to kill older flies quickly enough to reduce oviposition more than spinosad. Taken together, these conclusions imply that spinosad and spinetoram are equal for controlling R. indifferens infestations.

Key words: western cherry fruit fly, Entrust SC insecticide, Delegate WG insecticide

Western cherry fruit fly, Rhagoletis indifferens Curran (Diptera: In 2007, the semisynthetic spinosyn spinetoram, consisting of Tephritidae), is a major quarantine pest of cherries (Prunus spp.) spinosyns J and L in an approximate 3:1 ratio, was registered under in western North America, including the Pacific Northwest of Environmental Protection Agency’s Reduced Risk Pesticide Initiative the (, , , , and (Dripps et al. 2008a). Spinetoram is reportedly more potent, fast- ) and in Canada. In the Pacific Northwest er-acting, and longer-lasting than spinosad and controls a wider in 2017, the value of sweet cherry (Prunus avium (L.) L.) was range of insect pests in fruits and vegetables (Dripps et al. 2008a,b; US$1 billion (Northwest Horticultural Council 2018). The fly Bacci et al. 2016). However, spinetoram and spinosad are consid- must be controlled using insecticides because of the zero toler- ered toxicologically equivalent (EPA 2009) with apparently the same ance for its larvae in cherries destined for all domestic and for- mechanism of action (Shimokawatoko et al. 2012). This raises the eign markets (Smith 2005). The organic insecticide spinosad is question of whether spinetoram is more toxic to R. indifferens and often used to manage the fly in organic and conventional systems. better to use for controlling it. There are no studies comparing the Spinosad is derived from fermentation products of the bacterium two against R. indifferens. Saccharopolyspora spinosa Mertz & Yao and consists of spino- Work comparing spinosad with spinetoram against a wide range syns A and D in a 5.67:1 ratio (Dripps et al. 2008a). It is desir- of insect taxa has recently been published (e.g., Abdel-Latif and able to use due to its low mammalian toxicity, safe environmental Abdu-Allah 2011, Besard et al. 2011, Hamm et al. 2015, Bhatta profile (Dow AgroSciences 2001), high toxicity to R. indifferens et al. 2016, Siebert et al. 2016, Andreazza et al. 2017). However, (Yee 2011, 2015; Yee and Alston 2016), and its effectiveness in the little work in this area has been done for tephritid fruit flies. Against field (Flye Sainte Marie 2003). However, spinosad may not always apple maggot fly, Rhagoletis pomonella (Walsh), spinetoram aged eliminate infestations in trees when there are outside sources of for 7 d in the field caused less than or as much mortality as spinosad flies (Yee 2006). in GF-120 bait aged for 7 d (Yee et al. 2007), but it is unknown if the

Published by Oxford University Press on behalf of Entomological Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. 1 This Open Access article contains public sector information licensed under the Open Government Licence v2.0 (http://www.nationalarchives.gov.uk/doc/ open-government-licence/version/2/). 2 Journal of Insect Science, 2018, Vol. XX, No. X protein and sugar components in GF-120 increase or decrease the and possible efficacies of applications due to variations in seasonal toxicity of spinosad. Spinosad and spinetoram in soil drenches were temperatures. While it is unknown if kill caused by insecticides is similarly effective in preventing emergence of three tropical fruit fly affected by fly age, fly kill caused by spinosad is lower at cooler species (Stark et al. 2013). In a recent study, spinosad was found to than higher temperatures in the laboratory (Yee 2015, 2017). The be 2–36 times as toxic as spinetoram when combined with differ- mechanism for lower kill appears to be reduced movement by flies at ent hydrolyzed proteins against South American fruit fly Anastrepha cooler temperatures, such that these flies contact localized spinosad fraterculus (Wiedemann) (Schutze et al. 2018). However, as alluded residues less rapidly than more active flies at warmer temperatures to, the role of protein bait in affecting the efficacies of spinosyns is (Yee 2015). Fly contact with scattered spray residues through move- unknown. ment could determine control levels in the field, as insecticide spray For R. indifferens control, timings of spinosad and spinetoram coverage of cherry trees is not likely to be complete and is dependent applications during the cherry season from May to July are crit- on application method and row locations (Rothwell et al. 2017). ical. Timing of applications affects control of flies of different ages Whether temperature influences the effects of spinetoram on kill

A. 15.6°C, 3-4 d Old Flies B. 15.6°C, 14-18 d Old Flies 10 10 9 9 8 8 7 7 6 6 5 5 4 4

SE 3 3 ± 2 2 s 1

ie 1 0 0 Fl

le C. 22.5°C, 3-4 d Old Flies D. 22.5°C, 14-18 d Old Flies

ma 10 10 9 9 Fe 8 8 7 7 ead 6 6

. D Control 5 5 Low Spinosad No 4 4 High Spinosad 3 3 ve Low Spinetoram ti 2 2

la High Spinetoram 1 1

mu 0 0

Cu E. 29.4°C, 3-4 d Old Flies F. 29.4°C, 14-18 d Old Flies

an 10 10 9 9 Me 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 0 0 1 3 5 1 3 5 Day After Exposure

Fig. 1. Experiment 1: mean cumulative numbers of dead female Rhagoletis indifferens ± SE in controls and low and high rate spinosad and spinetoram treatments at 15.6°C for (A) 3–4 and (B) 14–18 d old flies, at 22.5°C for (C) 3–4 and (D) 14–18 d old flies, and at 29.4°C for (E) 3–4 and (F) 14–18 d old flies at 1, 3, and 5 d after exposure. Maximum is 10 females. Journal of Insect Science, 2018, Vol. XX, No. X 3 and oviposition of R. indifferens the same way it does of spinosad measured here. In addition, control flies laid many eggs in these cher- is unknown. Temperature effects on the more toxic malathion (vs ries. Finally, flies can survive >50 d when exposed to such cherries spinosad) were absent (Yee 2017). If spinetoram is more toxic than (Yee 2003), indicating no detrimental effect of using them. All cher- spinosad, then its effect on kill may be less impacted by temperature ries were firm at beginning of tests. than that of spinosad. For R. indifferens, oviposition is the measure of damage done Experiment 1: Spinosad and Spinetoram Effects at by the fly, assuming the eggs hatch and produce larvae that feed Three Temperatures, 3–4 and 14–18 d Old Flies on cherry flesh. Timing of and levels of oviposition by R. indif- This experiment was conducted in 2016 using 10 male (for mating) ferens are affected by fly age and temperature (Frick et al. 1954; and 10 female flies held inside a 1.9-liter container (same type as Yee 2015, 2017). At 26.7, 32.2, and 15.6–18.3°C in the labora- during pretesting with food and water as described above), five cher- tory, the minimum preoviposition periods of R. indifferens are 5–8 ries, and a dish with dry spinosad or spinetoram. Each container was (post-eclosion), 5–7, and 7–21 d, respectively (Frick et al. 1954). placed in a BOD low temperature incubator (VWR, Radnor, PA) set Thus, to prevent oviposition, toxicities of spinosad and spineto- to a constant 15.6, 22.5, or 29.4°C (±0.5°C, range). Controls and ram need only be high enough to kill flies before they reach 5 d low and high rates of spinosad and of spinetoram were tested (four old. Lower temperatures reduce oviposition levels by R. indiffer- insecticide treatments) at each of the three temperatures using 3–4 d ens exposed to spinosad, and they may also do this to flies exposed old and 14–18 d old flies. Five replicates of controls and treatments to spinetoram, potentially affecting fly control, although there are were set up. no data for this. Low or high rate spinosad or spinetoram solutions were applied The objective of this study was to determine spinosad versus as five 500-µl drops onto a 9.8-cm diameter Petri dish and then spinetoram effects on kill and oviposition of R. indifferens at differ- allowed to dry on the dish for 1 d at 21–22°C. The amount of both ing fly ages and temperatures. Four hypotheses were tested: 1) spin- dry spinosad and dry spinetoram per dish for the low rate was etoram is more toxic than spinosad to R. indifferens of all fly ages; 0.21 mg a.i.; for the high rate, 0.33 mg a.i. The 2,500 µl total vol- 2) for spinosad and spinetoram, lower temperatures result in lower ume was chosen because past work using 1.9-liter containers (Yee kill and oviposition; 3) the performance of spinosad is more affected 2015) suggested that lower volumes (maximum of 1,200 µl) were by temperature than that of spinetoram; 4) spinosad is toxic enough insufficient to cause most flies to contact spinosad quickly enough despite the greater toxicity of spinetoram to kill and prevent ovipo- to be killed. In the current study, volumes >2,500 µl were not tested sition by younger flies. Possible significance of results for control of R. indifferens is discussed.

Table 1. Experiment 1: analysis of treatment effects on 3–4 and 14–18 d old female Rhagoletis indifferens exposed to dry residues Materials and Methods of spinosad and spinetoram in dishes: three-way ANOVA for num- Flies and Pretest Conditions bers of dead flies (spinosad and spinetoram treatments only) and two-way ANOVA for oviposition (control, spinosad, and spineto- Flies used in experiments originated as larvae infesting cherries in ram treatments; fly age not included because 3–4 d old flies laid backyard trees in Kennewick in central Washington in June and July no eggs) 2015 and 2017. Puparia were held at 3–4°C for ~6 mo and then transferred to 21–22°C for adult emergence before testing. Flies Source F-value df P-value upon emergence were maintained inside 1.9-liter (10.2 cm diameter a × 16.2 cm high) paper containers with tulle fabric covering at a dens- No. dead females by day 1 (three-way ANOVA) Treatmenta 9.17 3, 96 <0.0001 ity of ~30 males and 30 females at ~23°C, 16:8 L:D h, and 20–35% Fly age 1.13 1, 96 0.2894 RH. Dry 80% sucrose and 20% yeast extract (by weight) (Hy-Yest Temperature 322.59 2, 96 <0.0001 412, powder, Sigma-Aldrich, St. Louis, MO) food on paper strips Treatment × fly age 0.43 3, 96 0.7349 and water in cotton wicks were provided to flies up to and during Treatment × temperature 0.86 6, 96 0.5259 testing. Fly age × temperature 1.00 2, 96 0.3709 Treatment × fly age × temperature 0.86 6, 96 0.5261 Insecticide Sources and Rates and Source of Oviposition—14–18 d old flies only (two-way ANOVA) Treatment 141.69 4, 60 <0.0001 Cherries Temperature 52.09 2, 60 <0.0001 The source of spinosad was Entrust SC (suspension concentrate) Treatment × temperature 15.18 8, 60 <0.0001 insecticide (22.5% spinosad; 77.5% other ingredients; 0.24 kg active Treatment × temperature effect sliced by temperatureb ingredient [a.i.] per liter); the source of spinetoram was Delegate 15.6°C 7.30 4, 60 <0.0001 WG insecticide (water dispersible granule) (25% spinetoram; 75% 22.5°C 65.75 4, 60 <0.0001 other ingredients) (both Dow AgroSciences, Indianapolis, IN). Low 29.4°C 99.00 4, 60 <0.0001 b and high rates tested were equivalent to label rates for Entrust of Treatment × temperature effect sliced by treatment 292 and 584 ml/ha and for Delegate of 315 and 490 g/ha, both Control 99.46 2, 60 <0.0001 at 935 liters water per hectare. Sweet cherries (~2.5 cm diameter) Low spinosad 2.91 2, 60 0.0624 High spinosad 3.83 2, 60 0.0272 used for tests originated from Chile and were purchased from a local Low spinetoram 5.71 2, 60 0.0054 market and were held in modified atmosphere packaging at 3°C to High spinetoram 0.90 2, 60 0.4108 retain their freshness. Cherries were not organic and were washed in water to remove potential insecticide residues and then dried for aExcluded control because there were zero deaths by day 1 in five of six 1–2 h at 21°C before use. Any insecticides that penetrated the fruit control treatments (three temperatures × two fly ages). cuticle would not have affected results, as insecticides inside fruit bSliced because of significant treatment× temperature interaction (terminol- would affect larval development rather than oviposition, which was ogy of Schabenberger et al. 2000). 4 Journal of Insect Science, 2018, Vol. XX, No. X because the aim was to tease apart any toxicity differences between Experiment 3: Spinosad and Spinetoram Effects at spinosad and spinetoram, which may not have been detectable had Three Temperatures: Cherries Dipped in Insecticides, higher volumes been used. 7–8 d Old Flies The control dish or dish with insecticide was placed on the To test if exposure of flies to dry spinosad or spinetoram residues on bottom of a container and then cherries placed around the dish. cherries results in the same kill and oviposition patterns as exposure Flies were introduced into the container, and the container placed to dry residues in dishes, cherries were dipped in spinosad or spin- in one of the three incubators. Each test ran for 10 d. A new set etoram in Experiment 3. Five cherries were dipped for ~3 s in a low of five cherries was placed into the container at day 3 and at day rate of spinosad or spinetoram solution and allowed to dry over 1 d 7. Numbers of dead flies were recorded on days 1, 3, 5, 7, 9, and at 21–22°C on a 9.8-cm diameter Petri dish. Only the low rate was 10. Flies were considered dead if they were unable to walk. All tested because Experiments 1 and 2 had shown there was little or no cherries upon removal from containers were preserved in 70% difference using the two rates. Ten each of 7–8 d old male and female ethanol. After at least 3 d, cherries were examined under a micro- flies were introduced into each container with the five treated cherries scope at 50× for eggs. Storage in ethanol removed the dark cherry at 15.6, 22.5, or 29.4°C. Tests ran for 4 d using the one set of five skin pigments, allowing the white eggs found below the surface cherries. Numbers of dead flies were recorded each day. Cherries were (≤1 mm) to be easily counted. collected at the end of testing and stored for later examination for eggs as before. Five replicates of controls and treatments were set up. Experiment 2: Spinosad and Spinetoram Effects at Three Temperatures, 7–10 and 14–18 d Old Flies Statistical Analyses An experiment similar to Experiment 1 was conducted in 2018. The Numbers of dead female flies and numbers of eggs laid were square- major difference was that 7–10 d old flies were tested in place of root transformed to normalize the data and homogenize the variances 3–4 d old flies. Flies 14–18 d old were tested as before. In addition, (males do no damage, so male data were not analyzed). Data of dead numbers of dead flies were checked on fewer days, at days 1, 3, 7, females by day 1 after exposure in Experiments 1 and 2 were analyzed and 10, because results of Experiment 1 indicated that most flies using a three-way analysis of variance (ANOVA), with treatment (con- in treatments (dependent on temperature) were dead by day 3. Five trol and the four insecticide treatments), temperature, and fly age as replicates of controls and treatments were set up. factors. In Experiment 1, there were no dead females in the five control

Fig. 2. Experiment 1: mean numbers of eggs laid by Rhagoletis indifferens ± SE starting at 3–4 d old for (A) controls and (B) low and high rate spinosad and spinetoram treatments at 15.6, 22.5, and 29.4°C and at 14–18 d old for (C) controls and (D) low and high rate spinosad and spinetoram treatments at 15.6, 22.5, and 29.4°C. Note 12 times greater y-axis scale for control than treatments. Journal of Insect Science, 2018, Vol. XX, No. X 5 groups, so controls had no variance and were not included in the ana- Pairwise comparisons were then conducted within significant slices lysis. Also, in Experiments 1 and 2, data of dead females by day 3 using one-way ANOVA and LSD tests. Analyses were performed using were analyzed using one-way ANOVA for just 15.6°C because all flies the PROC GLM procedure in SAS 9.4 (SAS Institute Inc. 2015). in some treatments at 22.5 and 29.4°C were dead by then (and after 3 d), resulting in no variance. Data of dead females in Experiment 3 for days 1–4 were analyzed using two-way ANOVA. Oviposition data Results from Experiment 1 were analyzed using a two-way ANOVA, drop- ping fly age as a factor because flies in the 3–4 d old treatment laid Experiment 1: Spinosad and Spinetoram Effects at no eggs. Oviposition data from Experiments 2 and 3 were analyzed Three Temperatures, 3–4 and 14–18 d Old Flies using a three-way and two-way ANOVA, respectively. When there Kill by insecticides at different fly ages were significant interactions, simple main effects were analyzed using Numbers of dead females are shown for days 1, 3, and 5 and not for slices following methods described in Schabenberger et al. (2000). days 7–10 (Fig. 1), as almost all flies across treatments were dead

A. 15.6°C, 7-10 d Old Flies B. 15.6°C, 14-18 d Old Flies 10 10 9 9 8 8 7 7 6 6 5 5 4 4

SE 3 3 ± 2 2 s 1

ie 1 0 0 Fl

le C. 22.5°C, 7-10 d Old Flies D. 22.5°C, 14-18 d Old Flies

ma 10 10 9 9 Fe 8 8 7 7 Control Dead 6 6 . 5 Low Spinosad 5 High Spinosad

No 4 4 Low Spinetoram 3 3 ve High Spinetoram

ti 2 2 la 1 1

mu 0 0

Cu E. 29.4°C, 7-10 d Old Flies F. 29.4°C, 14-18 d Old Flies

an 10 10 9 9 Me 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 0 0 1 3 7 1 3 7 Day After Exposure

Fig. 3. Experiment 2: mean cumulative numbers of dead female Rhagoletis indifferens ± SE in controls and low and high rate spinosad and spinetoram treatments at 15.6°C for (A) 7–10 and (B) 14–18 d old flies, at 22.5°C for (C) 7–10 and (D) 14–18 d old flies, and at 29.4°C for (E) 7–10 and (F) 14–18 d old flies at 1, 3, and 7 d after exposure. Maximum is 10 females. 6 Journal of Insect Science, 2018, Vol. XX, No. X by day 7. There was no fly age effect on kill (Table 1) and for both Table 2. Experiment 2: analysis of treatment effects on 7–10 and 3–4 and 14–18 d old flies the two spinetoram treatments killed more 14–18 d old female Rhagoletis indifferens exposed to dry resi- females by day 1 than the spinosad treatments at 15.6 and 22.5°C dues of spinosad and spinetoram in dishes: three-way ANOVA (Table 1, Fig. 1). In addition, the high spinosad treatment killed more for numbers of dead female flies (spinosad and spinetoram treat- ments only) and oviposition (control, spinosad, and spinetoram females by day 1 than the low spinosad treatment (critical t = 1.985; treatments) LSD = 0.2301; P < 0.05). By day 3, numbers of dead 3–4 d old females at 15.6°C (Fig. 1A) were greater in the high spinetoram than Source F-value df P-value both spinosad treatments (F = 17.37; df = 4, 20; P < 0.0001). By a day 3, numbers of dead 14–18 d old females at 15.6°C (Fig. 1B) No. dead females by day 1 (three-way ANOVA) a were greater in both spinetoram treatments than the low spinosad Treatment 15.00 3, 96 <0.0001 Fly age 0.53 1, 96 0.4683 treatment (F = 3.42; df = 3, 16; P = 0.0428; controls left out, no Temperature 327.03 2, 96 <0.0001 variance). However, by days 3 and 5 at 22.5 and 29.4°C, there were Treatment × fly age 0.32 3, 96 0.8114 >9 dead females (maximum of 10) across all insecticide treatments Treatment × temperature 2.02 6, 96 0.0702 (Fig. 1C–F), which can be considered similar and not significant. Fly age × temperature 0.97 2, 96 0.3820 Treatment × fly age × temperature 0.18 6, 96 0.9814 Kill among temperatures Oviposition (three-way ANOVA) Numbers of females in both age groups killed by spinosad and Treatment 182.33 4, 120 <0.0001 spinetoram by day 1 were lowest at 15.6°C, higher at 22.5°C, and Fly age 72.61 1, 120 <0.0001 Temperature 16.95 2, 120 <0.0001 highest at 29.4°C (critical t = 1.985; LSD = 0.1993; P < 0.05) (no Treatment × fly age 1.70 4, 120 0.1552 interactions between or among any factors, Table 1). Treatment × temperature 14.42 8, 120 <0.0001 Fly age × temperature 1.43 2, 120 0.2422 Oviposition Treatment × fly age × temperature 0.20 8, 120 0.9898 Control 3–4 d old females survived the 10 d of the experiment to Treatment × temperature effect sliced by temperatureb oviposit, even though they laid fewer eggs at lower than higher tem- 15.6°C 14.10 4, 120 <0.0001 peratures (Fig. 2A). However, 3–4 d old females were killed by both 22.5°C 80.15 4, 120 <0.0001 spinosad and spinetoram before they were old enough to oviposit 29.4°C 116.90 4, 120 <0.0001 (Fig. 2B). For 14–18 d old flies within each of the three temperatures a (penultimate subheading in Table 1, Fig. 2), oviposition was greater Excluded control because there were zero deaths by day 1 in four of six control treatments (three temperatures × two fly ages). in the control than insecticide treatments (only 14–18 d old flies: bSliced because of significant treatment× temperature interaction (terminol- 15.6°C: F = 9.00; P = 0.0003; 22.5°C: F = 66.61; P < 0.0001; 29.4°C: ogy of Schabenberger et al. 2000). F = 82.38; P < 0.0001; df for all = 4, 20). Within 15.6 and 22.5°C, there were no differences in oviposition among spinosad and spineto- ram treatments (P > 0.05). However, at 29.4°C, oviposition was lower Kill among temperatures in the low spinetoram than high spinosad treatment (F = 82.38; df = 4, By day 1 within fly age groups, kill by spinosad and spinetoram 20; P < 0.0001) (Fig. 2D), the only case in this study where spinetoram was lower at 15.6°C than at 22.5 and 29.4°C (critical t = 1.980; significantly reduced oviposition more than spinosad. LSD = 0.1914; P < 0.05) (Fig. 3). However, there was a near sig- Oviposition within control 14–18 d old females was lower nificant (P = 0.0702) treatment × temperature interaction by day at 15.6°C than at 22.5 and 29.4°C (no difference between 22.5 1 (Table 2), explained by kill among the three temperatures in the and 29.4°C) (F = 58.71; df = 2, 12; P < 0.0001) (last subheading spinosad treatment differing numerically, while kill by spinetoram in Table 1, Fig. 2C). Within the high spinosad treatment, ovipo- differed only between 15.6°C and the two high temperatures, which sition was lower at 15.6°C than at 29.4°C (F = 7.20; df = 2, 12; did not differ (Fig. 3). P = 0.0088). Within the low spinetoram treatment, oviposition was lower at 15.6°C than at 22.5°C (F = 5.78; df = 2, 12; P = 0.0175) Oviposition (Table 1, Fig. 2D). There were no significant differences in oviposition between spinosad and spinetoram within fly age groups. The only difference in oviposi- Experiment 2: Spinosad and Spinetoram Effects at tion was between the control and all insecticide treatments (Table 2, Three Temperatures, 7–10 and 14–18 d Old Flies Fig. 4) (7–10 d old: 15.6°C: F = 5.74; P = 0.0030; 22.5°C: F = 43.65; P < 0.0001; 29.4°C: F = 19.23; P < 0.0001; 14–18 d old: 15.6°C: Kill by insecticides at different fly ages F = 23.58; P < 0.0001; 22.5°C: F = 88.11; P < 0.0001; 29.4°C: Numbers of dead females are shown for days 1, 3, and 7 (Fig. 3) F = 99.39; P < 0.0001; df for all = 4, 20). For controls and insecti- and not for day 10 because most treatment flies were dead by then. cide treatments, oviposition within age groups was lower at 15.6°C There was no age effect on fly kill (Table 2). In both 7–10 and 14–18 than at 22.5 and 29.4°C (critical t = 1.980; LSD = 1.261; P < 0.05). d old flies by day 1, low and high spinetoram treatments killed more Importantly, 7–10 d old flies (Fig. 4A and B) oviposited at signifi- females than low and high spinosad treatments at all temperatures cantly lower levels (Table 2) than 14–18 d old flies (Fig. 4C and D). (no age × treatment interaction) (critical t = 1.980; LSD = 0.2211; P < 0.05) (Table 2, Fig. 3). By day 3 at 15.6°C, both spinetoram treatments had killed more Experiment 3: Spinosad and Spinetoram Effects at 7–10 d old females than both spinosad treatments (F = 4.43; df = 3, Three Temperatures: Cherries Dipped in Insecticides, 16; P = 0.0190; controls left out, no variance) (Fig. 3A), as was 7–8 d Old Flies the case at 15.6°C for 14–18 d old females (F = 26.04; df = 4, 20; Kill by insecticides P < 0.0001) (Fig. 3B). However, by day 3 at 22.5 and 29.4°C, there There was no difference in kill at 15.6°C by day 1 between the were no numerical differences in kill among treatments (Fig. 3C–F). control and spinosad and spinetoram treatments (P > 0.05) Journal of Insect Science, 2018, Vol. XX, No. X 7

Flies 7-10 d Old at Start of 10-Day Test 1800 150 A. Control, 7-10 d old B. Treatments, 7-10 d old flies 1600 flies 120 1400 15.6°C 1200 22.5°C 29.4°C Low Spinosad SE 90 1000 High Spinosad ± Low Spinetoram 800 High Spinetoram 60 erries 600

400 30

of 15 Ch 200 m 0 0 mu xi Flies 14-18 d Old at Start of 10-Day Test

Ma 1800 150 r C. Control, 14-18 d D. Treatments, 14-18 d old flies pe 1600 old flies gs 1400 120 Eg . 1200 No 90 1000 ean

M 800 60 600

400 30 200

0 0 Control 15.6°C 22.5°C 29.4°C

Fig. 4. Experiment 2: mean numbers of eggs laid by Rhagoletis indifferens ± SE starting at 7–10 d old for (A) controls and (B) low and high rate spinosad and spinetoram treatments at 15.6, 22.5, and 29.4°C and at 14–18 d old for (C) controls and (D) low and high rate spinosad and spinetoram treatments at 15.6, 22.5, and 29.4°C. Note 12 times greater y-axis scale for control than treatments.

(Table 3, Fig. 5A). However, at 22.5°C by day 1, spinetoram Oviposition killed more females than spinosad (both more than the control) There were differences in oviposition between the control and two (F = 28.67; df = 2, 12; P < 0.0001) (Fig. 5B), although at 29.4°C insecticide treatments, but there was also a significant interaction by day 1, there was no difference between spinosad and spin- (Table 3, Fig. 6). Specifically, there was no difference at 15.6°C etoram (although both more than the control; F = 80.33; df = 2, but there were significant differences at 22.5 and 29.4°C, but only 12; P < 0.0001) (Table 3, Fig. 5C). By days 2–4, there were no between the control and the two insecticide treatments (22.5°C: differences between spinosad and spinetoram at any temperatures F = 19.09; df = 2, 12; P = 0.0002; 29.4°C: F = 24.29; df = 2, 12; (P > 0.05). P < 0.0001) (penultimate subheading in Table 3, Fig. 6). Within the control (last subheading in Table 3, Fig. 6A), oviposition was greater Kill among temperatures at 22.5 and 29.4°C than at 15.6°C (F = 15.61; df = 2, 12; P = 0.005). By day 1, there was no difference in deaths within control flies However, no statistically significant temperature effects within each among the three temperatures (Table 3, Fig. 5), but kill by spinosad insecticide treatment were detected (P > 0.05), even though oviposi- at 15.6°C was lower than at 22.5°C, which was lower than at 29.4°C tion was numerically greater at higher temperatures (Fig. 6B). (F = 29.45; df = 2, 12; P < 0.0001). In contrast, for spinetoram by day 1, kill at 15.6°C was lower than at 22.5 and 29.4°C (F = 14.96; Discussion df = 2, 12; P = 0.0005), but kill at 22.5 and 29.4°C did not differ (Fig. 5). This same pattern was true by day 2, but by days 3 and 4, Results show that spinetoram is more toxic to R. indifferens than differences in kill by spinosad and spinetoram across temperatures spinosad, as measured by fly kill using the protocol here, and that did not differ significantly (P > 0.05). all age groups of flies are similarly affected. Spinetoram is also more 8 Journal of Insect Science, 2018, Vol. XX, No. X

Table 3. Experiment 3: analysis of treatment effects on 7–8 old 10 female Rhagoletis indifferens exposed to cherries with dry resi- 9 A. 15.6°C dues of spinosad and spinetoram: two-way ANOVA for numbers of 8 Control dead female flies and oviposition (control, spinosad, and spineto- 7 Low Spinosad ram treatments for both) 6 Low Spinetoram 5 Source F-value df P-value 4 SE 3

No. dead females by day 1 (two-way ANOVA) ±

Treatment 60.76 2, 36 <0.0001 s 2

Temperature 29.69 2, 36 <0.0001 ie 1

Treatment × temperature 10.01 4, 36 <0.0001 Fl 0

Treatment × temperature effect sliced by temperaturea le 10 9 B. 22.5°C 15.6°C 2.16 2, 36 0.1302 ma 22.5°C 23.40 2, 36 <0.0001 8 29.4°C 55.22 2, 36 <0.0001 Fe 7 Treatment × temperature effect sliced by treatmenta 6 ead Control 0.29 2, 36 0.7502 5 Low spinosad 26.84 2, 36 <0.0001 . D 4

No 3

Low spinetoram 22.58 2, 36 <0.0001 2

Oviposition (two-way ANOVA) ve

Treatment 40.23 2, 36 <0.0001 ti 1 Temperature 19.52 2, 36 <0.0001 la 0

Treatment × temperature 8.56 4, 36 <0.0001 mu 10 Treatment × temperature effect sliced by temperaturea 9 C. 29.4°C Cu 15.6°C 0.49 2, 36 0.6142 8 22.5°C 22.12 2, 36 <0.0001 an 7

29.4°C 34.74 2, 36 <0.0001 Me 6 Treatment × temperature effect sliced by treatmenta 5 Control 34.44 2, 36 <0.0001 4 Low spinosad 0.52 2, 36 0.5996 3 Low spinetoram 1.69 2, 36 0.1992 2 1 Only results of dead females by day 1 of the 4-d experiment are shown. 0 aSliced because of significant treatment× temperature interaction (terminol- 1234 ogy of Schabenberger et al. 2000). Day After Exposure toxic than spinosad against many (Dripps et al. 2008a,b; Fig. 5. Experiment 3: mean cumulative numbers of dead female Rhagoletis Abdel-Latif and Abdu-Allah 2011; Hamm et al. 2015; Siebert et al. indifferens ± SE in controls and low rate spinosad and spinetoram treatments 2016; Andreazza et al. 2017), but possibly not against all (Besard starting at 7–8 d old at (A) 15.6°C, (B) 22.5°C, and (C) 29.4°C at 1, 2, 3, and 4 d et al. 2011, Athanassiou and Kavallieratos 2014, Bhatta et al. 2016), after exposure. Maximum is 10 females. including fruit flies (Schutze et al. 2018). For nontarget insects, whether spinetoram is more toxic than spinosad appears to depend on the insect taxa (Lefkaditis et al. 2017). In sum, results here were Differences in kill of R. indifferens caused by spinosad versus not entirely predictable based on the literature. spinetoram by day 1 or 3 were most detectable at 15.6°C but less The reason for the greater toxicity of spinetoram against R. indif- or not detectable at 22.5 and 29.4°C. As alluded to in the previous ferens is not clear based on its reported mechanism of action. paragraph, at lower temperatures, flies probably contacted smaller Spinetoram affects nicotinic acetylcholine receptors and γ-amin- spinosad and spinetoram doses, allowing for a detectable separation obutyric acid receptors on postsynaptic membranes in insect nerv- due to differential toxicity. At higher temperatures, greater fly activ- ous systems, causing abnormal neural transmission and death ity levels may have resulted in contact with higher doses of spinosad, (Shimokawatoko et al. 2012). This is the same mechanism proposed making its effects on kill similar to that of spinetoram. earlier for spinosad (Salgado 1998; Orr et al. 2006, 2009). Spinetoram Temperature effects on kill seemed less important for spinetoram may disrupt the function of nicotinic acetylcholine and γ-aminobu- than spinosad. In Experiment 1, kill within spinosad and spinetoram tyric acid receptors in R. indifferens faster than spinosad does. was successively greater at each of the three temperatures, but in Cooler temperatures resulted in lower kill of R. indifferens exposed to Experiments 2 and 3 this was true only for spinosad. Possibly the both spinosad and spinetoram. Based on results at 18.3°C versus 23.9°C amount of spinetoram picked up by flies at 22.5°C, even if lower (Yee 2015), this was probably due to flies being less active at 15.6°C than at 29.4°C, reached a threshold that maximized kill. It would than at 22.5 and 29.4°C. Less active flies contacted the insecticides in the be valuable to determine if in general more toxic insecticides such dishes or on the cherries later or at lower doses than more active flies. as spinetoram, as well as malathion (Yee 2017), are less affected by An alternative explanation that toxicities of insecticides were reduced at temperature than less toxic insecticides. cooler temperatures is less likely. For example, the toxicity of spinosad In Experiment 1, unlike for kill, there was a clear age effect fed to (Musca domestica L.) (Diptera: Muscidae) decreased on oviposition of R. indifferens, in that 3–4 d old flies exposed to 3.16-fold as temperatures increased from 20 to 34°C (Khan and Akram both spinosad and spinetoram laid no eggs while 14–18 d old flies 2014). When pyrethroids were applied onto houseflies, kill was greater exposed to both laid many eggs in similar numbers. Despite its lower at 18°C than 32°C (Scott and Georghiou 1984). toxicity, spinosad was poisonous enough to kill 3–4 d old flies before Journal of Insect Science, 2018, Vol. XX, No. X 9

SE 200 12 A. Control B. Treatments ± 175 15.6°C 10 Low Spinosad 22.5°C

erries Low Spinetoram 150 29.4°C Ch

8 125 ve

r Fi 100 6 pe 75 4 ggs

. E 50

No 2 25 an

Me 0 0 15.6°C 22.5°C 29.4°C

Fig. 6. Experiment 3: mean numbers of eggs laid by Rhagoletis indifferens ± SE starting at 7–8 d old for (A) control and (B) low rate spinosad and spinetoram treatments at 15.6, 22.5, and 29.4°C. Note 17 times greater y-axis scale for control than treatments. egg development and oviposition occurred. Even at 15.6°C, the 3–4 2017a,b). Sprayed at these intervals, spinosad and spinetoram res- d old flies (Experiment 1) were apparently active enough to con- idues should be effective for preventing infestations of fruit when tact spinosad and suffer 100% mortality before they could oviposit, used against flies <4 d old. Because flies emerge from soil from late perhaps aided by delayed oviposition at that temperature. Thus, to May to early July in central Washington (Frick et al. 1954), weekly prevent oviposition, it did not matter if spinetoram killed all flies sprays need to be applied during this period up until 7 d (for both (exposed at 3–4 d old) 1–3 d earlier than spinosad. insecticides) before harvest. There may be no reason to use spineto- Significantly, oviposition within 7–10 and 14–18 d old flies exposed ram over spinosad when the 7-d spray interval is followed, except to spinosad and spinetoram did not differ at any temperature. Cooler if residual activity of spinosad is much <7 d. Residual activity dura- temperatures suppressed oviposition by both, but even spinetoram tions of spinosad and spinetoram may depend on heat, sun exposure, residues on cherries could not knock flies down quickly enough to rainfall, and residue amounts on surface versus bottom of leaves, and eliminate oviposition at the lowest temperature tested (although close will require field study. Preventing oviposition may be more difficult in Experiment 3, Fig. 6B). There was only one case where spinetoram or impossible when flies are approaching reproductive maturity even caused lower oviposition, at 29.4°C in Experiment 2. The lack of ovi- though it may be easier when it is cooler. Eliminating oviposition position differences caused by the two insecticides was unexpected, as by older (7–10 and 14–18 d) flies using spinosad and spinetoram greater (faster) kill by spinetoram should result in lower oviposition. may be possible only if one or the other is sprayed directly on the Conversely, more flies survived in the spinosad treatment at day 1, so flies (Yee and Alston 2012). Faster-acting knockdown insecticides oviposition should have been greater in this than in the spinetoram such as pyrethroids (Scott and Georghiou 1984) or some neonic- treatment. One possible explanation is that most flies that contacted otinoids (Yee 2010) may be better options to use than spinosyns spinosad and survived after day 1 were sickened and rendered inca- or should be included in spray programs when there is a threat of pable of ovipositing or oviposited only at low levels during the short many reproductively mature flies moving into cherry orchards from time they were alive. In a previous study, 20% of R. indifferens that outside infested trees. fed on a spinosad-protein/sugar bait (GF-120) oviposited, but they In summary, results provide the first comparisons of spinosad laid only 0.8 eggs per fly before dying within 1 d (Yee 2010). versus spinetoram effects on kill and oviposition of R. indifferens. Lack of oviposition differences in spinosad and spinetoram Results indicate spinetoram is more toxic to R. indifferens than spi- treatments could also be due to the very low oviposition levels in nosad. However, this higher toxicity is not needed to prevent ovi- treatments relative to controls, and the high variability inherent in position by younger flies. Furthermore, spinetoram residues are not oviposition levels among individual R. indifferens. The differences in sufficiently toxic to kill older flies quickly enough to reduce ovipo- fly kill, though significant, were not large enough to overcome this sition more than spinosad. Taken together, these conclusions imply variability. For instance, at day 1 in Experiment 2 within the high that spinosad and spinetoram are equal for controlling R. indifferens rate spinosad and spinetoram treatments (Fig. 3D), the difference in infestations, a hypothesis that needs to be tested in the field. numbers of live flies was about two. However, flies in these respec- tive treatments laid 6–43 and 7–122 eggs per replicate, contributing to the lack of significant differences. These ranges probably were Acknowledgments dependent in part on whether females that had a tendency to ovi- I thank Janine Jewett, Dana Jones, and Peter Chapman (United States posit at high levels, highly variable itself, were killed early or not. Department of Agriculture-Agricultural Research Service [USDA-ARS], Results have implications for control of R. indifferens and for Wapato, WA) for assistance during the study, Heidrun Vogt (Julius Kühn- further study. They support the specimen label instructions for spi- Institut Schwabenheimer, Dossenheim, Quedlinburg, Germany) and Scott nosad (Entrust SC) and spinetoram (Delegate WG) against R. indif- Ockey (Certis USA, L.L.C., Yakima, WA) for reviewing early drafts of the ferens to ‘maintain protective sprays at 7-day intervals while adults manuscript, Bruce Mackey (USDA-ARS, Albany, CA) for statistical advice, are present and fruit is susceptible to attack’ (Dow AgroSciences and United States Department of Agriculture - Foreign Agricultural Service 10 Journal of Insect Science, 2018, Vol. XX, No. X for partial funding for the project. Mention of product names are for research Orr, N., G. B. Watson, J. Hasler, J. Michael, C. Geng, K. R. Cook, V. L. Salgado, purposes only and do not constitute endorsements for their use by USDA-ARS. and S. Chouinard. 2006. Sequences of Drosophila melanogaster nicotinic receptor alpha-6 and alpha-7 subunits for bioassay. 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