Susceptibility of Different Instars of the Obliquebanded Leafroller (Lepidoptera: Tortricidae)

BIOLOGICAL AND MICROBIAL CONTROL Susceptibility of Different Instars of the Obliquebanded Leafroller (Lepidoptera: Tortricidae) to Bacillus thuringiensis var. kurstaki S. Y. LI,l S. M. FITZPATRICK, 1 AND M. B. ISMAN2 J. Econ. Entomol. 88(3): 610-614 (1995) ABSTRACT Susceptibility of different instal's of the obliquebanded leafroller, C1IOrislorwllra rosaceana (I'larris), to Bacillus thuringiensis Berliner var. kurstaki was determined in the laboratory by using a leaf dip bioassay. All larval stages were susceptible to B. thllringiensis; fourth and sixth instal's were the most and the least sensitive, respectively. A sublethal concentration of B. thuringiensis val'. kurstaki had different effects on the development of surviving huvae, depending on the stage treated. Treated first instal's weigbed significantly less and larval development time was longer than the controls, but pupal weight and adult emer'- gence were not affected. \Vhen fifth instal's were treated, however, pupal weight and adult emergence were significantly reduced. Treatment with B. thllringiensis val'. kurstaki did not affect the adult sex ratio of survivors in experiments with first or fifth instal's. KEY WORDS Bacillus tlwringiensis, Choristoneura rosaceana, larval susceptibility THE OBLIQUE BANDED LEAFROLLER, Choristoneura rosaceana (Harris), is a native species widely distributed throughout North America. This polyphagous species is a major pest of small fruits (Schuh & Mote 1948), tree fruits (Chapman et ai. 1968, Madsen & Madsen 1980, Pree & Roberts 1981), filberts (AIiNiazee 1986), and minor pest of forest trees (Prentice 1965). In recent years, infestation of commercial raspberries by this pest has become a serious problem in the Fraser Valley, British Columbia. C. rosaceana not only damages raspberry leaves, but larvae contaminate machineharvested fruit, resulting in downgraded berries. Thus, raspberry growers are facing a severe problem with this pest. Organophosphorous insecticides are currently recommended for control of leafrollers on raspberries in British Columbia (Anonymous 1992), but they cannot be used close to harvest and are harmful to beneficial species. Dipel WP (wettable powder) (Bacillus thuringiensis Berliner val'. kurstaki) is currently registered for control of leafrollers on raspberries in British Columbia. However, the relative susceptibility of different instal's of C. rosaceana to this formulation of B. thuringiensis val'. kurstaki has not been studied and is important for optimal timing of Dipel applications. Previous research demonstrated that early instal's of some lepidopterous species are more susceptible to different varieties of B. thuringiensis than later instal's (Afify & Merdan 1969, Altahtawy & Abaless 1973, I PaciRc A~riculture Research Centre, 6660 N.W. Marine Drivt', Vancouver, BC V6T lx2 Canada. 2 Dt'partll1ent of Plant Science, University of British Columbia, Vancouver, Be V6T lz4 Canada. Morris 1973, McGaughey 1978, Fast & Dimond 1984, Hornby & Gardner 1987). In other species, later instal's are more sensitive than early instal's (Rock & Monroe 1983, James et a1. 1993). Under field conditions, some individuals may only receive a subletllal amount of Dipe!. The sublethal effects of B. thuringiensis val'. kurstaki on the development of surviving C. rosaceana larvae have not been determined. The objectives of our study were to determine the relative sensitivities of different instal's of C. rosacearw to B. thuringiensis, and to determine the effects of a sublethal concentration of B. thuringiensis on the development of C. rosaceana, Materials and Methods Insects. C. rosaceana larvae used in this study were the progeny of overwintering larvae collected from raspberry plants in the Fraser Valley, British Columbia, in early May 1993. After collection, the larvae were maintained at ambient photoperiod from 15:9 (L:D) h in May to 16,5:7.5 (L:D) h in June. Room temperatures varied from 15 to 22 C. Approximately 300 newly emerged adult males and females from overwintering larvae were kept in pairs of five or six in inflated plastic bags (25-cm diameter by 60-cm depth) with a flask of 8% sugar solution, Egg masses deposited on the surfaces of the bags were collected; upon hatching, larvae were transferred to the leaves of fresh, pesticidefree raspberry branches in a cage (0.6 by 0.3 by 0.3 m), Foliage was replaced as needed. Under these rearing conditions, larval development times averaged 4.1, 3.6, 3.7, 3.7, 5.4, and 5.1 d for first through sixth instal's, respectively, Development 0022-0493/95/0610-0614$02.00/0 1995 Entomological Society of America

JIlJ1(' 1995 1.1 ET AI..: SUSCEPTIBILITY OF C. rosaceana TO B. thuringiensis 611 Tubl.' 1. SU.('t'l'tibility of difterent insturs of C. rosaceana ruspbt'rry foliu/(" to B. tllllringiensis vur. kurstaki when fcd tr"utetl Instar Sampl. siz.. Int"reept, Slope ± SEM LC50, mg Dipel/ml, t-ratio a (f3) 95% CI " First 360 6.045 1.323 ± 0.207 6.391 0.164 (O.130-O.218)b 3.97 S.cond 360 5.841 1.032 ± 0.214 4.822 0.153 (0.II1-O.226)b 9.40 Third 270 6.500 1.660 ± 0.195 8.513 0.12.5 (O.096-0.I60)b 1.65 Fourth 270 6.805 1.518 ± 0.201 7.552 0.065 (0.044-O.086)c 2.77 Fifth 270 6.145 1.405 ± 0.193 7.280 0.153 (0.111-0.215)b 4.87 Sixth 270 5.620 1.174 ± 0.178 6.596 0.297 (0.2 13-0.443)a 2.4.5 Pnol.d 1.355 ± O.oll 123.164 i" LC 5U s fnllnw.d hy tl", same letter are not signircantly different Pn'isI"r 1992). at the 5% signircance level (the lethal dose ratio test, Hobertson & times were used as a guide for distinguishing instars. Sm;ct~ptibility Tesls. The response of each instar to various concentrations of B. thuringiensis was determined by using a leaf dip bioassay technique. Dipe! WP (potency 16,000 IU/mg; Abbott, North Chicago, 11.)was diluted with water (ph = 5.57) to make a series of six or eight concentrations, ranging from 0.0375 to 1 mg of Dipel WP per milliliter of water. Controls were treated with water alone. First and second instars were exposed to eight concentrations; third, fourth, fifth, and sixth instars were exposed to six. Leaf dip bioassays for each instar were done as follows. Three replicates of 30 raspberry leaves were immersed in each concentration of B. thllringiensis or water for 5 s. Dipped leaws were a.llowedto air dry for 1 h, and were then placed in pairs in plastic Dixie cups (4- cm diameter by 4-cm depth; James River, Norwalk, CT). Larvae of appropriate stages were individually transferred into the cups. Thus, for each instar and concentration, three replicates of 15 larvae were tt'stt'd. Total sample sizes ranged from 270 to 360. The first instars chosen for bioassays were <12 h old. Larvae of all other instars had molted 1 or 2 d prt'vionsly. Larvae were allowed to feed on the treated leaws for 6 d before the leaves were remowd and replaced with fresh, untreated ones. Larvat' in tllp cups were maintained in an insectary at 21 ± 1 C with a photoperiod of 16:8 (L:D) h. Larval mortality was recorded at 3, 6, and 9 dafter treatmt'nt. Abbott's (1925) formula was used to correct for control mortality. If mortality in the control was >10%, the data were discarded and the experiment was repeated. The cumulative mortality data at 9 d were subjected to probit ana]- ysis (Morse et al. 1988). The chi-square goodnessof-fit was used to test whether each instar data set fits tlw probit model. The t ratio was used to test the significance (P = 0.05) of each regression line (Robertson & Preisler 1992). Regression lines Onstars 1-6) were subjected to a likelihood ratio test for the equality and parallelism (Morse et al. 1988). LC5(lSfor instars were respectively compared \vith each other for significance (P < 0.05) by using tilt' lethal-dose ratio test (Robertson & Preisler 1992). Sublethal Effects. A sublethal concentration of B. thuringiensis may affect the development of survivors differently, depending on the larval instar th,lt is treated. We treated first and fifth instars with a sublethal concentration of B. thuringiensi~ at 0.15 mg of Dipe] per milli]iter, which was expected to cause "".50% mortality. First and fifth instars were chosen because we wished to determine the sublethal effects of B. tlwringiensis on early and late larval stages. Water treatment served as the control. The treatment of raspberry leaves with B. thuringiensis or water was the same as described for the susceptibility tests. Newly hatched larvae «12 h old) in individual cups were fed raspberry leaves treated with Dipe! WP or water for 6 d and then fed fresh, untreated leaves. Survivors from treatment and control groups were individually weighed to the nearest 0.1 mg 16 d after hatching. Thereafter, the survivors were observed daily and time to pupation was recorded. Sex of pupae was determined, and they were weighed 3 d after pupation. Treatment groups were compared with the controls to assess effects of the sublethal concentration of B. thuringiensl~ on larval weight, larva] development time, pupal weight, pupal duration, adult emergence, and adult sex ratio by \Ising at-test (P < 0.05). Fifth instars (1-2 d after molting) received the same treatment as the first instars described above. However, only pupal weight, adult emergence, and.adult sex ratio were assessed. Susceptibility Results Tests. The results for diffcrent instars (Table 1) indicate that the responses of different instars to the range of concentrations of B. thuringiensis val'. kurstaki were satisfactorily described by the probit model (first: K- = 3.97, df = 7, P > 0.0.5; second: K- = 9.40, df = 7, P > 0.05; third: K- = 1.65, df = 5, P > 0.05; fourth: K- = 2.77, df = 5, P > 0.0.5; fifth: K- = 4.87, df = 5, P > 0.0.5; and sixth: K- = 2.45, df =.5, P > 0.05). The t ratio test suggests that all regression lines were significant (P < 0.05) because all t ratios were >1.96 (Table 1). Slopes of the regression lincs were parallel ({3= 1.355 ± a.on [± SEM]; K- =

612 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 88, 110. 3 ':l:$ ~ "" -< 0-..0.-., +1!; ti &J ""d'!"-'mr13 co -:ti ] "o U -:ti.-, M "" ci +I~+I~ "'d'!--\o- 0; r-..: '" '".<:> oj 00 -< o ~ +100 +1;:::- a>-c")- '" ~ ~ ~ '".<:> -< '""! ci -< +I~+I~ ~-oor-: ci '" "2 "Eo u C'l 6.60, df = 5, P > 0.05). However, intercepts differed (K- = 58.12, df = 5, P < 0.05). LCslIs for fourth instars were significantly lower than others, and those for sixth instars were significantly higher than others (Table 1), suggesting that fonrth and sb::th instars were the most and the least sensitive to B. thuringiensis var. kurstaki, respectively. No significant differences in LCso were found among the other instars. Sublethal Effects. When newly hatched larvae were fed B. thuringiensis-treated foliage, the weight of surviving larvae was significantly lower than the controls (t = 5.105, df = 127, P < 0.0,'5), and larval development time was prolonged (t = 3.712, df = 113, P < 0.05) (Table 2). However, we found no significant differences in pupal weight (I = 0.514, df = 113, P > 0.05), pupal dnratioll (t = 0.514, df = 107, P > 0.05), adult emergence (t = 0.546, df = 113, P > 0.05), or sex ratio (t = 0.:331, df = 11:3, P > 0.05) between treated and control larvae. When fifth instars were fed foliage treated with B. thuringiensis var. kurstaki, pnpal weight was significantly reduced (t = 3.712, df = 74, P < 0.05), as was adult emergence (t == 3.264, df = 74, P <0.05). We observed no significant differences in sex ratio between treated and control larvae in either experiment, indicating that B. thuringil'nsis var. kurstaki has equal toxicity to male and female larvae. Approximately 20% of 41 fifth instars treated with B. thuringiensis var. kurstaki emerged as adults with malformed wings, compared with 3% of 35 control larvae (t = 3.984, df = 74, P < 0.(5). The percentage of treated first instars that emerged malformed (6% of 67), was not significantly different than the percentage of control larvae (4% of 48, t = 0.461, df = 113, P > 0.05). Discussion Our tests indicate that all larval stages of C. moo saceana are susceptible to B. thuringiensis var. kurstaki, that fourth ins tars are the most easily killed, and that sixth instars are the most difficult to ki]1. Gangavalli & AliNiazee (1985) reported that fourth-instar C. rosaceana have a developmental temperature threshold that is lower than any other larva] stage. Their results and ours snggest physiological differences between the fourth and other instars, although the ecological significance of these differences is unclear. The ph and activity of digestive enzymes required to activate the deltaendotoxin (Falcon 1971) in the gut in C. msacem/(/ may also differ among instars. We cannot determine which instar is truly the most susceptible to B. thllringiensis var. kllrstaki because the actual amount ingested by individuals of each instar was not known. In our study and in other previous reports, the relationship bctw('pn ]arval stage and susceptibility is confounded by instar-specific feeding rates and feeding habits. For example, early instars may scrape the surface of

J1111(' 1995 Ll ET AI..: SUSCEPTIBILITY OF C. rosaceana TO B. thuringiensis 6]3 treated leaves and ingest more B. thuringiensis var. kllrstaki per unit of consumed food than older lar- Val'. In some species (e.g., Tyria jacobaeae L., James et a!. 1993), older larvae are more easily killed by B. tlwringiemis var. kurstaki than younger ones; in othf.'r spf.'cies (e.g., Choristoneura fulilifcnllla, Monis 1973), younger larvae are more easily killed. In the former species, older larvae may ingest more toxin tl1<ulyounger ones because relative food consumption increases with age. In the lattn species, larval tolerance to B. thuringiensis was speculated to increase with body weight. Studit's on the use of B. thuringiensis var. kurstaki against pest Lepidoptera focus primarily on larval mortality. Effects of sublethal amounts of B. tlmringic/lsis on surviving larvae are not well understood. Our results suggest that B. thuringiensis var. kllrstaki adversely affects surviving larvae of C. rosacemlll. \Vht'n newly hatched larvae were exposed, treatment \vith B. thuringiensis var. kurstaki resulted in increased larval development time and reduced larval weight of survivors. However, we found no significant differences in pupal weight and adult emergence between treated and untreated larvae. This suggests that larvae can totally recover from initial e:-,'"posure to sublethal amounts of B. tlmringic/lsis var. kllrstaki, as is the case for C. flllllifcrlllul (Fast & Regniere 1984). Prolonging larval development time may not be desirable if it incrf.'ases the feeding time for survivors, but it may be beneficial it larvae are exposed to otllt'r mortality factors (e.g., predators and parasites). Although the developmental time of fifth instars treated with a sublethal concentration was not assessed, we noticed that surviving larval' pupated at almost the same time as untreated larvae, suggesting that B. thllringiensis var. kurstaki did not prolong larval development when later instars were treated. A similar result was reported for other species treated with B. thttringicnsis var. krtrstaki (McGaughey 1978). However, van Frankenhuyzen & Nystrom (1987) reported that treatnlt'nt of sixth-instar C. fil1niferana with a sublethal amount of B. thttringiensis var. kttrstaki significantly delayed larval development. Exposure of fifth-instar C. rosacea/la to B. thttringiensis var. kllrstaki significantly reduced pupal size and adult emprgf.'ncf.' as compared with the untreated larvae (Table 2). Surviving larvae might have reduced tlll'ir feeding rate after ingesting B. thllringiensis, and partial starvation might have contributed to reduced pupal size and adult emergence. The reproductivt' potential of treated fifth instars might also have been affected because the fecundity is positively correlated with pupal size in this species (Carrihe 1992). Sublethal amounts of B. thttringie/lsis var. kllrstaki are known to reduce reproductivt' potential of other pest species (Harper 1974, Morris 1976, Hornby & Gardner 1987, Hafez et a!. 1993). Malformation of adult moths follo\ving ingestion of sublethal amounts of B. thuringiensis var. thuringiensis by the larvae has been reported previously (Ignoffo & Gregory 1972, Sebesta et al. 1981). M,llformations are most often associated with B. thuringiensis formulations that contain f3- exotoxin. However, Dipel WP docs not contain f3- exotoxin. Thus, wing malformations may occur even in the absence of this toxin. Acknowledgments We thank the following people at the Pacific Agriculture Research Centre, Vancouver: M. Evangelista, J. M. Tronbridge, and M. Soucie for technical assistancp; B. D. Frazer and B. S. 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