Response of Oriental Fruit Moth, Grapholita Molesta (Busck) (Lepidoptera: Tortricidae), Eggs to Gamma Radiation

Radiation Physics and Chemistry 79 (2010) 1063–1066

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Radiation Physics and Chemistry

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Response of oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), eggs to gamma radiation

W.D. Silva a,n, V. Arthur b, T. Mastrangelo b a Department of Entomology and Acarology, Laboratory of Chemical Ecology and Insect Behavior, University of Sao~ Paulo, ‘‘Luiz de Queiroz’’ College of Agriculture, Pa´dua Dias Avenue, 11, 13418-900 Piracicaba, Brazil b Food Irradiation and Radioentomology Laboratory, Center for Nuclear Energy in Agriculture (CENA/USP), Centena´rio Avenue 303, 13400-970 Piracicaba, Brazil article info abstract

Article history: As insects increase in radiotolerance as they develop and usually several developmental stages of the Received 31 March 2010 pest may be present in the fresh shipped commodity, it is important to know the radiation Accepted 30 May 2010 susceptibility of the stages of the target insect before the establishment of ionizing radiation quarantine treatments. This study was performed to determine the radiotolerance of eggs of the oriental fruit moth, Keywords: Grapholita molesta (Busck) (Lepidoptera: Tortricidae), to gamma radiation. This species is considered as Grapholita molesta one of the most serious worldwide pests for temperate fruits, especially peaches. Eggs (12 h old) were Irradiation exposed to 0 (control), 25, 35, 50, 75, 100, 125 and 150 Gy of gamma radiation. Surviving larvae were Moth allowed to feed on an artificial diet. Three days after irradiation, it was verified that larvae’s cephalic Eggs capsules were significantly affected by gamma radiation, and the estimated mean LD90 and LD99 were 66.3 Gy and 125.8 Gy, respectively. Oriental fruit moth eggs revealed to be quite radiosensitive and very low doses as 50 Gy were sufficient to disrupt G. molesta embryogenesis. At 25 Gy, only male adults originated from the surviving larvae and, after mating with untreated fertile females, shown to be sterile. & 2010 Elsevier Ltd. All rights reserved.

1. Introduction required to reach it, and any quarantine treatment must be virtually 100% effective. In international agricultural markets, the use of radiation as a As insects increase in radiotolerance as they develop (Bakri method for the prevention of quarantine insects represents an et al., 2005) and theoretically many developmental stages of the important alternative post-harvest pest control, reducing the pests may be present in the shipped commodity, many investiga- need for chemical fumigants and other similar toxic products. The tions have been conducted to know the radiation susceptibility of US Food and Drug Administration (FDA) has approved radiation the target insect life stages. The results from radiobiology studies up to 1 kGy to control insects in foods and to extend the shelf life can be useful for example to determine the effective radiation of fresh fruits and vegetables (Morrison, 1989; US FDA, 2004). dose against the most tolerant life stage of quarantine pests The advantages of radiation include the no resistance devel- (International Atomic Energy Agency (IAEA), 2009). opment by pest insects, the absence of residual radioactivity and As the effects of gamma radiation on the oriental fruit moth, few significant changes in the physicochemical properties or the Grapholita molesta (Busck) (Lepidoptera: Tortricidae) eggs remain nutritive value of the treated products (Lapidot et al., 1991; World largely unexplored, the current study was performed to investi- Health Organization (WHO), 1994; Kwon et al., 2004). A major gate the response of Grapholita molesta eggs to a range of doses of disadvantage is that it is the only commercially applied gamma radiation. The oriental fruit moth is considered to be one quarantine treatment that does not result in significant acute of the most important pests of peaches in several growing areas mortality (Hallman, 1998). (Rothschild and Vickers, 1991; Riga et al., 2006). It is also one of Hallman (2004) stated that the objective of irradiation is not the most serious worldwide pests for temperate fruits like stone acute mortality but prevention of development or reproduction, fruits (Prunus spp.), pomes (Malus, Pyrus and Cydonia) and some as most commodities do not tolerate the usual dose ranges species of Rosaceae (Pree, 1985). In Brazil, it causes losses to peach growers by three to five percent (Farias et al., 2003). Nonetheless, G. molesta is a quarantine pest for many export destinations (Hallman, 2004). n Corresponding author. Tel/fax: +55 19 3429 4199. Previous works have been done only for pupae and adults of E-mail address: [email protected] (W.D. Silva). the oriental fruit moth (Genchev, 2002; IAEA, 2009). Hallman

(2004) irradiated G. molesta ﬁfth instars and reported that no Larval development changes and egg hatch were recorded daily. fertile adults emerged after a dose of 200 Gy. Surviving larvae were allowed to feed on the artiﬁcial diet. Besides determining the minimum doses required to prevent Reproductive parameters of G. molesta adults originated from G. molesta eggs normal development, this study was also designed irradiated eggs were also evaluated. These adults were individu- to assess the effect of gamma radiation on the fertility of surviving ally paired with an opposite sex partner originated from non- insects under a sub-dose, since Lepidoptera may suffer great irradiated eggs in PVC cages. All adults were fed with a 10% honey sterility in subsequent generations (Bloem et al., 2003; Bakri et al., solution. Their reproductive parameters were compared to the 2005). control for seven replicates. The total number of eggs was recorded daily until the death of adult females and the viability of eggs was recorded daily until the end of hatching. 2. Materials and methods

2.1. Insects 2.3. Statistical analysis

The oriental fruit moths were obtained from a colony Probit regression analysis for the presence of cephalic capsule established from larvae collected from infested peach fruits and egg hatch (%) was performed and the doses that reduce both (Prunus persica L.) in Pelotas, southern Brazil. The larvae were variables in 90% and 99% (LD (Lethal Dose)90 and LD99, reared on an artificial diet modified from Ivaldi-Sender (1974). respectively) were also estimated (PROC PROBIT; SAS Institute, This diet consisted of agar, corn flour, wheat germ, brewer’s yeast, 2003). The analysis for the cephalic capsule variable used log10 of ascorbic acid, benzoic acid, formalin and water at a proportion of dose while for the egg hatch it was not necessary. To compare 4:28:7:7:1:0.1:0.1: 900 mL, respectively. Mature fifth instars were untreated adults with those from irradiated eggs regarding the allowed to pupate in boxes covered with cotton fabric. total number of eggs laid, fecundity (number of eggs/day/female) Pupae were transferred individually into glass tubes until the and fertility (egg hatch), the one-way analysis of variance F-test emergence of adults. Groups of fifteen pairs of virgin adults was calculated at the 5% level of significance and the Student’s were placed individually into cylindrical PVC cages, which were t-test (a¼0.05) was used to compare the means. The later covered with screen lids and lined with plastic film to provide an analyses were performed by the statistical program SAS 9.1. egg-laying substrate. Adults were fed with a 10% honey solution. (PROC GLM; SAS Institute, 2003). The food and film plastic containing eggs were replaced every 12 h. Both rearing and experiments were conducted in an acclimatized room at a temperature of 2571 1C, relative 3. Results humidity of 70710% and 16 h of photophase. The insects were reared for five generations before the tests. None of the dosimetry results differed at the 5% level from the target dose. The target dose values were, therefore, used 2.2. Bioassays with radiation throughout. Three days after irradiation, it was possible to visualize the The pieces of plastic film containing the eggs (12 h old) were formation of larvae’s cephalic capsules even at 125 Gy. The placed into plexiglass containers (10 8 6 cm) and exposed to a cephalic capsule formation was significantly affected by radiation 60 s Co source (Gamma Cell 220 irradiator , MDS Nordion Interna- (Table 1). The estimated mean LD90 and LD99 were 66.3 and tional Inc., Canada) with an activity of 34 TBq and a dose rate of 125.8 Gy, respectively. 0.51 kGy/h at the centerline. Radiation doses were 0 (control), 25, A significant reduction in egg hatch was observed that was 35, 50, 75, 100, 125 and 150 Gy. The doses were measured with a dependent on the radiation doses (Table 1). Eggs were unable to

Fricke Dosimeter (Fricke and Hart, 1966). For each treatment, hatch at very low doses as 50 Gy. The mean estimated LD90 and there were six replicates with 15 eggs. LD99 were 20.9 and 35 Gy, respectively. The 95% confidence After irradiation, the eggs were placed in Petri dishes with intervals for the percentage of eggs with visible cephalic capsule moistened filter paper. Three days after irradiation, the formation were wider. Wheeler et al. (2007) stated that the width of of larvae’s cephalic capsules inside the eggs was verified by a confidence intervals tends to increase as the number of tested micrometric ocular connected to a Wild M5 stereomicroscope. doses decrease or as the sample size per dose decreases.

Table 1 Response of Grapholita molesta eggs to gamma irradiation (mean7standard error).

Biological Doses (Gy) Prob.4ChiSq. df Slope7SE LD90(95% LD99(95% parameters CI) CI) 0 25 35 50 75 100 125 150

Percentage of 68.975.07a 58.976.1 46.773.85 16.775.64 8.972.2 2.271.41 1.170.1 0 0.06 5 3.7670.33 66.3 125.8 eggs with (59.9; (105.6; visible 75.3)b 159.9) cephalic capsule Egg hatch (%) 6078.43 5.5672.68 0.970.01 0 0 0 0 0 0.98 2 0.0747 0.008 20.9 35.0 (17.5; (29.8; 25.4) 42.9)

a Original means in this table. b Conﬁdence interval stated at 95% conﬁdence level. W.D. Silva et al. / Radiation Physics and Chemistry 79 (2010) 1063–1066 1065

Table 2 Comparisons between fertile adults (control) and fertile females mated with males from eggs irradiated at 25 Gy of Grapholita molesta (means7standard error).

Biological parameters Control Irradiated t-tests (LSD) C.V.(%)

a Total number of eggs 107.3718.1 a 78719.2 a F1,13 ¼1.5; P¼0.24 35.6

Fecundity 5.770.95 a 3.770.9 a F1,13 ¼2.8; P¼0.12 34.1 3 Fertility 60.372.93 a 0 b F1,13 ¼57.9; Po10 39.5

a Original means in this table. Means within lines followed by the same letter are not signiﬁcantly different, ANOVA at Pr0.05.

At 25 Gy, only male adults emerged from the larvae that resistant (Hallman, 2000; Bakri et al., 2005) and sex distortion survived and fed on the diet. Not a single female was able to appears to be a common phenomenon occurring in the progeny of complete the development. Those males mated with virgin irradiated male lepidopterans, probably because radiation induces females from non-irradiated eggs to observe the offspring sex-linked recessive lethal mutations in the Z chromosomes of (Table 2). Surviving adults from eggs irradiated at 35 Gy were females (Mittwoch, 1967; Marec and Mirchi, 1990). Tothova and not used. Marec (2001) discussed that Lepidopteran males are more able to The eggs were collected up to the 22nd day, which concurred survive with a higher number of chromosomal breaks than with the death of adult females. There was no signiﬁcant females. difference between the control and the males from the treated When the adult males obtained from the treated eggs at 25 Gy eggs at 25 Gy in regards to the total number of eggs laid mated with fertile females it resulted in the number of eggs laid

(F1,13¼1.5; P¼0.24) and fecundity (number of eggs/day/female) per female being unchanged. It has been reported that the most (F1,13¼2.8; P¼0.12). However, the males obtained from the eggs important factors for a normal fecundity are the successful mating irradiated at 25 Gy revealed to be sterile, since no egg hatched. and right stimulation of females, which depend on the transfer of The viability from control was about 60% and was significantly the spermatophores by males (Saour and Makee, 1997). Regard- different from the pairs with males from irradiated eggs less the amount of eggs laid by the females mated with treated 3 (F1,13¼57.9; Po10 ). males, the radiation-induced deleterious effects were verified by the F1 generation. No egg hatched and the offspring was completely unviable. Ayvaz et al. (2008) reported that the adults 4. Discussion of P. interpunctella from irradiated eggs at 200–300 Gy were not able to produce eggs. The current study explored the effects of exposing G. molesta A generic dose of 400 Gy was approved as a phytosanitary eggs to gamma radiation. Therefore, the evaluation of the treatment for all insects other than Lepidoptera for commodities acceptability of the radiation doses tested as a quarantine entering the United States (USDA-APHIS, 2006) and Hallman and treatment to disinfest peaches or other temperate fruits from G. Phillips (2008) suggested that a generic dose of 600 Gy for all molesta eggs was not the purpose of this study. insects in ambient atmospheres would be efficacious to attend The results showed that increasing radiation doses caused quarantine purposes. In practice, the goal of an irradiation reduced egg hatch and formation of cephalic capsule when 12 h quarantine treatment can be the induction of mortality of old eggs were irradiated. Although it was possible to visualize immature stages before the adult or inhibition of reproduction, larvae’s cephalic capsules inside the eggs up to 125 Gy, the in order to lower the risk of establishment of invasive pests G. molesta eggs showed to be quite radiosensitive and the (Ozyardimci et al., 2006). estimated mean dose to inhibit 99% hatching was about 35 Gy. Considering the lack of adult emergence as a criterion for The mode of action of the dominant lethal mutations induced effectiveness, relatively low doses of radiation were effective in by radiation differs considerably between Hemipteran, Dipteran inhibiting the normal development of G. molesta eggs. Damages to and Lepidopteran species (Bakri et al., 2005). In irradiated Diptera, fresh commodities could be significantly reduced at a dose range the dominant lethal is expressed very early in embryonic of 100–200 Gy when applied on a commercial scale. development and often provokes death during the first few However, as the eggs are not the most advanced stage of the cleavage divisions, but in Lepidoptera, the major lethality is insect and, therefore, the most radiotolerant, potentially found on expressed later when the larvae are virtually fully developed peaches and other fruit commodities (Hallman, 2004), doses (La Chance, 1974; La Chance and Graham, 1984). lower than 200 Gy should not be endorsed as a general quarantine Actually, in contrast to other insect orders, the Lepidopterans treatment against this pest. Furthermore, the percentage egg are a relatively radioresistant group (Bakri et al., 2005). Toba and hatch may be an inappropriate criterion for quarantine effects Burditt (1992) reported that 100 Gy was enough to completely when older Lepidoptera eggs are exposed to gamma radiation prevent adult emergence from irradiated codling moth Cydia (Burditt and Hungate, 1989; Saour and Makee, 2002). pomonella L. (Lepidoptera: Tortricidae) eggs. Mansour and The results of this work make part of the basic research in the Mohamad (2004) showed that one- and two-day old C. pomonella radiobiology of the oriental fruit moth. At last, the development of eggs failed to hatch following an 80 Gy dose. In Ephestia cautella any phytosanitary treatment must include a precise description of (Walker) (Lepidoptera: Pyralidae), the development of adults the response that achieves efficacy (Hallman and Phillips, 2008), from treated eggs and larvae can be prevented by 300 and 200 Gy, so regulatory agencies can know whether the threshold has been respectively (Cogburn et al., 1973; Ozyardimci et al., 2006). Doses crossed or not. of 200 Gy and above can prevent adult emergence from irradiated eggs of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) (Ayvaz and Tunc-bilek, 2006). The dose to inhibit 95% hatching of three- Acknowledgements day old Plodia interpunctella (Hubner)¨ (Lepidoptera: Pyralidae) eggs was found to be about 450 Gy (Aye et al., 2008). We thank to Dr. Jose´ Roberto Postali Parra, Department of A strong male-biased sex ratio was observed for the adults Entomology (ESALQ/USP), for providing us the facilities for the derived from treated eggs. Insect females are usually less radio- G. molesta rearing. We also thank Dr. Mauro Silveira Garcia, 1066 W.D. Silva et al. / Radiation Physics and Chemistry 79 (2010) 1063–1066

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