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Radiation Physics and Chemistry 96 (2014) 44 49

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Assessment of electron beam-induced abnormal development and DNA damage in Spodoptera litura (F.) (Lepidoptera: Noctuidae)
Seung-Hwan Yun, Seon-Woo Lee, Hyun-Na Koo, Gil-Hah Kim n
Department of Plant Medicine, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea



Electron beam irradiation induced abnormal development of the cutworm. Electron beam irradiation induced the sterility of the cutworm. Electron beam irradiation increased levels of DNA damage. DNA damage by high irradiation exposure was not completely repaired.

art ic l e i nf o
Article history: Received 14 February 2013 Accepted 17 August 2013 Available online 25 August 2013 Keywords: Electron beam Spodoptera litura Comet assay DNA damage Sterility

a b s t r a c t
The armyworm, Spodoptera litura (F.) is a polyphagous and important agricultural pest worldwide. In this study, we examined the effect of electron beam irradiation on developmental stages, reproduction, and DNA damage of S. litura. Eggs (024 h old), larvae (3rd instar), pupae (3 days old after pupation), and adults (24 h after emergence) were irradiated with electron beam irradiation of six levels between 30 and 250 Gy. When eggs were irradiated with 100 Gy, egg hatching was completely inhibited. When the larvae were irradiated, the larval period was signicantly delayed, depending on the doses applied. At 150 Gy, the fecundity of adults that developed from irradiated pupae was entirely inhibited. However, electron beam irradiation did not induce the instantaneous death of S. litura adults. Reciprocal crosses between irradiated and unirradiated moths demonstrated that females were more radiosensitive than males. We also conducted the comet assay immediately after irradiation and over the following 5 days period. Severe DNA fragmentation in S. litura cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. However, at more than 100 Gy, DNA damage was not fully recovered. & 2013 Elsevier Ltd. All rights reserved.

1. Introduction Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), also referred to as an armyworm or cutworm, is an economically important and regular polyphagous pest, which seriously harms plants in the families of Cruciferae, Solanaceae, Cucurbitaceae, and Leguminosae. High resistance against a wide variety of insecticides has been reported from South Asia, and as a consequence, the management of this pest has become increasingly difcult. As international agricultural trade is increasing, the risk of introducing exotic insects into new areas where they may become plant pests is also on the rise. Therefore, importing countries only permit the entry of pest-free and secured commodities. In order to satisfy quarantine requirements, the following methods of disinfestation are used: fumigation with chemicals such as methyl

Corresponding author. Tel.: 82 43 261 2555; fax: 82 43 271 4414 E-mail address: (G.-H. Kim).

bromide (MB), application of extreme temperatures (heat or cold), controlled atmospheres (level of CO2 or O2), irradiation, and combinations of these (Heather and Hallman, 2008). However, MB treatment is under regulation as a stratospheric ozone-depleting substance under the Montreal Protocol of substances that deplete the ozone layer (UNEP, 2009). Research is currently underway to develop alternatives to MB. Irradiation is the ideal technology for developing generic treatments because it is effective against most insects at dose levels that do not affect the quality of most commodities. The electron-beams are generated by electrical energy, which is thought to be safe and effective for rapid surface treatment, with no consideration of remaining radioactivity. Moreover, it is an environmental friendly control technique, and does not produce harmful materials, being currently used widely throughout various areas, including medical, food, agricultural products, semiconductor (Taniguchi, 2005; Park et al., 2006; Moon et al., 2010). In our previous research (Koo et al., 2011; 2012), electron beam

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irradiation induced abnormal development, sterility, and DNA damage of Plutella xylostella and Liriomyza trifolii. Therefore, electron beam irradiation-induced changes in DNA could be used as the basis for detecting irradiation treatment in insects (Imamura et al., 2004). The microgel electrophoresis assay of single cells (DNA comet assay) is a sensitive technique for detecting DNA damage (Todoriki et al., 2006; Hasan et al., 2008). The comet assay has also been used to observe DNA repair in irradiated cells (Trzeciak et al., 2008; Kameya et al., 2012). The comet assay is quick, simple, sensitive, reliable, and inexpensive method for measuring DNA damage. The objectives of the study are to examine the effects of electron beam irradiation on egg, larval, and pupal development, and on adult reproduction in S. litura, in order to identify a potential quarantine treatment dose. Also, the present study was designed to evaluate the levels of DNA damage and repair using the alkaline comet assay on S. litura adult after exposure to electron beam irradiation.

emergence, 20 unmated ies ( r 2 days old) were irradiated with 30200 Gy or left 20 untreated. Irradiated or untreated males and females in all combinations were then mated as pairs in individual Petri dishes. The number of eggs laid and the number of eggs hatched were recorded until all ies died. This test was done in three replicates. 2.4. DNA comet assays DNA damage in S. litura adults was determined under alkaline conditions using the Comet Assay Kit from Trevigen (Gaithersburg, MD) with slight modications. Another population of irradiated insects was sampled immediately after irradiation, and insects that would undergo a time course experiment of 1, 3, and 5 days after irradiation were instantaneously frozen with liquid nitrogen and subjected to the comet assay. Briey, after irradiation, adults were frozen in liquid nitrogen and then gently homogenized into a powder. The powder was suspended in cold phosphate-buffered saline (PBS) and ltered through a 125 m nylon mesh. The cells were combined with molten agarose and pipetted onto a comet slide. After solidication, the slides were immersed in lysis solution for 50 min at 4 1C. For alkaline single-cell electrophoresis which detects the combination of single- and double-strand DNA breaks, DNA cross-links, and base damage, the slides were placed in alkaline buffer and electrophoresed at low voltage for 30 min at 4 1C. Air-dried slides were stained with SYBR Green I and analyzed using a confocal laser scanning microscope (TCS SP2 AOBS; Leica). The cells with damaged DNA displayed migration of DNA fragments from the nucleus by forming a comet-like shape. The images were analyzed using CASP software (Comet Assay Software Project 1.2.2). At least 100 comets were analyzed for each sample. Comet assays were performed three times, each time in duplicate. 2.5. Data analysis Data were compared by one-way analysis of variance (ANOVA) followed by Tukeys studentized range test (SAS Institute, 2003) when signicant differences were found at P o 0.05. ED (effective dose) values were estimated by probit analysis. The regression analysis was carried out and the regression equation was obtained by iteration (standard probit analysis technique, Finney, 1964) by SPSS (1999).

2. Materials and methods 2.1. Test insects The cutworm, S. litura, larvae were collected from cucumber leaves in Gurye area (Republic of Korea) in 2010. The larvae were reared in the laboratory in plastic cages (25 19 8 cm3) with articial diet at 25 7 2 1C, 5060% RH and a 16 L:8D h photoperiod 2.2. Electron beam treatment Electron beam irradiation was conducted in the EB-Tech Co., Ltd. (Daejeon, Republic of Korea) using a high energy linear accelerator (UEL V10-10S, 10 MeV). The different developmental stages viz. eggs, larvae, pupae and adults of S. litura were treated with doses of 0 (control), 30, 50, 100, 150, 200, and 250 Gy. The doses used were based on our previous study (Koo et al., 2012). Target doses were monitored by dosimetry with a radiochromic lm dosimeter (GEX GAF3002DS, USA) (ISO/ASTM51275:2004(E)). Thirty females and 60 males S. litura adults were placed in a parchment paper envelope (27 50 cm) that contain cotton with sugar solution (10%). They were allowed to lay eggs for 24 h and then removed. Parchment paper piece with attached eggs (024 h old) was exposed to the electron beam. One hundred S. litura larvae (3rd instar) and pupae (3 days old after pupation) were placed in a plastic Petri dish with a net cover (10 cm diameter 4 cm height) and exposed to the electron beam. Sixty adults (20 females and 40 males, 024 h old after emergence) were placed in a glass vials and exposed to the electron beam. To investigate lethal effects on various developmental stages, we observed the rate of egg hatch inhibition and rate of emergence inhibition. The numbers of adults (24 h after emergence) surviving after 2 days were also counted. In electron beam-irradiated eggs, the emergence rate of larvae and the longevity and fecundity of emerged adults were recorded. In electron beam-irradiated larvae, emergence rate, longevity and fecundity of emerged adults and hatchability of the eggs were recorded. In electron beamirradiated adults (24 h after emergence), longevity and fecundity and hatchability of the eggs were recorded. Data on untreated controls were also recorded. Trials on the different stages of S. litura were done with three replicates. 2.3. Reciprocal crossing Adult sterility tests were conducted using a reciprocal cross design. Pupae were isolated in individual plastic Petri dishes. After

3. Results 3.1. Effects of electron beam irradiation on developmental stage At all stages, the rate of failure to develop increased with increasing doses of electron beam radiation from 30 to 250 Gy. Hatchability, pupation, emergence, longevity, and fecundity from irradiated eggs are shown in Table 1. When 1-day-old eggs were irradiated at more than 100 Gy, hatchability was completely inhibited. When third instar larvae were irradiated at 100 Gy and higher doses, pupation was signicantly affected (Table 2) (2 42.2; df 4; P o 0.05). The number of adults emerging from larvae was signicantly reduced as the dose was increased (2 4.5; df 2; P o 0.05). At 150 Gy and above, no adult emergence was observed. At 100 Gy, F1 eggs did not hatch. Electron beam irradiation of larvae, for all doses, had no effect on adult longevity. Developmental period (larvae to pupae) of S. litura electron beam irradiated larvae is also given in Table 2. There was a dosedependent delay in the developmental period. When third instar larvae were irradiated at 200 Gy, the developmental period was about 8 days longer than that of the untreated group. The results for electron beam-irradiated pupae are shown in Table 3. The mean number of adults that emerged from pupae irradiated with 30 and 50 Gy was not signicantly different from that of the untreated control, but doses of


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Table 1 Hatchability, pupation, emergence, adult longevity, and fecundity of electron beam-irradiated S. litura eggs (024 h old) a. Dose (Gy) 0 30 50 100 150 200 250

n 1288 832 1091 764 907 1078 869

Hatchability (%) 88.4 7 9.3a 87.2 7 4.7a 86.0 7 6.1a 0.0 7 0.0a 0.0 7 0.0b 0.0 7 0.0b 0.0 7 0.0b

Pupation (%) 91.6 7 6.7a 88.0 7 8.2a 85.7 7 9.5a

Adult emergence (%) 90.4 7 2.3a 88.4 7 4.4a 87.9 7 2.8a

Longevity (day) 9.1 7 3.1a 9.3 7 2.0a 8.7 7 2.8a

No. eggs (/day) 1134.4 7 210.8a 1053.9 7 232.0a 1064.6 7 185.7a

Hatchability (%) (F1) 88.5 7 4.1a 87.5 7 8.0a 86.4 7 5.1a

Means within each column followed by the same letter are not signicantly different at P o 0.05 by Tukey's studentized range test (SAS Institute, 2003).

Table 2 Pupation, larva period, emergence, adult longevity, fecundity, and hatchability of electron beam-irradiated S. litura larvae (3rd instar)a. Dose (Gy) 0 30 50 100 150 200 250 ED50 (95% FL) ED95 (95% FL)

n 150 150 150 150 150 150 150 121.5 Gy (84.2174.6) 238.3 Gy (139.6409.8)

Pupation (%) 90.0 7 2.0a 86.1 7 6.1a 85.1 7 6.2a 56.3 7 4.4b 32.7 7 8.5c 8.4 7 5.8d 2.2 7 1.9d 84.1 Gy (77.389.1) 165.4 Gy (146.3197.2)

Larva period (day) 14.6 7 1.7a 15.2 7 1.4a 15.3 7 1.7a 17.5 7 2.2b 22.0 7 1.1c 22.3 7 2.4c

Adult emergence (%) 90.5 7 6.1a 89.5 7 2.7a 87.6 7 3.9a 48.2 7 4.9b 0.0 7 0.0c 0.0 7 0.0c

Longevity (day) 9.1 7 2.9a 9.2 7 2.1a 8.9 7 1.6a 8.4 7 3.1a

No. eggs (/day) 1102.6 7 326.9a 1087.7 7 223.1a 1028.8 7 297.7a 941.0 7 181.5a

Hatchability (%) (F1) 87.2 7 7.1a 85.6 7 6.5a 84.5 7 6.0a 0.0 7 0.0b

Means within each column followed by the same letter are not signicantly different at P o 0.05 by Tukey's studentized range test (SAS Institute, 2003).

Table 3 Emergence, longevity, fecundity and hatchability of electron beam-irradiated S. litura pupae (3-days-old)a. Dose (Gy) 0 30 50 100 150 200 250 ED50 (95% FL) ED95 (95% FL)

n 150 150 150 150 150 150 150

Adult emergence (%) 89.8 7 8.9a 82.4 7 9.9a 77.8 7 9.6a 50.5 7 12.2b 34.2 7 12.2bc 30.3 7 11.3c 22.2 7 9.0c 116.2 Gy (102.6-130.7) 548.6 Gy (422.8-797.6)

Longevity (day) 9.4 7 2.2a 9.1 7 1.5a 9.2 7 3.1a 8.8 7 2.2a 6.8 7 1.7b 6.5 7 2.1b 5.8 7 1.8b

No. eggs (/total) 955.7 7 274.9a 973.4 7 242.8a 938.0 7 200.1a 878.4 7 280.0a 60.2 7 87.7b 0.0 7 0.0b 0.0 7 0.0b

Hatchability (%) (F1) 91.5 7 7.3a 89.3 7 6.1a 87.4 7 4.5a 2.9 7 4.3b 0.0 7 0.0b 62.0 Gy (57.766.1) 124.1 Gy (111.6143.1)

Means within each column followed by the same letter are not signicantly different at P o 0.05 by Tukey's studentized range test (SAS Institute, 2003).

100 Gy and above decreased adult emergence (2 2.0; df 4; P o 0.05). When pupae were irradiated with 200 Gy, emergence rate was 30.3%, however, adults from pupae irradiated with same dose were not able to produce eggs. Interestingly, F1 eggs did not hatch at 150 Gy (2 5.5; df 3; P o 0.05). No effect on adult longevity was detected at any doses. When adults were irradiated, fecundity was not signicantly affected by electron beam radiation doses administered to the adults, even at 250 Gy (Table 4). Adults irradiated with 200 Gy laid eggs (928.6/total), however, F1 eggs did not hatch. In addition, no effect on adult longevity was detected at any dose. 3.2. Effects of electron beam irradiation on reciprocal crosses To assess the sterility of S. litura female and male adults irradiated at six dose levels (30250 Gy), reciprocal crosses were performed. When irradiated females (IF) were mated with untreated males (UM), the mean number of eggs laid per female was similar to the untreated control (Table 5). However, the mean percentage of eggs hatching was dose-dependently inhibited compared with the control. The rate of egg hatching decreased by 3.7% at 150 Gy, and was completely inhibited at 200 Gy (2 0.4; df 1; P o 0.05). When untreated females (UF) were mated with males (IM) irradiated at 250 Gy, the mean number of

Table 4 Longevity, fecundity and hatchability of electron beam-irradiated S. litura adults (24 h old after emergence)a. Dose (Gy) 0 30 50 100 150 200 250 n 90 90 90 90 90 90 90 Longevity (day) 9.1 7 2.0a 8.9 7 1.5a 9.1 7 1.8a 8.5 7 3.1a 8.3 7 2.1a 8.0 7 1.5a 8.2 7 1.8a No. eggs (/total) 1035.3 7 267.2a 1025.7 7 304.8a 1012.0 7 327.0a 985.9 7 242.8a 931.6 7 214.5a 928.6 7 295.1a 911.6 7 252.6a Hatchability (%) (F1) 90.4 7 8.2a 88.5 7 8.1a 86.8 7 7.5a 2.1 7 2.6b 0.1 7 0.2b 0.0 7 0.0b 0.0 7 0.0b

a Means within each column followed by the same letter are not signicantly different at P o 0.05 by Tukey's studentized range test (SAS Institute, 2003).

eggs laid was 921.5 7 189.2 (total number and was similar to the untreated controls 1083.0 7 335.6). The proportions of egg hatching of F1 were 28.5%, 12.9%, 11.2%, and 3.7% at 100, 150, 200, and 250 Gy, respectively. However, hatching was not fully inhibited, even at 250 Gy (2 2.1; df 2; P o 0.05). The longevity of adults from reciprocal crosses showed no signicant differences. This result indicates that egg hatchability of the IF UM group was lower than that of the UF IM group. Therefore, females of S. litura

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adult were determined to be more sensitive to electron beam irradiation than males. 3.3. Effects of electron beam irradiation on DNA damage In this study, we have conducted a comet assay under alkaline conditions to observe DNA damage and repair. Electron beam
Table 5 Reproductive performance of S. litura when irradiated or unirradiated adults are mated in reciprocal crossesa. Pairing Dose (Gy) 0 100 150 200 250 n Longevity (day) 8.5 7 2.8a 8.7 7 2.8a 8.2 7 2.3a 9.3 7 3.1a 8.2 7 3.2a No. eggs (/total) 1083.0 7 174.9a 933.9 7 358.3a 1189.6 7 253.8a 832.8 7 191.2a 853.0 7 354.3a Hatchability (%) (F1) 90.4 7 8.2a 15.1 7 11.8c 3.7 7 1.8cd 0.0 7 0.0d 0.0 7 0.0d 53.6 Gy (18.973.7) 146.9 Gy (127.1197.4) 28.5 7 11.0b 11.2 7 3.7cd 12.9 7 6.3cd 8.6 7 7.6cd 53.9 Gy (17.978.9) 325.3 Gy (242.9 734.2)


90 90 90 90 90

ED50 (95% FL) ED95 (95% FL) UF IMe 100 150 200 250 90 90 90 90 7.9 7 2.5a 8.7 7 2.4a 8.1 7 1.8a 8.3 7 1.8a 825.2 7 335.6a 796.4 7 323.9a 914.4 7 456.0a 921.5 7 189.2a

irradiation induces DNA breakage, increasing the migration of short-chain fragments, and a tail of comet-like shape appears. Fig. 1 shows the comet images for cells obtained from S. litura adults. After electron beam irradiation, the comet tails were observed and were dose-dependently lengthened. In addition, comets with short or medium tails were also observed occasionally, indicating that DNA damage occurred irregularly in cells of electron beam-irradiated S. litura adults. Quantitation of these observations is depicted in the graphs (Fig. 2). The tail moment of the comet assay increased, indicating that DNA damage increased as the doses increased. Next, to observe the time-series repair of DNA damage induced by electron beam irradiation, the comet assay was conducted using S. litura adults. The cells from S. litura adults were harvested 1 h, 1, 3 and 5 days after electron beam irradiation and analyzed under alkaline conditions using the Comet Assay Kit. Frequency histograms of ratio of individual cells were prepared as shown in Fig. 3. The frequency of ratio becomes closer to 1 with increased radiation dose after 1 h of electron beam irradiation implying increased DNA damage. Generally, the frequency of ratio changed from 1 to 0 over days 1, 3 and 5 after electron beam irradiation.

ED50 (95% FL) ED95 (95% FL)

a Means within each column followed by the same letter are not signicantly different at P o 0.05 by Tukey's studentized range test (SAS Institute, 2003). b UF, untreated female. c UM, untreated male. d IF, irradiated female. e IM, irradiated male.

Fig. 2. Graphic depiction of the calculated tail length from analysis of alkaline comet assays. Data are shown for a representative experiment, where at least 100 comets were quantied for each sample.








Fig. 1. Representative images of comets from S. litura adults treated with electron beam irradiation at different doses. The cells were harvested 5 h after electron beam irradiation and analyzed under alkaline conditions using the Comet Assay Kit (magnication 100 ).


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Fig. 3. Frequency histograms for the parameter ratio at different time points before and after electron beam irradiation with various doses. The cells from S. litura adults were harvested 1 h, 1, 3 and 5 days after electron beam irradiation (0, 30, 50, 100, 200, and 250 Gy) and analyzed under alkaline conditions using the Comet Assay Kit. Ratio, a percentage determined by uorescence in the damaged area to overall luminance, including intact DNA (head) and the damaged area (tail) of a comet image. X-axis, ratio and y-axis, frequency (%).

Five days after electron beam irradiation, the frequency of ratio of the irradiation doses at 30 and 50 Gy become almost the same as the control. However, the frequency of ratio of irradiation dose at 100 Gy and above did not approach 0. This result suggested that DNA repair was not complete for a radiation doses of 100 Gy and above.

4. Discussion When S. litura eggs were irradiated with an electron beam, hatchability of the eggs was completely inhibited at doses of 100 Gy and above. However, electron beam irradiated eggs develop normally at low doses (30 and 50 Gy). Also, when S. litura larvae were irradiated, their emergence was fully controlled from 150 Gy. The number of eggs laid by adult stages irradiated with 100 Gy when a larva was similar to that of the control, but the F1 generation eggs were not able to hatch. In previous study, Dohino et al. (1996) investigated the effect of electron beam (2.5 MeV) irradiation on eggs and larvae of S. litura. The third instars were more radiosensitive than fth instars. Also, the fth instar feeding could not prevent even at 400 Gy. However, the electron beam used in this study was high energy linear accelerators (10 MeV). Due to differences in accelerating voltage of electron beams, it was difcult to make a direct comparison between previous reports and our data. Some S. litura pupae underwent emergence and spawning but their eggs did not hatch. Irradiation at 200 Gy and above perfectly impedes F1 generation hatching, but does not result in the immediate death of adults. As this shows, electron beam irradiation results in controlled hatching and the sterility of adults, rather than exerting an instantaneous lethal effect. Even though a pest is present in importing or exporting products, they are not capable of reproduction. Thus, it can be used as a quarantine technique. To ascertain the reason why the electron beam results in sterility, DNA damage and repair capability were examined. As we previously reported, electron beam irradiation also induces DNA damage in P. xylostella and L. trifolii (Koo et al., 2011; 2012). Todoriki et al. (2006) also reported that electron beam treated chestnut larvae showed typical DNA fragmentation. Results showed that DNA damage increases with increasing irradiation, and also, repair over time is

observed, though there is no recovery to its original condition. On 5th day after irradiation, DNA damage would not be completely repaired at doses of 100 Gy and above. However, at 30 and 50 Gy, it did not differ between the irradiated and the control groups. This nding suggested that partial DNA damage by low doses irradiation can be repaired by multiple DNA repair system. Therefore, low doses irradiation of P1 was not greatly affected hatchability of F1 generations. Female and male adults were irradiated with the electron beam after which they were bred with each other. S. litura adult females were determined to be more radiosensitive than male adults, which is generally the case among insect species (Bloem et al., 1999; Hallman, 2000). The study showed that female adults are more sensitive to the electron beam which implies that it causes fatal damage to the Z chromosome (Ayvaz and Tunbilek, 2006), though further studies of this matter are needed in order to conrm the modes of action of irradiation treatments. In conclusion, electron beam irradiation is recommendable as pest control techniques for phytosanitary treatments. However, the eggs, larvae, pupae and adults of S. litura developed normally by low-dose irradiation (below 100 Gy). Therefore, optimum-dose of electron beam irradiation that can inhibit all developmental stage should be treated.

Acknowledgments This study was supported by a Grant (Project no. 312045-03-1HD080) from Korea Institute of Planning and Evaluation for Technology of Food, Agriculture, Forestry and Fisheries (iPET). References
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