Confirmatory test of gamma irradiation against the larvae and pupae of Helicoverpa assulta (Lepidoptera: Noctuidae) in paprika

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  • ABSTRACT

    The oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), is classified as a quarantine pest that must be controlled for the exportation of Korean paprika to the USA. We performed a confirmatory test of gamma irradiation against the last-instar larvae and pupae of H. assulta in paprika. Previous, small-scale gamma irradiation at a dose of 100 Gy to the last-instar larvae allowed emergence of adults, though they were abnormal, but 200 Gy prevented adult emergence completely. For pupae, irradiation of 5- to 6-day-old pupae with 300 Gy prevented normal emergence completely. To gather confirmatory data applicable to phytosanitary quarantine regulations, larvae and pupae were placed inside paprika in a box and were irradiated with 200 Gy and 300 Gy, respectively. After irradiation with 200 Gy (measured doses 170–199 Gy) of 2,186 individuals of the last-instar larvae, 10.84% survived, but either formed abnormal pupae (7.57%) or died during the pupal stage (3.27%), resulting in no emergence of normal adults. For pupae, the dose of 300 Gy (measured doses 276–319 Gy) given to 1,200 pupae allowed 10.75% to survive and 9.17% to emerge with deformity. However, 1.58% of irradiated pupae emerged normally, requiring an increased dose for complete prevention of normal emergence. Subsequently, an increased dose of 400 Gy (measured doses 340–402 Gy) to 1,005 pupae allowed 88.35% to emerge, but all emerged with deformity. Thus, irradiation treatment with a minimum dose of 400 Gy will provide quarantine security for all premature H. assulta in exported paprika.


  • KEYWORD

    oriental tobacco budworm , Helicoverpa assulta , paprika , gamma radiation , quarantine , phytosanitary treatment

  • Introduction

    The oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), occurs in many areas of the world, including South Africa through the Middle East and central and southeast Asia to Australia. It does not occur in the USA. It feeds inside plants such as pepper, tobacco, corn, eggplant, and paprika (Hill, 1983), and is currently classified as a quarantine pest that must be controlled before Korean paprika (Capsicum annuum var. angulosu) may be exported to the USA (http://www.qia.go.kr/plant/pest/plant_insec_rule.jsp). We previously showed gamma irradiation to be an effective agent for quarantine purposes in place of methyl bromide, long considered the most effective quarantine treatment (Park et al., 2015). Specifically, in a small-scale experiment, we found that irradiation of last-instar larvae at a dose of 100 Gy caused 55.92% larval or pupal death. Some emergence of abnormal pupae or adults occurred (44.07% of last-instar larvae irradiated), but no adults developed normally. When irradiated at 200 Gy, 10.54% of the larvae survived, but they either formed abnormal pupae (7.51%) or died during the pupal stage (3.03%), resulting in complete prevention of adult emergence. Gamma irradiation of 5- to 6-day-old pupae with 200 Gy resulted in 1.52% of adults emerging normally, although no F1 eggs were produced. When pupae were irradiated at higher doses (300 to 500 Gy), there was a dose-dependent decrease in adult deformity and a dose-dependent increase in pupal mortality, with complete prevention of normal emergence after irradiation doses 300 Gy and above.

    According to the import-export phytosanitary regulation for quarantine in South Korea, a confirmatory test must be conducted with more than 1,000 individuals at the stage most resistant to phytosanitation (QIA, 2014). Among premature stages of H. assulta, the pupal stage is most resistant to gamma radiation; the adult stage is excluded as a target for quarantine purposes because physical removal for phytosanitation is feasible (Park et al., 2015). To validate the previous findings according to the phytosanitary regulation, this study administered gamma radiation (at doses of 300 and 400 Gy) to more than 1,000 pupae placed inside paprika. Although pupae are the most resistant of the stages that can occur inside paprika, the larval stage is, in fact, more likely to occur during exportation due to their ability to move and feed inside paprika. Therefore, we additionally performed the confirmatory test with larvae at the dose of 200 Gy.

    Materials and Methods

      >  Helicoverpa assulta culture

    The oriental tobacco budworms used in these experiments were reared at Chonnam National University using a general-purpose, artificial diet for Lepidoptera (Bio-Serv, Frenchtown, NJ, USA). Details of the rearing condition and method were previously reported (Park et al., 2015). Tobacco plants were grown in acrylic rectangular pots (6 × 6 × 10 cm) for 50 d after the appearance of seedlings, and were made available to the budworms to stimulate oviposition.

      >  Gamma irradiation

    As a confirmatory experiment, with replication, two groups (986 and 1,200 individuals) of final-instar larvae, 17–20 d of age and approximately 4 cm in length, were irradiated with 200 Gy based on the results of the previous study. Additionally, two groups of 5- to 6-day-old pupae, 600 individuals in each, were irradiated with 300 Gy. At the 300-Gy dose, emergence of normal adults occurred. Thus, two additional pupae groups (500 and 505 individuals) were irradiated with an increased dose of 400 Gy. A cobalt-60 gamma irradiator at the Korea Atomic Energy Research Institute, Jeongeup, Korea (150 TBq capacity; ACEL, MDS, Nordion, Canada), was used for irradiation.

    Paprika were halved using a sterilized knife, and five to six larvae or pupae were placed inside. Halves were then reassembled and secured with a rubber band to prevent escape. Boxes actually used for export (45 cm × 29.5 cm × 17.7 cm) were then filled with approximately 20 paprika (Fig. 1). After irradiation, to prevent damage from occasional paprika decay, the larvae and pupae were removed from the paprika and returned to their normal, artificial diet (Park et al., 2015). The effects of the radiation treatment were assessed by scoring for larval mortality, pupal mortality, abnormal/normal pupation, and abnormal/ normal emergence. Larvae and pupae were scored as dead if they made no detectable movement after being touched with a brush.

      >  Dose mapping

    To determine the absorbed doses of gamma radiation within the paprika box, dose mapping was carried out using 13-15 alanine dosimeters with a diameter of 5 mm (Bruker Instruments, Rheinstetten, Germany) and a Bruker EMS 104 EPR analyzer (Bruker Instruments). The dosimeters were attached in a randomly selected box (Fig. 2), and the free-radical signals were measured.

    Dose mapping results indicated that with irradiation of 200 Gy, the absorbed dose ranged from 170 to 199 Gy. The lowest and highest absorbed doses were found inside the paprika (no. 13) and in the upper region of the box (no. 4), respectively. The mean of the 13 alanine dosimeters was 188 Gy, and the dose uniformity ratio (represented by maximum per minimum) was 1.17. When the box was irradiated with 300 Gy, the absorbed dose ranged from 276 to 319 Gy. The lowest and the highest absorbed doses were found at the bottom of the box (no. 11) and the upper side of the box (nos. 1 and 2), respectively. The mean of the 15 alanine dosimeters was 297 Gy and the dose uniformity ratio (represented by maximum per minimum) was 1.16. When 400 Gy was used, the absorbed dose ranged from 340 to 402 Gy, with the lowest and highest absorbed doses found inside the paprika (no. 15) and at the right side of the box (no. 7), respectively. At this dose, the mean of the 15 alanine dosimeters was 376 Gy and the dose uniformity ratio (represented by maximum per minimum) was 1.14. Thus, overall, the absorbed doses inside the box are within the reasonable range, although local differences exist, as can be found in other studies (Follett and Lower, 2000; Zhan et al., 2014).

    Results and Discussion

    After irradiation with 200 Gy, 10.84% of larvae survived, but they either formed abnormal pupae (7.57%) or died during the pupal stage (3.27%) (Table 1). Consequently, no normal adults emerged from larvae that received 200 Gy. The previous small-scale experiment had shown that for last-instar larvae, a radiation dose of 100 Gy caused larval or pupal death or emergence of abnormal adults; no normal adults developed (Park et al., 2015).

    For pupae, a total of 1,200 individuals were irradiated with 300 Gy. On average, this led to 89.25% death and 9.17% emergence of abnormal adults. However, in 1.58%, adults emerged normally (Table 1). This result is in sharp contrast to the previous small-scale experiment in which complete prevention of adult emergence occurred at doses of 300 Gy or above (Park et al., 2015). Although the exact reason for the difference was not scrutinized, it was probably caused by the differing irradiation environment between the two experiments. The previous experiment was performed by placing individual pupae in an insect culture cage (diameter 4.5 cm, height 4 cm) for irradiation, whereas the current irradiation was performed by placing pupae inside paprika in export boxes (45 cm × 29.5 cm × 17.7 cm), resulting in slightly less irradiation inside paprika (279~297 Gy; Fig. 2). Alternatively, a lowered oxygen condition inside paprika may have caused emergence of normal adults. It has been shown that the oxygen concentration is lowered inside fruits (Hallman et al., 1994), and such a lowered condition increases resistance to irradiation (Earle et al., 1979; Alpen, 1998; Mansour, 2003).

    In the subsequent experiment, to determine the dosage for complete prevention of normal adult emergence, a total of 1,005 larvae were irradiated with 400 Gy. On average, this dosage led to 88.35% death, 11.65% abnormal emergence, and no normal emergence (Table 1). In comparison with the previous small-scale experiment, both experiments achieved complete prevention of normal emergence with 400-Gy irradiation. However, the ratio of adult deformity increased in the current, larger-scale experiment as compared to the previous small-scale experiment (11.65% vs. 4.55%). By contrast, the ratio of pupal mortality decreased in the current study (88.35% vs. 95.45%) (Park et al., 2015), indicating a lowered ratio of pupal mortality in the current confirmatory test. Regardless, it is obvious that irradiation treatment with a minimum dose of 400 Gy will provide quarantine security against H. assulta larvae and pupae in exported paprika.

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  • [Fig. 1.] Preparation of confirmatory test. (A) Paprika were inoculated with five to six H. assulta larvae. (B) After inoculation, rubber bands fixed paprika in a closed position to prevent escape. Pupae were infected using the same method.
    Preparation of confirmatory test. (A) Paprika were inoculated with five to six H. assulta larvae. (B) After inoculation, rubber bands fixed paprika in a closed position to prevent escape. Pupae were infected using the same method.
  • [Fig. 2.] Dose mapping. Sites where alanine dosimeters were attached within the box are indicated, and the doses of free-radical signals are shown on the right side of figure.
    Dose mapping. Sites where alanine dosimeters were attached within the box are indicated, and the doses of free-radical signals are shown on the right side of figure.
  • [Table 1.] Results of confirmatory test for irradiated paprika infected by last-instar larvae and pupae of Helicoverpa assulta
    Results of confirmatory test for irradiated paprika infected by last-instar larvae and pupae of Helicoverpa assulta