TY - JOUR
AU - Kim,, Gil-Hah
AB - Abstract The Sakhalin pine longicorn, Monochamus saltuarius (Gebler; Coleoptera: Cerambycidae), is an insect vector of the pine wilt nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, and is widely distributed in central Korea. M. saltuarius is a forest pest that seriously damages Pinus densiflora (Siebold et Zucc, Pinales: Pinaceae) and Pinus koraiensis (Siebold & Zucc, Pinales: Pinaceae) forests. We examined the effect of electron beam irradiation on the mating, DNA damage and ovarian development of M. saltuarius adults and sought to identify the optimal dose for sterilizing insects. When the adults were irradiated with electron beams, both females and males were completely sterile at 200 Gy. In a reciprocal crossing experiment between unirradiated and irradiated adults, the reproductive ability of wild adults was recovered by crossing with wild adults even after crossing previously with sterile adults. When a pair of unirradiated adults (♀− × ♂−) and 10 or 20 irradiated adults (♀+ or ♂+) were kept together, the control effect was as high as 80~90%. After electron beam irradiation at 200 Gy, the DNA of M. saltuarius adults was damaged, the ovarian development of female adults was inhibited, and the level of vitellogenin was significantly decreased compared with that in unirradiated female adults. These results suggest that pine wilt disease can be effectively controlled if a large number of sterilized M. saltuarius male adults are released into the field. electron beam, Monochamus saltuarius, sterile insect technique, DNA damage, ovarian development The pine wilt nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, was first discovered in 1988 in Busan, Republic of Korea. It was later discovered in the Pinus koraiensis forests of Gwangju, Gyeonggi-do, in December 2006. In 2007 and 2008, PWN was found in Wonju, Chuncheon, Namyangju, Pocheon, Sangju, Sancheong, and Seoul, Republic of Korea. This pest has resulted in damage to 7,800 hectares of Pinus densiflora Siebold et Zucc (Pinales: Pinaceae) and Pinus koraiensis Siebold & Zucc (Pinales: Pinaceae) forests (KFRI 2010). Pine wilt disease is distributed not only in North America and East Asia, including Japan, China, Taiwan and Korea, but also in Portugal and Spain (KFRI 2013). Monochamus saltuarius (Gebler; Coleoptera: Cerambycidae), a Sakhalin pine longicorn beetle, is widely distributed in central Korea. M. saltuarius and Monochamus alternatus Hope (Coleoptera: Cerambycidae), also referred to as Japanese pine sawyers, are the main insect vectors of PWN (Kobayashi et al. 2003). PWN is propagated and moved by insect vectors, as it does not have the ability to move to other trees by itself (KFRI 2013). This species is transferred to healthy trees through the wounds caused by feeding and spawning (Evans et al. 1996). Infected trees show wilting symptoms resulting from the blockage of the passage of water and nutrients. In severe cases, death can occur (KFRI 2010). Methods to control PWN have been developed, such as the aerial spraying of pesticides against longhorn beetles, the felling and crushing of diseased P. densiflora, trunk injection on diseased P. densiflora, and fumigation (KFRI 2007). In many countries, ionizing radiation (gamma ray, electron beam, X-ray) has recently been used in quarantine treatment as a means of disinfection technology (Hallman 2016). In addition, ionizing radiation is used in the sterile insect technique (SIT) to control insect pests (Dyck et al. 2005). The SIT relies on the release of sterile (usually male) insects in overflooding numbers to mate with wild females and thereby suppress the insect population in the targeted area. Many countries, including the United States and European countries, are studying the control of insect pests using SIT. One example is the complete removal of the melon fly, Bactrocera cucurbitae (Coquillett; Diptera: Tephritidae), from several groups of Japanese islands, including the southern-most Yaeyame Islands, Miyako Islands, and Okinawa, and the whole of Japan (Koyama et al. 2004). However, the SIT for M. saltuarius has not been examined. It is challenging to apply the SIT to M. saltuarius because individuals mate several times during their lifetime (Kim et al. 2006). Moreover, the major problem is artificial rearing of this species. Commonly, gamma-ray irradiation has been used for this purpose, but these materials are difficult to handle and transport because of increased controls on radioisotopes due to the fear of terrorism (Mastrangelo et al. 2010). Therefore, an X-ray and electron beam irradiator may offer a good alternative as it presents several positive characteristics: the discontinuous emission of radiation, no radioactive waste, and lower transport costs (Mastrangelo et al. 2010). Recently, Yamada et al. (2014) reported on the effects of X-ray irradiation on Aedes albopictus (Skuse; Diptera: Culicidae). Attempted pest control using ionizing radiation began with cigarettes in 1910 (Morgan and Runner 1913). Ionizing radiation causes electrons in atoms to be released from orbit, creating free electrons. The radicals formed by this action affect various biomolecules, including DNA, thereby disturbing the function of the body (Hallman 2011, Hamideldin and Hussien 2013). Previous studies have shown that the electron beam causes abnormal development, sterility, and DNA damage of Plutella xylostella, Liriomyza trifolii, and Spodoptera litura (Koo et al. 2011, 2012; Yun et al. 2014). In this study, the adults of M. saltuarius were irradiated with electron beams to select the suitable developmental stage and the optimal dose to induce the sterility. After electron beam irradiation, the degree of DNA damage and ovarian development in adults was evaluated. Moreover, semi-field experiments were carried out to investigate the possibility of control by releasing a large number of sterile M. saltuarius adults. This study provides the first meaningful report of M. saltuarius sterilization technology. Materials and Methods Test Insects M. saltuarius pupae were obtained from the Osang Kinsect Co., Ltd. (Yesan, Republic of Korea), and from the Chungcheongbuk-do Forest Environment Research Institute (Cheongju, Republic of Korea) and Gyeonggi Forest Environment Research Center (Gapyeong, Republic of Korea) in 2014–2016. After emergence, the adults were reared in a breeding dish (10 × 4 cm2) during the maturation feeding stage at 25 ± 2°C, 60 ± 1.5 RH, and with a 16:8 (L:D) h photoperiod. One-year-old P. koraiensis twigs were supplied as food because M. saltuarius have a high preference for feeding and oviposition (Han et al. 2016). P. koraiensis twigs were obtained from the Chungcheongbuk-do Forest Environment Research Institute (Cheongju, Republic of Korea). In a 1: 1 ratio (male: female adult) experiment, small-sized trees were used (r = 3–5.5 cm, h = 25–35 cm). In a 1: 10 or 1: 20 ratio (one unirradiated pair: irradiated 10 or 20 adults) experiment, larger-sized trees were used (r = 5.5–8.5 cm, h = 25–35 cm). In this experiment, the developmental stage of M. saltuarius adults was divided into the following three stages: early (1–3 d after maturation feeding), middle (4–6 d after maturation feeding), and late (7–9 d after maturation feeding). M. saltuarius adults are reproductively immature at emergence. They feed on the bark of pine twigs or other conifers for survival and sexual maturation. This is often referred to as ‘maturation feeding’. The pre-oviposition period is about 14 d. Therefore, we treated the electron beam before maturing and divided it into three stages as the effect would be better. Electron Beam Treatment Electron beam irradiation was conducted at the EB-Tech Co., Ltd. (Daejeon, Republic of Korea) using a high-energy linear accelerator (UEL V10-10S, 10 MeV) at ambient temperature. A Bruker EMS 104 EPR analyzer (Bruker Instruments, Rheinstetten, Germany) with an alanine pellets dosimeter (Bruker Instruments) was used to monitor the absorbed doses at the Advanced Radiation Technology Institute (Jeongup, Republic of Korea). Irradiation was performed in aluminum trays on a conveyor that transported the samples through the irradiation zone. The equipment was calibrated annually to ensure its integrity and conformance to nationally recognized standards, such as those of the Korea Testing Laboratory (Ansan, Republic of Korea). Each petri dish containing M. saltuarius adults (early, middle, and late stage) were exposed directly to electron beam at acceleration voltages of 0 (control), 100 (at a dose rate of 242.0 Gy/s), 150 (at a dose rate of 335.6 Gy/s), 200 (at a dose rate of 484.0 Gy/s), and 250 Gy (at a dose rate of 559.3 Gy/s). The thickness of the petri dish lid was 0.8 mm and the penetration error rate by electron beam irradiation was below 2%. M. saltuarius adults have no reproductive ability immediately after emergence. They do not feed for 2–3 d after emergence, but feed after the body is completely sclerotized. M. saltuarius males and females engage in maturation feeding for approximately 0–16 d and 5–24 d, respectively, at 25°C (Nakayama et al. 1998). The ovarian development of females is complete when female adults feed for more than 10 d (Yoon et al. 2011). After electron beam irradiation, M. saltuarius adults of each stage (early, middle, and late) were mated with unirradiated adults in plastic cages (16 × 30 × 19 cm3). The hatchability, number of eggs, and longevity of the adults were investigated in every P. koraiensis twig after 21 d. All experimental groups were provided with water and P. koraiensis twigs every 2 d. Reciprocal Cross Experiment According to the Ratio of Unirradiated and Irradiated Adults Newly emerged adults were fed P. koraiensis twigs as food for 7–9 d. The experimental groups were divided into five treatments as follows: Treatment 1, the unirradiated male (UM) and female (UF) groups (1: 1 ratio) were mated for 28 d; treatment 2, the UM and UF groups were mated for 7 d, after which the UMs were removed and the irradiated males (IMs) (with 200 Gy electron beam) were introduced and mated for 21 d; treatment 3, the IMs (with 200 Gy electron beam) were mated with UFs for 7 d, after which the IMs were removed and the UMs were introduced and mated for 21 d; treatment 4, the UMs were mated with UFs for 7 d, after which the UFs were removed, and the irradiated females (IFs) (with 200 Gy electron beam) were introduced and mated for 21 d; treatment 5, IFs (with 200 Gy electron beam) were mated with UMs for 7 d, after which the IFs were removed, and the UFs were introduced and mated for 21 d. The number and hatchability of the eggs were investigated in every group. All experiments were performed in plastic cages (16 × 30 × 19 cm3) with water and P. koraiensis twigs. New twigs were supplied every 7 d. Cross-mating experiments were replicated 20 or 30 times. For the semi-field experiment (1.2 × 1.2 × 1.2 m3), a pair of UM × UF adults was mated with 10 IF or IM (200 and 250 Gy) to perform a mating competition experiment between unirradiated and irradiated adults. In addition, a pair of UM × UF adults was mated with 20 IFs or IMs (only 200 Gy) to perform a mating competition experiment between unirradiated and irradiated adults. After 28 d, the adult longevity and the number of F1 larvae were measured. New P. koraiensis twigs were supplied every 7 d. The late stage of adults was used in this experiment. To distinguish the unirradiated adults from irradiated adults, natural fluorescent material (BioQuip, Compton, CA) of different colors was applied to the antennas of M. saltuarius adults. If unirradiated adults died during the experiment, they were replaced with new unirradiated adults that matched the longevity of irradiated adults. Ratio groups of one pair to 10 adults were examined with five replicates, and groups consisting of one pair to 20 adults were examined with six replicates. DNA Comet Assays DNA damage in M. saltuarius adults was determined under alkaline conditions using the Comet Assay Kit from Trevigen (Gaithersburg, MD) with slight modifications. After electron beam irradiation, the thorax of adults was ground with a grinding stick in PBS (pH 7.4, Gibco, NY) solution. After filtering with 125 μm nylon mesh, the cell solution was mixed with LMAgarose at a ratio of 1: 10, after which 50 μl was loaded onto a comet slide. After solidification, the slides were immersed in lysis solution for 50 min at 4°C. The slides were then treated with alkaline unwinding solution in dark conditions for 30 min at room temperature. The slides were placed in alkaline electrophoresis solution and electrophoresed for 30 min. After electrophoresis, the solution remaining on the slides was removed, washed with tertiary distilled water twice for 5 min, and then washed with 70% ethanol for 5 min. LMAgarose on the slides was dried at 45°C for 15 min. Then, 100 μl of SYBR Green I was applied to the slides that had been dried, and DNA was stained by treatment at 4°C for 5 min. After staining, the SYBR Green I remaining on the slides was removed 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 the 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. Ovarian Development All adult wings and legs were removed with dissecting scissors, and the abdomens were incised. The ovarian development of the M. saltuarius adults was examined at 0, 3, 6, 9, 12, and 15 d after electron beam irradiation with 200 Gy. The index values of ovarian development were based on our previous studies and are indicated by ODI (ovarian development index) (Yoon et al. 2011). The expression level of vitellogenin protein in irradiated and unirradiated groups was evaluated using polyacrylamide gel electrophoresis. After 100 μl of lysis buffer (iNtRON, Seongnam, Korea) was added to the ovarian samples and homogenized using sonication, the supernatant was collected after centrifugation at 13,000 rpm for 10 min at 4°C. The collected samples were electrophoresed at 100 V in 10% SDS-polyacrylamide gel. After electrophoresis, the gel was stained with staining solution (1 g brilliant blue, 450 ml methanol, 100 ml glacial acetic acid, and 450 ml distilled water) for 3 h at room temperature and destained with destaining solution (450 ml methanol, 100 ml glacial acetic acid, and 450 ml distilled water). Data Analysis Data on the ovarian development of electron beam-irradiated and unirradiated groups were compared with a t-test, and all other data were compared with regression analysis and one-way analysis of variance (ANOVA) followed by Tukey’s studentized range test (SAS Institute 2009) when significant differences were found at P < 0.1. Results Effects of Electron Beam Irradiation on 1 to 1 (Male to Female) Mating of M. saltuarius Adults Electron beams were irradiated in the early, middle, and late stages of M. saltuarius adults (male or female) to determine the optimum irradiation dose required for sterility. In all stages of adults, the longevity decreased in electron beam-IMs (early; R2 = 0.314, F = 69.87, P < 0.0001, middle; R2 = 0.309, F = 68.25, P < 0.0001, late; R2 = 0.316, F = 70.64, P < 0.0001) and females (early; R2 = 0.332, F = 75.89, P < 0.0001, middle; R2 = 0.214, F = 41.58, P < 0.0001, late; R2 = 0.126, F = 22.03, P < 0.0001) compared to the unirradiated adults (Table 1). However, the number of eggs in all stages in the IM × UF and IF × UM groups was not decreased by electron beam irradiation. As the doses of electron beam irradiation increased, the hatchabilities of the IM × UF group (early; R2 = 0.727, F = 407.72, P < 0.0001, middle; R2 = 0.712, F = 378.19, P < 0.0001, late; R2 = 0.716, F = 386.4, P < 0.0001) and the IF × UM group (early; R2 = 0.712, F = 378.16, P < 0.0001, middle; R2 = 0.72, F = 392.27, P < 0.0001, late; R2 = 0.725, F = 404.2, P < 0.0001) were significantly decreased. The hatchability in both groups was completely inhibited at over 200 Gy. There was no difference based on the maturation feeding period. However, we used a more stable late-stage adult for the experiment. Table 1. Adult longevity, fecundity, and hatchability of electron beam-irradiated M. saltuarius adults (n = 30)a Dose (Gy) Early stage Middle stage Late stage Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) ♂ ♀ ♂ ♀ ♂ ♀ IM × UFb 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3ab 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 26.6 ± 3.0b 35.3 ± 5.7a 23.6 ± 4.5a 12.8 ± 14.3b 27.1 ± 4.0b 34.9 ± 7.9a 22.2 ± 5.2a 9.1 ± 13.0b 27.2 ± 3.1b 35.1 ± 5.8b 21.7 ± 5.9a 9.1 ± 11.0b 150 26.2 ± 4.2b 33.7 ± 7.4a 20.7 ± 5.4ab 1.2 ± 2.9c 26.6 ± 2.3b 34.0 ± 7.9a 23.7 ± 4.6a 1.4 ± 3.1c 25.2 ± 4.0b 34.2 ± 6.1b 22.2 ± 6.2a 1.7 ± 4.6c 200 25.8 ± 2.4b 34.5 ± 6.4a 19.2 ± 6.4b 0.0 ± 0.0c 26.1 ± 4.3b 34.3 ± 7.4a 21.4 ± 4.2a 0.0 ± 0.0c 26.0 ± 2.8b 34.7 ± 5.3b 20.8 ± 6.3a 0.0 ± 0.0c 250 25.1 ± 4.4b 34.0 ± 7.7a 22.9 ± 3.4a 0.0 ± 0.0c 25.8 ± 4.2b 33.3 ± 6.2a 23.8 ± 6.2a 0.0 ± 0.0c 25.4 ± 4.4b 33.7 ± 5.2b 23.0 ± 5.0a 0.0 ± 0.0c UM × IF 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3a 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 34.3 ± 6.6a 27.4 ± 3.1b 22.9 ± 4.5a 8.2 ± 6.5b 33.8 ± 7.4a 28.3 ± 6.9b 22.7 ± 4.7a 8.5 ± 7.0b 33.1 ± 7.3a 28.8 ± 4.4b 22.9 ± 4.7a 9.4 ± 7.7b 150 32.9 ± 8.0a 26.8 ± 4.0b 21.8 ± 4.1a 1.5 ± 1.6c 32.2 ± 8.9a 27.7 ± 6.5b 22.4 ± 4.1a 1.7 ± 1.8c 33.6 ± 4.3a 28.1 ± 4.8b 22.7 ± 3.2a 2.0 ± 2.1c 200 32.5 ± 8.4a 26.5 ± 3.3b 21.2 ± 4.0a 0.0 ± 0.0c 33.1 ± 9.5a 27.4 ± 4.9b 22.0 ± 4.6a 0.0 ± 0.0c 32.5 ± 5.8a 27.2 ± 5.0b 22.3 ± 3.9a 0.0 ± 0.0c 250 33.1 ± 7.8a 25.7 ± 4.1b 20.9 ± 4.5a 0.0 ± 0.0c 34.1 ± 7.0a 26.2 ± 4.3b 21.8 ± 4.9a 0.0 ± 0.0c 32.9 ± 4.9a 26.5 ± 5.4b 21.6 ± 5.4a 0.0 ± 0.0c Dose (Gy) Early stage Middle stage Late stage Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) ♂ ♀ ♂ ♀ ♂ ♀ IM × UFb 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3ab 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 26.6 ± 3.0b 35.3 ± 5.7a 23.6 ± 4.5a 12.8 ± 14.3b 27.1 ± 4.0b 34.9 ± 7.9a 22.2 ± 5.2a 9.1 ± 13.0b 27.2 ± 3.1b 35.1 ± 5.8b 21.7 ± 5.9a 9.1 ± 11.0b 150 26.2 ± 4.2b 33.7 ± 7.4a 20.7 ± 5.4ab 1.2 ± 2.9c 26.6 ± 2.3b 34.0 ± 7.9a 23.7 ± 4.6a 1.4 ± 3.1c 25.2 ± 4.0b 34.2 ± 6.1b 22.2 ± 6.2a 1.7 ± 4.6c 200 25.8 ± 2.4b 34.5 ± 6.4a 19.2 ± 6.4b 0.0 ± 0.0c 26.1 ± 4.3b 34.3 ± 7.4a 21.4 ± 4.2a 0.0 ± 0.0c 26.0 ± 2.8b 34.7 ± 5.3b 20.8 ± 6.3a 0.0 ± 0.0c 250 25.1 ± 4.4b 34.0 ± 7.7a 22.9 ± 3.4a 0.0 ± 0.0c 25.8 ± 4.2b 33.3 ± 6.2a 23.8 ± 6.2a 0.0 ± 0.0c 25.4 ± 4.4b 33.7 ± 5.2b 23.0 ± 5.0a 0.0 ± 0.0c UM × IF 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3a 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 34.3 ± 6.6a 27.4 ± 3.1b 22.9 ± 4.5a 8.2 ± 6.5b 33.8 ± 7.4a 28.3 ± 6.9b 22.7 ± 4.7a 8.5 ± 7.0b 33.1 ± 7.3a 28.8 ± 4.4b 22.9 ± 4.7a 9.4 ± 7.7b 150 32.9 ± 8.0a 26.8 ± 4.0b 21.8 ± 4.1a 1.5 ± 1.6c 32.2 ± 8.9a 27.7 ± 6.5b 22.4 ± 4.1a 1.7 ± 1.8c 33.6 ± 4.3a 28.1 ± 4.8b 22.7 ± 3.2a 2.0 ± 2.1c 200 32.5 ± 8.4a 26.5 ± 3.3b 21.2 ± 4.0a 0.0 ± 0.0c 33.1 ± 9.5a 27.4 ± 4.9b 22.0 ± 4.6a 0.0 ± 0.0c 32.5 ± 5.8a 27.2 ± 5.0b 22.3 ± 3.9a 0.0 ± 0.0c 250 33.1 ± 7.8a 25.7 ± 4.1b 20.9 ± 4.5a 0.0 ± 0.0c 34.1 ± 7.0a 26.2 ± 4.3b 21.8 ± 4.9a 0.0 ± 0.0c 32.9 ± 4.9a 26.5 ± 5.4b 21.6 ± 5.4a 0.0 ± 0.0c aMeans within each column followed by the same letter are not significantly different at P < 0.05 by Tukey’s studentized range test (SAS Institute 2009). bI, irradiated; U, unirradiated; M, male; F, female. Open in new tab Table 1. Adult longevity, fecundity, and hatchability of electron beam-irradiated M. saltuarius adults (n = 30)a Dose (Gy) Early stage Middle stage Late stage Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) ♂ ♀ ♂ ♀ ♂ ♀ IM × UFb 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3ab 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 26.6 ± 3.0b 35.3 ± 5.7a 23.6 ± 4.5a 12.8 ± 14.3b 27.1 ± 4.0b 34.9 ± 7.9a 22.2 ± 5.2a 9.1 ± 13.0b 27.2 ± 3.1b 35.1 ± 5.8b 21.7 ± 5.9a 9.1 ± 11.0b 150 26.2 ± 4.2b 33.7 ± 7.4a 20.7 ± 5.4ab 1.2 ± 2.9c 26.6 ± 2.3b 34.0 ± 7.9a 23.7 ± 4.6a 1.4 ± 3.1c 25.2 ± 4.0b 34.2 ± 6.1b 22.2 ± 6.2a 1.7 ± 4.6c 200 25.8 ± 2.4b 34.5 ± 6.4a 19.2 ± 6.4b 0.0 ± 0.0c 26.1 ± 4.3b 34.3 ± 7.4a 21.4 ± 4.2a 0.0 ± 0.0c 26.0 ± 2.8b 34.7 ± 5.3b 20.8 ± 6.3a 0.0 ± 0.0c 250 25.1 ± 4.4b 34.0 ± 7.7a 22.9 ± 3.4a 0.0 ± 0.0c 25.8 ± 4.2b 33.3 ± 6.2a 23.8 ± 6.2a 0.0 ± 0.0c 25.4 ± 4.4b 33.7 ± 5.2b 23.0 ± 5.0a 0.0 ± 0.0c UM × IF 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3a 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 34.3 ± 6.6a 27.4 ± 3.1b 22.9 ± 4.5a 8.2 ± 6.5b 33.8 ± 7.4a 28.3 ± 6.9b 22.7 ± 4.7a 8.5 ± 7.0b 33.1 ± 7.3a 28.8 ± 4.4b 22.9 ± 4.7a 9.4 ± 7.7b 150 32.9 ± 8.0a 26.8 ± 4.0b 21.8 ± 4.1a 1.5 ± 1.6c 32.2 ± 8.9a 27.7 ± 6.5b 22.4 ± 4.1a 1.7 ± 1.8c 33.6 ± 4.3a 28.1 ± 4.8b 22.7 ± 3.2a 2.0 ± 2.1c 200 32.5 ± 8.4a 26.5 ± 3.3b 21.2 ± 4.0a 0.0 ± 0.0c 33.1 ± 9.5a 27.4 ± 4.9b 22.0 ± 4.6a 0.0 ± 0.0c 32.5 ± 5.8a 27.2 ± 5.0b 22.3 ± 3.9a 0.0 ± 0.0c 250 33.1 ± 7.8a 25.7 ± 4.1b 20.9 ± 4.5a 0.0 ± 0.0c 34.1 ± 7.0a 26.2 ± 4.3b 21.8 ± 4.9a 0.0 ± 0.0c 32.9 ± 4.9a 26.5 ± 5.4b 21.6 ± 5.4a 0.0 ± 0.0c Dose (Gy) Early stage Middle stage Late stage Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) Longevity (d) No. eggs/♀ Hatchability (%) (F1) ♂ ♀ ♂ ♀ ♂ ♀ IM × UFb 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3ab 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 26.6 ± 3.0b 35.3 ± 5.7a 23.6 ± 4.5a 12.8 ± 14.3b 27.1 ± 4.0b 34.9 ± 7.9a 22.2 ± 5.2a 9.1 ± 13.0b 27.2 ± 3.1b 35.1 ± 5.8b 21.7 ± 5.9a 9.1 ± 11.0b 150 26.2 ± 4.2b 33.7 ± 7.4a 20.7 ± 5.4ab 1.2 ± 2.9c 26.6 ± 2.3b 34.0 ± 7.9a 23.7 ± 4.6a 1.4 ± 3.1c 25.2 ± 4.0b 34.2 ± 6.1b 22.2 ± 6.2a 1.7 ± 4.6c 200 25.8 ± 2.4b 34.5 ± 6.4a 19.2 ± 6.4b 0.0 ± 0.0c 26.1 ± 4.3b 34.3 ± 7.4a 21.4 ± 4.2a 0.0 ± 0.0c 26.0 ± 2.8b 34.7 ± 5.3b 20.8 ± 6.3a 0.0 ± 0.0c 250 25.1 ± 4.4b 34.0 ± 7.7a 22.9 ± 3.4a 0.0 ± 0.0c 25.8 ± 4.2b 33.3 ± 6.2a 23.8 ± 6.2a 0.0 ± 0.0c 25.4 ± 4.4b 33.7 ± 5.2b 23.0 ± 5.0a 0.0 ± 0.0c UM × IF 0 34.3 ± 6.3a 34.8 ± 5.6a 22.4 ± 4.3a 98.3 ± 7.0a 34.6 ± 6.2a 35.7 ± 6.2a 23.2 ± 4.9a 99.6 ± 1.9a 35.3 ± 7.3a 33.8 ± 10.7a 23.1 ± 6.0a 98.0 ± 3.0a 100 34.3 ± 6.6a 27.4 ± 3.1b 22.9 ± 4.5a 8.2 ± 6.5b 33.8 ± 7.4a 28.3 ± 6.9b 22.7 ± 4.7a 8.5 ± 7.0b 33.1 ± 7.3a 28.8 ± 4.4b 22.9 ± 4.7a 9.4 ± 7.7b 150 32.9 ± 8.0a 26.8 ± 4.0b 21.8 ± 4.1a 1.5 ± 1.6c 32.2 ± 8.9a 27.7 ± 6.5b 22.4 ± 4.1a 1.7 ± 1.8c 33.6 ± 4.3a 28.1 ± 4.8b 22.7 ± 3.2a 2.0 ± 2.1c 200 32.5 ± 8.4a 26.5 ± 3.3b 21.2 ± 4.0a 0.0 ± 0.0c 33.1 ± 9.5a 27.4 ± 4.9b 22.0 ± 4.6a 0.0 ± 0.0c 32.5 ± 5.8a 27.2 ± 5.0b 22.3 ± 3.9a 0.0 ± 0.0c 250 33.1 ± 7.8a 25.7 ± 4.1b 20.9 ± 4.5a 0.0 ± 0.0c 34.1 ± 7.0a 26.2 ± 4.3b 21.8 ± 4.9a 0.0 ± 0.0c 32.9 ± 4.9a 26.5 ± 5.4b 21.6 ± 5.4a 0.0 ± 0.0c aMeans within each column followed by the same letter are not significantly different at P < 0.05 by Tukey’s studentized range test (SAS Institute 2009). bI, irradiated; U, unirradiated; M, male; F, female. Open in new tab Effects of Electron Beam Irradiation on Reciprocal Crosses Under Various Treatments The effect of sterility was investigated under various treatments. We used a late-stage M. saltuarius adult for the experiment. Treatment 1 was the unirradiated control group (UM × UF), and there was no change in hatchability over time (Table 2). The experimental results of treatment 2 showed that the hatchability was significantly decreased to 14.2% after 14 d (F = 748, df = 3, P < 0.0001). In the opposite case, the hatchability of treatment 3 was gradually increased with time and recovered in the unirradiated control group (F = 173.22, df = 3, P < 0.0001). In treatment 4, the hatchability was 94.3% after 7 d and then completely inhibited after 14 d (F = 6067.34, df = 3, P < 0.0001). In the opposite case, the hatchability of treatment 5 was 0% after 7 d but recovered to 93.4% after 28 d (F = 82.66, df = 3, P < 0.0001). The number of eggs of treatment 2 (addition of IMs) was smaller than in treatment 4 (addition of IFs). These results suggest that electron-beam-IMs are more effective in inducing sterility into the wild population. However, this study showed that females are also available. Table 2. Reproductive performance of M. saltuarius on reciprocal crosses under various treatmentsa Treatment Pairing Mating period (d) n Total no. eggs/30♀ Average no. eggs/♀ Hatchability (%) (F1) 1b UM × UF 7th 30 395 13.2 ± 2.3a 95.6 ± 8.4a 14th 30 333 11.1 ± 2.4b 91.0 ± 10.5a 21st 30 278 9.3 ± 3.7b 91.9 ± 20.7a 28th 30 124 4.1 ± 3.4c 86.7 ± 20.5a 2c UM × UF 7th 30 386 12.9 ± 3.5a 98.3 ± 5.4a IM × UF 14th 30 366 12.2 ± 4.1a 14.2 ± 18.1b 21st 30 282 9.4 ± 3.0b 0.0 ± 0.0c 28th 30 99 3.3 ± 1.1c 0.0 ± 0.0c 3d IM × UF 7th 30 404 13.5 ± 2.2a 0.0 ± 0.0c UM × UF 14th 30 343 11.4 ± 2.8b 27.9 ± 27.2b 21st 30 289 9.6 ± 2.3c 83.3 ± 25.2a 28th 30 108 3.6 ± 3.2d 97.4 ± 11.5a 4e UM × UF 7th 20 242 12.1 ± 2.2a 94.3 ± 5.4a UM × IF 14th 20 223 11.2 ± 4.2a 0.0 ± 0.0b 21st 20 119 6.0 ± 3.6b 0.0 ± 0.0b 28th 20 72 3.6 ± 3.3b 0.0 ± 0.0b 5f UM × IF 7th 20 213 10.7 ± 3.9a 0.0 ± 0.0a UM × UF 14th 20 266 13.3 ± 2.3a 36.3 ± 18.0b 21st 20 216 10.8 ± 4.2a 52.4 ± 32.1c 28th 20 116 5.8 ± 1.5b 93.4 ± 9.9d Treatment Pairing Mating period (d) n Total no. eggs/30♀ Average no. eggs/♀ Hatchability (%) (F1) 1b UM × UF 7th 30 395 13.2 ± 2.3a 95.6 ± 8.4a 14th 30 333 11.1 ± 2.4b 91.0 ± 10.5a 21st 30 278 9.3 ± 3.7b 91.9 ± 20.7a 28th 30 124 4.1 ± 3.4c 86.7 ± 20.5a 2c UM × UF 7th 30 386 12.9 ± 3.5a 98.3 ± 5.4a IM × UF 14th 30 366 12.2 ± 4.1a 14.2 ± 18.1b 21st 30 282 9.4 ± 3.0b 0.0 ± 0.0c 28th 30 99 3.3 ± 1.1c 0.0 ± 0.0c 3d IM × UF 7th 30 404 13.5 ± 2.2a 0.0 ± 0.0c UM × UF 14th 30 343 11.4 ± 2.8b 27.9 ± 27.2b 21st 30 289 9.6 ± 2.3c 83.3 ± 25.2a 28th 30 108 3.6 ± 3.2d 97.4 ± 11.5a 4e UM × UF 7th 20 242 12.1 ± 2.2a 94.3 ± 5.4a UM × IF 14th 20 223 11.2 ± 4.2a 0.0 ± 0.0b 21st 20 119 6.0 ± 3.6b 0.0 ± 0.0b 28th 20 72 3.6 ± 3.3b 0.0 ± 0.0b 5f UM × IF 7th 20 213 10.7 ± 3.9a 0.0 ± 0.0a UM × UF 14th 20 266 13.3 ± 2.3a 36.3 ± 18.0b 21st 20 216 10.8 ± 4.2a 52.4 ± 32.1c 28th 20 116 5.8 ± 1.5b 93.4 ± 9.9d aMeans within each column followed by the same letter are not significantly different at P < 0.05 by Tukey’s studentized range test (SAS Institute 2009). bTreatment 1, UM × UF for 28 d. cTreatment 2, UM × UF for 7 d → remove UMs and add IMs → mated for 21 d. dTreatment 3, IM × UF for 7 d → remove IMs and add UMs → mated for 21 d. eTreatment 4, UM × UF for 7 d → remove UFs and add IFs → mated for 21 d. fTreatment 5, UM × IF for 7 d → remove IFs and add UFs → mated for 21 d. Open in new tab Table 2. Reproductive performance of M. saltuarius on reciprocal crosses under various treatmentsa Treatment Pairing Mating period (d) n Total no. eggs/30♀ Average no. eggs/♀ Hatchability (%) (F1) 1b UM × UF 7th 30 395 13.2 ± 2.3a 95.6 ± 8.4a 14th 30 333 11.1 ± 2.4b 91.0 ± 10.5a 21st 30 278 9.3 ± 3.7b 91.9 ± 20.7a 28th 30 124 4.1 ± 3.4c 86.7 ± 20.5a 2c UM × UF 7th 30 386 12.9 ± 3.5a 98.3 ± 5.4a IM × UF 14th 30 366 12.2 ± 4.1a 14.2 ± 18.1b 21st 30 282 9.4 ± 3.0b 0.0 ± 0.0c 28th 30 99 3.3 ± 1.1c 0.0 ± 0.0c 3d IM × UF 7th 30 404 13.5 ± 2.2a 0.0 ± 0.0c UM × UF 14th 30 343 11.4 ± 2.8b 27.9 ± 27.2b 21st 30 289 9.6 ± 2.3c 83.3 ± 25.2a 28th 30 108 3.6 ± 3.2d 97.4 ± 11.5a 4e UM × UF 7th 20 242 12.1 ± 2.2a 94.3 ± 5.4a UM × IF 14th 20 223 11.2 ± 4.2a 0.0 ± 0.0b 21st 20 119 6.0 ± 3.6b 0.0 ± 0.0b 28th 20 72 3.6 ± 3.3b 0.0 ± 0.0b 5f UM × IF 7th 20 213 10.7 ± 3.9a 0.0 ± 0.0a UM × UF 14th 20 266 13.3 ± 2.3a 36.3 ± 18.0b 21st 20 216 10.8 ± 4.2a 52.4 ± 32.1c 28th 20 116 5.8 ± 1.5b 93.4 ± 9.9d Treatment Pairing Mating period (d) n Total no. eggs/30♀ Average no. eggs/♀ Hatchability (%) (F1) 1b UM × UF 7th 30 395 13.2 ± 2.3a 95.6 ± 8.4a 14th 30 333 11.1 ± 2.4b 91.0 ± 10.5a 21st 30 278 9.3 ± 3.7b 91.9 ± 20.7a 28th 30 124 4.1 ± 3.4c 86.7 ± 20.5a 2c UM × UF 7th 30 386 12.9 ± 3.5a 98.3 ± 5.4a IM × UF 14th 30 366 12.2 ± 4.1a 14.2 ± 18.1b 21st 30 282 9.4 ± 3.0b 0.0 ± 0.0c 28th 30 99 3.3 ± 1.1c 0.0 ± 0.0c 3d IM × UF 7th 30 404 13.5 ± 2.2a 0.0 ± 0.0c UM × UF 14th 30 343 11.4 ± 2.8b 27.9 ± 27.2b 21st 30 289 9.6 ± 2.3c 83.3 ± 25.2a 28th 30 108 3.6 ± 3.2d 97.4 ± 11.5a 4e UM × UF 7th 20 242 12.1 ± 2.2a 94.3 ± 5.4a UM × IF 14th 20 223 11.2 ± 4.2a 0.0 ± 0.0b 21st 20 119 6.0 ± 3.6b 0.0 ± 0.0b 28th 20 72 3.6 ± 3.3b 0.0 ± 0.0b 5f UM × IF 7th 20 213 10.7 ± 3.9a 0.0 ± 0.0a UM × UF 14th 20 266 13.3 ± 2.3a 36.3 ± 18.0b 21st 20 216 10.8 ± 4.2a 52.4 ± 32.1c 28th 20 116 5.8 ± 1.5b 93.4 ± 9.9d aMeans within each column followed by the same letter are not significantly different at P < 0.05 by Tukey’s studentized range test (SAS Institute 2009). bTreatment 1, UM × UF for 28 d. cTreatment 2, UM × UF for 7 d → remove UMs and add IMs → mated for 21 d. dTreatment 3, IM × UF for 7 d → remove IMs and add UMs → mated for 21 d. eTreatment 4, UM × UF for 7 d → remove UFs and add IFs → mated for 21 d. fTreatment 5, UM × IF for 7 d → remove IFs and add UFs → mated for 21 d. Open in new tab The Control Effect Based on the Ratio of Irradiated Adults When a pair of UM × UF adults was combined with 10 IFs, the control value was 81.0% at 200 Gy and 85.8% at 250 Gy (F = 57.04, df = 2, P < 0.0001) (Table 3). When a pair of UM × UF adults was combined with 10 IMs, the control value was 90.7% at 200 Gy and 95.3% at 250 Gy (F = 199.26, df = 2, P < 0.0001). However, there was no significant difference between the 10 IF and 10 IM addition groups or the 200 Gy and 250 Gy irradiation doses. Next, in the competition mating experiment of one pair to 20 IFs or IMs, only electron beams with 200 Gy were used. As a result, the control value in the addition of 20 IFs was 85.1% (F = 347.11, df = 1, P < 0.0001), and the control value in the addition of 20 IMs was 87.0% (F = 254.38, df = 1, P < 0.0001). The control effect of M. saltuarius was not significant between 200 Gy and 250 Gy doses, but the addition of the IMs was more effective than that of the IFs. Table 3. Control effect based on the ratio of irradiated M. saltuarius adultsa Pairingb Replicates Longevity (d) Average no. F1 larva/♀ Control value (%) ♀− ♂− ♀+ ♂+ UM × UF: 10 UF 5 34.3 ± 7.1a 32.2 ± 2.3a — — 74.8 ± 17.7a — UM × UF: 10 IF (200 Gy) 5 35.4 ± 2.1a 31.8 ± 2.2a 26.0 ± 4.0a — 14.2 ± 3.2b 81.0 UM × UF: 10 IF (250 Gy) 5 34.4 ± 2.9a 31.2 ± 1.8a 25.2 ± 4.3a — 10.6 ± 4.2b 85.8 UM × UF: 10 UM 5 34.6 ± 3.0a 31.0 ± 6.7a — — 43.0 ± 5.0a — UM × UF: 10 IM (200 Gy) 5 35.0 ± 3.4a 31.6 ± 2.4a — 26.5 ± 4.2a 4.0 ± 1.0b 90.7 UM × UF: 10 IM (250 Gy) 5 33.8 ± 3.1a 32.8 ± 3.1a — 25.6 ± 3.1a 2.0 ± 0.7b 95.3 UM × UF: 20 UF 6 33.9 ± 6.5a 30.9 ± 2.2a — — 146.2 ± 12.4a — UM × UF: 20 IF (200 Gy) 6 34.3 ± 2.6a 31.6 ± 2.2a 26.0 ± 3.9a — 21.7 ± 11.0b 85.1 UM × UF: 20 UM 6 34.3 ± 2.2a 30.6 ± 5.9a — — 48.3 ± 6.3a — UM × UF: 20 IM (200 Gy) 6 33.7 ± 2.8a 32.2 ± 2.3a — 26.2 ± 4.2a 6.3 ± 1.8b 87.0 Pairingb Replicates Longevity (d) Average no. F1 larva/♀ Control value (%) ♀− ♂− ♀+ ♂+ UM × UF: 10 UF 5 34.3 ± 7.1a 32.2 ± 2.3a — — 74.8 ± 17.7a — UM × UF: 10 IF (200 Gy) 5 35.4 ± 2.1a 31.8 ± 2.2a 26.0 ± 4.0a — 14.2 ± 3.2b 81.0 UM × UF: 10 IF (250 Gy) 5 34.4 ± 2.9a 31.2 ± 1.8a 25.2 ± 4.3a — 10.6 ± 4.2b 85.8 UM × UF: 10 UM 5 34.6 ± 3.0a 31.0 ± 6.7a — — 43.0 ± 5.0a — UM × UF: 10 IM (200 Gy) 5 35.0 ± 3.4a 31.6 ± 2.4a — 26.5 ± 4.2a 4.0 ± 1.0b 90.7 UM × UF: 10 IM (250 Gy) 5 33.8 ± 3.1a 32.8 ± 3.1a — 25.6 ± 3.1a 2.0 ± 0.7b 95.3 UM × UF: 20 UF 6 33.9 ± 6.5a 30.9 ± 2.2a — — 146.2 ± 12.4a — UM × UF: 20 IF (200 Gy) 6 34.3 ± 2.6a 31.6 ± 2.2a 26.0 ± 3.9a — 21.7 ± 11.0b 85.1 UM × UF: 20 UM 6 34.3 ± 2.2a 30.6 ± 5.9a — — 48.3 ± 6.3a — UM × UF: 20 IM (200 Gy) 6 33.7 ± 2.8a 32.2 ± 2.3a — 26.2 ± 4.2a 6.3 ± 1.8b 87.0 aMeans within each column followed by the same letter are not significantly different at P < 0.05 by Tukey’s studentized range test (SAS Institute 2009). bI, irradiated; U, unirradiated; M, male; F, female. Open in new tab Table 3. Control effect based on the ratio of irradiated M. saltuarius adultsa Pairingb Replicates Longevity (d) Average no. F1 larva/♀ Control value (%) ♀− ♂− ♀+ ♂+ UM × UF: 10 UF 5 34.3 ± 7.1a 32.2 ± 2.3a — — 74.8 ± 17.7a — UM × UF: 10 IF (200 Gy) 5 35.4 ± 2.1a 31.8 ± 2.2a 26.0 ± 4.0a — 14.2 ± 3.2b 81.0 UM × UF: 10 IF (250 Gy) 5 34.4 ± 2.9a 31.2 ± 1.8a 25.2 ± 4.3a — 10.6 ± 4.2b 85.8 UM × UF: 10 UM 5 34.6 ± 3.0a 31.0 ± 6.7a — — 43.0 ± 5.0a — UM × UF: 10 IM (200 Gy) 5 35.0 ± 3.4a 31.6 ± 2.4a — 26.5 ± 4.2a 4.0 ± 1.0b 90.7 UM × UF: 10 IM (250 Gy) 5 33.8 ± 3.1a 32.8 ± 3.1a — 25.6 ± 3.1a 2.0 ± 0.7b 95.3 UM × UF: 20 UF 6 33.9 ± 6.5a 30.9 ± 2.2a — — 146.2 ± 12.4a — UM × UF: 20 IF (200 Gy) 6 34.3 ± 2.6a 31.6 ± 2.2a 26.0 ± 3.9a — 21.7 ± 11.0b 85.1 UM × UF: 20 UM 6 34.3 ± 2.2a 30.6 ± 5.9a — — 48.3 ± 6.3a — UM × UF: 20 IM (200 Gy) 6 33.7 ± 2.8a 32.2 ± 2.3a — 26.2 ± 4.2a 6.3 ± 1.8b 87.0 Pairingb Replicates Longevity (d) Average no. F1 larva/♀ Control value (%) ♀− ♂− ♀+ ♂+ UM × UF: 10 UF 5 34.3 ± 7.1a 32.2 ± 2.3a — — 74.8 ± 17.7a — UM × UF: 10 IF (200 Gy) 5 35.4 ± 2.1a 31.8 ± 2.2a 26.0 ± 4.0a — 14.2 ± 3.2b 81.0 UM × UF: 10 IF (250 Gy) 5 34.4 ± 2.9a 31.2 ± 1.8a 25.2 ± 4.3a — 10.6 ± 4.2b 85.8 UM × UF: 10 UM 5 34.6 ± 3.0a 31.0 ± 6.7a — — 43.0 ± 5.0a — UM × UF: 10 IM (200 Gy) 5 35.0 ± 3.4a 31.6 ± 2.4a — 26.5 ± 4.2a 4.0 ± 1.0b 90.7 UM × UF: 10 IM (250 Gy) 5 33.8 ± 3.1a 32.8 ± 3.1a — 25.6 ± 3.1a 2.0 ± 0.7b 95.3 UM × UF: 20 UF 6 33.9 ± 6.5a 30.9 ± 2.2a — — 146.2 ± 12.4a — UM × UF: 20 IF (200 Gy) 6 34.3 ± 2.6a 31.6 ± 2.2a 26.0 ± 3.9a — 21.7 ± 11.0b 85.1 UM × UF: 20 UM 6 34.3 ± 2.2a 30.6 ± 5.9a — — 48.3 ± 6.3a — UM × UF: 20 IM (200 Gy) 6 33.7 ± 2.8a 32.2 ± 2.3a — 26.2 ± 4.2a 6.3 ± 1.8b 87.0 aMeans within each column followed by the same letter are not significantly different at P < 0.05 by Tukey’s studentized range test (SAS Institute 2009). bI, irradiated; U, unirradiated; M, male; F, female. Open in new tab Effects of Electron Beam Irradiation on DNA Damage There are many reports already but DNA damage of M. saltuarius adults after 5 h of electron beam irradiation was analyzed using a DNA comet assay. In this assay, the strand break of individual cells appears like the tail of a comet. In the unirradiated group (0 Gy), the comet tail is short and barely present, so there was little damage to the DNA (Fig. 1A). When the electron beam was applied at 150 Gy, the length of the tail was longer than that in the unirradiated group, and DNA damage was found to be at a certain level. At a dose of more than 200 Gy, the length of the tail was remarkably increased compared to the unirradiated group and increased as the doses increased. Quantitation of these observations is depicted in the graphs shown in Fig. 1B. This result suggested that the electron beam dose-dependently leads to DNA damage in M. saltuarius adults. However, it slowly recovered over time because the DNA repair system is functional. Fig. 1. Open in new tabDownload slide Representative images (A) of comets from M. saltuarius 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 (magnification 100×). (B) Graphic depiction of the calculated tail length from an analysis of alkaline comet assays. Data are shown for a representative experiment, where at least 100 comets were quantified for each sample. Fig. 1. Open in new tabDownload slide Representative images (A) of comets from M. saltuarius 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 (magnification 100×). (B) Graphic depiction of the calculated tail length from an analysis of alkaline comet assays. Data are shown for a representative experiment, where at least 100 comets were quantified for each sample. Effects of Electron Beam Irradiation on Ovarian Development To observe the degree of ovarian development during the maturation feeding period after electron beam irradiation in M. saltuarius adults, visual observation was performed according to our previous reports (Yoon et al. 2011). The abdomens of M. saltuarius female adults were incised at 0, 3, 6, 9, 12, and 15 d after electron beam irradiation with 200 Gy. Most UF M. saltuarius adults completed ovarian development at 9 d after maturation feeding. However, the ovarian development of irradiated M. saltuarius adults was significantly inhibited. The mean values of the ODI in UF adults were 0.0 at 0 d, 1.2 at 3 d, 2.1 at 6 d, 2.8 at 9 d, 3.0 at 12 d, and 3.0 at 15 d (Fig. 2). Those of the IF adults were 0.0 at 0 d, 0.2 at 3 d, 0.9 at 6 d, 1.1 at 9 d, 1.4 at 12 d, and 1.7 at 15 d. Therefore, when the M. saltuarius female adults were irradiated with electron beam irradiation, the ovaries did not sufficiently develop, and this seems to be the reason for the reduced spawning of females after electron beam irradiation. Next, the total protein of UF and IF M. saltuarius adults was analyzed by SDS-PAGE, and we observed the levels of vitellogenin protein at approximately 180 kDa and 40 kDa (the arrow in Fig. 3). Vitellogenin protein was markedly decreased in the irradiated M. saltuarius adults compared to the unirradiated group. Fig. 2. Open in new tabDownload slide Ovarian development index (ODI) of electron beam-irradiated M. saltuarius adults. ODI 0, follicle growing; ODI 1, pre-vitellogenic; ODI 2, mid-vitellogenic; and ODI 3, post-vitellogenic and mature egg (Yoon et al. 2011). Each point indicates the mean ± SE of 20 replicates. The significance of the difference was determined between 0 Gy and 200 Gy. Value is indicated by *P < 0.05, **P < 0.01 (SAS Institute 2009; t-test). Fig. 2. Open in new tabDownload slide Ovarian development index (ODI) of electron beam-irradiated M. saltuarius adults. ODI 0, follicle growing; ODI 1, pre-vitellogenic; ODI 2, mid-vitellogenic; and ODI 3, post-vitellogenic and mature egg (Yoon et al. 2011). Each point indicates the mean ± SE of 20 replicates. The significance of the difference was determined between 0 Gy and 200 Gy. Value is indicated by *P < 0.05, **P < 0.01 (SAS Institute 2009; t-test). Fig. 3. Open in new tabDownload slide Changes in ovarian protein level of M. saltuarius female adults by electron beam irradiation. The ovaries were harvested 0, 3, 6, 9, 12, and 15 d after electron beam irradiation and were analyzed by 10% SDS-PAGE. Panel A, 0 Gy and panel B, 200 Gy. Fig. 3. Open in new tabDownload slide Changes in ovarian protein level of M. saltuarius female adults by electron beam irradiation. The ovaries were harvested 0, 3, 6, 9, 12, and 15 d after electron beam irradiation and were analyzed by 10% SDS-PAGE. Panel A, 0 Gy and panel B, 200 Gy. Discussion This study was the first to apply the SIT on M. saltuarius. We investigated the longevity, the number of eggs, and the hatchability following electron beam irradiation (100–250 Gy) according to developmental stage. All the treatment groups showed slightly decreased longevity. When female or male adults were irradiated with electron beams, there was no effect on the number of eggs. However, the hatching rate of the treatment group was reduced. The hatching rate of eggs in both female and male adults reflected complete sterility at an electron beam of 200 Gy or more. The longevity and the fecundity of M. saltuarius females varied as a function of the age of P. koraiensis logs. The longevity and number of eggs decrease with older twigs (Yoon et al. 2011). Therefore, in the present study, we used 1-yr-old P. koraiensis twigs as food. If sterile adults are released into the field, there is a high possibility of mating again with the normal adults from the wild. Therefore, we set up a probable situation and experimented under various treatments. When unirradiated adults were mated with sterile adults, the hatchability was decreased over time, and the hatching rate of eggs was completely inhibited by the end of the experiment. In contrast, if the unirradiated adults were mated with sterile adults and then mated again with unirradiated adults, the hatchability gradually increased and recovered to normal. Therefore, it is necessary to increase the probability that sterile adults will mate with normal adults by releasing a large number of sterile adults into the field. When the pairing of UM × UF was assumed to be wild and they were mated with 10 sterile females or males that had been irradiated with a 200 or 250 Gy electron beam, a high control effect of 81–95% was obtained. In the subsequent experiment, a pair of UF and UM adults was mated with 20 female or male sterile adults irradiated with a 200 Gy electron beam. As a result, the control effect was 85–87%. Therefore, SIT is considered to be effective even in the case of long-horned beetles mating several times during their lifetime. However, field experiments of a larger scale should be performed to verify the control effect. If both female and male sterile adults are released into the field, they can mate with each other, reducing the chance that sterile adults will mate with wild adults. Therefore, when a large number of sterile male or female adults are released, they can mate with many wild female or male adults by competing with wild male adults in field. The comet assay revealed that DNA damage increases with increasing irradiation dose in M. saltuarius adults. However, it recovered itself over time via the DNA repair system. However, DNA damage would not be completely repaired at high radiation doses (Kameya et al. 2012; Yun et al. 2014, 2015). Previous studies reported that electron beam irradiation induces DNA fragmentation in Curculio sikkimensis (Todoriki et al. 2006), Plutella xylostella, Aphis gossypii, and Spodoptera litura (Koo et al. 2011, Yun et al. 2014, 2015). Ionizing radiation also affects gene expression (Chang et al. 2015, Yun et al. 2015, Sachdev et al. 2017). When M. saltuarius was irradiated at more than 200 Gy, the DNA was damaged, resulting in abnormal reproductive induction. In addition, ovarian development was inhibited. The level of ovarian vitellogenin was significantly reduced compared to that in unirradiated adults. Vitellogenin is an energy source for eggs that is used when eggs are developed (Chen et al. 2004, Oliveira et al. 2012). If the vitellogenin supply is not sufficient, eggs do not develop properly and do not hatch. In conclusion, the optimal dose to induce sterility in M. saltuarius is 200 Gy, it adds another data point to doses needed for phytosanitary irradiation (PI), especially to support generic PI doses. Additionally, these results showed that not only males but also females are useful for sit technology. Sterile male or female adults should be released in large numbers to compete with wild M. saltuarius adults. The SIT will have a better chance of succeeding if pest populations are first suppressed using whatever tools are available. The electron beam irradiator, therefore, has been shown to provide a practical and effective tool for sterilization for SIT purposes. Acknowledgments This study was supported by a Grant (Project No. S111414L060140) from the Korea Forest Service. 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Google Scholar Crossref Search ADS WorldCat Yun , S.-H. , H.-N. Koo , S.-W. Lee , H. K. Kim , Y. Kim , B. Han , and G.-H. Kim . 2015 . A comparative study on the effects of electron beam irradiation on imidacloprid-resistant and -susceptible Aphis gossypii (Hemiptera:Aphididae) . Radiat. Phys. Chem 112 : 151 – 157 . Google Scholar Crossref Search ADS WorldCat Author notes Co-first author. © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - Electron Beam-Induced Sterility and Inhibition of Ovarian Development in the Sakhalin Pine Longicorn, Monochamus saltuarius (Coleoptera: Cerambycidae)
JF - Journal of Economic Entomology
DO - 10.1093/jee/tox306
DA - 2018-04-02
UR - https://www.deepdyve.com/lp/oxford-university-press/electron-beam-induced-sterility-and-inhibition-of-ovarian-development-CDL99CXALf
SP - 725
VL - 111
IS - 2
DP - DeepDyve
ER -