Research Article Received: 13 March 2015
Revised: 8 May 2015
Accepted article published: 2 June 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/ps.4053
Detrimental effects of electron beam irradiation on the cowpea bruchid Callosobruchus maculatus Wen Sang,a,b,c Mickey Speakmon,d Lan Zhou,e Yu Wang,b,c Chaoliang Lei,a Suresh D Pillaid and Keyan Zhu-Salzmanb,c* Abstract BACKGROUND: Electron beam (eBeam) irradiation technology is an environmentally friendly, chemical-free alternative for disinfesting insect pests of stored grains. The underlying hypothesis is that specific doses of eBeam will have defined detrimental effects on the different life stages. We evaluated the effects of eBeam exposure in a range of doses (0.03–0.12 kGy) on the development of the cowpea bruchid (Callosobruchus maculatus) at various stages of its life cycle. RESULTS: Differential radiosensitivity was detected during egg development. Early and intermediate stages of eggs never hatched after exposure to a dose of 0.03 kGy, whereas a substantial portion of black-headed (i.e. late) eggs survived irradiation even at 0.12 kGy. However, further development of the hatched larvae was inhibited. Although midgut protein digestion remained intact, irradiated larvae (0.06 kGy or higher) failed to develop into normal living adults; rather, they died as pupae or abnormally eclosed adults, suggesting a detrimental effect of eBeam on metamorphosis. Emerged irradiated pupae had shorter longevity and were unable to produce any eggs at 0.06 kGy or higher. At this dose range, eggs laid by irradiated adults were not viable. eBeam treatment shortened adult longevity in a dose-dependent manner. Reciprocal crosses indicated that females were more sensitive to eBeam exposure than their male counterparts. Dissection of the female reproductive system revealed that eBeam treatment prevented formation of oocytes. CONCLUSION: eBeam irradiation has very defined effects on cowpea bruchid development and reproduction. A dose of 0.06 kGy could successfully impede cowpea burchid population expansion. This information can be exploited for post-harvest insect control of stored grains. © 2015 Society of Chemical Industry Keywords: electron beam; irradiation; Callosobruchus maculatus; emergence; longevity; reproductive system
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INTRODUCTION
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out. Phosphine, the other most commonly used fumigant, has been challenged by the development of pest resistance.5 Furthermore, registration of new fumigants for use on foods is becoming increasingly difficult with increasing awareness of environmental and human health concerns associated with synthetic pesticides. Therefore, it is imperative to develop alternative approaches that are environmentally sound and economically feasible.
∗
Correspondence to: Keyan Zhu-Salzman, Department of Entomology, Texas A&M University, College Station, TX 77843, USA. E-mail: [email protected]
a Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, Huazhong Agricultural University, Wuhan, Hubei, China b Department of Entomology, Texas A&M University, College Station, TX, USA c Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA d National Center for Electron Beam Research, Texas A&M University, College Station, TX, USA e Department of Statistics, Texas A&M University, College Station, TX, USA
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Cowpea (Vigna unguiculata) is a legume crop planted worldwide owing to its tolerance to heat, drought and poor soil conditions. Cowpea seeds are rich in easily digestible proteins and carbohydrates and are a staple food for people living in many rural areas, particularly in Africa and Asia.1,2 The cowpea bruchid (Callosobruchus maculatus) is a common post-harvest insect pest of cowpea and other legumes. Originating in West Africa, this insect has now moved throughout the world with the trade of legumes, causing heavy losses of stored grains each year.3,4 A female adult can lay 50–100 eggs on the surface of legume seeds, after which hatched larvae burrow into the seeds. Larval development and pupation occur inside the seeds. Emerged adults mature rapidly, and, after mating, another cycle of infestation starts again. Because of high fecundity and a short generation time, heavy losses (up to 100%) may occur within a few months of storage. Seed damage resulting from larval feeding also reduces germination rate and market value. Chemical fumigation is currently the most widely used control measure for storage pests. However, owing to its damaging effects on the ozone layer, methyl bromide is gradually being phased
www.soci.org Irradiation technology can be used to disinfest a variety of stored products.6 – 8 Ionizing radiation can be produced either through radioactive isotopes or through linear accelerators. Ionizing radiation using electron beam (eBeam) technology, X-rays or gamma radiation is damaging to cellular macromolecules such as DNA and RNA at doses that are normally employed for phytosanitary treatment. In recent years, ionizing radiation treatment of agricultural commodities to disinfest insect pests has expanded rapidly. The volume of irradiated agricultural produce imported into the United States has increased approximately 60-fold since 2007.9 This technology is effective for all insect developmental stages. As acute mortality is not necessary for pest control efficacy, detailed studies are needed to evaluate the impact of specific irradiation doses on specific insect life cycle stages. This will help to identify the optimal treatment strategy. Such research has been conducted on a number of storage insects. For instance, 350 Gy of 𝛾-ray can inhibit egg hatch and adult emergence of Indian meal moth Plodia interpunctella.10 X-ray at 120 Gy arrests the development of rice weevil Sitophilus oryzae and sterilizes its adults, thus providing quarantine security.11 Studies of radiosensitivity of the cowpea bruchid revealed an increasing progression in the lethal dose of 𝛾-ray as bruchids grow older, from 20 Gy for eggs to 120 Gy for pupae.12 eBeam is a stream of electrons generated by heat, bombardment of charged particles or strong electric fields. The eBeam technology employs linear accelerators to generate a highly planar stream of energetic electrons from commercial electricity.13 This technology is currently employed worldwide for food pasteurization,14 medical product sterilization,15 decontamination16 and crosslinking of polymers.17 eBeam can inactivate insect development at all stages, as shown in the diamondback moth (Plutella xylostella),18 the American serpentine leafminer (Liriomyza trifolii)19 and the armyworm (Spodoptera litura).20 Very limited information is available thus far with regard to the efficacy of eBeam in the disinfestation of pests of stored grains.21,22 The hidden life stages of the within-seed feeders represent a further challenge to determine how sublethal doses would affect insect physiological functions. In this study, we performed dose–response analyses of eBeam irradiation on mortality of cowpea bruchids at all developmental stages. We examined effects of eBeam on insect digestion, metamorphosis and reproduction. In addition, we identified the minimum effective eBeam radiation dose potentially useful for disinfestation of this storage pest.
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MATERIALS AND METHODS
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2.1 Insects Cowpea bruchids were maintained on cowpea seeds (purchased from a local grocery store) in 500 mL wide-mouth glass bottles in an environmental chamber (27 ∘ C, 60% RH). To obtain an age-synchronized bruchid culture, adults (∼200, 1–4 days old) were introduced into wide-mouth glass bottles containing approximately 150 seeds, and were allowed to lay eggs on the seeds for only 1–2 h. We defined eggs of 0–24 h, 24–120 h and 120–156 h as early, intermediate and black-headed (or late) stage respectively. Owing to the unique within-seed life style, the precise larval and pupal developmental stages were determined as previously described.23 Briefly, infested seeds were broken open 3 times a day to trace larval development, and head capsule size was used as a standard to distinguish larval stages. Successful egg hatch was recognized by change of the egg color. The number of larvae or pupae in the seeds was determined on the basis of the number of successfully hatched eggs. To collect adults 2 h old or younger, we took
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Table 1. Time period for eBeam treatment at each developmental stage/substage of cowpea bruchids Developmental stage/substage Egg Early Intermediate Black-headed Larva First instar Second instar Third instar Fourth instar Pupa Adult
Time interval for eBeam treatment (h)a
4–6 52–54 148–150 196–198 244–246 292–294 436–438 532–534 4–6
a Stages of egg, larva and pupa were calculated from the time of egg laying; adult stage was from the time of emergence.
a cowpea bruchid culture at a stage where many bruchids were emerging, and discarded all adults present. New adults emerging over the following 2 h were collected and separated to prevent mating. Time intervals of eBeam treatment for eggs, larvae, pupae and adults are shown in Table 1. 2.2 eBeam treatment and dosimetry eBeam irradiation was conducted in the National Center for Electron Beam Research at Texas A&M University (College Station, TX) using a high-energy linear accelerator (10 MeV, 18 kW; L-3 Pulse Sciences, San Leandro, CA) at room temperature. Infested seeds bearing eggs, larvae or pupae were sealed in plastic bags (16.5 × 4.5 cm) with small holes punched for air movement. Each treatment contained approximately 150 eggs of each egg stage, 150 larvae of each instar and 150 pupae. The bags were then placed in a high-density polyethylene (HDPE) cassette (12 × 12 × 1/8 in) and irradiated at target doses of 0.03, 0.06, 0.09 and 0.12 kGy respectively. For adults, 90 males and 90 females (free from cowpea seeds) were subjected to treatments of 0.06, 0.15, 0.50, 1.0, 1.5 and 2.0 kGy respectively. For every treatment, an alanine dosimeter was placed among the seeds or insects to be irradiated, and read by a Bruker eScan spectrometer (Bruker, Billerica, MA) to verify the absorbed dose. The actual measured dose ranges for each target dose is shown in Table 2. Control samples were subjected to the same handling procedures but without exposure to eBeam doses. 2.3 Assessment of effect of eBeam on insect survival and development After eBeam treatment, samples were immediately removed from the plastic bags and returned to the laboratory chamber. Treated and control insects were examined daily for egg hatch, adult emergence and survival. Eggs whose shells turned cream white were considered to be alive, whereas those remaining transparent or appearing shriveled or wrinkled were considered to be dead.23,24 For larvae and pupae, individuals that failed to emerge were assumed to be dead, as were adults remaining motionless after being stimulated by turning the container several times. Egg hatch rate, adult emergence rate and adult acute mortality (i.e. death within 2 h after irradiation) were calculated. For longevity, pupae
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Detrimental effects of electron beam irradiation on the cowpea bruchid
Table 2. Actual absorbed dose range at each target dose
Target dose (kGy) 0.03 0.06 0.09 0.12 0.15 0.50 1.00 1.50 2.00
Actual absorbed dose range (kGy) 0.027–0.037 0.057–0.072 0.085–0.096 0.123–0.130 0.150–0.166 0.500–0.550 0.990–1.023 1.420–1.541 1.969–1.990
were subjected to eBeam treatment at doses of 0.03, 0.06 and 0.09 kGy respectively. Irradiated pupae were continuously incubated. The doses for adults were 0.06, 0.15, 0.50, 1.00, 1.50 and 2.00 kGy respectively. Treated and control insects were examined twice daily for adult survival. Acute adult mortality was recorded 2 h after irradiation. 2.4 Reciprocal crossing Pupae used for reciprocal crossing were subjected to eBeam treatment at doses of 0.03, 0.06 and 0.09 kGy. Irradiated pupae were continuously incubated in the chamber until they developed into adults. Upon emergence, male and female adults (less than 2 h old) were identified and separated. In a 30 mL plastic cup with holes on the side and in the lid for air movement, one irradiated but unmated male was paired with one non-irradiated and unmated female, and vice versa. There were 20 replicates for each combination at every dose except the highest (0.09 kGy) which caused a low emergence rate. Non-irradiated male–female pairs subjected to the same handling procedures served as the negative control, and irradiated male–female pairs served as the positive control. Ten cowpea seeds were placed in the cup for egg deposition. Fecundity and egg viability were recorded. For adult reciprocal crossing, male and female adults (less than 2 h old) were separated and irradiated at doses of 0.06, 0.15, 0.50, 1.00, 1.50 and 2.00 kGy prior to mating. Fecundity and egg hatch rate were determined as above. Controls included male–female pairs with both irradiated and with both non-irradiated (respectively).
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Table 3. Hatch and emergence rates of eBeam-irradiated eggsa Substage
Dose (kGy)
Hatch rate (%)
Emergence rate (%)
Early
0.00 0.03 0.06 0.09 0.12
80.7 ± 3.2 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
96.4 ± 1.6 n.a.b n.a. n.a. n.a.
Intermediate
0.00 0.03 0.06 0.09 0.12
88.0 ± 2.7 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
97.2 ± 1.4 n.a. n.a. n.a. n.a.
Black-headed
0.00 0.03 0.06 0.09 0.12
94.0 ± 1.9 a 88.7 ± 2.6 a 77.3 ± 3.4 b 63.3 ± 3.9 c 58.0 ± 4.0 c
98.2 ± 1.0 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
a
Values (means ± SE) followed by different lower-case letters were significantly different (Pearson’s 𝜒 2 -test, P < 0.05). b Not applicable.
ethanol, from which 500 μL was used to measure the absorbance at 440 nm using a Genesys spectrophotometer (Thermo Fisher Scientific, Inc., Pittsburgh, PA). Control insects went through the same handling procedures but without eBeam exposure. 2.6 eBeam effects on metamorphosis and oocyte development The fourth-instar larvae were irradiated at a dose of 0.12 kGy, followed by continued incubation in the chamber. Two weeks after the non-irradiated fourth-instar control eclosed to adults, the irradiated seeds were opened, and insects, all confirmed to be dead, were collected, surface-cleaned in 0.1 M PBS buffer (pH 7.0) and photographed with a EOS T3i digital camera (Canon, Tokyo, Japan). Likewise, pupae irradiated at 0.03, 0.06 and 0.09 kGy were allowed to grow to adult stage. Two days after emergence, the female reproductive system was dissected in the PBS buffer under a SZH-ILLD microscope (Olympus Optical Co., Tokyo, Japan) and photographed as above. Control reproductive systems were obtained from the non-irradiated individuals subjected to the same rearing and handling procedures. 2.7 Statistical analysis Data analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL) and R v.3.1.2 (R Foundation for Statistical Computing, Vienna, Austria). Pearson’s 𝜒 2 -test was used to determine whether significant differences (P < 0.05) existed between treatments in hatch rate of irradiated eggs, emergence rate of larvae and pupae and acute mortality of adults. One-way ANOVA was used to analyze longevity and midgut proteolytic activity. Tukey’s multiple range test was used for comparison of the differences between treatments for mean separation (P < 0.05). Exponential dose–response models were fitted to the reciprocal crossing data, i.e. fecundity and arcsine-square-root-transformed hatch rate, and likelihood ratio tests were used for model testing (P < 0.01).
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2.5 Gut proteolytic analysis Midguts were dissected from the fourth-instar larvae 24 or 48 h after irradiation at 0.12 kGy. Total midgut proteolytic activity was measured using the azocasein assay as described previously,25 with six replicates. Specifically, two midguts were placed in one Eppendorf tube in 30 μL of prechilled dissection buffer (100 mM sodium acetate buffer with 1 mM of EDTA, pH 5.5). Samples were homogenized and centrifuged at 10 000 × g at 4 ∘ C for 10 min. The supernatant (0.5 gut equivalent) was incubated with 20 μL of assay buffer [100 mM sodium acetate, pH 5.5, 5 mM of L-cysteine, 0.1% triton 100 (v/v)] and 60 μL of 2% (w/v) azocasein (dissolved in assay buffer) for 6 h in a 37 ∘ C water bath. The reaction was stopped by the addition of 300 μL of 10% TCA. After centrifugation at 10 000 × g at room temperature for 5 min, the supernatant (350 μL) was transferred to a new tube and mixed with 200 μL of 50%
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Table 4. Emergence rate of eBeam-irradiated larvae and pupaea Larvae (%) Dose (kGy)
First instar
Second instar
Third instar
Fourth instar
Pupae (%)
0.00 0.03 0.06 0.09 0.12
97.8 ± 1.3 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
97.0 ± 1.5 a 1.4 ± 1.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
97.8 ± 1.2 a 2.8 ± 1.4 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
99.2 ± 0.8 a 9.6 ± 2.4 b 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c
98.2 ± 1.0 a 34.5 ± 3.6 b 9.0 ± 2.3 c 5.3 ± 1.7 c 1.3 ± 0.9 d
a
Values (means ± SE) followed by different lower-case letters were significantly different (Pearson’s 𝜒 2 -test, P < 0.05).
Figure 1. Shortened longevity of unmated adults emerged from eBeam-irradiated pupae (days, means ± SE). Data were analyzed by one-way ANOVA (F 7,119 = 20.16, P < 0.05). Tukey’s multiple range test was used to compare the difference between treatments; means followed by different letters are significantly different.
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RESULTS
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3.1 eBeam irradiation impeded cowpea bruchid development To begin to understand the impact of eBeam irradiation on cowpea bruchids, initial experiments were performed to determine and refine the optimal eBeam dose range for all developmental stages based on observed fecundity, hatch rate, emergence rate and longevity. The ranges were 0.03–0.12 kGy for eggs, larvae and pupae and 0.06–2.00 kGy for adults. Eggs were divided into early, intermediate and black-headed age groups. No early or intermediate eggs hatched after irradiation even at the lowest dose of 0.03 kGy (Table 3). This radiosensitivity decreased at later developmental stages, as egg hatches were observed at the black-head stage at all doses ranging from 0.03 to 0.12 kGy. Nevertheless, the deleterious treatment effect was apparent because the hatch rate decreased when eBeam dosage increased. Furthermore, no emergence was detected at any tested doses, indicating the inability of hatched larvae from irradiated eggs to complete their life cycle (Table 3). Indeed, when seeds were subsequently cracked open, we found that none lived beyond the first-instar larval stage (data not shown). Sensitivity of larvae to eBeam treatment is reflected by the low emergence rate (Table 4). A very small portion of the second-, third- and fourth-instar larvae survived a dose of 0.03 kGy and emerged as adults. The older larvae had a higher emergence rate and thus may be more tolerant than younger ones. At a dose of 0.06 kGy or higher, however, all larvae
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Figure 2. Negative effects of eBeam on cowpea bruchid adults. (A) Acute mortality (%, means ± SE) was analyzed by Pearson’s 𝜒 2 -test (P < 0.05). (B) Longevity (days, means ± SE) was analyzed using a one-way ANOVA (F 13,419 = 93.31, P 0.05), suggesting that eBeam did not directly interfere with protein digestion, a basic physiological function. Maintaining seemingly normal digestive function did not ensure successful transformation from immature larvae to adults. Although most eBeam-treated fourth-instar larvae molted to pupae, they eventually died as either pupae (data not shown) or as abnormal adults (Fig. 3). The head, antennae, thorax and legs of the eclosed adults appeared to develop normally. The abdomen, however, exhibited mixed traits of pupae and adults; the segments were covered by alternating adult and pupal-like cuticles. Another striking phenotype was the deformed wing structure held laterally.
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3.3 Reciprocal crosses eBeam irradiation at sublethal doses may potentially affect insect reproduction. We performed reciprocal crosses using emerged adults from irradiated pupae that survived eBeam exposure from 0.03 to 0.09 kGy, and observed significantly reduced fecundity and hatch rate (Table 5). When virgin males from irradiated pupae were paired with unirradiated virgin females (UF × IM), effects were dose dependent. Males derived from irradiated pupae were still fertile at 0.09 kGy. In contrast, in the reciprocal cross (i.e. IF × UM), female adults became sterile when their pupae were irradiated at 0.06 kGy or higher doses. Egg hatch rate in the IF × UM cross was lower than
DISCUSSION
The eBeam technology relies on electricity to generate a highly planar stream of high-energy electrons. The initial investment cost of a linear accelerator for eBeam is higher than cobalt-60 as the radiation source for 𝛾-ray, but unit costs of the two types of irradiator are comparable, ranging from 0.5 to 7 cents lb−1 .26 Compared with chemical fumigation, irradiation is twice as expensive or more.27 However, the technology has a number of salient advantages: the process is extremely fast (measured in seconds), does not involve the use of any chemicals or radioactive isotopes and the technology is scalable.6,7,16,21,28 In this study, we have demonstrated that eBeam inhibited cowpea bruchid development at all stages and caused morphological abnormalities. The harmful effects appeared to be dose and developmental stage dependent. Further investigation suggested that not all physiological functions were affected equally; the larval protein digestion was not immediately affected, but reproduction was severely impaired, leading to extinction of the irradiated population at certain doses. Radiosensitivity was apparent at the egg stage, but this sensitivity decreased as eggs continued to develop (Table 3). During early embryogenesis, fertilized insect eggs undergo active mitosis to form a blastoderm, and blastoderm cells further multiply and differentiate, generating ectoderm and mesoderm. DNA deletion, mutation and breakage caused by eBeam ionization, particularly double-strand breaks, could overwhelm the DNA repair system and become lethal to this process. Higher radiosensitivity in the early stage of the embryos has also been observed in hermaphroditic fish (Kryptolebias marmoratus)29 and in tardigrade (Milnesium tardigradum), a small aquatic invertebrate.30 A weak antioxidant defense system of the former at this stage seems to explain the higher developmental impairment relative to later stages, whereas for the latter, vulnerability to radiation is suspected to be attributable to rapid mitotic activity and ineffective
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Table 5. Non-linear regression analyses of fecundity and hatch rate of eBeam-irradiated pupae in reciprocal crosses Pairinga
Dose (kGy)
Fecundityb(eggs female−1 )c
Hatch rate (%)c
UF × UM
0.00
74.4 ± 2.2
96.2 ± 0.4
UF × IM
0.03 0.06 0.09
35.8 ± 4.5 36.8 ± 4.6 26.2 ± 12.7
67.4 ± 6.7 70.2 ± 5.6 41.7 ± 4.9
IF × UM
0.03 0.06 0.09
27.9 ± 5.4 0.0 ± 0.0 0.0 ± 0.0
49.4 ± 6.1 n.a.d n.a.
IF × IM
0.03 0.06 0.09
12.3 ± 2.5 0.0 ± 0.0 0.0 ± 0.0
5.6 ± 1.7 n.a. n.a.
Fecundity R2 = 0.684, A = 71.57 ± 3.42
UF × IM IF × UM IF × IM
B = −14.42 ± 1.78f B = −37.93 ± 4.53 B = −60.45 ± 9.81
Hatch rate, R2 = 0.703, A = 1.35 ± 0.05
UF × IM IF × UM IF × IM
B = −6.28 ± 1.17 B = −18.14 ± 3.25 B = −69.53 ± 13.03
Non-linear regression analyses Y = A* exp(B* X)e
a
UF, untreated female; UM, untreated male; IF, irradiated female; IM, irradiated male.
b Of the entire reproduction period. c Means ± SE. d Not applicable. e Y represents the fecundity or hatch
rate (arcsine square root transformed); X represents the dose; A (means ± SE) is the fecundity or hatch rate of untreated insects; B (means ± SE) is the exponential rate of decrease in fecundity or hatch rate. f The likelihood ratio test was used for pairwise comparisons of the exponential rate of decrease among treatments, P < 0.01.
Table 6. Non-linear regression analyses of fecundity and hatch rate of eBeam-irradiated adults in reciprocal crosses Pairinga
Dose (kGy)
Fecundityb(eggs female−1 )c
Hatch rate (%)c
UF × UM
0.00
71.0 ± 2.1
94.4 ± 0.7
UF × IM
0.06 0.15 0.50 1.00 1.50 2.00
40.8 ± 7.2 43.5 ± 6.6 37.2 ± 5.5 15.3 ± 5.0 11.5 ± 5.0 9.7 ± 5.9
32.3 ± 7.5 29.8 ± 6.5 32.1 ± 7.7 28.8 ± 12.4 19.9 ± 9.3 17.0 ± 9.9
IF × UM
0.06 0.15 0.50 1.00 1.50 2.00
38.9 ± 4.6 30.9 ± 3.5 11.3 ± 1.9 1.8 ± 0.6 0.0 ± 0.0 0.0 ± 0.0
0.8 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 n.a.d n.a.
IM × IF
0.06 0.15 0.50 1.00 1.50 2.00
18.4 ± 3.6 14.7 ± 3.3 2.4 ± 0.7 0.7 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 n.a. n.a.
= 0.551 A = 55.26 ± 2.37
UF × IM IF × UM IF × IM
B = −1.10 ± 0.14f B = −3.86 ± 0.63 B = −13.90 ± 2.26
Hatch rate R2 = 0.495 A = 0.91 ± 0.06
UF × IM IF × UM IF × IM
B = −0.99 ± 0.22 B = −55.66 ± 44.10 n.a.
Non-linear regression analyses Y
= A* exp(B* X)e
Fecundity R2
a UF, untreated female; UM, untreated male; IF, irradiated female; IM, irradiated male. b Of the entire reproduction period. c Means ± SE. d Not applicable. e Y represents the
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fecundity or hatch rate (arcsine square root transformed); X represents the dose; A (means ± SE) is the fecundity or hatch rate of untreated insects; B (means ± SE) reflects the exponential rate of decrease in fecundity or hatch rate. f The likelihood ratio test was used for pairwise comparisons of the exponential rate of decrease among treatments, P < 0.01.
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Detrimental effects of electron beam irradiation on the cowpea bruchid
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Figure 4. eBeam inhibits oocyte development. Shown are female reproductive systems of adults emerged from irradiated pupae. Scale bars: 1 mm. oo, oocyte; ov, ovary; gc, genital chamber.
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their midgut epithelia, which may contribute to the decreased competitiveness of the flies in nature, affecting the efficacy of SIT programs.38 In cowpea bruchids, however, larval protein digestion apparently was unaffected by irradiation. Different treatment regimes may provide some insight into the disagreeing results; the fruit flies were irradiated at pupal stage and their midgut structure was examined after adult eclosion, whereas the eBeam treatment and proteolytic assay of cowpea bruchids occurred within the same larval stage. It is known that irradiation, particularly at low dose ranges, has a stronger impact on the formation of a tissue than on existing tissue. Thus, the irradiated bruchid larvae most likely maintained similar levels of food consumption until they approached their next molt. For non-feeding adults, 2 kGy only caused approximately 50% acute mortality. However, 0.06 kGy drastically reduced fecundity, and no eggs were viable. For stored grain protection, sterilizing insects with low-dose radiation to impede reproduction and prevent maturation ought to be a better strategy than attempting to achieve 100% mortality at a substantially higher dose. Our results indicated that 0.06 kGy should be sufficient to prevent propagation of the irradiated cowpea bruchids. For practical applications, however, this recommended dose has to be verified by trials with much larger insect populations. In addition, different bruchid strains or the same strain under different rearing conditions may vary in their radiosensitivity, similarly to our observations in a number of life-history and genetic parameters.39 Although post-irradiation feeding by developmentally blocked yet live larvae is an unwanted effect, early irradiation when pest numbers are low presumably could minimize the grain damage while avoiding alteration of the physical, physicochemical and nutritional properties of the stored grains.
5
CONCLUSION
As an alternative to chemical fumigation, eBeam could become an important component in post-harvest technologies to protect stored grains. Appropriate irradiation doses are only available for some storage insects,11 necessitating more studies to expand the information. Generic radiation doses have to be carefully established because different insect species and different developmental stages often exhibit differing radiosensitivity.37,40 – 43 To achieve high confidence, environmental factors that may affect irradiation efficacy, such as temperature, host and oxygen content,20,44 – 48
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DNA repair processes associated with the early egg stage. While no egg hatches occurred at any doses during early and intermediate developmental stages, a substantial portion of black-headed eggs hatched apparently normally (Table 3). Presumably, major DNA replication related to embryogenesis has completed at this stage. However, erroneous repairs may explain the fact that eventually no hatched larvae completed their life cycle. Larval development involves increases in the cell number and cell size of the epidermis and internal structures. Failing to reach adult stage from irradiated larvae most likely was due to unsuccessful mitosis and cell division.31 The fact that younger insects had to go through more mitoses could contribute to the higher susceptibility in earlier developmental stages. Damage resulting from the electron shower or radicals would likely be amplified in each subsequent DNA replication and cell division. During pupation, more profound changes, including histolysis and phagocytosis of the larval tissues and reconstruction of adult tissues, occur in the transformation from larvae to adults. eBeam interfered with the metamorphosis of cowpea bruchids, which was reflected by the forming of physically deformed adults (Fig. 3). A pupal-like cuticle could result from unwanted juvenile hormone (JH) action at this stage.32,33 It has been shown in the red flour beetle that disruption of pupal specifier broad (br) gene caused the formation of abnormal wings and the appearance of pupal traits in adults.34 The expression of the br gene requires JH, and their coordination is necessary to turn off the JH system during the pupal–adult molt.35 It is possible that eBeam disrupted hormone signaling pathways, and that failure to shut down the JH system resulted in a pupal-like cuticle. Wing deformities may also result from eBeam damage of the imaginal discs. A healthy reproductive system is essential for success of any insect species in nature. Although irradiated pupae exhibited a higher emergence rate relative to larvae, eBeam disrupted the development of the reproductive systems and interfered with germline stem cell division which leads to the formation of oocytes (Fig. 4). Furthermore, in spite of the intact appearance of ovaries and oocytes (data not shown), the reproductive function of irradiated adults was sufficiently destroyed by eBeam at 0.06 kGy or above (Table 6). Reciprocal crossing revealed that females are more susceptible to eBeam than males in terms of reproduction, which is consistent with observations in other species.19,20,36,37 It has been shown that irradiation of Mediterranean fruit fly (Ceratitis capitata) and Mexican fruit fly (Anastrepha ludens) damages
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ACKNOWLEDGEMENTS We would like to thank Dr Ron Salzman for his critical review of the manuscript. This project was supported by USDA AFRI grant 2014-67013-21781 and by the China Scholarship Council.
REFERENCES
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1 Sanon A, Ba NM, Binso-Dabire CL and Pittendrigh BR, Effectiveness of spinosad (Naturalytes) in controlling the cowpea storage pest, Callosobruchus maculatus (Coleoptera: Bruchidae). J Econ Entomol 103:203–210 (2010). 2 Zhu-Salzman K and Murdock LL, Cowpea: insects, ecology and control, in The Encyclopedia of Pest Management. Taylor & Francis, New York, NY, pp. 1–3 (2006). 3 Jackai L and Daoust R, Insect pests of cowpeas. Annu Rev Entomol 31:95–119 (1986). 4 Loganathan M, Jayas DS, Fields PG and White NDG, Low and high temperatures for the control of cowpea beetle, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), in chickpeas. J Stored Prod Res 47:244–248 (2011). 5 Wong-Corral FJ, Castane C and Riudavets J, Lethal effects of CO2 -modified atmospheres for the control of three Bruchidae species. J Stored Prod Res 55:62–67 (2013). 6 Kume T, Furuta M, Todoriki S, Uenoyama N and Kobayashi Y, Status of food irradiation in the world. Radiat Phys Chem 78:222–226 (2009). 7 Salimov RA, Cherepkov VG, Kuksanov NK and Kuznetzov SA, The use of electron accelerators for radiation disinfestation of grain. Radiat Phys Chem 57:625–627 (2000). 8 Hallman GJ, Control of stored product pests by ionizing radiation. J Stored Prod Res 52:36–41 (2013). 9 Jeffers LA, Hands-on Workshop on Electron Beam Technologies. [Online]. Texas A&M University, College Station, TX (2014). Available: https:// agriliferegister.tamu.edu/dropinn/materials/material_517.pdf [4 April 2014]. 10 Ayvaz A, Albayrak S and Karaborklu S, Gamma radiation sensitivity of the eggs, larvae and pupae of Indian meal moth Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). Pest Manag Sci 64:505–512 (2008). 11 Follett PA, Snook K, Janson A, Antonio B, Haruki A, Okamura M et al., Irradiation quarantine treatment for control of Sitophilus oryzae (Coleoptera: Curculionidae) in rice. J Stored Prod Res 52:63–67 (2013). 12 Diop YM, Marchioni E, Ba DD and Hasselmann C, Radiation disinfestation of cowpea seeds contaminated by Callosobruchus maculatus. J Food Process Pres 21:69–81 (1997). 13 Hamm RW and Hamm ME, Introduction to the beam business, in Industrial Accelerators and their Applications, ed. by Hamm RW and Hamm ME. World Scientific Publishing Co., Singapore, pp. 1–8 (2012). 14 Pillai SD and Braby LA, Electronic pasteurization, in Advances in Thermal and Non-Thermal Food Preservation, ed. by Tewari G and Juneja V. Blackwell Publishing Ltd, Ames, IA, pp. 195–202 (2007). 15 Dziedzic-Goclawska A, Kaminski A, Uhrynowska-Tyszkiewicz I and Stachowicz W, Irradiation as a safety procedure in tissue banking. Cell Tissue Bank 6:201–219 (2005). 16 Pillai SD, Blackburn C and Bogran C, Ionizing irradiation for phytosanitary applications and fresh produce safety, in Global Safety of Fresh Produce: a Handbook of Best Practice, Innovative Commercial Solutions and Case Studies, ed. by Hoorfar J. Woodhead Publishing Ltd, Cambridge, UK, pp. 221–232 (2014). 17 Cleland MR, Electron beam materials irradiators, in Industrial Accelerators and their Applications, ed. by Hamm RW and Hamm ME. World Scientific Publishing Co., Singapore, pp. 87–137 (2012).
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18 Koo HN, Yoon SH, Shin YH, Yoon C, Woo JS and Kim GH, Effect of electron beam irradiation on developmental stages of Plutella xylostella (Lepidoptera: Plutellidae). J Asia-Pacif Entomol 14:243–247 (2011). 19 Koo HN, Yun SH, Yoon C and Kim GH, Electron beam irradiation induces abnormal development and the stabilization of p53 protein of American serpentine leafminer, Liriomyza trifolii (Burgess). Radiat Phys Chem 81:86–92 (2012). 20 Yun SH, Lee SW, Koo HN and Kim GH, Assessment of electron beam-induced abnormal development and DNA damage in Spodoptera litura (F.) (Lepidoptera: Noctuidae). Radiat Phys Chem 96:44–49 (2014). 21 Cleghorn DA, Nablo SV, Ferro DN and Hagstrum DW, Electron beam treatment parameters for control of stored product insects. Radiat Phys Chem 63:575–579 (2002). 22 Imamura T, Miyanoshita A, Todoriki S and Hayashi T, Usability of a soft-electron (low-energy electron) machine for disinfestation of grains contaminated with insect pests. Radiat Phys Chem 71:213–215 (2004). 23 Cheng WN, Lei JX, Ahn JE, Liu TX and Zhu-Salzman K, Effects of decreased O2 and elevated CO2 on survival, development, and gene expression in cowpea bruchids. J Insect Physiol 58:792–800 (2012). 24 Shazali MH, Imamura T and Miyanoshita A, Mortality of eggs of the cowpea bruchid, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), in carbon dioxide under high pressure. Appl Entomol Zool 39:49–53 (2004). 25 Zhu-Salzman K, Koiwa H, Salzman RA, Shade RE and Ahn JE, Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor. Insect Mol Biol 12:135–145 (2003). 26 Morrison RM, An Economic Analysis of Electron Accelerators and Cobalt-60 for Irradiating Food. Economic Research Service, US Department of Agriculture, Washington, DC (1989). 27 Nelson HC, A cost analysis of alternatives for methyl bromide for postharvest and quarantine treatment of apples and cherries. Am J Agric Econ 78:1424–1433 (1996). 28 Ershov BG, Radiation technologies: their possibilities, state, and prospects of application. Her Russ Acad Sci 83:437–447 (2013). 29 Rhee JS, Kim BM, Kang CM, Lee YM and Lee JS, Gamma irradiationinduced oxidative stress and developmental impairment in the hermaphroditic fish, Kryptolebias marmoratus, embryo. Environ Toxicol Chem 31:1745–1753 (2012). 30 Beltran-Pardo E, Jonsson KI, Wojcik A, Haghdoost S, Harms-Ringdahl M, Bermudez-Cruz RM et al., Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development. PLoS ONE 8 (2013). 31 Chapman RF, The Insects: Structure and Function. Cambridge University Press, New York, NY (1998). 32 Riddiford LM, Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions. Adv Insect Physiol 24:213–274 (1994). 33 Riddiford LM, Hiruma K, Zhou X and Nelson CA, Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol 33:1327–1338 (2003). 34 Suzuki Y, Truman JW and Riddiford LM, The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa. Development 135:569–577 (2008). 35 Erezyilmaz DF, Riddiford LM and Truman JW, The pupal specifier broad directs progressive morphogenesis in a direct-developing insect. P Natl Acad Sci USA 103:6925–6930 (2006). 36 Burditt AK, Jr, Hungate FP and Toba HH, Gamma irradiation: effect of dose and dose rate on development of mature codling moth larvae and adult eclosion. Radiat Phys Chem 34:979–984 (1989). 37 Huang F, Li WD, Li XQ, Bei YW, Lin WC, Lu YB et al., Irradiation as a quarantine treatment for the solenopsis mealybug, Phenacoccus solenopsis. Radiat Phys Chem 96:101–106 (2014). 38 Lauzon CR and Potter SE, Description of the irradiated and nonirradiated midgut of Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Anastrepha ludens Loew (Diptera: Tephritidae) used for sterile insect technique. J Pestic Sci 85:217–226 (2012). 39 Cheng W, Lei J, Fox CW, Johnston JS and Zhu-Salzman K, Comparison of life history and genetic properties of cowpea bruchid strains and their response to hypoxia. J Insect Physiol 75:5–11 (2015).
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Detrimental effects of electron beam irradiation on the cowpea bruchid 40 Abbas H and Nouraddin S, Application of gamma radiation for controlling the red flour beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae). Afr J Agr Res 6:3877–3882 (2011). 41 Hallman GJ, Ionizing irradiation quarantine treatment against Plum curculio (Coleoptera: Curculionidae). J Econ Entomol 96:1399–1404 (2003). 42 Hallman GJ and Thomas DB, Ionizing radiation as a phytosanitary treatment against fruit flies (Diptera: Tephritidae): efficacy in naturally versus artificially infested fruit. J Econ Entomol 103:1129–1134 (2010). 43 The DT, Khanh NT, Vo TKL, Chung CV, Tran TTA and Nguyen HHT, Effects of gamma irradiation on different stages of mealybug Dysmicoccus neobrevipes (Hemiptera: Pseudococcidae). Radiat Phys Chem 81:97–100 (2012).
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44 Hallman GJ, Irradiation disinfestation of apple maggot (Diptera: Tephritidae) in hypoxic and low-temperature storage. J Econ Entomol 97:1245–1248 (2004). 45 Hallman GJ, Levang-Brilz NM, Zettler JL and Winborne IC, Factors affecting ionizing radiation phytosanitary treatments, and implications for research and generic treatments. J Econ Entomol 103:1950–1963 (2010). 46 Follett PA and Snook K, Cold storage enhances the efficacy and margin of security in postharvest irradiation treatments against fruit flies (Diptera: Tephritidae). J Econ Entomol 106:2035–2042 (2013). 47 Follett PA, Wall M and Bailey W, Influence of modified atmosphere packaging on radiation tolerance in the phytosanitary pest melon fly (Diptera: Tephritidae). J Econ Entomol 106:2020–2026 (2013). 48 Hallman GJ, Guo K and Liu TX, Phytosanitary irradiation of Liriomyza trifolii (Diptera: Agromyzidae). J Econ Entomol 104:1851–1855 (2011).
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Revised: 8 May 2015
Accepted article published: 2 June 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/ps.4053
Detrimental effects of electron beam irradiation on the cowpea bruchid Callosobruchus maculatus Wen Sang,a,b,c Mickey Speakmon,d Lan Zhou,e Yu Wang,b,c Chaoliang Lei,a Suresh D Pillaid and Keyan Zhu-Salzmanb,c* Abstract BACKGROUND: Electron beam (eBeam) irradiation technology is an environmentally friendly, chemical-free alternative for disinfesting insect pests of stored grains. The underlying hypothesis is that specific doses of eBeam will have defined detrimental effects on the different life stages. We evaluated the effects of eBeam exposure in a range of doses (0.03–0.12 kGy) on the development of the cowpea bruchid (Callosobruchus maculatus) at various stages of its life cycle. RESULTS: Differential radiosensitivity was detected during egg development. Early and intermediate stages of eggs never hatched after exposure to a dose of 0.03 kGy, whereas a substantial portion of black-headed (i.e. late) eggs survived irradiation even at 0.12 kGy. However, further development of the hatched larvae was inhibited. Although midgut protein digestion remained intact, irradiated larvae (0.06 kGy or higher) failed to develop into normal living adults; rather, they died as pupae or abnormally eclosed adults, suggesting a detrimental effect of eBeam on metamorphosis. Emerged irradiated pupae had shorter longevity and were unable to produce any eggs at 0.06 kGy or higher. At this dose range, eggs laid by irradiated adults were not viable. eBeam treatment shortened adult longevity in a dose-dependent manner. Reciprocal crosses indicated that females were more sensitive to eBeam exposure than their male counterparts. Dissection of the female reproductive system revealed that eBeam treatment prevented formation of oocytes. CONCLUSION: eBeam irradiation has very defined effects on cowpea bruchid development and reproduction. A dose of 0.06 kGy could successfully impede cowpea burchid population expansion. This information can be exploited for post-harvest insect control of stored grains. © 2015 Society of Chemical Industry Keywords: electron beam; irradiation; Callosobruchus maculatus; emergence; longevity; reproductive system
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INTRODUCTION
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out. Phosphine, the other most commonly used fumigant, has been challenged by the development of pest resistance.5 Furthermore, registration of new fumigants for use on foods is becoming increasingly difficult with increasing awareness of environmental and human health concerns associated with synthetic pesticides. Therefore, it is imperative to develop alternative approaches that are environmentally sound and economically feasible.
∗
Correspondence to: Keyan Zhu-Salzman, Department of Entomology, Texas A&M University, College Station, TX 77843, USA. E-mail: [email protected]
a Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, Huazhong Agricultural University, Wuhan, Hubei, China b Department of Entomology, Texas A&M University, College Station, TX, USA c Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA d National Center for Electron Beam Research, Texas A&M University, College Station, TX, USA e Department of Statistics, Texas A&M University, College Station, TX, USA
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Cowpea (Vigna unguiculata) is a legume crop planted worldwide owing to its tolerance to heat, drought and poor soil conditions. Cowpea seeds are rich in easily digestible proteins and carbohydrates and are a staple food for people living in many rural areas, particularly in Africa and Asia.1,2 The cowpea bruchid (Callosobruchus maculatus) is a common post-harvest insect pest of cowpea and other legumes. Originating in West Africa, this insect has now moved throughout the world with the trade of legumes, causing heavy losses of stored grains each year.3,4 A female adult can lay 50–100 eggs on the surface of legume seeds, after which hatched larvae burrow into the seeds. Larval development and pupation occur inside the seeds. Emerged adults mature rapidly, and, after mating, another cycle of infestation starts again. Because of high fecundity and a short generation time, heavy losses (up to 100%) may occur within a few months of storage. Seed damage resulting from larval feeding also reduces germination rate and market value. Chemical fumigation is currently the most widely used control measure for storage pests. However, owing to its damaging effects on the ozone layer, methyl bromide is gradually being phased
www.soci.org Irradiation technology can be used to disinfest a variety of stored products.6 – 8 Ionizing radiation can be produced either through radioactive isotopes or through linear accelerators. Ionizing radiation using electron beam (eBeam) technology, X-rays or gamma radiation is damaging to cellular macromolecules such as DNA and RNA at doses that are normally employed for phytosanitary treatment. In recent years, ionizing radiation treatment of agricultural commodities to disinfest insect pests has expanded rapidly. The volume of irradiated agricultural produce imported into the United States has increased approximately 60-fold since 2007.9 This technology is effective for all insect developmental stages. As acute mortality is not necessary for pest control efficacy, detailed studies are needed to evaluate the impact of specific irradiation doses on specific insect life cycle stages. This will help to identify the optimal treatment strategy. Such research has been conducted on a number of storage insects. For instance, 350 Gy of 𝛾-ray can inhibit egg hatch and adult emergence of Indian meal moth Plodia interpunctella.10 X-ray at 120 Gy arrests the development of rice weevil Sitophilus oryzae and sterilizes its adults, thus providing quarantine security.11 Studies of radiosensitivity of the cowpea bruchid revealed an increasing progression in the lethal dose of 𝛾-ray as bruchids grow older, from 20 Gy for eggs to 120 Gy for pupae.12 eBeam is a stream of electrons generated by heat, bombardment of charged particles or strong electric fields. The eBeam technology employs linear accelerators to generate a highly planar stream of energetic electrons from commercial electricity.13 This technology is currently employed worldwide for food pasteurization,14 medical product sterilization,15 decontamination16 and crosslinking of polymers.17 eBeam can inactivate insect development at all stages, as shown in the diamondback moth (Plutella xylostella),18 the American serpentine leafminer (Liriomyza trifolii)19 and the armyworm (Spodoptera litura).20 Very limited information is available thus far with regard to the efficacy of eBeam in the disinfestation of pests of stored grains.21,22 The hidden life stages of the within-seed feeders represent a further challenge to determine how sublethal doses would affect insect physiological functions. In this study, we performed dose–response analyses of eBeam irradiation on mortality of cowpea bruchids at all developmental stages. We examined effects of eBeam on insect digestion, metamorphosis and reproduction. In addition, we identified the minimum effective eBeam radiation dose potentially useful for disinfestation of this storage pest.
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MATERIALS AND METHODS
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2.1 Insects Cowpea bruchids were maintained on cowpea seeds (purchased from a local grocery store) in 500 mL wide-mouth glass bottles in an environmental chamber (27 ∘ C, 60% RH). To obtain an age-synchronized bruchid culture, adults (∼200, 1–4 days old) were introduced into wide-mouth glass bottles containing approximately 150 seeds, and were allowed to lay eggs on the seeds for only 1–2 h. We defined eggs of 0–24 h, 24–120 h and 120–156 h as early, intermediate and black-headed (or late) stage respectively. Owing to the unique within-seed life style, the precise larval and pupal developmental stages were determined as previously described.23 Briefly, infested seeds were broken open 3 times a day to trace larval development, and head capsule size was used as a standard to distinguish larval stages. Successful egg hatch was recognized by change of the egg color. The number of larvae or pupae in the seeds was determined on the basis of the number of successfully hatched eggs. To collect adults 2 h old or younger, we took
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Table 1. Time period for eBeam treatment at each developmental stage/substage of cowpea bruchids Developmental stage/substage Egg Early Intermediate Black-headed Larva First instar Second instar Third instar Fourth instar Pupa Adult
Time interval for eBeam treatment (h)a
4–6 52–54 148–150 196–198 244–246 292–294 436–438 532–534 4–6
a Stages of egg, larva and pupa were calculated from the time of egg laying; adult stage was from the time of emergence.
a cowpea bruchid culture at a stage where many bruchids were emerging, and discarded all adults present. New adults emerging over the following 2 h were collected and separated to prevent mating. Time intervals of eBeam treatment for eggs, larvae, pupae and adults are shown in Table 1. 2.2 eBeam treatment and dosimetry eBeam irradiation was conducted in the National Center for Electron Beam Research at Texas A&M University (College Station, TX) using a high-energy linear accelerator (10 MeV, 18 kW; L-3 Pulse Sciences, San Leandro, CA) at room temperature. Infested seeds bearing eggs, larvae or pupae were sealed in plastic bags (16.5 × 4.5 cm) with small holes punched for air movement. Each treatment contained approximately 150 eggs of each egg stage, 150 larvae of each instar and 150 pupae. The bags were then placed in a high-density polyethylene (HDPE) cassette (12 × 12 × 1/8 in) and irradiated at target doses of 0.03, 0.06, 0.09 and 0.12 kGy respectively. For adults, 90 males and 90 females (free from cowpea seeds) were subjected to treatments of 0.06, 0.15, 0.50, 1.0, 1.5 and 2.0 kGy respectively. For every treatment, an alanine dosimeter was placed among the seeds or insects to be irradiated, and read by a Bruker eScan spectrometer (Bruker, Billerica, MA) to verify the absorbed dose. The actual measured dose ranges for each target dose is shown in Table 2. Control samples were subjected to the same handling procedures but without exposure to eBeam doses. 2.3 Assessment of effect of eBeam on insect survival and development After eBeam treatment, samples were immediately removed from the plastic bags and returned to the laboratory chamber. Treated and control insects were examined daily for egg hatch, adult emergence and survival. Eggs whose shells turned cream white were considered to be alive, whereas those remaining transparent or appearing shriveled or wrinkled were considered to be dead.23,24 For larvae and pupae, individuals that failed to emerge were assumed to be dead, as were adults remaining motionless after being stimulated by turning the container several times. Egg hatch rate, adult emergence rate and adult acute mortality (i.e. death within 2 h after irradiation) were calculated. For longevity, pupae
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Detrimental effects of electron beam irradiation on the cowpea bruchid
Table 2. Actual absorbed dose range at each target dose
Target dose (kGy) 0.03 0.06 0.09 0.12 0.15 0.50 1.00 1.50 2.00
Actual absorbed dose range (kGy) 0.027–0.037 0.057–0.072 0.085–0.096 0.123–0.130 0.150–0.166 0.500–0.550 0.990–1.023 1.420–1.541 1.969–1.990
were subjected to eBeam treatment at doses of 0.03, 0.06 and 0.09 kGy respectively. Irradiated pupae were continuously incubated. The doses for adults were 0.06, 0.15, 0.50, 1.00, 1.50 and 2.00 kGy respectively. Treated and control insects were examined twice daily for adult survival. Acute adult mortality was recorded 2 h after irradiation. 2.4 Reciprocal crossing Pupae used for reciprocal crossing were subjected to eBeam treatment at doses of 0.03, 0.06 and 0.09 kGy. Irradiated pupae were continuously incubated in the chamber until they developed into adults. Upon emergence, male and female adults (less than 2 h old) were identified and separated. In a 30 mL plastic cup with holes on the side and in the lid for air movement, one irradiated but unmated male was paired with one non-irradiated and unmated female, and vice versa. There were 20 replicates for each combination at every dose except the highest (0.09 kGy) which caused a low emergence rate. Non-irradiated male–female pairs subjected to the same handling procedures served as the negative control, and irradiated male–female pairs served as the positive control. Ten cowpea seeds were placed in the cup for egg deposition. Fecundity and egg viability were recorded. For adult reciprocal crossing, male and female adults (less than 2 h old) were separated and irradiated at doses of 0.06, 0.15, 0.50, 1.00, 1.50 and 2.00 kGy prior to mating. Fecundity and egg hatch rate were determined as above. Controls included male–female pairs with both irradiated and with both non-irradiated (respectively).
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Table 3. Hatch and emergence rates of eBeam-irradiated eggsa Substage
Dose (kGy)
Hatch rate (%)
Emergence rate (%)
Early
0.00 0.03 0.06 0.09 0.12
80.7 ± 3.2 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
96.4 ± 1.6 n.a.b n.a. n.a. n.a.
Intermediate
0.00 0.03 0.06 0.09 0.12
88.0 ± 2.7 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
97.2 ± 1.4 n.a. n.a. n.a. n.a.
Black-headed
0.00 0.03 0.06 0.09 0.12
94.0 ± 1.9 a 88.7 ± 2.6 a 77.3 ± 3.4 b 63.3 ± 3.9 c 58.0 ± 4.0 c
98.2 ± 1.0 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
a
Values (means ± SE) followed by different lower-case letters were significantly different (Pearson’s 𝜒 2 -test, P < 0.05). b Not applicable.
ethanol, from which 500 μL was used to measure the absorbance at 440 nm using a Genesys spectrophotometer (Thermo Fisher Scientific, Inc., Pittsburgh, PA). Control insects went through the same handling procedures but without eBeam exposure. 2.6 eBeam effects on metamorphosis and oocyte development The fourth-instar larvae were irradiated at a dose of 0.12 kGy, followed by continued incubation in the chamber. Two weeks after the non-irradiated fourth-instar control eclosed to adults, the irradiated seeds were opened, and insects, all confirmed to be dead, were collected, surface-cleaned in 0.1 M PBS buffer (pH 7.0) and photographed with a EOS T3i digital camera (Canon, Tokyo, Japan). Likewise, pupae irradiated at 0.03, 0.06 and 0.09 kGy were allowed to grow to adult stage. Two days after emergence, the female reproductive system was dissected in the PBS buffer under a SZH-ILLD microscope (Olympus Optical Co., Tokyo, Japan) and photographed as above. Control reproductive systems were obtained from the non-irradiated individuals subjected to the same rearing and handling procedures. 2.7 Statistical analysis Data analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL) and R v.3.1.2 (R Foundation for Statistical Computing, Vienna, Austria). Pearson’s 𝜒 2 -test was used to determine whether significant differences (P < 0.05) existed between treatments in hatch rate of irradiated eggs, emergence rate of larvae and pupae and acute mortality of adults. One-way ANOVA was used to analyze longevity and midgut proteolytic activity. Tukey’s multiple range test was used for comparison of the differences between treatments for mean separation (P < 0.05). Exponential dose–response models were fitted to the reciprocal crossing data, i.e. fecundity and arcsine-square-root-transformed hatch rate, and likelihood ratio tests were used for model testing (P < 0.01).
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2.5 Gut proteolytic analysis Midguts were dissected from the fourth-instar larvae 24 or 48 h after irradiation at 0.12 kGy. Total midgut proteolytic activity was measured using the azocasein assay as described previously,25 with six replicates. Specifically, two midguts were placed in one Eppendorf tube in 30 μL of prechilled dissection buffer (100 mM sodium acetate buffer with 1 mM of EDTA, pH 5.5). Samples were homogenized and centrifuged at 10 000 × g at 4 ∘ C for 10 min. The supernatant (0.5 gut equivalent) was incubated with 20 μL of assay buffer [100 mM sodium acetate, pH 5.5, 5 mM of L-cysteine, 0.1% triton 100 (v/v)] and 60 μL of 2% (w/v) azocasein (dissolved in assay buffer) for 6 h in a 37 ∘ C water bath. The reaction was stopped by the addition of 300 μL of 10% TCA. After centrifugation at 10 000 × g at room temperature for 5 min, the supernatant (350 μL) was transferred to a new tube and mixed with 200 μL of 50%
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Table 4. Emergence rate of eBeam-irradiated larvae and pupaea Larvae (%) Dose (kGy)
First instar
Second instar
Third instar
Fourth instar
Pupae (%)
0.00 0.03 0.06 0.09 0.12
97.8 ± 1.3 a 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
97.0 ± 1.5 a 1.4 ± 1.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
97.8 ± 1.2 a 2.8 ± 1.4 b 0.0 ± 0.0 b 0.0 ± 0.0 b 0.0 ± 0.0 b
99.2 ± 0.8 a 9.6 ± 2.4 b 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c
98.2 ± 1.0 a 34.5 ± 3.6 b 9.0 ± 2.3 c 5.3 ± 1.7 c 1.3 ± 0.9 d
a
Values (means ± SE) followed by different lower-case letters were significantly different (Pearson’s 𝜒 2 -test, P < 0.05).
Figure 1. Shortened longevity of unmated adults emerged from eBeam-irradiated pupae (days, means ± SE). Data were analyzed by one-way ANOVA (F 7,119 = 20.16, P < 0.05). Tukey’s multiple range test was used to compare the difference between treatments; means followed by different letters are significantly different.
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3.1 eBeam irradiation impeded cowpea bruchid development To begin to understand the impact of eBeam irradiation on cowpea bruchids, initial experiments were performed to determine and refine the optimal eBeam dose range for all developmental stages based on observed fecundity, hatch rate, emergence rate and longevity. The ranges were 0.03–0.12 kGy for eggs, larvae and pupae and 0.06–2.00 kGy for adults. Eggs were divided into early, intermediate and black-headed age groups. No early or intermediate eggs hatched after irradiation even at the lowest dose of 0.03 kGy (Table 3). This radiosensitivity decreased at later developmental stages, as egg hatches were observed at the black-head stage at all doses ranging from 0.03 to 0.12 kGy. Nevertheless, the deleterious treatment effect was apparent because the hatch rate decreased when eBeam dosage increased. Furthermore, no emergence was detected at any tested doses, indicating the inability of hatched larvae from irradiated eggs to complete their life cycle (Table 3). Indeed, when seeds were subsequently cracked open, we found that none lived beyond the first-instar larval stage (data not shown). Sensitivity of larvae to eBeam treatment is reflected by the low emergence rate (Table 4). A very small portion of the second-, third- and fourth-instar larvae survived a dose of 0.03 kGy and emerged as adults. The older larvae had a higher emergence rate and thus may be more tolerant than younger ones. At a dose of 0.06 kGy or higher, however, all larvae
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Figure 2. Negative effects of eBeam on cowpea bruchid adults. (A) Acute mortality (%, means ± SE) was analyzed by Pearson’s 𝜒 2 -test (P < 0.05). (B) Longevity (days, means ± SE) was analyzed using a one-way ANOVA (F 13,419 = 93.31, P 0.05), suggesting that eBeam did not directly interfere with protein digestion, a basic physiological function. Maintaining seemingly normal digestive function did not ensure successful transformation from immature larvae to adults. Although most eBeam-treated fourth-instar larvae molted to pupae, they eventually died as either pupae (data not shown) or as abnormal adults (Fig. 3). The head, antennae, thorax and legs of the eclosed adults appeared to develop normally. The abdomen, however, exhibited mixed traits of pupae and adults; the segments were covered by alternating adult and pupal-like cuticles. Another striking phenotype was the deformed wing structure held laterally.
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3.3 Reciprocal crosses eBeam irradiation at sublethal doses may potentially affect insect reproduction. We performed reciprocal crosses using emerged adults from irradiated pupae that survived eBeam exposure from 0.03 to 0.09 kGy, and observed significantly reduced fecundity and hatch rate (Table 5). When virgin males from irradiated pupae were paired with unirradiated virgin females (UF × IM), effects were dose dependent. Males derived from irradiated pupae were still fertile at 0.09 kGy. In contrast, in the reciprocal cross (i.e. IF × UM), female adults became sterile when their pupae were irradiated at 0.06 kGy or higher doses. Egg hatch rate in the IF × UM cross was lower than
DISCUSSION
The eBeam technology relies on electricity to generate a highly planar stream of high-energy electrons. The initial investment cost of a linear accelerator for eBeam is higher than cobalt-60 as the radiation source for 𝛾-ray, but unit costs of the two types of irradiator are comparable, ranging from 0.5 to 7 cents lb−1 .26 Compared with chemical fumigation, irradiation is twice as expensive or more.27 However, the technology has a number of salient advantages: the process is extremely fast (measured in seconds), does not involve the use of any chemicals or radioactive isotopes and the technology is scalable.6,7,16,21,28 In this study, we have demonstrated that eBeam inhibited cowpea bruchid development at all stages and caused morphological abnormalities. The harmful effects appeared to be dose and developmental stage dependent. Further investigation suggested that not all physiological functions were affected equally; the larval protein digestion was not immediately affected, but reproduction was severely impaired, leading to extinction of the irradiated population at certain doses. Radiosensitivity was apparent at the egg stage, but this sensitivity decreased as eggs continued to develop (Table 3). During early embryogenesis, fertilized insect eggs undergo active mitosis to form a blastoderm, and blastoderm cells further multiply and differentiate, generating ectoderm and mesoderm. DNA deletion, mutation and breakage caused by eBeam ionization, particularly double-strand breaks, could overwhelm the DNA repair system and become lethal to this process. Higher radiosensitivity in the early stage of the embryos has also been observed in hermaphroditic fish (Kryptolebias marmoratus)29 and in tardigrade (Milnesium tardigradum), a small aquatic invertebrate.30 A weak antioxidant defense system of the former at this stage seems to explain the higher developmental impairment relative to later stages, whereas for the latter, vulnerability to radiation is suspected to be attributable to rapid mitotic activity and ineffective
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Table 5. Non-linear regression analyses of fecundity and hatch rate of eBeam-irradiated pupae in reciprocal crosses Pairinga
Dose (kGy)
Fecundityb(eggs female−1 )c
Hatch rate (%)c
UF × UM
0.00
74.4 ± 2.2
96.2 ± 0.4
UF × IM
0.03 0.06 0.09
35.8 ± 4.5 36.8 ± 4.6 26.2 ± 12.7
67.4 ± 6.7 70.2 ± 5.6 41.7 ± 4.9
IF × UM
0.03 0.06 0.09
27.9 ± 5.4 0.0 ± 0.0 0.0 ± 0.0
49.4 ± 6.1 n.a.d n.a.
IF × IM
0.03 0.06 0.09
12.3 ± 2.5 0.0 ± 0.0 0.0 ± 0.0
5.6 ± 1.7 n.a. n.a.
Fecundity R2 = 0.684, A = 71.57 ± 3.42
UF × IM IF × UM IF × IM
B = −14.42 ± 1.78f B = −37.93 ± 4.53 B = −60.45 ± 9.81
Hatch rate, R2 = 0.703, A = 1.35 ± 0.05
UF × IM IF × UM IF × IM
B = −6.28 ± 1.17 B = −18.14 ± 3.25 B = −69.53 ± 13.03
Non-linear regression analyses Y = A* exp(B* X)e
a
UF, untreated female; UM, untreated male; IF, irradiated female; IM, irradiated male.
b Of the entire reproduction period. c Means ± SE. d Not applicable. e Y represents the fecundity or hatch
rate (arcsine square root transformed); X represents the dose; A (means ± SE) is the fecundity or hatch rate of untreated insects; B (means ± SE) is the exponential rate of decrease in fecundity or hatch rate. f The likelihood ratio test was used for pairwise comparisons of the exponential rate of decrease among treatments, P < 0.01.
Table 6. Non-linear regression analyses of fecundity and hatch rate of eBeam-irradiated adults in reciprocal crosses Pairinga
Dose (kGy)
Fecundityb(eggs female−1 )c
Hatch rate (%)c
UF × UM
0.00
71.0 ± 2.1
94.4 ± 0.7
UF × IM
0.06 0.15 0.50 1.00 1.50 2.00
40.8 ± 7.2 43.5 ± 6.6 37.2 ± 5.5 15.3 ± 5.0 11.5 ± 5.0 9.7 ± 5.9
32.3 ± 7.5 29.8 ± 6.5 32.1 ± 7.7 28.8 ± 12.4 19.9 ± 9.3 17.0 ± 9.9
IF × UM
0.06 0.15 0.50 1.00 1.50 2.00
38.9 ± 4.6 30.9 ± 3.5 11.3 ± 1.9 1.8 ± 0.6 0.0 ± 0.0 0.0 ± 0.0
0.8 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 n.a.d n.a.
IM × IF
0.06 0.15 0.50 1.00 1.50 2.00
18.4 ± 3.6 14.7 ± 3.3 2.4 ± 0.7 0.7 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 n.a. n.a.
= 0.551 A = 55.26 ± 2.37
UF × IM IF × UM IF × IM
B = −1.10 ± 0.14f B = −3.86 ± 0.63 B = −13.90 ± 2.26
Hatch rate R2 = 0.495 A = 0.91 ± 0.06
UF × IM IF × UM IF × IM
B = −0.99 ± 0.22 B = −55.66 ± 44.10 n.a.
Non-linear regression analyses Y
= A* exp(B* X)e
Fecundity R2
a UF, untreated female; UM, untreated male; IF, irradiated female; IM, irradiated male. b Of the entire reproduction period. c Means ± SE. d Not applicable. e Y represents the
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fecundity or hatch rate (arcsine square root transformed); X represents the dose; A (means ± SE) is the fecundity or hatch rate of untreated insects; B (means ± SE) reflects the exponential rate of decrease in fecundity or hatch rate. f The likelihood ratio test was used for pairwise comparisons of the exponential rate of decrease among treatments, P < 0.01.
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Detrimental effects of electron beam irradiation on the cowpea bruchid
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Figure 4. eBeam inhibits oocyte development. Shown are female reproductive systems of adults emerged from irradiated pupae. Scale bars: 1 mm. oo, oocyte; ov, ovary; gc, genital chamber.
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their midgut epithelia, which may contribute to the decreased competitiveness of the flies in nature, affecting the efficacy of SIT programs.38 In cowpea bruchids, however, larval protein digestion apparently was unaffected by irradiation. Different treatment regimes may provide some insight into the disagreeing results; the fruit flies were irradiated at pupal stage and their midgut structure was examined after adult eclosion, whereas the eBeam treatment and proteolytic assay of cowpea bruchids occurred within the same larval stage. It is known that irradiation, particularly at low dose ranges, has a stronger impact on the formation of a tissue than on existing tissue. Thus, the irradiated bruchid larvae most likely maintained similar levels of food consumption until they approached their next molt. For non-feeding adults, 2 kGy only caused approximately 50% acute mortality. However, 0.06 kGy drastically reduced fecundity, and no eggs were viable. For stored grain protection, sterilizing insects with low-dose radiation to impede reproduction and prevent maturation ought to be a better strategy than attempting to achieve 100% mortality at a substantially higher dose. Our results indicated that 0.06 kGy should be sufficient to prevent propagation of the irradiated cowpea bruchids. For practical applications, however, this recommended dose has to be verified by trials with much larger insect populations. In addition, different bruchid strains or the same strain under different rearing conditions may vary in their radiosensitivity, similarly to our observations in a number of life-history and genetic parameters.39 Although post-irradiation feeding by developmentally blocked yet live larvae is an unwanted effect, early irradiation when pest numbers are low presumably could minimize the grain damage while avoiding alteration of the physical, physicochemical and nutritional properties of the stored grains.
5
CONCLUSION
As an alternative to chemical fumigation, eBeam could become an important component in post-harvest technologies to protect stored grains. Appropriate irradiation doses are only available for some storage insects,11 necessitating more studies to expand the information. Generic radiation doses have to be carefully established because different insect species and different developmental stages often exhibit differing radiosensitivity.37,40 – 43 To achieve high confidence, environmental factors that may affect irradiation efficacy, such as temperature, host and oxygen content,20,44 – 48
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DNA repair processes associated with the early egg stage. While no egg hatches occurred at any doses during early and intermediate developmental stages, a substantial portion of black-headed eggs hatched apparently normally (Table 3). Presumably, major DNA replication related to embryogenesis has completed at this stage. However, erroneous repairs may explain the fact that eventually no hatched larvae completed their life cycle. Larval development involves increases in the cell number and cell size of the epidermis and internal structures. Failing to reach adult stage from irradiated larvae most likely was due to unsuccessful mitosis and cell division.31 The fact that younger insects had to go through more mitoses could contribute to the higher susceptibility in earlier developmental stages. Damage resulting from the electron shower or radicals would likely be amplified in each subsequent DNA replication and cell division. During pupation, more profound changes, including histolysis and phagocytosis of the larval tissues and reconstruction of adult tissues, occur in the transformation from larvae to adults. eBeam interfered with the metamorphosis of cowpea bruchids, which was reflected by the forming of physically deformed adults (Fig. 3). A pupal-like cuticle could result from unwanted juvenile hormone (JH) action at this stage.32,33 It has been shown in the red flour beetle that disruption of pupal specifier broad (br) gene caused the formation of abnormal wings and the appearance of pupal traits in adults.34 The expression of the br gene requires JH, and their coordination is necessary to turn off the JH system during the pupal–adult molt.35 It is possible that eBeam disrupted hormone signaling pathways, and that failure to shut down the JH system resulted in a pupal-like cuticle. Wing deformities may also result from eBeam damage of the imaginal discs. A healthy reproductive system is essential for success of any insect species in nature. Although irradiated pupae exhibited a higher emergence rate relative to larvae, eBeam disrupted the development of the reproductive systems and interfered with germline stem cell division which leads to the formation of oocytes (Fig. 4). Furthermore, in spite of the intact appearance of ovaries and oocytes (data not shown), the reproductive function of irradiated adults was sufficiently destroyed by eBeam at 0.06 kGy or above (Table 6). Reciprocal crossing revealed that females are more susceptible to eBeam than males in terms of reproduction, which is consistent with observations in other species.19,20,36,37 It has been shown that irradiation of Mediterranean fruit fly (Ceratitis capitata) and Mexican fruit fly (Anastrepha ludens) damages
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ACKNOWLEDGEMENTS We would like to thank Dr Ron Salzman for his critical review of the manuscript. This project was supported by USDA AFRI grant 2014-67013-21781 and by the China Scholarship Council.
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