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Measurement of absorbed doses of gamma radiation delivered by the commercial MDS Nordion 60Co irradiator, which was used for irradiation of Aspidiotus.

Mortality and growth inhibition of γ-irradiated Aspidiotus destructor (Hemiptera: Diaspididae) on mango (Sapindales: Anacardiaceae) plantlets Inamullah Khan1,*, Bitani Salahuddin2, and Habib Ur Rahman3 Abstract Aspidiotus destructor Signoret (Homoptera: Diaspididae) is a quarantine pest of mango (Mangifera indica L.; Sapindales: Anacardiaceae) and many other tropical crops. Irradiation was examined as a potential phytosanitary treatment to control A. destructor. Dose response tests were conducted with eggs, 1st and 2nd instars, pre-ovipositing adult females and ovipositing females with a series of radiation doses between 100 and 300 Gy to determine the most tolerant stage. The egg was found to be the most susceptible and adult the most tolerant stage. From probit analysis a dose of 217.7 Gy was estimated as the effective dose to completely stop scale development to subsequent stages. In large-scale validation tests 40,531 ± 79 scales in all stages were tested with 200 Gy and 51,101 ± 117 scales in all stages with 220 Gy (total = 91,633 scales). Scales did not develop to the subsequent stage when exposed to a dose of 220 Gy. We therefore propose a dose of 220 Gy for complete inhibition of development of all life stages of A. destructor that would provide quarantine security for A. destructor on exported commodities. Key Words: scale insects; phytosanitary; growth inhibition; export; quarantine pests; mango scale

Resumen Aspidiotus destructor Signoret (Hemiptera: Diaspididae) es una plaga cuarentenaria de mango (Mangifera indica L.; Sapindales: Anacardiaceae) y muchos otros cultivos tropicales. Se examinó la irradiación como un tratamiento fitosanitario potencial para controlar A. destructor. Se realizaron pruebas de respuesta a la dosis con los huevos, las ninfas del primero y segundo estadio, las hembras adultas del tercer estadio sin huevos, y las hembras adultas con huevos en una serie de dosis de radiación entre 100 y 300 Gy para determinar el estadio más tolerante. Se encontró que el estadio de huevo fue el más susceptible y las ninfas de segundo estadio fue el más tolerante. De un análisis del Log dosis, se determinó que una dosis de 217.7 Gy es la dosis eficaz para detener completamente el desarrollo de la escama a los estadios posteriores. En pruebas de validación de gran escala, se analizaron con todas los estadios de escamas, 40,531 ± 79 escamas a 200 Gy y 51,101 ± 117 a 220 Gy (total = 91,633 escamas). Las escamas no se desarrollaron más cuando fueron expuestas a una dosis de 220 Gy. Por lo tanto, proponemos una dosis de 220 Gy para la inhibición completa del desarrollo de todos los estadios de vida de A. destructor y su reproducción con lo que se proveerá la seguridad cuarentenaria contra A. destructor sobre los productos básicos exportados. Palabras Clave: cochinillas; fitosanitaria; inhibición del crecimiento; exportación; plagas cuarentena; escama de mango

Mango (Mangifera indica L.; Sapindales: Anacardiaceae) is grown in more than 100 countries of the world (Galán Saúco 2002) and is considered as the “king of fruits” (Tharanathan et al. 2006; Singh & Singh 2011). It is an important fruit of tropical areas, and in South Asia it has been known for the last 4,000 yr (Salunkhe & Desai 1984). Mango is a rich source of carbohydrates, vitamins A and C, iron, potassium, calcium and protein (Baloch 1994). In Pakistan, mango is ranked as the second most important fruit after citrus and the third most important cash crop after cotton and rice. During 2010–2011, the area under mango crops was 117.9 km2 and its total production was 1,885,900 t. Pakistan is ranked fourth among mango producing and exporting countries (Anonymous 2005). Mango is mainly exported to Dubai, Saudi Arabia, Oman, the United Kingdom, Kuwait, Bahrain, France, and Germany. Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are very important sucking pests of mango and many other plant taxa. Scale insects feed on the various parts of their host plants such as leaves,

twigs, branches and roots (Salahuddin et al. 2015; Suomi 1996; Nabity et al. 2009). They cause severe damage to mango trees and depress photosynthesis and plant respiration, inject toxins, transmit viruses and excrete honey dew, which serves as a growth medium for fungi causing sooty mold. In heavily infested trees the yield is reduced mainly by reduced photosynthesis and respiration (Elwan 1990). Scale insects may be quarantine pests and fruits infested with scale insects may be rejected at the port of entry. Recently irradiation has been adopted as a safe measure for the disinfestation of quarantine pests and has gained much importance in the export of agricultural fresh commodities (Heather & Hallman 2008). Phytosanitary irradiation has advantages over other postharvest treatments, e.g., phytosanitary irradiation can be developed for a pest species irrespective of the host. Furthermore, irradiation does not affect the quality of commodities at doses that nullify the pest.

Nuclear Institute of Food and Agriculture (NIFA), Tarnab, Peshawar, Pakistan Department of Entomology, Faculty of Agriculture, Gomal University, Dera Ismail Khan, Pakistan 3 Department of Horticulture, Faculty of Agriculture, Gomal University, Dera Ismail Khan, Pakistan *Corresponding author; E-mail: [email protected]; mailto:[email protected] Copyright © International Atomic Energy Agency 2016. Published by the Florida Entomological Society. All rights reserved. 1 2

2016 — Florida Entomologist — Volume 99, Special Issue 2



2016 — Florida Entomologist — Volume 99, Special Issue 2

Limited data are available on disinfestation of scale insects (Follett 2006a, b). Currently, the Government of Pakistan has adopted a radiation dose of 400 Gy for disinfestations of fruit flies and other insects including armored scales (Diaspididae). Specific studies on irradiation doses that would inhibit the development of scales, reduce the risk of damage to fresh commodities as well as reduce costs and duration of treatment are required and could be used to develop a generic phytosanitary irradiation dose for use against scale insects. The current study aims to determine the minimum effective irradiation dose to inhibit the development of nymphs and the emergence and reproduction of adults of the coconut scale, Aspidiotus destructor Signoret (Hemiptera: Diaspididae). Follett (2006a) developed a phytosanitary irradiation treatment against A. destructor on fruits of Cucurbita moschata Duchesne ex Poir. (Cucurbitales: Cucurbitaceae).A dose of 150 Gy was supported via irradiation of 32,716 gravid adult females resulting in no gravid F1 adults, although one sterile non-gravid F1 adult developed for every 23 parent generation gravid female adults irradiated. This dose was accepted by APHIS (2016). The present study will use an end point that does not allow for significant F1 development, as that might not be acceptable to some plant protection organizations (Hallman et al. 2010).

Materials and Methods ESTABLISHMENT OF A. DESTRUCTOR CRAWLERS ON NURSERY PLANTS Mango plants of uniform age and size in plastic pots of 30 × 35 cm were placed on mango infested plants. Branches and leaves of the nursery plants were attached to the infested leaves of field plants. Some heavily scale-infested leaves from surrounding branches were cut off and wrapped around the upper portions of nursery plants. When crawlers moved and settled on the nursery plants, the newly infested potted mango plants were removed from the field and placed in a lath house. Batches of 6–7 plants per dose were infested in the same way at 4–7 d intervals. Scales on prior infested plants developed to the next stage while freshly infested plants were added into the lot. Plant infestation continued until at least 6–7 plants were infested with each stage of the scale. Leaves on the abaxial side were marked on plants with the specific stage of the scale. The infested area of the plant was marked and divided into square centimeters and scale counts were taken from at least 4 squares and values were extrapolated for the whole treatment area.

IRRADIATION All infested plants were transferred to the commercial irradiator at the Pakistan Radiation Services facility, Lahore, about 500 km away, where irradiation studies were conducted using a product overlap source 60Co irradiator (MDS Nordion, Ottawa, Ontario, Canada) with an activity of approximately 1.74 PBq. Dose mapping of the area within

the chamber where samples were placed was conducted to verify the accuracy and range of the doses applied. The absorbed dose was measured using Fricke dosimeters at 3 irradiation positions (top, bottom, and center) of infested plants (Table 1). Responses to irradiation doses between 100–300 Gy of 1st and 2nd instars and adult females either pre-ovipositional or gravid were examined. Individual plants with a specific stage of the scale served as replicates and each treatment was replicated 3–4 times depending on the number of scales on the plants. After irradiation, plants were transported to the Nuclear Institute for Food and Agriculture and kept in the greenhouse at 30 ± 2 °C, and 60 ± 5% RH. Scales on plants were examined twice per week to determine the number that had developed to the next stage. As the scale body is translucent and eggs are visible under the scale cover, the eggs under the body cover of 10 gravid females were counted using a magnifying lens. Recording of data was discontinued when scales atrophied and no longer progressed to the next stage. Untreated controls from each stage were held under identical conditions and examined similarly.

LARGE SCALE VALIDATION TESTS Large scale confirmatory tests were conducted to validate the estimated dose to prevent reproduction of the most tolerant stage, i.e., the adult female. A total of 40,531, 51,101, and 43,274 adult female scales were irradiated with 200 and 220 Gy and non-irradiated control, respectively. Number of 1st instars and adults at F1 generation was noted for each treatment.

DATA ANALYSIS Data on the effect of gamma irradiation on mortality/survival were subjected to analysis of variance (ANOVA) after testing for equal variances and normality. Means separations were done using Tukey’s test. All statistical analyses were done using Statistix 8.1 (Analytical Software, Tallahassee, Florida). In addition, percentage mortality and adult inhibition data were subjected to probit analysis using PoloPlus (LeOra Software, Petaluma, California) to estimate the dose response of exposed eggs, nymphs, and adults.

Results Table 2 shows the responses of scale insects irradiated as 1st instars (white cap) stage. When irradiated with 100 Gy, 7.9% of scales matured to gravid female and laid eggs while scales irradiated with doses ≥150 Gy did not survive to develop to subsequent instars. No acute mortality in control treatments was recorded. Table 3 shows the response of irradiated 2nd instars. Second instars were relatively tolerant and reached the gravid stage except when irradiated at 250 Gy. When irradiated with 100 Gy, 68% of scales developed to become gravid females. Twenty five and 22 percent of scales developed to gravid females when irradiated with 150 and 200 Gy, respectively. All insects stopped growing and died after few d when irradiated at doses ≥ 250 Gy. Mortality in control treatments was 16%.

Table 1. Measurement of absorbed doses of gamma radiation delivered by the commercial MDS Nordion 60Co irradiator, which was used for irradiation of Aspidiotus destructor scale insects on mango plantlets. Target dose (Gy) 100 150 200 250 300

Minimum dose (Gy) 93.33 140.60 191.00 243.66 281.33

Maximum dose (Gy) 99.67 150.00 202.60 252.67 300.03

Average dose (Gy)

Dose uniformity ratio

95.00 145.30 196.50 248.16 290.68

1.06 1.06 1.06 1.04 1.06

Khan et al.: Effect of gamma irradiation on Aspidiotus destructor on mango


Table 2. Effect of γ-irradiation of Aspidiotus destructor 1st instars on their development and transformation into subsequent life stages.

Target dose (Gy) 0 100 150 200 250 300

Number of 1st instars

Number of 2nd instars

Number of adult females (non-gravid)

14,158 1,105 0 0 0 0

14,158 1,007 0 0 0 0

14,158 1,007 0 0 0 0

Number of gravid females

Gravid females* (%)

8,979 87 0 0 0 0

53.8 a 7.87 b 0c 0c 0c 0c

*Approximately 46.2% of scales were males. No mortality was recorded in control treatments. Different letters within a column indicate significant differences using Tukey’s HSD test at (P >/0.05) among treatments (0, 100, 150, 200, and 250 Gy).

Tables 4 and 5 show the response to irradiation of female scales with and without eggs. They exhibited a relatively higher tolerance to gamma irradiation. When irradiated with 100 Gy, 54% of scales developed to become gravid females. Forty four and 34 percent of scales developed to gravid female when irradiated with 150 and 200 Gy respectively. No acute mortality in control treatments was recorded. As shown in Table 6, probit analysis for 99.99% prevention of development to the next stage provided estimated doses of 156.9 Gy for ir-

radiation of eggs and 178.9 Gy for irradiation of 1st instars, respectively. Second instars were comparatively more tolerant and 217 Gy was estimated to cause 99.99% inhibition. In large scale confirmatory tests (Table 7), 220 Gy to gravid females completely prevented development of F1 scales beyond the egg stage, i.e., no production of F1 generation 1st instars. The number of 1st instars per female in the control—i.e., 553,727 1st instars from 43,274 P generation adult females (Table 7) = 12.79/ female—was less than the 28-65 eggs/female reported by Sala-

Table 3. Effect of γ-irradiation of Aspidiotus destructor 2nd instars on their development and transformation into subsequent life stages. Target dose (Gy)

Number of 2nd instars

0 100 150 200 250 300

Number of adult females

1,568 1,770 1,402 1,359 1,426 1,020

Number of gravid females

1,551 1,325 385 332 9.47 0

Gravid females (%)

1,314 1,208 344 302 0 0

83.80a 68.20b 24.54c 22.22c 0d 0d

Different letters within columns indicate significant differences using Tukey’s HSD test at P >/0.05 among treatments (0, 100, 150, 200 and 250 Gy).

Table 4. Effect of γ-irradiation of Aspidiotus destructor pre-ovipositional adult females on their maturation into gravid females. Dose (Gy)

Number adult females

0 100 150 200 250 300

Number gravid females

2,116 3,076 2,992 2,456 2,670 2,007

Adult females that became gravid (%)

1,778 1,661 1,324 840 0.00 0.00

No. eggs per female

84.02a 54.01b 44.24bc 34.2c 0.0d 0.0d

47.6 ± 05 33.5 ± 04 7.0 ± 11 1.0 ± 01 0.0 0.0

Different letters within a column indicate significant differences using Tukey’s HSD test at P >/0.05 among treatments (0, 100, 150, 200 and 250 Gy).

Table 5. Effects of various γ-irradiation doses applied to the eggs under Aspidiotus destructor gravid females showing the subsequent numbers of F1 1st instars, 2nd instars, and adult pre-ovipositional and gravid females.

Dose (GY) 0 100 150 200 250 300

No. parent females

Approx. no. eggs under female

1st instars

2nd instars


Total gravid females

355 311 385 272 289 221

16,685 14,611 15,091 7,184 7,656 7,211

14,158 1,105 0 0 0 0

14,158 1,007 0 0 0 0

14,158 1,007 0 0 0 0

8,979 87 0 0 0 0

Gravid females (%)* 53.8a 27.97b 0c 0c 0c 0c

*Approximately 55% of progeny were females. Different letters within the right column indicate significant differences using Tukey’s HSD test at (P > 0.05) among treatments (0, 100, 150, 200 and 250 Gy).


2016 — Florida Entomologist — Volume 99, Special Issue 2

Table 6. Probit analysis for percent inhibition of development of various developmental stages of Aspidiotus destructor at 60 d after various γ-irradiation doses were applied to each of the indicated life stages. Inhibition of development to next stage (%) Dose (Gy) Development stage irradiated








1st instar




2nd instar




adult female







Slope ± SD

ED99.99 (GY)* (95% CI)




12.32 ± 0.6




7.25 ± 0.6




11.90 ± 0.8




19.77 ± 0.95

156.93 (151.1–164.3) 176.88 (161.2–204.7) 217.66 (206.9–233.4) 215.08 (208.3–223.7)

*ED means effective dose, e.g., ED99.99 means the dose that is effective in to causing 99.99% inhibition of development. CI means confidence interval.

Table 7. Large-scale validation tests on effects of γ-irradiation of Aspidiotus destructor gravid females on mango plantlets.

Target dose (Gy) Control 200 220

No. of infested plants

Measured dose (Gy)

No. of P generation adult scales

12 10 12

— 190–202 210–224

43,274 40,531 51,101

No. of F1 generation 1st instars* 553,727 201 0

No. of F1 generation adults 531,939 3 0

*Average number of F1 generation 1st instars per P generation female = (553727/43274) = 12.79.

huddin et al (2015). However, in the current study, a count of 1st instars in the control was only taken once to determine that the females were reproducing, and not as a measure of total reproduction.

Discussion In our study A. destructor scales were irradiated as eggs, 1st instars, 2nd instars, and adult females—both pre-ovipositional and gravid. Follett (2006a) irradiated the same species as 2nd instars and adult females—both pre-ovipositional and gravid. In our study 2nd instars appeared to be considerably more radiotolerant when measured as irradiated 2nd instars that developed into gravid adults, as 22% did so when irradiated with 200 Gy vs. 0.36% in Follett (2006a). The opposite was observed for the conversion of irradiated non-gravid female adults to gravid ones, since irradiation with 200 Gy cause 11.9% conversion in Follett (2006a) vs. 0% in our study. However, since the most radiotolerant stage of A. destructor of phytosanitary concern is the gravid adult female, it does not matter for phytosanitary purposes if the preceding and more susceptible instars of the 2 populations differ in radiotolerance. At the common doses used against gravid adults between the 2 studies (100 and 200 Gy), Follett (2006a) found that when gravid females were irradiated with 100 Gy, 18.2% of the F1 eggs developed to 2nd instars, while that corresponding fraction in the present study was 7.6%. Further development to F1 adults at that dose was 0.0035% per irradiated (100 Gy) parent adult in Follett (2006a) and 0.28% for the present study. At 200 Gy both studies found no F1 2nd instars. Based on Follett (2006a) APHIS (2016) accepted a phytosanitary irradiation dose of 150 Gy for hosts of A. destructor with the measure of efficacy being prevention of gravid F1 females. The present study uses a more conservative measure of efficacy, i.e., the prevention of F1 1st instars, because some plant protection organizations may not agree with a measure of efficacy that might allow for F1 adult formation. We found that F1 1st instars were prevented with 220 Gy.

Acknowledgment This work was part of the FAO/IAEA Coordinated Research Project D62008 on Development of Generic Irradiation Doses for Quarantine Treatments. The author would like to thank Dr. Ihsanullah, Director, Nuclear Institute for Food and Agriculture, and Tariq Nawaz for their help in irradiation of research samples, and Sulman Shah, Research Associate, for data recording. This research was financially supported by IAEA Research Contract No. PAK/16894. We are thankful for this financial contribution and for technical guidance during the various coordination meetings.

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