Mortality and growth inhibition of γ-irradiated destructor (: ) on mango (Sapindales: Anacardiaceae) plantlets Inamullah Khan1,*, Bitani Salahuddin2, and Habib Ur Rahman3

Abstract 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 ; 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 twigs, branches and roots (Salahuddin et al. 2015; Suomi 1996; Nabity in more than 100 countries of the world (Galán Saúco 2002) and is con- et al. 2009). They cause severe damage to mango trees and depress sidered as the “king of fruits” (Tharanathan et al. 2006; Singh & Singh photosynthesis and plant respiration, inject toxins, transmit viruses 2011). It is an important fruit of tropical areas, and in South Asia it has and excrete honey dew, which serves as a growth medium for fun- been known for the last 4,000 yr (Salunkhe & Desai 1984). Mango is a gi causing sooty mold. In heavily infested trees the yield is reduced rich source of carbohydrates, vitamins A and C, iron, potassium, calci- mainly by reduced photosynthesis and respiration (Elwan 1990). Scale um and protein (Baloch 1994). In Pakistan, mango is ranked as the sec- insects may be quarantine pests and fruits infested with scale insects ond most important fruit after citrus and the third most important cash may be rejected at the port of entry. crop after cotton and rice. During 2010–2011, the area under mango Recently irradiation has been adopted as a safe measure for the crops was 117.9 km2 and its total production was 1,885,900 t. Paki- disinfestation of quarantine pests and has gained much importance stan is ranked fourth among mango producing and exporting countries in the export of agricultural fresh commodities (Heather & Hallman (Anonymous 2005). Mango is mainly exported to Dubai, Saudi Arabia, 2008). Phytosanitary irradiation has advantages over other posthar- Oman, the United Kingdom, Kuwait, Bahrain, France, and Germany. vest treatments, e.g., phytosanitary irradiation can be developed Scale insects (Hemiptera: : Coccoidea) are very for a pest species irrespective of the host. Furthermore, irradiation important sucking pests of mango and many other plant taxa. Scale does not affect the quality of commodities at doses that nullify the insects feed on the various parts of their host plants such as leaves, pest.

1Nuclear Institute of Food and Agriculture (NIFA), Tarnab, Peshawar, Pakistan 2Department of Entomology, Faculty of Agriculture, Gomal University, Dera Ismail Khan, Pakistan 3Department 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.

2016 — Florida Entomologist — Volume 99, Special Issue 2 125 126 2016 — Florida Entomologist — Volume 99, Special Issue 2 Limited data are available on disinfestation of scale insects (Follett the chamber where samples were placed was conducted to verify the 2006a, b). Currently, the Government of Pakistan has adopted a radia- accuracy and range of the doses applied. The absorbed dose was mea- tion dose of 400 Gy for disinfestations of fruit flies and other insects sured using Fricke dosimeters at 3 irradiation positions (top, bottom, including armored scales (Diaspididae). Specific studies on irradiation and center) of infested plants (Table 1). Responses to irradiation doses doses that would inhibit the development of scales, reduce the risk of between 100–300 Gy of 1st and 2nd instars and adult females either damage to fresh commodities as well as reduce costs and duration of pre-ovipositional or gravid were examined. Individual plants with a treatment are required and could be used to develop a generic phyto- specific stage of the scale served as replicates and each treatment was sanitary irradiation dose for use against scale insects. replicated 3–4 times depending on the number of scales on the plants. The current study aims to determine the minimum effective irra- After irradiation, plants were transported to the Nuclear Institute for diation dose to inhibit the development of nymphs and the emergence Food and Agriculture and kept in the greenhouse at 30 ± 2 °C, and 60 ± and reproduction of adults of the scale, Aspidiotus destructor 5% RH. Scales on plants were examined twice per week to determine Signoret (Hemiptera: Diaspididae). Follett (2006a) developed a phyto- the number that had developed to the next stage. As the scale body is sanitary irradiation treatment against A. destructor on fruits of Cucur- translucent and eggs are visible under the scale cover, the eggs under bita moschata Duchesne ex Poir. (Cucurbitales: Cucurbitaceae).A dose the body cover of 10 gravid females were counted using a magnifying of 150 Gy was supported via irradiation of 32,716 gravid adult females lens. Recording of data was discontinued when scales atrophied and resulting in no gravid F1 adults, although one sterile non-gravid F1 adult no longer progressed to the next stage. Untreated controls from each developed for every 23 parent generation gravid female adults irradi- stage were held under identical conditions and examined similarly. ated. This dose was accepted by APHIS (2016). The present study will use an end point that does not allow for significant F1 development, as LARGE SCALE VALIDATION TESTS that might not be acceptable to some plant protection organizations Large scale confirmatory tests were conducted to validate the es- (Hallman et al. 2010). timated 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 Materials and Methods scales were irradiated with 200 and 220 Gy and non-irradiated con- trol, respectively. Number of 1st instars and adults at F1 generation was noted for each treatment. ESTABLISHMENT OF A. DESTRUCTOR CRAWLERS ON NURSERY PLANTS DATA ANALYSIS Mango plants of uniform age and size in plastic pots of 30 × 35 Data on the effect of gamma irradiation on mortality/survival were cm were placed on mango infested plants. Branches and leaves of the subjected to analysis of variance (ANOVA) after testing for equal vari- nursery plants were attached to the infested leaves of field plants. ances and normality. Means separations were done using Tukey’s test. Some heavily scale-infested leaves from surrounding branches were All statistical analyses were done using Statistix 8.1 (Analytical- Soft cut off and wrapped around the upper portions of nursery plants. ware, Tallahassee, Florida). In addition, percentage mortality and adult When crawlers moved and settled on the nursery plants, the newly inhibition data were subjected to probit analysis using PoloPlus (LeOra infested potted mango plants were removed from the field and placed Software, Petaluma, California) to estimate the dose response of -ex in a lath house. Batches of 6–7 plants per dose were infested in the posed eggs, nymphs, and adults. 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 Results 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 Table 2 shows the responses of scale insects irradiated as 1st instars was marked and divided into square centimeters and scale counts were (white cap) stage. When irradiated with 100 Gy, 7.9% of scales matured taken from at least 4 squares and values were extrapolated for the to gravid female and laid eggs while scales irradiated with doses ≥150 whole treatment area. Gy did not survive to develop to subsequent instars. No acute mortal- ity in control treatments was recorded. Table 3 shows the response of irradiated 2nd instars. Second instars were relatively tolerant and IRRADIATION reached the gravid stage except when irradiated at 250 Gy. When irra- All infested plants were transferred to the commercial irradiator diated with 100 Gy, 68% of scales developed to become gravid females. at the Pakistan Radiation Services facility, Lahore, about 500 km away, Twenty five and 22 percent of scales developed to gravid females when where irradiation studies were conducted using a product overlap irradiated with 150 and 200 Gy, respectively. All insects stopped grow- source 60Co irradiator (MDS Nordion, Ottawa, Ontario, Canada) with an ing and died after few d when irradiated at doses ≥ 250 Gy. Mortality in activity of approximately 1.74 PBq. Dose mapping of the area within control treatments was 16%.

Table 1. Measurement of absorbed doses of gamma radiation delivered by the commercial MDS Nordion60 Co irradiator, which was used for irradiation ofAspidiotus destructor scale insects on mango plantlets.

Target dose (Gy) Minimum dose (Gy) Maximum dose (Gy) Average dose (Gy) Dose uniformity ratio 100 93.33 99.67 95.00 1.06 150 140.60 150.00 145.30 1.06 200 191.00 202.60 196.50 1.06 250 243.66 252.67 248.16 1.04 300 281.33 300.03 290.68 1.06 Khan et al.: Effect of gamma irradiation on Aspidiotus destructor on mango 127

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

Number of Number of Number of adult females Number of Gravid Target dose (Gy) 1st instars 2nd instars (non-gravid) gravid females females* (%) 0 14,158 14,158 14,158 8,979 53.8 a 100 1,105 1,007 1,007 87 7.87 b 150 0 0 0 0 0 c 200 0 0 0 0 0 c 250 0 0 0 0 0 c 300 0 0 0 0 0 c

*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 radiation of eggs and 178.9 Gy for irradiation of 1st instars, respective- with and without eggs. They exhibited a relatively higher tolerance to ly. Second instars were comparatively more tolerant and 217 Gy was gamma irradiation. When irradiated with 100 Gy, 54% of scales devel- estimated to cause 99.99% inhibition. In large scale confirmatory tests oped to become gravid females. Forty four and 34 percent of scales (Table 7), 220 Gy to gravid females completely prevented development developed to gravid female when irradiated with 150 and 200 Gy re- of F1 scales beyond the egg stage, i.e., no production of F1 generation spectively. No acute mortality in control treatments was recorded. 1st instars. The number of 1st instars per female in the control—i.e., As shown in Table 6, probit analysis for 99.99% prevention of devel- 553,727 1st instars from 43,274 P generation adult females (Table 7) = opment to the next stage provided estimated doses of 156.9 Gy for ir- 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 Number of adult females Number of gravid females Gravid females (%) 0 1,568 1,551 1,314 83.80a 100 1,770 1,325 1,208 68.20b 150 1,402 385 344 24.54c 200 1,359 332 302 22.22c 250 1,426 9.47 0 0 d 300 1,020 0 0 0 d

Different letters within columns indicate significant differences using Tukey’s HSD test atP >/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 Number gravid females Adult females that became gravid (%) No. eggs per female 0 2,116 1,778 84.02a 47.6 ± 05 100 3,076 1,661 54.01b 33.5 ± 04 150 2,992 1,324 44.24bc 7.0 ± 11 200 2,456 840 34.2c 1.0 ± 01 250 2,670 0.00 0.0d 0.0 300 2,007 0.00 0.0d 0.0

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

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

No. Approx. Total Gravid Dose (GY) parent females no. eggs under female 1st instars 2nd instars Adults gravid females females (%)* 0 355 16,685 14,158 14,158 14,158 8,979 53.8a 100 311 14,611 1,105 1,007 1,007 87 27.97b 150 385 15,091 0 0 0 0 0c 200 272 7,184 0 0 0 0 0c 250 289 7,656 0 0 0 0 0c 300 221 7,211 0 0 0 0 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). 128 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 0 100 150 200 250 300 Slope ± SD ED99.99 (GY)* (95% CI) Egg 18.08 85.83 99.02 100 100 100 12.32 ± 0.6 156.93 (151.1–164.3) 1st instar 17.80 87.75 98.27 100 100 100 7.25 ± 0.6 176.88 (161.2–204.7) 2nd instar 19.35 27.62 81.63 96.36 100 100 11.90 ± 0.8 217.66 (206.9–233.4) adult female 16.91 28.73 63.47 96.33 100 100 19.77 ± 0.95 215.08 (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.

No. of Measured No. of No. of No. of

Target dose (Gy) infested plants dose (Gy) P generation adult scales F1 generation 1st instars* F1 generation adults Control 12 — 43,274 553,727 531,939 200 10 190–202 40,531 201 3 220 12 210–224 51,101 0 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 Acknowledgment in the control was only taken once to determine that the females were reproducing, and not as a measure of total reproduction. This work was part of the FAO/IAEA Coordinated Research Project D62008 on Development of Generic Irradiation Doses for Quarantine Discussion 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 In our study A. destructor scales were irradiated as eggs, 1st instars, Associate, for data recording. This research was financially supported 2nd instars, and adult females—both pre-ovipositional and gravid. by IAEA Research Contract No. PAK/16894. We are thankful for this Follett (2006a) irradiated the same species as 2nd instars and adult financial contribution and for technical guidance during the various females—both pre-ovipositional and gravid. In our study 2nd instars coordination meetings. 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 References Cited was observed for the conversion of irradiated non-gravid female adults to gravid ones, since irradiation with 200 Gy cause 11.9% conversion in Anonymous. 2005. Fruit, Vegetable and Condiment Ministry of Food, Agricul- Follett (2006a) vs. 0% in our study. ture and Livestock, Islamabad. However, since the most radiotolerant stage of A. destructor of APHIS [ and Plant Health Inspection Service]. 2016. Treatment Manual. phytosanitary concern is the gravid adult female, it does not matter for US Department of Agriculture, Washington, DC, USA. Baloch AF. 1994. Horticulture-Mango, National Book Foundation, Islamabad. phytosanitary purposes if the preceding and more susceptible instars Elwan E. 1990. Ecological and biological studies on certain pests of Coc- of the 2 populations differ in radiotolerance. At the common doses coidea (Homoptera) infesting mango trees. Ph.D. Thesis, Faculty of Agricul- used against gravid adults between the 2 studies (100 and 200 Gy), ture, Al-Azhar University, Egypt. 175 pp. Follett (2006a) found that when gravid females were irradiated with Follett PA. 2006a. Irradiation as a phytosanitary treatment for Aspidiotus de- structor (Homoptera: Diaspididae). Journal of Economic Entomology 99: 100 Gy, 18.2% of the F eggs developed to 2nd instars, while that corre- 1 1138-1142. sponding fraction in the present study was 7.6%. Further development Follett PA. 2006 b. Irradiation as a phytosanitary treatment for white peach scale to F1 adults at that dose was 0.0035% per irradiated (100 Gy) parent (Homoptera: Diaspididae). Journal of Economic Entomology 99: 1974-1978. adult in Follett (2006a) and 0.28% for the present study. At 200 Gy both Galán Saúco V. 2002. Mango production and world market: Current situation studies found no F 2nd instars. and future prospects. VII International Mango Symposium 645. pp. 107-116. 1 Hallman GJ, Levang-Brilz NM, Zettler JL, Winborne IC.2010. Factors affecting Based on Follett (2006a) APHIS (2016) accepted a phytosanitary ir- ionizing radiation phytosanitary treatments, and implications for research radiation dose of 150 Gy for hosts ofA. destructor with the measure of and generic treatments. Journal of Economic Entomology 103: 1950-1963. Heather NW, Hallman GJ. 2008. Pest management and phytosanitary trade bar- efficacy being prevention of gravid F1 females. The present study uses a more conservative measure of efficacy, i.e., the prevention of F 1st riers. CABI, Wallingford, UK. 1 Nabity PD, Zavala JA, Delucia EH. 2009. Indirect suppression of photosynthesis instars, because some plant protection organizations may not agree on individual leaves by herbivory. Annals of Botany 103: 655-663. with a measure of efficacy that might allow for F1 adult formation. We Salahuddin, ur Rahman H, Khan I, Daud, MK, Rashid MM. 2015. Biology of co- found that F1 1st instars were prevented with 220 Gy. conut scale, Aspidiotus destructor Signoret (Hemiptera: Diaspididae), on Khan et al.: Effect of gamma irradiation on Aspidiotus destructor on mango 129

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