Introduction. Materials and methods. An Open Access Journal published by ICRISAT

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1 Use of indices based on consumption and utilization of food as a criterion to evaluate putative transgenic pigeonpea plants for resistance to pod borer Helicoverpa armigera SVS Gopala Swamy 1, 2, HC Sharma 1 *, C Siva Kumar 1, KK Sharma 1 and GV Subbaratnam 2 1. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru , Andhra Pradesh, India 2. Acharya NG Ranga Agricultural University, Rajendranagar , Andhra Pradesh, India *Corresponding author: h.sharma@cgiar.com Citation: Gopala Swamy SVS, Sharma HC, Siva Kumar C, Sharma KK and Subbaratnam GV Use of indices based on consumption and utilization of food as a criterion to evaluate putative transgenic pigeonpea plants for resistance to pod borer Helicoverpa armigera. Journal of SAT Agricultural Research 8. Introduction The increasing need for food can be met through an increase in crop productivity and reduction in pest associated losses. Recombinant DNA technology has created new avenues for genetic enhancement of crops through the development of transgenic plants with resistance to insects. Bacillus thuringiensis (Bt) genes have been widely used as candidate genes for genetic transformation of crop plants for resistance to insect pests (Hilder and Boulter 1999, Sharma et al. 2004). In addition, non-bt genes such as soybean trypsin inhibitor (SBTI), cowpea trypsin inhibitor (CpTI) and lectin genes have also been exploited to produce insect-resistant plants. Soybean trypsin inhibitor and plant lectins have been found to reduce larval growth and disrupt normal development of H. armigera in artificial diet (Johnston et al. 1993, Shukla et al. 2005). Transgenic pigeonpea (Cajanus cajan) plants with Bt and SBTI genes have been recently developed to reduce the losses due to H. armigera (Sharma et al. 2006). There is a need to develop standardized protocols for evaluation of transgenic plants for resistance to insect pests, and their interaction with other non-target herbivores, and the natural enemies of crop pests. The present studies were therefore conducted to assess the usefulness of indices based on consumption and utilization of food and diet incorporation assay to assess the usefulness of putative transgenic plants of pigeonpea for resistance to H. armigera. Materials and methods Two pigeonpea varieties, and, transformed using the constructs phs 723: Bt Cry1Ab and phs 737: SBTI by Agrobacterium tumefaciensmediated transformation (Sharma et al. 2006) were raised in a containment (P 2 level) greenhouse at C, 70 80% RH, and a natural photoperiod of 12:12 (light:dark) h. The plants were analyzed for the presence of transgene by polymerase chain reaction (PCR), and only PCR positive plants were selected for further evaluation. The H. armigera culture was maintained under laboratory conditions on artificial diet (Armes et al. 1992). Helicoverpa armigera population from the field was introgressed into the laboratory culture every five generations to maintain the heterogeneity of the insect population. For assessing the biological activity of the test material, insect survival and development were studied on artificial diet impregnated with lyophilized powder of leaves, flowers and pods of transgenic and non-transgenic plants. Plant parts (leaves, flowers and pods) collected from the plants grown under greenhouse conditions were lyophilized, and the lyophilized material was incorporated into the artificial diet to study the effect on growth and development of H. armigera larvae. The consumption and utilization of food was studied by feeding the third-instar H. armigera larvae on the pods of transgenic and non-transgenic pigeonpea plants under laboratory conditions. Fully expanded pigeonpea leaves from the top (third and fourth leaves from the tip), flowers and pods were collected from plants grown in the glasshouse in 10 cm 15 cm Zip-lock sample bags, and immediately frozen at 20 C. Non-transformed and plants grown under similar conditions were used as controls. The samples were lyophilized in a freeze drier system (Thermo Savant TM ) to minimize denaturation of sensitive proteins and secondary metabolites. First, the condenser temperature of the lyophilizer was allowed to reach 40ºC. The pre-frozen plant samples were then placed in trays, and covered with an acrylic chamber, and fixed with vacuum grease to ensure a leak proof system. The samples were freeze-dried for 48 h. The samples were then ground at room temperature using a grinder. The dried plant material was ground to a fine powder and passed through a 40-mesh sieve. The samples were stored SAT ejournal ejournal.icrisat.org December 2010 Volume 8

2 in labeled polythene bags, and kept in desiccators maintained at room temperature. The optimum amount of pigeonpea leaf/flower/pod powder needed to be incorporated in artificial diet to measure antibiotic effects on H. armigera larvae was quantified using dose-mortality response. For this purpose, leaf or pod powder was used as a substitute for chickpea (Cicer arietinum) flour in the artificial diet (leaf or pod powder : chickpea flour 5:70, 10:65, 15:60 and 20:55 g for 300 ml of artificial diet) (Armes et al. 1992) using the diet incorporation assay (Narayanamma et al. 2008, Sujana et al. 2008). For studying the antibiotic effects of putative transgenic plants on H. armigera, 10 g of sample was added into the artificial diet. The leaf powder was blended with other ingredients of the artificial diet in warm water (100 ml) for 2 minutes. Boiled agar-agar (100 ml) was added to the constituents and blended for 2 minutes. Standard artificial diet (without any pigeonpea leaf or pod powder) was used as a control. Each treatment was replicated thrice (n = 30 larvae). Diet aliquots (20 ml) were poured into small cups (30 ml capacity), and one neonate larva was released in each cup with the help of a camel hair brush. All the operations were carried out under a laminar air flow. Survival and development of H. armigera were studied in terms of post-embryonic development and fecundity. After releasing the larvae on artificial diet the cups were kept under laboratory conditions (10 to 28 C), and data were recorded on larval survival and larval weights on the 10 th day after initiating the experiment, larval period, pupation and adult emergence. Pupal weights were recorded one day after pupation. For computing the indices based on consumption and utilization of food, tender pods of pigeonpea were obtained from both transgenic and non-transgenic plants, and placed singly in plastic cups after recording their fresh weight. A single, pre-weighed, 4-h starved, thirdinstar larva was released in each cup. After three days of larval feeding on pods, the weights of larvae and the uneaten food were recorded. The larval feces was collected in individual vials. The uneaten food, H. armigera larvae and the feces were dried in a hot air oven at 55 C for three days, and their dry weights were recorded. The experiment was replicated three times in a randomized block design under laboratory conditions. Various indices of food consumption and utilization were calculated as proposed by Waldbauer (1968), and as used by Sharma and Norris (1991). Weight of food consumed Consumption index = 100 Duration of feeding period Mean larval weight Efficiency of conversion of ingested food into body matter = Weight gained by larvae during the feeding period 100 Weight of food consumed Efficiency of conversion of digested food into body matter = Weight gained by larvae during the feeding period 100 Weight of food consumed Weight of feces Approximate digestibility Weight of food consumed Weight of feces = 100 Weight of food consumed SAT ejournal ejournal.icrisat.org December 2010 Volume 8

3 Statistical analysis Data were subjected to analysis of variance (ANOVA) using Genstat Release 8.2. The significance of differences between the treatments was judged by F-test, while the treatment means were compared using least significant difference (LSD) at P Results and discussion Growth and development of H. armigera. The weights of H. armigera larvae at 10 days after initiating the experiment on artificial diet impregnated with lyophilized leaves of transgenic pigeonpea lines were not significantly different from the larvae grown on diets with leaves of non-transgenic plants (Table 1). However, Williams et al. (1998) observed significant mortality and reduced weights of surviving Helicoverpa zea larvae when fed on lyophilized leaf and silk tissue from Bt transgenic maize (Zea mays) hybrids incorporated into artificial diet. Diatraea grandiosella larvae fed on diet containing Bt Cry1Ab maize tissue weighed less and were shorter in length than larvae fed on diet containing conventional maize (Allen and Pitre 2005). Lyophilized leaf tissues from Bt soybean (Glycine max) incorporated into artificial diet at 25-fold dilution of fresh tissue caused complete mortality of Anticarsia gemmatalis and Pseudoplusia includens neonates after 11 days (MacRae et al. 2005). In the present studies, the larvae reared on Bt , Bt , SBTI and SBTI showed prolonged larval developmental period compared to those reared on control diets. However, the effects of putative transgenic pigeonpea plants on H. armigera were not as great as observed with transgenic plants in other crops possibly because of very low amounts of Btproteins present in the transgenic plants. There were no adverse effects of transgenic pigeonpea plants on pupation and adult emergence of H. armigera. Similarly, no adverse effects were observed in terms of larval and pupal weights, and larval and pupal periods (Table 2). Gore et al. (2001) observed better bollworm survival on floral bodies of transgenic cotton (Gossypium sp) than on other plant parts. They indicated that this may be due to lower levels of expression of Bt toxin proteins, and higher nutritional value of flowers. However, there was a significant reduction in larval weight on artificial diet impregnated with lyophilized pods of Bt (22.2 mg) plants as compared to the larvae grown on diet containing pods of non-transgenic plants (39.5 mg) (Table 3). Pupal weight was lower on diets with Bt (277.8 mg) plants than that on the non-transgenic plants (316.4 mg). The larval and pupal duration, and percentage pupation and adult emergence did not differ significantly between the diets with leaf tissue of Table 1. Development and survival of H. armigera on artificial diet impregnated with lyophilized leaf powder of transgenic and non-transgenic pigeonpea plants 1. Larval weight Larval period Pupal weight Pupal period Pupation 2 Adult emergence 2 Genotype (mg) at 10 DAI (days) (mg) (days) (%) (%) Bt (57.0) 66.7 (54.8) Bt (61.2) 70.0 (56.8) Bt (61.9) 63.3 (52.9) Bt (57.0) 60.0 (50.8) Non-transgenic control (61.2) 66.7 (54.8) SBTI (57.0) 60.0 (50.9) SBTI (66.1) 66.7 (54.8) SBTI (63.9) 66.7 (54.8) Non-transgenic control (57.0) 63.3 (52.8) Standard artificial diet (68.9) 70.0 (56.8) Fp < SE± LSD at P= NS NS NS 1. DAI = Days after initiation of the experiment; NS = Not significant. 2. Figures in parentheses are angular transformed values. SAT ejournal ejournal.icrisat.org December 2010 Volume 8

4 Table 2. Development and survival of H. armigera on artificial diet impregnated with lyophilized flower powder of transgenic and non-transgenic pigeonpea plants 1. Larval weight Larval period Pupal weight Pupal period Pupation 2 Adult emergence 2 Genotype (mg) at 10 DAI (days) (mg) (days) (%) (%) Bt (61.2) 73.3 (59.2) Bt (61.9) 63.3 (52.8) Bt (63.9) 73.3 (59.2) Non-transgenic control (66.1) 80.0 (63.4) SBTI (59.0) 70.0 (57.0) Non-transgenic control (66.1) 73.3 (59.2) Standard artificial diet (68.9) 80.0 (63.9) Fp < SE± LSD at P= NS NS NS NS NS 1. DAI = Days after initiation of the experiment; NS = Not significant. 2. Figures in parentheses are angular transformed values. Table 3. Development and survival of H. armigera on artificial diet impregnated with lyophilized pod powder of transgenic and non-transgenic pigeonpea plants 1. Larval weight Larval period Pupal weight Pupal period Pupation 2 Adult emergence 2 Genotype (mg) at 10 DAI (days) (mg) (days) (%) (%) Bt (61.2) 66.7 (54.8) Bt (68.9) 76.7 (61.2) Bt (63.4) 70.0 (57.0) Non-transgenic control (59.0) 63.3 (52.8) Standard artificial diet (61.9) 70.0 (57.0) Fp SE± LSD at P= NS NS NS 1. DAI = Days after initiation of the experiment; NS = Not significant. 2. Figures in parentheses are angular transformed values. transgenic and non-transgenic plants. The duration of pupal period was prolonged by nearly one week because of cooler climate during the experimental period (10 to 28 C) than that observed on pigeonpea by Sujana et al. (2008). The Cry1Ab toxin in MON810 maize resulted in prolongation of pre-pupal and pupal periods of H. zea, and also resulted in lower moth emergence (Horner et al. 2003); while SBTI in artificial diet affected the growth and development of S. litura (McManus and Burgess 1995) and H. armigera adversely (Johnston et al. 1993, Wang et al. 1995, Shukla et al. 2005). Lack of variation in larval and pupal weights and their duration, and adult emergence on artificial diet impregnated with lyophilized flowers or pods of putative transgenic pigeonpea plants indicated that the toxin levels in flowers and pods were too low to cause any adverse effect on growth and development of H. armigera. Consumption and utilization of food by the thirdinstar larvae of H. armigera. Consumption index of food per unit of body weight of H. armigera larva varied from 2.04 to 9.55 mg on transgenic plants compared to 3.47 mg on non-transgenic and 2.26 mg on (Table 4). Approximate digestibility ranged from to 96.85% on the transgenic lines as compared to and 70.43% on control plants of SAT ejournal ejournal.icrisat.org December 2010 Volume 8

5 Table 4. Consumption and utilization of pods of transgenic (T 5 ) and non-transgenic pigeonpea plants by the thirdinstar larvae of H. armigera (2003 rainy season) 1. AD ECI ECD Genotype CI (%) (%) (%) Bt Bt Bt Non-transgenic control SBTI SBTI SBTI SBTI Non-transgenic control Fp <0.001 <0.001 <0.001 <0.001 SE± LSD at P= CI = Consumption index; AD = Approximate digestibility; ECI = Efficiency of conversion of ingested food into body matter; ECD = Efficiency of conversion of digested food into body matter. Table 5. Consumption and utilization of pods of transgenic (T 5 ) and non-transgenic pigeonpea plants by the thirdinstar larvae of H. armigera (2004 rainy season) 1. AD ECI ECD Genotype CI (%) (%) (%) Bt Bt Bt Bt Bt Non-transgenic control SBTI SBTI SBTI Non-transgenic control Fp SE± LSD at P= NS NS CI = Consumption index; AD = Approximate digestibility; ECI = Efficiency of conversion of ingested food into body matter; ECD = Efficiency of conversion of digested food into body matter; NS = Not significant. and, respectively. However, none of the transgenic lines showed a significant reduction in amount of food consumed and approximate digestibility of the H. armigera larvae. The larvae fed on Bt showed a significantly lower efficiency of conversion of ingested food into body matter (12.89%) than those fed on the pods of non-transgenic plants of (24.93%). Similarly, the larvae fed on SBTI (13.00%) and SBTI (13.37%) pods had lower efficiency of conversion of ingested food into body matter than the larvae fed on the pods of non-transgenic plants of (24.79%). The efficiency of conversion of digested food into body matter was lower in the larvae fed on Bt (10.54%), SBTI (10.21%) and SBTI (10.84%) pods compared to the larvae fed on the pods of non-transgenic (14.98%) and (17.52%) plants. The consumption index varied from to on transgenic plants compared to and in the larvae fed on the pods of control plants of and (Table 5). Larvae fed on SBTI (15.93) showed a significant reduction in consumption index compared to the larvae fed on pods of the nontransgenic plants of (18.05). However, none of the transgenic lines showed a significant reduction in approximate digestability, efficiency of conversion of ingested food and efficiency of conversion of digested food into body matter by the third-instar larvae of H. armigera. Although there was no significant reduction in consumption index and approximate digestibility of H. armigera larvae on transgenic plants, the efficiency of conversion of ingested as well as digested food into body matter were lower on pods of Bt , SBTI and SBTI as compared to the larvae fed on the pods of non-transgenic plants. Relative growth rate of H. armigera fifth-instar larvae on CpTI-Bt transgenic cotton or Bt-transgenic cotton has been reported to be significantly lower than in the larvae fed on nontransgenic cotton (Zhang et al. 2004). Sareen et al. (1983) observed a decrease in consumption index and growth rates of larvae of Spodoptera litura in a dose-dependent manner, while Wang et al. (2004) observed higher approximate digestibility in Mythimna separata larvae fed on Bt maize, which was associated with low levels of food consumed or reduced metabolic efficiency in larvae surviving B. thuringiensis var kurstaki (Gujar et al. 2001). The results of the present studies indicated that the available transgenic pigeonpea plants exhibited little biological activity against H. armigera possibly because of very low amounts of Bt toxin and SBTI. Indices based on consumption and utilization of food and incorporation of lyophilized leaf or pod powder into the artificial diet SAT ejournal ejournal.icrisat.org December 2010 Volume 8

6 did differentiate between transgenic and non-transgenic plants. However, there is a need to develop transgenic pigeonpea plants with optimum level of expression of Bt toxins in reproductive parts for effective control of H. armigera. References Allen KC and Pitre HN Toxicity of transgenic corn tissues expressing insecticidal protein of Bacillus thuringiensis Berliner to southwestern corn borer (Lepidoptera: Crambidae) in the laboratory. Journal of Entomological Science 40: Armes NJ, Bond GS and Cooters RJ The laboratory culture and development of Helicoverpa armigera. Natural Resources Institute Bulletin No. 57. Chatham, UK: Natural Resources Institute. Gore J, Leonardo BR and Adamczyk JJ Bollworm (Lepidoptera: Noctuidae) survival on Bollgard and Bollgard II cotton flower bud and flower components. Journal of Economic Entomology 94: Gujar GT, Kalia V and Archana K Effect of sublethal concentration of Bacillus thuringiensis var kurstaki on food and developmental needs of the American bollworm, Helicoverpa armigera (Hübner). Indian Journal of Experimental Biology 39: Hilder VA and Boulter D Genetic engineering of crop plants for insect resistance a critical review. Crop Protection 18: Horner TA, Dively GP and Herbert DA Development, survival and fitness performance of Helicoverpa zea (Lepidoptera: Noctuidae) in MON810 Bt field corn. Journal of Economic Entomology 96: Johnston KA, Gatehouse JA and Anstee JH Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera. Journal of Insect Physiology 39: MacRae TC, Baur ME, Boethel DJ, Fitzpatrick BJ, Gao AG, Gamundi JC, Harrison LA, Kabuye VT, McPherson RM, Miklos JA, Paradise MS, Toedebusch AS and Viegas A Laboratory and field evaluations of transgenic soybean exhibiting highdose expression of a synthetic Bacillus thuringiensis cry1a gene for control of Lepidoptera. Journal of Economic Entomology 98: McManus MT and Burgess EPJ Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura. Journal of Insect Physiology 41: Narayanamma LV, Sharma HC, Gowda CLL and Sriramulu M Incorporation of lyophilized leaves and pods into artificial diets to assess the antibiosis component of resistance to pod borer, Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea. International Journal of Tropical Insect Science 27: Sareen V, Rathore YS and Bhattacharya AK Influence of Bacillus thuringiensis var. thuringiensis on the food utilization of Spodoptera litura (Fabricius). Journal of Applied Entomology 95:253. Sharma HC and Norris DM Comparative feeding preference and food intake and utilization by the cabbage looper (Lepidoptera: Noctuidae) on three legume species. Environmental Entomology 20: Sharma HC, Sharma KK and Crouch JH Genetic transformation of crops for insect resistance: Potential and limitations. Critical Reviews in Plant Sciences 23: Sharma KK, Lavanya M and Anjaiah V Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L. Millsp.) expressing the synthetic Bt cry1ab gene. In Vitro Cell & Developmental Biology Plant 42: Shukla S, Arora R and Sharma HC Biological activity of soybean trypsin inhibitor and plant lectins against cotton bollworm/legume pod borer, Helicoverpa armigera. Plant Biotechnology 22:1 6. Sujana G, Sharma, HC and Manohar Rao D Antixenosis and antibiosis components of resistance to pod borer, Helicoverpa armigera in wild relatives of pigeonpea. International Journal of Tropical Insect Science 28: Waldbauer GP The consumption and utilization of food by insects. Advances in Insect Physiology 5: Wang CZ, Xiang XF, Zhang SF and Qin JD Effect of soybean trypsin inhibitor on the growth and digestive physiology of Helicoverpa armigera larvae. Acta Entomologica Sinica 38: Wang DY, Wang ZY, He KL, Cong B, Wen Li and Shu BX Food consumption and utilization of the SAT ejournal ejournal.icrisat.org December 2010 Volume 8

7 fifth instar larvae of Mythimna separata (Walker) feeding on the leaves of transgenic Bacillus thuringiensis corn expressing Cry1Ab protein. Acta Entomologica Sinica 47: Williams WP, Buckley PM, Sagers JB and Hanten JA Evaluation of transgenic corn for resistance to corn earworm (Lepidoptera: Noctuidae), fall armyworm (Lepidoptera: Noctuidae), and southwestern corn borer (Lepidoptera: Crambidae) in a laboratory bioassay. Journal of Agricultural Entomology 15: Zhang JH, Wang CZ and Guo SD Effects of CpTI-Bt transgenic cotton and Bt transgenic cotton on survival, growth and nutrition utilization of Helicoverpa armigera (Hübner). Acta Entomologica Sinica 47(2): SAT ejournal ejournal.icrisat.org December 2010 Volume 8