SURVIVAL AND DEVELOPMENT OF AMERICAN BOLLWORM (HELICOVERPA ARMIGERA HUBNER) ON TRANSGENIC BT COTTON

Indian J. Agric. Res.., 48 (3) 177-184, 2014 doi:10.5958/j.0976-058x.48.3.030 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com SURVIVAL AND DEVELOPMENT OF AMERICAN BOLLWORM (HELICOVERPA ARMIGERA HUBNER) ON TRANSGENIC BT COTTON M.S. MahaLakshmi* and N.V.V.S.D. Prasad Regional Agricultural Research Station, Lam, Guntur -522 034, India Received: 05-09-2012 Accepted: 19-03-2013 ABSTRACT A laboratory bioassay was conducted to study the impact of Cry 1 Ac protein on survival and development of Helicoverpa armigera (Hubner.) by feeding larvae with Bt cotton plant parts. During the study, terminal leaves and squares of two Bt hybrids, their corresponding non Bt hybrids and Bunny as check were fed to larvae. Among the plant parts tested, mortality of H.armigera larvae was higher on leaves compared to squares of both the Bt hybrids. Further, the mortality of early larval instars was higher compared to older instar larvae on both leaves and squares of Bt hybrids. Though, the later instar larvae were able to survive when exposed continuously to Bt cotton plant parts, but reduction larval and pupal weight, increase in larval developmental period, malformations in pupae and adults, reduction in adult emergence, less fecundity, low growth and survival indices was observed on both the Bt hybrids compared to their corresponding non Bt hybrids and check hybrid, Bunny. Key words: American bollworm, Bt cotton, Larval mortality, Survival index. INTRODUCTION The unfocussed and unrestricted use of various toxic agrochemicals not only caused adverse effects on the environment, but also lead to the development of resistance and resurgence of insect pests. The cotton growers witnessed failures in control of American bollworm and whiteflies by insecticides in Andhra Pradesh in past. Under these circumstances, Bt cotton emerged as a credible tool with inherent resistance against bollworms. After the release of Bt cotton in India for cultivation during 2003, the area under transgenic Bt cotton increased year by year, indicating the economic advantage of Bt cotton like reducing the usage of insecticides by 60-80 per cent and increasing natural enemies population. Many scientists reported the efficacy of Bt cotton against Helicoverpa armigera larvae (Murugan et al., 2003; Gore et al. 2003). But, the sensitivity of H.armigera larvae towards the cry toxin was varied among the different instars (Murugan et al, 2003). Further, the level of cry protein is also not consistent throughout the season, since the cry protein content decreases as the plant age get advanced and the quantity of cry protein is also not uniform in all the plant parts (Greenplate, 1996). The quantity of Bt protein was high in young leaves of the top canopy compared to squares, flowers and other parts (Kranthi et al., 2005). Keeping the above points in view, the present study was conducted to assess the sensitivity of different larval instars of H. armigera to different plant parts of Bt cotton. MATERIALS AND METHODS A laboratory bioassay was conducted in a completely randomized block design with 5 replications each with 20 larvae to test the impact of Bt cotton on mortality and development of H.armigera larvae at Regional Agricultural Research Station, Lam, Guntur, Andhra Paradesh. A pair of adults soon after emergence was kept in big plastic containers having a small twig erected in small plastic cups with moistened soil from non Bt cotton plant to facilitate egg laying and the entire setup was covered with a fine muslin cloth and moth diet as suggested by Kranthi (2004) was prepared and used. The eggs laid on muslin cloth, cotton swab and twig were removed with camel hair brush after four days and the neonates emerged subsequently from the eggs were used for bioassay studies. * Corresponding authore s e-mail: msmlaxmi@gmail.com.

178 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Fully expanded terminal leaves and squares are the mostly preferred plant parts for egg laying by H.armigera, hence they were selected for bioassay studies. Plant parts were hand picked from 70-100 DAS crop of MECH 12 Bt, RCH 2 Bt, their corresponding non Bt versions and Bunny hybrid from unsprayed experimental plots and were brought to the laboratory in polythene covers to avoid drying. They were cleaned with sterile water and wiped with blotting paper to remove excess moisture. Later, the leaves were placed individually in small plastic jars while squares were placed in small plastic vials into which first instar larvae were released with the help of camel hairbrush. Similarly, II to V instars reared previously on artificial diet (Kranthi, 2004) were also used for testing the sensitivity of different instars against cry protein. All the larval instars were exposed continuously to plant parts of Bt hybrids till their pupation. The average initial weight of larvae was recorded with an electronic balance of 1 mg sensitivity before release on to the plant parts for all the larval instars. The first instar larvae were weighed as a whole (20 larvae) for a treatment while the remaining instars were weighed individually. The plant parts were changed daily to avoid death or growth reduction due to tissue drying or nutritional deterioration. Daily larval mortality and duration to pupate were recorded for all the larval instars until pupation. The average weight of survived larvae from different treatments was recorded after 5 days of larval exposure to Bt plant parts and weight of pupae was also recorded from each treatment. The adult emerged from different larval instars and from both the plant parts were allowed separately for mating and fecundity was observed. The growth index and survival index were calculated for H.armigera on different treatments using the formulae given by Vennila et al. (2006). Growth index : Per cent pupation / L arval developmental period (days) Survival index : Number of moths emerged / Total number of neonates used RESULTS AND DISCUSSION Effect on survival of larvae: The bioassay studies clearly indicated that none of the first instar larvae were survi ved beyond five days when fed continuously with top young leaves of Bt hybrids, while 76.0 and 74.0 per cent mortality was recorded when fed with squares of MECH 12 Bt and RCH 2 Bt, respectively (Table.1). Similarly, the II instar larvae recorded 100 per cent mortality on leaves, while it was only 32.0-30.0 per cent on squares of Bt hybrids. The mortality of subsequent instars such as third and fourth instars was comparatively low, since some of the larvae were able to survive though they were exposed continuously to leaves and squares of Bt hybrids. However, the mortality of both the third and fourth instars was higher on leaves compared to flower buds of Bt hybrids. The V instar larvae showed tolerance to cry protein since all the larvae TABLE 1: Mortality of larval instars of H.armigera on test hybrids Per cent mortality of larvae exposed to leaves MECH 12 Bt 100 (90) a 100 (90) a 82 (64.90) a 30 (33.21) a 0 (0) MECH 12 non Bt 4 (11.54) b 2 (8.13) b 0 (0) b 0 (0) b 0 (0) RCH 2 Bt 100 (90) a 100 (90) a 82 (64.90) a 28 (31.95) a 0 (0) RCH 2 non Bt 4 (11.54) b 2 (8.13) b 0 (0) b 0 (0) b 0 (0) Bunny 6 (14.48) b 2 (8.13) b 0 (0) b 0 (0) b 0 (0) F test Sig Sig Sig Sig - CD 11.54 9.42 5.86 4.60 - Per cent mortality of larvae exposed to squares MECH 12 Bt 76 (60.67) a 32 (34.45) a 16 (23.58) a 0 (0) 0 (0) MECH 12 non Bt 0 (0) b 0 (0) b 0 (0) b 0 (0) 0 (0) RCH 2 Bt 74 (59.34) a 30 (33.21) a 12 (20.27) a 0 (0) 0 (0) RCH 2 non Bt 0 (0) b 0 (0) b 0 (0) b 0 (0) 0 (0) Bunny 0 (0) b 0 (0) b 0 (0) b 0 (0) 0 (0) F test Sig Sig Sig - - CD 9.04 4.56 7.74 - - * Figures in parentheses are arcsine transformed values ** Numbers followed by same superscript are not statistically different

survived on both the leaves and flower buds of both Bt hybrids (Table.1). The findings are in agreement with Zhao et al. (1998) who reported that 5 th instar larvae were able to survive and pupate normally on Bt plants. Murugan et al. (2003) also reported that the survival of neonate and four days old larvae was significantly low, while older larvae were tolerant without significant reduction in survival on exposure to Bt cotton. The larval mortality of H.armigera ranged between 0 to 100 per cent on leaves while it was 0 to 76 per cent on squares of transgenic cultivars (Table.1). The mean per cent mortality for all the larval instars was high when fed with top leaves compared to squares of both the Bt hybrids which can be contributed to the higher content of Cry 1 Ac toxin protein in leaves when compared to the squares. The present findings are in accordance with Henneberry et al. (2001) also reported that the mortality of neonate larvae was 100 per cent on leaves when compared to 96 per cent mortality on squares. Kuijun et al. (2004) reported that the declining order of Bt protein content was leaf > square > boll > flower and fall in susceptibility was I instar > II instar > III instar > IV instar > V instar of H. armigera. Effect on larval D evelopment: There was considerable reduction in larval weights of all the Vol. 48, No. 3, 2014 FIG.1: Weight of larvae at 5 DAE on leaves of test hybrids 179 instars when fed with leaves and squares of Bt plants compared to non Bt plants and control. Mortality was low on squares compared to leaves of Bt plants (Fig.1&2). The weight of first instar larvae was not recorded since all the neonate larvae died with in 3-4 days when fed with leaves of Bt cotton hybrids continuously. But, the first instar larvae were able to survive when fed with squares to some extent (Table.1) but the weights of surviving larvae at 5 days after exposure (DAE) were far below compared to their corresponding non Bt s and check hybrid, Bunny. The weight of II instar larvae was 78.2 and 81.5 mg only which were fed with leaves of MECH 12 Bt and RCH 2 Bt hybrids, respectively at 5 DAE where as it was 326.7 and 362.4 mg on their corresponding non Bt hybrids and 358.8 mg on check hybrid, Bunny (Fig.2). The V instar larvae pupated by 2 nd day when fed on Bt hybrids and by 3 rd to 4 th day on non Bt s and Bunny, hence the data on larval weights of V instar were not considered. The percentage of pupation was low in the larvae fed on leaves compared to the larvae that were fed on squares of Bt plants for all the larval instars (Table.2). The pupal weight when the larvae fed with Bt plant parts as compared to those of non Bt hybrids and Bunny (Fig.3 & 4). Further, the larvae exposed continuously to Bt plant parts were unable to pupate normally and many aberrant pupae were observed. The present findings are analogous with Weight (mg)

180 INDIAN JOURNAL OF AGRICULTURAL RESEARCH FIG.2: Weight of larvae at 5 DAE on squares of test hybrids Weight (mg) TABLE 2: Pupation of surviving larvae of H.armigera on test hybrids Per cent pupation on leaves MECH 12 Bt 0 (0) b 0 (0) b 18 (25.10) b 70 (56.79) b 100 (90) MECH 12 non Bt 96 (78.46) a 98 (81.87) a 100 (90) a 100 (90) a 100 (90) RCH 2 Bt 0 (0) b 0 (0) b 18 (25.10) b 72 (58.05) b 100 (90) RCH 2 non Bt 96 (78.46) a 98 (81.87) a 100 (90) a 100 (90) a 100 (90) Bunny 94 (75.82) a 98 (81.87) a 100 (90) a 100 (90) a 100 (90) F test Sig Sig Sig Sig NS MECH 12 Bt MECH 12 Non Bt RCH 2 Bt RCH 2 Non Bt Bunny CD 11.54 9.42 5.85 4.60 - Per cent pupation on squares MECH 12 Bt 24 (29.33) b 68 (55.55) b 84 (66.42) b 100 (90) 100 (90) MECH 12 non Bt 100 (90) a 100 (90) a 100 (90) a 100 (90) 100 (90) RCH 2 Bt 26 (30.66) b 70 (56.79) b 88 (69.73) b 100 (90) 100 (90) RCH 2 non Bt 100 (90) a 100 (90) a 100 (90) a 100 (90) 100 (90) Bunny 100 (90) a 100 (90) a 100 (90) a 100 (90) 100 (90) F test Sig Sig Sig NS NS CD 8.68 4.56 7.73 - - * Figures in parentheses are arcsine transformed values ** Numbers followed by same superscript are not statistically different Murugan et al. (2003), Liu et al. (2004) and Men et al. (2005) who reported significant reduction in the larval and pupal weights when fed on different parts of Bt cotton plants. There was marked difference in larval growth since the survived larvae remained in first or second instar itself even after 4-5 days of release on both the Bt cotton cultivars while those reared on non Bt s and check hybrid moulted to subsequent instars with in 2-3 days after release. Further, the larvae taken almost double the time to complete their development on Bt cottons than on non Bt cottons. The present findings are concomitant with Fitt et al. (1994), Stewart et al. (2001), Men et al. (2005) and Vennila et al. (2006) who reported the longer developmental periods of larvae on Bt cotton cultivars compared to non Bt cultivars. Effect on adult emergence: T he adul t emergence was also adversely affected due to feeding of larvae on leaves and squares of Bt hybrids and it was clearly evident from V instar larvae. Since, V instar larvae were able to survive

Vol. 48, No. 3, 2014 181 FIG.3: Weight of pupae reared on leaves of test hybrids Weight (mg) Weight (mg)

182 INDIAN JOURNAL OF AGRICULTURAL RESEARCH and pupate normally when fed with leaves and squares of Bt hybrids but, the adults were unable to emerge normally from pupae. There was incomplete emergence of adults from pupae formed from Bt exposed larvae and malformed adults were also observed which were survived only for few hours after emergence. Further, the adult emergence was low from the larvae that were fed on leaves compared to squares of both MECH 12 Bt and RCH 2 Bt hybrids which can be attributed to higher content of Cry 1 Ac toxin (Table.3). Effect on fecundity: The adults that emerged from the larvae reared on Bt and non Bt hybrids were collected separately and two pairs of adults were released in individual jars for egg laying from each test hybrid and the fecundity was recorded. The pupation was nil from I instar larvae when fed with leaves of Bt hybrids hence there was no adult emergence for further study. The fecundity was nil from adults emerged from the surviving I instar larvae and pupae when fed with squares of both the Bt hybrids, while there was a great reduction in fecundity of adult that emerged from subsequent larval instars which were fed with Bt plant parts (Fig.5 & 6). Though the IV and V instar larvae were able to survive when exposed to leaves and squares of Bt cotton cultivars, they could not develop themselves normally with significant reduction in larval and pupal weight, development of malformed pupae and adults, decreased adult emergence and low fecundity in Bt cotton than on non Bt cottons and check hybrid. Present findings are in conformity with Cui and Xia (1999) and Fitt et al. (1994). Effect on Growth and Survival indices: The growth and survival indices for H. armigera were very low when reared on Bt cotton plant parts than on non Bt and Bunny hybrids. The growth index values for H. armigera larvae reared on leaves were only 1.06 and 1.05 on MECH 12 Bt and RCH 2 Bt, respectively as against 5.81, 6.17 and 6.15 on MECH 12 non Bt, RCH 2 non Bt and Bunny hybrids, respectively. The survival index values for H.armigera larvae reared on leaves were only 0.34 and 0.32 on MECH 12 Bt and RCH 2 Bt, respectively against 0.99 on non Bt hybrids and 0.98 on Bunny hybrid. Both the growth index and survival index were high for the larvae reared on flower buds of Bt hybrids compared to leaves. The low growth and survival indices can be attributed to longer developmental period, low per cent pupation and less adult emergence on Bt hybrids. The present findings are in conformity with Stewart et al. (2001) who reported 0.74 survival rate on Bt cotton compared to 1.0 on non Bt cotton. Men et al. (2005) reported that only 8.3 per cent individuals fed with flowers and bolls of TABLE 3: Adult emergence from surviving pupae of H.armigera on test hybrids Per cent adult emergence from larvae exposed to leaves MECH 12 Bt 0 (0) b 0 (0) b 12 (20.27) b 66 (54.33) b 90 (71.56) b MECH 12 non Bt 96 (78.46) a 98 (81.87) a 100 (90) a 100 (90) a 100 (90) a RCH 2 Bt 0 (0) b 0 (0) b 10 (18.44) b 62 (51.94) b 88 (69.73) b RCH 2 non Bt 96 (78.46) a 98 (81.87) a 100 (90) a 100 (90) a 100 (90) a Bunny 94 (75.82) a 98 (81.87) a 100 (90) a 100 (90) a 100 (90) a F test Sig Sig Sig Sig Sig CD 11.54 9.42 9.64 8.37 6.89 Per cent adult emergence from larvae exposed to squares MECH 12 Bt 10 (18.44) b 44 (41.55) b 76 (60.67) b 90 (71.56) a 98 (81.87) a MECH 12 non Bt 100 (90) a 100 (90) a 100 (90) a 100 (90) a 100 (90) a RCH 2 Bt 8 (16.43) b 40 (39.23) b 72 (58.05) b 88 (69.73) a 96 (78.46) a RCH 2 non Bt 100 (90) a 100 (90) a 100 (90) a 100 (90) a 100 (90) a Bunny 100 (90) a 100 (90) a 100 (90) a 100 (90) a 100(90) a F test Sig Sig Sig Sig NS CD 10.24 7.66 5.56 13.70 - * Figures in parentheses are arcsine transformed values ** Numbers followed by same superscript are not statistically different

Vol. 48, No. 3, 2014 183 Fig. 5: Fecundity of adults emerged from larvae fed on leaves Fig.6: Fecundity of adults emerged from larvae fed on squares 600 500 No. of eggs/female No. of eggs/female 400 300 200 100 0 I II III IV V Larval instar MECH 12 Bt MECH 12 Non Bt RCH 2 Bt RCH 2 Non Bt Bunny Bt cotton could develop from neonate to pupa. Vennila et al. (2006) reported lower growth and survival indices for H.armigera on Bt cotton. The present study clearly indicated that the Cry 1 Ac toxin had highly detrimental effects on survival and development of H. armigera.

184 INDIAN JOURNAL OF AGRICULTURAL RESEARCH REFERENCES Cui, J. J and Xia, J. Y. (1999) Effects of transgenic Bt cotton on development and reproduction of cotton bollworm. Acta Agriculturae Universitatis Henanensis 33(1): 20-24. Fitt, G. P.; Mares, C. L. and Llewellyn, D. J. (1994) Field evaluation and potential impact of transgenic cottons (Gossypium hirsutum) in Australia. Biocontrol Sci. and Tech. 4: 535-548. Gore, J.; Leonard, B. R. and Jones, R. H. (2003) Influence of agronomic hosts on the susceptibility of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) to genetically engineered and non-engineered cottons. Environ. Entomol. 32 (1): 103-110. Greenplate,.T J. (1996) Quantification of Bacillus thuringiensis insect control protein Cry 1 Ac over time in bollgard cotton fruit and terminals. J. Eco. Entomol. 92(6):1377-1383. Henneberry, T. J.; Jech, L. F. and Torre, T. (2001) Effects of transgenic cotton on cabbage looper, tobacco budworm and beet armyworm (Lepidoptera: Noctuidae) larval mortality and development and foliage consumption in the laboratory. Southwestern Entomologist. 26(4): 325-338. Kranthi, K. R. (2004) Diet preparation for H.armigera and S.litura. in: Insecticide Resistance: Monitoring, Mechanisms and Management Manual. Central Institute for Cotton Research, Nagpur, p.15. Kranthi, K. R.; Dhawad, C. S.; Naidu, S.; Mate, K.; Patil, E. and Kranthi, S. (2005) Bt cotton seed as a source of Bacillus thuringiensis insecticidal Cry1Ac toxin for bioassays to detect and monitor bollworm resistance to Bt cotton. Cur. Sci. 88(5): 796-800. Kuijun, Z.; Zhao, J. Z.; Lu, M. G.; Fan, X. L. and Li, G. X. (2004) A systematic evaluation of the effects of Bt transgenic cotton on the growth and development of cotton bollworm. Acta Phytophylacica Sinica. 27(3): 205-209. Liu, X. X.; Wen, Z. Q.; Cai, Q. N.; Li, J. C. and Jie, D. (2004) Effect of Bt protein on development of different strains of the cotton bollworm, Helicoverpa armigera (Hubner) and the parasitoid, Microplitis mediator (Haliday). Acta Entomologica Sinica. 47(4): 461-466. Men, X. Y.; Ge, F.; Yardim, E. N. and Parajulee, M. N. (2005) Behavioral response of Helicoverpa armigera (Lepidoptera: Noctuidae) to cotton with and without expression of the Cry l Ac delta endotoxin protein of Bacillus thuringiensis Berliner. J. Insect Behavior. 18(1): 33-50. Murugan, M.; Sathiah, N.; Dhandapani, N.; Rabindra, R. J. and Mohan, S. (2003) Laboratory assays on the role of Indian transgenic Bt cotton in the management of Helicoverpa armigera (Hubner) (Noctuidae: Lepidoptera). Indian J of Pl. Prot. 31: 1-5. Stewart, S. D.; Adamczyk, J. J.; Knighten, K. S. and Davis, F. M. (2001) Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis on growth and survival of noctuiid larvae. J. Econ. Entomol. 94(3):752-760. Vennila, S.; Panchbhai, P. R. and Biradar, V. K. (2006) Growth and survival of Helicoverpa armigera (Hub.) and Spodoptera litura (Biosd.) on transgenic cotton. J. Cot. Res. and Develop. 20(1):131-133. Zhao, J. Z.; Lu, M. G.; Fan, X. L. and Xie, F. Z. (1998) Survival and growth of different instar larvae of Helicoverpa armigera (Hubner) on transgenic Bt cotton. Acta Entomologica Sinica. 41(4): 354-358.