The dynamics of cotton aphids Aphis gossypii Glover and its predator viz., Cheilomenes sexmaculata Fab., Chrysoperla carnea Steph. and Ischiodon scutellaris Fab. was studied in RCH-2Bt and non-Bt cotton hybrids. The mean incidence of aphids was 23.82 and 21.37 per leaf in RCH-2 Bt and non Bt respectively indicating no significant variation. The dynamics of predators was density dependent on aphids in both Bt and non Bt hybrids. Mean population of coccinellids, chrysoperla and syrphids was 0.89, 0.78 and 1.0 per plant in RCH-2 Bt which was almost similar to the incidence on RCH-2 non Bt. There was strong and positive correlation between incidence of predators and aphid on both Bt and non Bt cotton. The ‘r’ value for syrphids v/s aphids was 0.94 in RCH -2 Bt and 0.96 in non Bt. Laboratory feeding experiments using Bt and non Bt cotton was carried out to study the effect of Bt fed aphids on predator C carnea indicated no difference in incubation period, longevity of grubs and adults, fecundity and aphid consumption potential indicating safety of Cry1Ac to C. carnea through intoxicated aphid host.

Key words: Bt cotton, Cry1Ac, Aphis gossypii, Cheilomenes sexmaculata, Chrysoperla carnea and Ischiodon scutellaris

Introduction

Biological control programmes those based on insect pathogens particularly bacterium, Bacillus thuringiensis (Berliner) have shown phenomenal feasibility and success mitigating the resistance problem. Since last 20 years several research organizations are engaged in exploiting the insecticidal properties of this gram positive soil bacterium in meaningful way. Thus ultimately, GMO’s (Genetically modified organisms) were developed to which class Bt (Bacillus thuringiensis) genotypes also belongs and have in built ability to express or produce crystal (Cry) protein toxic to the target pests. The largely exploited protein is Cry1 Ac having specific action against lepidopteron insects and most widely to Heliothine. Transgenic technologies have proven to be one of the fastest and most effective means of insect control ever developed and Bt considered to be a natural choice for this role, as it produces a large variety of toxins very specific for certain orders of insect pests. Hence, there were continuous efforts to develop Bt transgenic crops viz., potato, rice, maize, canola, cotton, brinjal, tobacco etc to combat dreaded pests belonging to Lepidoptera, Diptera and Coleopteran orders. Past or present commercialized Bt crop and their respective genes include cotton (Cry1Ac, Cry2Ab2, Cry1Fa2), maize (Cry1Ab, Cry1Ac, Cry1Fa2, CryBb1, Cry9 c) and potato (Cry3Aa). Thus for commercial transgenic including Bt cotton have only one gene to produce Cry toxin to contain the target pest. Thus Bt technology has been regarded as most significant development in the field of agricultural science after green revolution. Since loss of biodiversity threatens such benefits, transgenic Bt technology is increasingly scrutinized for its potential environmental impact despite fast spreading of Bt cotton world wide.

Adoption rates of Bt transgenic insect resistance cotton genotypes producing activated -endotoxin from Cry1Ac gene are quite high in almost all cotton growing countries and at present more than 90 m ha area in world (James, 2006). With approval of GEAC during 2002 India also resorted for Bt cotton cultivation based on demand for a full proof and viable solution to the quite intense bollworm problem. Thus India gained status of mega biotech country with 7.8 m ha coverage under Bt cotton (Anon,2006). Nearly 64 Bt cotton cultivars are being grown in the country at present. Transgenic plants produced Cry protein in high doses and in most of their tissues through out the season. The insecticidal toxin could become available to the predatory insect in a new and modified form through non susceptible or sublethally effected non target herbivores prey feeding on Bt plants (Jepson et al., 1994) this could constitute an important pathway for ecological impacts of Bt cotton (Andow and Hilbeck, 2004) and several experiments have sort to test such potential impacts (Lovie and Arpaia, 2005). Therefore, the objective of the present investigation was to assess the impact of Bt cotton hybrids on predatory insects which are native and quite effective in natural as well as biological control of cotton pests particularly aphids Aphis gossypii(Glover) and eggs/ neonates of bollworms.

Material and methods

The studies on natural incidence of insect predators viz., lady bird beetles/ Coccinellids (Cheilomenes sexmaculata Fab. Coccinellidae: Coleoptera) green lacewing (Chrysoperla carnea Steph. Chrysopidae : Neuroptera) and hover flies / syrphids (Ischiodon scutellaris Fab. Syrphidae :Diptera) carried out through field experiments conducted during 2004-05 and 2005-06 at Agricultural Research Station, Dharwad, using RCH-2Bt intraspecific hybrid. The crop was sown on 16.06.2004 and 24.06.2005 for respective seasons in un replicated block of 10.8 X 6.0 m² size with 90 cm x 60 cm spacing. Similarly a block of RCH-2 non-Bt was sown at the same time for comparative study. Including seed treatment no plant protection measures were imposed on any block or any period of crop growth during both the seasons. Between Bt and Non-Bt blocks an isolation distance of 10 meters was maintained to avoid migration effect. Observations were recorded from 10 randomly selected plants at weekly interval from July to December (28^th to 51^st ISW) for incidence of aphids (host insect) as well as predators. The population of aphids was recorded by counting the nymphs on three leaves, one each from top, middle and bottom portion of plant and then averaged as population per leaf. Similarly adults and nymphs of coccinellids, grubs of C. carnea and maggots of syrphids were recorded on whole plant basis and averaged to population per plant. The seasonal mean incidence of aphid as well as predators population in RCH-2 Bt and non-Bt was compared through paired t-test to assess the impact. The correlation between incidence of predators and aphid population was assessed in both Bt and non Bt crop.

A laboratory study was done during 2004 for assessing the impact of Bt cotton on C. carnea. The seedlings of RCH-2Bt and non Bt cotton were raised by sowing seeds (without treatment of imidacloprid) in pots on 20.08.2004. Ten pots were maintained for both RCH-2 Bt and Non Bt. Late sowing was done to coincide the aphid incidence on the plants with optimum expression period for Cry 1 Ac i.e. 50-80 DAS to have proper impact (if any) on aphids. The aphid colonies developed on these plants were used to feed C. carnea grubs in the laboratory. Feeding potentiality and impact on growth and development was assessed by rearing C. carnea from hatching to pupation on aphids infesting potted plants of RCH-2 Bt and non-Bt. All the instars were reared in specimen tubes (1.0 x3.5 cm) till pupation by feeding known number of nymphs of aphids each day. For each treatment there were 10 grubs and each set was replicated five times. After every 24 hours, grubs were shifted with brush tip to new specimen tube marked for same treatment and replication. The old specimen tube was taken for counting the aphids remaining in the tube to know each day feeding potential. Host material i.e. nymphs of aphids from RCH-2 Bt and non-Bt plants for respective treatments were increased gradually as instar advanced. After pupation and emergence of adults, pairs of males and females for each ten treatment were isolated (2 pairs from each replication) and released separately for mating and oviposition. Adult food (10% honey) was provided in each oviposition cage. The observations for days taken to complete different instar stage, prepupal stage, adult longevity, sex ratio, fecundity, incubation period and total aphid consumed were recorded and presented as comparative figures between two hybrids.

Results and Discussion

The relative abundance of aphids and three predators viz., coccinellids, chrysoperla and syrphids could not vary much between Bt and non Bt crops during both years of observation as well as pooled analysis (Table 1). The population of aphids ranged between 8.58/ leaf (34^th ISW) to 42.15/ leaf (50^th ISW) with a mean of 23.82/ leaf in RCH-2 Bt. Since beginning of the incidence at 28^th ISW (July) population remained above ETL (>10.0/ leaf) till end of the season and with increasing trend and heavy buildup was noticed during December. Population dwindled to below ETL during 31^st, 34^th and 35^th ISW. This incidence appeared to be numerically more in respective weeks compared to the incidence in non-Bt crop, which supported the buildup of aphids to thresholds and above from September onwards only. Prior to that the incidence was found to vary much on weekly observation basis. The range of incidence was 7.43/ leaf (36^th ISW) to 37.08/ leaf (46^th ISW) with a mean of 21.37/ leaf. Despite numerical variations to limited extent, RCH-2 Bt supported the incidence of aphids as well as predators similarly to that of its non-Bt version. The statistical analysis of all these parameters (Table-2) could not show any significant difference in a paired row t-test. Thus the theoretic possible impact of Cry protein on predators through passive exposure was not supported by the two season and pooled data as for as the way in which naturally they occur in the cotton ecosystem. Similar to the present findings Bt cotton did not affect the population dynamics of aphids (Reed et al., 2000, Wu and Guo, 2003 and Hegde et al., 2004 and Venkateshalu, 2005). Therefore, the likely adverse effect of Bt protein on aphid incidence and further on its predators was not evident in the present investigation.

The population of coccinellids, chrysoperla and syrphids were also observed throughout the cropping season. The incidence of predators also followed the trend of two seasons upon pooled analysis. A density dependent variation with respect to prey was shown by all the three predators after their appearance in the season (Fig.1). Mean population of coccinellids, Chrysoperla and syrphids was 0.89, 0.78 and 1.0 per plant in RCH-2 Bt and 0.91, 0.75 and 1.04 per plant in RCH-2 non Bt respectively. There was no significant difference between the predatory population on Bt and non Bt cotton crops (Table 2). The population of predator always depends on its prey. As there was no variation in the host population between two types of cotton, the dependent predator also did not show any difference and there was strong and positive correlation between aphid and predatory population (Table 3). Amongst three predators syrphids exhibited a high degree of host dependent, population build up (r = 0.94 and 0.96) followed by chrysoperla and coccinellids. This clearly indicated that Bt toxin has no effect on major predators on cotton crop. The dynamics of major predation and conventional cotton fields were almost same in a similar study conducted by Wang and Xia (1997) however there was reduction in eggs parasitisation of third generation noctuid eggs. As cry1Ac has target specific action against selected lepidopteron pests the safety of insects belonging to other orders is naturally endorsed. Xia et al, (1999) also observed no change in dominant predators species. There was no significant difference among Bt and non Bt cultivars with respect to seasonal mean incidence of major insect predators viz., coccinellid, chrysoperla, syrphids and spiders as reported by Hegde et al, (2004) and Udikeri et al., (2003).

Further, the most potential predator Chrysoperla carnea was found to remain unaffected in terms of its potentiality when fed with aphids infesting RCH-2 Bt. The figures of bio-potentiality (Table-4) parameters of C. carnea remained statistically on par for two batches reared on aphids that colonized on Bt and non Bt plants. The incubation period was 3.22 and 3.72 day respectively when aphids host was RCH-2 and RCH-2 non Bt. The time spent in each instar stage was slightly more for the C. carnea reared on aphids with non Bt cotton host. Similarly pupal period (10.65 days), adult longevity (47.23 and 50.17 days for male and female respectively) was more in non Bt.sThe fecundity was also high (102.90 / female) in C. carnea population reared on non-Bt crop colonized aphids. Thus total aphid consumption was more (523.22) in this treatment. However none of the bio-potential parameter recorded significant variation. Thus bio-potentiality of C. carnea remained same irrespective of aphid host. Similarly, Pilcher (1997), Mascarenhas and Luttrel (1997), Hilbeck et al, (1998) and Hilbeck (2001) also demonstrated that the Chrysoperla fed on intoxicated aphids survived and continued its progeny as good as Chrysoperla fed on non toxicated aphids. However, the negative effect of mortality and delayed development in predatory stages were observed when C. carnea was fed on intoxicated early instar larvae of lepidopteran pests viz., H. armigera and syrphids larvae was well documented by Mascarenhas and Luttrel (1997) and Dutton et al. (2002). Thus there is every chance of bio magnification of cry proteins over the generations and affect predatory population through intoxicated hosts which need continuous monitoring and necessary action for their sustainability in the eco system.

References

ANDOW, D. A., AND HILBECH, A. 2004. Sciences-based risk assessment for non-target effects of transgenic crops. Bio-sciences. 54: 637-649.

ANONYMOUS., 2006, Project Co-ordinator’s report, All India Co-ordinated Cotton Improvement Project. CICR, Coimbatore.

DUTTON, A., KLEIN, H., ROMEIS, J. AND BIGLER, F., 2002, Uptake of Bt-toxin by herbivores feeding on transgenic maize and consequences for the predator Chrysoperla carnea. Ecological Entomology, 27 : 441-447.

HEGDE, M., NIDAGUNDI, J.M., BIRADAR, D.P., UDIKERI, S.S. AND KHADI, B.M., 2004, Performance of Bt and non-Bt cotton hybrids against Insect pests under irrigated condition. In: International symposium on “Strategies for Sustainable Cotton Production - A Global Vision”. 3. Crop Protection, 23-25 November 2004, UAS, Dharwad, Karnataka (INDIA), pp. 143-145.

HILBECK, A., 2001, Implications of transgenic, insecticidal plants for insect and plant biodiversity. Perspective Plant Ecological Evolutionary System, 4: 43-61.

HILBECK, A., BAUMGARTNER, M., FRIED, P.M. AND BIGLER, F., 1998, Effects of transgenic Bacillus thuringiensis corn fed prey on mortality and development time of immature Chrysoperla carnea (Neuroptera: Chrysopidae). Environmental Entomology, 27 : 480-487.

JAMES, C., 2006, Global review of commercialized transgenic crops: 2006. International Service for the Acquisition of Agribiotech Applications, Briefs No. 29: Preview. ISAAA: Ithaca, NY.http//www..isaaa.org.

JEPSON, P. C., CROFT, B.A, AND PRATT. G.E. 1994. test systems to determine the ecological risks posed by toxin release from Bacillus thuringiensis genes in crop plants. Molecular Ecology.3: 81-89.

LOVEI, G.L. AND ARPAIA, S. 2005, The impact of transgenic plants on natural enemies: a critical review of laboratory studies. Entomologia Experimentalis et Applicate. 114 : 1-14.

MASCARENHAS, V.J., AND LUTTRELL, R.G., 1997, Combined effect of sub-lethal exposure to cotton expressing the endotoxin protein of Bacillus thuringiensis and natural enemies on survival of bollworm (Lepidoptera: Noctuidae) larvae. Environmental Entomology, 26: 939-945.

PILCHER, C, D., OBRYEKI, J.J., M.F. AND LEWIS, L.C. 1997, Pre imaginal development, survival and field dominance of insect predators on transgenic B. thuringencies corn. Environmental Entomology, 26 (2) : 446-454.

REED, J.T., STEWART, S., LAUGHLIN, D., HARRIS, A., FURR, R., RUSCOE, A. AND DUGGER, P., 2000, Bt and conventional cotton in the hills and delta of Mississippi: 5 years of comparison. Proceedings of Beltwide Cotton Conferences, San Antonio, USA, January 4-8, 2000, 2: 1027-1030.

UDIKERI, S.S., PATIL, S.B., NADAF, A.M. AND KHADI. 2003, Performance of Bt-cotton genotypes under unprotected conditions. Proceeding of World Cotton Research Conference-3. 9-13 March, 2003, Cape Town, South Africa : 1282-1286.

VENKATESHALU, 2005, Utilization of Bt cotton hybrids in IPM and their impact on non-target insects. Ph D Thesis University of Agricultural Sciences, Dharwad., p. 254

WANG, C.Y. AND XIA, J.Y., 1997, Differences of population dynamics of bollworms and of population dynamics of major natural enemies between Bt transgenic cotton and conventional cotton. China Cotton, 24: 13-15.

WU, K. AND GUO, Y., 2003, Influences of Bacillus thuringiensis Berliner cotton planting on Population dynamics of the cotton aphid, Aphis gossypii Glover, in Northern China. Environmental Entomology, 32: 312-318.

XIA, J.Y., CUI, J.J., MA, L.H., DONG, S.X. AND CUI, X.F., 1999, The role of transgenic Bt cotton in integrated pest management. Acta Gossypii Sinica, 11 : 57-64.

Table 1: Relative season long abundance of Aphids and predatory insects on RCH-2 Bt and Non- Bt cotton hybrid (pooled).

Month	ISW	Aphids/leaf		Coccinellids/plant		Chrysoperla / plant		Syrphids / plant
		Bt	NBt	Bt	NBt	Bt	NBt	Bt	NBt
Jul	28	10.69 (3.42)	9.67 (3.27)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)
	29	17.92 (4.35)	15.65 (4.08)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)
Aug	30	26.04 (5.20)	24.73 (5.07)	0.70 (1.30)	0.62 (1.27)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)	0.00 (1.00)
	31	9.87 (3.30)	8.17 (3.03)	0.45 (1.20)	0.47 (1.21)	0.03 (1.01)	0.02 (1.01)	0.00 (1.00)	0.00 (1.00)
	32	15.93 (4.11)	12.57 (3.68)	0.60 (1.26)	0.50 (1.22)	0.01 (1.00)	0.03 (1.01)	0.00 (1.00)	0.00 (1.00)
	33	12.00 (3.61)	8.18 (3.03)	0.25 (1.12)	0.40 (1.18)	0.02 (1.01)	0.01 (1.00)	0.00 (1.00)	0.00 (1.00)
	34	8.58 (3.10)	6.22 (2.69)	0.55 (1.24)	0.48 (1.21)	0.06 (1.03)	0.04 (1.02)	0.00 (1.00)	0.00 (1.00)
Sep	35	5.94 (2.63)	7.67 (2.94)	0.60 (1.26)	0.52 (1.23)	0.14 (1.07)	0.14 (1.07)	0.00 (1.00)	0.00 (1.00)
	36	10.55 (3.40)	7.43 (2.90)	0.70 (1.30)	0.74 (1.32)	0.33 (1.15)	0.25 (1.12)	0.00 (1.00)	0.00 (1.00)
	37	13.29 (3.78)	9.92 (3.30)	0.46 (1.21)	0.80 (1.34)	0.54 (1.24)	0.52 (1.23)	0.00 (1.00)	0.00 (1.00)
	38	14.03 (3.88)	11.87 (3.59)	0.29 (1.14)	0.65 (1.28)	0.70 (1.30)	0.61 (1.27)	0.00 (1.00)	0.00 (1.00)
Oct	39	17.05 (4.25)	13.10 (3.75)	0.41 (1.19)	0.52 (1.23)	0.46 (1.21)	0.53 (1.23)	0.32 (1.15)	0.40 (1.18)
	40	19.26 (4.50)	17.13 (4.26)	0.33 (1.15)	0.30 (1.14)	0.47 (1.21)	0.50 (1.22)	0.46 (1.21)	0.53 (1.23)
	41	25.77 (5.17)	25.45 (5.14)	0.49 (1.22)	0.44 (1.20)	0.48 (1.22)	0.56 (1.25)	1.01 (1.42)	0.91 (1.38)
	42	31.43 (5.69)	28.30 (5.41)	0.84 (1.36)	0.76 (1.32)	0.67 (1.29)	0.68 (1.30)	1.15 (1.47)	1.27 (1.50)
	43	35.91 (6.08)	32.73 (5.81)	1.09 (1.45)	0.83 (1.35)	0.95 (1.39)	1.12 (1.46)	1.65 (1.63)	1.64 (1.62)
Nov	44	34.03 (5.92)	33.22 (5.85)	1.13 (1.46)	0.97 (1.40)	1.36 (1.54)	1.28 (1.51)	1.88 (1.70)	2.05 (1.74)
	45	30.75 (5.63)	34.58 (5.97)	1.27 (1.51)	1.18 (1.48)	1.56 (1.60)	1.45 (1.56)	1.69 (1.64)	1.86 (1.69)
	46	37.91 (6.24)	37.08 (6.17)	1.39 (1.55)	1.37 (1.54)	1.78 (1.67)	1.59 (1.61)	2.07 (1.75)	2.06 (1.75)
	47	33.51 (5.87)	33.70 (5.89)	1.76 (1.66)	1.53 (1.59)	1.91 (1.71)	1.81 (1.67)	2.32 (1.82)	2.39 (1.84)
Dec	48	39.23 (6.34)	36.17 (6.10)	1.71 (1.65)	1.83 (1.68)	2.04 (1.74)	2.10 (1.76)	2.75 (1.94)	2.77 (1.94)
	49	38.25 (6.26)	33.62 (5.88)	1.75 (1.66)	2.26 (1.81)	1.88 (1.70)	1.71 (1.64)	2.80 (1.95)	2.99 (2.00)
	50	42.15 (6.57)	32.17 (5.76)	2.06 (1.75)	2.71 (1.93)	1.66 (1.63)	1.47 (1.57)	3.17 (2.04)	2.99 (2.00)
	51	41.62 (6.53)	33.62 (5.88)	2.62 (1.90)	2.06 (1.75)	1.65 (1.63)	1.67 (1.63)	2.80 (1.95)	3.10 (2.02)
Mean		23.82 (4.98)	21.37 (4.73)	0.89 (1.38)	0.91 (1.38)	0.78 (1.33)	0.75 (1.32)	1.00 (1.41)	1.04 (1.43)

Figures in the parentheses are √x + 1 transformation

Table 2: Test statistics for relative abundance of Aphids and insect predators on RCH-2 Bt and Non-Bt cotton.

Test of significance between Bt and Non-Bt cultivar for incidence of
Year	Aphids /plant		t-test	Coccinellids/plant		t-test	Crysoperla / plant		t-test	Syrphids / plant		t-test
	RCH2 Bt	Non-Bt		RCH2 Bt	Non-Bt		RCH2 Bt	Non-Bt		RCH2 Bt	Non-Bt
2004	23.35 (4.93)	21.74 (4.77)	NS	0.95 (1.40)	1.02 (1.42)	NS	0.85 (1.36)	0.88 (1.37)	NS	1.02 (1.42)	1.05 (1.43)	NS
2005	24.28 (5.03)	21.01 (4.69)	NS	0.83 (1.35)	0.80 (1.34)	NS	0.68 (1.29)	0.66 (1.29)	NS	0.99 (1.41)	1.03 (1.42)	NS
Pooled	23.82 (4.98)	21.37 (4.73)	NS	0.89 (1.38)	0.91 (1.38)	NS	0.78 (1.33)	0.75 (1.32)	NS	1.00 (1.41)	1.04 (1.43)	NS

Table t = 2.08 Figures in the parentheses are √x + 1 transformation

Table 3: Correlation matrix for aphid incidence and predatory population on Bt and Non Bt cultivar (r value).

Year	Coccinellids/plant		Crysoperla / plant		Syrphids / plant
	RCH2 Bt	Non-Bt	RCH2 Bt	Non-Bt	RCH2 Bt	Non-Bt
2004	0.62*	0.50*	0.72*	0.70*	0.74*	0.73*
2005	0.78*	0.79*	0.81*	0.85*	0.94*	0.96*

* Significant at P = 0.01

Table 4: Comparative biology of Chrysoperla carnea reared on cotton aphids.

Aphids raised on	Days								Aphids consumed per grub.
	Incubation period	I instar	II instar	III instar	Pupal period	Adult longevity		Fecundity
						Male	Female
RCH-2 Bt	3.22	2.95	3.73	4.10	10.27	45.30	48.13	97.50	510.80
RCH-2 Non Bt	3.72	3.27	3.51	4.35	10.65	47.23	50.17	102.90	523.22
t-test	NS	NS	NS	NS	NS	NS	NS	NS	NS

n = 20

Fig 1: Dynamics of predatory insect population in Bt and non- Bt cotton