Effect of dietary antioxidants on growth and development of Taro caterpillar, Spodoptera litura (Fab.)

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1 Indian Journal of Experimental Biology Vol. 56, February 2018, pp Effect of dietary antioxidants on growth and development of Taro caterpillar, Spodoptera litura (Fab.) Kirti Sharma 1 *, Sridhar J 2 & RK Sharma 1 1 Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi , India 2 ICAR-National Institute of Biotic Stress Management (ICAR-NIBSM), Baronda, Raipur , Chhattisgarh, India Received 27 June 2015; revised 15 Jan 2016 In India, crop yield losses due to various factors including insect-pests range from 15-25% amounting to INR lakh crores annually. IPM including on-line pest monitoring and real time advisory dissemination system may help minimizing such yield losses. With regard to insect pests, it becomes imperative to understand their biology, life history, behaviour and feeding habits, susceptibility and resistance to insecticides. On the other hand, insect diets in rearing room are highly susceptible to microbial growth as well as oxidative destruction by reactive oxygen species (ROS) which can damage macromolecules, such as DNA, RNA and proteins, and thereby check the growth and development of insects. However, not much attention has been paid on the role and nature of antioxidants in rearing insect. Free radicals have been implicated as relevant players in the biology of the insect midgut and as mediators of the relation with the indigenous micro biota. Furthermore, insect feeding contributes to acceleration of oxidative deterioration of diets due to introduction of extra-oral digestive secretions and increased surface area of the diet that is directly exposed to atmospheric oxygen. Here, we studied the effects of five dietary antioxidants in various combinations on the biological attributes like developmental period, survival, fecundity and growth index of tobacco caterpillar Spodoptera litura (Fab.) (Lepidoptera: Noctuidae) using meridic diet. The larval weight at 14 th day of development, total development period and per cent survival were mg, days and 88.0% in modified diet as compared to mg, days and 84.1%, in control. The combinations of α-tocopherol at 1500 ppm and ascorbic acid at 3200 ppm enhanced the survival by 1.33%, fecundity by 73 eggs per female and larval growth index (LGI) by 0.31 points. It also enhanced the reproductive potential of S. litura in combination with β-carotene at 200 ppm by enhancing survival to 89.33%, fecundity by 139 eggs per female, LGI by 5.04 and total developmental index (TDI) by 3.24 which were significantly higher over all other treatments. Keywords: Cotton leaf worm, Fecundity, Growth index, Meridic diet, Tobacco cutworm Crop yield losses in India due to various factors such as insect-pests, diseases, weeds, nematodes and rodents has been estimated to range from 15-25% amounting to 0.9 to 1.4 lakh crore rupees annually 1. Integrated pest management (IPM) including online monitoring of common pests of economic crops may help to contain such yield losses. The tobacco cutworm, Spodoptera litura (Fab). (Lepidoptera: Noctuidae), is one such major pest affecting crop plants. Since cutworm larvae can defoliate many economically important crops possessing a high dispersal capability, this pest often leads to high levels of agricultural losses 2,3. The epidemic of S. litura in Maharashtra during in an area of 1.46 million ha of soybean and cotton based cropping system caused yield and monetary loss of 0.85 million tonnes and 1.39 billion rupees, respectively 1. S. litura is now under *Correspondence: kirtisharma2@yahoo.com CROPSAP e-pest surveillance and monitored regularly for outbreaks 1. Its wide dissemination and key pest status has been attributed to polyphagous nature and its ability to undergo both facultative diapause and seasonal migration 4. Hence, it is important to understand its biology, life history, behaviour and feeding habits, susceptibility and resistance to chemical. Artificial diet is nutritionally balanced insect food prepared from conveniently available substances for mass culturing insects round the year and to produce uniform and healthy insect for reducing experimental error. However, the insect diets in rearing room are highly susceptible to microbial growth as well as undergo oxidative destruction by reactive oxygen species (ROS) which are biologically active substances that can damage macromolecules such as DNA, RNA and protein 5,6 unfavourable for the growth and development of insect rearing. The insects are known to be adversely affected specifically by free radicals but less attention has been paid on the role and

2 130 INDIAN J EXP BIOL, FEBRUARY 2017 nature of antioxidants in rearing insect 5,7. Free radicals have been implicated as relevant players in the biology of the insect midgut and as mediators of relation with the indigenous microbiota 8. Furthermore, insect feeding contributes to acceleration of oxidative deterioration of diets due to introduction of extra-oral digestive secretions and increase of surface area of the diet that is directly exposed to atmospheric oxygen. Hence, antioxidants which protect diets and insects from the attack of ROS are added to insect diets to prevent the oxidative destruction 9. There has been a growing interest in the functional significance of ROS and antioxidant responses in growth, development and differentiation of insects and plants 7,10. The essentiality of β-carotene, ascorbic acid and α-tocopherol in insects have been known for decades but the potential function of these vitamins as antioxidants in insects and in the diets themselves has been recognised by Cohen 7. The antioxidants, namely ascorbic acid, glutathione, and α-tocopherol that protect the cells of other eukaryotes from oxidative stress are also present in insect cells 11. The eggs of Schistocerca gregaria (Forsk) normally contain adequate β-carotene to allow the growth of the larvae and also for normal body colouration. Lack of β-carotene inhibits development of their normal yellow and green colour and melanisation is also reduced 12. The potential of these antioxidants has greater application in insect diets. In this context, it is important to understand the possible effects of diet antioxidants on growth and developmental parameters of insects. In this study, we examined the effects of antioxidants of artificial diet and its dose on growth and developmental parameters of the tobacco caterpillar Spodoptera litura. Apart from modifying concentrations of ascorbic acid and α-tocopherol of control diet, β-carotene, glutathione reduced and gallic acid were studied with a purpose to find out more suitable combination of antioxidants. Materials and Methods The egg patches of S. litura were collected from cauliflower fields of Indian Agricultural Research Institute, New Delhi, to start the nucleus colony. The insect culture was maintained in the laboratory from a single pair and one egg patch on improved artificial diet under controlled conditions i.e. temperature 27±1 C, relative humidity 65±5% and a scoto/photo phase regime of 16:8 h. Antioxidants All antioxidants used in the study viz. ascorbic acid, α-tocopherol, β-carotene, glutathione reduced and gallic acid were procured from Himedia Laboratories Pvt. Ltd. The antioxidants were studied alone as well as in combination with other antioxidants. In the treatments D1-D13, the antimicrobial agents used were same as in the control diet (Table 1). In diets D14-D17, the best suited antioxidants were combined in optimal concentrations of antimicrobial agents worked out in our laboratory to know their combined effect 13. The diet preparation and insect rearing were done as per standard methodology 14. Most of the diet ingredients were obtained from Himedia Laboratories Pvt. Ltd. except boric acid (BA) (Qualigens Fine Chemicals), castor oil (Thomas Baker (Chemicals) Ltd., multivitamin and multimineral drops (Alken Laboratories Ltd.). Twenty five neonate larvae were inoculated in sterilised glass vials, one each. The larval weight was recorded on 8 th and 14 th day of the larval period, standardized as optimal time to measure the growth. The one day old pupae were weighed and transferred to glass jars covered with cloth kept at ambient conditions. One pair of newly emerged adults were transferred to ovipositional jars provided with 10% Table 1 The antioxidant combinations of various diet treatments Diets Diet antioxidants (ppm) Ascorbic α tocopherol β carotene Glutathion Gallic acid reduced acid D1* D2* D3* D4* D5* D6* D7* D8* D9* D10* D11* D12* D13* D14# D15# D16# D17# Control [*Antimicrobial agents of control diet: methyl paraben, sorbic acid, streptomycin sulphate and formaldehyde (1800, 1300, 200 and 2000 ppm, respectively). #Antioxidant diet treatments in combination with modified concentrations of antimicrobial agents. Modified concentrations of antimicrobial agents: methyl paraben, sorbic acid, streptomycin sulphate, either formaldehyde or boric acid (2500, 1300, 400, 2500 and 200 ppm respectively)]

3 SHARMA et al.: EFFECT OF DIETARY ANTIOXIDANTS ON SPODOPTERA LITURA 131 sucrose solution (fortified with 2% vitamin solution) as adult diet to study adult longevity and fecundity. The effects of the test diets on larval and pupal weight (mg), larval, pupal and total developmental periods, per cent pupation, adult emergence, per cent survival and fecundity were recorded. The data was subjected to appropriate statistical tool, OPSTAT using Completely Randomized Design. The growth and development of the larvae were assessed using the following formulae. Per cent pupation Larval Growth index LGI = Average larval period days Total Developmental Index TDI = Larval Pupal Period days In the concluding experiment, the antioxidants were combined with optimal concentration of antimicrobial agents keeping all the other ingredients of diet as same. Per cent survival Results The experiment was conducted in three sets (Set I: D1-D7; Set II: D8-D13; and Set III: D14-D17) for assessing the effect of dietary antioxidants and antimicrobials on growth and development of S. litura and separate control was maintained for each set of experiment and the results are presented in Table 2. Two antioxidant combinations of ascorbic acid α-tocopherol 1000 ppm (D1) and ascorbic acid α-tocophrol 1000 (D2) significantly yielded low survival i.e and 76.00%, respectively, as compared to 84.0% in control. The developmental period was lengthened by 3.5 days. The larval growth index (LGI) and total developmental index (TDI) were statistically lower in these diets as compared to test diet, indicating that these dietary concentrations significantly affected the post larval developmental stages of S. litura (Fig. 1). The combinations of α-tocopherol at 1500 ppm and ascorbic acid at 3200 ppm enhanced the survival, fecundity, LGI by 1.33%, 73, and 0.31, respectively and the developmental period was shortened by 1.45 days. Surprisingly, β-carotene at 200 ppm (D5) enhanced survival rate to 89.33%, fecundity by 139 (Table 2 and Fig. 1). LGI and TDI were significantly higher over all other treatments (5.04 and 3.24). At this dose, the larval weights on 8 th and 14 th day increased by 29.3 and 20%, respectively as compared to control. The larval and total developmental periods were shortened by 3 and 3.77 days, respectively. The ascorbic acid and α-tocopherol were found effective at 3200 and 1500 ppm, respectively and these concentrations were used in further treatments. The diet antioxidant combinations of β-carotene with ascorbic acid and α-tocopherol (D8 and D11), supported the growth and development of S. Litura (Table 3). The larval weights on 14 th day were and mg, respectively which were 23 and 19.4% higher as compared to control diet. The LGI and TDI in these were enhanced by 4 and 2.65%, respectively indicating that β-carotene can be used to protect diet from oxidative stress. The higher fecundity was recorded in D11 ( ) containing α-tocopherol as compared to control diet (Fig. 1). The effects of glutathione reduced with ascorbic acid and α-tocopherol (D9 and D12) showed the moderate effect on the biological attributes tested. However, the gallic acid in both combinations (D10 and D13) yielded low larval weights ( and mg), longer developmental periods (36.36 and days) with poor per cent survival (54.67 and 52.0%) as well as very low fecundity which was almost one fourth of the control diet (Table 3). The LGI and TDI of diets incorporated with gallic acid declined by approximately 50 per cent (Fig. 1). Fig. 1 Effect of dietary antioxidants on growth index and fecundity of Spodoptera litura

4 132 INDIAN J EXP BIOL, FEBRUARY 2017 The third set of the experiment (i.e., Set III), we evaluated the combined effects of antioxidants and antimicrobials on the biological attributes of S. litura. Set I Diet antioxidants (ppm) Formaldehyde was included as an antimicrobial agent in D14 while it was substituted with boric acid in diets D15, D16 and D17. The antioxidant and Table 2 Effect of antioxidants added to standard diet on growth and development of Spodoptera litura LW 8 th day (mg) LW 14 th day (mg)* LP Pupation (%)** PW (mg)* PP DP AE (%)** AL Survival (%)** D1- AA 4000+α tocopherol b b d b bc c d b 7.50 (21.45) (64.43) (19.82) (73.56) c (59.80) D2- AA 2500+α tocopherol c b c b ab 9.96 b c bc 7.50 (21.98) (66.53) (20.12) (72.44) c (60.72) D3- α tocopherol 1500+AA bc ab b a ab 9.51 b b ab 8.17 (22.67) (71.01) (20.32) (77.78) ab (67.53) D4- α tocopherol 500+AA c b b b bc 9.91 ab bc a 8.33 (21.23) (65.43) (19.59) (81.30) bc (63.51) D5- β carotene a a a a a 9.28 a a a 8.50 (24.54) (73.92) (20.52) (81.87) a (71.01) D6- Glutathione reduced bc b c a ab b cd ab 8.00 (21.27) (71.01) (20.19) (76.03) b (66.53) D7- Gallic acid d c e c c c e c 6.33 (12.07) (56.39) (16.74) (65.41) d (49.22) Control- AA α tocopherol bc b c a ab 9.77 ab c ab (21.94) (71.01) (20.07) (76.16) b (66.53) SE (m)± CD (P=0.05) Set II D8-AA 3200+β carotene a a a a a 9.09 ab a a 8.87 a a (25.00) (73.92) (20.68) (77.96) (69.91) D9- AA 3200+Glutathione reduced b b b a a 9.40 ab b a 8.17 ab ab 250 (22.53) (71.01) (20.17) (78.11) (66.82) D10- AA 3200+Gallic acid c c c b b c c b 6.50 c c (12.59) (55.58) (16.63) (64.05) (47.68) D11- α tocopherol 1500+β carotene a a ab a a 8.93 a b a 8.50 ab ab 200 (24.69) (73.92) (20.61) (76.29) (68.63) D12- α tocopherol 1500+Glutathione b b b a a 9.66 b b a 8.17 ab b reduced 250 ppm (22.03) (69.73) (20.23) (74.20) (64.43) D13- α tocopherol 1500+Gallic acid 9.46 c c c b b c c b 6.33 c c 200 (11.85) (54.75) (16.61) (62,10) (46.15) Control- AA 3200+α tocopherol b b b a a 9.58 b b a 8.00 b ab 1000 (22.16) (69.91) (20.12) (79.99) (66.52) SE (m) ± CD (P=0.05) Set III D14- β carotene 200+α tocopherol 1500+MP 2500+SA 1300+SS 400+For 2500 D15- β carotene 200+AA 3200+MP 2500+SA 1300+SS 400+BA 200 D16- β carotene 200+α tocopherol 1500+MP 2500+SA 1300+SS 400+BA 200 D17- β carotene 200+glutathion reduced 250+MP 2500+SA 1300+BA 200+SS 400 D18 Control- AA3200+α tocopherol 1000+MP 1800+SA 1300+SS 250+For a a (26.94) a a (26.59) a a (26.46) b b (22.52) b b (22.33) a (75.55) ab (73.92) ab (73.92) b (69.91) c (68.81) a (20.72) a (20.66) a (20.93) b (20.46) c (19.93) 8.50 a a (81.96) 8.79 ab a (77.96) 8.84 ab b (80.11) 9.10 b a (81.51) 9.83 c b (83.90) 8.63 a (72.29) 8.44 ab (69.91) 8.53 ab (69.91) 8.11 b (67.81) 7.83 b (66.42) SE (m) ± CD (P=0.05) NS NS 0.47 NS [The experiment was conducted in three sets (Set I : D1-D7; Set II: D8-D13; Set III: D14-D17) and separate control was maintained for each set of experiment. * Values in the parenthesis are square root transformed. ** Values in the parenthesis are arc sin transformed. LW- Larval weight; LP- Larval period; PW- Pupal weight; PP- Pupal period; DP- Developmental period; AE- Adult emergence; AL- Adult longevity; Diet antioxidant treatments incorporated with antimicrobial agents (Methyl paraben-1800, Sorbic acid-1300, Streptomycin sulphate-250 and Formaldehyde-2000 ppm)]

5 SHARMA et al.: EFFECT OF DIETARY ANTIOXIDANTS ON SPODOPTERA LITURA 133 Table 3 Effect of different concentrations of antimicrobials (suggested) in diet on growth and development of S. litura Treatments (Antimicrobial agents in ppm) Survival (%) LGI PGI TDI MP SA SS FOR (73.79) 5.16 a a 3.50 a MP SA BA SS (70.19) 4.88 b a 3.26 ab Control diet- MP SA SS FOR 2000 ppm (69.87) 4.51 b 9.61 b 2.99 c CD (0.05) 3.88 NS Table 4 Increase in bacterial population of different diets at various durations recorded at rearing room (27 o C) Treatments Bacterial population/(log CFU/g dry weight) 1 h 12h 24h 48 h 72 h 96 h 120h Control diet- MP SA SS For 2000 ppm (1.49) (1.30) (1.29) (1.35) (1.58) (1.82) (1.96) Suggested diet- MP SA SS BA (1.44) (1.30) (1.12) (1.08) (1.50) (1.75) (1.89) Values in the parenthesis are Log CFU/gm dry weight antimicrobial combinations of diet D14 (containing formaldehyde as one antimicrobial agent) recorded significantly higher larval weight ( mg), pupal weight ( mg), survival rate (90.67%), fecundity ( ), LGI (5.26) and TDI (3.35) (Table 2). The diets, D15 and D16 enhanced the larval and pupal weights. The survival was increased by 4% in these two diets compared to control. The developmental period was shortened nearly by 3 days while the fecundity was increased by 2.8 and 5 per cent in D15 and D16 respectively, as compared to control. The LGI and TDI were significantly higher than control. The antioxidant combinations of β-carotene with α-tocopherol and ascorbic acid significantly enhanced the biological attributes of insect when compared to control (Table 3). The best performing diets were where antioxidants were combined with optimum concentration of antimicrobial agents i.e. D14, D15 and D16 because they enhanced the diet quality which resulted in high per cent survival and fecundity over control (Table 2 and Fig. 1). The suggested antimicrobials were able to control the bacterial colonies better than the control diet. The CFU/g of dry diet was recorded (Table 4). The overall best performing diet was D14 although D15 and D16 were statistically at par with it. Discussion Reactive oxygen species (ROS) are natural products of oxidative metabolism and play important roles in the growth, development, and defense responses of most aerobic organisms. However, excessive accumulation of ROS can cause severe oxidative damage to organisms and may lead to cell damage, aging, and death 6,15. To protect cells from oxidative damage caused by excessive ROS, organisms have developed a complete defense system to maintain the intracellular redox balance 16. In addition, the insects reared under laboratory are to be supplanted through artificial diets to protect insects from oxidative stress. The main objective of this study was to investigate the effects of dietary antioxidants on developmental period, survival and fecundity and growth index of S. litura. Discussion on vitamins A, C and E is ambiguous, largely because of multiple functions, including antioxidant activity. Furthermore, recognition of other compounds as antioxidants has emerged as a challenging component of human food science and human nutrition, and these compounds also has great potential as antioxidants in insect diets. These deliberately added antioxidants improve the nutritional quality of diet by scavenging the oxygen free radicals and enhance the absorption of nutrients in insect gut. It was also proved that addition of the antioxidants to the diet ameliorate oxidative stress in the gut of the insects 17. The results have shown significant variation in the possible effects of antioxidants. The diet antioxidant combinations of α-tocopherol and ascorbic acid (1500 and 3200 ppm) improved the biological attributes of S. litura. The ascorbic acid concentration of 2500 and 4000 ppm were not found suitable for the development of S. litura as they have also reduced survival, fecundity and growth index. Higher dietary ascorbic acid concentration (0.4%) did not support the larval weights, and survival besides inflicting considerable mortality in the aphid, Myzus persicae (Sulzer) 18. It is opined that 0.4% ascorbic acid might have been detrimental to the larval growth. However, our results show that ascorbic acid at 3200 ppm has significantly supported the growth and development of S. litura. Each molecule of ascorbic acid has two

6 134 INDIAN J EXP BIOL, FEBRUARY 2017 redox-active hydrogen atoms in its structure which carries, two high-energy electrons, whose distinct functions make it a premier antioxidant in the biological system. Barbehenn et al. 19 have found that the dietary ascorbic acid together with glutathione were the most efficient in scavenging the reactive oxygen species in the midgut lumen of tent caterpillar, Malacosoma disstria Hubner by recycling the ascorbate for better performance of the larvae. These findings are in agreement with Zhang and his coworkers 16 who observed that ascorbate, protein carbonyl and total GSH could remove excess ROS and may be involved in antioxidant defense. Antioxidant, α-tocopherol when used in the diets at 1500 ppm in combination with either ascorbic acid (3200 ppm) or β-carotene significantly increased the rate of development of insect, so as to increase the overall survival. Vitamin E (α-tocopherol) can be included in the diet of some insects for its antioxidant activities to protect the integrity of fatty acids and perhaps some other substances. The possible effect of vitamin E came to light clearly in the reproductive activities of the adult, and is needed in the food of the nymphs and larvae of the parasitoid, Agria affinis. The growth and development of A. affinis increased when reared on the diets containing α-tocopherol and β-carotene 20. The higher fecundity and fertile egg laying of A. affinis was because of the vitamin E 19 and in its absence insect produced only 0.6 larvae per female as compared to 7.2 per female. The addition of vitamin E to the diets enhanced the fecundity of Spilosoma obliqua Walker up to 92.6, and on 0.02, 0.04 and 0.06% fortification, respectively, as compared to the diet lacking this vitamin 21. However, our data show that vitamin E promotes growth of S. litura better than vitamin A. The fecundity of S. litura was observed to be significantly better when larvae were reared on diets containing α-tocopherol at 1500 ppm. β-carotene in combination with either α-tocopherol or ascorbic acid exhibited significant positive effect on the biological attributes of S. litura. The performance of larval and post larval developmental stages was better. Vitamin A acetate was found to accelerate the growth of A. affinis and seemed to enable reproduction to occur 22. Interestingly, the moths of Manduca sexta (L.) reared on β-carotene supplemented artificial diet were 2-3 times more likely to feed and escape behaviour than were the moths from other adult diet treatments 23. The optimum concentrations of antimicrobials were also tested in combination with antioxidants to study their cumulative effect. It was observed that the developmental parameters were better than control in all the combinations except glutathione reduced. The better performance of the larvae could be because small amount of oxygen left after reduction found as superoxides, hydrogen peroxide and oxyl radicals, which selectively damage amino acids, fatty acids or nucleotides, are taken care by added antioxidants 6. The improved quality and shelf life of the diet as the modified concentrations of antimicrobial agents prevent or delay microbial contamination of the diet favours insect growth and survival. Hence, the antioxidants, β-carotene, ascorbic acid and α-tocopherol can be used in combinations with antimicrobial agents in the diet for the successful mass rearing of S. litura. The diet D14 as compared with diet D15-D16 better supported the biological attributes of the insect but diet D15 and D16 were statistically on par with D14. The boric acid is an effective antimicrobial agent, cheap, easily available and here used in 1/12 fraction as compared to formaldehyde in the diet. The formaldehyde which is carcinogenic, the merits of the diets D15 and D16 increase. It is inferred from the current study that antioxidant and antimicrobial combinations of diets D14 (β-carotene α-tocopherol MP SA SS For 2500), D15 (β-carotene AA MP SA BA SS 400) and D16 (β-carotene α-tocopherol MP SA SS BA 200) showed additive effect on the growth and development of S. litura. The culturable bacterial population at rearing room temperature (27 C) was less in the diet D15 (new suggested diet) as compared to control diet. Therefore, it can be concluded that for mass culturing of healthy insects both antioxidants and antimicrobials in optimum concentration should be added to insect diet as deliberately added protective ingredients to save the diet from microbial deterioration and oxidation. References 1 Vennila S, Lokare R, Singh N, Ghadge SM & Chattopadhyay C, Crop pest surveillance and advisory project of Maharashtra - A role model for an e-pest surveillance and area wide implementation of integrated pest management in India. (ICAR-National Research Centre for Integrated Pest Management, New Delhi, India), 2016, 56p.

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