Nutritional Responses and Digestive Physiology of Chrysodeixischalcites (Esper) (Lepidoptera: Noctuidae) on Four Commercial Tomato Cultivars.

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1 Academia Journal of Agricultural Research 4(6): , June 2016 DOI: /ajar ISSN: Academia Publishing Research Paper Nutritional Responses and Digestive Physiology of Chrysodeixischalcites (Esper) (Lepidoptera: Noctuidae) on Four Commercial Tomato Cultivars. Accepted 9 th May, 2016 ABSTRACT Malek Azizi 1, Maryam Khosravi 1 and Bahram Naseri 2* 1 Department of Biology, Faculty of Biological Sciences, Islamic Azad University- Tehran North Branch, Tehran, Iran. 2 Department of Plant Protection, Faculty of Agricultural Sciences, University of MohagheghArdabili, Ardabil, Iran. *Corresponding author. bnaseri@uma.ac.ir Chrysodeixis chalcites (Esoer) is one of the important pests of various crops in many countries of the world. Nutritional responses and enzymatic activity of C. chalcites on different tomato cultivars were evaluated at 25± 1 C, 65 ± 5% RH, and photoperiod of 16: 8 (L: D) h. Our results showed that although the lowest food consumption of whole fourth and fifth instars was observed when larvae were fed on cultivar SUN 6108 f1, the mean larval weight during feeding period and weight gain of larvae were the highest when fed on this cultivar. Fifth instar fed on SUN 6108 f1 and Hed Rio Grande showed the highest proteolytic activity. Amylolytic activity of fifth instar was the lowest on Hed Rio Grande. Our findings revealed thatsun 6108 f1 was a suitable cultivar and Hed Rio Grande and Cal.JN3 were unsuitablecultivars for feeding of C. chalcites. Key words: Chrysodeixis chalcites, nutritional response, digestive enzymes, tomato. INTRODUCTION Tomato (Lycopersicon esculentum Mill.) (Solanaceae) is an important vegetable crop grown in Iran and many other countries of the world. The tomato looper, Chrysodeixis chalcites (Esper) (Lepidoptera: Noctuidae), is a highly polyphagous pest of agricultural crops such as vegetables, ornamentals and greenhouse plants (Van Oers et al., 2004). The larvae of C. chalcites, especially in penultimate and ultimate instars, can cause economic damages by feeding on host plant leaves (Harakly and Farag, 1975). Adverse effects resulting from widespread application of broad-spectrum insecticides on various crops have complicated management programs of this pest (Alonso, 2009). and utilization by herbivores rely on both host plant quality and insect nutritional performance (Singh and Mullick, 1997). On the other hand, variation in food source quality can change the growth and development of arthropods (Waldbauer, 1968). Numerous studies have, therefore, been focused on the nutritional responses and digestive physiology of insects in response to feedingon nutritive compounds or secondary metabolites of host plants (Taylor, 1989). The understanding of crop resistanceas aneconomical methodis important in detecting the anti-digestive compounds and their further use in integerated pest management (IPM) programs (Panda and Khush, 1995). The alimentary canal and digestive enzymes of insect pests (especially, proteases) have been selected as targets for pest management programs (Bigham and Hosseininaveh, 2010). For example, the potential application of digestive enzymes inhibitors to control lepidoteran larvae has been documented (Brito et al., 2001; Telang et al., 2003). In this work, to investigate the effects of four commonly grown tomato cultivars on feeding potential of C. chalcites, the nutritional responses and digestive enzymatic activity of this insect wasstudied. Since tomato cultivars examined in our study have different nutritional values for C. chalcites larvae, it is hypothesized that thenutritional performance of larvae feedingon some tomato cultivars will be better than the other tested cultivars. Moreover, it

2 Academia Journal of Agricultural Research; Azizi et al. 340 is hypothesized that the larvae feeding on nutrient-poor cultivars will show lower digestive enzymatic activity than those feeding on nutrient-rich cultivars. Although, C. chalcites is an important pest of tomato in Iran, no published papers are available regarding nutritional physiology of this pest on eithertomato or other host plants. However, two papers were recently published concerning the two-sex life history of C. chalcites on different tomato (Nouri-Ganbalani et al., 2015) and bean (Alami et al., 2014) cultivars. The cultivation of C. chalcites-resistant tomato cultivars is very limited in Iran because of the lack of information regarding susceptibility/resistance of tomato cultivars to this pest. Therefore, the objective of this study was to compare the nutritional responses and enzymatic activityof C. chalcites larvae on four tomato cultivars to provide helpful information for designing comprehensive management options against this pest. MATERIALS AND METHODS Tomato sources Seeds of four tomato cultivars including Cal.JN3, Hed Rio Grande, SUN 6108 f1 and Super crystalwere obtained from the Plant and Seed Modification Research Institute (Karaj, Iran), and sown under field conditions. The selected tomato cultivars were economically grown cultivars in many provinces of Iran and some other countries of the world (Nouri-Ganbalani et al., 2015). For this study, the leaves of the four tomato cultivars were used for feeding of C. chalcites larvae. Laboratory rearing of C. chalcites Originally, fifth instar larvae of C. chalcites were collected from tomato fields located in the Moghan region in northwestern Iran during July, The insects tested on the various tomato cultivars had already been reared for one generation on the same cultivars they were reared during the experiments. All experiments were conductedat 25± 1 C, 65 ± 5% RH, and photoperiod of 16: 8 (L: D) h. Chemicals All enzyme substrates, dinitrosalicylic acid (DNSA) and Bradford reagent were purchased from Sigma Chemical Co., St Louis, USA. Bovine serum albumin (BSA) was purchased from Roche Co., Germany. Nutritional responses of C. chalcites Fifty neonate larvae of C. chalcites were transferred into plastic containers (diameter 16.5 cm and depth 7.5 cm) with four outlets covered by a fine mesh net for larval ventilation. The equal sizes of fresh leaves of each tested cultivar were placed into the containers. The petioles of excised leaves were inserted in water soaked cotton to maintain freshness. Nutritional responses (larval weight, food consumption and feces produced) were calculated on the dry weight basis, using fourth and fifth instar larvae following the gravimetric method (Waldbauer, 1968). The weight of fourth instar larvae was measured, and then they were reared on the leaves of different tomato cultivars.the weights of the larvae were recorded daily before and after feeding until they stopped feeding and reached the prepupal stage. The initial fresh leaves and the leaves and feces remnant at the end of each experiment were weighed daily. The feces produced by fourth and fifth instar larvae reared on different tomato cultivars were also weighed daily. To obtain the dry weights of the leaves, feces, and larvae, 50 specimens for each were weighed, oven-dried at 60 C for48 h and then re-weighed. Preparation of digestive enzymes The rearing procedures ofc. chalcites larvae were similar to those noted in Laboratory rearing of C. chalcites section.the actively feedinglarvae of C. chalcites (fourth and fifth instar) on different tomato cultivars were anesthetized on ice and dissected under a stereoscopic microscope. After the removal of the whole guts, they were cleaned by removal of unnecessary tissues, and then the pooled midguts including contents (5 midguts/ml for the fourth instar and 4 midguts/ml for the fifth instar) were collected in 1.5 ml micro tubes. A hand-held homogenizer was used to homogenize the midguts. The midgut homogenates were then centrifuged at g for 10 min at 4 C and the resulting supernatants used as enzyme sources. Assay of midgut proteolytic activity According to the method of Elpidina et al. (2001), general proteolytic activity of C. chalcites larvae feeding on different tomato cultivars (for 24 h feeding) was measured using an azocasein substrate (at optimal ph). The universal buffer system (50 mm sodium phosphateborate) was utilized to determine the optimal ph of midgut proteolytic activity. Assay of midgut amylolytic activity Digestive amylolytic activity of C. chalcites larvae that fed on different tomato cultivars (24 h feeding) was determined using Bernfeld method (1955) at optimal ph.

3 Academia Journal of Agricultural Research; Azizi et al. 341 Table 1. Mean (± SE) nutritional responses of fourth instar larvae of Chrysodeixis chalcites on different tomato cultivars. Cultivar Larval weight Larval weight gain SUN 6108 f ± 1.13 ab ± 0.63 a 3.10 ± 0.30 b 4.11 ± 0.05 b Hed Rio Grande ± 1.08 bc 7.65 ± 0.95 a 3.12 ± 0.40 b 2.16 ± 0.05 d Cal.JN ± 1.31 a 8.69 ± 0.60 a 2.32 ± 0.13 c 3.45 ± 0.05 c Super crystal ± 0.63 c 7.97 ± 0.53 a 4.74 ± 0.32 a ± 0.20 a Table 2. Mean (± SE) nutritional responses of fifth instar larvae of Chrysodeixis chalcites on different tomato cultivars. Cultivar Larval weight Larval weight gain SUN 6108 f ± 1.66 a ± 0.65 a ± 0.79 c 3.02 ± 0.08 b Hed Rio Grande ± 1.55 b 8.55 ± 1.31 b ± 7.26 a 2.29 ± 0.27 d Cal.JN ± 0.79 b 7.75 ± 0.36 b ± 3.20 b 2.41 ± 0.08 c Super crystal ± 1.03 b 9.67 ± 0.99 b ± 3.05 a 3.71 ± a Statistical analysis Prior to analysis, the experimental data were tested for normality using the Kolmogorove-Smirnov test. Nutritional responses and digestive enzymatic activity of C. chalcites on four tomato cultivars were analyzed based on a completely randomized design (CRD) through One-way analysis of variance (ANOVA) using the statistical software Minitab 16. Least significant difference (LSD) test was used at α = 0.05 to evaluate statistical differences among the means. RESULTS Nutritional responses of C. chalcites Tables 1, 2 and 3 showed the data of nutritional responses inthe fourth and fifth instars of C. chalcites that fed on different tomato cultivars. Nutritional responses of fourth instar larvae of C. chalcites, except larval weight gain (F= 2.7; df = 3, 77; P = 0.051) were significantly different on tested tomato cultivars. The mean weight of the fourth instar (F= 4.97; df = 3, 77; P<0.01) was the heaviest on cultivar Cal.JN3 and the lightest on cultivar Super crystal. However, the highest food consumption (F= 15.43; df = 3, 77; P<0.01) and feces produced (F= ; df = 3, 78; P<0.01) by larvae were seen on cultivar Super crystal (Table 1). The Fifth instar larvae reared on cultivar SUN 6108 f1 showed the heaviest weight (F= 22.03; df = 3, 77; P<0.01) compared with the other cultivars. Although, larvae consumed less food on cultivar SUN 6108 f1 (F= ; df = 3, 78; P<0.01), the highest larval weight gain was observed when larvae were fed on this cultivar (F= 9.59; df = 3, 78; P<0.01). The results of this study indicated that the fifth instar larvae fed on cultivars Super crystal and Hed Rio Grande consumed more food and larvae reared on Hed Rio Grande produced less feces (F= 38.99; df = 3, 78; P<0.01) than those reared on the other tomato cultivars (Table 2). For whole instar larvae, the mean larval weight (F= 4; df = 3, 78; P<0.05) and weight gain of larvae (F= 10.96; df = 3, 78; P<0.01) showed significant differences, being highest on cultivar SUN 6108 f1 and lowest on other tested cultivars. Furthermore, the highest value of food consumption was on cultivars Super crystal and Hed Rio Grande, and the lowest value was on cultivar SUN 6108 f1 (F= 56.81; df = 3, 78; P<0.01). The highest and lowest weights of feces produced (F= ; df = 3, 78; P<0.01) were on cultivarssuper crystal and Hed Rio Grande, respectively (Table 3). Digestive enzymatic activity of C. chalcites Proteolytic and amylolytic activity in midgut extracts from C. chalcites fourth and fifth instar larvae fed on different tomato cultivars (for 24 h feeding) are shown in Table 4. The highest proteolytic activity of fourth instar was observed when larvae were reared on cultivar Cal.JN3 (F = 46.48; df = 3, 8; P<0.01). The highest proteolytic activity of fifth instar was seen when larvae were reared on cultivars SUN 6108 f1 and Hed Rio Grande (F = 26.29; df = 3, 8; P<0.01), whereas the lowest activity was in larvae fed on cultivars Cal.JN3 and Super crystal. The lowest amylolytic activity of fourth (F = 41.94; df = 3, 8; P<0.01) and fifth (F = 66.50; df = 3, 8; P<0.01) instar larvae were on cultivars

4 Academia Journal of Agricultural Research; Azizi et al. 342 Table 3. Mean (± SE) nutritional responses of whole fourth and fifth instar larvae of Chrysodeixis chalcites on different tomato cultivars. Cultivar Larval weight * Larval weight gain SUN 6108 f ± 1.21 a ± 0.47 a ± 1.98 c 3.56 ± 0.05 b Hed Rio Grande ± 2.21 b 8.10 ± 0.78 b ± 7.42 a 2.23 ± 0.13 d Cal.JN ± 0.89 b 8.22 ± 0.29 b ± 2.27 b 2.93 ± 0.04 c Super crystal ± 0.97 b 8.81 ± 0.58 b ± 2.39 a 9.36 ± 0.10 a The means followed by different letters in the same column are significantly different (P<0.01, P<0.05 *, LSD). Table 4. Mean (±SE) proteolytic and amylolytic activity of midgut extracts from fourth and fifth instar larvae of Chrysodeixis chalcites on different tomato cultivars. Cultivar Proteolytic activity (U/mg) Amylolytic activity (mu/mg) Fourth instar Fifth instar Fourth instar Fifth instar SUN 6108 f ± 0.28 b 4.09 ± 0.36 a ± c ± b Hed Rio Grande 2.31 ±0.42 b 4.02 ±0.66 a ±0.002 b ±0.003 c Cal.JN ± 0.31 a 3.02 ± 0.20 b ± a ± ab Super crystal 3.24 ±0.48 b 2.94 ±0.38 b ±0.004 a ±0.005 a SUN 6108 f1 and Hed Rio Grande, respectively. DISCUSSION The results of this study showed that different tested tomato cultivars have significant influence on the nutritional responses and digestive enzymatic activity of C. chalcites. The insect s growth directly correlated with nutrient input and lepidopteran larvae fed on nutrient-rich plants can increase population growth higher than those larvae fed on nutrient-poor plants (Hwang et al., 2008). In compatibility to other reports (Naseri et al., 2010; Namin et al., 2014), we showed that the nutritional responses between fourth and fifth instars of C. chalcites were different from each other. This is owing to the fact that the nutritional requirements of the insects are altered during their development (Barton, 1995) and are positively correlated with body weight over the period (Phillipson, 1981; Fathipour and Naseri, 2011). Food consumption in the fifth instar of H. armigera fed on a navy bean-based diet was greater than in the fourth, mainly owing to the greater rate of ingestion (Barton and Raubenheimer, 2003). Another possible reason for this variation could be the larval age in a particular stadium at the time of weighing. For example, the weights of either the fourth or fifth instars are expected to be lower when the larvae are near to the next instar as compared with the larvae in the middle age of any instars (Fathipour and Naseri, 2011). A significant difference in the insect's ability to utilize different host-plant cultivars suggests some intrinsic variations between these cultivars (Samraj and David, 1988). The nutritional responses of C. chalcites reared on different tomato cultivars were significantly different, suggesting that tested tomato cultivars had various nutritional values. The body weight is known as an important fitness index of insect population (Liu et al., 2004). The larval weight during the feeding period and weight gain of larvae were the highest on cultivar SUN 6108 f1 (Table 3) that might be due to highly-nutritious food. However, the other tested cultivars showed the lowest values of these indices that might be due to having low-nutritious food. As reported by Nouri-Ganbalani et al. (2015), the highest larval mortality, the longest developmental time and the longest larval period of C. chalcites were on cultivars Hed Rio Grande and Cal.JN3, suggesting that these cultivars might have some properties with negative effects on the growth and development of this pest. According to Alami (2014), the larval weight of C. chalcites is affected by different bean cultivars used for larval feeding. Although, the range of larval weight (fifth instar) of C. chalcites on four tomato cultivars is correlated to the data reported by Alami (2014) on different bean cultivars; however, the mean weight gain of larvae on tested tomato cultivars is lower. Therefore, C. chalcites larvae reared on bean cultivars can increase their body weight more efficiently than those fed on tomato cultivars, suggesting that bean leaves are more suitable than tomato leaves for feeding of C. chalcites larvae. Among the different tomato cultivars examined in our study, the highest digestive proteolytic activity of fourth and fifth instar of C. chalcites was seen on cultivars Cal. JN3 and SUN 6108 f1, respectively. Perhaps, the higher

5 Academia Journal of Agricultural Research; Azizi et al. 343 digestive enzymatic activity in larvae fed on these cultivars might be attributed to the variations in the protein content of these tomato cultivars, or to the response of C. chalcites larvae to digestive enzyme-inhibitors (Naseri et al., 2014). The highest proteolytic activity of the fifth instar larvae fed on SUN 6108 f1(4.09 U/mg) was approximately similar to that reported by Naseri et al., (2014) for sixth instar larvae of H. armigera fed on the tomato cultivar Cal.JN3 (4.86 U/mg). However, it was approximately 30-fold higher than that noted by Kotkar et al. (2009) for H. armigera on tomato (0.16 U/mg). Variations in the tested insect species, or physiological differences of the tomato cultivars used for larval feeding could explain this discrepancy. As seen in Table 4, the highest amylolytic activity of the fifth instar larvae on cultivar Super crystal was approximately 4.5-fold lower than that reported for the sixth instar larvae of H. armigera on the tomato cultivar Korral (Naseri et al., 2014), and higher than that reported by Kotkar et al. (2009) for the fifth instar larvae of H. armigera on tomato. These differences could be attributed to variations in the tested insect species, differences in the procedures used for the amylolytic assay and physiological differences among host-plant cultivars used (Khedr et al., 2015). By combining the results of previous study (Nouri- Ganbalani et al., 2015) with the findings of the current research about nutritional physiology of C. chalcites on tested tomato cultivars, it is concluded that cultivar SUN 6108 f1 was suitable and cultivars Hed Rio Grande and Cal.JN3 were unsuitable hosts for feeding and development of C. chalcites. For the future, it would be suggested to study the demographic parameters of C. chalcites on a wide range of tomato cultivars and other host-plants in order to design more practical techniques in the IPM program of this pest. ACKNOWLEDGEMENT We are grateful to M. Mardani-Talaei (from University of Mohaghegh Ardabili) for her assistance in this research. REFERENCES Alami S (2014). Effect of different bean cultivars on some ecophysiological aspects of Chrysodeixischalcites (Esper) (Lepidoptera: Noctuidae). MSc Thesis. University of MohagheghArdabili, Ardabil, Iran (in Persian with English summary). pp. 84. Alami S, Naseri B, Golizadeh A, Razmjou J (2014). Age-stage, two-sex life table of the tomato looper, Chrysodeixischalcites (Lepidoptera: Noctuidae), on different bean cultivars. Arthropod-Plant Interact. 8: Alonso A (2009). 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