EFFECT OF VITAMIN B6 AND MELATONIN ON THE BIOASSAY OF VARIOUS ECONOMIC CHARACTERS OF SILKWORM, BOMBYX MORI L. (LEPIDOPTERA: BOMBYCIDAE) 1 IBRAHIM A. I. SAAD1 AND USAMA M. GHAZY2 Department of Sericulture, Plant Protection Institute, Agricultural Research Center, Sakha, Egypt 2 Department of Sericulture, Plant Protection Institute, Agricultural Research Center, Giza, Egypt (Received 30-11-2008) INTRODUCTION Much research has been done on the diet supplementation of mulberry leaves fed to silkworms in order to improve the commercial characters of silkworm including vitamins, sugars, proteins, amino acids, minerals etc. which are responsible for the proper growth and development of the silkworm, B. mori. The vitamins such as ascorbic acid, thiamin, niacin, folic acid and multi-vitamins were studied by (Etebari 2002 and Etebari et al., 2004.) It has been reported that the vitamins of B-complex group and certain essential sugars, proteins, amino acids, minerals etc. are responsible for the proper growth and development of the silkworm, B. mori (Khan and Saha, 1997 and Faruki, 1998). Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine originally identified in the pineal gland, where it is synthesized enzymatically from serotonin (5- hydroxytryptamine) (Namboodiri et al., 1987; Sugden et al., 1987). The activity of Melatonin was studied in the mulberry silkworm, B. mori by some researchers (Sehadová et al., 2004). The present study was attempted to supplement the mulberry leaves with Melatonin and Vitamin B6 at different concentrations to determine their effect on the different biological aspects of silkworm B. mori. MATERIAL AND METHODS The mulberry silkworm used for the present study was the Chinese hybrid (9F7X). The larvae were reared under the laboratory hygrothermic conditions of 25±2°c and 65-70% R.H., according to the conventional method in trays and provided with suitable amounts of fresh mulberry leaves. Bull. Ent. Soc. Egypt, 86, 2009 (109-1 14) 110 The experimental study was performed on the last larval instar. All larvae which molted to the last instar at the same time were grouped and used for the experimentation. The last larval instar was determined by (0 hour) from the last larval ecdysis (Peferoen and Deloof, 1980). Tablets of commercial compound (Viva-Max) were used in this study. Each Tablet Contain: N-Acetyl 5 Methoxy Tryptamine (Melatonin 3mg) and Pyridoxine hydrochloride (Vitamin B6 10mg) manufactured by Amoun Pharmaceutical Co.(APIC) S.A.E, Egypt. Tablets were offered to the larvae by feeding method after dissolved in distilled water at different concentrations i.e. 3, 6, 9, 12, 15 and 18 ppm. The clean mulberry leafs were dipped in different concentrations for 5 minutes and left to dry then provided to larvae, while untreated larvae fed on clean mulberry dipped in distilled water only . Each treatment and control has 20 replicates. The daily mean weight of larvae was recorded. The silk glands, fresh pupae, fresh cocoons and cocoon cortex's mean weight were also recorded. RESULTS AND DISCUSSION 1- Effect of Melatonin and Vitamin B 6 on the larval growth. Data in Fig (1) show a gradual daily increase in the mean weights of last larval instar during the feeding period from the beginning of the first day until reaching to its maximum mean weights at 8th day (mature larva), then the larval weights showed a gradual decrease in weights after the 8th day (beginning of spinning period) and during the pre pupal stage. Table (1) showed that larvae treated with Melatonin and Vit.B6 presented an increase in the mean weights of mature larvae at all concentrations, but the only significant increase was 10.3% in the mean weight of treated larvae at concentration of 6 ppm compared with untreated one. The present results are in agreement with the findings of Saha and Khan (1996) who determined that mulberry silkworm reared on food supplemented with Vita-Plus (a multivitamin + multimineral capsule) enhanced the larval growth. 2- Effect of Melatonin and Vitamin B6 on the silk gland. Data in table (2) showed also that larvae treated with Melatonin and Vit.B6 presented a significant increase in the silk glands mean weight at concentration of 6 ppm (17%) in comparison to control. These results are in agreement with the findings of ELKaraksy et al., (1983) who observed that B. mori silk glands were highly affected by supplementing mulberry leaves by B-complex vitamins; and Saad (2005) who studied that 111 mulberry silkworm reared on food supplemented with minerals at low concentrations gave the highest mean weight of silk glands. Table (I) Effect of Melatonin and Vitamin B6 on the mean weights of mature larvae. Treatments Mean Weight (gm) ± SD 2.5989 ± 0.380568 2.7306 ± 0.189169 2.8655 ± 0.160516 2.6948 ± 0.365655 2.6325 ± 0.275986 2.6685 ± 0.231983 2.6433 ± 0.233524 Control 3ppm 6ppm 9ppm 12ppm 15ppm 18ppm  Increase Or Decrease % b ab a b b b b 5.1 10.3 3.7 1.3 2.7 1.7 There are no significant differences among the means with the same litters at 5% level according to LSD test. Mean Weight (gm) Fig:1 The Mean weights of mulberry silkworm after treatment with Melatonin and Vitamin B6 during the last larval instar. 3.5 3 2.5 2 control 3ppm 1.5 6ppm 9ppm 1 12ppm 15ppm 0.5 18ppm 0 1 2 3 4 5 6 7 8 9 10 11 12 Days 3- Effect of Melatonin and Vitamin B 6 on the Silk Production. Data in table (3) showed that larvae treated with Melatonin and Vit.B6 presented an increase in the fresh cocoon mean weights at all concentrations except at 12 ppm where an insignificant decrease (0.6%) was observed. Larvae treated with Melatonin and Vitamin B 6 at concentrations of 3 ppm gave an insignificant increase of fresh cocoon mean weights (4.2%), while significant increases of 11.6, 9.0 and 6.7% were observed at the concentrations of 6, 9 and 15 ppm respectively. An increase in the cocoon shell mean weight were also observed when larvae were treated with Melatonin and Vit.B6, these increases ranged from 2.5 to 112 7.9%, but the only significant increase 7.9 % occurred in larvae treated with Melatonin and Vit.B6 at the concentration of 6 ppm. The percentage of cocoon shell mean weight to the pupal mean weight (Cocoon Shell Ratio) was calculated as 21.6 % in larvae treated with Melatonin and Vit.B6 at concentration of 6 ppm without significant difference in comparison to control larvae. These results are in agreement with the findings of ELKaraksy and Idriss (1990) who observed that B. mori silk production was highly affected by supplementing mulberry leaves by B-complex vitamins.  Table (II) Effect of Melatonin and Vitamin B6 on the mean weights of Fresh Silk Glands. Increase Or Decrease Treatments Mean Weight (mg)±SD % 653 ± 11 d Control 700 ± 11 bc 7.2 3ppm 764 ± 15 a 17.0 6ppm 716 ± 10 b 9.7 9ppm 648 ± 33 d -0.8 12ppm 665 ± 17 c 1.8 15ppm 655 ± 23 d 0.3 18ppm There are no significant differences among the means with the same litters at 5% level according to LSD test. Table (III) Effect of Melatonin and Vitamin B6 on the mean weights of Fresh Cocoon and Cocoon Shell. Treatments Fresh Cocoon Mean Weight (gm)±SD Increase Or Decrease % Cocoon Shell Mean Weight (mg)±SD Increase Or Decrease % Cocoon Shell Ratio % Control 3ppm 6ppm 9ppm 12ppm 15ppm 18ppm 1.2424 ± 0.042535 c 1.2945 ± 0.148988 bc 1.3869 ± 0.077913 a 1.3541 ± 0.132362 ab 1.2352 ± 0.086138 c 1.3255 ± 0.227839 ab 1.29545 ± 0.05872 bc 4.2 11.6 9.0 -0.6 6.7 1.0 278 ± 16 b 293 ± 40 ab 300 ± 16 a 289 ± 40 ab 287 ± 25 ab 285 ± 19 ab 289 ± 25 ab 5.4 7.9 4.0 3.2 2.5 4.0 22.4 22.6 21.6 21.3 23.2 21.5 22.3 * There are no significant differences among the means with the same litters at 5% level according to LSD test. 113 4- Effect of Melatonin and Vitamin 86 on the pupal weight. As shown in table (4), larvae treated with Melatonin and Vit.B6 at all concentrations except at 12 ppm presented an increase in the pupal mean weights. The highly and significant increase of pupal weights were 12.7 and 10.4 % when larvae were treated with the concentrations of 6 and 9 ppm, respectively. Table (IV) Effect of Melatonin and Vitamin B6 on the mean weights of Pupae. Treatments Control 3ppm 6ppm 9ppm 12ppm 15ppm 18ppm Mean Weight (gm)±SD 0.9643 ± 0.039313 1.0015 ± 0.126277 1.0866 ± 0.075798 1.0647 ± 0.132776 0.9481 ± 0.084865 1.0407 ± 0.236365 1.0063 ± 0.060512 cd bcd a ab d abc bcd Increase Or Decrease % 3.9 12.7 10.4 -1.7 8.0 4.4 * There are no significant differences among the means with the same litters at 5% level according to LSD test. These results are in agreement with the findings of Saha and Khan (1996) who determined that mulberry silkworm reared on leaves supplemented with Vita-Plus (a multivitamin + multimineral capsule) enhanced the pupal weight. The present study showed that mulberry leaves supplemented with 6ppm of Melatonin and Vitamin B6 had great effect on growth of the silkworm larvae and silk production. SUMMARY Addition of Melatonin 3mg and Vitamin B6 10mg to mulberry leaves of silkworm, Bombyx mori L. at 3, 6, 9, 12, 15 and 18 ppm concentrations was studied during the last larval instar. The results indicated that larvae treated with low concentration (6 ppm) gave the highest parameters and improvement in larval weight, cocoon weight, cocoon shell weight and pupal weight. The significance of Melatonin and Vitamin B6 in the development of silkworm with the economic characters is discussed. 114 REFFRNCES EL-KARAKSY, I. A.; A. HOSNY and H. A. MESBAH (1983): Effect of vitamin Bcomplex on silkworm Bombyx mori L. (Proc. 5th Arab Pesticide Conf Tanta Univ., 1: 153-159). EL-KARAKSY, I. A. and M. IDRISS (1990): Ascorbic acid enhances the silk yield of the mulberry silkworm Bombyx mori L. (J. Appl. Entomol., (109):81-86). ETEBARI, K. (2002): Effect of enrichment of mulberry leaves (Morus alba) with some vitamins and nitrogenous compounds on some economic traits and physiological characters of silkworm Bombyx mori (Lep., Bombycidae), (Unpublished M. Sc. Theses, Isfahan Univ. of Technology, Isfahan, Iran, 150 pp). ETEBARI, K.; R. EBADI and L. MATINDOOST (2004): Effect of feeding mulberry’s enriched leaves with ascorbic acid on some biological, biochemical and economical characteristics of silkworm Bombyx mori L.; (Int. .J. Indust. Entomol. 8: 81—8 7). FARUKI, S. 1. (1998): Nutritive effects of thianomin enriched mulberry leaves on the silkworm, Bombyx mori L. ( Univ. J Zool. Rajshahi Univ. 17: 39-44). KHAN, A R. and B. N. SAHA (1997): Nutrition of the mulberry silkworm, Bombyx mori on feed supplemented with calcium lactate. (J. Ecobiol. 9: 53-58). NAMBOODIRI, M. A. A.; R. DUBBELS and D. C. KLEIN (1987): Arylalkylamine Nacetyltransferasc from mammalian pineal gland. (Methods Enzymol., 142,83—590.) PEFEROEN, M. and A. 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