Effect of Phytoecdysteroid on Incubation Period and Per Cent Fertilized Egg of Multivoltine Mulberry Silkworm Bombyx mori Linn.

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1 World Journal of Zoology 8 (2): , 2013 ISSN IDOSI Publications, 2013 DOI: /idosi.wjz Effect of Phytoecdysteroid on Incubation Period and Per Cent Fertilized Egg of Multivoltine Mulberry Silkworm Bombyx mori Linn. K. Srivastava and V.B. Upadhyay Silkworm Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur , India Abstract: Effect of phytoecdysteroid on incubation period and per cent fertilized egg of multivoltine mulberry silkworm Bombyx mori was studied. Variation in e phytoecdysteroid concentration and number of larval treatment(treated e larvae) influenced e incubation period and per cent fertilized egg of Bombyx mori eggs. In 40, 50 and 60% phytoecdysteroid concentration, e incubation period decreased slowly and slowly, in single(treated e V instar larvae onlyone times just before two days of larval spinning) and double treatment(in first treatment treated e IV instar larvae just before two days of IV moulting furer, second treatment for e same larvae was given to e V instar just before two days of spinning). Seventy per cent phytoecdysteroid concentration in all e treatment caused considerable increase in e incubation period of eggs. The minimum incubation period was noticed (7.95 ± 0.180) days in case of double treatment by 60% phytoecdysteroid concentration. Maximum per cent fertilized egg was noticed (94.75 ± 2.03) in case of double treatment by 60% phytoecdysteroid concentration. Experiments were performed by different concentration of phytoecdysteroid obtained from Achyranes leaf extract. A control set was always maintained wi each set of experiment. Number of larval treatment and variation in e phytoecdysteroid concentration did not cause significant effect on e incubation period of egg as e economy of Bombyx mori. Two-way ANOVA indicates at number of larval treatment significantly (P 2 < 0.01) influenced e percentage of fertilized egg. Phytoecdysteroid treatment, if applied tactfully, may be useful for boosting up e sericulture industry as well as e economy of silkworm rearing. Key words: Bioactive Hormone IV Instar Larvae Eggs Moulting INTRODUCTION The silk industry has developed as a popular cottage industry providing self employment to more an ten million rural persons in e unorganized sector. It is well known for its low investment and quick and high return which make it an ideal industry fitting well in to e socioeconomic frame of India. An analysis of e international trends in e silk production suggests at sericulture has better prospects for grow in e developing countries an in e developed countries. The efforts are being made to evolve new technologies at are cast effective, labour saving and ecofriendly. In order to increase e production of silk efforts have been made to study e effect of temperature [1], relative humidity [2], photoperiod [3], artificial diet [4], X rays [5] etc on e performance of silkworm. The magnetization of egg influences silk producing potential [6,7] and incubation period of eggs[8,9]. Ecdysone and juvenile hormone are e two major circulating hormones in insects which control e grow and development in insects. The plant at produce insect moulting hormone termed phytoecdysteroids (PES), functions as strategic defenses for plants against insects by acting as eier feeding deterrents or rough developmental disruption in insects. The response of silkworm to e small quantities of ese phytoecdysteroids or its analogues may hasten e larval maturation events and also influenced e spinning process of e several silkworm larvae may prove to be very much useful for e management of rearing program and economics of silkworm industry. Ecdysteroids play key role in moulting and metamorphosis in insects. Many plants are known to contain ecdysteroids wi high moulting hormone activity. The plant like Achyranes aspera (Lat jeera) and Cassia tora (Choti chakwar) have been identified to Corresponding Auor: Kanchan Srivaastava, Department of Zoology, D.D.U. Gorakhpur University, Gorakhkpur (UP), India. 147

2 have phytoecdysteroids [10]. Keeping is in view, an attempt has been made to investigate e influence of phytoecdysteroid on incubation period of multivoltine mulberry silkworm (B. mori). MATERIALS AND METHODS Seed Cocoon: The seed cocoons (pupa enclosed in silken case) of multivoltine mulberry silkworm (Bombyx mori nistari), a native of West Bengal in India, were obtained from e silkworm grainage Behraich, Directorate of Sericulture Uttar Pradesh and, maintained in e plywood trays (23 x 20 x 5 cm) under e ideal rearing conditions [11] in e silkworm laboratory, department of Zoology, D.D.U. Gorakhpur University, Gorakhpur. The temperature and relative humidity were maintained at 26 ± 1 C and 80 ± 5 % RH respectively till e emergence of mos from e seed cocoons. The mo emerged generally in e morning at around 4 am. The tray in which e seed cocoons were kept was suddenly illuminated by light in e morning at 4 o clock on 9 and 10 day of spinning. The newly emerged mos from seed cocoon were quickly picked up and kept sexwise in separate trays to avoid copulation. The male mos were smaller in size but more active an e female mos which were comparatively larger and less active. Copulation: Mos have a tendency to pair immediately after e emergence. Therefore, e female mos required to copulate wi e male mos, were allowed eir mates for copulation. Three hundreds sixty pairs, each containing one male and one female from newly emerged mos were allowed to mate at 26 ± 1 C, 80 ± 5% RH and 12 hour / day dim light condition. After four hours of mating, e paired mos were decoupled manually by holding e female mo between e umb and middle finger gently and pushing e male away by e fore finger. The male mos were discarded while e female mos were allowed to egg laying. Oviposition: Just after separation, e gravid females laid eggs on e sheet of paper in dark condition at 26 ± 1 C and 75 ± 5% RH. The egg laying mos were covered by open plastic cellules to prevent intermixing of egg masses deposited by different mos. After 24 hours of egg laying, e female mos were individually examined for eir disease freeness. The females were crushed individually in mortar wi pestles and blood smears were examined by microscope under 15 x 45 magnifications for e detection of bacterial and protozoan paogens. s The disease free layings (D.F.L ), us prepared, were treated wi 2% formalin for 15 min to increase e adhesiveness of egg of e paper sheet and surface disinfection. Thereafter, e egg sheets wi egg laid on were oroughly washed wi running water to remove formalin and eggs were dried in shade. The dried eggs, us obtained, were taken for phytoecdysteroid treatment under various experimental conditions. Experimental Design: To observe e influence of bioactive phytoecdysteroid hormone on e performance of B. mori, e experiments were performed wi different concentrations of phytoecdysteroid hormone wi respect rd to e treatment of III, IV and V instar larvae. For extraction of phytoecdysteroid, e leaves of Achyranes aspera were collected, washed oroughly wi distilled water and dried in incubator at 37 C. The dried leaves were powdered wi e help of mechanical device. Furer, 50 g powder, us obtained was subjected to extraction rough soxlet apparatus wi 250 ml distilled water for 40 h. After 40 h of extraction a little amount of concentrated solution was obtained which was dried and 6.75 g powdered material was obtained. The dried powder was dissolved in distilled water as 5 g in 25 ml water and used is solution for furer experiment as 100% concentration of phytoecdysteroid. For furer experiment e suitable narrow range of Achyranes phytoecdysteroid concentration viz; 40, 50, 60 and 70 % were taken. Thus, four phytoecdysteroid concentrations were applied topically by spraying as 10 ml on 100 g mulberry leaves and e larvae were fed on e treated leaves. Three set of experiments were designed viz single, double and triple treatment of larvae. Single Treatment: Single treatment of larvae was performed wi e V instar larvae just before two days of e beginning of larval spinning. 100 larvae were taken out from e Biological Oxygen Demand(BOD) incubator and e mulberry leaf, treated wi 40% concentration of Achyranes leaf extract, was given as food. Furer, e treated larvae were given normal mulberry leaves as food. Double Treatment: Double treatment of larvae was started from e final stage of IV instar larvae. In e first treatment, 100 larvae of IV instar were treated just before two days of IV moulting, by providing treated mulberry leaf wi 40% solution of Achyranes leaf extract. The treated larvae were en transferred in BOD incubator for furer rearing and development. Furer, second treatment for e same larvae was given at e final stage of V instar ie just before two days of spinning. 148

3 Triple Treatment: For triple treatment, e ird instar rd larvae just before III moulting, were separated from BOD rd incubator. In e first treatment 100 larvae of III instar were treated by providing extract treated mulberry leaf and kept in BOD incubator for rearing. The second treatment of same larvae was done just before two days of IV moulting ie at e final stage of IV instar larvae and transferred in BOD incubator for rearing. Third treatment was given to V instar larvae, two days before e start of rd spinning. Thus, in e triple treatment III, IV and V instar larvae were treated. Similar experiments were performed by 50, 60 and 70% concentration of phytoecdysteroid obtained from Achyranes leaf extract. A control set was always maintained wi each set of experiment. Incubation Period of Egg: The eggs (resulted from treatments) were transferred chronically to BOD incubator maintained at e optimum condition of 26 ± 1 C temperature,80 ± 5% RH and 12 h of photoperiod a day. The time required for e incubation before e hatching of larvae was calculated for each set of experiment separately. The average of eggs was counted from 8 pm on e day of decoupling. Three replicates of 10 laying, in each replicate, were made. The average time taken for incubation by e eggs of different experimental conditions was calculated by taking e mean value of e data obtained. Percent Fertilized Eggs: For determining e effect of phytoecdysteroid on per cent fertilized eggs, obtained from e mos of treated larvae were considered and e eggs us obtained, were incubated at optimum conditions of temperature relative humidity and photoperiod of 26 ± 1 C, 80 ± 5% and 12 ± 1 h, respectively. At e head pigmentation stage e eggs were counted. Thirty layings (3 batches of 10 laying in each batch) were counted for each replicate. The percent of e fertilized eggs was calculated as follows No of eggs fertilized* Percent fertilized egg = x 100 Total No. of egg *Eggs were counted at e head pigmentation stage, i.e. eggs having black spots are fertilized eggs. RESULTS Incubation Period of Egg: The data presented in table 1a shows at change in e phytoecdysteroid concentration and e number of larval treatment influenced e incubation period of egg. Wi e increasing number of larval treatment by phytoecdysteroid from one to two times, e incubation period of egg decreased in case of 40, 50 and 60% phytoecdysteroid treatment but triple treatment caused notable increase in e incubation period in all e above concentrations. 70% phytoecdysteroid treatment caused notable increase in e incubation period of egg wi e increase in number of treatment from single to triple. The trend of decrease in e incubation period of egg wi e increasing number of treatment has been recorded to be almost same in case of 40, 50 and 60% phytoecdysteroid treatment. The minimum incubation period was noticed to be 7.95 ± days in case of double treatment by 60% phytoecdysteroid concentration and e maximum ± days was recorded in case of triple treatment by 70% phytoecdysteroid concentration. Two-way ANOVA indicates at number of larval treatment and variation in e phytoecdysteroid concentration did not cause significant effect on e incubation period of egg of Bombyx mori. The Post hoc test (Table 1b) shows significant group difference in e incubation period of eggs in e double treatment of larvae, in between control and 60%, 40 and 60% and 60 and 70% concentration of phytoecdysteroid treatment. Table 1a: Effect of phytoecdysteroid treatment on e incubation period (days) of eggs of Bombyx mori. Phytoecdysteroid concentration (%) Number of treatment F 1 ratio (Larval instar) Control(X1) 40(X2) 50(X3) 60(X4) 70(X5) n 1 = 4 Single (V) 9.50± ± ± ± ± * Double (IV-V) 9.50± ± ± ± ±0.690 Triple (III-V) 9.50± ± ± ± ±0.746 F 2 ratio = 4.07* n 2 = 2 *Non-significant Each value represents mean ± S.E. of ree replicates. X 1, X 2, X 3, X 4 and X 5 are e mean values of e incubation period (days ) in control, 40%, 50%,60% and 70% phytoecdysteroid concentration respectively. 149

4 Table 1 b: Post-hoc test showing group difference in e effect of phytoecdysteroid treatment on e incubation period (days) of Bombyx mori Number of treatment Mean difference in between groups Single Double Triple X1~X X1~X X1~X * X1~X X2~X X2~X * X2~X X3~X X3~X X4~X * q Honestly significant difference (HSD) = v MS wiin / 5.05 n = v / 3 = 1.11 MS = Mean square values of ANOVA table q = Studentized range static n = Number of replicates * Showing significant group difference X 1, X 2, X 3, X 4and X 5are e mean values of e e incubation period (days) in control, 40%, 50%, 60% and 70% phytoecdysteroid concentration respectively. Table 2 a: Effect of phytoecdysteroid treatment on e per cent fertilized eggs of Bombyx mori Number of treatment Phytoecdysteroid concentration (%) F 1 ratio (Larval instar) Control(X1) 40(X2) 50(X3) 60(X4) 70(X5) n 1 = 4 Single (V) 88.25± ± ± ± ± * Double (IV-V) 88.25± ± ± ± ±2.04 Triple (III-V) 88.25± ± ± ± ±1.74 F 2 ratio = 11.38** n 2 = 2 *Non-significant **P 2 <0.01 Each value represents mean ± S.E. of ree replicates. X 1, X 2, X 3, X 4 and X 5 are e mean values of e e percent fertilized eggs in control, 40%, 50%,60% and 70% phytoecdysteroid concentration respectively. Table 2 b: Post-hoc test showing group difference in e effect of phytoecdysteroid treatment on e per cent fertilized eggs of Bombyx mori Number of treatment Mean difference in between groups Single Double Triple X1~X X1~X X1~X X1~X *11.50 X2~X X2~X X2~X *8.50 *9.75 X3~X X3~X *10.20 *8.00 X4~X5 *8.25 * q Honestly significant difference (HSD) = v MS wiin / 5.05 n = v / 3 = 7.60 MS = Mean square values of ANOVA table q = Studentized range static n = Number of replicates * Showing significant group difference X 1, X 2, X 3, X 4 and X 5 are e mean values of e percent fertilized eggs in control, 40%, 50%, 60% and 70% phytoecdysteroid concentration respectively. 150

5 Per Cent Fertilized Egg: The data presented in table 2a mori eggs [12]. The role of temperature and relative shows at change in e phytoecdysteroid concentration humidity on oviposition and incubation of eggs was and e number of larval treatment influenced e reported by several workers [13-15]. The activity of percentage of fertilized egg. Wi e increasing number juvenile hormones considerably influenced e incubation of larval treatment from one to two times, e percentage period of Bombyx mori eggs [16]. The low magnetic field of fertilized eggs increased in case of 40, 50 and 60% acts on enzyme molecules and produces conformational phytoecdysteroid treatment but triple treatment caused changes in enzyme which is concerned for e activation notable decline in e percentage of fertilized eggs in all of enzyme system, whereas, at higher streng of e above concentrations. Seventy percentage magnetic field, e change brought about e negative phytoecdysteroid treatment caused notable decline in e hence e enzyme get inhibited [17].The functional percentage fertilized egg wi e increase in number of significance of sequence homology can be illustrate by treatment from single to triple. The trend of increase in e cytochrome, an iron containing mitochondrial protein at percentage of fertilized egg wi e increasing number of transfer electron during biological oxidation in eukaryotic treatment has been recorded to be almost same in case of cells [18] and e magnetic field below 0.03 T caused an 40, 50 and 60% phytoecdysteroid treatment. The maximum increase in e oxidation rate due to enhance reducing percentage of fertilized egg was noticed to be ± 2.03 power of cytochrome C in cell [19]. The present in case of double treatment by 60% phytoecdysteroid investigation shows positive correlation between e concentration and was minimum ± 1.74 in case of reproductive potential and larval treatment wi triple treatment by 70% phytoecdysteroid concentration. phytoecdysteroids. The exogenous hormones influenced Two-way ANOVA indicates at number of larval only e choriogenic follicle cell [20] supported by e fact treatment significantly (P 2 < 0.01) influenced e at more eggs were clustured wiin a limited region percentage of fertilized egg. The Post-hoc test (Table 2b) along wi a single ovariole [21]. Eggs containing different shows significant group difference in e percentage of amounts of ecdysteroids showed different level of fertilized eggs in between 60 and 70% concentration of embryonic development and maternal ecdysteroids appear phytoecdysteroid in case of single treatment of larvae. In to be required at different tiers for fertilization, e double treatment of larvae, significant group embryogenesis and hatching of e silkworm larvae [22]. difference in e percent fertilized egg was determined in Most ecdysone 20-monooxygenase activity was between 40 and 70%, 50 and 70% and 60 and 70% associated wi microsomes and at ere was little concentration of phytoecdysteroid treatment. In e triple intrinsic mitochondrial ecdysone 20-monooxygenase [23]. treatment of larvae, significant group difference in percent fertilized egg was noticed in between control and 70%, 40 Per Cent Fertilized Egg: The variation in e and 70% and 50 and 70% concentration of phytoecdysteroid concentration and number of larval phytoecdysteroid treatment. treatment caused notable influence on e per cent fertilized eggs. Wi e increasing number of larval DISCUSSION treatment from one to two times, e hatchability increased from 40 to 60% concentration of Incubation Period: Variation in e phytoecdysteroid phytoecdysteroid but a considerable decline was noticed concentration and number of larval treatment influenced in triple treatment in all e concentration. Maximum per e incubation period of Bombyx mori eggs. In 40, 50 and cent fertilized eggs were noticed in double treatment wi 60% phytoecdysteroid concentration, e incubation 60% phytoecdysteroid concentration. Maternal period decreased slowly and slowly, in single and double ecdysteroids appear to be required at different titers for treatment. 70% phytoecdysteroid concentration in all e fertilization and embryogenesis in e silkworm [22]. treatment stages caused considerable increase in e Developmental response and fertility under various incubation period of eggs. Thus, e trend of variation of conditions have studied in different insects [24, 25]. Yolk e incubation period in 40, 50 and 60% phytoecdysteroid proteins of e silkworm, B. mori are important sources of concentration is almost of similar pattern in single and nutrition and energy during embryo development [26]. double treatment of larvae while in 70% phytoecdysteroid Temperature stress caused delay and poor hatching, concentration, e trend of variation wi e change in depression of eggs and dea of fully formed number of treatment is different. The mating duration has larvae inside egg [27]. Large and heavy eggs resembling been noticed to affect e incubation period of Bombyx e giant egg can be induced by e administration of 151

6 20- hydroxyecdysone in to normal female pupae [28]. In 4. Iwanvat, Y. and Y. Ono, Rearing experiments insects, e steroid hormone 20- hydroxyecdysone (20 E) plays a major role in activating vitellogenesis, a process required for egg development [29]. The occurrence of fertilized and unfertilized eggs is not affected by 5. on artificial diet composed mainly on e mulberry beat powder harvested in e late autumn and after. J. Seric. Sci., 38: Kanarev, G. and G.T. Cham, Effect of laser periodicity of egg laying [30].At higher temperature (30 C irradiation of silkworm eggs on silkworm and above) bo fecundity and fertility are severely Bombyx mori development and productivity. affected [31]. KK-42 is a novel agent to suppress e ecdysteroid accumulation in eggs and 80% eggs containing less an 10 µg free ecdysteroids/g eggs were not fertilized [18]. 6. Zhivotnov Nauki., 22: Upadhyay, V.B. and S.K. Tripai, Effect of e magnetization of eggs on e silk producing potential of multivoltine mulberry silkworm leaves on economic Thus, it is concluded at incubation period and per characters of silkworm (Bombyx mori Linn.) cent fertilized eggs, obtained from e larvae treated wi Sericologia, 46: low phytoecdysteroid concentration may be due to ultra 7. Upadhyay, V.B. and S. Prasad, structural changes in e cell contents and enzyme Magnetization for e improvement of silk producing activity during larval and pupal stages caused positive potential in multivoltine mulberry silkworm effect on e incubation period and per cent fertilized eggs (Bombyx mori Linn). The Bioscan, 5: resulting increase in e per cent fertilized eggs but 8. Tripai, S.K. and V.B. Upadhyay, decrease e incubation period of Bombyx mori eggs. The Magnetization of eggs influences e incubation higher concentration of phytoecdysteroid caused adverse period of multivoltine mulberry silkworm Bombyx effect on e cellular level, erefore, incubation period of mori Linn Eggs. J. Adv. Zool., 26: Bombyx mori eggs increased and e per cent fertilized 9. Upadhyay, V.B. and S. Prasad, eggs declined. Biotechnological importance of cocoon ACKNOWLEDGEMENT magnetization wi particular reference to e reproductive potential of multivoltine mulberry I would like to express e deep sense of gratitude towards my supervisor, Prof. V.B. Upadhyay for e valuable guidance, keen interest, kind altitude and e total training at is imparted on various aspects of e scientific research. I am grateful to Prof. C.P.M. Tripai Head, Department of Zoology, for providing necessary facilities to carry out e investigation. I am also ankful for e valuable help received from Behraich Centre, Directorate of Sericulture, Uttar Pradesh and e oer scientists, working in e field of sericulture. REFERENCES 1. Upadhyay, V.B. and A.B. Mishra, Nutritional ability of bivoltine silkworm (Bombyx mori Linn.) larvae at high temperature regimes. J. Adv. Zool., 12: Mishra, A.B. and V.B. Upadhyay, Nutritional efficiency of bivoltine silkworm (Bombyx mori Linn.) larvae at high regimes of relative humidity. J. Adv. Zool., 13: Mishra, A.B. and V.B. Upadhyay, Nutritional efficiency of bivoltine Bombyx mori Linn. larvae at different photoperiod. Proc. 80 session Indian Science Congress Assoc. Goa, pp: silkworm (Bombyx mori Linn). Sericologia, 50: Lafont, R. and D.H.S. Horn, Ecdysone from chemistry to mode of action. Ed. J. Koolmann heor Thieme Verlag, pp: Krishnaswamy, S., M.N. Narasimhanna, S.K. Suryanaryan and S. Kumar Raja, Sericulture Manual 2 Silkworm Rearing F.A.O. Agric Serves Bull. Rome, 15: Reddy, K.V.R., K.S. Nair, S.B. Magadum and R.K. Dutta, Anti juvenoid indiced modulation on e tissue grow rate pattern and biochemical components of silkworm Bombyx mori L Bangladesh Journal of Zoology, 23: Ahsan, M.M., M.S. Jolly and R.K. Khatri, Some preliminary studies on various aspects of grainage in tasar silkworm (Aneraea mylitta Drury). Proceeding of 1st International Seminar in Non-Mulberry Silks, October 3-4, 1974, Central Tasar Research Station, Ranchi, India, pp: Yamaoka, K. and T. Hirao, Mechanism of ovipositional behaviour of Bombyx mori, time gating and accumulation of e internal factor. Int. J. Invertbr. Reprod., 4:

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