(Larva to Pupa) in the Tropical Tasar Silkworm Anthe

Barsagade et al. Int. J. Res. Chem. Environ. Vol. 4 Issue 4 (69-74) October 2014

International Journal of

Research in Chemistry and Environment Available online at: www.ijrce.org ISSN 2248-9649

Research Paper

Hormonal Regulation of Metamorphosis (Larva to Pupa) in the Tropical Tasar Silkworm Antheraea mylitta (D.) Eco-race Bhandara *Barsagade D. D. and Gharade S. A. PGTD Zoology, M. J. F. Educational Campus, RTM Nagpur University, Nagpur (MS), INDIA (Received 02nd July 2014, Accepted 29th July 2014)

Abstract: Juvenile hormone (JH-III) and 20-hydroxyecdysone (20-HE) regulate metamorphosis in tropical tasar silkworm Antheraea mylitta (D.) eco-race Bhandara. The fluctuation in the concentration of JH and 20-HE hormones in the silkworm, results changes in moulting and metamorphosis. The JH-III treated larvae undergo prolonged larval period while, 20-HE treated larvae pupate early. The topical application of JH-III on larvae show increased protein and carbohydrate concentration in haemolymph and fat body because of its growth stimulating effect and the synthesis of protein and carbohydrates while in ecdysone treated larvae protein and carbohydrate concentration decreased significantly in haemolymph and fat body due to increase in energy demand during cocoon formation . Keywords: Juvenile hormone, 20-hydroxyecdysone, Antheraea mylitta, metamorphosis, spinning. © 2014 IJRCE. All rights reserved

applications of JH-III and 20-HE on the fifth instar larva to pupa metamorphosis in tropical tasar silkworm A. mylitta.

Introduction In insects juvenile hormone (JH) and 20hydroxyecdysone (20-HE) regulate metamorphosis, reproductive maturation, behavior, diapause, stress resistance, immune mechanism, and aging [7,9,23]. The hormone ecdysone (hydroxylated steroid) and juvenile hormone (sesquiterpene) are involved in metamorphosis [5] .

Material and Methods Tasar silkworm, Antheraea mylitta (D) eco-race Bhandara is the principle non-mulberry silk producing insect and is cultivated in the tropical forest of India including the region of Bhandara, Chandrapur and Gadchiroli District of Vidarbha in the Maharashtra state. The non-dipausing cocoons of Antheraea mylitta (D) ecorace Bhandara were brought from the forest of Gadchiroli to the laboratory and rearing was carried out at the insectaries of the Department of Zoology, RTM Nagpur University, Nagpur, India. Healthy fifth instar larvae were selected for the experiment.

Juvenile hormone is secreted by the corpora allata and ecdysone is initially produced by the prothoracic glands and later on converted into 20hydroxyecdysone in peripheral tissues, especially the midgut [5]. Both, the ecdysteroids and juvenile hormone circulate in the haemolymph throughout the insect’s body and get exposed to all the tissues at the same concentration of ecdysteroids and juvenile hormone at the same time [5].

Hormonal treatment: the fifth instar larvae were topically treated with Juvenile hormone (JH-III) and 20hydroxyecdysone (20-HE) 1. JH-III: Juvenile hormone- III (JH-III from Sigma, USA Cat. No. J2000) 1mg of JH-III was dissolved in 1 ml of cold acetone and 20µl was applied to each larva. 2. 20-hydroxyecdysone: 1mg of 20-hydroxyecdysone (Sigma, USA Cat. No.H5142) was dissolved in 1 ml of cold acetone alone and 20µl was applied to each larva. The 15th day old healthy fifth instar larvae were selected and divided into three groups. Each group consists of 25 larvae. Larvae of one group treated with JH-III, second group with 20-HE and third group with cold acetone only.

In the holometabolous insects, like Lepidoptera, the development from juvenile to adult depends on an appropriate action of juvenile hormone (JH) [12]. The hormonal control of insect metamorphosis was studied long back in Rhodnius prolixus by Wigglesworth [39]. The hormonal regulation of metamorphosis in Cecropia, Lymantria dispar, Trichoplusia ni. and Bombyx mori was described erliar [24,28,29,30,36]. However, there are no reports on the effect of JH-III and 20-HE on the metamorphosis in A. mylitta from larvae to pupae. Therefore the present study is undertaken to find out the effect of topical 69

Nandhakumar et al. Int. J. Res. Chem. Environ. Vol. 4 Issue 3 (1-9) October 2014

1 mg of JH-III and 1 mg of HE diluted in 1 ml of cold acetone and topically applied on the 4- 6th abdominal intersegmental area with the help of microsyrynge. Equal quantity of cold acetone treated larvae was served as controls. Larvae of all three groups were maintained until the pupa formation.

Effect of JH- III and HE on Behavior: The treatment of JH –III and 20-HE was given to the 15th day old healthy larvae and monitored accordingly. The JH-III treated larvae continued feeding up to 7 days after treatment and underwent spinning on 9th day since treatment. The 20HE treated larvae stopped feeding after 48 hours of treatment and underwent spinning. The control larvae stopped feeding on 5th day and underwent spinning (Table 1). This experiment indicates that the larval life span extends after JH-III treatment while the larval life span reduced after 20-HE treatment as compared to the control larvae.

After 6, 12, 18, 24 and 36 hours, haemolymph was collected by pricking the prolegs of instars larvae in small vials precoted with phenylthiourea to prevent melonization. From the larvae the fat body were dissected out gently at each interval in ice cold Ringer’s solution from equal number of JH-III, 20-HE and control larvae. The haemolymph were centrifuge immediately at 4500 rpm for 15 min. and supernatant was stored at -20ºC. The tissues were homogenized using pestle and mortar for 5 min at room temperature in Ringer’s solution and centrifuge at 4500 rpm for 15 minutes. The supernatant were used for biochemical analysis of total soluble protein and carbohydrate. Estimations were repeated thrice to confirm the result.

Effect of JH-III and 20-HE on haemolymph protein and carbohydrate: The concentration of protein in haemolymph increases in the JH-III treated larvae from 75.02 ±0.92 to 85.32 ±0.50 µg/ml and decreases in 20HE treated larvae from 57.4 ±0.92 to 50.4±0.79 µg/ml after 6th, 12th, 18th, 24th and 36th hours treatment in comparison to the control larvae (Fig. 1). The carbohydrate concentration in the haemolymph in the JHIII treated larvae increases from 3.30 ±0.93 to 14.87 ±0.75 µg/ml and decreases from 3.074±0.03 to 2.98±0.02µg/ml in 20-HE treated larvae after 6th, 12th, 18th, 24th and 36th hours of treatment in comparison to the control larvae (Fig. 2).

The bimolecular concentration was measured by different methods, the total protein concentration in the haemolymph and tissue of larva and pupa were estimated by method of Lowry et al., [20]. The blue colour intensity was observed at 650 nm on the Spectrophotometers (ELICO LTD, SL 177). The total carbohydrate concentration in the larval and pupal haemolymph and tissue were estimated by method of Dubois et al., [8]. The colour intensity with yellowish brown colour was observed at 490 nm on the spectrophotometer. The standard graph was plotted for known samples of BSA and sugar. Finally, the actual amount of protein and carbohydrate was determined from the extracted sample.

Effect of JH-III and 20-HE on fat body protein and carbohydrate: The concentration of protein and carbohydrate in the fat body increased in the JH-III treated larvae from 57.74±0.03 to 57.74±0.03 µg/mg and decreased in 20- HE treated larvae from 43.8±0.73 to 50.8±0.80 µg/ mg after 6th, 12th, 18th, 24th and 36th hours in comparison to the control larvae (Fig. 3- 4).

Statistical methods: All statistics presented in this study are mean ± standard error use for testing significance of difference between the means of reading of experimental and control groups.

Discussion In insect, many physiological events throughout its life cycle including larval growth, metamorphosis, ecdysis, reproduction, diapause and behavior, are regulated by the combined actions of 20hydroxyecdysone (20E) and juvenile hormone (JH) [7, 9, 11, 23] .

Results Hormonal control of metamorphosis of the fifth instar larva

Table 1: Effects of JH- III and 20- HE on metamorphosis of the fifth instar larvae of A. mylitta Age of larvae (days)

23

15 16 17 18

Age of larvae after treatment (hrs) 0 24 48 72

19 20 21

96 120 154

22

178 202

Control larvae Continued feeding Continued feeding Continued feeding Continued feeding Continued feeding Stopped feeding Larvae started spinning cocoon Cocoon formed 70

JH-III treated larvae Continued feeding Continued feeding Continued feeding Continued feeding Continued feeding Continued feeding Continued feeding Stopped feeding Larvae started spinning cocoon

20-hydroxyecdysone treated larvae Continued feeding Continued feeding Stopped feeding Larvae started spinning cocoon Cocoon formed -


Effects of JH on the larval-pupal transformation of Lepidoptera was first demonstrated by Piepho [25, 26] by the implantation of active corpora allata into final instar larvae of the wax moth Galleria mellonella. The effect of JH on the larval and pupal metamorphosis of Cecropia was studied earlier while, the administration of juvenile hormone (JH) prolongs the larval duration and results in a significant increase in cocoon weight and cocoon shell weight in Bombyx mori (L) [1].

The moulting hormone ecdyosteriod (20-HE) serve for regulating the onset of moulting cycle. The juvenile hormone and juvenoids regulate the quality of the moult [16,27]. During the last stadium of holometabolous insects like silkworm, Bombyx mori (L), reduction in the titer of juvenile hormone (JH) and increased ecdysone in haemolymph is necessary step in the initiation of metamorphosis [21]. In the present investigation, application of 20-HE resulted in the early metamorphosis from larvae to pupa confirmed the role of 20-HE in initiation of metamorphosis in A. mylitta larvae supporting earlier findings. The treatment with JH or JHanalogue reported to increase the silk protein synthesis, replication of DNA and accumulation of RNA in silkgland Bombyx mori [1, 3, 6,13,34]. Glycogen (multibranched polysaccharide of glucose) in fat body decreses towards larval-pupal ecdysis to meet the energy demands required for cocoon formation and chitin synthesis in Achaea janata [4] and also observed in Galleria mellonella and Bombyx mori [19, 35].

In the present study it has been observed that the life span of A. mylitta larva treated with JH-III were increased and in the 20-hydroxyecdysone treated larval life span decreased significantly confirmed stimulatory role of JH-III and inhibitory role of 20-HE hormone during larval metamorphosis. Since, several investigators have shown that the treatment with JH-analogues including Manta increased the larval duration [2, 15, 17, 31, 32, 38] and larval weight [18, 33]. After JH treatment the larval growth is prolonged by 2 to 3 days and silk protein biosynthesis was increased with corresponding increase in cocoon and shell weights [2, 5, 14, 21, 22, 27, 33, 37] . Prolongation of the larval period is directly proportional to the concentration of JH [10]. During present study it has been observed that life duration of JH-III treated larvae was increased by 2 to 3 days, confirming the stimulatory role of JH during larval growth and prolonged metamorphosis.

In the present study the concentration of protein and carbohydrate increases after the treatment of JH-III because of its growth stimulating effects while, reduction in the protein as well as carbohydrates after the 20-HE treatment indicates its utilization for the various processes of cocoon formation and chitin synthesis as the early larva –pupa metamorphosis is occurred.

Figure 1: Protein concentration of haemolymph in the fifth instar larva after treatment of Juvenile hormone (JHIII) and 20-hydroxyecdysone (20-HE)

Figure 2: Carbohydrate concentration of haemolymph in the fifth instar larva after treatment of Juvenile hormone (JH-III) and 20-hydroxyecdysone (20-HE) 71


Figure 3: Protein concentration in the fat body of fifth instar larva after treatment of Juvenile hormone (JH-III) and 20-hydroxyecdysone (20-HE)

Figure 4: Carbohydrate concentration in the fat body of fifth instar larva after treatment of Juvenile hormone (JH-III) and 20-hydroxyecdysone (20-HE) M., Hassell A., Wisely B., Caravella J.A., Lambert M.H., Reinking J.L., Krause H., Thummel C.S., Willson T.M. and Mangelsdorf D.J., The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signalling pathway, Cell., 113 (6), 731-42 (2003)

Acknowledgement We thankful to University Grants Commission for providing financial support for major research project {F. No. 36-202/2008(SR)}, Rajiv Gandhi National Fellowship and Rajiv Gandhi Science and Technology commission (RGSTC) Government of Maharashtra for financial assistance.

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