ELECTROPHORETIC PROTEIN PATTERNS DURING LARVAL DEVELOPMENT OF CHILO PARTELLUS ( SWINHOE)

Journal of Cell and Tissue Research Vol. 12(2) 3163-3168 (2012) ISSN: 0974-0910 (Available online at www.tcrjournals.com) Original Article ELECTROPHORETIC PROTEIN PATTERNS DURING LARVAL DEVELOPMENT OF CHILO PARTELLUS ( SWINHOE) NALAWADE, S. P. AND BAKARE, R. V. 1 Department of Zoology, Yashawantrao Chavan Institute of Science, Satara. 415001 (M.S.); 1 Kisan Veer Mahavidyalaya, Wai. Dist. Satara. (M.S.). E. mail: drsavita73@indiatimes.com Received: April 2, 2012; Accepted: May 28, 2012 Abstract: Chilo partellus is one of the most serious maize stem borer. The electrophoretic protein patterns during larval development of Chilo partellus have been studied. The larval developmental period is of 28 days. Gradual increase in no of protein fractions from 1 to 2 day and decrease from 2 to 4 day larvae was observed. With the onset of second instar sharp increase in no. of protein fractions was observed and after 5 day it decline and remain constant up to 10 day larvae. With the onset of third instar larva gradual increase in number of protein fractions was observed up to 13 day and then sharp decrease in number of protein fractions were observed upto end of the instar. Sharp increase in protein fractions were observed with the onset of fourth instar and sharp decrease was observed at the end of fourth instar larvae. In the fifth instar larvae decrease in number of protein fractions were observed from 24 to 28 day. The physiological significance of proteins during larval development of Chilo partellus is discussed. Key words: Electrophoresis, Proteins, Larval development, Chilo partellus.? INTRODUCTION The maize stem borer Chilo partellus ( Lepidoptera- Crambidae) is very serious pest in India. In early stages of crop growth this insect larvae attacks the leaves and central shoot. The larvae feed and damage the inner vascular tissue of stem to form elongated tunnel leading to great loss in yield. The newly hatched larva of Chilo partellus is tiny, cylindrical and pale brown in colour. The first instar larvae feed on young tender leaves near the base of whorl. From the second instar onwards the larvae feeds and damage inner vascular tissue of the stem to form elongated tunnel. The larva of Chilo partellus is caterpillar type having mandibulate type of mouthparts, three pairs of jointed thoracic appendages with claws and five pairs of prolegs. The larval developmental period is of 28 days with five instars which lasts for 4, 6, 7, 6 and 5 days respectively. The moulting is observed in 4, 10, 17, 23 and 28-day larvae. The larval growth was computed from mean time of egg hatching to the pupal state. Within the egg as a whole there is no growth. Only a kind of inner growth occurs in the egg representing a transformation of stored yolk components into active protoplasm. The growth during insect development is restricted to the larval development and during this feeding period there will be deposited all the mass necessary for final adult [1]. In insects the larval form is highly variable in structure and is adapted for life in different environments. The entire postembryonic development of insects is punctuated by moults during which the old cuticle is replaced by a new one. The food requirements for normal growth and development are essential. A number of substances, particularly amino acids and vitamins, are essential for any development. The balance between different constituents is also important [2]. Dipteran larvae are known to accumulate lipids, glycogens and proteins during development [3]. The reasons for storing these constituents in larva is fairly obvious, this material later on can be used during metamorphosis. 3163

J. Cell Tissue Research Proteins provide chief structural elements of muscles, glands and other tissues. A certain amount of protein is stored in fat bodies and much is deaminated or converted into carbohydrate or fat body and used for energy production [3]. During larval development fat body responsible for the synthesis of various major haemolymph proteins and serves at the same time as a place of storage of these components, in addition to carbohydrates and lipids. Insect larvae depend on uptake and utilization of exogenous proteins for both growth and development. The ingested proteins must be broken down into amino acids before being absorbed. The proteolytic enzymes are responsible for the degradation of proteins (4). In general all amino acids, which are commonly contained in the proteins, have been indentified, either in tissue extracts or in the haemolymph. It has been claimed that a number of amino acids, such as arginine, cysteine, glycine, proline, tryptophan, tyrosine, phenylalanine are especially concerned with moulting, differentiation, pupation or emergence of adult [5]. The developing insect larva, in contrast to egg and pupa, depends on a continuous supply of food of energy production and growth. The major mechanism underlying the growth in larval development is protein synthesis, which is of course directly related to both amino acids and proteins in diet. Few studies have been carried out in rate of proteins during larval development of various insects [7-13]. However, there exists a lacuna in the field of proteins during larval development of Chilo partellus. Therefore, present attempt provides information on proteins during larval development of this species which may be useful in controlling this most serious pest of maize. MATERIAL AND METHODS The culture of Chilo partellus was maintained in the laboratory on natural food of cut pieces of fresh maize stems. Egg masses were put in tender maize shoots for hatching. The young larvae were trapped in tender maize shoots. The later instars were inserted in cut pieces of fresh maize stems. The pupae were collected and kept in glass jar lined with white paper at its inner side for emergence of moths. The eggs were laid in bunches and larvae hatched within six days. The newly hatched larva of Chilo partellus is tiny, cylindrical and pale brown in colour. The first instar larvae feed on young tender leaves near the base of whorl. From the second instar onwards the larvae feeds and damage inner vascular tissue of the stem to form elongated tunnel. The larva of Chilo partellus is caterpillar type having mandibulate type of mouthparts, three pairs of jointed thoracic appendages with claws and five pairs of prolegs. The larval developmental period is of 28 days with five instars which lasts for 4, 6, 7, 6 and 5 days respectively. The moulting is observed in 4, 10, 17, 23 and 28-day larvae. The larval growth was computed from mean time of egg hatching to the pupal state. In case of larval developmental stages from 1-day to 28-day were taken for study of electrophoretic protein patterns and proteases activity. The larvae were isolated, cleaned with distilled water weighed and homogenized in chilled buffer solution. The homogenates were diluted with chilled buffer solution so as to get various concentrations for electrophoresis activity. The electrophoresis of proteins was carried out by method of Laemlli (14) and the relative mobility ( Rm) and molecular weights of separated proteins were calculated by method of Weber and Osborn [15]. RESULTS The electrophoretic protein patterns during first instar larvae was represented in figure 1. Gradual increase in number of protein fractions from 1 to 2 day and decrease from 2 to 3 day larvae was observed. After 3 day it decline upto 4 day larvae. Maximum protein fractions were recorded in 2day larvae and minimum in 4 day larvae. The electrophoretic protein patterns during second instar larvae was represented in figure 2. With the onset of second instar sharp increase in number of protein fraction was observed in 5 day larvae. After 5 day it decline upto 6 day and remains constant upto 9 day larvae. Slight decline in number of protein fractions was observed on 1-day larvae. Maximum protein fraction were observed in 5 day larvae and minimum in 10 day larvae. The electrophoretic protein patterns during third instar larvae was represented in figure 3. Gradual increase in number of protein fractions in third instar from 11 to 13 day larvae was observed. After 13 day gradual decrease in number of protein fractions from 13 to 14 day, sharp increase from 14 to 15 day and 3164

Nalawade and Bakare Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 1: Electrophoretic protein patterns during first instar larva of Chilo partellus Fig. 2: Electrophoretic protein patterns during second instar larva of Chilo partellus Fig. 3: Electrophoretic protein patterns during third instar larva of Chilo partellus Fig. 4: Electrophoretic protein patterns during fourth instar larva of Chilo partellus Fig. 5: Electrophoretic protein patterns during fifth instar larva of Chilo partellus 3165

J. Cell Tissue Research sharp decrease from 15 to 16 day larvae was observed and after 16 day slight increase in number of protein fractions was observed upto 17 day larvae. Maximum protein fractions were recorded in 15 day larvae and minimum in 11 day and 16 day larvae. The electrophoretic protein patterns during fourth instar larvae was represented in figure 4. With the onset of fourth instar sharp increase in number of protein fractions from 17 to 18 day and sharp decrease from 18 to 19 day larvae was observed. After 19 day the number of protein fractions remains almost constant upto 23-day larvae except slight increase in 22 day larvae. Maximum protein fractions were observed in 18 day larvae and minimum in 21 and 23 day larvae. The electrophoretic protein patterns during fifth instar larvae was represented in figure 5. In last (fifth) instar slight increase in number of protein fractions 23 from 24 day larvae was observed. After 24 day it remains almost constant upto 28 day larvae. Maximum protein fractions were observed in 24 day larvae and minimum in 25 day larvae. DISCUSSION The developing insect larva, in contrast to egg and pupa, depends on a continuous supply of food for energy production and growth. The major mechanism underlying the growth in larval development is protein synthesis, which in of course directly related to both amino acid and proteins in the diet. Following the uptake of the components they may enter diverse metabolic pathways and exert a profound influence on the composition and size of the amino acid pool. At the initiation of larval development growth is the predominant phenomenon. In Drosophila the increase in total protein content parallels closely that tin both wet and dry weight during the first 72 hrs of development [16]. Another important aspect of protein metabolism during larval development is the synthesis of haemolymph protein [17]. In general, the protein concentration in the haemolymph increases rapidly during the later half of larval development, falls at metamorphosis, the declines to zero in early adult life. In Calliphora, the most prominent fraction is calliphorin, which accounts for about 75% of the total haemolymph protein in the 6 day-old larvae [18]. Accumulation of haemolymph protein occurs in developing Drosophila larvae [19]. It is now generally accepted that nearly all these proteins are synthesized in the fat body [20]. Reugg [21] demonstrated that in vitro conditions the specific rate of protein synthesis in larval fat body of Drosophila declines rapidly between 65 hrs in pupariation. Thus the increase in the hemolymph protein concentration is accompanied by a fall in the synthetic capacity of the fat body. Starvation and feeding exert a significant alteration in the free amino acid composition in insect haemolymph, though the influence may be more pronounced in some components and less in others. However, as compared to other drastic metabolic changes following starvation, such as the drop in respiratory rate and the total free amino acid appears to be maintained at a fairly constant level. This implies the existence of certain regularly mechanisms. In Drosophila larvae starvation results in a rapid decrease of most amino acids which then remains at low level for several days [22]. On the other hand, one peptide, which results apparently from degradation of tissue proteins, shows as steady increase. Firling [8] states that in Chironomus tentans another depteran insect, the concentration of haeolymph amino acids increases between the early and middle fourth instar larvae followed by a decline in the late fourth instar larvae. As in Drosophila, alkaline and glutamine are the predominant components at all stages. It should be emphasized that the pattern described is by no means valued for other insect species. For example, there is no readily discernible alteration of the total free amino acids pool during the larval development of Culex pipiens [23] and Aedes aegypti, Ephestia kuhniella, Sechistocerca gregaria, Rhodnius prolixus and Periplaneta americana [24]. Jindra and Sehnal [11] worked on larval growth, food consumption and utilization of dietary protein and energy in Galleria mellonella. Growth rates of caterpillars on diets differing in their water, energy and nitrogen contents varied within 30% and depended primarily on the amount of food ingested. Protein synthesis is necessary for maintenance of the body growth and reproduction. It is obvious that feeding provides the raw materials and energy for protein synthesis but the relationship between feeding and protein synthesis may be complex. 3166

Verma and Nath [13] studied changes in haemolymph protein during late larval stage of Spodoptera litura. One observation in S. litura which gets support from earlier workers in Lepodaptera [7] is the fact that there is a gradual increase in the haemolymph protein concentration in the successive larval instars which reaches peak during quiescent phase i.e. pharate pupal period. It seems probable that during the larval period of endoterygote insects, proteins needed by forthcoming quiescent pupal stage when for reaching changes involving reconstruction and differentiation of organs and tissue take place are synthesized and get accumulated because of gradual increase in haemolymph protein concentration during the larval period. This results into an abrupt accumulation of large quantity of protein in the haemolymph, which is subsequently utilized during the metamorphosis. One of the important reason for the abrupt rise in the haemolymph protein concentration during larval pupal transition could be loss of large quantity of body fluid prior to wandering sixth instar larvae entering quiescence for forming the pharate pupal stage, which results in haemo concentration [20]. In the present work gradual increase in the number of protein fractions from 1 to 2 day larvae of Chilo partellus indicates that during this early feeding period of larval development, the larval growth is predominant phenomenon and such fast growing larva require more protein as structural components for growth and different enzymes for fraction from 2 to 3-day larvae and decline from 3 to 4-day larvae suggests the larva entering in the first moult and gradually seize feeding. With the onset of second insar sharp increase in the number of protein fraction was observed in 5 day larvae. This suggest active feeding. After 5 day the number of protein fraction decline upto 6 day and then it remained constant upto 9 day larvae. This suggests steady accumulation of protein in growing larva. Decline in number of protein fractions from 9 to 10 day larvae suggests second moult requiring proteins. Gradual increase in number of protein fractions in third instar from 11 to 13-day decrease from 13 to 14 day and increase from 14 to 15 day larvae was observed. It indicates storage of different metamorphosis. Sharp decrease in the number of protein fraction from 15 to 16 day larvae and slight Nalawade and Bakare increase from 16 to 17 day larvae suggests larva entering in the third moult. Sharp increase in the number of protein fractions from 17 to 18 day larvae suggests active feeding and accumulation of proteins in the haemolymph and in the fat body. Sharp decrease in number of protein fractions from 18 to 23 day larvae suggests utilization of proteins for growth, enzymes, hormones and other purposes. Decrease in number of protein fractions from 24 to 28-day larvae suggests the feeding and ingestion of some proteins after moulting. After 24-day number of protein fractions remains constant upto 28-day larvae suggests the inactive prepupal stage of larva show no change in storage proteins in haemolymph and fat body. Our results are in good agreement with the findings of earlier workers [ 21,23,24]. REFERENCES [1]. Agrell, I.P.S. and Lundquist, A.M.: Physiological and biochemical changes during insect development. In: Physiology of Insecta.(Rockstein, M. ed.) Acad. press, New York and London, pp 159-248 (1973). [2]. Chapman, R.F. In: The insects. The English Univ. Press Ltd. (1969) [3]. Chen, P.S. : Protein synthesis in relation to cellular activation and deactivation. In : Biochemistry of Insect. ( Rockstein, M. ed.). Acad. Press, New York and London. pp 145-203 (1978). [4]. Wigglesworth, V.B.: The principles of insect physiology. Chapman and Hall, London. pp 593-662 (1972). [5]. Chen, P.S.: Free amino acid in insects. In: Amino acid pools (Holden, J. T. ed) Elsevier, Amsterdam London and New York, pp 115-138 (1962). [6]. Marshal, A.T.: J. Insect. Physiol., 86: 1973-2317 (1973). [7]. Whitmore, E. and Gilbert, L.T.: Comp. Biochem. Physiol., 47 B: 63-78 ( 1974). [8]. Firling, C.E.: J. Insect Physiol., 23: 17-23 (1977). [9]. Tysell, B. and Butterworth, F.: J. Insect Physiol. 24: 201-227 (1978). [10].Tojo, S., Morio, M., Agui, N. and Hiruma, K.:. J. Insect Physiol., 31: 283--286 (1985). [11].Jindra, M. and Sehnal, F.: J. Insect Physiol. 35: 719-724 (1989). [12].Sinha, U. Sinha, A. and Sinha, S.: Indian J. Sericul., 30(2): 155-156 ( 1991). [13].Verma, A. and Nath, G.: Indian J. Entomol., 53(4): 597-602 (1991). [14].Laemlli, U. K.: Nature, 227: 681-685 (1970). 3167

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