VARIATIONS INDUCED IN ELECTROPHORETIC PATTERN OF HAEMOLYMPH PROTEINS OF FLESH FLY, SARCOPHAGA ARGYROSTOMA (DIPTERA: SARCOPHAGIDAE) LARVAE CHALLENGED WITH HYDROGEN PEROXIDE

S.H. Mahmoud 1 , W.A. Moselhy 2 , L.A.A. El-Khashab 2 , B. Z. Abdelbaset 2 and A.M. Seufi 3 . 1. Zoology Department, Faculty of Science, Menoufia University, Menoufia, Egypt. 2. Zoology Department, Faculty of Science, Al-Azhar University (Girls branch), Nasr city, Cairo, Egypt. 3. Entomology Department, Faculty of Science, Cairo University, Giza, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Insects like all aerobic organisms are continuously exposed to oxidative stress due to products produced during oxygen metabolism and via the metabolism of the encountered toxins including allelochemicals and pesticides.
This study was conducted to determine the variation induced in electrophoretic pattern of haemolymph proteins of third instar larvae of flesh fly, Sarcophaga argyrostoma challenged with hydrogen peroxide. Haemolymph was collected every four hours interval for seventy two hours postinjection with hydrogen peroxide. Total soluble protein of the haemolymph was extracted and separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Hydrogen peroxide challenge induced differences in number of electrophoretic protein bands with different molecular weights as compared to control. These results clearly showed that larval challenge with hydrogen peroxide could evoke the haemolymph to synthesize new proteins to overcome such stress. The quantitative analysis also clearly indicated variations in the number as well as intensity of the protein bands.  (Byrd, 1998(Byrd, & 2001. Larvae of these flies are closely associated with human and are known to cause animal tissue myiasis. Such severe myiasis caused by this fly, is a grave problem in terms of both the animal welfare and economic loss (Sotiraki et al., 2010). It is known that various chemicals, physical and physiological stressors can result in a stress situation that may upset functional homeostasis which is termed oxidative stress (OS), and is characterized by enhanced production of reactive oxygen species (ROS) with the simultaneous impairment of their scavenging systems. Increased concentrations of ROS result in oxidative damage to proteins, lipids, and nucleic acids, and thus the functions of cells, organs, or the whole organism may be seriously disrupted, leading to death (Kodrik et al., 2015). Hydrogen (Hassan, 1997). Hydrogen peroxide is better known for its cytotoxic effects and it has also become established as an important regulator of eukaryotic signal transduction. Insects are subjected to various environmental stressors that lead to the generation of deleterious reactive oxygen species (Felton, 1995). Oxidative stress reflects the disruption of an intricate balance between the formation and clearance of highly-reactive free radicals in living organisms. The increased production of reactive oxygen species that exceeds the capacities of cellular defense systems leads to oxidative stress in the cell and to the oxidation of proteins, lipids and nucleic acids Haemolymph collection:-Haemolymph was pooled by cutting off the anterior tip of the larvae with sterile fine scissors. Haemolymph was collected every four hours post injection for three days (5 μl each), in an ice-cold Eppendorf containing few crystals of phenylthiourea to prevent melanization. A haemolymph sample was collected from untreated larvae as control one. All haemolymph samples were preserved in liquid nitrogen till analysis.

Protein extraction and gel preparation:-
Proteins were extracted from the samples by using Tris buffer system as described by (Dunn, 1993), with 2% (w/v) [SDS] and 5% (w/v) 2-mercaptoethanol to cleave the disulphide bonds. The slurry was cooling centrifuged for 20 min. at 12000 rpm. The samples were heated in a boiling water bath for 15 minutes before loading to ensure dissociation. Preparation of the gels followed the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method described by (Laemmli, 1970). Resolving gel (15%) was used according to (Hames, 1981). Bromophenol blue (0.001%) tracking dye was used for marking the buffer front during electrophoresis. The gel was electrophoresed at 25-30 mA constant current, at 200V. Staining was done using Coomassie brilliant blue, and a solution of 10% acetic acid and 45% methanol was used for destaining. Protein standards (markers) were used to estimate the molecular weights of the separated bands. Figures (1-3) and Tables (1-3) illustrated haemolymph proteins of control and treated larvae that electrophoretically separated by SDS-PAGE using 15% polyacrylamide gel.

Results:-
The results of protein banding pattern in Fig. 1        38 Table 2:-The molecular weight analysis of haemolymph proteins of third larval instar of S. argyrostoma challenged by hydrogen peroxide using SDS-PAGE. LaneM: Marker, C :control and lanes L7-L12 treated larvae at 28, 32, 36, 40, 44 and 48 h post injection.
The variations that were observed at 40, 44 and 48 h post injection changed on the third day of treatment (Fig. 3 and Table 3). Most of disappeared protein bands were observed again at 52, 56, 60 and 64 h post injection, while some of these bands disappeared again at 68 and 72 h post injection. Another unique protein band with molecular weight 39.154 kDa was observed at 72 h post injection. It was clear from the overall results of SDS-PAGE that larvae challenged with hydrogen peroxide could evoke the haemolymph to synthesise new proteins.  40 Table 3:-The molecular weight analysis of haemolymph proteins of third larval instar of S. argyrostoma challenged by hydrogen peroxide using SDS-PAGE. LaneM: Marker, C :control and lanes L13-L18 treated larvae at 52,56, 60, 64, 68 and 72 h post injection.

Discussion:-
Protein is necessary for various biological activities during development, metamorphosis and maintenance of various physiological functions in different tissues (Kumar et al., 2011). The various aspects of protein metabolism including quantitative changes in haemolymph protein synthesis and metabolic activity of specific enzymes have attracted the interest of many insect biochemists. The proteins play an important role in the haemolymph of insects not only in specific transport functions, but also in their enzyme action. The synthesis and utilisation of haemolymph proteins are controlled by genetic and hormonal factors (Hurlimann et al., 1974). Proteins could be antibacterial agents (Riddiford et al., 1983), detoxifying agents (Furukawa et al., 1999), hormone carriers (Fengwu et al.,  1997), morphogenesis proteins (Kiheung et al., 1998) or even similar to some human proteins. As the haemolymph composition of the insects reflects the nature and degree of metabolism of the tissue suffused in this fluid, changes in the proteins of the haemolymph may show the level of modification in the organism. The qualitative variations in the protein bands of different treatments and in different days during the larval life indicate both utilities of the specific proteins as well as the synthesis of new proteins by the insect (Lokesh et al., 2006) changes in the protein pattern of the challenged larvae as compared to control. Thus, H 2 O 2 was capable of changing the profiles of haemolymph proteins qualitatively. The appearance of different bands in challenged larvae may be attributed to the induction of new proteins. The synthesis of new proteins may be a result of simultaneous induction of challenging with hydrogen peroxide. Such change in protein profile between normal and challenged larvae may be attributed to the increased production of antioxidants and repair proteins that allow adaptation to these oxidative conditions (Storz & Tartaglia, 1992; Jamieson, 1998) and may reflect specialization and adaptation in the organisms based on subtle metabolic alterations (Witmore & Gilbert, 1974). Total protein changes and the electrophoretic protein bands studies in insects after various treatments were recorded by many authors (Latha et  al., 1996; Zidan et al., 1996; Chau faux et al., 1997; Kawaski, 1998; Salama et al., 1999). Till now there have not been any published reports regarding variations induced in protein profiles of Sarcophaga sp following hydrogen peroxide challenge. However, some studies are available with other chemical and physical stressors.The present results agreed with Amin (2010) who determined the variation induced in electrophoretic protein pattern of first and third instar larvae of flesh fly, S. bullata irradiated with Gamma rays (physical stressor) comparing with unirradiated larvae and found that the challenging with radiation resulted in appearance of unique protein bands in the treated sample by using SDS-PAGE and disappearance of protein bands as compared with control. Also, Bedenarova et al. found that the protein profiles of S. littoralis treated with five profenofos formulations, after stored at cold and hot conditions, were differently changed and distinguished into several separated protein bands in the range of 3.6 to 195.5 kDa. It can be concluded that the proteins play an important role in the haemolymph of insects not only in specific transport functions, but also in their enzyme and hormone action as antibacterial agents or detoxifying agents. This conclusion was reinforced by Ahmad (1992) who reported that oxidative stress may lead to membrane or the whole cell damage by lipid peroxidation and protein oxidation resulting in uncontrolled apoptosis. Eukaryotes, including insects, possess a suite of antioxidant enzymes that protects their cells from oxidative radicals.