Electrophoretic Analysis of Salivary Gland Proteins of Adult Culex antennatus (Diptera: Culicidae)

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.8(1)pp1-9(2016) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net Provided for non-commercial research and education use.

Adult mosquito salivary glands are paired organs located on either side of the thorax flanking the oesophagus (Dhar andKumar, 2003, Jariyapan, et al., 2007).The female gland has three lobes, including two lateral lobes, with distinct proximal and distal portions, and a medial lobe.Salivary glands contain several kinds of protein (Siriyasatien, et al., 2005), but female glands contain approximately 10 times more protein than male glands.Mosquitoes feed on blood as quickly as possible to avoid haemostasis processes consisting of platelet aggregation, vasoconstriction and blood coagulation.Proteins are the most complex compounds and at the same time, the most characteristic components of living matter.Insect proteins are numerous and present in all viable cells (Kyung and Kim, 1990).Each protein is considered as reflect to the activity of specific gene through the production of enzyme which act as catalyst to produce protein responsible for specific biological character (Cersa, 2003).
Polyacrylamide gel electrophoresis (PAGE) has been extensively used as an excellent tool for the separation of proteins from all living organisms (Zacharius et al., 1969).The vast majority of recent studies on insect proteins have used electrophoretic techniques.Polyacrylamide gel, with the advantages of high sensitivity and resolving power, is generally the most efficient medium (Wyatt and Pan, 1978).In the present study, salivary gland proteins of Cx. antennatus were electrophoretically separated by native and SDS-PAGE at different time intervals; un-fed stage, 0-, 3-, 6-, 12-and 24-h.after sugar feeding, starved stage and at probing time, partial engorgement, full engorgement, 3-, 6-, 12-, 24-, 48-and 72-h.after blood meal and after oviposition.
Taking all of these considerations into account, it should be of great importance to identify protein contents in the salivary glands of males and females of Cx. antennatus qualitative (native and SDS-PAGE).

MATERIALS AND METHODS Insect colonization:
A laboratory colony of the Cx.antennatus, used for experiments was obtained from the field (Shubramunt, Giza, Egypt), and maintained in the insectary of the Department of Zoology, Faculty of Science, Al-Azhar University.This colony was maintained under laboratory conditions of 27 + 2 °C, 60-70% RH and 10L: 14D photoperiod for supplying clean adults of known ages, according to the method described by (El-Bokl and Moawad, 1996).

Mosquito Salivary Gland Dissection:
Mosquitoes were anesthetized by subjecting to a temperature of 4°C, until immobilized.Salivary glands of adult mosquitoes (20 pairs of salivary glands were used) were dissected using fine entomological needles under a stereomicroscope at 4X magnification in phosphate-buffered saline [PBS;10 mM Na2SO4, 145 mM NaCl (pH 7.2)] and stored at −80°C prior to SDS-PAGE (Cotama et al., 2013).

Native polyacrylamide gel electrophoresis (PAGE):
Native-PAGE was used to analyze protein content of the salivary glands of males and females of Cx. antennatus at the above mentioned intervals.15 % polyacrylamide gels pH 4, using a discontinuous buffer system was employed (Gabriel, 1971).Acrylamide/ bisacrylamide ratio was 60:0.8.The gels were run at 100 V until the tracker dye (Bromophenol blue) was running off the gel (approximately 4 h).

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE):
SDS-PAGE was used to analyze protein content of salivary glands of males and females of Cx. antennatus at the above mentioned intervals.15% polyacrylamide gels pH 8.8, in a discontinous buffer system was employed (Maizel and Jr, 1971).Acrylamide/ bisacrylamide ratio was 50: 1.The gels contained no SDS before electrophoresis.Protein samples were pretreated with 1% SDS and 1% βmercaptoethanol for 5-10 min at 100 °C.The gels were run at 100 V until the tracker dye was leaving the gel (approximately 4 h.).All gels were fixed in 20% 5-sulfosalicylic acid, stained with Coomassie Brilliant Blue R250, destained in 7% acetic acid and photographed using gel-documentation system with a 20-Mp camera.

Data analysis:
The data obtained from the scanning process of each gel were analyzed using Gel pro analyzer (Ver.31 Media cybernetics, USA) and Alpha Ease FC stand alone for windows 2000/XP.

SDS-PAGE:
Salivary gland proteins of males and females of Cx. antennatus mosquito were electrophoretically separated by SDS-PAGE using 15% acrylamide gel (Figs. 5,6,7 and 8).Protein standard (marker) was used to estimate the molecular weights of the separated bands.
Figure ( 5) showed changes in salivary gland protein banding patterns of males and females of Cx. antennatus at un-fed stage and after 0-, 3-and 6 h sugar feeding.The numbers of observed bands were 25 and 22 bands (M.wt.ranged from 289.21 to 10.91 KDa) in the case of un-fed females and un-fed males, respectively.23 and 29 bands (M.wt.ranged from 317.36 to 10.91 KDa) were observed after 0 h from sugar feeding in females and males, respectively.22 and 27 bands (M.wt.ranged from 289.21 to 10.91 KDa) were observed after 3 h from sugar feeding in females and males, respectively.19 and 25 bands (M.wt.ranged from 317.36 to 10.91 KDa) were observed after 6 h sugar feeding in females and males, respectively.
Figure ( 6) showed protein banding patterns of male and female Cx.antennatus at 12, 24 h after sugar feeding and starved stage using 15% SDS gel.21 and 18 bands (M.wt ranged from 284.81 to 11.35 KDa) were observed in the salivary glands of female and male 12 h after sugar feeding (Fig. 6), 18 and 21 bands (ranged from 284.81 to 12.7 KDa) were recorded in female and male 24 h after sugar feeding (Fig. 6), 16 and 21 bands (M.wt ranged from 284.81 to 11.35 KDa) in the case of starved female and male mosquitoes (Fig. 6).
Figure ( 7) demonstrated changes in salivary gland protein patterns of control and female mosquito after blood meal.It was obvious that the females exhibited different number of protein bands after feeding on the host blood.21 and 16 bands (M.wt.ranged from 243.38 to 11.35 KDa) were detected in females 0 h after sugar feeding and starved stage, respectively (Fig. 7), 16 bands (M.wt.ranged from 169.86 to 15.4 KDa) were present in probing time, 15 bands (M.wt.ranged from 146.24 to 15.4 KDa) were found in partially engorged females, 21 bands (M.wt.ranged from 243.38 to 15.4 and KDa) were distinguished in both fully engorged females and 3 h after blood meal females (Figs. 7 and 8).
Figure ( 8) showed protein banding of females after blood meal resolved in 15 % SDS gel.It was clear that females exhibited different number of protein bands after feeding on the host blood.14 bands (M.wt.ranged from 238.55 to 15.4 and KDa) were distinguished in each of females 6, 12 and 24 h after blood meal, 15 bands (M.wt.ranged from 204.11 to 15.4 and KDa) were found in females 48 h after blood meal, 16 bands (M.wt.ranged from 204.11 to 15.4 and KDa) were detected in females 72 h after blood meal and 14 bands (M.wt.ranged from 243.38 to 15.4 KDa) were detected in females after oviposition (Fig. 8).
Native and SDS-PAGE of salivary glands of male and female Cx.antennatus at different time intervals demonstrated many changes in bulk and denatured proteins.The appearance of different bands in sugar fed and blood fed may be attributed to the induction of new proteins in a specific feeding stage.
Similar results were recorded on the salivary gland proteins of sugar and normal blood-fed An. gambiae mosquitoes.Salivary glands of blood-fed An. gambiae showed high and low molecular mass proteins 1 h postfeeding.The most notable difference was expression of 100 and 29 kDa proteins in response to a blood meal when compared to sugar-fed mosquitoes (Brennan, et al., 2000).In addition, salivary gland proteins profile of Aedes aegypti, Armigeres subalbatus and Culex quinquefasciatus mosquito were studied by (Siriyasatien et al., 2005).SDS-PAGE analysis demonstrated 8 major polypeptides of 20, 35, 37, 42, 45, 47, 70 and > 118 kDa in female Ae.aegypti preblood feeding.After a blood meal, depletion of major peptide bands of 35,37,45,47,70 kDa and high molecular weight band> 118 kDa was observed.In the case of Cx. quinquefasciatus, nine major polypeptides were observed with molecular weights of 20,25,36,38,45,47,49 and two bands of > 118 kDa.The bands of 20, 26, 36 and 38 kDa were depleted after a blood feeding.Similar results were observed in other blood sucking insects e.g.Thyrsopelma guianense, the vector of onchocerciasis, in Brazil.The salivary glands of this species begin synthesizing proteins soon after adult emergence, similar to the protein expression observed in mosquitoes.The levels of soluble proteins present in the salivary secretions are comparable to other anthropophilic species already studied, such as Simulium metallicum and Simulium ochraceum (Cross et al., 1993 andAbebe et al., 1994).Protein level reflects the size of the salivary glands of each species.Electrophoretic analysis of T. guianense showed similar profile to other black fly species in regard to the number of polypeptides (10-12 bands).Meanwhile, the expression profile changes with salivary gland maturation (Jariyapan et al., 2006).Cross et al. (1993) analyzed four black flies species and found that zoophilic species had more bands (19-20 molecular bands) than anthropophilic species (11-12 molecular bands).This observation suggested that the difference in salivary protein composition may represent different evolutionary adaptations in anthropophilic species to aid feeding from human hosts.
In addition, the differences in protein level at different developmental stages, as visualized by SDS-PAGE, reflect an increase in the amount of protein in the glands of older black flies.This observation may be associated with the fact that efficient blood feeding occurs mainly during maximum salivary secretions, at 48 h following emergence for this black fly species (Cross et al., 1993).In sand flies, which are pool feeders like black flies, the number of protein components gradually increases with age and depends not only on sex but also on physiological state of the female (Volf et al., 2000).In contrast, our results showed that the protein concentration of the black fly salivary glands did not vary qualitatively during the first three days of female adult life.Jariyapan et al. (2006) showed that all black fly species had a small number of major proteins and some of the major proteins differed in molecular mass between species.The proteins responsible for inhibiting coagulation cascade are secreted soon after emergence.It is possible that the anticoagulation activity may be even greater than that shown here if the flies are measured at the peak of salivation.after sugar fed female, L4: after sugar fed male, L5: 3 h after sugar fed female, L6: 3 h after sugar fed male, L7: 6 h after sugar fed female, and L8: 6 h after sugar fed male.