Introduction

Mosquitoes are still the world’s number one vectors of human and animal, transmit a broad range parasitic and viral diseases.  Dengue fever is now endemic in more than hundred countries in Africa, Mediterranean region, South America and South East Asia (WHO ,200,2003 ,Whitehorn and Farrar, 2010).). Until the 1960s, systematic data were generally collected from morphological and behavioral variations. However, after the 1960s biological macromolecules gained an increasingly important role in evolutionary and systematic studies. Early molecular studies with systematic purposes were concerned largely with proteins. The electrophoresis of proteins is an effective technique for generating systematic data from macromolecules. This method has become increasingly popular among systematists (Onaric and Sumer, 2003).

Electrophoresis has been proven useful in insect taxonomy where conventional taxonomic methods of identification of immature stages of related species have been inadequate (Berlocher, 1980). In the last 20 years, electrophoresis has been widely used in population genetics studies of more than 1100 species of animals and plants (Nevo et al., 1984). The electrophoresis of proteins is an effective technique for generating systematic data from macromolecules.

Gel electrophoresis technique of proteins is the most widely use molecular technique in insect systematic. This technique relies on the fact that identical proteins migrate the same distance under the electrical force applied to an electrophoretic gel while non-identical proteins usually migrate different distances. The proteins used in insect systematics are usually enzymes (Sheppard and Smith 2000). An accurate identification of the species of mosquitoes  is required to determine whether it belongs to a species group that poses a potential risk (Gupta  and Preet 2012). Recently, several studies have been  isolated and described total , specific protein  and gene expression from  mosquitoes  such as Aedes, Culex and Anopheles   from immature  stages  and specific parts of adult antenna, salivary glands   and protein  bindings with dengue virus (Ishida  et al ., 2002, Chee  and AbuBakar  2004 , Hung et al. 2004 , Rohani, et al ., 2005 and Popova-Butler and Dean 2008 )

The present study conducted to identify the total  protein profile of different  developmental stages of Ae.aegypti dengue fever vector using SDS-PAGE analysis.

Materials and methods

Study sites

The presented investigation was conducted using Ae. aegypti collected from 5 sites  of Jeddah  provinces in western  Saudi Arabia(Table1). The dengue cases and the re-epidemics  were continuously reported  in the selected location during the past six years.

Table 1: Coordinate of study locations in Jeddah governorate.

Where:

A= Al-Balad

B= Al-Nazlah Al-Yamaneyyah

C= Ghuleel

D= Al-Jameiah

E= Al-Safa

Collection and identification of mosquito

Adults of  Ae. aegypti were collected from  five different localities of Jeddah governorate. Adults and larvae stages were identified by using standard taxonomic keys ( Wood et al. , 1979 and  Darsie and  Ward, 2005 ). Briefly  Ae. aegypti adults can be identified throughout  the white scales on the dorsal (top) surface of the thorax that form the shape of a violin or lyre. The larvae stages  can be identified using the comb scales on the eighth segment of the abdomen and the shape of the pectin teeth on the siphon . In larvae, the comb teeth have well developed lateral dentiles but the pectin teeth have less defined denticles.

Mosquito

Ae. aegypti (L.) larvae were reared under the laboratory conditions for several generations . Adults, larvae and pupae were  used  to isolate total protein in this study. A field strain of Ae. aegypti larvae were collected from Jeddah governorate. This stock colony was maintained at a room temperature at 27±l°C and 70±5% R.H., with a 14:10 (L: D) photoperiod.

Mosquito Total Protein Electrophoresis

Sodium dodecylsulfate polyacrylamide gel electrophoresis was used to identify Ae. aegypti  by their protein analysis  according to the method of Laemmli  1970 , and modified by Studier (1973).

Protein extraction

Adults or immature stages of  Ae. aegypti were extracted according to Abo-El-Saad. and  Ajllan. (2003).  Mosquito stages were  finely homogenized together in eppendorf tubes containing 200 µl of the extraction buffer by a handle plastic homogenizer and  left in refrigerator at 4°C over night then vortex for 15 seconds and  centrifuged at 12,000 rpm at 4°C for 15 minutes.  The supernatants were transferred to new eppendorf  tubes and kept in deep – freezer until use for electrophoretic analysis.

Preparation of protein samples

A 20 µl of the samples as extracted previously from sample with 5 µl of 2 % SDS-sample buffer. The mixture was incubated in a boiling water bath for 5 min before application.

Application of protein samples

The protein samples of 25 µg protein were loaded into the wells using a Hamilton syringe. Protein fractionated by SDS polyacrylamide gel electrophoresis (PAGE) as described by Smith (1976); using slab gel that consist of a 4% polyacrylamide stacking gel and an acrylamide (12%) gel. High and low MW standards were used for the determination of protein profiles of all fractionated samples.

Electrophoretic analysis

Protein fractionated by SDS polyacrylamide gel electrophoresis (PAGE) as described by Smith (1976). High and low MW standards were used for  the determination of protein profiles of all fractionated samples.

Results and discussion

The protein samples were purified from the larva, pupa and adults(lab and field stain) stages. The protein banding of lab and field strain adult and immature stages of dengue fever vector, were electrophoretic. The  Coomassie-stained SDS-PAGE was applied to analysis the purified protein sample of the different insect  stages. Two gels were obtained for different  developmental stages of Ae.aegypti as shown in Fig.(1 and 2).The protein purified samples were quantified using bovine serum albumin (BSA) marker. Fig.(1). High and low  molwcular weight as protein marker were sued to estimate several major and minor bands Table (2)

The results revealed that  the molecular weight of protein samples  were around 66 kDa. Six protein bands were identefied ranged from 16.6 to 75kDa in molecular weight, Fig(2) and Table (3). A single protein pattern with a molecular weight of 16.6 kDa  was observed for all insect stages. The lab strain adults revealed two protein patterns with molecular weight of 19.1 and 52 while the field strain adult   produced a total protein of 52 and 16.6 kDa. Four  protein bands with molecular weight of 16.6, 58.8, 69  and 75.6 kDa were identified for pupa (Table 3). This data consistent with other data reported by Lee et al.,2009 who found similar protein bands, which fall in the range of approximately 200 kDa to less than 24 kDa. This result  in agreement with Rohani, et al ., 2005  who mentioned that the protein bands of Ae.aegypti were within the range of 14 – 80 kDa. Study of  Junsuo  and Jianyong 2006 described total and specific protein isolated from matures and immature stages of Ae.aegypti, Another researcher revealed that the isolated   haemolymph  proteins profiles   of   adult female Aedes togoi  varied from 10-80 kDa (Jariyapan et al., 2011) Proteins with molecular weights of  240 and 70 kDa were reported ( Capurro et al., 1994; Ford and Van Heusden, 1994; Van Heusden et al., 1998).

.Several  studies about mosquitoes protein have been reported  in different parts in the world (Biessmann   et al., 2002 , Sun et al., 2000. Von-Dungern and  Briegel,  2000, 2001 Pimsamarn , et al.,  2009 , Jariyapan et al., 2007 and Valenzuela et al., 2002).

As showed in Fig.(1) several major and minor bands were appeared with high resolution comparing with bands in  Fig(2) which appeared to be slightly poor in resolution (fainted or invisible band) and number of bands, this might be due to volume of  extracted protein sample (15 µl) that was loaded as compared to 25 µl in Fig(2) Moreover the electrophoresis conditions in Fig (1) such as  the  concentration of Bromophenol blue dye, stained  and the running time which  was 2hrs in Fig(1)  compared to 6 hrs. in Fig(2)

Figure 1: Protein quantification using SDS electrophoresis (PAGE) of four  development stages of Ae. aegypti.    Click here to View figure

Where:

M Marker

1 =  larvae,

2 = Lab adult,

3 = Pupae,

4 and 5 = Field  adult,

6 = Pupae

Figure 2: Protein identification  using SDS electrophoresis analysis  of four  development stages of Ae. aegypti.    Click here to View figure

Where:

L M W Low molecular weight kDa,

H M W High molecular weight kDa,

1 =  larvae

2 = Lab adult,

3 = Pupae,

4 and 5 = Field  adult

,6 = Pupae

Table 2: Molecular weight of protein marker Used in electrophoretic SDS –PAGE

Log molecular weight

Figure 3: Molecular weight values of Ae. aegypti as calculated for calibration of SDS-PAGE protein analysis.   Click here to View figure

The  calibration  curve was  constructed by plotting the electrophoresis mobility of standard proteins  versus the  logarithms of  their corresponding  molecular weight values. Unknown molecular weight of total protein of  Ae. agyptian development stages were  determined by measuring the electrophoretic mobility of  the  unknown total protein  and the corresponding molecular weight  from the curve  as showed in  Fig.(3) and table (3)

Table 3; Total protein of different developmental stages (larvae, pupae and adults) using SDS-PAGE  electrophoresis

Conclusion

This study described and identified ten distinct  of Ae. aegypti developmental stages as a total protein . These results provide basic information  and  an initial step for  identification of differentially specific proteins of Ae. aegypti.. Further studies are needed for   identifying specific  proteins and their function  and biological roles in dengue fever infections. Proteomics study is necessary for understanding  developing novel vector control strategies and parasite-vector interactions, gene expression which will be dominate post- research.

Acknowledgments

We gratefully acknowledge  the king Abdulaziz city for science and technology( kacst) for their financial support  under grand No.( A-S-10-0013)   .We would also  like to express our deep appreciation  to Prof. Dr Mahmoud Abo-El – Saad  for his helping  in practical work. I deeply thank Dr. Magdi  Mosa for  valuable guidance and  reviews of  part of  draft as well as Dr. Ahmed Bakhashwain for generous support of requirements  chemicals in biotechnology lab.

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