INTRODUCTION:
Culex, a mosquito belongs to the phylum Arthopoda, is an important factor causing filariasis [1] St. Louis encephalitis [2]West Nile virus [3] Avion malaria (4). C.quinquefasciatus also called the Southern house mosquito is extensively studied as it transmits crucial diseases [5]. C. pipiens found in the urbans (6) is because of swift urbanization uncanny city growth which eventually resulted in the progression the disease transmittors (7,8,9). Lymphatic filariasis is spread worldwide, affecting 120 million people [10]. They bite on the foot regions of humans (11) causing skin allergy associated with irritation (12).Since 1979 there are reports Japanese encephalitis in India, AP and was again reported in 2003 [13]. Known as a cosmopolitan mosquito, acts as vector for protozoan parasites (14 ),filarial worms (15), and for arboviruses(16 ,17). Cx. quinquefasciatus is the prime vector in India causing filariasis (18) which involve 91% caused by Wuchereria bancroftiCobbold [19]
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Hence, therefore, it is very important to eradicate this for the human welfare for which it is very essential to understand them at their molecular levels. The genome sequence of Culex quienquefasciatus [20]triggered a new hope for the researchers. However, the researchers are still involved in identifying and characterization of these species causing diseases (21).
The objective of the present article is to access the evolutionary relationship among the culex species with the DNA barcoding and to assess the evolutionary relationship with Tamura 3 Parameter. To accomplish the objective, it is important to use the advanced techniques like the computerized data assessments (22) which could have an immense impact on the health care S. Morio, Computers in Biology and Medicine, 9: 295 (1989)(BOOK) (23) system as the data could be accurate (22). The use of DNA barcoding has been in practice over the conventional 16s ribosomal DNA (24) which is more advantageous and promising (25) by playing a pivotal role in identification between the species (26).
2. MATERIALS AND METHODS:
2.1 Larvae collection and Characterization:
Culex larvae were sampelled from various parts of Hyderabad during the breeding season from different locations, say stagnant water, coconut shells, tires etc. Thus collected larvae were reared and were identified morphologically for Culex quienquefasciatus under the microscope. Microscopic analysis revealed the following characteristics, Short and stout head, yellow long mouth brushes. It was also observed that the abdomen has eight segments, the saddle and the siphon, which is four times longer that its width, securing the larvae to be Culex quinquefasciatus. The larvae were allowed to grow into an adult (27). Further study was performed from the F1 generation of the pure culture after they were identified morphologically for adult.
2.2 DNA EXTRACTION:
From the 4th instar larvae, the total DNA was extracted and was then finely ground in 50 µl of homogenate buffer (28). The homogenate was left in the thermo cycler for 30 minutes at 60oC with a quick addition of 7 µl of 8M CH3COOK. Incubate the tubes in ice for 30 minutes and centrigugate them for at a maximum speed for 15 minutes. Transfer the supernatant into fresh tubes. To precipitate the DNA, incubate the tubes for 15minutes after adding 100 µl of 95 % ethanol. DNA pellet was collected after a rapid centrifugation for 15 minutes at maximum speed and decay the supernatant. Air dry the pellet and suspend it in Tris buffer. Store it at 20oC till further experimental procedure is carried.
COI Partial sequencing, amplification, Sequencing and Alignment:
The isolated DNA was further amplified on PCR by mitochondrial Cytochrome c Oxidase I (COI) gene, which can differentiate between the diversity of taxa . The mitochondrial COI gene of ~ 500 bp was amplified by forward and reverse primers (28) which were developed on the spanning, ~700 bp of Aedes aegypti, Anapheles stepfheni and Cx. Quienquefasciatus. The reaction mixture, 25 µl, consisting of 10-50 ng of DNA template, 200µM of dNTPS’ 1U of Taq DNA polymerase, 1X assay buffer and 5p mol of primer was then subjected to amplification for 2 minutes at 94o C initiation, denaturation of DNA template for 35 cycles for 30 seconds at 95 o C, followed by primer aliening -30 seconds at 55 o C, extension- 70 o C for 30 seconds and final extension at 70 o C for 10 minutes.
The amplified sequence ,thus, was run on Agarose gel electrophoresis and then the sequencing was perfoemed by Bioserver Biotechnologies Pvt. Ltd. The sequence was submitted to NCBI. The accession number assigned was JX08870 (501bp).
Further multiple sequence alignment was performed for partial COI gene sequence of 501bp with the default parameters. Sequence alignment studies elucidate the similarity and differences among different species in India along with other parts of the world.
Further, the results of the DNA barcoding were made increasingly vivid with the phylogenetic analysis by the construction of phylogenetic tree. The analysis of the phylogeny was attained by maximium likelihood method (29) with the deletion of gaps and missing data. Bootstrap replication was used to validate the tree.
RESULTS AND DISCUSSIONS:
Known as the ‘Southern House Mosquito’, Cx. Quienquefasciatus a vector which played a prime role in filariasis and the incidences have seen in different parts of the world. Hence, there is an utmost need to address this problem with priority. In order to snub the prognosis of this vector, it is very essential to take certain control measures. To attain this, the mosquito larvae were collected and grown into an adult. The DNA was isolated and amplified and run on the agarose gel electrophoresis. The agarose wells comprised of sample from different locations along with 200bp marker DNA. The amplicons were seen between 400bp and 600bp of the DNA marker and were then subjected to sequencing. It was noted that the sequence was identified to be novel and was submitted to NCBI-gene bank nucleotide database and the accession number assigned was JX088701.
Further, the multiple sequence alignment was performed to understand the evolutionary relationship among the species of the world.. Using the Maximum Likelihood Method, which creates a tree of highest Likelihood from the given data. The Maximum Likelihood tree elucidates that the Culex species of Hyderabad was closely related to the UK species and hence both emerged as out group in the phylogenetic tree.
CONCLUSION:
The medium sized brown mosquito, Culex quinquefasciatus, predominantly exists all through the tropics acts as a vector causing several parasitic diseases. Usually active at night, it is an opportunistic blood feeder which allows the parasites to use the humans as hosts. It is hence important to address their control with priority. The present article the DNA sequence was successfully isolated, sequenced and was submitted to NCBI-Gen Bank nucleotide database. Further, to understand its evolutionary relationship the phylogenetic analysis was performed. It was noticed that the DNA sequence of Hyderabad Culex quinquefasciatus was different from other species and can be used as DNA barcode to identify the organism
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