Evolutionary Analysis of Culex Species

Evolutionary Analysis of Culex SpeciesINTRODUCTIONCulex, a mosquito belongs to the phylum Arthopoda, is an grave factor causing filariasis 1 St. Louis phrenitis 2West Nile virus 3 Avion malaria (4). C.quinquefasciatus also c completelyed the Southern ho apply mosquito is extensively studied as it transmits crucial diseases 5. C. pipiens found in the urbans (6) is because of swift urbanization uncanny city developing which eventually resulted in the progression the disease transmittors (7,8,9). Lymphatic filariasis is spread worldwide, affecting 120 million people10. 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 200313. 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% cause by Wuchereria bancroftiCobbold19Hence, therefore, it is very important to eradicate this for the human eudaemonia for which it is very essential to understand them at their molecular levels. The genome installment of Culex quienquefasciatus20triggered a new hope for the researchers. However, the researchers are still involved in come outing and characterisation of these species causing diseases(21).The objective of the present article is to access the evolutionary relationship among the culex species with the desoxyribonucleic acid 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 medicament, 9 295 (1989)(BOOK) (23) system as the data could be accurate (22). The use of desoxyribonucleic acid barcoding has been in practice over the conventional 16s ribosomal deoxyribonucleic acid (24) which is more advantageous and promising (25) by playacting a pivotal role in identification between the species (26).2. MATERIALS AND METHODS2.1 Larvae collection and CharacterizationCulex larvae were sampelled from various parts of Hyderabad during the breeding season from varied locations, rank 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, yellowed 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). set ahead study was performed from the F1 generation of the pure culture afterward they were identified morphologically for adult.2.2 DNA EXTRACTIONF rom the 4th instar larvae, the total DNA was extracted and was then finely scope in 50 l of homogenate buffer (28). The homogenate was left in the thermo cycler for 30 minutes at 60oC with a quick addition of 7 l of 8M CH3COOK. breed 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 l of 95 % ethanol. DNA pellet was collected after a rapid centrifugation for 15 minutes at maximum speed and decomposition reaction 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, elaborateness, Sequencing and AlignmentThe 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 r everse primers (28) which were authentic on the spanning, 700 bp of Aedes aegypti, Anapheles stepfheni and Cx. Quienquefasciatus. The reaction mixture, 25 l, consisting of 10-50 ng of DNA template, 200M 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 alliance 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 a long with other parts of the world.Further, the results of the DNA barcoding were made increasingly vivid with the phyletic 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 DISCUSSIONSKnown 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 potency 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 4 00bp 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 well related to the UK species and hence both emerged as out group in the phylogenetic tree.CONCLUSIONThe medium sized brown mosquito, Culex quinquefasciatus, predominantly exists all through the tropics acts as a vector causing several parasitic diseases. 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