Journal of Entomology and Zoology Studies 2017; 5(4): 301-305 E-ISSN: 2320-7078 P-ISSN: 2349-6800 JEZS 2017; 5(4): 301-305 Studies on morphology and molecular © 2017 JEZS characterization of oriental cat flea infesting Received: 07-05-2017 Accepted: 08-06-2017 small ruminants by barcoding Ashwini MS MVSc Scholar, Department of Veterinary Parasitology, Ashwini MS, Puttalakshmamma GC, Mamatha GS, Rakshith Ojha, Veterinary College Bengaluru, Chandranaik BM, Thimmareddy PM, Placid E D’souza, Jalali SK and India Venkatshan T Puttalakshmamma GC Associate Professor, Department Abstract of Veterinary Parasitology, The study was carried out to identify the flea species infesting small ruminants in Karnataka state, India. Veterinary College Bengaluru, The morphological differentiation of species of genus Ctenocephalides was often confused due to loss of India specific characters. Therefore, DNA barcoding of COX1 mitochondrial gene was carried and PCR product yielded a specific amplicon at 700 bp. The COX1 sequences of Ctenocephalides orientis from Mamatha GS sheep were showing 99-100% homology to C. orientis from goats whereas, C. felis felis from dogs Assistant Professor, Department of Veterinary Parasitology, showed 99% homology to C. felis felis from cat. The accession number of KX467332 and KX467333 for Veterinary College Bengaluru, C. orientis from Sheep and goats, KX467334 and KX467335 for C. felis felis from dog and cats India respectively was allotted by NCBI. In the phylogenetic analysis, C. orientis formed a separate cluster to the subspecies of C. felis and clustered as sister species with C. canis. Thus, supporting C. orientis as Rakshith Ojha separate species as previously suggested by morphological and molecular approaches. Senior Research Fellow, NBAIR, Hebbal, Bengaluru, India, India Keywords: Small ruminants, C. orientis, COX1 gene, Barcoding Chandranaik BM Introduction Scientists, IAH &VB Small ruminants are the major source of income to the farmers through milk, meat, wool and Hebbal, Bengaluru hide production which has positive impact on their socio-economic life. Presently, India has Karnataka, India, 65.07 million sheep (Ovis aries) and 135.17 million goat (Capra hircus) population out of Thimmareddy PM which Karnataka contributes 9.58 and 5.27 million of sheep and goat population respectively. Professor, Department of Fleas of the genus Ctenocephalides are the important group of ectoparasites infesting small Veterinary Parasitology, ruminants in tropical and subtropical countries like India causing dermatitis, severe anaemia Veterinary College Bengaluru, and deaths especially in young animals. Fleas are important since they act as a vector of India zoonotic pathogens such as Rickettsia felis and Bartonella species [1]. However, small [2] Placid E D’souza ruminants like sheep and goats do not have their own species of flea . The cat flea, Professor cum Director Ctenocephalides felis has a wide host range including small ruminants, distributed worldwide CAFT Bengaluru and formerly consisted of four sub species viz., Ctenocephalides felis felis, Ctenocephalides Karnataka, India felis orientis, Ctenocephalides felis strongylus and Ctenocephalides felis damarensis[3]. But, Jalali SK morphological revision of the phallosome (male reproductive organ) proposed for the [4, 5] Principal Scientist, NBAIR, elevation of C. orientis and C. damarensis to full species level . However, the Hebbal, Bengaluru, India morphological differentiation of species of genus Ctenocephalides is often confused due to loss of specific characters like legs, head and minute morphological differences like head Venkatshan T shape, chaetotaxy and manubrium of clasper. Therefore, molecular characterization is a boon Principal Scientist NBAIR, Hebbal, Bengaluru, for exact identification and classification of fleas of the genus Ctenocephalides. DNA India barcoding using COX1 mitochondrial gene as a universal marker is the most reliable method as COX1 mitochondrial gene are in abundance (1000 identical copies per cell), is highly conserved functional domains and variable region [6]. Hence, a barcode of fleas infesting small ruminants, dogs and cats of Karnataka was undertaken. Correspondence Materials and Methods Ashwini MS Study area, sample and identification MVSc Scholar, Department of The study was carried out during January 2015 to June 2016 in private farms, household pens Veterinary Parasitology, 0 0 Veterinary College Ben22galuru, and nomadic flocks of sheep and goats in Karnataka state located between 11.30 and 18.30 0 0 India north latitude and 74 and 78.30 east longitude in India. Fleas were collected from both sheep ~ 301 ~ Journal of Entomology and Zoology Studies and goats and also from dog and cats closely associated with ruminants whereas cat flea, C. felis felis was found on dogs these flocks. Initially, animals were restrained and examined and cats associated with infested small ruminants farms. C. in detail. Later fleas were collected from randomly selected orientis (C. felis orientis) was previously reported on Goats infested animals in a flock using fine toothed metal comb and from Karnataka [13] supporting that C. orientis is the dominant placed in a plastic vial containing absolute ethanol. The flea species found in India. Previously, four subspecies viz., collected fleas were brought to the laboratory and identified Ctenocephalides felis felis, Ctenocephalides felis orientis, based on the standard keys [7]. Ctenocephalides felis strongylus and Ctenocephalides felis damarensis were classified under the species C. felis [14, 15]. DNA extraction, PCR amplification and Sequencing However, C. orientis and C. damarensis as a separate species The genomic DNA of adult Ctenocephalides was extracted based on the revision of the morphological characters of the using Qiagen DNeasy Blood and Tissue Kit (Germany) as per male reproductive organ [4]. The presence of both genal and the manufactures protocol. The COX1 mitochondrial gene of pronotal combs confirmed the identity of the genus Ctenocephalides species viz., C. orientis and C. felis felis Ctenocephalides. In C. orientis flea, the anterior most genal were targeted and subjected for PCR by using Phire animal spine was shorter than subsequent spine. The genal ctenidium direct Kit using the universal primers: forward 5’- consisted of seven to eight pairs of spines and the pronotal GGTCAACAAATCATAAAGATA TTGG-3 and reverse 5’- ctenidium has got totally 15 to16 spines. The length of head TAAACTTCAGGGTGACCAAAAAATCA-3’[8]. Reaction was twice as long as height and the frons were elongate and for the COX1F/COX1R primers were performed in a total broadly rounded at anterior end (Fig. 1). Metepisternum or volume of 50µl consisting 2X PCR buffer 25 µl, 0.5µl lateral metanotal area (LMA) had two bristles. Females had a COX1F (20ppm), 0.5 µl COX1R(20ppm), DNA polymerase row of one to 12 short spiniform bristles behind the antennal 1µl, flea DNA, NFW to make final volume 50µl under the groove (Fig. 2). Dorsal margin of hind tibia had seven notches following cycling conditions: an initial denaturation stage at with stout and single bristles on third and sixth notches. 980 C for 5min, then 40 cycles of denaturation at 980 C for 5 Chaetotaxy formula of metatibial bristles was 2-2-1-2-2-1-3 sec, annealing at 450 C for 10 sec and 720 C for 30 sec (Fig. 3). In males, the manubrium of the clasper was widened followed by a final extension phase at 720 C for 1 min[9]. Once at the apex (Fig. 4). the PCR reaction were carried out, 1µl of DNA release buffer and gel loading dye were subjected for electrophoresis in 1.5% agarose gel and the images were captured using gel documentation system. The amplified products were sent for DNA sequencing to M/s Chromous Bengaluru, India. The sequences obtained were subjected to Basic Local alignment search tool (BLAST) analysis at NCBI-BLAST. The sequences were edited and submitted to National Centre for Biotechnology Information (NCBI) GeneBank. The BoldSystem V3 was used to generate barcode for the two species of Ctenocephalides. The DNA sequences of the both C. orientis and C. felis felis from the study and other DNA sequences of Ctenocephalides species uploaded from GenBank database were assembled and subjected to MEGA 5.0 software for Phylogenetic tree construction. The tree obtained by applying Neighbor-joining (NJ) method with 1000 bootstrap support. The evolutionary history was inferred [10] using the Neighbor-joining method . The optimal tree with Fig 1: C. orientis: a. Elongate and broadly rounded head; b. LMA the sum of branch length = 0.50258811 is shown. The with two bristles and c. 1st comb is shorter than 2nd comb percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [11]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2- parameter method [12] and are in the units of the number of base substitutions per site. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). The analysis involved 9 nucleotide sequences. Codon positions included were 1st+2nd+3rd. All positions containing gaps and missing data were eliminated. There were a total of 601 positions in the final dataset. Results and Discussion Morphological identification In the present study, two species of fleas in the genus