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Home , Barbarea orthoceras, Coliadinae, Crambe, Erysimum, Pierinae, Pieris (butterfly)

Pakistan J. Agric. Res. Vol. 29 No.3, 2016 PIERID (: ) PESTS AND THEIR NEW CRUCIFERS HOSTS IN POTHWAR REGION OF PAKISTAN

Syed Waqar Shah* and Muhammad Ather Rafi*

ABSTRACT:- Field surveys were conducted in Pothwar region during January 2012-December 2013 to find out the pierids pests on cultivated and non-cultivated cruciferous , the known cultivated hosts such as Raphnus sativus,, Eruca sativa Brassica napus ,, B. oleraceae B. oleracea var.italica and B. campestris were attacked by brassicae , P. canidia andPontia daplidice . Among above reported Pieridae P. rapae is reported for the first time however, daplidice is also a new record from districts Rawalpindi and Chakwal andP. canidia from Jhelum, Rawalpindi and Chakwal. However, the non-cultivated host plants in the region wereCaspsella bursa-postoris, Coronopus didymus , Lepidium pinnatifidium, Arabidopsis thaliana and B. officinalis . Among non- cultivated hostsL. pinnatifidium was the new host for P. daplidice . C. didymuswas found common host for P. brassicae , P. canidia and P. daplidice from the study area. Among non-cultivated host plants A. thalianawas found for P. canidia and B. officinalis for P. brassicae and P. daplidice. All the non-cultivated host plants were new records from Pakistan however,L. pinnatifidium was new host of P. daplidice from the world, which was not reported earlier.

Key Words: Crucifers; Host Plants; Lepidoptera; Pieridae; Pests; Pakistan.

INTRODUCTION in make it extraordinarily curative food for treating cancer Family Cruciferae is a large fami- (USDA, 2009). ly comprising 400 genera and 3000 Cruciferous plants are attacked species, mostly herbaceous by a number of pests which rarely woody (Vaughan et al., 1976). cause severe damage to cauliflower, The cultivated crucifers are impor- cabbage and many other cruciferous tant vegetables; hence widely grown plants. The most important pests in all parts of the world. Among these from Order Lepidoptera belong to fa- cabbage,B. oleracea L., is rich with milies such as Pieridae, Noctuidae, important nutrients and is good Putellidae and Pyralidae. Among source of fibers, vitamins and mine- these families, family Pieridae rals, low in caloric food containing belongs to yellow and white high percentage of calcium, iron, , is a cosmopolitan family iodine, potassium, sulphur, and pho- comprising 83 genera under four sphorus. The anti-cancer properties subfamilies (Ehrlich and Raven,

* National Insect Museum. Department of Plant and Environmental Protection, PARC Institute of Advance Studies in Agriculture, National Agricultural Research Centre, Islamabad, Pakistan. Corresponding author: [email protected]

273 SYED WAQAR SHAH AND MUHAMMAD ATHER RAFI 1965; Vane-Wright, 1978; Courtney, thern and north-eastern parts of the 1986; Ackery, 1991; Janz and Nylin; Pothwar division are situated with 1998). Out of four subfamilies its two Himalayan foothills with an average sub-families Coliadinae and Pierinae height of 450 to 900 m. Biogeogra- represent their taxon in Pakistan. phically the Pothwar region is Orien- Their caterpillars feed upon a number tal and species those belonging to the of plants belonging to families Com- Oriental occur primarily in the Indus positae, Leguminosae, Rhamnaceae, plain, the Himalayan foothills and Zygophilaceae and Cruciferae continuous with those of Indian (Robert, 2001). For example Pierids Punjab and Rajasthan (Qadri, 1968). speciesAnaphaeis aurota Cram., A. mesentinaCram., P. brassicae, P. MATERIALS AND METHOD kruperiStdjr ., P. rapae L ., and P. deplidice L. feed on cauliflower, To explore the cultivated and cabbage and other cruciferous plants non-cultivated host plants and their (Alam,1969) and cause severe dam- pierids pests from Pothwar region, 75 age.P. brassicae has been reported as surveys were conducted in different major pest of cabbage and cauliflower localities of five districts i.e., Jhelum, (Ghouri, 1960; Mohyuddin, 1981) Rawalpindi, Chakwal, Attock and and in severe infestation plants often Islamabad during 2012-2013. From completely skeletinized to stalk and each district 15 localities of variable veins (Jones and Jones, 1974) be- habitats were visited such as natural cause both cauliflower and cabbage vegetation, cropped and urban areas. are the most preferred host of P. Each locality was visited fortnightly brassicae (Mushtaque and Mohyu- for collecting plant samples, egg, din, 1984; Rizvi et al., 2009; Yadav larvae, pupae, and adults butterflies and Barwal, 2008). From Pakistan starting from January 2012 till the reported cultivated cruciferous host end of December 2013. plants ofPieridae species were Brassicasp., B. oleraceae, B.napus, Larval Collection and Rearing B. campestrisand R. sativus while Larval host plant flora was re- non-cultivated were Capsella bursa viewed, from published records (Bin- postoris, Caronopus didymus, Sisym- gham, 1905; Talbot, 1939; Robert, briumirioand Lepidium repens 2001; and Robinson et al., 2001). (Alam,1969). Random search was made to During this study both cultivated observe and record data for the imm- and non-cultivated cruciferous host ature stages i.e., number of eggs and plants and their pierids pests were larvae (with their respective stages) explored from Pothwar region of Paki- on their host plants. Some eggs or stan. Pothwar division of Pakistan is larvae were collected with their host politically a part of the Punjab pro- o plants for rearing in laboratory for vince of Pakistan, located at 32.5 N to further confirmation of after ooo 34.0 N latitude and about 72 E to 74 emergence. All collection were initia- 2 E longitude, covering 23161 km and lly placed in small plastic petri dishes comprising five districts namely Att- of 55 mm × 30 mm diameter the base ock, Chakwal, Jhelum, Rawalpindi of the or branch containing eggs and Islamabad (GoP, 1997). The nor- was secured with moistened cotton to

274 PIERID PESTS AND THEIR NEW CRUCIFERS HOSTS reduce desiccation. Petri dishes were RESULTS AND DISCUSSION placed in small cages (6”×6”×6”) for further rearing. All rearing cages were Four species of subfamily Pie- observed at least after 12 h daily to rinae belonging to two genera were observe egg, and larval developments. identified which were found in study The cages were regularly cleaned by areas. Among four reported pierids removing frass. After emergence of species three species belong to adults, the butterflies were identified. Pieris, P. brassicae, P. canidia and P. rapaewhile, one species P. daplidice Identification was recorded from genusPontia . The identification of the collected Three species P. brassicae, P. canidia specimens of pierids and their host andP.daplidice were reported on six plants were made to possible taxo- cultivated host plants such as B. nomic taxa by using literature of pre- napus, B.oleraceae, B. oleraceae var. vious workers like Bingham (1905); italica, B. campestris, R. sativus, and Talbot (1939); Kitamura (1964); E.sativa and, four on non-cultivated Stewart (1982); Haribal (1992); Gay et crucifers plants i.e., C. didymus, B. al. (1992); Gunathilagaraj et al. officinalis, L. pinnatifidiumand A. (1998); Naz et al. (2001); Feltwell thaliana. However, among non- (2001); Bhaskaran and Eswaran cultivated hostL. pinnatifidium was a (2005); Jafri (2011); Robinson et al. new host forP. daplidice . While cruci- (2012); Olive et al. (2012); Braby fers plantC. didymus was common (2012); Kaneria et al.(2013). host forP. daplidice, P. canidia and P. brassicae, plant A. thaliana was Relative Abundance and Perce- found host forP. canidia while B. ntage officinalisfor P. brassicae and P. The relative abundance and per- daplidice. All the non-cultivated host centage of each species along with plants were new records from their host plants were calculated by Pakistan, however; L. pinnatifidium using formula: was new host ofP. daplidice which was not earlier reported from any part ni of the globe as a host for pierid Relative Abundance = species. However, adults of P. rapae N were collected from the study areas where, but its larvae were not found on any th above reported host plant of the areas ni = Number of individuals in i species and ().Table 1 N = Total number of individuals in the sample. (Lannaeus, 1758) % = R x 100 Distribution Pothwar Through relative abundance and During current study P. daplidice percentage the most common and the was collected from Jhelum, Rawal- least common insect were deter- pindi, Chakwal, Attock and Islam- mined. abad districts.

275 SYED WAQAR SHAH AND MUHAMMAD ATHER RAFI Table 1. Number of different stages of pierids species on cultivated and non- cultivated Crucifer plants in 2012 and 2013

Butterflies Name Pontia Pieris Pieris Pieris daplidice brassicae canidia rapae

Host Plants ELPAELPAELPAELPA

2012 Coronopus didymus* 5 3 22060 12 4 5 22 --- Lepidium 7 2 1 65 15 3 6 11 --- pinnatifidium* 40 25 2 Arabidopsis thaliana* --- - 221 --- Brassica officinalis* --- 55 16 7 ------B.napus 12 4 2 31 85 20 6 55 9 5 2 43 ---4 Raphanus sativus 15 5 1 57 17 81143 --- Eruca sativa 6 1 2 59 12 7 ------Brassica oleraceae --- 49 15 5 ------B. oleracea var. --- 55 21 8 ------italica B.campestris -- 72 18 4 ------2013 Coronopus didymus* 10 5 4 50 75 6 2241 --- Lepidiumpinnatifidium* 8 6 3 70 8320 33135 --- 3 Arabidopsis thaliana* --- - - 2 6 0 --- Brassica officinalis* --- 60 5 2 ------5 B.napus 15 9 6 87 80 6 4 42 55242 --- Raphanussativus 18 8 4 75 7 2 6 7 1 --- Eruca sativa 13 9 5 82 5 2 ------Brassica eoleraceae --- 62 7 3 ------B. oleraceavar. italica --- 90 7 3 ------B. campestris --- 86 6 2 ------

E= Egg, L= , P= Pupa, A=Adult, *= non cultivated

Roberts, 2001), and Jhelum River (Steven, 2001; Khan et al., 2007).

Previously Reported Cruciferous Host Plants

Female Male Crucifer cultivated plants such Pontia daplidice Pontia daplidice as mustard andBrassica spp., and non-cultivated plants are Sysimbrium Remarks sp.S. altissimum , S. irio (Alam, 1969; New for Pothwar districts Rawal- Hassan, 1994; Roberts, 2001). pindi and Chakwal. However, earlier it is reported from Attock (Hasan Newly Reported Cruciferous Host Abdal) and Islamabad (Malik, 1970; Plants Arshad et al., 1983; Hassan, 1997; During the current study re-

276 PIERID PESTS AND THEIR NEW CRUCIFERS HOSTS ported cultivated host plants are B. Remarks napus, E.sativa, R. sativus and non- Earlier this species was reported cultivated host plants are L. pinnatifi- throughout the Pothwar region dum, C. didymus. Although Sysm- (Malik, 1970; Ahsan and Iqbal, 1975; brium sp. were found in the region Roberts, 2001; Tshikolovets, 2001). extensively but no egg, larva, pupa were found on these plants in two Previously Reported Cruciferous years study. Host Plants Cultivatedcrucifer like R. sativus , Pest Status Brassicasp., Brassica juncea, B. It was observed as most common napus, B. nigra, B. oleracea, B. rapa, pest of crucifer in the study area, B. oleraceaevar. botrytis, B. olearacea feeds on , and fruits. var.capitata, B. oleraceae var. Slowly progressed and destroy whole gongylods, while non-cultivated plant. Initially feeds upon leaves and crucifersare Arabis allionii , Armoracia in later stages shifted to inflorescen- rusticana, stricta, Berteroa ces. Rarely found in fruits. Highest incana, Bunias orientalis, percentage recorded on cultivated maritime, Capsella bursa-pastoris, hosts i.e.,73.4% and 71.8% as com- pared to non-cultivated host plants Crambe maritime, Draba incana, i.e.,26.7% and 28.1% (Table 2 ). , E. hieraciifolium, E. scoparium, Hesperis matronalis, (Linnaeus, 1857) Arabisspp., Lepidium repens, L. Large White draba, Lunaria, Rorripa, Sysimbrium austriacum, S. irio. (Alam, 1969; Distribution Mohyuddin, 1981; Hassan, 1994; Pothwar Roberts, 2001; Robinson et al., 2001). Common in all districts of study Newly Reported Cruciferous Host area. Plants During the current study cultiva- ted reported host plants are Brassica napus, B. oleraceae , B. oleracea var. italica, R. sativus , B. campestris and E. sativa and non-cultivated host plants areB. officinalis , C. didymus , Female Male Pieris brassica Pieris brassicae and L. pinatifidium.

Table 2. Percentage abundance of pierids on crucifer plants during 2012 and 2013

Host Plants Pontia daplidice Pieris brassicae

Years 2012 2013 2012 2013 2012 2013 2012 2013 Non-cultivated 26.7 28.1 32.3 30.1 39.4 38.9 -- Cultivated 73.4 71.8 67.7 69.8 60.6 61.1 --

277 SYED WAQAR SHAH AND MUHAMMAD ATHER RAFI Pest Status ever non-cultivated are L.virginicum, P. brassicae is a major pest; feed Rorippa indica, Cardam ineflexuosa, on leaves, particularly cabbage and C. escutata (Robinson et al., 2001). broccoli. Mostly whole plant is skeletized. Eggs are laid in bunches Newly Reported Cruciferous Host so attack is severe and rapid. In Plants present study unusual behaviour of During the current study repor- host trend of this butterfly has been ted cultivated host plants are observed i.e., laying a single egg on Brassica napusand R. sativus and non-cultivated host plant C. didymus non-cultivated host plants are A. the egg successfully reared and male thaliana, Coronopus didymusand L. butterfly was emerged. Similar high pinatifidium. percentage was observed in culti- vated plants i.e.,67.7% and 69.8% as Pest Status compare to non-cultivated host It was observed comparatively as plants which is 32.3% and 30.1% minor pest of crucifer cultivated (Table 2 ). plants i.e., 60.9% and 61.1%, non cultivated 39.4% and 38.9% (Table Pieris canidia (Sparrman, 1768) 2) mainly attack on leaves and . Mostly remained un- Distribution noticeable so a type of hidden pest. Pothwar Least observed and has highly During current study P. canidia selective host range. was collected from all districts of study area. Pieris rapae (Linnaeus, 1758) - Small White

Distribution Pothwar During current study P. rapae was collected from district Rawal-

Female Male pindi and Islamabad. Pieris canidia Pieris canidia

Remarks New to Jhelum, Rawalpindi and Chakwal districts of Pothwar. Earlier it has been reported from districts Islamabad and Attock (Campbellpur) (Hassan, 1994; 1997; Iqbal, 1978; Female Male Roberts, 2001). Pieris rapae Pieris rapae

Previously Reported Cruciferous Remarks Host Plants P. rapae is new for Pothwar dis- Cultivated areB. oleracea , how- tricts like Rawalpindi and Islamabad.

278 PIERID PESTS AND THEIR NEW CRUCIFERS HOSTS Earlier Reported Cruciferous Host was observed during the study. Plants However, Jogar et al., (2005) Cultivated host plants are Bra- described this species as a major pest ssica caulorapa, B. alboglabra, B. of crucifer crops. A single female lays juncea, B. napus, B. oleracea, B. 400 eggs; on average 300 which may oleraceaevar. botrytis, B. olearacea approaches up to 1,000 young larvae var.capitata, B. oleraceae var. feed upon outer leaf of cabbage. When gongylods, Raphanus raphanistrum, larvae become old they move into R. sativus, and non-cultivated are inner leaves of cabbage and finally , Arabis alpine, into cabbage head and eating outer Armoracia rusticana, Aurinias leaves of the cabbage head. axatilis, Barbarea orthoceras, B. The study is significant because verna, B. vulgaris, Biscutella it provides detail regarding new auriculata, B. parachi-nensis, B. rapa, crucifer cultivated and non- B. chinensis, B. nigra, Cakile cultivated host plants of pierid pests edentula, Capsella bursa pastoris, from Pothwar region of Pakistan. The Cardamine diphylla, C. pratensis, non cultivated host plants provide Caulanthu scooperi, Descura opportunities to the former to manage iniasophia, Diplotaxis virgate, Eruc pest status of these butterflies by avesicaria, , shifting their host trends from Hesperis matronalis, Hirschfeldi cultivated to non-cultivated host. aincana, Lepidium densiflorum, L.campestre, L. virginicum, Lobularia LITERATURE CITED maritime, Lunaria annua, Matthiola incana, Rorippa aquatic, R. Ackery, P.R. 1991. Host plant curvisiliqua, R. indica, R. microphylla, utilization by African and R. nasturtium aquaticum, R. Australian butterflies. Biol. J. sylvestris, Sinapis alba, S. arvensis, Linn. Soc. 44: 335-351. Sisymbriu maltissimum, S.irio, Ahsan, M. and J. Iqbal. 1975. A S.officinale, tortuosus contribution to the butterflies of andThlaspi arvense (Mohyuddin, Lahore with addition of new 1981; Robinson et al., 2012). records. Biologica. 21(2): 143- 158. Expected Future Threat of Alam, M.M. 1969. Survey of parasites Crucifer Plants of insect pests of cultivated and During the current study althou- useful plants and survey of gh no egg, larvae and pupae were destroying weeds and found over cultivated and nonculti- their parasites. Final report, vated host plants however, adults UBC, Rawalpindi. were reported hovering around the Arshad, M., A.G. Jan and S. Parveen. fields of host plants like cauliflower, 1983. A checklist of butterflies of B. napusand R. sativus (Table 2 ). N.W.F.P. Reprint from J. Sci. and Tech. 7(1-2):1-3. Pest Status Bhaskaran, S. and R. Eswaran. 2005. Reported as major pest of crucifer Status and Distribution of specially cauliflower. No pest status Butterfly species in Sivakasi

279 SYED WAQAR SHAH AND MUHAMMAD ATHER RAFI Taluk, Tamil Nadu. J. Insect Sci. Islamabad and Muree hills. 18(1): 134-136. Asian Study Group, Islamabad. Bingham, C.T. 1905. The fauna of 68p. British India including Ceylon Hassan, S.A. 1997. Biogeography and and Burma: Butterflies vol.1, Diversity of Butterflies of North Taylor and Francis Ltd. London. West Himalaya In: Mufti S.A., UK. 15(1-2): 1-528. Wood, C.A. and Hassan, S.A. Braby, M.F. 2012. New larval food (eds.). Biodiversity of Pakistan. plants and biological notes for Pakistan Museum of Natural some butterflies (Lepidoptera: History, Islamabad. Florida Papilionoidea) from eastern Museum of National History, Australia. Australian Entom- Gainesville, USA. p. 181-204. ology. 39(2): 65-68. Iqbal, J. 1978. A Preliminary report Courtney, S.P. 1986. The ecology of on butterflies of district pierid butterflies: Dynamics and Rawalpindi and Islamabad. interactions. Adv. Ecol. Res. 15: Biologia. 24(2): 238-247. 51-131. Jafri, S.M.H, 2011. Family Ehrlich, P.R. and P.H. Raven. 1965. Burmett outline of Butterflies and plants: A study in botany p. 854, 1093, 1123. 1835. coevolution. Evolution. 18: 586- (Oultine Bot.). 608. Janz, N. and S. Nylin. 1998. Feltwell, J. 2001. The illustrated Butterflies and plants: A encyclopedia of butterflies. phylogenetic study. Evolution. Chartwell Books, New Jersey, 52: 486-502. USA. 288 p. Jogar, K., L. Metspalu and K. Gay, T., I.D. Kehimkar and J.C. Hiiesaar. 2005. Influence of food Punetha. 1992. Common plants on the development of butterflies of India. Oxford large white butterfly (Pieris University Press, Bombay. 67 p. brassicae) larvae. Transactions Ghouri, A.S.K. 1960. Insect pests of of the Estonian Agriculture, Pakistan. Reg. Tech. Doc. 8: 31. University, Agronomy. 220: 201- GoP. 1997. Economic Survey 1996- 203. 97. Government of Pakistan. 398 Jones, F.G.W. and M.G. Jones. 1974. p. Pests of field crops. (2nd edn.) St. Gunathilagaraj, K., T.N.A. Perumal, Martin Press, New York. p. 100- K. Jayaram and M.G. Kumar. 101. 1998. Field guide. Some South Kaneria, M. M. Kaneria and V. Indian butterflies. Nilgiri Wildlife Kushwah. 2013. Diversity of and Environment Association, butterflies (Lepidoptera) in Udhagamandalam, India. 274p. Bilaspur District, Chhattisgarh. Haribal, M. 1992. The butterflies of India. Asian. J. Exp. Biol. Sci. Sikkim Himalayas and their 4(2): 282-287. . Sikkim Nature Khan, R.M., A.M. Rafi, M. Munir, S. Conservation Foundation Hussan, W.M. Baig and W.M. (SNCF), Gangtok. 217 p. Khan. 2007. Biodiversity of Hassan, S.A. 1994. Butterflies of Butterflies from districts Kotli,

280 PIERID PESTS AND THEIR NEW CRUCIFERS HOSTS Mirpur and Bhimber, Azad Pakistan, Oxford University Kashmir. Pakistan J. Zool. 39(1): Press, Mass Printer, Karachi, 27-34. 200 p. Kitamura, S. 1964. Plants of West Robinson, G.S., P.R. Kitching, I.J. Pakistan and Afghanistan. Beccaloni, G.W.Hernandez. Tengyosha Printing Co. Ltd., 2001. Host plants of the Japan. 3: 283. and butterflies caterpillar of the Malik, M.J. 1970. Notes on the Oriental Reigon, Southdene sdn butterflies of Pakistan in the Bhd, Kuala Lampoor. 744 p. collection of Zoological Survey Steven, C. 2001. Bristish butterflies. Department. Report of Zoological http://www.britishbutterflies.co Survey Department, Pakistan. .uk. 2(2): 24-54. Stewart, R.R. 1982. History and Mohyuddin, A.I. 1981. A review of exploration of plants in Pakistan biological control in Pakistan. and adjoining areas. In: Nasir, E. Proc. 2nd Pakistan Congr. Zool. p. and Ali, S.I. (eds.). Flora of 31-79. Pakistan. Published by authors. Mushtaque, M. and A.I. Mohyuddin. 186 p. 1984.Pieris brassicae (Pieridae: Talbot, G. 1939. The fauna of British Lepidoptera): A pest of crucifers India, including Ceylon and and its control by parasites. Burma. Butterflies, Vol. I. Pakistan J. Agric. Res. 5(3): 165- Papilionidae, Pieridae. Taylor 169. and Francis Ltd., London. 600 p. Naz, F., M.A. Rafi and M. Inyatullah Tshikolovets, V. V. 2001. Insecta. - In: and V. Tuzove 2001. The A catalogue of the type- butterflies of the Buner district, specimens in the zoological North-West Friontier Province, museum, Nat. Mus. Hist., Pakistan. In: Helios. Collection of Ukrainian Aca. Sci. 1: 57-86. Lepidopterological Articles, USDA, 2009. National Nutrient Moscow sovetsky sport, Russia. Database for Standard 2:123-224. Reference, Release 22. Olive, B., C. Udipta, D. Aditya, C. Vane-Wright, R.I. 1978. Ecological Sujan, D. Swati, and M. Bidisha and behavioural origins of 2012. Lepidoptera and Araneae diversity in butterflies. In: diversity of Salt lake City, Mound, L. A. and Waloff, N. Kolkata. Bionotes, 14(3):95-96. (eds.). Diversity of Insect Fauna. Qadri, M.A.H. 1968. Zoogeography of Symposia of the Royal Pakistan. Central Urdu Board, Entomological Society of London: Lahore. No. 9 Blackwell Scientific Rizvi, P.Q., A. Ali and S. Khalid. 2009. Publications, Oxford.p. 56-70. Age and stage specific life table of Vaughan, J.G., Phelan, J.R. and cabbage butterfly Pieris Denford, K.E. 1976. studies brassicae L. (Lepidoptera: in the Cruciferae. In: Vaughan, Pieridae) on various cole crops. J. J.G., Macleod, A.J. and Jones, Plant Protec. Res. 49: 145-150. B.M.G. (eds.). The biology and Robert, T. J. 2001. The butterflies of chemistry of the Cruciferae.

281 SYED WAQAR SHAH AND MUHAMMAD ATHER RAFI

Academic Press, London. 119 – F1 hybrids of cauliflower, 144p. Brassica oleraceavar. botrytis L. Yadav, J.L. and R.N. Barwal. 2008. against cabbage butterfly. Veg. Evaluation of some and Sci. 35: 69-71.

AUTHORSHIP AND CONTRIBUTION DECLARATION

S.No Author Name Contribution to the paper

1. Mr. Syed Waqar Shah Conceived the idea, Wrote abstract, Methodology, Conclusion, Technical input at every step, Data collection, Data entry in SPSS and analysis, Results and Discussion, Introduction, References. 2. Dr. Muhammad Ather Rafi Conceived the idea, Wrote abstract, Methodology, Technical input at every step, Overall management of the article, Results and Discussion, Introduction, References.

(Received September 2016 and Accepted November 2016)

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