front page 08.BMP - Pakistan Journal of Entomology Karachi
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front page 08.BMP - Pakistan Journal of Entomology Karachi
Pakistan j. entomol. Karachi. 23 (1&2): 2008 CONTENTS DETERMINATION OF MAMMALIANS TOXICITY OF CEDRUS DEODARA ROOT OIL, AGAINST ALBINO RATS (WISTAR STRAIN). PERVEEN, R., NAQVI, S.N.H., M. AHMED AZMI, AHMED, M. & SAIMA MEHMOOD………………………………………………………………………………………........ 01-04 TAXONOMIC STUDIES OF SERGENTOMYIA FREETOWNENSIS SINTON, 1930, SINDHICUS N. SUB. SP. (DIPTERA: PSYCHODIDAE) IN SINDH, PAKISTAN. JUMA KHAN KAKARSULEMANKHEL…………………………………………………………….. 05-10 COMPARATIVE TOXICITY OF ORGANIC SOLVENTS AND INSECTICIDES AGAINST CABBAGE BUTTERFLY. SEEMA TAHIR, TAHIR ANWAR, MUHAMMAD SAMIULLAH CHANNA, IMTIAZ AHMAD & YOUSEF HAYAT KHAN…………………………………………………………………………….. REDESCRIPTION OF CAYSTRUS PYGMEAUS LINNAVUORI PENTATOMIDAE) AN ISOLATED SPECIES FROM ETHIOPIAN REGION. 11-14 (HEMIPTERA: MUHAMMAD ZAHID & IMTIAZ AHMAD…………………………………………………………... STUDY OF RESISTANCE IN SITOPHILUS ORYZAE AGAINST CYPERMETHRIN AND PHOSPHINE ON THE BASIS OF TOXICITY VALUES. 15-17 BIOSAL, S.M. NAUSHAD ZAFAR, NAQVI, S.N.H., TARIQ, R.M. & REHANA PERVEEN …………….. 19-26 EFFICACY OF IMIDACLOPRID AND ENDOSULFAN IN COMPARISON WITH BIOSAL (BIOPESTICIDE) AGAINST MYZUS PERSICAE (SULZER) ON MUSTARD CROP. MUHAMMAD FAHEEM AKBAR, NIKHAT YASMIN, FARAH NAZ & NAZIA QURESHI……………………………………………………………………………………………… 27-30 REVISION OF THE GENUS ISOMETRUS HEMPRICH & EHRENBERG (SCORPIONIDA: BUTHIDAE: CENTRURINAE) WITH DESCRIPTION OF TWO NEW SPECIES AND CLADISTIC RELATIONSHIP FROM PAKISTAN. RAFAT AMIR & SYED KAMALUDDIN …………….. …………………………………………….. 31-40 TAXONOMIC STUDIES OF SERGENTOMYIA BAGHDADIS (ADLER & THEODOR) IN SINDH AND PUNJAB AND ITS PHYLOGENETIC RELATIONSHIPS. JUMA KHAN KAKARSULEMANKHEL ……………………………………………………………. 41-46 POTENTIAL OF SWEET FLAG RHIZOME OIL AND CUSTARD APPLE SEED OIL AGAINST THE MAJOR SUCKING PESTS OF COTTON, AS COMPARED WITH CONFIDOR + DELTAPHOS, AT ARI-TANDOJAM, SINDH-PAKISTAN. RAJPUT MUHAMMED TARIQ, S. NAIMUL HASAN NAQVI, S.M. NAUSHAD ZAFAR & ABDUL SATTAR BURRERO……………………………………………………………………….. 47-50 STUDIES ON SAMPLING TECHNIQUES TO MONITOR ADULT POPULATION OF WHITEFLY, BEMISIA TABACI (GENN.) IN CUCURBIT FIELD. ABDUL GHANI LANJAR, MUHAMMED KHAN LOHAR, HAKIM ALI SAHITO, ASHFAQUE AHMED NAHIYOON AND NAHEED BALOCH…………………………………………………… 51-54 POPULATION DYNAMICS OF GROUND WATER BREEDING OF CULEX MOSQUITOES OF KARACHI AND THATTA DISTRICT. TANVEER FATIMA SIDDIQUI……………............................................................................... LEVELS OF DENGUE FEVER VIRUS CONTROL: THE FFECTIVENESS AND VASTNESS OF CONTROLLING POWER BOUNDARIES OF THESE LEVELS RAJPUT MUHAMMED TARIQ & S. SALAHUDDIN QADRI…………………………………….. 55-60 61-62 ENTOMOLOGICAL SOCIETY OF KARACHI, PAKISTAN (1971) Office Bearers and Council for the year 2008-2009 President S.N.H. Naqvi: D.Sc.: HEC Eminent Professor, Department of Zoology, University of Karachi, Gulshan-e-Iqbal Town, Karachi-Sindh. Vice-Presidents Abdul Sattar Burrero: Ph.D. (Sindh), Director General, Agricultural Research Institute, Tandojam, Sindh. General Secretary M. Arshad Azmi: Professor, Department of Zoology, University of Karachi, Karachi. Joint Secretary Tahir Anwer: Incharge, Pesticide Research Institute, SARC (PARC), University Campus, Karachi. Treasurer Masarrat J. Yousuf: Professor, Department of Zoology, University of Karachi, Karachi. Councilors S. Amin ullah Khan: Ph.D., M. Ahmed Azmi: Ph.D., Rukhsana Perveen: Ph.D., Rahila Tabassum: Ph.D., M.S. Wagan: Ph.D., Nasreen Memon: Ph.D., Muhammed Zahid: Ph.D., S. Salahuddin Qadri: Ph.D. Entomological Society of Karachi was established in 1971, with the object of promoting Entomological Science and a closer cooperation between entomologists of Pakistan. Pakistan J. Entomol. Karachi (Biannual) is available in exchange or by subscribing the cost, from the Society’s office at the Department of ZoologyEntomology, University of Karachi, Karachi-75270, Pakistan. The Journal is being abstracted by Biological Abstracts, Entomological Abstracts, Chemical Abstracts and Current Contents and recognized by HEC in “Y” category. Due to high cost of publication the page charges are Rs.2500.00 for 1-4 pages and Rs.500.00 per page for extra pages or US $ 10.00 per page from members and Rs.700.00 per page or US $ 15.00 per page from non-members. Subscription Price Pakistan Rs.400.00 per copy/number Other Countries US $ 40.00 per copy/number Composed and Designed at Muhammed Afzal Husain Qadri Biological Research Centre, University of Karachi, Gulshan-e-Iqbal Town, Karachi-75270, Sindh-Pakistan Pakistan J. Entomol. Karachi, 23 (1&2): 2008 Foreign & National Editorial Board Carl Schaefer, Ph.D. Michael Breuer, Ph.D. University of Connecticut, Storrs, Conn. (USA) Zoological Institute, Catholic University of Leuven (Belgium). A.R. Shakoori, Ph.D. Kehkashan Akhter, Ph.D. University of the Punjab, New Campus, Lahore (Pakistan) Department of Zoology, University of Karachi, Karachi-75270, Sindh-Pakistan. M.F. Khan, Ph.D. M.A. Matin, Ph.D. Department of Zoology, University of Karachi. National Agricultural Research Centre (NARC), Park Road, PO NIH, Islamabad (Pakistan). S. Anser Rizvi, Ph.D. R.C. Saxena, Ph.D. Department of Zoology, University of Karachi. Chairman, Neem Foundation, Mumbai, India. Feyzi Onder, Ph.D. M. Ather Rafi, Ph.D. Department of Plant Protection, Agric. Faculty, Ege Univ. Bornova, Azmir (Turkey). National Agricultural Research Centre (NARC), Park Road, PO NIH, Islamabad (Pakistan). Seema Tahir, Ph.D. Dr. Jumakhan Kakarsulemankhel, Ph.D. Department of Zoology, University of Karachi. Department of Zoology, University of Balochitan, Saryab Road, Quetta, Pakistan. Foreign Advisory Board U.S.A. Asia Alfred Wheeler Jr. Ph.D. Cornell University, Itacha, N.Y. Chiu, Shin-Foon, Ph.D. South China Agriculture Guangzhou (Peoples Republic of China) T.J. Henry, Ph.D. US National History Museum Washington, D.C. (USA) V.K. Ganesalingam, Ph.D. University of Jaffna (Sri Lanka) J. Koolman, Ph.D. Philips Universitat Marburg (Germany) B.N. Islam, Ph.D. BAU, Mymensingh, University (Bangladesh) R.W. Mwangi, Ph.D. University of Nairobi P.O. Box 72913, Nairobi (Kenya) K. Sombatsiri, Ph.D. Karetsart University Bangkok (Thailand) J.I. Olaifa, Ph.D. University of Technology Ogbomoso (Nigeria) R.P. Singh, Ph.D. Entomology Div., IARI New Delhi 10013 (India) Errol Hasan, Ph.D. University of Queensland, Gattons College, Lawes, QLD. Absar Mustafa Khan, Ph.D. Department of Zoology M.U. Aligarh (India) Europe Africa Australia INSTRUCTIONS TO CONTRIBUTORS (For Research and Review Articles) 1. Manuscripts should be in English, typed and double spaced, on one side of the paper. There should be an abstract, not exceeding 200 words, which will be printed in small type before the introduction. Nothing in the text, except the scientific name will be italicized. Tables should be typed on separate sheets. Footnotes should be avoided as much as possible. 2. Illustrations should be in black Indian ink preferably on white or transparent paper. All letterings and numericals should be in light pencil for insertion in uniform style. The author’s name and the number of the figures should be written on the back of each drawing. The size of the illustration after reduction will not exceed 4x6 inch. The legends should be typed on a separate piece of paper. 3. The author should submit a hard copy in original and a photocopy of the original and soft copy in re-writable C.D. 4. Photographs should be glossy black and white prints. They should be numbered separately from the Text-figures. The cost of the coloured plates will be met by the author. 5. Reference should be cited in the text by giving the author’s name followed by the year. Papers and books should be listed in the alphabetical order. Journals should be abbreviated according to the latest edition of the World List of Scientific Periodicals e.g. DREYER, M. (1984). Effects of aqueous neem extracts and nee mol on the main pests of Cucurbita pepo in Togo. Proc. 2nd Int. Neem Conf. (Rauischholzhausen, 1983), pp. 435-443. EDWARDS, C.A. AND HEATH, G.W. (1964). The Principles of Agricultural Entomology. Chapman and Hall, London, 418 pp. NAQVI, S.N.H., ASHRAFI, S.H. AND QADRI, M.A.H. (1968). Acid phosphatase activity in the digestive system of the desert locust, Schistocerca gregaria (Forskål). Aust. J. Biol. Sci. 21: 1047-52. RAUPP, M.J. AND DENNO, R.F. (1983). Leaf age as a predictor of herbivore distribution and abundance. In: Denno, R.F. and McClure, M.S. (eds.): Variable Plants and Herbivores in Natural and Managed Systems. Academic Press, New York, USA, pp. 91-124. 6. Page-proofs will be sent to the authors for correction which should be returned within 7 days. Authors may be required to pay for alternations in proofs other than those needed to correct printer’s errors. The responsibility of the results will be on authors(s). 7. Key words/Index of maximum 10 words and short title of 8 words be mentioned at the proper place. 8. Authors may order for purchase of extra reprints when proofs are returned to the Editor. After acceptance of paper the amount of publication charges should be paid before publication of the paper. 9. All correspondence should be addressed to the Executive Editor, C/o Office of the Entomological Society of Karachi, Department of Zoology, University of Karachi, Karachi-75270, Pakistan, or Assistant Editor, MAH Qadri Biological Research Centre, University of Karachi. 10. Paper(s) for publication may be emailed at [email protected] or naeemnaqvi.life.com. Pakistan j. entomol. Karachi 23(1&2): 1-4, 2008 DETERMINATION OF MAMMALIAN TOXICITY OF CEDRUS DEODARA ROOT OIL, AGAINST ALBINO RATS (WISTAR STRAIN) PERVEEN1, R., NAQVI1, S.N.H., M. AHMED AZMI1, TARIQ2, R.M., AHMED3, M AND MEHMOOD1, S. 1 Baqai Institute of Pharmaceutical Sciences, Baqai Medical University, Super Highway, Karachi. 2 M.A.H. Qadri Biological Research Centre, University of Karachi, Karachi-Sindh, Pakistan. 3 Aga Khan Medical University, Stadium Road, Karachi. ABSTRACT Mostly Cedrus deodara root oil has been analyzed for sesquiterpenes and hydrocarbons, However, in the present case GC-Mass analysis and spectral studies were done on root oil. This oil is being used orally as anti-ulcer agent by Hakeems. However, scientifically it is necessary that the toxicity level be determined against mammals. During present investigations mammalian toxicity was determined, by oral administration, against albino rats (Wister strain). The LD50 by probit mortality graph was found to be 34.4 gm/kg. This is quite safe as compared to Neem oil LD50 (5gm/kg). Key words: Mammalian toxicity, Cedrus deodara root oil, albino rats. INTRODUCTION Essential oils are volatile and hydrophobic. They are used in various industries, household cosmetics and medicine etc. The production of Cedrus deodara oil is about 2600 tons per year. The oral LD50 of various essential oils various from 0.5-5gm/kg (Tisser and Robert 1995). According to MSDS (Material Safety Data Sheet) reported by Environmental Health and Safety Department, USA. Cedar wood oil may cause irritation to skin, eyes and respiratory tract (Case No. 8000-27-9). The oral LD50 for rat is 5gm/kg, for rabbit skin 5gm/kg and for draize test 500mg (MSDS) (Anonymous 2003) GC mass analysis of oil showed that C. deodara root oil contains deodarone and atlantone (Sankarananyan et al., 1973). Agarwal and Rastogi (1981) reported presence of himachalol, himasecollone and cendarol in trunk oil. Avcibasi et al. (1987) reported four terpenoids i.e. α torosol, βtorosol, andirolactone and transatlantone. Khan and Naheed (1990) reported a new sesquiterpene, himachanene from bark of Cedrus deodara. Molluscicidal activity of cedar wood oil, neem oil was reported by Rao and Singh (2001). Dimiri and Sharma (2004) reported acaricadal effect of Cedrus deodara oil, Pomgamnia glabra oil, Jatropa curcus oil and Benzyle benzoate, on sheep mange mite. This indicates that these oils though not toxic to mammals but are toxic to insect and acarines and control the ectoparasites. As cedar wood root oil was being used for healing peptic ulcer in North Western Province of Pakistan, therefore, it was necessary to find the mammalian toxicity of this oil, which will help in adjusting the therapeutic dose. For this reason LD50 of the root oil was determined. MATERIALS AND METHOD For toxicity determination albino rats (Wistar strain) were taken. Rats were starved for 12 hours. The weight of the rats was 250gm ± 5gm. Ten rats were not treated (control) while 5 batches of 10 rats each (5 male+5 female) were treated with oil. Then 2.2ml, 4.4ml, 6.6ml, 8.8ml and 11.1ml, oral dose was given orally by special gauze syringe. After 24 hours mortality readings were taken. The volume was converted to grams by multiplying volume with specific gravity of oil (0.91). Five replications were done and readings were subjected to Abbot's formula (1925). The rats were allowed to feed on normal diet after experiments. Experiments were conducted under controlled condition at a temperature of 25°C + 2 and.relative humidity as 50 + 2. The probit morality graph was drawn and LD50 was noted. Perveen et al. 2 RESULTS Whenever, a new compound or drug is to be used for clinical purpose, it is necessary that toxic dose for mammals be determined. During present experiments, the toxic dose of Cedrus deodara root oil was determined, as it is being used as anti ulcer drug in NWFP by Hakeems. Toxicity experiments were done under controlled conditions as mentioned above, on standard Wistar Strain, being breed in animal house of Baqai Medical University. The rats were treated with 2.2, 4.4, 6.6, 8.8 and 11.1 ml dose/rat. The average mortality after 24 hours was found to be 10, 22, 40, 49 and 68% respectively. In the control batch (untreated) no mortality was recorded i.e. zero. According to the graph the LD50 was found to be 34.4gm/kg + 1.2 gm (Fig. 1) standard deviation, and range at 95% confidence limit was also determined (Table 1). DISCUSSION When a plant product is found effective against any disease/disorder. The product acquires the status of medical importance and termed as medicinal plant. There are some barriers for using and commercialization of these type of plant products as reported by Isman (1997). This is because of its acute toxicity, piscean toxicity, as reported by Tariq et al. (2004), avian toxicity as reported by Tariq & Naqvi (2006) and mammalian toxicity as reported by Naqvi & Tariq (2007). After the satisfaction or low toxicity of the candidate plant product, it is tested against the small mammals e.g. on rats, mice and rabbits before going on direct application to human beings as Shirwaikar et al. (2004) reported anti-diabetic activity of aqueous leaf extract of Annona squamosa in streptozotocin-nicotinamide type 2 diabetic rats. Therefore in the present work, root oil of C. deodara was tested against rats, as the oil acts as anti-ulcer in human beings. The LD50 (34.4 gm/kg) of the cedar wood root oil is very safe. According to the classification of Casarette and Doull (1992) any dose above 15gm/kg is very safe. Most of the essential oils, cotton seed oil, sunflower oil, clove oil, corn oil etc. are very safe or approximately non-toxic. The LD50 of there oils varies from 4-5 gm/kg or more (Tisser and Robert 1995). The oral LD50 of cedar wood oil (red juniper) has been reported as 5 gm /kg. However, in the present case the root oil of Cedrus deodara is much safer than juniper oil. (MSDS) (Material and Safety Data Sheet) issued by Department of Environmental Health and Safety, USA. The oral LD50 of neem oil has been reported as 4.2 gm/kg which is close to the LD50 of red cedar wood oil, but this is also in safe category. Acute toxicity of neem oil has reen reported as 3540mg/kg to 5000 mg/kg (Farm Chem. Hand book 1995), and US EPA (1993). During present analysis of root oil three compounds were isolated viz, himachalol, allchimachalol and trans-atlantone which are already reported from trunk oil (Perveen 2006). Toxic/ therapeutic affect of crude oil and isolated compounds were also observed which have been reported elsewhere. It has been found that the Cedrus deodara root oil is quite safe and may be used for therapeutic use and pest control as reported by Dimiri and Sharma (2004). However, some mild pathological effects were observed during present investigation. Naqvi & Tariq (2007) reported the mammalian toxicity of two medicinal local plants, Acorus calamus and Annona squamosa, against white rats. They reported 8.7 ml = 38.37 gm/kg LD50 of calamus oil. Whereas the LD50 of squamosa was reported as 7.7 ml = 33.95 gm/kg against the same wistar strain of albino rats. The LD50 value of squamosa is very close to the LD50 value of deodara in present findings. Whereas the LD50 of calamus is more lower as compared to deodara, being more safer than deodara, but the LD50 of three local plant falls within the safe limits that is above 15 gm/kg. Therefore the root oil of C. deodara may be used safely as antiulcer. CONCLUSION The LD50 dose of C. deodara against small mammal, Wistar strain (Albino rats) has been found 34.4 gm/kg, which is above 15 gm/kg dose. Therefore it will also be safe for human beings as well and may be used safely as anti-ulcer as concluded experimentally in the present work. Beside this, the oil of Acorus calamus which is also used as medicine for stomach complaints, snake bite, for remittent fevers Nasir (1978), and insect repellent Tariq & Qadri (2001). The Acorus is locally found in Pakistan and may be experimented for above disorders in human beings. Further more the leaves of Annona squamosa are reported having anti-diabetic activity, by Shirwaikar. This plant is also frequently available in Pakistan especially in Karachi. It may also be taken into practical use after research as the diabetic problem is a major problem in Pakistan. The main active ingredient of Acorus oil is the β-asarone. Streloke et al. (1989) and neoanonin (squamosin) is the main active ingredient in squamosa seed oil. Kawazu et al. (1989). 3 Determination of Mammalian Toxicity of Cedrus deodara root oil Table 1 DETERMINATION OF TOXICITY OF CEDRUS DEODARA OIL AGAINST ALBINO RATS Obs. No. Dose per rat (ml) Mean % mortality S.D. + S.E. + Range at 95% confidence limit 0 Control Zero Zero Zero - 1 2.2 10 4.4 2.01 6.2-14.35 2 4.4 22 4.47 2.20 18.08-25.90 3 6.6 40 6.7 3.02 33.80-45.19 4 8.8 49 7.01 3.16 43.70-55.20 5 11.1 68 7.20 3.12 61.10-74.90 N = 5 range = mean + S.E. x significant value at 95% confidence limit. Perveen et al. 4 REFERENCES ABBOT W.S. (1925). A method of computing effectiveness of an insecticide. J. Econ. Entomol. 18:265-67. AGARWAL, P.K. AND RASTOGI R.P (1981). Terpenoids from Cedrus deodara. Phytochemistry 20(6); 1319-21 ANONYMOUS (2003). Material Safety Data Sheet issued by Environmental Health and Safety Department, USA. AVCIBASI, H., ANIT, H. AND TOPRAK, M. (1987). Four ter-penoids from Cedrus libanotica. Phytochemistry 26(10): 2852-54. CASARETT, L.J. AND DOULL, M.D. (1992). Toxicological evaluation: The Basic Science of Poisons. Published by Macmillan Publishing Co. Inc. New York. Pp. 11-25. DIMIRI, V. AND SHARMA, M.C. (2004). Effects of Sarcoptic mange mite and its control with oil of Cedrus deodara, Pangamia glabra, Jatropa curcu’s and Benzyl Benzoate, both with and without ascorbic acid on growing sheep, assessment of weight gain, liver function, nutrient digestion ability, wool production and meat quality. J. Vet. Med. A (Physiol. Pathol: Chem. Med) 51 (2): 79 - 84. FARM CHEMICALS HANDBOOK (1995), Meister, Publishing Co. Willough by, OH. ISMAN, M.B. (1997). Neem and other botanical insecticides: Barriers to commercialization. Phytoparasitica 25(4): 339-344. KAWAZU, KAZUYOSHI, JOCELYN, P., ALCANTARA AND KOBAYASHI, A. (1989). Isolation and structure of neoanonin, a novel insecticidal compound from the seeds of Annona squamosa. AGRIC BIOL CHEM 53(10): 2719-2722. KHAN, K., AND NAHEED, S. (1990). Chemical investigations of Cedrus deodara. Stem bark Isolation and identification of some sesquiterpene hydrocarbons. J. Chem. Soc. Pak. 12(4): 282-84. NAQVI, S.N.H. AND TARIQ, R.M. (2007). Mammalian toxicity of Acorus calamus (AC) and Annona squamosa (AS) oil against Albino rats. Pakistan j. entomol. Karachi. 22 (1&2): 37-38. NASIR, Y.J. (1978). Araceae. Flora of Pakistan N o. 120. In E. Nasir and S.I. Ali (eds). Department of Botany, University of Karachi and National Herbarium, Agricultural Research Council, Islamabad. PERVEEN, R. (2006). Analysis of Cedrus deodara root oil and its Pharmacokinetic and Pharacodynamic studies, with reference to anti ulcer and anti fungal effects. Ph.D. Thesis, Baqai Medical University, Super Highway, Karachi, Pakistan. RAO, I.G. AND SINGH, D.K. (2001). Combination of A. indica and C. deodara oil with PBO, MGK 264 an Embolia ribs against acuminata. Chemosphere. 44 (8): 1691-95. SANKARANARAYAN, R., KRISHAHAPA, S., BISARYA, S.C. AND DEV, S. (1973). Pryone from Cedrus deodara essential oil. Tetrahedron Letters (6): 427 1981 428. SHIRWAIKAR, A., RAJENDRAN, K., KUMAR, C.D. AND BODLA, R. (2004). Antidiabetic activity of aqueous leaf extract of Annona squamosa in streptozotocin-nicotinamide type 2 diabetic rats. (online) Journal of Ethnopharmacology. Pp. 1-8. STRELOKE, M., ASCHER, K.R.S., SCHMIDT, G.H. AND NEUMANN (1989). Vapor pressure and volatility of ß-asarone, the main ingredient of an indigenous stored-product insecticide, Acorus calamus oil. PHYTOPARASITICA 17(4): 299314. TARIQ, R.M. AND NAQVI, S.N.H. (2006). Avian toxicity of Acorus calamus (AC) and Annona squamosa (AS) oil against chicken (poultry). Pakistan j. entomol. Karachi 21 (1&2): 29-30. TARIQ, R.M. AND QADRI, S.S. 2001. Repellent activity of some local plant’s oil, two commercial repellents, di-methyl phthalate and non-alcoholic itter against dengue vector mosquitoes. Pakistan j. entomol. Karachi, 16 (1&2): 7-10. TARIQ, R.M., NAQVI, S.N.H., ZAFAR, S.M.N. AND SAQIB, T.A. (2004). Piscean toxicity of rhizome stem oil of Acorus calamus and seeds oil of Annona squamosa against Labeo rohita and Cyprinus carpio. Pakistan j. entomol. Karachi, 19 (1 & 2): 33-34. TISSER, R AND ROBERT, M.C. (1995). Essential oil safety. A guide for Health Care Professional. Churchill Livingstone Publications. USA (ISBN 0443052603). US ENVIRONMENTAL PROTECTION AGENCY (1993). Azadirachtin tolerance Exemption, Federal Register Vol. No. 30 Rules and Regulations. Pakistan j. entomol. Karachi 23 (1&2): 5-10, 2008 TAXONOMIC STUDIES OF SERGENTOMYIA FREETOWNENSIS SINTON 1930, SINDHICUS n. sub sp. (DIPTERA: PSYCHODIDAE) IN SINDH, PAKISTAN JUMA KHAN KAKARSULEMANKHEL Investigator of Sand flies, Leishmaniases, Helminths, Mosquitoes & Ticks, Department of Zoology, University of Balochistan, Saryab Road, Quetta, Pakistan [email protected], Cell. # 0333-7860240 ABSTRACT In the survey, the work was done to develop taxonomic record of sand fly Sergentomyia (Parrotomyia) freetownensis Sinton, 1930, sindhicus n. sub sp. collected for the first time from new epidemic localities of cutaneous leishmaniasis in Sindh Province (Pakistan). Since the specimens of the new sub species were collected first time from the Sindh Province, therefore, it is named sindhicus due to its collection site. In view of the published reports about the detection of encephalitis viruses from the species of the genus Sergentomyia Franca and Theodor as well as from genus Phlebotomus Rondani and Berte, from the Indian localities and European countries, the correct identification of the sand fly species becomes of significant value in the study of epidemiology of leishmaniases and other viral diseases. The new sub species sindhicus differs from the species S. freetownensis Sinton, 1930 in number of cibarial teeth, presence of 1 dozen punctiform fore teeth at the base of parallel teeth and ratio of anterior part of pharynx to its posterior part. Therefore, in order to facilitate Zoologists and Medical researchers in correct identification, taxonomic characters of S. freetownensis Sinton, sindhicus n. sub sp. were studied in detail with special reference to its mouth parts, male and female genitalia and findings are presented in the present paper. Key to the already known sub species of S. freetownensis Sinton is also formulated in order to distinguish the new sub species. Key words: Sand flies, Sergentomyia freetownensis Sinton 1930, sindhicus n. sub sp., Sindh. INTRODUCTION Phlebotomine sand flies are the proven vectors of leishmaniases, sand fly fever and viral diseases. There are about 700 species of sand flies of which about 70 are considered to transmit diseases to people. Pakistan has several endemic foci of leishmaniases and the disease is spreading continuously (Rowland et al., 1999; Kolaczinski et al., 2004; Kakarsulemankhel, 2004; Hashiguchi et al., 2005; Khan, 2005; Kolachi et al., 2005; Wakil, et al., 2006., Ahmad et al., 2008., Bari and Rahman, 2008). Previous studies of the sand fly fauna of Pakistan have been fragmentary. No comprehensive taxonomic work exists in facilitating the identification of Pakistani sand fly species except a few localized surveys (Nasir,1958; Barnett and McDonald,1964; Aslamkhan and Barnett, 1966,1967; Aslamkhan and Rafiq, 1980; Burney and Lari 1986; Rab et al. 1986; Safi, 1993; Aslamkhan 1996; Aslamkhan et al. 1997,1998). They have mentioned only the names of the species they collected. Lewis (1967) while studying west Pakistani sand flies, could not record Sergentomyia freetownensis Sinton from Pakistan. Species of genus Sergentomyia Franca and Parrot are prevalent in the country. There are published reports of transmission of encephalitis viruses (Toscana virus-TOSV-Family Bunyaviridae, genus Phlebovirus) from the species i. e. Phlebotomus pernicious Newstead and Phlebotomus perfiliewi Parrot of the genus Phlebotomus Rondani & Berte from Italy (Verani et. al.,1988), in Spain (Sanbonmatsu-Gamez et al., 2005), in southern France in Sergentomyia minuta (Rondani) (Charrel et al., 2006), Sabova virus in Phlebotomus of Senegal (Bay et al.,1999), and in India also (Geevarghese et al., 2005). In the light of these findings, the correct identification of the species of sand fly becomes of significant value in the study of epidemiology of leishmaniases and other viral diseases. In addition, the modern interest in zoonoses, animal reservoirs of leishmaniases and the role of sand flies as vectors, all have greatly been focused and the significance of the correct identification of sand flies is highlighted. During the routine entomological collection, specimens of S. freetownensis Sinton sindhicus n. sub sp. were captured by the present author from human residences of new endemic places of leishmaniases (Jacobabad and Dadu) in Sindh Province. Taxonomic characters of this fly especially mouth parts and genitalia were studied and results are given in the present paper. Key is also formulated of subspecies of S. freetownensis Sinton and relationships with its closest allies are also briefly discussed. This attempt hopefully, will not only serve as base line study of the taxonomy of Pakistani sand flies but will also guide researchers in correct identification of these flies. MATERIALS AND METHOD The present investigation was carried out on the materials (18 specimens of S. freetownensis Sinton sindhicus n. sub. sp. collected from the field, human residences, colleges, schools and hostels by means Juma Khan Kakarsulemankhel 6 of sucking tubes and sticky papers in day and night times in Sindh Province (Jacobabad and Dadu) during May, 2006. Collected flies were transferred in vials containing 70% alcohol and shifted to Zoology Department, University of Balochistan, Quetta, where descriptive observational study was carried out. Each fly was processed and dissected following the conventional techniques (Young and Duncan, 1994). Characteristics structures were studied, measured, taxonomic notes were prepared and their photographs were taken through camera mounted Olympus microscope (BX41). Most structures were measured with magnification of X100. All given measurements are in mm. The data of specimens critically examined for the description and measurements are designated under “Material examined”. Measured taxonomic characters are those suggested by Dedet et al. (1991). Identification of specimens was carried out with the help of available literature (Kirk and Lewis, 1951; Lewis, 1967,1978). Prepared permanent slides were deposited with the author’s collection of sandflies, Department of Zoology, University of Balochistan, Quetta. pharynx with posterior width twice anterior width, strongly constricted posteriorly, armature fairly long, denticles stronger and more numerous…freetownensis var. niger Parrot and Schwetz (Belgian Congo, Nigeria, Ivory Coast, Uganda). - 3. ♀ pharyngeal teeth much smaller, some times appearing as rows of points………freetownensis var. sudanicus Theodor (Sudan and Kenya) - 48-50 parallel narrow buccal teeth arranged on an arc slightly concave posteriorly, with 10-12 anterior slightly pointed denticles………….........4 4. ♀ pigmented area brown, covering the whole width of buccal cavity and prolonged anteriorly by a clear triangular extension, pharynx a little less than twice as wide posteriorly as anteriorly with numerous short pointed teeth……….…...freetownensis var. longoir Parrot (Abyssinia) - 60-65 parallel, straight buccal teeth, at the bases of these teeth a row of about a dozen punctiform denticles……………………………………………5 RESULTS S. freetownensis Sinton is known from one female, from Sierra Leone. However, in the work of Kirk and Lewis (1951) the name of africanus was regarded as synonym of P. (S.) squamipleuris Newstead and P. (S.) freetownensis Sinton becomes the typical example of the various forms previously known as P. africanus Newstead and its varieties. Sinton (1930) discussed basic characters of S. freetownensis. Buccal cavity has about 60 narrow parallel teeth on an arc concave posteriorly, flask shaped pharynx, about 2.5 times as wide posteriorly as anteriorly, armed with numerous backwardly directed armature, spermatheca an oval capsule twice as long as wide, ending in a narrow duct. Key to the sub species of S. freetownensis Sinton 1. ♀ antennal formula 2/3-15, A3= 0.135-0.17, cibarial teeth 40-50 parallel arranged on a concave arc, pigmented plate sausage shaped, concave posteriorly and with a paler, triangular, pointed, forward extension, pigmented plate triradiate with thickened lateral arms, pharyngeal spines backwardly directed, spermatheca an elongated capsule opening into a relatively narrow duct…freetownensis var. magnus Sinton (Belgian Congo and Transvaal). - 60-70 uniform parallel buccal teeths on an arc slightly concave, no anterior denticles….………2 2. ♀ pigmented plate black, and sausage shaped, occupying nearly the whole width of the cavity, with an anterior sub triangular pale prolongation, 30-33 buccal teeth, and a small point like denticle at the base of every second tooth in the armature……………………………………………3 5. ♀ dark brown pigmented plate in the form of crescent slightly concave possteriorly, not occupying whole width of buccal cavity, a paler anterior triangular prolongation, pharynx with well developed pointed spines freetownensis var. eremitis Parrot and de Joliniere (Sudan). - 46- 48 straight long, parallel buccal teeth on an arc…………………………………………………..6 6. ♀ pigmented plate black, not occupying the whole width of buccal cavity and with a small triangular anterior prolongation, pharynx constricted posteriorly, armed with moderately long denticles……….……freetownensis var. ater Parrot (Abyssinia) - 20 large, pointed buccal teeth with their bases nearly in straight line, about three median and one or two of the extreme lateral teeth shorter than the rest, a row of fine punctiform teeth……7 7. ♀ pigmented plate large, mushroom shaped, pharynx armed with numerous sharply pointed teeth posteriorly.......................freetownensis var. meridianus De Meillo and Lavoipierre (Transvaal, South Africa) Sergentomyia freetownensis Sinton, sindhicus n. sub. sp. (Figures 1A–1G) Phlebotomus minutus var africanus, Newstead, 1912, Bull. ent. Res., 3: 363; Adler & Theodor, 1926, Bull. ent. Res., 16: 401, Ann trop. Med. Parasit., 21: 63. P. africanus, Adler et al. 1929, Rev. Zool. Bot. Afric. 18:72. P. fensis Sinton, 1930, Ind. J. med. Res., 18: 171-193. Taxonomic studies of Sergentomyia freetownensis Sinton 1930, Sindhicus n. sub sp. Wings (X100) Distribution Narrow (1.50 long, 0.35 broad) and pointed, α/β=0.66, δ=0.05, П=0.10, gamma= 0.25. Pakistan: Sindh: Jacobabad and Dadu. 7 Comparative note Palps and antennal segments (X100) Palps 0.53 long, ratio 1, 2.6, 3.3,4, 7, formula 1, 2, 3, 4, .5. 3rd antennal segment 0.13 long, segments 4th and 5th each 0.07 long. Position of ascoid on A3 0.76, and at A4 and A5 each 0.28 of the segments. Mouth parts, Cibarium and Pharynx (X100) (Figs.1A–F) Proboscis 0.20 long. Labrum 0.16 long, strong chitinous structure, sides parallel, apex bluntly pointed, and margins furnished by a series of long very short leaf like sensillae closely together and numbering about eight on either side. Mandible 0.16 long, blade like structure, outer edges markedly serrated with sharp but minute teeth (Fig. 1A). Dental depth of 0.05. Maxilla 0.15 long, basally stout but narrows very much towards its apex (Fig.1B), much narrower than mandibles, one edge is pointed with about five relatively larger dot like chitinous lateral teeth, widely separated, the opposite edge with smaller ones set closely together (Fig.1C) and numbering about 34 ventral teeth. Dental depth 0.09. Hypopharynx 0.16 long, marginal leaf like sensillae very much shorter, apical part is broadly concave and in its center a salivary duct runs. Cibarium 0.05 broad, comb-like cibarial armature (Fig.1D) consisting of about 65 parallel, straight teeth, the extreme lateral ones are slightly bigger in length than the median ones, teeth standing on a slightly curved arch, anterior to it, there is a row of about dozen punctiform denticles, pigment patch 0.04 long and 0.01 broad (Fig.1E), brown in color with a yellowish short triangular extension, pigmented area not occupying the whole of the cibarium. Pharynx 0.13 long, flask shaped, slightly widened at its posterior half but narrowing again towards base, posterior breadth is 1.66 of the anterior breadth. The anterior edge of armature forms an almost straight line, most of the armature confined to the basal part of the pharynx (Fig.1F). Female genitalia (X100) Spermatheca (Fig.1G) cylindrical, barrel-shaped, 0.08 long, 0.03 broad, narrow duct leaves from each spermatheca and at some distance unite with one another to form a joint single duct finally open in to genital atrium. ♂ not known from Sindh during present survey. Material examined 8 ♀, Sindh, May, 2006. This new sub species shares some of its characters such as comb-like buccal armature, flask shaped pharynx, constricted posteriorly and oblong spermatheca with magnus, ater, eremitis, longior, niger, sudanicus, meridianus. It is distinct however, in its group in having brown colored pigment patch, about 65 parallel straight buccal teeth, with extreme lateral ones slightly bigger in length than the median ones, and with a row of about one dozen punctiform denticles anterior to larger teeth, pharynx 1.6 X as wide posteriorly as anteriorly. DISCUSSION According to Lewis (1978) S. africana complex is mainly African and includes a number of forms which have a comb-like cibarial armature and oblong spermatheca and have been variously treated as several species or as one by Quate (1964). Lewis (1974) pointed out that due to some degree of intergradation between some named forms, various local variants have come into existence. Therefore, Lewis (1978) treated it as S. africana magna (Sinton) form asiatica Theodor stat. n. The cibarium of ♀ has 4550 teeth in a concave row and no central patch of fore teeth, no notch in ventral plate, pigment patch concave posteriorly and has a simple forward process, pharynx with long teeth and spermatheca is oblong (Lewis (1967, 1978) and for these reasons, Lewis (1974) regarded S. a. magna as a sub species. However, ♀ S. freetownensis Sinton have a triradiate pigmented area with thickened lateral arms, cibarial teeth about 60, arranged on a concave line, pharynx flask shaped, the broadest posterior part being 2.50 times as broad as its anterior part (Sinton, 1930).Since Pakistani specimens was not available even Lewis (1978) suggested that Pakistani specimens collected from Kandhkot and Shikarpur by Lewis (1967) probably belongs to a small northern form found in Morocco, Israel and India, which Abonnenc and Yvore (1969) and Abonnenc (1972) known as S. africanus Newstead and its varieties. Kirk and Lewis (1951) while discussing Ethiopian sand flies, they suggested that P. africanus Newstead (Adler et al. 1929) from Belgian Congo is a synonym of P. squamipleuris Newstead 1912, and P. freetownensis Sinton, 1930 becomes the typical example of various forms. Two species of africanus group i. e. S. a. magna Sinton form asiatica Theodor stat. n. which was reported and described from Sindh (Kandhkot and Shikarpur) by Lewis (1967) 8 Juma Khan Kakarsulemankhel having ♀ cibarium 45-50 parallel teeth in a concave row no punctigorm fore teeth, and oblong spermatheca and S. africana Newstead reported from Uthal, Balochistan by Rab et al.(1986), details of figures and diagnostic characters were not supplied. The present new sub species i. e. S. freetownensis Sinton sindhicus, n. sub sp. is quite differentiated from the above mentioned sub species in characteristics like more number (65) of cibarial teeth, parallel, and straight, extreme lateral ones slightly larger in size than the middle ones, and with a row of about 12 punctiform denticles at the base of larger buccal teeth, pharynx 1.6 time as wide posteriorly as anteriorly, pigmented area not occupying the whole of the cibarium. Therefore, in the light of these characters, present specimens of Sindh fully deserve to be treated as a new sub species. Its name sindhicus is given due to its locality (Sindh). Morph metric measurements and morphology of labrum, mandible, mxilla, hypopharynx, and cibarium of the of the new sub species can not be compared with the other sub species as the same has not been furnished by the earlier workers like Sinton (1930), Lewis (1967, 1974, 1978) and Rab et al. (1986). However, the new sub species has a very localized and restricted distribution in Sindh and specially its presence in the human residences of the endemic foci of leishmaniases and its non established vectorial capacity clearly demands to initiate a comprehensive program for the control of sand flies and leishmaniases. It is hoped that the present findings would provide a basis for the further research on sand flies taxonomy. 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AND RASHTI, M.A. S. (1986). Cutaneous leishmaniasis in Balochistan: Reservoir hosts and sand fly vectors in Uthal-Lasbella. J. Pak. Med. Assoc. 36: 134-138. ROWLAND, M., MUNIR, A., DURRANI, N. NOYES, H. AND REYBURN, H. (1999). An out break of cutaneous leishmaniasis in settlements of Afghan Refugees in North West Pakistan. Trans. R. Soc. Trop. Med. Hyg. 93: 133-136. SAFI, F.S. (1993). A contribution to the sand fly fauna of Peshawar. M.Sc. Thesis. Government College Lahore, University of Punjab, Pakistan, 61 pp. SANBONMATSU-GAMEZ, S., PEREZ-RUIZ, M., COLLAO, X., SANCHEZ, F., ROSA-FRAILE, M. DE-LA. et al. (2005). Toscana virus in Spain. Emerg. Infect. Dis.11: 1701-1707. SINTON, J. A. (1930). Some new species and records of Phlebotomus from Africa. Ind. J. Med. Res. 18: 171-193. VERANI, P., CIUFOLINI, M. G., CACIOLLI, S., RENZI, A., NICOLLETTI, L., SABATINELLI, G. et al., (1988). Ecology of viruses isolated from sand flies in Italy and characterization of a new Phlebovirus (Arbo-virus). Am. J. Trop. Med. Hyg. 38: 433-439. WAKIL, A., BILQEES, F. M. AND SALIM, (2006). Cutaneous leishmaniasis in Dadu district during 2001-2004. Proc. Parasitol. 41: 19-39. YOUNG, D. G. AND DUNCAN, M. A. (1994). Guide to the Identification and Geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Mem. Am. Entomol. Inst. 57: 1881. Pakistan j. entomol. Karachi 23 (1&2): 11-14, 2008 COMPARATIVE TOXICITY OF ORGANIC SOLVENTS AND INSECTICIDESAGAINST CABBAGE BUTTERFLY SEEMA TAHIR1, TAHIR ANWAR1, MUHMMED SAMIULLAH CHANNA1, IMTIAZ AHMED2 AND YOUSAF HAYAT KHAN3 1. Pesticide Research Institute, Southern-Zone Agricultural Research Pakistan Agricultural Research Council, University Campus, Karachi-75270, Pakistan. 2. Department of Zoology, University of Karachi, Karachi-75270, Pakistan 3. Ecotoxicology Research Institute, National Agricultural Research Centre, Islamabad. ABSTRACT Under dose response observation chloroform was found to be more toxic with 60% mortality after 24 hours followed by the methanol (56.6%), benzene (53.3%) and acetone (40%) when applied topically on the 3rd instar larvae of cabbage butterfly. DDT was found to be toxic when tested against CBF caterpillar with other pesticides like ϒ-BHC and chlorpyriphos. KEYWORDS: Insecticides, solvents, toxicity. INTRODUCTION Large cabbage butterfly, P. brassicae L. is one of the greatest pests of cruciferous plants in the peak periods of its abundance, causing serious change on cabbage cultivation. Their larvae attach the leaves stems; inflorescence pots of the host plant and lower the yield of the seed. These are in delay distribute throughout the Asia and Europe. In Pakistan it is reported as a major pest of cauliflower (Ghouri 1960) and causes heavy losses to cabbage and cauliflower (Mushtaq and Mohuddin, 1984). Atwal (1976) recommended Malathion (0.05%) Diazinon (0.02%) or carbaryl (0.02%) for the control of this pest. In Pakistan the chemical control of this pest has been reported by many workers. (Gupta et al. 1985; Anwar et al. 1989 & 1995). The sub lethal, anti-feedant & repellants effects of insecticides have been studied by Blackwell (1988) & Ten (1981). Insecticides used to maintain the population at less than damaging levels when received little attention as to their role in a management scouting program utilizing the threshold concept. Growers presently used many different insecticides to control lepidpoterous pest of cabbage. The objective of our study to evaluate the efficacy of commonly used and nonregistered material and banned chemicals (usually smuggled from neighbour countries and used in remote areas of Sindh and Punjab). With toxicity of solvents also. The present investigations showed that DDT and Chlorpyriphos were equally toxic when applied topically on the thoracic region of 3rd instars larvae of cabbage butterfly (CBF) after 24 hours of treatment. MATERIALS AND METHOD The larvae of cabbage butterfly (CBF) were collected from the cabbage field from Sialkot. Larvae were brought to Entomological Research Laboratory, National Agriculture Research Centre, Islamabad. The larvae were fed on fresh cauliflower leaves. Different larval stages of cabbage butterfly were held in cage of 45 x 45 x 45cm made up of wooden frame from sided were provided with wire screen of 20 mesh and bottom were made up of ply wood. One side of cage had on opening provided with sieve of mouslin cloth for cleaning and feeding purpose, temperature and relative humidity were maintain at 25oC and 50% respectively. Different concentrations 0.01, 0.025, 0.05 and 0.1% of DDT and Chlorpyriphos were prepared using acetone as solvent. Three replicates with batches of 10 insects were used for each treatment. A batch of control (no treatment) and check (acetone) were also kept. Treated insects were keep in petridishes (90mm) and provided fresh leaves of cabbage with control and check. They were placed in rearing room at temperature of 25oC and 50% humidity. Observations for mortality were Tahir et al. 12 taken (14.1 LD) at 6 hrs, 12 hrs, 24 hrs and 48 hrs after treatment. DDT was tested against 2nd instar larvae (0.2g/10 larvae). 1µl of the test concentration was topically applied on dorsal side of thoracic region with the help of hand-mounted micro-applicator. Chlorpyriphos was tested against 3rd instar larvae (2.0g/10 larvae). 0.1µl of the test concentration ranging from 0.01 to 0.1% was injected on lateral side of thoracic region with the help of electrical micro-applicator (Bukard, Manufacturing Co. Ltd., Reckmons Worth, England. Variations in number of mortality for treatment were analyzed by means of Analysis of Variance; more were separate (P=0.05) by Ducans (1955) multiply range test Probit analysis of C50 and LC90 after 24 hours and 48 hours of the treatment. RESULTS AND DISCUSSION The present investigation showed that acetone, methanol and chloroform and benzene which are known to be used as solvents also possess toxic properties at different concentration when applied topically on the thoracic region of 3rd instar larvae of Pieres brassicae. Chloroform found to be more toxic with 60% mortality after 24 hours followed by the methanol (56.6%), benzene (53.3%) and acetone (40%) while mortality evaluation against other pest species has shown acetone to be more toxic than methanol. The results are given in Table-1 & Fig.1. Topical bioassay application has been used successfully on different test species evaluation of toxicity of different insecticides by topical application of the larvae of P. brassicae has been reported (Choudhary et al., 1988, Durha and Hameed, 1988, Kasana et al., 1996, Tahir et al., 1999). Endosulfan was found to be more toxic than other tested insecticides when applied topically CBF larvae (Mahabin et al., 1992) which is in favor with the present result that topical application of DDT and Chlorpyriphos was found to be less toxic. Sharma and Nath (1992) evaluated the bio efficacy of insecticide against CBF larvae and observed that toxicity of fenvalerate, HCH, methyl parathion, and DDT is almost same. Similarly in the present study the toxic effects of both insecticides against 3rd instar larvae of CBF were found. Sharma and Nath (1992) observed that the larvae of CBF were found to be more susceptible to DDT when applied topically which is fully compatible with present findings. Anwar et al. (1995) observed the maximum larvae mortality i.e. 87.50% after 24 hours and 92.5% after 48 hours of treatment when Dimlore (Chlorpyriphos and Dimethoate) the applied on CBF larvae as chlorpyriphos is also found to be more toxic to CBF in the present study. Singh and Jain (1987) observed the toxicity of various solvents against housefly when screening plant products as insecticides. It was found that acetone, methanol, benzene and chloroform when applied topically on thoracic region were found more toxic, while it was noted that as the carbon chain of solvent increased the toxicity also increased as tested Hydrocarbon solvent on the toxicity of Cypermethrin to 3rd instar larvae of S. Litura (Jaglan and Sircar 2003). In past the DDT was commonly used against the larvae of P. barssicae. Ripper et al. (1948) observed the toxicity of DDT against P. barssicae larvae when applied DDT particles with substance digestible only by specific insects which may be useful in acquiring the destruction of beneficial insects at the same time penetration of DDT through cabbage butterfly eggs killed the larvae first after hatching (Beament & Lal, 1957). Similarly the DDT was found to be toxic when tested against CBF caterpillar with other pesticides like ϒ-BHC diazinon, dichlorvos & carbaryl (Verma et al., 1969). These pervious findings are in full agreement with present results that relative toxicity of DDT is also like other pesticides i.e. Chlorpyriphos against CBF. In the lab experiments it was found that acetone and acrolein vapors could penetrate into the wheat mass and kill concealed insects in interkernel spaces while they were harmless to wheat seed viability (Poumirza & Tajbakhsh 2002 & 2008). While in present study acetone was found to be toxic when applied topically on CBF. Similarly the acetone toxicity LC50 was found to be 15.40, 15.51, 17.55 & 18.26 µl/L respectively against Oryzaephilus surinamensis (L), Callosobruchus maculates (F), Tribolium confusum (Duv.) and Sitophilus granaries (L.) respectively. Comparative toxicity of organic solvents and insecticides 13 …. Fig. 1. Time course study of DDT and Chlorpyrifos (CHP) applied topically at the rate of 0.1µl on the thoracic region against 3rd instar larvae of cabbage butterfly, Pieris brassicecae. Table 1. Lethal Doses of DDT and chlorpyrifos at 95% confidence level after 24 and 48 hours of treatment against larvae of P. barssicae (cabbage butterfly). S. No. Insecticides 1. DDT 2. 3. CHF 4. Time (hrs) 24 LD50s Range Regression line (non weighted) 0.36 0.022-0.058 Y=4.945 + 1.416 (X-8.524) 48 0.020 0.011-0.028 Y=5.394 + 1.828 (X-8.524) 24 0.040 0.021-0.067 Y=4.781 + 1.954 (X-8.524) 48 0.016 0.000-0.035 Y=5.287 + 0.958 (X-8524) LD90s 5. DDT 6. 7. 8. CHF 24 0.279 0.130-2.417 Y=4.945 + 1.416 (X-8.524) 48 0.099 0.062-0.269 Y=5.394 + 1.828 (X-8.524) 24 0.222 0.109-3.060 Y=4.781 + 1.954 (X-524) 48 0.358 0.104-0.245 Y=5.287 + 0.958 (X-8.524) Tahir et al. 14 REFERENCES Indian Journal of Plant Protection, 20: 2, 202204. ANWAR, T., S. TAHIR, A. JABBAR AND A.A. HASHMI (1995). Field evaluation of novel pesticides against cabbage butterfly, Pieris barssicae (L.) (Pieridae: Lepidoptera). MUHAMMAD ZAMAN (1986). Effect of four pyrethroids on the insect pests of rape. Pakistan J. Sci. Ind. Res., Vol. 29, No.3. ANWAR, T., A. JABBAR, A. MAJID, S. TAHIR AND S.N.H. NAQVI (1989). Antifeedant activity of juliflorine against larvae of brassicae (L.). J. Pak. Entomol. Kararachi, 41(1-2): 25-32. MUHAMMAD ZAMAN (1989). Evaluation of some insecticides against the mealy cabbage aphid and cabbage butterfly on rape in Swat. Sarhad J. Agric., Vol.5, No.4. ATWAL, A.S. (1976). Agricultural pest of India South East Asia. Kalyani Publishers, Delhi, India 479 p9. MUSHTAQUE, M. AND MOHYUDDIN, A.I. (1984). Pieris barssicae (Piendae Lepidoptera), a pest of crucifers and its control by parasites. Pak. J. Agric. Res. 5(3): 165-169. BEAMENT, J.W.L.; AND LAL, R. (1957). Penetration through the egg shell of Pieris barssicae. Bulletin of Entomol. Res. 48 109-25. BLACKWELL, A. (1988). A several and development of Pieris barssicae larvae upon application of sublethal doses of chlordimeform. Entomologia Experimentalis at Applicata, 48(2): 149-156. CHOUDHARY, S.K.; SINGH, A.K.; SINGH, S.P.; (1988). Evaluation of toxicity of some newer insecticides against the larvae of cabbage butterfly, Pieris barssicae L. Journal of Research, 6:, 1-2, 88-90. DUHRA, M.S.; HAMEED, S.F.; (1988). Toxicity of some insecticides to the cabbage butterfly on cauliflower. Journal of Research, 6: 1-2, 17-20. DUNI CHAND SHARMA; MAHABIR SING; AMIT NATH; SHARMA, D.C.; SINGH, M; NATH, A; (1993). Synergistic properties of hexachlorocyclohexane. Indian Journal of Entomology, 55: 4, 368-374. GHOURI, A.S.K. (1960). Insect pest of Pakistan. FAOPL. Prot Comm. S. Asia Pacific. Reg Tech. Doc. 8:31. GUPATA, P.R., VERMA, A.K. MISHRA, R.C. (1985). Field efficacy of some insecticide against caterpillars and thrips on cauliflowers crop. Vegetable Science 12(1): 49-54. JAGLAN, R.S.; SIRCAR, P. (2003). Effect of carbonchain of solvents on the toxicity of Cypermethrin emulsion formulation against larvae of Spodoptera litura (Fab.). Annals of Biology (Hisar, India), 19(1), 81-83. KASANA, A; MATIN, MA; RAFI, M.A. (1996). Efficacy of Malathion against larvae of Pieris barssicae. Pakistan Journal of Zoology, 27: 4, 359-361. MAHABIR SINGH; CHANDEL, R; SHARMA, D.C.; SINGH, M.; (1992). Toxicity of insecticides to different larval instars of Pieris barssicae L. POURMIZA, A.A.; TAJBAKHSH, M. (2002). Effect of acetone in controlling stored pest insect. Majallah-I Ulum-I Kishavarzi va Manabi-I Tabi Danishgah-I Sanati-I Isfahan, 6(3), 229-239. POURMIRZA. A.A. AND TAJBAKHSH. M. (2008). Studies on the Toxicity of Acetone and Carbon Dioxide on Stored-Poduct Insects and Wheat Seed. Pak. Jour. Biol. Sci. 11(7): 953-963. RIPPER, W.E.; GREENSLADE, R.M.; HEALTH, J.; BARKER, K. (1948). New formulation of DDT with selective properties. Pest Control Ltd., Harston, Cambridge, UK. Nature (London, United Kingdom). SHARMA, D.C., NATH, A; (1992). Effect of hexachlorocyuclohexane on the bio-efficacy of insecticides. Pesticide Research Journal, 4: 2, 111-115. SINGH, D.; JAIN, D.C. (1987). Relative toxicity of various organic solvents generally used in screening plant products for insetticidal activity against the house fly Musca domestica L. Indian Journal of Experimental Biology, 25(8), 569-70. TAHIR, S., ANWAR, T., KHAN, M.R., AZIZ, S., ILYAS, M. AND RAFI, M.A. (1999). Toxicological of Dimlor (A Mixture of Eimethoate and Chlorpyrifos against corn leaf Aphid, Rhopalosiphum maidis (Fitch) under different Micro-Climatic conditions. Pakistan J. of Biological Sciences, 2(2): 315-317. TEN, K.H. (1981). Antifeeding effect of Cypermethrin and permethrin at sublethal levels against Pieris barssicae Larvae. Pesticide Science, 12(6): 619626. VERMA, A.N.; SHARMA, P.D.; SARAMMA, P.U. (1969). Relative toxicity of some contact insecticides against cabbage caterpillar, Pieris barssicae (Pieridae; Lepidoptera) Journal of Research (Punjab Agricultural University). 6(1) (Suppl.), 197-9. Pakistan j. entomol. Karachi, 23 (1&2): 15-17, 2008 REDESCRIPTION OF CAYSTRUS PYGMEAUS LINNAVUORI (HEMIPTERA: PENTATOMIDAE) AN ISOLATED SPECIES FROM ETHIOPIAN REGION MUHAMMED ZAHID1 AND IMTIAZ AHMED2 1) Department of Zoology, Federal Urdu University of Arts, Sciences and Technology, Gulshan-e-Iqbal Campus, Karachi. 2) Department of Zoology, University of Karachi, Karachi. ABSTRACT The species belonging to the stink bug genus Caystrus Stål pygmeaus Linnavuori is redescribed here with reference to its colouration, head, pronotum, scutelum, hemelytra and important measurements specially male genitalia from Barkina faso (earlier name upper Volta) south of Senegal in the western Africa is considered here entirely isolated in its subclade C. trivalis (Gerstaecker) group. Key words: Redescription, Caystrus pygmeaus, Ethiopian region. INTRODUCTION Linnavouri (1972) described C. pygmaeus Linnavuori with reference to some of the external characters of head & pronotum including some measurements and male genitalia including some of the characters of pygophore and paramere. He also keyed his new species rather close to another of his new species parviceps but considered pygmaeus very distinctive in the genus. He described his new species from Poundou, upper Volta (present name Barkina Faso), south of Senegal in the western Africa), on the basis of one male specimen which was designated as holotype. The present 2nd author had an opportunity to examine this species in American Museum of Natural History (AMNH), New York, by the courtesy of Dr. Toby Schuh incharge entomological collections. Presently this species is redescribed with special reference to its characters of prnotum, scutelum, colouration & male genitalia. It is considered entirely isolated in its C. trivalis (Gerstaecker) group and to date its female genitalia is unknown. Its relationship is described here for the first time in its trivalis group. MATERIALS AND METHOD The holotype of this species was examined by the 2nd author of the present paper during the visit of American Museum of Natural History, New York (AMNH) by the courtesy of Dr. Toby Schuh incharge entomological collections of that museum. For the study of male genitalia the entire specimen with pygophore was plunged in the boiling water for five minutes. The pygophore in the softened specimen was extracted and placed in warm 10% KOH solution for a few minutes following the technique of Ahmad (1986) & Ahmad and McPherson (1990, 1998) and the male genitalia specially the pygophore and paramere were dissected and illustrated. The male genitalial capsule with paramere and aedeagus was then glued with the specimen. RESULTS Caystrus pygmaeus Linnavuori (Figs. 1-3) Caystrus pygmaeus Linnavuori 1972: 400-402; 1974: 402; 1982: 76. Coloration and general shape Body generally yellowish brown with brown punctures; eyes brownish black; ocelli pinkish; membrane of hemelytra hyaline; ovate. Head Slightly broader than long, anteocular distance longer than remainder of head; paraclypei narrowed, slightly longer than clypeus but not enclosing the later; antennae short with second segment much shorter than third, antennal formula 1<2<3<4<5; labium reaching hind coxae. Thorax Pronotum about 2x broader than its length, anterior margin slightly broader than head width across the eyes, anterior angles acute, humeral angles subacutely produced, lateral margins sinuate, posterolateral margin distinctly sinuate.; scutellum with elongated subrounded apical lobe. 16 Zahid & Ahmed Taxonomic studies of Sergentomyia freetownensis Sinton 1930, Sindhicus n. sub sp. 17 Key to close allies of C. pygmaeus Male genitalia Pygophore (Fig. 2) some what quadrangular, dorsomedian surface deeply concave, ventroposterior margin medially knobed, lateral lobe prominent, truncately produced; proctiger rectangular; paramere (Fig. 3) somewhat F- shaped, outer margin convex, apex of blade beak shaped, inner lobe broad with hairs. Material Examined One male 19. X 1927; Olsufjev; American Museum of Natural History (AMNH) Comparative note This species is most closely related to C. deserticolus Linnavuori in having head shorter and lateral margins of pronotum narrowly lamellate but it can easily be separated from the same in having shorter length (9.0 mm), antennae short and incrassate and puncturing of pronotum very coarse and dense. 1. Puncturing of pronotum sparse and fine………………………………………………….2 - Puncturing of pronotum very coarse and dense……………………………………………….3 2. Head much reduced in length (0.73 X as broad as long), scutellum sharply triangular…………………………………edentatus - Head not much reduced as above, scutellum not as above……………………....…………parviceps 3. Body of medium to large size, antennae incrassate…………………..…….….mawambinus - Body of small size, antennae not as above…….………………………………………...4 4. Lateral margins of pronotum straight only extreme margin upcurved……...……deserticolus - Lateral margins of pronotum distinctly sinuate……………….……………….…pygmaeus DISCUSSION Linnavouri (1972) gave the illustrations of head, pronotum, pygophore and paramere but he considered it most closely related to C. parviceps although he considered it distinctive. C. pygmeaus appears much smaller (9.0 mm in length) as compared to C.parviceps (11.5 mm in length). The tips of paraclypei appear separated in C. pygmeaus but they clearly enclose clypeus in C. parviceps, pronotum appears narrowish in C. pygmeaus as compared to broader pronotum in C. parviceps, lateral margins of pronotum appears distinctly concave in the middle with posterolateral margins distinctly sinuate in C. pygmeaus as compared to lateral margins of pronotum slightly sinuate in C. parviceps. We consider this species most closely related with Linnavuori’s species C. mawambinus, C. deserticolus and C. edentatus in having lateral margin of abdominal venter less contrastedly pale with brown punctures and body of small to medium size but it appears entirely isolated in its clade in having antennae short and incrassate, pronotum very coarse and dense and paramere with apex of blade subacute. We also have given here the key to separate the above species to make the identification of the presentspecies, much easier. Linnavuori (1982) also gave some illustrations of C. pygmaeus but his long key to the species of Caystrus did not include only some of the above species. REFERENCES AHMAD, I. (1986). A fool-proof technique for inflation of male genitalia in Hemiptera (Insecta). J. ent. Soc. Kar. 1 (2): 111-112. AHMAD, I. AND MCPHERSON, J.E. (1990). Male genitalia of the type species of Corimelaninae White, Cydnoides Malloch and Galgupha Amyot and Serville (Hemiptera: Cydnidae: coriomelaeniae) and their bearing on classification. Ann. Entomol. Soc. Am. 83 (2): 162- 170. AHMAD, I. AND MCPHERSON, J.E. (1998). Additional information on male and female genitalia of Parabrochymena Lariviere and Brochymena Amyot and Serville (Hemiptera: Pentatomidae). Ann. Entomol. Soc. Am. 91 (6): 800-807. LINNAVUORI, R. (1972). Studies on African Pentatomoidea. Arquivos do Museu Bocage (2) 3 (15): 395-434. LINNAVUORI, R. (1974). Hemipterological studies. Ann. Naturhist. Mus. Wien 78:393-413. LINNAVUORI, R. (1982). Pentatomidae and Acanthosomatidae (Heteroptera) of Nigeria and the Ivory Coast, with remarks on species of the adjacent countries in West and Central Africa. Acta Zoologica Fennica, no. 163: 176 pp. 18 Zahid & Ahmed Pakistan j. entomol. Karachi 23 (1&2): 19-26, 2008 STUDY OF RESISTANCE IN SITOPHILUS ORYZAE AGAINST BIOSAL, CYPERMETHRIN AND PHOSPHINE ON THE BASIS OF TOXICITY VALUES S.M. NAUSHAD ZAFAR1, TARIQ2, R.M. ASLAM3, M., BREUER4, M., NAQVI5, S.N.H. & PERVEEN6, R. 1 2 Department of Zoology, University of Karachi, Karachi-75270, Sindh-Pakistan. M.A.H. Qadri Biological Research Centre, University of Karachi, Karachi-75270, Sindh-Pakistan. 3 Department of Department of Statistics, University of Karachi, Karachi-75270, Sindh-Pakistan. 4 Department of Ecology, Enology Section, Mehrhauser. Str. 119 Frieburg, Germany 5, 6 Department of Pharmacology, Baqai Medical University, Toll Plaza, Karachi, Sindh-Pakistan. ABSTRACT In the present work, the experiments were conducted for the resistance estimation in susceptible strain (SS), Karachi strain (KS) and Lahore strains (LS) of Sitophilus oryzae L. (Rice weevil). The three strains (populations) of S. oryzae were exposed to a neem formulation, Biosal (10 EC) available locally in the market, a synthetic pyrethroid, Cypermethrin (10 EC) and a fumigant, Phosphine (PH3) used worldwide. The resistance was estimated by filter paper impregnation contact method. The LD50 values were calculated by impregnated surface area as μg/cm2. The LC50 values of Biosal against three strains (populations) SS, KS & LS were computed as 18.600 47.1354 and 51.96 μg/cm2, respectively. Whereas the LC50 values of Cypermethrin against SS, KS and LS were -4 -4 -4 2 computed as 29.08x10 , 40.40x10 and 44.33x10 μg/cm , respectively. While the LC50 values of Phosphine (PH3) against SS, KS and LS were computed as 0.166429, 0.698113 and 0.84574 g/2.8m3, respectively. Thus it may be concluded that, in three strains (populations), the increase in resistance may be shown as KS is < LS. Whereas the toxicity (LD50) is in the following sequence, Biosal < Phosphine and < Cypermethrin. As a whole the Karachi strain (KS) and Lahore strain (LS) were compared with that of susceptible strain and both KS & LS were found more resistant to Cypermethrin and Phosphine. Lahore strain was found to be more resistant as compared to Karachi strain, but least or negligible resistance was found against Biosal (neem formulation = phytopesticide) which seems to be correct on the basis of LC50 values. Key words: Resistance, Toxicity, S. oryzae, Biosal, Cypermethrin, Phosphine. INTRODUCTION Pakistan is an agricultural country; it depends mainly on agricultural products. The cotton, paddy, wheat and others are the main crops. Cereals have an important nutritional value and are one of the principal diet worldwide and among them rice has a remarkable position. During storage, products may be infested by insect pests causing deterioration of the grain. This makes a treatment necessary for their control. The rice weevils, Sitophilus oryzae (L.), the granary weevil, Sitophilus granarius (Linnaeus), the maize weevil, Sitophilus zeamais Motschulsky, (Coleoptera: Curculionidae) and the broadnosed grain weevil, Caulophilus oryzae (Gyllenhal) are responsible for most of the insect-related damage of stored rice and grain. These insects are called primary insect pest, or internal feeder, because the adults attack whole kernels and larvae feed and develop entirely within the kernels. Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) is a secondary pest that feeds on damaged kernels. The Araecerus fasciculatus (De Geer) is 5th weevil which is primary insect pest but is found in coffee beans. To control stored grain pests different methods have been used from time to time, e.g. by spray method, fumigation method and biological method. The fumigation method was more adopted as compared to spray and biological control, due to easy, effective and vast area covering control. Indiscriminate and repeated use of the pesticides and fumigants (Phosphine etc.) produced hazardous effects specially, the resistance in the stored grain pests. Many workers have reported resistance against pesticide some of them are as follows (Stone 1968, W.H.O. 1970, Georghiou and Mellon 1983, Saleem & Shakoori 1989, Preisler et al., 1990, Zettler, 1991, Arthur and Zettler 1991, Chilcutt and Tabashnik 1995, Grafius 1997, Mostafa and Zaved 1999, Fragoso et al., 2002, Asidi et al., 2005, Akiner and Caglar 2006, N’Guessan et al., 2007, Khaliq et al., 2007) they evaluated the resistance in stored grain pest against pesticides. Whereas resistance against fumigants, have been reported by a number of researchers. Some of them are as follows (Winks 1969, Tyler et al., 1983, Taylor and Halliday 1986, White and Lambkin 1990, Irshad and Iqbal 1994, Bengston et al., 1999, Chaudhry 2000, Rajendram and Gunasekaran 2002, 20 S. M. N. Zafar et al. Falaksher & Shakoori 2004. Amount of the fumigants that may replace the ozone-depleting methyl bromide, which is to be taken off the market, are phosphine, ethyl formate, carbonyl sulfide, sulfuryl fluoride, etc. The expected loss of methyl bromide for the treatment of buildings, stored products and soil is still regarded as a major problem, particularly in the United States. Fumigants are respiratory poisons and upon exposure damage nervous system and muscles. Lack of breathing may also cause accumulation of CO2 which inturn keeps the spiracles open to allow more fumigants to enter body cells as well as extra cellular fluid and haemolymph. The acidosis due to CO2 retention may effect the proteins and enzymes in cells and haemolymph (Pratt and Reuss, 2004). As it is well known that the use of persistent organochlorines like DDT and the acute organophosphours compounds has led to hazardous effects on environment and human beings. In response, efforts were made to strengthen the Integrated Pest Management (IPM) approach where there is chemical control (Schmutterer 1995), if at all necessary, should be combined with other methods like crop rotation, resistant varieties and biological control. In addition, attention was directed towards the development of alternative chemicals. The neem tree, Azadirachta indica A. Juss is so far the most promising example of plants currently used for pest control but Pruthi (1937) was the first scientist who used neem as pesticide in India and Siddiqui (1942) from Indo-Pak was the scientist who isolated three bitter principles from neem oil for the first time. This holy tree in Indo-Pak, from where it originates, now has a global distribution throughout the tropics. It is used for many purposes such as shade tree, poles for construction, medicine, tooth sticks and as a source of insecticide (National Research Council, 1992). Since the early seventies much research has been carried out on the pesticidal properties of the neem tree and the results have been published in proceedings from three International Neem Conferences (Schmutterer and Ascher, 1982; 1984; 1987). A summary of how neem products are used as bio-pesticides, the mode of action, effects on pests and natural enemies has been reviewed by Schmutterer (1997). Although the active ingredients in the neem, such as the azadirachtin, are known, it has not been possible to synthesize these complex compounds. Stable formulations of purified extracts are commercialized (Margosan-O and others) and distributed in several countries. The botanical pesticides prepared from neem tree, other trees and plants are safer in use. It has been proved that very little or no resistance develop against phytopesticides, or developes after very long time. Naqvi (1987) discussed resistance in various insect species due to indiscriminate use of pesticides or constant use of the same pesticide in the field or selection pressure in laboratory. These factors increases the level of tolerance in the particular pest strain to a great limit. The phenomenon is biochemical and genetical. Hundreds of species and strains have been reported to be resistant to various pesticides or groups of pesticides. Sometimes crossresistance also develops. Here in Pakistan the same problem of resistance is being experienced by researchers and fumigators. Therefore, the present work is a step to the solution of this problem in Pakistan. Toxic effect of these pesticides i.e – Phosphine (PH3), Pyrethroid (Cypermethrin 10 EC) and Neem preparation (Biosal) on S. oryzae were estimated. The work was aimed to evaluate the level of resistance in S. oryzae against these pesticides. MATERIALS AND METHOD Pest collection and rearing technique Stored grain insect pest Sitophilus oryzae adults and its immature biological stages were collected for experimental purpose. Samples were collected from various grain warehouses at different localities of Karachi i.e. various rice warehouses in Korangi, Landhi, SITE area, TPX and Maripur Road. Insects were collected separately in plastic bags along with the rice on which they were feeding. They were called as S. oryzae Karachi strain (KS). They were then reared in the laboratory as a single population / strain in University of Karachi. Sitophilus oryzae Lahore strain (LS) were collected from Rice warehouses in different localities of Lahore and reared in the laboratory of SGS Agricultural Laboratory, PECHS, Karachi under 28°C + 2°C temperature and at 65 + 5% r.h. Whereas susceptible strain is being reared in the insectory of Zoology Department, since 15 years. The susceptible strain was given the code as (SS). Rearing of the pest and its biological stages was done in the insectory of Zoology Department, University of Karachi. Insects were reared in glass jars filled with rice in twelve (12) separate jars. Upper quarter of each jar was left empty in order to provide space for free movement of insects. Upper end of each jar was covered by muslin cloth for ventilation and to avoid escape of insects. In order to avoid fungal attack due to high humidity, grains were shaked and replaced by few fresh grains from time to time and or whenever required. Egg laying was allowed directly in the food medium. Mature adults were introduced for this purpose in the glass jars. Hatching of eggs was observed regularly and required number of insects were taken out and kept in the “Petri dishes” of 9 Study of resistance in Sitophilus oryzae against Biosal, Cypermethrin & Phosphine Preparation of chemicals The Biosal (10 EC) Neem formulation was directly used in the present work, due to having low toxicity against stored grain pest. Initially after trials 11.795 μg/cm2 to 27.522 μg/cm2 dose was selected for susceptible strain and 15.726 μg/cm2 to 78.634 μg/cm2 dose was selected for both Karachi and Lahore strain. Whereas of cypermethrin 0.1% stock solution was prepared by dissolving 0.1 ml of cypermethrin into 100 ml. bidistilled deionized water and then different doses in ml were applied in the experiment. After preliminary tests 0.15 ml to 1.00 ml doses were used in the experiment and then selected finalized doses were converted into μg/cm2 by using formula: μg/cm2 = concentration x vol x 1000 x _1_ 100 Area While in the case of fumigant phosphine tablets were used in the experiments. For this purpose from 0.125-2.00 tablets were kept in the artificial fumigation chamber. This was done by breaking the tablet into two parts and then in the four parts and then it was weighed approximately as 0.125, 0.25 gm, 0.5 gm, 1.00 gm, and 2.00 gm. Method of Treatment for Toxicity Determination: A group of hundred (100), adult weevils having uniform age and size was released in petri dishes having 9 centimeter diameter (= 4.5cms radius) with impregnated paper. The insects were released with respective neem formulation (Biosal) and standard (cypermethrin) having different concentrations. A set of ten petri-dishes were set up for neem formulation, in duplicate, nine for five different concentrations, and one for control. For standard (cypermethrin) also, six petri dishes were set up, nine for nine different concentrations and one for control. Mortality counts were made after 24 hours in each experiment. Each experiment was repeated 5 times. If in any experiment mortality rate increased more than 10% in the case of control the experiment was discarded. The observations were analyzed according to Abbot’s formula (1925) and recorded in the form of Tables. Lethal Concentration (LC50) Values: Average values were calculated and mortality curve was drawn on log-log graph paper to find out LC50 by taking dose on x-axis whereas the percent mortalities on y-axis for Biosal, “Cypermethrin and Phosphine following Raymond et al., (1993). Statistical analysis was done on computer program by fitting actual readings, which were transformed by computer and the LC50 of Biosal, Cypermethrin and Phosphine against three strains were calculated. RESULTS Toxicity The Biosal 10 E.C (Neem formulation) was used in the present work, due to its low toxicity against stored grain pests especially S. oryzae. After preliminary tests, nine concentrations were selected for each strain i.e. for susceptible (SS), Karachi (KS) and Lahore strain (LS). The concentrations of Biosal in ml was selected for susceptible strain. LC50 determination doses were 1.500, 1.750, 2.000, 2.250, 2.500, 2.75, 3.00, 3.25 and 3.50ml/4.5cm2. These concentrations were calibrated into 11.795, 13.761, 15.726, 17.692, 19.658, 21.624, 23.590, 25.556 and 27.522 μg/cm2 for quantification. The mean mortalities % with S.D value caused by these concentrations were 22 + 4.47, 28 + 8.36, 34 + 8.94, 42 + 4.47, 48 + 8.36, 56 + 5.48, 68 + 8.36, 82 + 4.47, and 96 + 8.94%, respectively. Whereas for Karachi and Lahore strains, the concentrations of Biosal selected for LC50 determination were 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 ml/4.5cm2. These ml concentrations were them converted into 15.726, 23.590, 31.543, 39.317, 47.181, 55.044, 62.907, 70,771 and 78.634 μg/cm2. The mean mortalities % with S.D value caused by these concentrations in the case of Karachi strain (KS) were 16 + 5.48, 20 + 7.07, 28 + 8.36, 36 + 5.48, 44 + 8.94, 52 + 4.47, 64 + 5.48, 78 + 8.36 and 96 + 8.94%, respectively. While the mean mortalities % with S.D value produced by these concentrations in the case of Lahore strain (LS) were 14 + 5.48, 18 + 4.47, 26 + 5.48, 32 + 4.47, 40 + 7.07, 48 + 8.36, 58 + 8.36, 72 + 4.47 and 88 + 8.36%, respectively. The LC50 value against susceptible strain, Karachi strain and Lahore strain of S. oryzae has been found as 18.600, 47.135 and 51.96 μg/cm2, respectively, and are shown in Fig. 1,2, and 3, respectively. Toxicity of Biosal (10 EC) against Sitophilus oryzae Susceptible strain (SS) showing LC50 = 18.600 µg/cm2 Transform Mortality cms diameter (= 4.5 cms radius) for treatment. In order to determine the efficiency of insecticides and fumigants artificial infestation was created in the required commodity. Samples were then treated with insecticides, i.e. Neem formulation (Biosal), Cypermethrin and Fumigant (Phosphine). 21 1.5 1 0.5 0 -0.5 0 -1 -1.5 5 10 15 20 Concentrations Figure 1 25 30 22 S. M. N. Zafar et al. 2 1 0 -1 0 20 40 60 80 100 -2 The LC50 values of cypermethrin against SS, KS and LS were found as 29.08 x 10-4, 40.4 x 10-4 and 44.33 x 10-4 μg/cm2 as shown in Fig. 4, 5, and 6, respectively. Toxicity of Cypermethrin (10 EC) against Sitophilus oryzae susceptible strain (SS) showing LC50 = 0.002908 μg/cm2. Concentrations Figure 2 Toxicity of Biosal (10 EC) against Sitophilus oryzae Lahore strain showing LC50 =51.96 μg/cm2 1.5 1.5 1 0 0.00E+00 -0.5 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 -1 -1.5 Concentrations Figure 4 1 0.5 -1 -1.5 20 40 60 80 100 Concentrations Figure 3 Toxic effects of cypermethrin 10 EC (Pyrethroid) were directly concentrations dependant. The selected concentrations against the susceptible strain (SS) of S. oryzae were 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 and 0.55 ml / 4.5cm2. These concentrations were then converted into, 11.795 x 10-4, 15.726 x 10-4, 19.658 x 10-4, 23.590 x 10-4, 27.522 x 10-4, 31.453 x 10-4, 35.385 x 10-4, 39.317 x 10-4 and 432.249 x 10-4 μg/cm2, respectively. The mean mortalities percentage with S.D value produced by these concentrations were 18 + 8.36, 26 + 7.07, 32 + 4.47, 44 + 8.94, 52 + 4.47, 66 + 5.48, 78 + 8.36, 84 + 5.48 and 96 + 8.94%, respectively. Whereas the selected ml concentrations used against both for Karachi strain (KS) and Lahore strain (LS) were 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90 and 1.00 ml / 4.5 cm2. These ml concentrations were then converted into 15.72 x 104 , 23.59 x 10-4, 31.45 x 10-4, 39.31 x 10-4, 47.18 x 104 , 55.04 x 10-4, 62.90 x 10-4, 70.77 x 10-4 and 78.63 x 10-4 μg/cm2. The mean mortalities % with S.D value produced by these concentrations in the case of Karachi strain (KS) were 24 + 5.48, 26 + 5.48, 34 + 8.94, 48 + 8.36, 56 + 5.48, 68 + 8.36, 76 + 5.48, 84 + 5.48 and 96 + 8.94%, respectively. Toxicity of Cypermenthrin (10 EC) against Sitophilus oryzae L. Karachi strain (KS) showing LC 50= 0.00404 μg/cm2. Transform Mortality 0 -0.5 0 2 1.5 1 0.5 0 -0.5 0.00E+00 2.00E-03 4.00E-03 6.00E-03 8.00E-03 1.00E-02 -1 -1.5 Concentrations Figure 5 Toxicity of Cypermentrin (10 EC) against Sitophilus oryzae L. Lahore strain (LS) showing the LC50 = 0.004433 μg/cm2 Transform Mortality Transform Mortality While by applying the above concentrations same as in KS, the mean mortalities % with S.D value shown against Lahore strain (LS) were 12 + 4.47, 24 + 5.48, 30 + 7.07, 44 + 5.48, 52 + 4.47, 64 + 5.48, 70 + 7.07, 82 + 4.47 and 96 + 8.94%. Transform Mortality Transform Mortality Toxicity of Biosal (10 EC) against Sitophilus oryzae L. Karachi stain (KS) showing the LC50 =47.1354 μg/cm2 2 1 0 0.00E+00 -1 -2 2.00E-03 4.00E-03 6.00E-03 8.00E-03 1.00E-02 Concentrations Figure 6 Toxic effects of phosphine against S. oryzae L. were noted at 5 different selected dose by applying in the fumigation chamber constructed especially for Study of resistance in Sitophilus oryzae against Biosal, Cypermethrin & Phosphine The dose of 0.0.125 g / 2.8 m3 caused no mortality after 24 hours but after 48 hours the mortality was 40% and after four days (96 hours) 100% mortality was noted. While at the dose of 0.25 g / 2.8 m3, 19% insects were killed after 24 hours exposure and 60% mortality was noted after 48 hours and the same mortality was observed after three days (72 hours). All the insects died after 96 hrs and showed 100%, mortality. Moreover, 56% and 50% mortality was found at 0.5 g / 2.8 m3 after 24 hours in the case of Karachi and Lahore strains, respectively. The fumigational effects of above doses in the fumigation chamber were noted after 24,48,72 and 96 hours. The 100% mortality was found at 0.500 g / 2.8 m3, 1.00 gm, 2.00 gm, in susceptible strain while in the case of Karachi and Lahore strains only at 1.00 and 2.00 g / 2.8 m3 100% mortality was found. The LC50 of phosphine against three strains (SS, KS and LS) was found to be 0.166429, 0.698113 and 0.84574 g/2.8m3, respectively as shown in Fig. 7, 8 and 9. Transform Mortality Toxicity of Phospine gas against S. oryzae L. susceptible strain (SS) showing the LC50= 0.166429 g/2.8m3 3 2 1 0 -1 0 0.5 1 1.5 Concentrations Figure 7 2 2.5 Transform Mortality The dose 0.125 g / 2.8m3 was applied at the temperature of 24 + 2 oC and 66 + 2 r.h., while the dose of 0.25 g / 2.8m3 was applied at the temperature of 26 + 2 oC and 64 + 2 r.h. The dose of 0.50 g / 2.8m3 was applied at the temperature of 28 + 2 oC and 62 + 2 r.h., whereas 1.00 g / 2.8 m3 dose was applied at 30 + 2 and 60 + 2 r.h. The dose of 2.00 g / 2.8m3 was applied at the temperature of 32 + 2 oC and 58 + 2 r.h. Toxicity of Phosphine gas against Sitophilus oryzae L. Karachi strain (KS) showing the LC50 =0.698113 g/2.8m3 3 2 1 0 -1 0 0.5 1 1.5 2 2.5 -2 Concentrations Figure 8 Toxicity of Phosphine gas against Sitophilus oryaze L. Lahore strain (LS) showing LC50= 0.84574 g/2.8m3 Transform Mortality this research work. The selected doses for treatment of susceptible (SS) Karachi (KS) and Lahore strain (LS) were 0.125, 0.250, 0.500, 1.00 and 2.00 g / 2.8 m3, respectively. 23 3 2 1 0 -1 0 0.5 1 1.5 2 2.5 -2 -3 Concentrations Figure 9 DISCUSSION In the present study, the experiments were carried out for the estimation of resistance against susceptible strain (SS), Karachi strain (KS) and Lahore strain (LS) of Sitophilus oryzae L. (Rice weevil) adult weevils. These strains were exposed to a neem based formulation, Biosal (10 EC) locally available in the market, a synthetic pyrethroid, cypermethrin (10 EC) and a fumigant used worldwide, phosphine (PH3). The resistance was estimated on the basis of calculating the LC50 values (contact method). Some other parameters may also be used in this connection, as are being used in different parts of the world. Using the above parameter the following results have been obtained which may be compared, with the results obtained by other researchers and workers. The results are as follows. The LC50 values of Biosal against SS, KS and LS were computed as 18.600 47.1354 and 51.96 μg/cm2, respectively. Whereas the LC50 value of Cypermethrin against SS, KS and LS were computed as 29.08x10-4, 40.4x10-4 and 44.33x10-4 24 S. M. N. Zafar et al. μg/cm2, respectively. While the LC50 value of Phosphine against three strains were computed as 0.166429, 0.698113 and 0.84574 g/2.8m3, respectively. On the other hand if the pesticide and fumigant are compared with each other, used in this study, then the sequence of increasing resistance may be shown as follows: Biosal < Phosphine and < Cypermethrin, which means the least tolerance have been found against the biopesticide. Biosal falls in phytopesticide group whereas Phosphine (fumigant) has shown greater resistance in the present study. On the other hand Cypermethrin showed the greatest resistance in the present study, which belongs to the synthetic pyrethroid group. The same type of study on resistance was studied by Georghiou and Mellon (1983). They studied the resistance among the mosquitoes species, and reported that only among the mosquitoes 96 species were found resistant to one or more groups of pesticides. As there are five groups of pesticide: Carbamate (CB), Organophosphate (OP), Organochlorine (OC), Synthetic pyrethroid (SP) and plant originated Biopesticides (BP). Among these 36 species were resistant to one group, 32 species to two groups, 19 species to three groups, 8 species to four groups and only one species to all five groups of pesticide. The last part of the above report is exactly in line with the present study, in which least/negligible resistance has been found against S. oryzae (SS) by paper impregnation method. Khan and Naqvi (1985) reported the effectiveness and penetration of Phosphine gas (PH3) by using a fixed dose of 30 grams = 10 tablets/1000 ft3 at temperature ranging 28 to 31oC and humidities at 76-82%, upto the penetration of 4,8,12 feet and in paper bags of 4 kgs each, having rice in them. Results indicated that 100% mortality of cigarettes weevils was obtained at the rate of 10 tablets/1000 ft3 upto the 100 ft3 in small stocks, godowns and warehouses, the minimum exposure of 48 to 72 hours or more was required depending upon temperature, humidity and quality of grains to be fumigated. In the present investigation it was carried out against rice weevils instead of cigarette weevils, using the same phosphine gas but on different dose. In the above report 10 tablets/1000 ft3 gave 100% mortality upto 100 ft3 against cigarette weevils in 48 to 72 hours, which mean 1 tablet/100 ft3, whereas in the present work 0.25 tablet/100 ft3 gave 100% mortality in 48 to 72 hours in susceptible strain, which is 75% low dose as compared to above report, while 0.5 tablet gave 100% mortality in rice weevil in 48 to 72 hours against Karachi and Lahore strains which is also 50% low as compared to above report. The difference in LC50 value against susceptible, Karachi and Lahore strains of S. oryzae due to PH3 is because of different insects in the two reports, but other parameters are in line. Vollinger (1987) reported comparative experimental study for possible development of resistance against neem seed kernel extract (NSKE) and deltamethrin in two genetically different strains of diamond back moth, the Plutella xylostella (L.) upto 42 generations. Response to NSKE and to deltamethrin was compared with that of untreated, susceptible lines. The larvae were treated during 3rd and 4th instar stage. NSKE-treated lines showed no sign of resistance in feeding and fecundity tests, while deltamethrin treated lines developed X20 and X35 resistance. There was no cross-resistance between deltamethrin, NSKE and diflubenzuron (Dimilin, a synthetic molting inhibitor). Beside this, activity of esterase enzyme did not change significantly, over the 35 generations. Similarly Naqvi and Tabassum (1992) reported about probable resistance by experiments in the two separate lines of Musca domestica (PCSIR-strain) which were subjected to the pressure of neem extract (RB-a) and cyfluthrin (Solfac 10% EC). Their studies indicated that after 35 generations 12 fold increase in LC50 of cyfluthrin occurred, whereas in the case of RB-a slight increase was found, this showed that possibly resistance against neem fraction will not appear or will develop after a long time. Ahmad et al. (1998) reported the LC50 as 19μg/cm2 of Cypermethrin against S. oryzae (Karachi strain) by paper impregnation method. While in the present work it has been found 40.4x104 μg/cm2, which is many times high and show higher resistance against Cypermethrin in last 15 years. Ivania et al. (1998) have reported the higher frequencies and high levels of resistance to Phosphine (PH3) against S. oryzae population as also detected by the Food and Agricultural Organization (FAO), with 10% and 20%CO2. While in the present work, higher resistance in S. oryzae has been found against Phosphine gas without using CO2 gas. Swain and Baral (2004) treated Sitophilus oryzae and Callosobruchus chinensis, with seven plant species leaf powder, to compare efficacy as pest control agent. They reported that Sitophilus oryzae was reasonably controlled by neem leaf powder and C. chinensis was controlled by begunia leaf dust powder. In the present case the neem product controlled the Sitophilus oryzae and lesser resistance was found against this product as compared to cypermethrin and phosphine as evident from LC50 for SS, KS and LS strains (18.6, 47.1 and 59.6μg/cm2). It also indicated that phosphine and cypermethrin are more toxic at a comparatively lower dose. 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Resistance to the fumigant phosphine in a strain of Tribolium castaneum (Herbst). Insect Toxicol. Info. Serv. 12: 178. WORLD HEALTH ORGANIZATION (1970). Insecticide resistance and vector control: 17th Report of WHO Expert Committee on insecticides: WHO Tech. Report, Ser. No. 443. ZETTLER, J.L. (1991). Pesticide resistance in Tribolium castaneum and T. confusum (Coleoptera: Tenebrionidae) from flour mills in the United States. J. Econ. Entomol. 84: 763-767. Pakistan j. entomol. Karachi 23 (1&2): 27-30, 2008 EFFICACY OF IMEDACLOPRID AND ENDOSULFAN IN COMPARISON WITH BIOSAL (BIOPESTICIDE) AGAINST MYZUS PERSICAE (SULZER) ON MUSTARD CROP MUHAMMED FAHEEM AKBAR1, NIKHAT YASMIN2, FARAH NAZ2 AND ABDUL HAQ3 1 Department of Agriculture and AgriBusiness Management, Karachi. Deparment of Zoology, University of Karachi, Karachi-75270, Karachi-Pakistan. 3 Department of Food Science & Technology, University of Karachi, Karachi 75270, Pakistan. 2 ABSTRACT Efficacy of different insecticides as foliar application was studied against mustard aphid Myzus persicae (Sulzer) in Malir field (Karachi). The insecticides imidachloprid 25 WP (80 gramsl/acre), endosulfan (800 ml/acre) and Biosal (2000 ml/acre) were sprayed at one week interval using Knapsack hand sprayer and a check plot was also maintained for comparison. Efficacy was sassessed by counting the aphid mortality in mustard field plots. All tested insecticides performed better against aphid as compared to untreated plots. Imidacloprid proved as the best with 86 percent aphid population reduction after 1st nd spray and 83 percent after 2 spray followed by Endosulfan and Biosal with 73, 70 and 56, 57 percent reduction respectively. Key words: Myzus persicae, Insecticides, Efficacy, Mustard. INTRODUCTION Next to Cotton and Rape, Mustard (Brassica campestris) occupies the maximum area among oilseed crops in Pakistan. Deficit of edible oil in Pakistan is a chronic problem, so the importance of brassica crop cannot be ignored. Due to this importance the area under mustard crop is increasing gradually from 227,000 hectares in 2005-06 to 256,000 hectares in 2006-07 (Source: Ministry of Food, Agriculture and Cooperatives) with increasing trend in the yield as well, which was 212,000 tonnes in 2006-07 as compared to 81,000 tonnes in 2005-06. One of major constraints in less production of Brassica campestris is damage due to insect pests. A number of insect pests attack this crop including Myzus persicae (Sulzer) which causes a major loss in crop yield due to severe infestation in brassica. Over 250 species of the superfamily Aphidoidea feed on agricultural and horticultural crops throughout the world (Blackman & Eastop 1984). Aphids as pests tend to have a wider host range than economically unimportant species. The green peach aphid, Myzus persicae, has an extremely wide host range of over 100 plants including a wide variety of vegetable and ornamental crops (Baker 1982). Aphids suck sap from plant (phloem) tissues using mouthparts modified for piercing and sucking. Some aphids feed on foliage while others feed on twigs, limbs, branches, fruits, flowers or roots of plants. Some species inject toxic salivary secretions into plants during feeding. If left unchecked, aphids can stunt plant growth, deform and discolor leaves and fruit or cause gall formation on leaves, stems and roots (Hamman 1985). In case of heavy attack, plants wither resulting in a drastic loss in seed yield and oil contents. Keeping in view the importance of mustard crop and pest infestation present studies were carried out to control the aphid Myzus persicae (Sulzer) through conventional and biopesticides to evaluate the efficacy of Biosal which is environmentally friendly. MATERIALS AND METHOD The experiment was conducted at the grower’s field in Malir (Karachi) in a randomized complete block design (RCBD) with three replicates having four treatments including control plot. Three insecticides viz. Imidachloprid 25 WP (80gm/acre) Endosulfan 35 EC (800 ml/acre) and Biosal (2000 ml/acre) were tested. After pest infestation, the crop was sprayed twice at one week interval with hand operated knapsack sprayer. Pre-treatment data was collected before 24 hours of each spray and post-treatment counts were made after 24 hours, 72 hours and 168 hours. The effectiveness of respective treatments was computed through Henderson and Tilton formula (1955). The data recorded was subjected to statistical analysis using ANOVA and Duncan’s multiple range Test (DMRT). Akbar et al. 28 RESULTS Results showed variation in relative toxicity and persistence among all pesticide treatments. The effectiveness however reduced with time interval in case of Imedacloprid and Endosulfan, while Biosal showed moderate result being less toxic initially, gradually increasing after 72 hours with a decline in effectiveness after 168 hours of each spray. After 1st spray Imidacloprid was found to be more toxic showing percent efficacy (90) after 24 (1st Spray) Table 1 Insecticides Dose hours, (85) after 72 hours and (83) after 168 hours followed by Endosulfan (81) after 24 hours, (72) after 72 hours and (66) after 168 hours, whereas Biosal showed less toxic effect (51) after 24 hours, (61) after 72 hours and (58) after 168 hours (Table 1). Similar trend was observed after 2nd spray showing effectiveness of Imedacloprid after 24, 72 & 168 hours as (88), (81) & (80), followed by Endosulfan (76),(69) & (64) and biosal as (58),(60) & (55) respectively, (Table 2). % Efficacy After different Time Intervals ml/Acre 24 Hours 72 Hours Average 168 Hours Imidacloprid 80 Grams 90 85 83 86 Endosulfan 800 ml 81 72 66 73 Biosal 2000 ml 51 61 58 56 8.7 LSD 0.05 (2nd Spray) Table 2 Insecticides Dose ml/Acre % Efficacy After different Time Intervals 24 Hours 72 Hours Average 168 Hours 80 Grams 88 81 80 83 Endosulfan 800 ml 76 69 64 70 Biosal 2000 ml 58 60 55 57 Imidacloprid 7.5 LSD 0.05 Population Reduction %age Mustard Aphid DISCUSSION 100 80 60 Imidacloprid 40 Endosulfan Azadirachtin 20 0 1 Day 3 Days 1st Spray 7 Days 1 Day 3 Days 2nd Spray 7 Days Efficacy of Imidacloprid and Endosulfan in comparison with Biosal (Biopesticide) against Aphid 29 DISCUSSION profenophos being moderate but yet more effective than Azadirachtin. According to the results, relative effectiveness of the pesticides varied significantly after different time intervals. Imedacloprid and Endosulfan were comparatively more effective than biosal, which being a neem based compound showed less toxic effect may be due to strong antifeedant, insect growth regulatory effect of neem (Azadirachta indica) as referred by Mordue and Blackwell (1993). Similar studies on relative efficacy of different insecticides have shown variable results. Higher persistence and effective control of mustard aphid has been reported (Zheng et al. 1997) by using carbofuran and Endosulfan under field conditions (Begum et al. 1991, Brown et al. 1999). Akbar et. al. (2006; 2007), evaluated the effectiveness of Biosal (neem formulation) in comparison with endosulfan and profenophos against jassid on okra and brinjal at different time intervals and found moderate effect of Biosal against jassid while on okra profenophos found to be more effective than endosulfan and vice versa on brinjal crop against jassid. Hussain et. al. (1992); Akbar et. al. (2005), tested organophosphate(OPs) and pyrethroid insecticides against white fly and jassid on soybean and found OPs more effective than pyrethroids against both insects; as pyrethroids lost their effectiveness after 7 days while OPs were persistent up to 14 days. Naqvi et al. (2006) used Acorus Calmus (AC) rhizome oil and Annona Squamosa (AS) seed oil against sucking pests of cotton as compared to Mospilan and Temaron. AC and AS were less effective than Mospilan and Temaron but with higher dose they gave 20-70% control of thrips, jassid and white fly as compared to control plot. Tariq et al., (2007) reported toxic effects against cotton bollworms using AS and AC as botanical pesticides in comparison with Karate, Polytrin-C and Curacron and found as effective as standards even better against Heliothis armigera after 2 weeks. In case of spotted and pink boll worm AS and AC gave moderate result as compared to Confidor + Deltafos used standard. AS and AC seems less effective than standards used but still AS and AC are safe to use as eco-friendly biopesticides. Narottam (2006) studied the effect of endosulphan and azadirachtin when used alone existed in middle order of effectiveness, whereas it varied in efficacy when used in combination as endosulphan + Bt (Bacillus thurigenesis) and azadirachtin + Bt, however in either case azadiractin was comparatively less effective than endosulfan. Misrah (2002) also used similar combinations of pesticides along with profenophos against aphids and jassids of Okra and observed significant superiority of some pesticides among others, REFERENCES AKBAR, M. F., YASMIN, N., KHAN, QURESHI, M. H. AND NAZ, F. Relative toxicity and persistence of insecticides against jassid, devastans dist. on soybean. Pakistan j. Karachi, 20(1&2): 1-3 M. F., (2005). different amrasca entomol. AKBAR, M. F., YASMIN, N., KHAN, M. F., NAZ, F. AND LATIF, T. A. 2006. Effectiveness of profenophos in comparison with endosulfan against amrasca devastans dist. on okra crop. Pakistan j. entomol. Karachi, 21(1&2): 25-27 AKBAR, M. F., YASMIN, N., NAQVI, S. N. H., KHAN, M. F. AND NAZ, F. (2007). Relative efficacy of biopesticide in comparison with conventional pesticides against Amrasca devastans dist. on brinjal crop. Pakistan j. entomol. Karachi,22 (1&2):1-3 BAKER J. R. (1982). Insect and Related Pests of Flowers and Foliage Plants. The North Carolina Agricultural Extension Service. 75 pp. BEGUM M., HUSSAIN M. AND TALUKDER F.A. (1991) “Relative effectiveness of some granular insecticides against mustard aphid”, Bang. J. Agric. Sci., 18, 49-52. BROWN J.J., MCCAFFEREY P., HARMON B.L., DAVIS J.B., BROWN A.P. AND ERICKSON D.A. (1999) “Effect of late season insect infestation on yield, yield components and oil quality of Brassica napus, B. rapa, B. juncea and Sinapis alba in the Pacific North West region of theUnited States”, J. Agric. Sci., 132, 281-288. HAMMAN P. J. (1985). Aphids on trees and shrubs. L-1227. Texas Agricultural Extension Service House and Landscape Pests. College Station, Texas. 2 pp. HENDERSON C.F. AND TILTON E.W. (1955). Tests with acaricides against the brown wheat mite. Journal of Economic Entomology 48:157-161. HUSSAIN, T., KHAN, M. M., AKBAR, M. F., NAQVI, S. M. S. H. AND RAJPUT, M. A. (1992). Relative toxicity and persistence of different insecticides against white fly, Bemisia tabaci Genn. on soybean. Proc. Pakistan Congr. Zool., vol. 12, pp. 295-297. 30 Akbar et al. MISRAH, P. (2002). Field evaluation of some newer insecticides against aphids (Aphis gossypii) and jassids (Amrasca biguttula biguttula) on okra. Ind. J. Entol. 64(1): 80-84. MORDUE, A.J. AND BLACKWELL (1993). Azadiractin: an update. J. Ins. Physiol., 39: 903924. NAQVI, S. N. H., TARIQ, R. M., ZAFAR, S. M. N., AND ATTIQUE, M. R. (2006). Efficacy of Acorus calamus (AC) rhizome oil and Annona squamosa (AS) seed oil against sucking pests of cotton at CCRI-Multan (Punjab) as compared to Mospilan and Temaron. Pakistan j. entomol. Karachi,21 (1&2):23-27. NAROTTAM, K. M. (2006). Evaluation of some new insecticides and biopesticides against jassid, Amrasca biguttula biguttula on okra, abelmoschus esculentus. Ind. Jor. App. Entol.20: 1. TARIQ, R. M., NAQVI, S. N. H., ZAFAR, S. M. N. AND BURRERO, A. S. (2007). Toxic effects of botanical pesticide, from Acorus calamus(AC) and Annona squamosa (AS) against bollworms at ARI-Tandojam, Sindh-Pakistan, Pakistan j. entomol. Karachi,22 (1&2):31-36. ZHENG B.Z., GAO X.W., ZHAO G.Y. AND CAO B.J. (1997) “Insecticide resistance in turnip aphids, Lipaphis erysimi (Kaltenbach), from Beijing and suburbs”, Resis. Pest. Mngt., 9, 27-28. Pakistan j. entomol. Karachi 23 (1&2): 31-40, 2008 REVISION OF THE GENUS ISOMETRUS HEMPRICH & EHRENBERG (SCORPIONIDA: BUTHIDAE: CENTRURINAE) WITH DESCRIPTION OF TWO NEW SPECIES AND CLADISTIC RELATIONSHIP FROM PAKISTAN RAFAT AMIR AND SYED KAMALUDDIN Department of Zoology Govt. Degree Girls College, 11-B, North Karachi. (R.A), Federal Urdu University of Arts, Science and Technology Gulshan-e-Iqbal Campus (S.K) ABSTRACT The genus Isometrus Hemprich & Ehrenberg is revised to accommodating two new species from Pakistan with special reference to their morphological characters, male genitalia, electrophoresis and gel filtration chromatography of the venom. A key to the sub-genera and 12-species of the genus Isometrus is also formulated. The new species are also compared to their closest allies. Key Words: Isometrus, New species, Buthidae, Key, electrophoresis, chromatography, cladistic relationship, Pakistan. INTRODUCTION The representatives of the genus Isometrus are disturbed in Tropical areas of the world like India, Pakistan, Srilanka, Burma, Java, Australia, Africa, South America and South Sea Islands. Both new species are recorded from Pakistan, Sindh. Pocock (1900) has been described the genus Isometrus, later Vachon (1969 & 1972) worked on Isometrus. Tikader and Bastawade (1983) have been described the genus Isometrus from India and proposed two genus on the basis of trichobothrial pattern on pedipalpi. They also formulated a key to the sub-genera and a key of nine species from India. MATERIALS AND METHOD (1972), Cah. Pacif, 16: 169-180; Stahnke (1972), Ent. News. 83: 121-133. Diagnostic features: Carapace sometimes with only median carina, mesosomal tergites monocarinated, inferior surface of cheliceral fixed finger with one tooth, cephalothracic sternum small, triangular and not as long as genital operculum, caudal region slender, vesicle as wide as metasomal segment V and with a strong, triangular subaculeus spine at the base of aculeus. Pedipalpi tarsus (movable finger) with 5-6 distinct median oblique rows of granules (nonimbricated) and a short apical depressed and fringed with setae. Trichobothria dorsal 1, dorsal 3 and dorsal 4 on femur from β angle. Comparative note: The animals were collected from the Korangi, Sindh Pakistan and were killed with the help of formalin and preserved in 70% Alcohol. For the sudy of male genitalia the specimens were dissected out by removing the tergites of mesosoma. After dissection the aedeagus was mounted on slide then taken the photograph using microscope and photographic camera. For the study of electrophoresis and chromatography the technique generally followed by Amir et al. (1994 a,b,c,d, 1995, 2003 and 2004). RESULTS Genus: Isometrus Hemprich and Ehrenberg Ismetrus Hemprich and Ehrenberg (1829). Phys. Scorp., 3; Kraepelin (1891), Jb. Hamb. Wiss. Anst. 8: 244; Pocock (1900), Fauna Brit. India, Arachn., 4445; Vachon (1969), Senckenberg Biol. 50: 417-420; The genus Isometrus is the only genus of the sub-family Centrorinae Kreplain. The members of this genus can easily be separated in having mesosomal tergites monocarinated, inferior surface of cheliceral fixed finger with one tooth, trichobothria dorsal 1, dorsal 3 and dorsal 4 on femur from β angle and by the other characters as noted in the description. Key to the Subgenera and species of the genus Isometrus Hemprich and Ehrenberg. 1. Trichobothria db placed always proximal to et but distal to est, vesicle is not bulbous...I. Raddyanus Vachon............................................2 - Trichobothria db placed always distal to et, vesicle is more bulbous..................................................................................I. Closotrichus.........................................................................11 32 Rafat Amir & Syed Kamaluddin segments II and III may or may not be ending posteriorly into an upstanding procurved spines, sub-aculeus spine provided with three pairs of small denticulate granules on inner margin..............................................................10 2. Metasoma 5 to 6 times as long as carapace, vesicle twice as long as aculeus...............................................................3 - Metasoma 7 to 8 times as long as carapace, vesicle more than twice as long as aculeus...............................................................8 - 3. Metasoma always 5 times as long as carapace, sub-aculeus spine provided with one pair of small denticulate granules on inner margin................................................................4 - Metasoma always 6 times as long as carapace, sub-aculeus spine provided with one or three pairs of small denticulate granules on inner margin................................................................5 4. Mesosomal tergites with 5 black and 6 yellow bands, vesicle globular and as long as wide……………………......I. (R) rigidulus Pocock - Mesosomal tergites with a pair of conspicuous median yellow bands, vesicle elongated and longer than wide.................................................6 10. Ratio of median eyes to anterior and posterior margins of carapace 1:1.9, vesicle tapering distally and much granules on ventral surface, fingers of pedipalpi short and stout, trichobothria ei placed proximally to d5 on femur, dorsal carinae of metasomal segments II and III ending posteriorly into very strong, procurved and upstanding spines….... /. (R) acanthurus Pocock - 5. Entire surface very weakly and finely granular, pectinal teeth 12-13............ /. (R) vittatus Pocock - Entire surface of carapace weakly granular, 32 pectinal teeth...........................I. (R) atherii sp. n. 6. Subaculeus spine provided with only one pair of small denticulate granules on inner margin, manus of pedipalpi smooth and acarinated...........................................................7 - Subaculeus spine provided with three pairs of small denticulate granules on inner margin, manus of pedipalpi granular and strongly carinated..................I. (R) brachycentrus Pocock 7. Pectinal teeth 15/16, body size more than 35 mm in length, body colour yellowish variegated with blakish brown patches, manus of pedipalpi short, stout in male as well as in female.............................. I. (R) thrustoni Pocock - Pectinal teeth 11/12, body size not more than 30mm in length, body color brownish variedated with yellow patches, manus of pedipalpi very long in male.................. I. (R) isadensis Tikader & Bastawade 8. Metasoma 8 times as long as carapace, subaculeus spine provided with one pair of small denticulate granules on inner margin, manus of pedipalpi very long and thin in male, pectinal teeth 17/17............................ /. (R) europaeus L. - Metasoma 7 times as long as carapace, subaculeus spine provided with two or three pairs of small denticulate granules on inner margin, manus of pedipalpi short and stout in male, pectinal teeth less than 17/17.............................9 9. Aculeus very short and less than one third of vesicular length, dorsal carinae of metasomal Aculeus slightly longer and less than half the length of vesicle, dorsal carinae of metasomal segments II and III posteriorly spiniform, subaculeus spine provided with two small denticulate granules on inner margin………………...............assamensis Oates Ratio of median eyes to anterior and posterior margins of carapace 1:2.25, vesicle globular on distal portion and very weakly granular on ventral surface, fingers of pedipalpi thin and long, trichobothria e1 placed in a same plane to d5 on femur, dorsal carinae of metasomal segments II and III ending posteriorly into a very small sub-denticular granules……...I. (R) corbeti Tikader and Bastawade 11. Body variegated with black to brown bands and spots, entire surface coarsely granular, pectinal teeth 15-16….…….I. (C) sankeriensis Tikader and Bastawade - Body generally mustered yellow, entire surface granular, pectinal teeth 29….. I (C) liaqatii sp. n. Isometrus (Raddyanus) atherii sp.n. (Figs. 1-11) Colouration: Body generally yellow. Prosoma (Fig.3): Entire surface of carapace weakly granular, all carinae granular, ocular tubercles reddish brown and granular, anterior margins smooth and provided with 30-33 small reddish setae, lateral margins granular but more granular on anterior portion. Pedipalp (Figs. 4a-4c): Manus stout, longer than femur, shorter than carapace, almost all carinae strongly granular and outer and anterior side provided with a crenulated crest, 16-19 denticular tubercles, patella longer than femur but shorter than carapace, carinae on outer side granular, inner or anterior surface provided almost granular strong crest with 16 sub-denticular tubercles, manus or hand flat and stout, length of underhand longer than femur, fixed finger almost as long as femur but Revision of the genus Isometrus Hemprich & Ehrenberg (Scorpionida: Buthidae: Centrurinae) movable finger longer than carapace, dentition on the fingers consisting of two rows of imbricated teeth, granular on the fixed and movable finger, trichobothrial pattern of pedipalp 'A' type. Legs (Fig 5): Femur, patella smooth and carinae crenulated, tibiae with strong tibial spurs, on the legs III and IV, size 0.10 cm., pedal spurs spiny, tarsomere I laterally smooth, tarsomere II smooth and furnished below with one row of bristles on ventral surface. Pectin (Fig. 7): Pectin well developed and almost two times longer than wide, seven middle lamellae present, fulcra nearly triangular, pectin pale yellow with 32 teeth. Genital operculum (Fig. 8): Genital operculum wider than long and sclerites slightly divided on posterior portion from which small genital papillae produced in male, sternum small and triangular. Mesosoma: All tergites more smooth on posterior portion of each tergite, sternites I-IV smooth and each provided with slit-like stigmata for book lungs. Metason: Cauda four times as long as carapace, first segment shorter than wide, segments I-IV with dorsal carinae crenulated, dentiform on posterior portion, much more elevated on segment III and IV, dorsolateral carinae evenly crenulated, lateral carinae weakly developed only on posterior portion of segment III-IV. Telson (Fig. 6): Telson with vesicle not as wide or deep as segment V, ventral surface densely smooth, ventral median crest developed, sub-aculeus nodule absent, aculeus weakly curved, as long as vesicle. Male genitalia (Fig. 9): Flagellum 0.71 mm long, elongated, elastic and flageller-like, trunk 0.52 mm long, 0.12 mm wide, trunk cylindrical basally dilated, pedicel very flat, 0.2 mm long, 0.1 mm wide, sperm spine blunt, sperm tube narrowed sclerotized. Material examined: Holotype, Male, Pakistan, Shikarpur (Sindh), 7.12.93, leg. Rafat Amir, lodged at MEMUK No. 86. Paratypes: 7 females, other data same as holotype, lodeged at ZMUK. Comparative note: This new species is most closely related to Isometrus (Raddyanus) vittatus (Pocock) in having mesosomal tergites with a pair of conspicuous median yellow bands, vesicle elongated and longer than wide but it can easily be separated from the same in having entire surface of carapace weakly granular, 32 pectinal teeth are present and 33 by the other characters as noted in the key and description. Isometrus (Closotrichus) liaqatii sp.n. (Figs. 12-22) Colouration: Body generally mustered colour. Prosoma (Fig. 14): Entire surface of carapace granular, all carinae weakly granular, ocular tubercles dark brown and black, anterior margins smooth and provided with 23-44 small brownish setae, lateral margins crenulated on anterior portion. Pedipalp (Figs. 15a-15c): Manus slender shorter than femur and carapace, almost all carinae weakly granular, outer and anterior side provided with a crenulated crest of 23-25 denticular tubercles, patella longer than femur but always shorter than carapace, inner or anterior surface provided almost granular crest with 22 sub-denticular tubercles, manus or hand slender and length of underhand longer than femur, fixed finger almost as long as femur but movable finger shorter than carapace, dentition on the fingers consisting of three rows of imbricated teeth, granular on the fixed and movable finger, trichobothrial pattern of pedipalp 'A' type. Legs (Fig. 16): Femur weakly granular, patella smooth and carinae crenulated, tibiae with two strong tibial spurs on the legs III and IV, size 0.1 cm, pedal spurs spiny, tarsomere I laterally smooth, a pair of pedal spurs present, tarsomere II flanked below, distal portion with a few bristles. Pectin (Fig. 18): Pectin well developed and almost two and one fourth times longer than wide, eight middle lamellae present, fulcra nearly triangular, fulcra and lamellae clothed with microscopic hairs, pectin light yellow with 29 teeth. Genital operculum (Fig. 19): Genital operculum wider than long and sclerites slightly divided on posterior portion from which small genital papillae produced in male, sternum small and triangular. Mesosoma: All tergites granular but more granular on posterior portion of each tergite, sternites I-V granular and each provided with slit-like stigmata for book lungs. Metasoma: Cauda four times as long as carapace, first segment shorter than wide, segments I-IV with dorsal carinae crenulated, dentiform on posterior portion, much more elevated on segment III and IV, dorso-lateral carinae evenly crenulated, lateral carinae strongly developed only on posterior protion of segment III-IV. Rafat Amir & Syed Kamaluddin 34 Telson (Fig. 17): Telson with vesicle not deep as segment V, ventral surface densely granular, ventral median crest not developed, sub-aculeus nodule absent, aculeus not curved, as long as vesicle. Male genitalia (Fig. 20): Flagellum 0.6 mm long, membranous, trunk cylindrical and blunt, 0.4 mm long and 0.15 mm wide, sperm spine typically blunt, sperm tube narrowed and short. Material examined: Holotype, Male, Pakistan: Korangi, (Karachi), 9.7.1993, leg. Rafat Amir, lodged at MEMUK No. 148. Paratypes: 19 females, other data same as holotype, lodged at ZMUK. Comparative note: This new species is most closely related to Isometrus (Closotrichus) sankeriensis in having entire trichobothria db placed always distal to et and vesicle is more bulbous but it can easily be separated from the same in having entire surface of carapace granular, 29-pectinal teeth are present and by the other characters an noted in the key and description. Polyacrylamide Gel Electrophoresis of Scorpion Venom Polyacrylamide gel electrophoresis of scorpion venoms were performed under denaturing condition using SDS-70L standard protein markers were used to prepare standard curve. The marker include bovine serum albumin (66.0 KDa), egg albumin (45.0 KDa), glyceraldehydes β phosphate, dehydroginase (36.0 KDa), carbonic anhydrase (29.0 DKa), trypsinogen (24.0 KDa), trypsin inhibiter (20.1 KDa), and X- lactalbumin (14.2 KDa). All venom samples showed multiple bands in the molecular weight range of (70.0 to 14.0 KDa). Venom of Isometrus species (Figs. 10 & 21) showed few broad bands. Gel filtration venom: chromatography of scorpion 1000 mg of each of two Isometrus atherii sp.n., and /. iqbalis Sp,n., venoms were loaded on sephadex G-50 column (2.5x90.0 cm) and eluted with 0.1 M ammonium acetate buffer (Ph 6.90). Both venoms were resolved into four peaks, representing the presence of high to moderate molecular weight components. Isometrus atherii (Fig. 11) posses two major (I and III) peaks, whereas Isometrus liaqatii (Fig. 22) into three major (I to III) and one moderate peak (IV). TABLE 1 Measurement in cm/mm meristic characters of the male holotype Isometrus (Raddyanus) atherii sp.n. Characters Total length Carapace length Mesosoma length Metasoma length I segment length/wide II segment length/wide Ill segment length/wide IV segment length/ wide V segment length/wide Telson length Vesicle length/wide Aculeus length Pedipalp length Femur length/wide Patella length /wide Chela length/wide Fixed finger length Movable finger length Chelicera, Chela length/wide Fixed finger length Movable finger length Pectinal tooth count Male genitalia Holotype Male 4.5 cm. 0.6 cm. 1.4 cm. 2.5 cm. 0.4/0.26 cm. 0.45/0.30 cm. 0.5/0.33 cm. 0.55/0.36 cm. 0.6/0.4 cm. 0.6 cm. 0.3/0.3 cm. 0.3 cm. 1.7 cm. 0.5/0.2 cm. 0.6/0.3 cm. 0.9/0.3 cm. 0.5 cm. 0.52 cm. 0.2/0.1 cm. 0.11 cm. 0.12 cm. 32 1.25 mm TABLE 2 Variation in tarsomere II spine counts in Isometrus (Raddyanus) atherii sp.n. on each specimen, the spine of the left and right legs of each pair were counted Legs Margin 4 5 6 7 8 I Prolateral 5 8 4 7 5 Retrolateral 5 7 6 7 7 Prolateral 4 4 6 9 5 Retrolateral 5 5 6 8 6 Prolateral 5 7 5 4 6 Retrolateral 6 6 5 5 7 Prolateral 5 5 5 7 5 Retrolateral 5 5 4 4 7 II III IV Revision of the genus Isometrus Hemprich & Ehrenberg (Scorpionida: Buthidae: Centrurinae) 35 TABLE 3 Measurement in cm/mm meristic characters of the male holotype Isometrus (Closotiichus) liaqatii sp.n. Characters Total length Carapace length Mesosoma length Metasoma length I segment length/wide II segment length/wide Ill segment length/wide IV segment length/ wide V segment length/wide Telson length Vesicle length/wide Aculeus length Pedipalp length Femur length/wide Patella length /wide Chela length/wide Fixed finger length Movable finger length Chelicera, Chela length/wide Fixed finger length Movable finger length Pectinal tooth count Male genitalia Holotype Male 4.6 cm. 0.65 cm. 1.2 cm. 2.76 cm. 0.50/0.2 cm. 0.53/0.22 cm. 0.55/0.25 cm. 0.58/0.27 cm. 0.6/0.3 cm. 0.6/0.3 cm. 0.3/0.3 cm. 0.3 cm. 1.7 cm. 0.45/0.2 cm. 0.5/0.25 cm. 0.85/0.25 cm. 0.45 cm. 0.50 cm. 0.35/0.2 cm. 0.13 cm. 0.15 cm. 29 1.3 mm. DISCUSSION The genus Isometrus Hemprich and Ehrenberg, is an only genus of the sub-family Centrurinae Kraepelin, and distributed in Oriental, Australian, Ethiopean and Neotropical regions. The sub-family Centrurinae plays sister group relationship with Buthinae by their synapomorphies like dorsal arm of movable fringes of chelicerae furnished with four minute teeth on inner margin and trichobothrial pattern is A-type, but plays out group relationship by its autapomorphy III and IV pairs of leg without tibial spur. Presently (Fig. 23) the genus Isometrus includes twelve species which fall into two groups. The first group includes only two species viz. sankeriensis and liaqatii which plays sister group relationship with each other and out group relationship with second group, which includes ten species viz. rigidulus, vittatus, atherii, bradycentrus, thrustoni, isadensis, europaeus, corbeti, acanthurus and assamensis. The second group further fall into two sub-groups. Among first sub-group the corbeti and acanthurus play sister group relationship with assamensis, further all these three species play sister group relationship with each other and out group relationship with europaeus. TABLE 4 Variation in tarsomere II spine counts in Isometrus (Raddyanus) liaqatii sp.n. on each specimen, the spine of the left and right legs of each pair were counted Among second subgroup, the thrustoni plays sister-group relationship with isadensis and out group relationship with bradycentrus. All these play sister group relationship with rigidulus, vittatus and atherii play sister group relationship with each other and out group relationship with rigidulus. ILLUSTRATION OF FIGURES Legs Margin 4 5 6 7 8 I Prolateral 6 4 5 4 4 Retrolateral 5 5 5 5 5 Prolateral 6 3 5 7 5 Retrolateral 4 3 4 5 3 Prolateral 7 5 5 5 6 Retrolateral 4 4 7 4 3 Prolateral 7 6 8 5 5 Retrolateral 5 3 4 4 4 II III IV Figs. 1-11. Isometrus atherii (sp. n.): 1. entire, dorsal view; 2. same, ventral view; 3. prosoma, dorsal view; 4a-4c. pedipalp, lateral view, a. femur, b. patella, c. hand; 5. leg, lateral view; 6. telson, lateral view; 7. pectin, ventral view; 8. genital operculum, ventral view; 9. aedeagus, lateral view, 10. showing electrophoresis of venom; 11. showing chromatography of venom. Figs. 12-22. Isometrus liaqatii (sp.n.): 12. entire, dorsal view; 13. same, ventral view; 14. prosoma, dorsal view; 15a-15c. pedipalp, lateral view; a. femur, b. patella, c. hand; 16. leg, lateral view; 17. telson, lateral view; 18. pectin, ventral view; 19. genital operculum, ventral view; 20. aedeagus, lateral view; 21. showing electrophoresis of venom; 22. showing chromatography of venom. Fig. 23. Cladogram showing relationship of the included taxa. 36 Rafat Amir & Syed Kamaluddin Revision of the genus Isometrus Hemprich & Ehrenberg (Scorpionida: Buthidae: Centrurinae) 37 38 Rafat Amir & Syed Kamaluddin Revision of the genus Isometrus Hemprich & Ehrenberg (Scorpionida: Buthidae: Centrurinae) 39 Rafat Amir & Syed Kamaluddin 40 REFERENCES AMIR, R., ALAM. J.M. AND KHAN, M.A.J. (l994a). comparative study of two toxins with phospalipase A^ activity isolated from the venom of Androctonus australis. Pakistan J. Zool. 26 (2): 127-133. AMIR, R., ALAM. J.M. AND KHAN, M.A.J. (l994b). Investigation on scorpion venoms as naval insecticides. Pakistan J. entomal Karachi, 9: 109-114. AMIR, R., ALAM. J.M. AND KHAN, M.A.J. (l994c). proloagulent property of venoms of some medically important scorpion from Sindh region. Pakistan J. Zool. 26(3): 261- 263. AMIR, R., ALAM. J.M. AND KHAN, M.A.J. (l994d). Comparative studies on the enzymatic content of venom from fifteen scorpion species from Sindh region. Pakistan J. Zool. 26 (1): 77-79. AMIR, R., ALAM. J.M. AND KHAN, M.A.J. (1995). Insecticidal activity of Toxic components from scorpion venoms and their effects on cationic concentration of Haemalymph of Periplanata Americana. Pakistan j. entomol. Karachi, 70:45-49. AMIR, R., KAMALUDDIN, S. AND KHAN, M.A.J. (2003). Redescription of Androctonus sp. (Sorpionida: Buthidae) from Sindh, Pakistan with special reference to its genitalia and chemical analysis of venom. J. nat. hist. wildl. 2 (2): 21-25. AMIR, R., KAMALUDDIN, S. AND KHAN, M.A.J. (2004). Redescription of Odontobuthus doriae odonturus (Pocock), (Arachnida: Scorpionida: Buthidae) from Pakistan with special reference to its genitalia, chromatography and electrophoresis of venom. J. nat. hist. Wildl. 3 (1): 17-21. POCOCK, R. L. (1900). Fauna of British India, Arachnida, London: 1-279. STAHNKE, H. I. (1972). A nomenclatural conundrum. Ent. News. 83:121-133. TIKADER, B.K. AND BASTAWADE, D.B. (1983). The fauna of India. Scorpion (Scorpionida & Arachnida). Zool. Ind., 3: 1-671. VACHON, M. (1969). Complement a la description C’ Isometrus madagassus Roewer 1943 (Scorpiones: Buthidae). Sencken-berg. boil. 50 (5-6): 417-420. VACHON, M. (1972). Sur l’ establissement d'une nomenclature trichobothriale uniforme convenout existence de trios types distinct de trichobothriotaxie, C. r. hebd. Seunc. Acad. Sci. paris, 275D (18): 2001-2004. Pakistan j. entomol. Karachi 23 (1&2): 41-46, 2008 TAXONOMIC STUDIES OF SERGENTOMYIA BAGHDADIS (ADLER AND THEODOR) (DIPTERA: PSYCHODIDAE) IN SINDH AND PUNJAB AND COMPARISION WITH OTHER PAKISTANI SPECIES OF THE SUBGENUS PARROTOMYIA THEODOR JUMA KHAN KAKARSULEMANKHEL Investigator of Sandflies, Leishmaniases, Helminths, Ticks & Mosquitoes, Department of Zoology, University of Balochistan, Saryab Road, Quetta, Pakistan. [email protected] Cell # 0333-7860240 ABSTRACT During the routine sandfly survey, Sergentomyia (Parrotomyia) baghdadis (Adler and Theodor) was collected for the first time from new epidemic localities of cutaneous leishmaniasis in Sindh and Punjab In view of the published reports about the detection of encephalitis viruses from the species of the genus Sergentomyia Franca and Theodor, from the Indian localities, the correct identification of sand fly species, becomes of significant value in the study of epidemiology of leishmaniases and other viral diseases. Therefore, in order to facilitate Zoologists and Medical researchers in correct identification, taxonomic characters of S. baghdadis were studied in detail with special reference to its mouth parts, male and female genitalia and findings are presented in the present paper. A comparative note of taxonomic characters of this species with its closest allies has also been given. Key words: Sandflies, Sergentomyia baghdadis, Parrotomyia, Sindh, Punjab. INTRODUCTION Pakistan has several endemic forms of leishmaniases, a protozoan disease transmitted by the bite of infected sand flies. The disease is spreading continuously and sand flies are being recorded from new localities. Lewis (1967,1978) reported the prevalence of five species of sand flies of the subgenus Parrotomyia Theodor of the genus Sergentomyia Franca and Theodor in the country viz. S. africana (Sinton), S. shorttii (Adler and Theodor), S. grekovi (Khodukin) and S. palestinensis (Adler and Theodor) including the present one. However, S. grekovi, S. palestinensis and S. baghdadis (Adler & Theodor) have also been reported from the Balochistan Province (Kakarsulemankhel, 2004,2006). Previously, several viruses have been found in sand flies (Lewis, 1978). In view of the recently published reports about the detection of encephalitis viruses from the species of the genus Sergentomyia Franca and Theodor from the Indian localities and their possible role in kala-azar transmission (Geevarghese et al., 2004), the correct identification of the species becomes of significant value in the study of epidemiology of leishmaniases and other viral diseases. Lewis (1967) and Artemiev (1978) while studying Pakistani and Afghani sand flies respectively, did not furnish morphometric measurements of mouth parts (proboscis, mandible, hypopharynx, maxilla, buccal cavity, pharyngeal armature), female genitalia and male terminalia. Sketches of mandible, hypopharynx and maxilla were also not furnished. Aslamkhan et al. (1997, 1998) reported S. baghdadis from Lehri, Sangsila and Dera Bugti areas of Balochistan province but they neither described nor illustrated morphology of taxonomic characters. Therefore, to fill this gap, morphometric measurements and photographs of diagnostic structures of S. baghdadis as additional information, are being given here. A comparative note of its taxonomic characters with its closest allies is also given. MATERIALS AND METHODS During the routine entomological collection of sand flies in Sindh & Punjab Provinces in 2006 the author has collected several hundred Phlebotmine sandflies including S. baghdadis and its closest allies. The present investigation was carried out on the materials (35 specimens of S. baghdadisi) collected from Sindh and Punjab Provinces during May, 2006 with sucking tubes and sticky traps. The collected materials was preserved, processed and dissected by conventional methods (Young and Duncan, 1994). Identification of specimen was carried out with the help of available literature (Lewis, 1967, 1978; Artemiev, 1978). Morphometric measurements and photographs were taken from camera mounted Olympus microscope (BX41). Most of the structures were measured with a low magnification (X100) whereas spermatheca, ducts and furca were examined under high magnification Juma Khan Kakar 42 (X400). All given measurements are in mm. Prepared permanent slides were deposited with the author’s collection of sand flies, Department of Zoology, University of Balochistan, Quetta. RESULTS The subgenus Parrotomyia Theodor includes babu (Annandale), barraudi (Sinton), grekovi, palestinensis, shortii (Adler and Theodor), montana (Sinton), bailyi (Sinton) and the present one. However, Kakarsulemnkhel (2004a, 2006) reported babu, grekovi, and palestinensis from Balochistan Province and examined them. The sub genus is defined on the basis of following characters: Comb-like buccal armature, lamp-glass shaped pharynx, and elliptical capsular spermatheca, male terminalia with 2 apical and two sub apical spines on style or all spines apical. 1978). Mandible (X100) also blade like, 0.16-0.17 long, distal end of the mandible sharply pointed and its inner margin carries small teeth, dental depth 0.015. Maxillary blade (X100) 0.16-0.17 long, stout based, but gradually narrows towards its apex, maxilla containing two rows of teeth, about 11 widely spaced lateral teeth on outer edge near the apex and its dental depth 0.035 and on inner edge after some distance from the apex there is another row of about 30 ventral teeth, its dental depth 0.08. Cibarial cavity (Fig. 1A) (X100) 0.05 broad, 12-14 comparatively larger and pointed horizontal teeth arranged in a concave line and 3-4 smaller median teeth. Pigment patch of varying shape and size, also present. Buccal plate shows a marked notch of varying shape in the middle line. Posterior to the buccal cavity, lies a large chitinous structure, pharynx (X100) 0.13-0.15 long, anterior breadth 0.04-0.05, and 0.06-0.07 posterior breadth, several toothed lines in the form of armature present at the base of the pharynx. Female genitalia (X400) (Fig. 1B) Sergentomyia (Parrotomyia) baghdadis Adler and Theodor, 1929 (Fig. 1A- 1B, 2A-2F) Phlebotomus baghdadis Adler and Theodor, 1929, Ann. Trop. Med. Parasit., 23: 281. Sinton, 1932. Indian J. Med. Res., 20: 60; 1933.21: 422-423. Sergentomyia (Parrotomyia) baghdadis Adler and Theodor, Lewis, 1967, Bull. Brit. Mus. Nat. Hist. (Ent.)19: 30, Artemiev, 1976. Medskaya Parazit. 45: 424; Artemiev, 1978. Ministry of Health, Afghanistan, Kabul, 29. Lewis, 1978, Bull. Brit. Mus Nat. Hist. (Ent.), 37: 258. Female Wings, Palps, Antenna (X100) Wings 1.3-1.35 long, breadth 0.3-0.33, α / β=0.38, δ=0.05, gamma=0.25, П=0.13. Palps (X100) 0.62-0.65 long, ratio 1,2.6,4,3.2,8.3, formula 1,2,3,4,5. Antennal segment (X100) A3 0.14-0.16 and A4 and A5 each 0.08-0.09. Ascoid formula 2 over A3-A15. Ascoids comparatively shorter (X400) 0.12 long and their positions (X400) on A3 0.71, on A4 and A5 0.36. Papilla formula 1over A3-A5. Positions of ascoids 0.79 and 0.75 respectively on A3 and on A4 and A5 each. Mouth parts, Cibarium, Pharynx (X100) (Fig. 1A) Proboscis 0.2-0.21 long. Labrum hard sword shaped structure, (X100) 0.15-0.16 long, relatively narrow, sides parallel, apex bluntly pointed, margins furnished with a series of long leaf like sensillae closely together and numbering about 10 on either side. Hypopharynx (X100) 0.15-0.16 long, also a blade like structure, perforated by a narrow salivary duct, apical end pointed, margins with short, thin and soft serrations. Hypopharynx with soft serrations is a character of the genus Sergentomyia (Artemiev, Spermatheca (Fig.1B) 1.08-1.16 long, its head is hard. 0.24 broad and plug-shaped while its base is quite broader (0.64). Individual spermathecal duct (X400) 1.2 long, arising from each spermatheca and combine with one another to form a common duct (1.1) finally falling in genital atrium (breadth 0.72). Furca length 1.4. Male Wings, Palps and Antenna (X100) Wings 1.35-1.37 long, 0.3-0.32 broad, length /breadth 4.28--4.5, α / β=0.46, δ=0.02, gamma=0.23, П=0.09. Palpas (X100): 0.48-0.5 long, ratio 1,3.5,4,5,10 and formula 1,2,3,4,5. Antennal segment 3 (X100) 0.15-0.16 long, A4 0.08 and A5 0.09 long, ascoid formula 1 over A3-A15, ascoid (X400) very small in length (0.36 long), position of ascoid on A3 0.65 and on A4 and A5 0.28. Mouth parts, Cibarium, Pharynx (X100) (Fig. 2A) Proboscis (X100) 0.17 long. Labrum: 0.10-0.11 long. Hypopharynx and maxilla each 0.1-0.11. Mandible absent. Cibarium (X100) (Fig.2A): cibarial cavity sharply angular, 0.03 long, below the ventral plate a row 13-15 of minute and weak horizontal teeth present arranged on a concave line, teeth some times invisible. Pharynx (X100): 0.1-0.12 long, anterior 0.03 and 0.05 posterior breadth, armature very weak and confined at base of pharynx. Male genitalia (X100) (Fig. 2B) Coxite 0.2-0.22 long, 0.06-0.07 broad, style 0.1 long, 0.03 broad, a small ventral seta at 0.7 on style. Paramere (Fig.2C) with blunt end, 0.12-0.13 long, 0.03 broad, narrow neck starts at 0.83 of length of paramere, lower corners of paramere extending downwards. Surstyle (Fig.2D) 0.16-0.17 long, apical ends of paramere and surstyle almost at same level. Taxonomic studies of Sergentomyia baghdadis (Adler and Theodor) in Sindh & Punjab Filaments (F) with transverse striations. Sperm pump (P) head (Fig.2E) like a semicircular funnel. F/ sperm pump=2.5. Aedeagus sheath (Fig.2F) short, 0.070.08 long, apical end of aedeagus sheath rounded, colorless but slightly curved ventrally. Material examined 18 ♀, 15 ♂, Sindh, Punjab, May, 2006. Distribution Iraq, Iran, southern Afghanistan, Pakistan. Present study, new record: Sindh: Dadu, Jacobabad, Khairpur Nathan Shah, Mehar, Multan, Nau Shehro Feroz, Qambar Ali Khan, Sehwan Shareef, Sukkur. Punjab: Dera Ghazi Khan, These are endemic foci of cutaneous leishmaniasis (CL). British Museum (Natural History): Dera Ismail Khan, Jhelum, Kandhkot, Lahore, Lyallpur, Pano Aqil, Peshawar, Sargodha, Tank. Landi Kotal, Mir Mohammad, Rawalpindi, Said Pur, Taxilla (Lewis, 1967). Comparative note The babu group (babu and baghdadis) is entirely isolated with apomorphies of presence of hind notch in the ventral plate of cibarium which is of particular importance because both species have better developed characters. Their oblong spermatheca with broader distal end isolates them among its subgroups. They also share synapomorphies of hind notch at ventral plate of cibarium. The two species, however, are clearly separated on the bases of buccal teeth. S. baghdadis appears unique in this group with aut-apomorphy of presence of smaller median teeth in the cibarium. The grekovi and palestinensis-group maintain their separate identity but having synapomorphies of buccal teeth arranged on a straight line, hind notch at ventral plate lost, presence of large triangular pigment patch, almost spherical spermathecae and smooth genital filaments. S. grookovi can be easily differentiated for its autapomorphies of short genital filaments and pharyngeal armature directed obliquely down. The palestinensis can be discriminated by its autapomorphic characters of about 15-18 parallel, arrow like buccal teeth, each with nodular thickening near its center and also with a second row of about 19-21 punctiform denticles at the base of the longer ones and with pharynx much dilated posteriorly. However, S. baghdadis shares some of its characters such as buccal cavity with 20 hardly visible teeth arranged in concave lines, pigment patch very pale or absent, genital filaments with transverse striations in male, comb-like cibarial armature, lamp-glass shaped pharynx, and long spermathecal capsule, ventral plate of cibarium with angular deep notch at center. It is distinct, however, in its group in having 15-18 buccal teeth including 34 very small median teeth and weak pharyngeal 43 armature, comparatively longer spermatheca with larger head and basal part of capsule much broader is closely related to S. babu (Annandale) in which male cibarium with about 20 hardly visible uniform teeth arranged in concave line, there is additional row of smaller denticles at their bases, pigment patch very small or absent, paramere with slightly beaked end and shorter than the length of surstyle, lower corners of paramere extending downwards, in female ventral plate of cibarium with angular deep notch and about 28 sharply pointed uniform horizontal teeth arranged on a concave row but S. baghdadis can easily be separated from the same in having 12-14 lateral cibarial teeth and 3-5 very small in the center, pharyngeal armature weak, comparatively longer spermathecal capsule with larger head and basal part of capsule much broader. DISCUSSION Results of the present study were compared with the published data of S. baghdadis (Adler and Theodor) from other territories (Table 1). Length / breadth ratio of the wings of our specimens (♀= 4.04.3; ♂=4.2-4.5) were observed to be shorter than of the results (♀= 4.5-4.7; ♂=4.7-4.9) reported by Lewis (1967). Similarly, A3 of present samples were found a little smaller (♀= 0.14-0.15; ♂=0.15-0.16) than of the findings (♀= 0.15-0.17; ♂=0.14-0.19) given by Lewis (1967). Number of cibarial teeth in Pakistani flies whether in present study or in the past study conducted by Lewis (1967), were almost same and less in number (about 18) however, more cibarial teeth (20-25) were reported by Artemiev (1978) in Afghanistan S. baghdadis. F/ sperm pump ratio in Pakistani ♂ S. baghdadis were observed almost similar whether in present study or in past study of Lewis (1967). Though minor variations in morphometric measurements of taxonomic characters were found when were compared with published data from other territories, however, present work is in conformity with the findings of Lewis (1967) and Artemiev (1978). The effect of different ecological factors like temperature, relative humidity ecological niche on the growth and size of structures of flies can not be ruled out. Belazzoug et al.(1982) while working in different ecological zones of Algeria has shown that number of cibarial teeth varies according to certain climatic factors (mainly humidity). It is hoped that present findings would provide the basis for further research on sand flies taxonomy in the country and indirectly also on other aspects that are essential for the control of sand flies and the disease leishmaniases. Keeping in view of its presence in human residences in the areas of C.L. in Sindh and Punjab Provinces, vectorial role of S. baghdadis needs to be further investigated. 44 Juma Khan Kakar Fig.1. Female Sergentomyia (Parrotomyia) Baghdadis (Adler and Theodor): A, Cibarium (X400); B, Spermatheca (X400). Fig.2. Male Sergentomyia (Parrotomyia) baghdadis (Adler and Theodor): A, Cibarium (X400); B, Male Genitalia (X200); C, Paramere (X200); D, Surstyle (X200); E, head of spermathecal pump (X400); F, Paramere (X200) and Aedeagus (X400). Taxonomic studies of Sergentomyia baghdadis (Adler and Theodor) in Sindh & Punjab Table-1. Comparison of Taxonomic Characters (mm) of S. baghdadis (Adler & Theodor) Characters Sindh Sand flies Present Study Sinton (1932) Lewis (1967) Afghanistan (Artemiev, 1978) ♀ ♂ 1.30 – 1.35 1.35 – 1.37 - 1.51- 1.72 1.39 – 1.64 - ♀ ♂ 4. 0 – 4. 3 4. 2 – 4.5 - 4. 5 – 4. 7 4. 5 – 4. 9 - ♀ ♂ 0. 38 0. 46 - 0. 3 – 0.8 - - ♀ ♂ 0. 14 – 0. 15 0. 15 – 0. 16 - 0. 15 – 0. 17 0. 14 – 0. 19 - ♀ ♂ 0. 93 – 1. 0 1. 45 – 1. 50 - 1.0 – 1. 1 1. 0 – 1. 2 - ♀ ♀ 11 30 - - - ♀ 12-14 larger + 3-4 smaller, median 13-15 hardly visible 16-18 7 large, each side + 4 small in centre, 16-20 +4-5 central, Scarcely visible 14- 16 2. 5 2. 5 Beaked, Lower corners extending downwards Apical end rounded, but slightly curved ventrally colorless - - - - Wing Length Wing L / Breadth Alar Index A3 Length A3 / Labrum Maxilla Teeth Lateral Ventral Cibarial Teeth ♂ Filament /Pump Paramere ♂ ♂ Adeagus ♂ Beaked, Lower corners extending downwards Apical end rounded, but slightly curved ventrally colorless 45 Juma Khan Kakar 46 REFERENCES ARTEMIEV, M.M. (1978). Sand flies (Diptera, Psychodidae, Phlebotominae) of Afghanistan. iv+87 pp. Kabul. ASLAMKHAN, K., ASLAMKHAN, M. AND AZIZULLAH. (1997). The distribution records of sand flies (Phlebotominae) of Pakistan and Kashmir from 1908 to 1996. Pakistan J. Zool., 29: 351-360. ASLAMKHAN, K., ASLAMKHAN, M. AND AZIZULLAH. (1998). Biodiversity of sand flies of Pakistan and Kashmir. Pakistan J. Zool., 30: 13-21. BELAZZOUG, S., MAZHOUL, D., ADDADI, K. AND DEDET, J.-P. (1982). Sergentomyia minuta parroti (Adler and Theodor, 1927) en Algierie (Diptera: Psychodidae). Ann. Parasit. Hum. Comp., 57: 621-630. GEEVARGHESE, G., AMAKALLE, V.A., JADI, R., KANOJIA, P.C. JOSHI, M. AND MISHRA, A.C. (2004). Detection of Chandipura Virus from Sand Flies in the Genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar, District, Andhra Pradesh, India. J. Med. Entomol. 10: 495-496. KAKARSULEMANKHEL, J.K. (2004). Composition of the Phlebotominae Fauna (Diptera:Psychodidae) in Balochistan, Pakistan. J. Biol. Sci., 4 (3): 391-392. KAKARSULEMANKHEL, J.K. (2006). Sand flies of Pakistan (Diptera: Psychodidae), pp.87-98. In:Role of Insect Taxonomy, Systematic in sustainable Agriculture. Proc. Natl. Workshop.PARC/NARC. Feb. 13-14, 2006, Islamabad. LEWIS, D.J. (1967). The phlebotomine sand flies of west Pakistan (Diptera: Psychodidae). Bull.Brit. Mus. Nat. Hist. (Ent.), 19: 1-57. LEWIS, D.J. (1978).The phlebotomine sand flies (Diptera, Psychodidae) of the Oriental region. Bull. Brit. Mus. Nat. Hist. (Ent.): 37: 217-343. THEODOR, O. (1948). Classification of the Old World species of the sub family Phlebotominae. Bull. Ent. Res., 39: 85-111. YOUNG, D.G. AND DUNCAN, M.A. (1994). Guide to the Identification and Geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Mem. Am. Entomol. Inst., 54: 1- 881. Pakistan j. entomol. Karachi 23 (1&2): 47-50, 2008 POTENTIAL OF SWEET FLAG RHIZOME OIL AND CUSTARD APPLE SEED OIL AGAINST THE MAJOR SUCKING PESTS OF COTTON, AS COMPARED WITH CONFIDOR + DELTAPHOS, AT ARI-TANDOJAM, SINDH-PAKISTAN TARIQ*, R.M., NAQVI**, S.N.H., ZAFAR*, S.M.N. AND BURRERO***, A.S. *Department of Zoology, University of Karachi, Karachi-75270, Pakistan. **Department of Pharmacology, Baqai Medical University, Super Highway, Toll Plaza, Karachi-Pakistan. ***Entomology Section, Agriculture Research Institute, Tandojam, Sindh-Pakistan. ABSTRACT The two local plants, Acorus calamus (Sweet flag/Batch) rhizome oil and Annona squamosa (Custard apple/Seeta phall/Shareefa) seeds oil was tested against three major sucking pests, whitefly (nymph & Adult) (Bemisia tabaci), Thrips (Thrip tabaci) and Jassids (Amrasca devastant). The results of these were compared with that of Confidor + Deltaphos (CD) used as standard. The controlling power (potential) of the two candidate tested plants and standard was 65.02, 63.23 and 78.02%, respectively by A. calamus (AC) and A. squamosa (AS) and standard (CD) after two weeks, against Adults of whitefly. Whereas 70.00, 66.86 and 80.47% against whitefly nymph, in the same sequence. While the potentiality of AC, AS and CD, against Thrips was, 46.93, 46.61 and 69.92%, respectively after two weeks. Whereas against Jassids the efficacy of AC, AS and CD was found to be 60.22, 62.43 and 82.32%, respectively after two weeks. Overall the phytopesticides AC and AS were found having less effective than Confidor + Deltaphos, but even that the candidate oils gave remarkable good results. More research, struggle and results are needed to prove them as pesticides. Key words: Acorus calamus, Annona squamosa, Confidor + Deltaphos, Major sucking pests, Cotton. INTRODUCTION Phytopesticides are eco-friendly and safe from the pollution point of view. Whereas the commercial and synthetic pesticides such as pyrethroids are reported hazardous because their poisoning syndromes, synergies and therapy has been reported by Ray and Philip (2000). The pyrethroid induced paresthesia, a central or local toxic effect have been reported by Wilks (2000). Therefore, to chalk out new alternates of commercial and synthetic pesticides the researchers are busy in testing different plants extracts, such as the neem which is providing a good alternate as a phytopesticide and several formulations are being used in different parts of the developed and developing countries. Several plants and trees are tested such as marrango tree by Ermel et al. (1991), Cameroon by Kouninki et al. (2005), Cerrado Plant extract by Rodngues et al. (2006), Pamp and Occimum by Verma et al. (2006), but more attention have been given to Acorus calamus (AC) and Annona squamosa (AS), such as Ahmed et al. (2000), Alesso et al. (2003), Johri et al. (2004), Morio and Kuriyama (2005), Miguel et al. (2006). Naqvi, et. al. (2006), Akbar et al. (2007), Tariq et al. (2007). Therefore keeping in mind this strategy we have tested the oil formulation from Acorus and Annona against the pests of cotton crop at field level in agriculture sector. MATERIALS AND METHODS The extracted oil from the dried rhizome of Acorus calamus was coded as “AC” whereas the oil of Annona squamosa, coded as AS was extracted from the seeds. Both oil formulations contained 2% emulsifier (Twin-80) and after preparing layout the candidate pesticides were sprayed on cotton crop after taking pre-treatment observation in all the cases. Other precautions were also taken in agriculture treatments, such as the humidity, the wind, the rain, the temperature etc. The observations were recorded after 24, 72 hours, and one week. In the case of whitefly nymph only the observations were recorded after 72 hours and one week. The results were analyzed on standard pattern and compared with standards Confidor + Deltaphos. The experiments were conducted on cotton crop at Integrated Pest Management (IPM) Laboratory, Agriculture Research Institute (ARI) Tandojam, in the year 2001. The Randomized Complete Block Design (RCBD) method was adopted. Six replications of 4 treatments was done and the average value was used in the analyses. RESULTS Four treatments were designed T1 (AC), T2 (AS), T3 (Confidor + Deltaphos) and T4 (Control). After taking pre-treatment observation, the designed plots for AC, AS & C+D were sprayed at the dose of 1250ml/acre of AC & AS, whereas the doe of C+D was 800ml/acre in (1:1) ratio. While the control plot was not sprayed, the experiments were set at six places as replicates at a time, on a cotton crop shown on 31-03-2001. The experiments were done in July 2001. All the plots designed for experiments of AC, AS, C+D, were sprayed on same day after pre- Tariq et al. 48 treatment observation. The observations were taken after 24, 48, 72 hours, one week and two weeks. The reduction percentage after 24, 48, 72 hours, one week and two weeks each by AC, AS and C+D against whitefly adults per leaf has been shown in Table-1. Whereas against whitefly nymphs per leaf has been shown in Table-2. The pre-treatment observation and observation after spray of 24, 48, 72 hours, one week and two weeks, with reduction percentage of the above duration against Thrips/leave have been shown in Table-3, whereas against Jassids/leaf has been shown in Table-4. It may be noted in all four case i.e. whitefly adults, while fly nymphs, Thrips and Jassids that the efficacy of AC & AS is less as compared to Confidor + Deltaphos (C+D), but even that they (AC & AS) are remarkably comparable with Confidor + Deltaphos after 2 week. It means that the phytopesticide may give control of insects upto 15 days (2 weeks) effectively which is less than C+D, but is more safer and beneficial as compared these synthetic and conventional pesticides. Table-1. Pre-treatment observation & average reduction percentage of whitefly adults/leaf. No. of Treatments T1 Acorus calamus T2 Annona squamosa T3 Confidor + Deltaphos T4 Control* PreTreatments 1.25 24 Hrs. 48 Hrs. 72 Hrs. One week Two week 0.63 0.81 0.96 1.86 0.78 1.29 0.62 0.74 1.01 1.19 0.82 1.16 0.40 0.31 0.40 0.54 0.49 1.65 2.15 2.68 2.73 2.23 24 Hrs. 70.69 71.16 81.39 48 Hrs. 67.77 72.38 88.43 2.71 Reduction % 72 Hrs. 64.57 62.73 85.24 One week 31.86 56.41 80.22 Two week 65.02 63.23 78.02 Table-2. Pre-treatment observation & average reduction percentage of whitefly nymphs/leaf. No. of Treatments T1 Acorus calamus T2 Annona squamosa T3 Confidor + Deltaphos T4 Control* PreTreatments 0.93 24 Hrs. 0.69 0.62 0.80 0.67 0.49 1.00 0.95 0.53 0.74 0.63 0.56 0.87 0.33 0.27 0.38 0.27 0.33 1.38 1.96 2.26 2.43 Reduction % 72 Hrs. 67.08 69.54 84.36 2.07 1.69 24 Hrs. 64.79 51.53 83.16 48 Hrs. 48 Hrs. 72.56 76.54 88.05 72 Hrs. One week One week 67.63 69.56 86.95 Two week Two week 70.00 66.86 80.47 Table-3. Pre-treatment observation & average reduction percentage of Thrips /leaf. No. of Treatments T1 Acorus calamus T2 Annona squamosa T3 Confidor + Deltaphos T4 Control* PreTreatments 4.46 24 Hrs. 1.63 1.77 1.73 3.22 3.37 4.34 1.49 1.42 1.62 3.71 3.39 4.01 1.05 0.75 0.70 1.73 1.91 4.27 4.27 4.14 3.95 Reduction % 72 Hrs. 56.20 58.98 82.27 7.22 6.35 24 Hrs. 61.82 65.10 75.41 48 Hrs. 48 Hrs. 57.24 65.70 81.88 72 Hrs. One week One week 55.40 48.61 76.04 Two week Two week 46.93 46.61 69.92 Potential of sweet flag rhizome oil and custard apple seed oil against the major sucking pests 49 Table-4. Pre-treatment observation & average reduction percentage of Jassids/leaf. No. of Treatments T1 Acorus calamus T2 Annona squamosa T3 Confidor + Deltaphos T4 Control* PreTreatments 1.30 24 Hrs. 0.62 0.78 0.66 0.79 0.72 0.99 0.64 0.59 0.48 0.59 0.68 1.05 0.24 0.26 0.23 0.34 0.32 1.51 2.23 2.25 2.43 Reduction % 72 Hrs. 72.84 80.24 90.53 2.44 1.81 24 Hrs. 73.27 72.41 89.65 48 Hrs. 48 Hrs. 65.33 73.77 88.44 72 Hrs. One week One week 67.62 70.82 86.06 Two week Two week 60.22 62.43 82.32 *The reduction% in control was nil, due to which it has not been shown in reduction columns. DISCUSSION Johri et al. (2004) reported comparative toxicity of seven indigenous botanical extracts against the infestative stage of three insect pests of agricultural importance. The plants used by them were Tephrosia vogelii Hook F. (leaves & seeds – rotenoids), Annona squamosa Linn (seed-oil), Tribulus terrestris Linn (leaves-saponins), Pongamia glabra Vent (leaves & seed-oils) were all above extracted in petroleum ether, and ethyl alcohol extract of Thevetia nerifolia Juss (fresh fruit-Thevetin glycoside) and Duranta repens Linn (fruit-alkaloids) while both the solvents were applied in two steps for Caesalpinia crista Linn. seed-glycoside and fatty oil) were screened for their toxicity against three phytophagous insect pests, viz. Painted bug Bagrada cruciferarum Kirk on mustard and cabbage, white butterfly Pieris brassicae Linn. on cabbage and blister beetle, Mylabris pustulata Thunb on mung. The T. nerifolia showed higher toxicity followed by A. squamosa, T. vogelii and C. crista. In the present work Acorus calamus (AC) & Annona squamosa (AS) were used to control whitefly (nymphs + Adults), Thrips & Jassids on cotton crop. Both AC & AS gave more than 60% control in the case of whitefly (N+A), whereas standard gave more than 70% control after two weeks. Whereas in the case of Thrips AC, AS & C+D gave 46.93, 46.61 & 69.92% control respectively. While in the case of Jassids AC, AS & C+D gave 60.22, 62.43 & 82.32% control respectively after two weeks. Shuijin et al. (2006) reported the toxicity more than of 10 insecticides by two methods, the topical application and the leaf dipping method. The aim of this study was to monitor the resistance against these insecticides in common cutworm. Spodoptera litura (Fabricius). The topical application method was found more sensitive for monitoring the resistance to contact insecticides whereas the leaf dipping method was more sensitive for monitoring resistance to insecticides with stronger stomach action. The results were conducted by using susceptible strain and were compared with two field populations of S. litura. The two field populations were shown to have high resistance to organophosphates and carbamates. However, the Nanjing population had low resistance to profenofos and methomyl. No resistance to monosultap, abamectin, hexaflumuron and fipronil was found in these populations. They proposed to avoid the use of pyrethroids organophosphates and the carbamates should be limited and the other insecticides with no resistance should be used rationally and in rotation to delay resistance development. Whereas in the present work keeping in mind the above resistance problem against the pyrethroids, organophosphates, organochlorinates, and carbamates, the botanical (plant origin)/phytopesticides are used against sucking pest of cotton crop in field, i.e. Ac & AS. Naqvi et al. (2006) reported the efficacy of Annona oils as biopesticide against the major sucking pests (whitefly nymphs + Adults, Jassids & Thrips) of cotton on field level at Central Cotton Research Institute (CCRI) Multan (Punjab-Pakistan). The results were compared with Mospilan & Tamaron. The results were based on 24, 72 hours & one week observation. They used 1500ml/acre dose. But in the present work 1250ml/acre dose was used for the experiments against the above major sucking pests of cotton i.e. whitefly nymphs + Adults, Jassids & Thrips. They sued 125ml/acre of Mospilan against whitefly (N+A) and 500ml/acre of Tamaron against Thrips & Jassids. They concluded that AC & AS proved less effective as compared to Mospilan & Tamaron but may be successfully used against these pests. The same conclusion was drawn in the present work after the experimentation at ARITandojam. Akbar et al. (2007) reported the efficacy of Biosal a neem product of HEJ, Karachi-Pakistan against Amrasca devastans, the Jassids on brinjal crop. They concluded that the Biosal being a neem based formulation showed less toxic effects as compared to Endosulfan & Profenophos, may be due to strong anti-feedant, insect growth regulatory Tariq et al. 50 (IGR) effect of neem. In the present work, as well, the Calamus & Squamosa oil proved less effective against whitefly (N+A), Thrips & Jassids as compared to confidor + Deltaphos, preferably due to the same above reasons. Tariq et al. (2007) reported the toxic effects of A. calamus & A. squamosa against the bollworms (American, spotted & pink bollworm) on cotton crop. They reported 45.76, 57.20, 48.30, 58.05 & 47.03% control of American bollworm by AC, AS, Karate, Polytrin-C & Curacron respectively, upto two weeks. Whereas against spotted bollworm, the control after two weeks by AC, AS & Confidor + Deltaphos was 41.12, 58.15 & 78.66% respectively. While the control after two weeks in the cast of pink bollworm by AC, AS & C+D was 51.54%, 58.17 & 81.94% respectively. But in the present work the experiments were done against the major sucking pests of cotton by Acorus calamus (AC) & Annona squamosa (AS) to control whitefly (nymphs + Adults), Thrips & Jassids on cotton crop. Both AC & AS gave more than 60% control in the case of whitefly (N+A), whereas standard gave more than 70% control after two weeks. Whereas in the case of Thrips AC, AS & C+D gave 46.93, 46.61 & 69.92% control respectively. While in the case of Jassids AC, AS & C+D gave 60.22, 62.43 & 82.32% control respectively after two weeks. REFERENCES AHMED, I., AHSAN, T., TABASSUM, R., AZMI, A. AND NAQVI, S.N.H. (2000). Effects of Acorus calamus extract and cypermethrin on enzymatic activities in Sitophilus oryzae. J. Exp. Zool. India. Vol. 3(2): 169-173. AKBAR, M.F., YASMIN, N., NAQVI, S.N.H., KHAN, M.F. & NAZ, F. (2007). Relative efficacy of biopesticide in comparison with conventional pesticides against Amrasca devastans Dist. on Brinjal crop. Pakistan j. entomol. Karachi, 22 (1&2): 1-3. ALESSO, E., TORVISO, R., LANTANO, B., ELRICH, M., LILIANA, M., FINKIELSZTEIN, MOLTRASIO, G., AGUIRRE, J.M. AND BRUNET, E. (2003). Synthesis of 1-ethyl-2methyl-3-arylindanes. Stereochemistry of finemembered ring formation (online) ARKIVOC 2003 (X): 283-297. ERMEL, K., KALINOWSKI, H.O. AND SCHMUTTERER, H. (1991). Isolation and characterization of narrangin, a new insect growth regulating (IGR) substance from the seed kernels of the marrango tree, Azadirachta excelsa (Jack). J. Appl. Entomol. 112(5): 512-519. JOHRI, P.K. MAURYA, R., SINGH, D., TIWARI, D. AND JOHRI, R. (2004). Comparative toxicity of seven indigenous botanical extracts against the infestative stage of three insect pests of agricultural importance. Journal of Applied Zoological Researches. 15(2): 202-204. KOUNINKI, H., HAUBRUGE, E., NOUDJOU, F.E., LOGNAY, G., MALAISSE, F., NGASSOUM, M.B., GOUDOUM, A., MAPONGMETSEM, P.M., NGAMO, L.S.T. AND HANCE, T. (2005). Potantial use of essential oils from Cameroon applied as fumigant or contact insecticides against Sitophilus zeamais Motsch (Coleoptera: Curculionidae). Communications in Agricultural and Applied Biological Sciences. 70(4): 787-792. MIGUEL, B., ZAVALA, F., SISNIEGAS, M., ZAVALETA, G., MOSTACERO, J. AND TARAMONA, L. (2006). Larvicidal evaluation of aqueous suspensions of Annona muricana Linnaeus “custard apple” against Aedes aegypti Linnaeus (Diptera, Culicidae). REVISTA PERUANA DE BIOLOGIA 12(1): 145-152. MORIO, T. AND KURIYAMA, M. 2005. Pests of Annona cherimola (Annonaceae) in a newly introduced area, with respect to their host range. Journal of the Entomological Research Society. 7 (Part 3): 1-12. NAQVI, S.N.H., TARIQ, R.M., ZAFAR, S.M.N. & ATTIQUE, M.R. (2006). Efficacy of Acorus calamus (AC) rhizome oil and Annona squamosa (AS) seed oil against sucking pests of cotton at CCRI-Multan (Punjab) as compared to Mospilan & Tamaron. Pakistan j. entomol. Karachi, 21 (1&2): 23-27. RAY, D.E. AND PHILIP, J.F. (2000). Pyrethroid insecticides: Poisoning syndromes, synergies and therapy. Journal of Toxicology Clinical Toxicology. 38(2): 95-101. RODNGUES, A.M.S., PAULA, J.E.D., DEGALHER, N., MOLEZ, J.F. AND ESPINDOLA, L.S. (2006). Larvicidal activity of some Cerrado plant extracts against Aedes aegypti. Journal of the American Mosquito Control Association. 22(2): 314-317. SHUIJIN, H., XU, J. AND HAN, Z. (2006). Baseline toxicity data of insecticides against the common cutworm Spodoptera litura (Fabricius) and a comparison of resistance monitoring methods. International Journal of Pest Management 52 (3): 209-213. TARIQ, R.M., NAQVI, S.N.H., ZAFAR, S.M.N. & BURRERO, S. (2007). Toxic effects of botanical pesticide, from Acorus calamus (AC) and Annona squamosa (AS) against bollworms at ARITandojam Sindh-Pakistan. Pakistan j. entomol. Karachi, 22 (1&2): 31-36. VERMA, P.R., SUBBURAJU, T. AND BALAKRISHNAN, N. (2006). Larvicidal activity of Artemisia nilagirica (Clarke) Pamp. and Ocimum sanctum Linn. – A preliminary study. JOURNAL OF NATURAL REMEDIES. 6(2): 157-161. WILKS, M.F. (2000). Pyrethroid induced paresthesia: A central or local toxic effect? Journal of Toxicology Clinical Toxicology. 38(2): 103-105. Pakistan j. entomol. Karachi 23 (1&2): 51-54, 2008 STUDIES ON SAMPLING TECHNIQUES TO MONITOR ADULT POPULATION OF WHITEFLY, BEMISIA TABACI (GENN.) IN CUCURBIT FIELD ABDUL GHANI LANJAR*, MUHAMMED KHAN LOHAR*, HAKIM ALI SAHITO*, NAHEED BALOCH** AND ASHFAQUE AHMED NAHIYOON* *Department of Entomology, Sindh Agriculture University, Tandojam **Department of Zoology, University of Sindh, Jamshoro ABSTRACT A half - acre field of melon, Cucumis melo L. was selected at Tandojam during spring, 2000 for sampling adult population of whitefly, Bemisia tabaci (Genn.). The methods of sampling were direct and indirect count. In the direct count, 30 leaves were randomly selected and examined from 2nd, 3rd and 5th nodes of the creepers. In the indirect count, yellow sticky traps of different shapes, i.e., flat, cylindrical and round were used. The traps were mounted at 6", 12", and 18" above ground. Highest population of B. tabaci 6.56 ± 1.34 per leaf was recorded on the 2nd- node leaves as compared to 3rd, 2.62 ± 0.52 and 5th-node leave 2.52 ± 0.56. More activity 6.56 ± 1.34 per leaf was recorded at 8.00 a.m. than 4.03 ± 0.83 at 12.00 noon. Highest catches 22.87 ± 2.69 per trap were recorded on cylindrical and lowest 9.24 ± 1.39 on round traps. Traps mounted at 6" above ground level gave 14.37 ± 2.02 catches of B. tabaci adults. Population counts in all three techniques were highly significant (P<0.01). Key words: Bemisia tabaci, Melon, Population and Sampling. INTRODUCTION placed parallel to plant height catch more whiteflies (Malamed-Madjar et al. 1982). The whitefly, Bemisia tabaci (Gennadius) is one of the most important members of Family Aleyrodidae for its grave ravages on tropical and subtropical agriculture. It is a polyphagus insect pest recorded on more than 506 species of plants and field crops (Mound and Halsey, 1978). Greathead (1986) updated this information, who listed the host range up to 540 species belonging to 77 plant Families. In Pakistan, it was recorded on 160 plant species belonging to 113 genera of 42 families including crops, ornamentals, fruit and forest trees, and weeds (Attique et at., 2003; Lanjar and Sahito, 2005). In a cotton field, the sample unit per plant consisted of first two fully expanded main terminal leaves (Musuna, 1986). The sampled leaves with high and low infestations of B. tabaci were found between 8th leaves from top to bottom (Fowler, 1956; Mound, 1965). Melamed-Madjar et al. (1982) reported that the most infested leaves were between 5th and 6th nodes. Gusmao et al. (2005) mentioned the most appropriate sampling methods were beating one apical leaf in the tray to assess adults and by direct counting of nymphs on a basal leaf of tomato plants. Sequeira and Naranjo (2008) recommended that minimum sample of 20 leaves at nodes 3, 4 or 5 below the terminal as the most parsimonious and practical sampling protocol for cotton fields. B. tabaci count on plant leaves was considerably high early in the morning, because the adults were less mobile (Gerling and Horowitz, 1984). The work of the previous authors demonstrate that satisfactory sampling method to manage B. tabaci on cucurbit crops is utmost important. Proper sampling is essential for estimating the number of insect pests in a field crop. Various Sampling methods have been used to monitor B. tabaci population. The immature stages of whitefly are hard to detect (Ohnesorge and Rapp, 1986). The adult dispersed in field for a considerable distance (Byrne, 1999). They are characteristically attracted to yellow surfaces (Cohen, 1982). The use yellow sticky trap is a major tool in monitoring adult population (Berlinger, 1980; Gerling and Horowitz, 1984; Butler et al. 1985). Various shapes and sizes of the traps such as flat and cylindrical have been satisfactorily used to monitor B. tabaci population (Byrne et al. 1986; Youngman et al. 1986). Traps MATERIALS AND METHOD Observations The investigations on the sampling techniques for monitoring adult population of B. tabaci were conducted in a melon field at Tandojam during spring Lanjar, A.G. et al. 52 season. Melon crop was sown on an area of 1/2 acre. The whitefly adult populations on melon were sampled by using direct and indirect counts. Indirect Count Yellow traps of different shapes namely flat, cylindrical and round were placed, two near the sides and one trap in center of the melon field. Three cylindrical shaped traps were placed above ground at different heights of 6", 12" and 18". The traps were coated with adhesive grease. Observations on population count were taken twice a week. After each observation the grease on yellow traps was changed. There were three (trap sizes and heights) replications of each sampling technique. Trap Size Flat trap: Made of a plastic sheet (12" x 12") fitted into one meter long wooden stick. Cylindrical trap: Made of a plastic pipe of 12" length and 6" diameter filled with plaster of paris to support onemeter long wooden stick. Round trap: Made of a glass globe of 8" diameter filled with plaster of Paris to support one -meter long wooden stick. 4th week of April, where 42.33±3.56 adult B. tabaci were recorded on cylindrical trap, 26.33±4.00 on flat and 18.66±3.13 on round trap. The average adult B. tabaci count on cylindrical trap was 22.87±2.68, on flat trap was 14.37±2.02 and on round trap was 9.24±1.39. Results show that the use of cylindrical traps is better than flat and round traps. B. tabaci adults were trapped from all directions on the cylindrical traps in the melon crop. This indicates that B. tabaci population was equally distributed in the melon field. The results showed that population of adult B. tabaci caught on three traps were highly significant. The present results are in agreement with the findings of earlier authors. Berlinger (1980) reported that B. tabaci was strongly attracted to yellow color surface. Butler et al. (1985) stated that yellow sticky traps became a major tool in monitoring of B. tabaci population. Flat traps of various sizes have generally been used (Butler et al. 1986). However, cylindrical types have given better results (Byrne et al. 1986, Youngman et al. 1986). 30 Direct Count Population densities of adult B. tabaci were estimated by direct count on melon leaves. The sampling unit per leaf comprised of fully expanded leaves of 2nd, 3rd and 5th nodes of melon creepers. Thirty leaves of different creepers were randomly selected and examined twice a week for B. tabaci population. The counting of B. tabaci was carried out between 8.00 a.m. and 12.00 noon. 25 20 15 10 5 0 Fl at cy l i ndr i cal r ound Fig.1. Mean catches per trap of Bemisia tabaci on various types of yellow sticky traps mounted in melon field during spring season. Whitefly Activity at Different Ground Levels RESULTS AND DISCUSSION Indirect Count Population of B. tabaci was estimated on melon from germination until harvest of the crop. Figure -1 shows that at initial growth stage of the creepers the number of adults caught in the traps was very low. The first adult catches on the different traps were recorded during 1st week of March just after crop germination. During initial catches 5.00±0.94, 2.66±0.72 and 1.00±0.47 adults per trap were recorded on cylindrical, flat and round traps, respectively. Population of B. tabaci fluctuated later on and showed two peaks. The first population peak was observed during 4th week of March, where the adults caught on cylindrical, flat and round traps were 50.00±4.11, 38.00±4.32 and 26.33±2.59 per trap, respectively. The second peak was recorded during Figure 2 shows that the population of B. tabaci varied at the different ground levels. At initial growth stage, activity of B. tabaci was higher at 6" above ground level than at12" and 18". The activity of B. tabaci depended proportionately on the growth of creeper. Two peaks namely, the 1st during 4th week of March, and the 2nd during 4th week of April were observed. At 1st peak the count of adult B. tabaci was 38.00±4.32, 25.00±3.09, and 7.00±0.94 adults/trap at 6", 12" and 18" respectively. At the second peak the count of adult B. tabaci was 26.33±4.00, 17.33±4.83 and 13.33±2.88 per trap respectively. The mean total count of adult B. tabaci at 6", 12" and 18" was 14.37±2.02, 7.81±1.43, and 3.61±0.68, respectively. The result of this experiment indicates that in melon field the flight and dispersal activities of B. tabaci adults were maximum at 6" above ground level, and lower at 18". It clearly shows that B. tabaci retain Studies on sampling techniques to monitor adult population of whitefly, Bemisia tabaci (Genn.) 53 their activities mostly up to the height of melon creepers. The present results are in agreement with the findings of earlier researchers. Sharaf (1982) reported that horizontally placed traps gave high catches per trap than those placed vertically. Traps fixed at ground level gave higher catches in fallow than in a cotton field (Gerling and Horowitz, 1984). Melamed Madjar et al. (1984) reported that whitefly catches within field and 4 meter away from border were the same, when the traps were placed at plant height. Suitable Timing of B. tabaci Sampling Direct Count In the direct count, numbers of B. tabaci on 2nd, 3rd and 5th node on leaves were recorded (Figure 3). In this experiment, adult B. tabaci were counted on melon leaves from early March till 20th May. The population of B. tabaci increased with the growth of melon creepers. Two population peaks, during 4th week of March and 3rd week of April, were observed. The highest mean density was recorded during the 2nd peak. Density of adults B. tabaci was significantly higher at the 2nd node with 14.90±1.54 individuals/node than 6.36±0.94 at 3rd node and 6.54±0.75 at 5th node. Mean total density of the adults was 6.56±1.34 at 2nd node, while lower number 2.52±0.55 individuals at 5th node. The result indicates that adults prefer 2nd nodes of leaves, because they are fresh and broader in size. Naranjo and Flint (1995) reported that B. tabaci adults on the top stratum of the plant were fairly uniformly distributed over leaves from main stem of 2-7 nodes, but adults were abundant at 5th node leaves. Musuna (1986) stated that B. tabaci adult sampling unit comprises the first two fully expanded main terminal leaves and one leaf at mid level of the plant. Like the indirect and direct count of adult B. tabaci on the melon leaves, the data in Table-I shows similar trend of population growth, i.e., two population peaks: first in 4th week of March and the 2nd in 3rd week of April. The data of the number of B. tabaci adults was significantly different at the two timings (T=3.73, P<0.01). Mean total density of adult fly at 8.00 am was 6.56±1.34, while 4.03±0.83 at12.00 noon. The present results are in agreement with those of Gerling and Horowitz (1984) They reported that direct counting should be made early in the morning when adults are least mobile. Musuna (1986) found similar trend of B. tabaci adult sampling. According to him B. tabaci adult count was higher during 9.00-12.00h than during 17.00- 18.00h, although the differences were not significant at P=0.05. Table-1. Mean density/leaf of B. tabaci recorded on 2nd node leaves at 8.00 a.m. and 12.00 noon on melon creeper during spring season. Observation Date 04- March 11 18 25 01- April 08 15 22 29 06- May 13 20 Mean SE 2nd node 8.00 a.m. Mean ± SE 2.00 ± 0.29 4.18 ±0.61 8.54 ±0.88 13.45 ±1.86 4.00 ±0.52 9.81 ±0.96 11.81 ±1.56 14.90 ±1.54 3.54 ±0.67 2.72 ± 0.47 2.00 ± 0.35 1.81 ±0.31 6.56 1.34 2nd node 12.00 noon Mean ± SE 1.27 ±0.24 2.72 ±0.38 4.90 ± 0.74 7.45 ± 0.89 2.90 ±0.58 8.09 ±0.61 4.36 ±0.56 10.1 ±0.82 2.54 ±0.35 1.45 ±0.23 1.45 ±0.23 1.09 ±0.20 4.03 0.83 Lanjar, A.G. et al. 54 REFERENCES ATTIQUE, M.R., MUHAMMED RAFIQ, ABDUL GHAFFAR, ZAHOOR AHMAD AND A.I. MOHYUDDIN (2003). Hosts of Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) in cotton areas of Punjab, Pakistan. Crop Protection 22 (5): 715-720. LANJAR, A.G. AND H.A. SAHITO (2005). Effect of some spring hosts on the life cycle of whitefly, Bemisia tabaci (Genn.) Pakistan j. entomol. Karachi 20 (1-2): 13-18. MELAMED-MADJAR, V., S. COHEN, M. CHEN, S. TARN AND D. ROSILIO (1982). 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Impact of trap design and placement when monitoring for the bandedwinged whitefly, and sweet-potato whitefly (Homoptera: Aleyrodidae). Environ. Ent. 15: 300-304. COHEN, S. (1982). Control of whitefly vector of virus by color mulching. In pathogens, Vectors and Diseases, approaches to Control ( Harris K.F and K. Maramoroches, Eds.). Academic Press, New York. pp. 45-56. FOWLER, H.D. (1956). Some physiological effects of attack by whitefly (Bemesia gossypiperda) and of spraying parathion on cotton in the Sudan Gezira. Empire Cotton Growing Review. 33: 288-299. GERIING, D. AND A.R. HOROWITZ (1984). Yellow traps for evaluating the population levels and dispersal patterns of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae). Ann. Ent. Soc. Amer. 77: 753-759. GREATHEAD, A.H. (1986). Host plants. In Bemesia tabaci. A Literature Survey (M.J.W.Cock, Ed.). C.A.B. Intern. Inst. Biolo. Cont, U.K. pp.17-25. GUSMÃO, M.R., M. C. PICANÇO, J.C. ZANUNCIO, D.J.H. SILVA AND J.A.F. BARRIGOSSI (2005). Standardised sampling plan for Bemisia tabaci (Homoptera: Aleyrodidae) in outdoor tomatoes. Scientia Horticulturae 103 (4):403412. MUSUNA, A.C.Z. (1986). A method for monitoring whitefly, Bemisia tabaci (Genn.) in cotton in Zimbabwe. Agric. Ecosys. Environ. 17: 29-35. NARANJO, S.E AND H.M. FLINT (1994). Spatial distribution of pre-imaginable Bemisia tabaci (Homoptera: Aleyrodidae) in cotton and development of fixed precision sequential sampling plants. Environ. Ent. 23 (2): 254-266. OHNESORGE, B., AND G. RAPP (1986). Monitoring Bemisia tabaci: A review. Agric. Ecosys. and Environ. 17 (1/2): 21-27. SEQUEIRA, R.V AND S.E. NARANJO (2008). Sampling and management of Bemisia tabaci (Genn.) biotype B in Australian cotton. Crop Protection 27 (9): 1262-1268 SHARAF, N.S. (1982). Determination of the proper height, direction, position and distance of a yellow sticky trap for monitoring adult sweet potato whitefly populations Bemisia tabaci Genn., (Homoptera: Aleyrodidae). Dirasat. 9: 169-182. YOUNGMAN, R.R., N.C. TOSANCO, V.P. JONES, K. KIDO AND E.T. NATWIK (1986). Correlation of seasonal trap counts of Bemisia tabaci (Homoptera: Aleyrodidae) in southeastern California. J. Econ. Ent. 79: 67-70. Pakistan j. entomol. Karachi 23 (1&2): 55-60, 2008 POPULATION DYNAMICS OF GROUND WATER BREEDING OF CULEX MOSQUITOES OF KARACHI AND THATTA DISTRICT TANVEER1 FATIMA SIDDIQUI AND NAQVI2, S.N.H. 1 Department of Zoology, Federal Urdu University of Arts, Sciences and Technology, Gulshan-e-Iqbal Campus, Karachi-Pakistan. 2 Department of Pharmacology, Baqai Medical University, Super Highway, Toll Plaza, Karachi-Pakistan. ABSTRACT A general survey of population of mosquito larvae commonly found in Karachi and Thatta district was carried out. The mosquito larvae of six (6) different species were collected from different ground water habitats, during January 2004 to December 2004. Samples were collected, identification, progressive changes, their abundance and faunistic association noted, are included. It is to be noted that larval fauna depends not only on the type of the habitat but also on the changes in the seasons, resulting in the replacement of summer with winter species. Key words: Population, Dynamics, Breeding, Culex Mosquito, Karachi, Thatta District. INTRODUCTION Mosquitoes are very important vectors and ectoparasite for human and animals. Insect-born diseases, such as malaria, yellow-fever, lymphatic filariasis, dengue, encephalitis, are transmitted by mosquitoes. Up till now little systematic efforts has been made to study the culicidae of Pakistan. The fauna of British India dealing with anopheline (Christophers 1933) and culicine (Barnard 1934) mosquitoes still remains the standard reference work. An excellent survey work done on the culicidae of Ceylon, India, and Pakistan has been given by Qutubuddin (1960). The culicidae of Pakistan remain poorly known except for the anopheline mosquitoes, which are better known because of their involvement in the transmission of malaria. Information about the culicine mosquitoes of Pakistan, special reference to Karachi is still very meager. However studies on bionomics, filariasis and cytogenetics have added some information concerning the distributional record of mosquitoes Nasiruddin 1952; Aslamkhan and Salman 1969; Baker and Aslamkhan 1969; Aslamkhan and Wolf 1971). Adequate knowledge of disease-vector population dynamics is of paramount importance in the understanding of the epidemiology of any insectborn disease. The purpose of present survey of larval breeding site in the Karachi and Thatta District of Pakistan was performed to delineate the range of conditions under which ground water mosquitoes breed, thus providing some indication of the oviposition preferences of the females and population of various prevailing species. MATERIALS AND METHOD Water samples were collected from different localities of all five districts of Karachi and Thatta district. All promising sites were first inspected for the presence of larval mosquitoes, negative samples were not analyzed. Larval abundance was estimated by counting the number of culicine larvae found in 10 to 20 dips with standard dipper. The number of dips varied with the size of the habitat, the density and dispersion of the larvae. Less dips were taken in small homogenous habitats such as ground pools, while more dips were taken in heterogenous environments such as large ponds etc., where mosquito abundance varied considerably from dip to dip. All collection were taken back to the laboratory where a maximum of 10 slides of “different looking” larvae were made and identified (WHO 1975). The remainder of the collection was reared to adult, identified and counted. The percentage composition of the total specimens identified including both larval slides and emerging adults was used to estimate the mean number of larvae per dip for each species of Culex mosquitoes. Temperature (ºC), pH, dissolved oxygen of each sample were also recorded. Temperature were taken 56 Tanveer Fatima Siddiqui with an ordinary thermometer at the edge of the habitat where larvae were collected. pH was recorded by pH meter, Dissolved oxygen mg/L was estimated by Wrinklers method in accordance with the procedure of Taras et al. (1971). RESULTS AND DISCUSSION Mosquito larvae were sampled throughout the year 2004; the immature stages of Culex genus were the most abundant mosquito larvae in the study areas. Six (6) Culex mosquito species were collected in the study area. Both immature and adult of these species were identified; these species are Culex bitaeniorhynchus, Culex fusco, Culex p. fatigans, Culex tritaeniorhynchus, Cx. pseudorishnui and Cx. vagans. In the study area different breeding habitats were characterized. For convenience, the habitats samples have been classified into six categories defined below. 1. Pond – Large body of water, usually permanent, adjacent to some small pockets of population. 2. Pool – Small body of standing water, probably temporary (i.e., dries during the hot months or in dry season), filled by rain or run-off water. 3. Borrow pit – Small depression, excavated for construction materials; difficult to tell old borrow pit from ground pools. 4. Ditches – Road side drain to carry off rain water or any other sources. 5. Catch basin – Cement tanks, usually filled with water, when sufficient water was not available. 6. Artificial container – Tyre puncture tubs, overhead tanks, buckets, flower pots etc. Physico-chemical conditions (Temp, pH, and Dissolved oxygen) for each of the six habitats have been analyzed. Temperature varied mostly due to seasonal changes. In general, smaller bodies of water showed the greatest variation than larger bodies. Temperature has two peaks with higher temperature in May-June and September-October. Slight changes in pH and dissolved oxygen were also observed due to change in the weather conditions, or filling of habitats by rain or sewerage water. A total of 19414 mosquito larvae were collected and examined during 2004, collectively from all six Districts out of 19414 larvae 15512 larvae were from Culex genus and rest belong to Anopheles and Aedes genus Table-1 shows that Culex population is most prevalent among all the three genera. Detail population is given in (Table-2, and Figure-1 and 2). A total of 15512 Culex mosquito larvae were collected from all Districts, comprising six different specimens (Table-2, and Figure 1 and 2). In the genus Culex, the Cx. p. fatigans was the most common, and showed highest percentage, i.e. 47.43%. The second highest was of Cx. tritaeniorhynchus i.e. 34.20%. Culex bitaeniorhynchus and Culex vagan were found in minimum percentage with 3.27% and 2.68%, respectively. Seasonal fluctuation of different Culex species during twelve months, i.e. from January 2004 to December 2004 (Graph and Data-1). It shows that Culex p. fatigans was the most common throughout the year, but inversely related to temperature, while Culex tritaeniorhynchus, Culex pseudovishnui and Culex fuscocephalus directly related to temperature. According to data and graph population of Cx. p. fatigans increased during November 2004 and January 2004. As temperature decreases population frequently increases. With the analysis of different species and breeding habitats, it was found that Cx. bitae collected from ponds, pools and ditches. Sirivanakarn (1976) has elevated the dark form of Cx. bitae. In the present survey, light and dark forms were not routinely separated. This species mostly breed in clean water with spirogyrs. Cx. fuscocephalus (Theobald, 1902) collected only from ponds and pools with a dense cover of duckweed (Lemnaceae) or dark-coloured water with decaying leaves. Cx. p. fatigans (Wiedemann, 1928) mostly collected from ponds, pools and catch basins, this mosquito species is inversely correlated with temperature during winter, population densities frequently exceeded 400 larvae/dip in polluted pool habitats. Cx. pseudorishnui (Colles, 1957) breed almost all different type of habitats except catch basin and polluted water. This species is positively correlated with temperature. This species frequently associated with Cx. tritaeniorhynchus (e.g., Colless, 1957; Reuben 1971). This species mostly occurs in clean-water habitats. Cx. tritaeniorhynchus was most frequently collected species during monsoon and post monsoon seasons. Positively related to temperature and inversely related to dissolved oxygen. During the summer Cx. tritaeniorhynchus breeding in all ground water habitats, especially with grasses and decaying vegetation were high. Cx. vegans mostly found in permanent ponds. This species mostly found in Lahore (Barnard 1934), but in few cases in Karachi. In all District of Karachi and Thatta District definite mosquito-habitat associations were determined which were modified by increases in temperature, and organic pollution. Seasonal temperature changes resulted in the shift of fauna e.g., Cx. p. fatigans increases in abundance during winter mean cold weather and Cx. tritaeniorhynchus increased during summer-season. Population dynamics of ground water breeding of Culex mosquitoes of Karachi & Thatta District 57 58 Tanveer Fatima Siddiqui Population dynamics of ground water breeding of Culex mosquitoes of Karachi & Thatta District Graph and Data-1 59 Tanveer Fatima Siddiqui 60 REFERENCES ASLAMKHAN, M., AND BAKER, R.H., (1969). Karyotypes of some Anopheles, Filalbia, and Culex mosquitoes of Asia. Pakistan J. Zool., 1: 107. ASLAMKHAN, M., AND SALMAN, C., (1969). The bionomics of the mosquitoes of the Changa Manga National Forest, West Pakistan. Pakistan J. Zool. 1: 183-205. ASLAMKHAN, M. & WOLF, M.S. (1971). Rural bankroftian filariasis in two villages in Dinajpur District, East Pakistan, II. Entomological investigation. Am. J. Trop. Med. Hyg., 20. REISEN, W.K. AND SIDDIQUI, T.F., (1977). The influence of consepecific immature on the oviposition preferences of Anopheles stephensi Liston and Culex tritaeniorhynchus Giles (Diptera: Culicidae). J. Med. Ent. REUBEN, R. (1971). Studies on the mosquitoes of North Arcot District, Madras State, India. Part 5. Breeding places of the Culex vishnui group of species. J. Med. Ent., 8: 363-66. SIRIVANAKARN, S., (1976). Medical entomological studies III. A revision of the subgenus Culex in the oriental region (Diptera: Culicidae). Contr. Amer. Ent. Int., 12: 1-72. BAKER, R.H., AND ASLAMKHAN, M., (1969). Karyotype of some Asian mosquitoes of the subfamily Culicinae (Diptera; Culicidae). J. Med. Ent., 6: 44-52. TARAS, M.J., GREENBERG, A.E., HOAK, R.D. AND RAND, M.C., (1971). Standard methods for the examination of water and waste water. 13th Edition, Amer. Publ. Hlth. Assoc. Washington, D.C. p. 874 p. BARNARD, P.J., (1934). The fauna of British India, including Ceylon and Burma. Diptera Vol. 5, Family Culicidae. Tribes Megarhinini and Culicini. London, XXX VIII + 463 pp. London. THEOBALD, F.V. (1902). A short description of the Culicidae of India, with description of new species of Anopheles. Proc. Roy. Soc., 69, 367394. CHRISTOPHERS, S.R., (1933). The fauna of British India, including Ceylon and Burma. Anophelini, London, XII + 371 pp. London. WADA, Y., M. MOGI, AND J. NISHIGAKI, (1971). Studies on the population estimation for insects of medical importance. IV, A method for the estimation of relative density of Culex tritaeniorhynchus larvae in the whole paddyfields of an area. Trop. Med. 13 (2): 86-93. COLLESS, D.H., (1957). Note on the Culicine mosquitoes of Singapore. III. Larval breeding places. Ann. Trop. Med. Parasit. 51: 102-16. NASIRUDDIN, M. (1952). Mosquitoes breeding in tree-holes and bamboo stumps in Dacca (East Pakistan, Bangladesh). Pakistan J. Hlth. 2: 110112. QUTUBUDDIN, M., (1960). The mosquito fauna of Kohat-Hangu Valley, West Pakistan. Mosquito News, 20: 355-361. WHO (1975). Manual of practical entomology in malaria. Part II. Methods and techniques. Wld. Hlth. Org., Geneva, Switzerland, 191 pgs. Pakistan j. entomol. Karachi 23 (1&2): 61-62, 2008 LEVELS OF DENGUE FEVER VIRUS CONTROL: THE FFECTIVENESS AND VASTNESS OF CONTROLLING POWER BOUNDARIES OF THESE LEVELS RAJPUT MUHAMMED TARIQ1 & S. SALAHUDDIN QADRI2 1 MAH Qadri Biological Research Centre, Room No.11, University of Karachi, Karachi-75270, Sindh-Pakistan 2 Department of Zoology, Jamia Millia Degree College, Malir Karachi-Pakistan ABSTRACT Each (any) problem has some levels (steps) to solve that problem. Usually a problem is solved step by step, but some times, in special and the most important case the particular problem is solved by taking all steps simultaneously and not by step by step. This type of situation happens there, where the boundaries of vastness of that problem grows by the passage of time. It means when all steps are necessary to be taken and there is a shortage of time to solve that problem, especially when the problem is growing with the passage of time. The Dengue Fever Virus (DFV) problem is being spread by a mosquito genus, the Aedes. The problem is increasing day-by-day not only in Pakistan but also in the world by the passage of time. Therefore the levels (steps) with their controlling power and effectiveness boundaries are discussed in detail. Key words: Aedes, DFV, Levels, Control, Effectiveness, Boundaries. INTRODUCTION & DISTRIBUTION The Aedes aegypti & Ae. albopictus have the global distribution and both are the confirmed vector (causative agents) of DFV. Barraud (1934) has reported Aedes aegypti in Pakistan since last 75 years. Naqvi (1992) presented a survey report of mosquitoes and houseflies of Karachi regions from 12 stations selected in the region of Karachi. Suleman et al. (1993) reported 8 species of Aedes in Pakistan from Peshawar, Charsadda, Mardan & Swabi viz. Ae. albopictus, capius, lineatopenis, pseudotaeniatus, pulchriventer, vittatus, uniliniatus & walbus. The ninth species of Ae. aegypti has been present long before in different areas of Pakistan as reported by Barraud (1934), especially in Punjab & Sindh. Kamimura et al. 1986, WHO (1989) also reported by Chan et al. (1995). Suleman (1996), Naqvi, et al. (1997), Tariq & Zafar (2000), Tariq & Qadri (2001), Qadri et al. (2007). MATERIALS & METHODS Literature survey, observations for controlling the vector of DFV, Medical strategies in Hospitals of Karachi in 2006, 2007 & 2008. RESULTS & DISCUSSIONS There are three basic levels to control the Dengue & Malaria vector and other mosquitoes as well. (1) Ground level: (Larval stage/Breeding stage). This stage is restricted to some/few places where they (Larvae) breed they may be control and destroyed easily, as they are non-flyer, remain at particular place in limited area. They may be controlled by destroying water in which they are contained, by putting Bt., fishes & Lemna plants in large area water which can not be destroyed or dried up containing these larvae. These larvae usually breed in small pots, plastic containers, cans, drums, earthen pots, over-head & ground water tanks, present in the human populated area. As they are present in hundreds in small plastic containers & in thousands number in the stored water for checking the puncture tubes at the puncture tyre shops as reported by Tariq & Zafar (2000). According to their report 11.2% puncture tyre shops were recorded positive for Aedes mosquito eggs larvae, pupae & adults. If awareness is created among the public through media then the destruction of these breeding points & places is sure. These breeding points are easy to destroy and cheep or less expensive as compared to spray and other methods. Only the manpower is needed to do work against breeding points themselves, after awareness by media and other sources. (2) Air level: Adult stage/flying stage of mosquito. In the adult stage the mosquitoes are flyer and spread far and wide in the area, they are breeding. As they fly from one place to another place their boundaries of presence increase, but the power of control through spray method decreases, due to which the effectiveness of the control strategy also decrease. At the same time expenses to control them increases ultimately due to the use of insecticide, spray machine, sprayer, vehicle, driver, spray-man, diesel, etc. for spraying. The spray is not Tariq et al. 62 done simultaneously in the area (Say Karachi city) it is done area by area, stepwise due to this reason the mosquitoes shift from the sprayed area to the other areas, due to which they are not killed but remain alive. The other reason is that the only selected area is sprayed not the whole city or area. Thus the spreading boundaries (as they are flyer) of mosquitoes in this stage is increased, with the increased expenses but the boundaries of controlling power to these flying mosquitoes is decreased resulting in less effectiveness of control, as compared to ground level control of mosquitoes and few mosquitoes may be killed in this level by spray. All the people or most of the people may be infected in this stage. (3) Clinical/Hospital level: Infection stage by mosquitoes. This is the stage in which only infected persons are brought to Clinic/Hospital, after the diagnostic report and tests, the patients are given platelets in points. The expenses in this stage increases more and more as compared to second stage due to the high cost of platelet bags, platelets, Clinic/Hospital charges, diagnostic test charges, etc. In this level only few hundred people are given treatment, only those who can reach Clinic or Hospital. The transmission of virus by the mosquitoes is continue and is increasing day-by-day by the passage of time during infection season (out break) Jawad et al. (2001), Kautner et al. (1997). The number of patients increases an daily basis, because only the patients are given concentration but not a single mosquito is killed in this level. The mosquito which is infecting the person to be patient is left alive, free and uncontrolled, to save the life of the patient is left the only option for us. So here the infective boundaries by the mosquitoes are unlimited, the expenses are very high as compared to second level and the control of the causative agents is zero. CONCLUSION It may be concluded that all 3-steps are necessary to control the situation of this problem currently. Unfortunately no concentration is given to the 1st level, only formalities are being completed for the second level and full/all concentration is being given to the 3rd level in which causative agent control is zero. 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