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Pakistan j. entomol. Karachi, 02 (1&2), 2005. CODEN: PJENEL ISSN: 1018-1180

Chief Editor Publication Incharge Executive Editor Imtiaz Ahmad, D.Sc.

Eminent Professor Deptt. of Zoology, Univ. of Karachi

Azher A. Khan Department of Zoology University of Karachi

S.N.H. Naqvi, D.Sc. (Founder Editor)

HEC Eminent Professor Deptt. of Zoology, Univ. of Karachi

Managing Editor Assistant Editor S.M. Naushad Zafar, Ph.D.

S.G.S. Pakistan (Pvt.) Ltd. Korangi, Karachi

M. Tariq Rajput, Ph.D. M.A.H.Q. Biological Res. Centre

University of Karachi Editorial Board

Carl Schaefer, Ph.D. University of Connecticut, Storrs,

Conn. (USA)

Michael Breuer, Ph.D. Zoological Institute, Catholic

University of Leuven (Belgium) A.R. Shakoori, Ph.D.

University of the Punjab New Campus (Pakistan).

Kahkashan Akhter, Ph.D. Department of Zoology, University of Karachi.

M.F. Khan, Ph.D. Department of Zoology, University of Karachi.

M.A. Matin, Ph.D. NARC, Islamabad,

(Pakistan). S. Anser Rizvi, Ph.D.

Department of Zoology, University of Karachi.

R.C. Saxena, Ph.D. Chairman, Neem Foundation,

Mumbai. Feyzi Onder, Ph.D.

Department of Plant Protection, Agric. Faculty, Ege Univ. Bornova,

Azmir (Turkey).

M. Ather Rafi, Ph.D. NARC, Islamabad,

(Pakistan).

Advisory Board U.S.A.

Alfred Wheeler Jr. Ph.D. Cornell University,

Itacha, N.Y.

Asia 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).

Europe J. Koolman, Ph.D. Philips Universitat,

Marburg (Germany).

B.N. Islam, Ph.D. BAU, Mymensingh,

University (Bangladesh)

Africa 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)

Australia Errol Hasan, Ph.D.

University of Queensland Gattons College, Lawes, QLD.

Absar Mustafa Khan, Department of Zoology,

M.U. Aligarh (India).

Pakistan j. entomol. Karachi, Vol. 20 (2005)

CONTENTS Charges 9. RELATIVE TOXICITY AND PERSISTENCE OF DIFFERENT INSECTICIDES

AGAINST JASSID, AMRASCA DEVASTANS DIST. ON SOYBEAN MUHAMMAD FAHEEM AKBAR, NIKHAT YASMIN, M. F. KHAN, M.HANIF QURESHI2 AND FARAH NAZ………………………………………………………..

1-3

600/-

10. TOXICITY DETERMINATION OF PHYTOPESTICIDES, ARGEMONE MEXICANA AND LANTANA CAMARA (LEAVES EXTRACT) AGAINST SITOPHILUS ORYZAE AND THEIR EFFECTS ON ENZYMES AND PROTEINS HIRA AYUB, M. ARSHAD AZMI AND KAHKASHAN AKHTER………................

5-9

1200/-

11. A CLADISTIC ANALYSIS OF THE GENERA OF FAMILY CURCULIONIDAE (COLEOPTERA) FROM SINDH, PAKISTAN ZUBAIR AHMAD, SYED ANSER RIZVI AND SAIMA NAZ……………...............

11-15

1000/-

12. EFFECT OF SOME SPRING HOSTS ON THE LIFE CYCLE OF WHITEFLY, BEMISIA TABACI (GENN.) ABDUL GHANI LANJAR AND HAKIM ALI SAHITO……………………………….

17-23

1400/-

13. HYDROPHILUS PICEUS L., (COLEOPTERA: POLYPHAGA: HYDROPHILIDAE) RECORDED FIRST TIME AND DESCRIBED IN DETAIL WITH ITS CLADISTIC RELATIONSHIP TABINDA ATTIQUE AND SYED KAMALUDDIN…………………………………..

25-28

1000/-

14. PANCREATIC GLUCAGON IN CERTAIN UNGULATES: COMPARATIVE STUDY OF EXTRACTION AND BIOASSAY M. AHMED AZMI, S.N.H. NAQVI, REHANA PERVEEN, M. USMAN, M. ALI SHERAZ AND SOFIA AHMED……………………………….

29-35

1400/-

15. A REVIEW COMPARISON OF NEEM FRUIT SEED EXTRACT (RB-a) AND NEEM FRUIT COAT EXTRACT (RB-b) AGAINST VARIOUS INSECTS R.M. TARIQ…………………………………………………………………...............

37-42

1200/-

Pakistan j. entomol. Karachi, 20(1&2): 1-3, 2005

RELATIVE TOXICITY AND PERSISTENCE OF DIFFERENT INSECTICIDES AGAINST JASSID, AMRASCA DEVASTANS DIST.

ON SOYBEAN

MUHAMMAD FAHEEM AKBAR1, NIKHAT YASMIN2, M. F. KHAN2, M.HANIF QURESHI2 AND FARAH NAZ2

1Department of Agriculture, 2Deparment of Zoology, University of Karachi,

Karachi 75270, Pakistan.

ABSTRACT

The studies on relative toxicity and persistence of Methyl parathion 50 EC, Monocrotophos 40 EC, Fenvalerate 20 EC and Cypermethrin 10 EC against jassid, Amrasca devastans. on soybean were carried out. All the insecticides were effective against jassids after 24 hours of spray; however toxicity reduced with the time interval. The pyrethroids were less toxic and less persistent than organophosphate insecticides. The pyrethroids lost their toxicity after 7 days of application, whereas organophosphates were persistent up to 14 days of spray. The implications in the application of more persistent insecticides on soybean are discussed.

Key words: Relative toxicity, persistence, Amrasca devastans, soybean.

INTRODUCTION

Soybean (Glycine max L. Merrill) is believed to be among the oldest plants cultivated by man. Due to its good quality oil for human consumption, rich protein source for livestock feed and soil fertility improving properties, it is gaining popularity in the agriculture sector of Pakistan. The foliar application of insecticides has proved to be effective in controlling the insect pests of soybean. The lipophyllic action of insecticides has made it necessary to test suitability of insecticides from safety point of view to consumer as greens of crop itself are used as vegetable and its seeds as such or oil and milk. (Hussain et al., 1992). In Tandojam, soybean in addition to thrips is also attacked by jassids and whiteflies (Arain, 1983). Similarly in Peshawar, the insects were found feeding on soybean with white fly Bemisia tabaci (Genn), Thrips flavus Schr, jassids Amrasca biguttula (Gen)., painted bug Piezodorus sp., hairy caterpillar Euproctus sp., chrysomellids, Systena sp. and different species of grasshoppers including Acrida exalta Walk, Truxalix nasuta (Lin.), Euprepocnemis alacris slecris (Ser.) and Attractomorpha acutipennis (Guer). as well the relative abundance of each of these species on

different cultivars of soybean. (Javed et al., 2000).

MATERIALS AND METHODS

Soybean Variety William-82 was sown in a completely randomized block design with three replicates. Each replicate had five sub plots for respective treatment (insecticides) including control. The commercial formulations of Methyl parathion, Monocrotophos, Fenvalerate and Cypermethrin were diluted in water to get the recommended doses for spray. Spraying in each case was made separately in respective soybean plots with a high volume sprayer. Care was taken to provide uniform coverage of the insecticide dilutions on the leaves.

The pest adults of uniform age (3-5 days) and size (from the culture maintained in pot house) were isolated for release in glass jars (1.5 litre capacity) containing soybean leaves treated with different insecticides. The jars were covered with muslin cloth held in position with rubber bands. In this way the jassid adults were released and fed on soybean leaves with insecticidal film at different time intervals. A similar set was maintained without any treatment. Mortality counts were recorded after 24 hours of

release and moribund adults were considered as dead. The experiment in each case was replicated thrice. The data on mortality of jassids was corrected by Abbot’s formula (1925) and the figures thus obtained were subjected to statistical analysis.

RESULTS

The results on mortality of jassid adults (Table 1) revealed remarkable variations in relative toxicity and persistence of the insecticides and relative performance of different insecticidal treatments. The jassid mortality recorded after 24 hours of spray was significantly higher (96.78) in the plots

sprayed with Methyl parathion followed by Monocrotophos (93.32), Fenvalerate (85.78) and Cypermethrin (83.18).

The analysis of variance and application of CD values to mean reduction percentages divided the insecticides into two distinct groups i.e. organophosphates (Methyl parathion and Monocrotophos) were significantly more effective than pyrethroids (Fenvalerate and Cypermethrin) against jassids on soybean. However, the differences within each group were statistically non-significant.

TABLE 1. Relative persistent toxicity of some insecticides

against Amrasca devasans

Corrected percent mortality after Insecticides Dose (ml/acre) 24 hr 48 hr 72 hr 7 days 14 days

Methyl parathion

450 96.78a 92.03a 82.07a 68.78a 57.87a

Monocrotophos 550 93.32a 87.77b 80.78a 65.10b 55.60a Fenvalerate 500 85.78b 81.79c 70.50b 56.84c 42.89b

Cypermethrin 450 83.18b 79.93c 68.59b 54.03d 36.84c CD1 3.58 2.46 2.72 1.21 2.77 CD2 5.43 3.73 4.13 1.83 4.19

DISCUSSION

A critical review of the data showed that the effectiveness of the insecticides varied significantly after 24 hours of spray. Awasthi et al. (1978) applied foliar sprays of methyl demeton -6 and monocrotophos for the control of sucking pests of soybean particularly Amrasca biguttula biguttula. Similarly many other researchers (Corso 1984, Watson et al. 1985 and Umrani 1988) used different insecticides for the control of sucking complex on soybean and they reported the differential toxicity and persistence of the chemicals employed. Hussain et al. (1990) showed a similar impact of spray schedules using Navacron 40 EC on sucking complex of soybean. Hussain et al. (1992) reported a similar trend with similar insecticides in results on the mortality of white fly adults, showing variations in relative toxicity, persistence and performance of different insecticides.

REFERENCES ABBOTT, W.S., (1925). A method for

computing the effectiveness of insecticides. J. econ. Ent., 18: 265-267.

ARAIN, S. A., (1983). Insect complex associated with soybean crop, Glycine max L. Merrill. M.Sc. (Agric.) Mons., Thesis Sindh Agric. Univ. Tandojam. 57.

AWASTHI, M.D., S.K. HANDA, A.K. DIKSHIT and N.S. BHATTACHARYA, (1978). Dissipation of Metasystox and monocrotophos on soybean crop. Indian J. Agric. Sci. 48(4): 245-247.

CORSO, I. C., (1984). Tests with systemic granular insecticides for the control of thrips attacking soybean. Anais da socieedade Entomolegica do Brasil 12 (1): 107-115.

HUSSAIN, T., CHEEMA, M. N. M., KHAN, M. M. MUNSHI, G.H. and RAJPUT, M. A. (1990). Impact of spray schedules of Nuvacron 40 EC on sucking complex of soybean. Proc. Pakistan Congr. Zool., 10: 25-32.

HUSSAIN, T., M.M. KHAN, M.F. AKBAR, S.M.S.H. NAQVI and M.A, RAJPUT (1992). Relative toxicity and persistence of different insecticides against white fly, Bemisia Tabaci Genn. on soybean. Proc. Pakistan Congr. Zool., 12: 295-297.

IQBAL, J., M. SHAHID, N. AKHTAR and M. HASSAN (2000). Diagnosis of important insect pests of soybean in Peshawar. Pak. J. Bio. Sci. 3(6): 1014-1015.

UMRANI, G.H., (1988). Relative toxicity of different insecticides to thrips, thrips tabaci Lind. And whitefly, Bemisia tabaci Genn. on soybean. M.Sc. (Agric.) Hons. Thesis Sindh Agric. Univ. Tandojam: 38.

WATSON, W.M., M.M. EL-BOHEIRY and M.W. GUIRGUIS, (1985). Laboratory and field studies on the effect of sequential application of pesticides on susceptibility some biological aspects of mite, Tetranychus cinnabarinus Brisd. Acarologia. 26(1): 17-23.

Pakistan j. entomol. Karachi, 20 (1&2): 5-9, 2005

TOXICITY DETERMINATION OF PHYTOPESTICIDES, ARGEMONE MEXICANA AND LANTANA CAMARA

(LEAVES EXTRACT) AGAINST SITOPHILUS ORYZAE AND THEIR EFFECTS ON ENZYMES AND PROTEINS

HIRA AYUB, M. ARSHAD AZMI AND KAHKASHAN AKHTER Toxicology Laboratory, Department of Zoology, University of Karachi,

Karachi-75270, Pakistan.

ABSTRACT

Present investigations have been carried out to determine the toxicity of phytopesticides, Argemone mexicana and Lantana camara (leaves extract) against adults of Sitophilus oryzae by glass film exposure method and their effects on enzymes and proteins. The LC50 were calculated to be 2651 μg/cm2 for A. mexicana and 3182.42 μg/cm2 for L. camara. Biochemical estimation revealed that inhibition of alkaline phosphatase was 50% by A. mexicacana and 33.33% by L. camara where as inhibition of acid phosphatase was 80% by both phytopesticides. Inhibition of total protein contents was 40.6% by A. mexicana and 65.14% by L. camara.

KEY WORDS: Sitophilus oryzae, Argemone mexicana, Lantana camara, Toxicity, enzymes.

INTRODUCTION Rice weevil is the most destructive grain insect

pest having cosmopolitan distribution. It causes heavy devastation of stored grains specially of rice and wheat. For the management of this pest conventional (synthetic) pesticides are used. The OP compounds were most effective in preventing the population growth, reducing the damage and weight loss (Shahoo and Rout 1990). Tahir, et al., (1992), Azmi, et al., (1998), Kishore, et al., (1999), Rizwan et al., (2000) and Ahmed et al., (2001), Athanassiou et al., (2004), reported the disturbance of activities at enzyme level in this insect against different groups of pesticides throughout the world. Toxic residues of most of the pesticides create so many problems like pollution, resistance and cumulative residues. They reported resistance and deposition of residues against different pesticides.

Plant products kill insects, however they have low mammalian toxicity, leave no toxic residues and do not pollute the environment. Gupta et al., (1991), Sharaby (1991), Su (1991), Tabassum et al., (1998), Sridevi and Dhingra (1999) and Khan et al., (2000) worked on plant products and reported their less toxic effects enable them to use as safe and friendly pesticides against indiscriminate synthetic insecticides.

Extracts from the leaves of Argemone mexicana and Lantana camara can be used effectively to protect stored grains from insect

infestation as revealed by experiments. As the synthetic insecticides are toxic and hazardous, so, phytopesticides which are much less toxic, biodegradable and economically favourable, the present extracts were used against S. oryzae.


Sitophilus oryzae were obtained from PARC, TARC, University campus, Karachi, reared under controlled conditions i.e., 27-31oC and 60-66% R.H. Sterilized rice were used as rearing medium. All experiments were performed on newly hatched adults of uniform size. Extraction of leaves of both Argemone mexicana and Lantana camara were carried out in the laboratory. Extraction of sample; 20g of Argemone mexicana leaves and 20gm of Lantana camara leaves, collected from Karachi University campus, were washed for the preparation of extract. Leaves were macerated in 50% methanol (1:1) H2O and CH3OH). The macerated leaves of both plants were left for 24h in 200ml of 50% methanol separately. Maceration of leaves was done in Ultra Turax grinder and homogenized for 30 min. Finally, it was filtered twice and stored in refrigerator at 10oC.

For the treatment of insects Glass film method was employed. After preliminary tests 1060.47, 1590.7, 2120.9, 2651 and 3181 μg/cm2 doses of Argemone mexicana and 795.3, 1590.7, 2386.06, 3181.42 and 3976.77 μg/cm2 doses of Lantana camara were selected. These doses were applied


on petri dishes of 2.5 cm diameter with the help of pipette. Ten S. oryzae adults of same age were released in each petri dish separately. For the estimation of acid phosphatase, alkaline phosphatase and protein, 200 adults were treated with LC50 dose of both phytopesticides separately, a day prior to enzyme assay. Thereafter treated insects were crushed in 1ml of distilled water with the help of mortar and pestle and then homogenized for five min at 1,000 rpm at 4oC. The homogenate were centrifuged in labofuge 15,000 at 2000 rpm for 10 min, placed in cold chamber. Supernatants were taken in separate tubes and used for biochemical estimation of alkaline phosphates activity by colorimetric kit method of Randox cat No. 307, acid phosphatase activity by colorimetric kit method of Randox cat No.1011 and Total protein estimation by Biuret method, using Randox diagnostic kit cat No. TP 245.

RESULTS AND DISCUSSION Toxicity of extracts of A. mexicana and L.

camara was determined by using five different doses of each pesticide against S. oryzae. It was observed that the rate of mortality gradually increased with the increase in dose of each pesticide (Table 1-2). LC50 was calculated by using log-log graph paper. By plotting the mean mortality values against the dose of the pesticide. LC50 of A. mexicana was found to be 2651 μg/cm2.

S. oryzae treated with L. camara showed mean mortality of 18.571, 27.142, 38.571, 51.428 and 55.714%, respectively at 24h by plotting the mean mortality values against the dose, the LC50 of L. camara was found to be 3181.42 μg/cm2.

Activity of alkaline phosphatase was found to be inhibited by 50% (when treated with L. camara at dose of LC50 i.e. 2551μg/cm2 and 33.33%

(when treated with L. camara at dose of LC50 i.e. 3181.42μg/cm2 (Table 3).

Activity of acid phosphatase was found to be inhibited by 80% at the respective LC50 doses of each A. mexicana and L. camara (Table 4). Protein activity was reduced up to 40.6% with A. mexicana at a dose of LC50 2651μg/cm2 and up to 65.14% with L. camara treatment at a dose of 3181.42μg/cm2 (Table 5).

In present study LC50 of A. mexicana and L. camara was found to be 2651μg/cm2 and 3181.42μg/cm2 respectively for S. oryzae. These results are in dose conformity with those of Gul-e-Rukhsana et al., (1993) and Ahmed et al., (1998) for pyrethroid and OP compounds.

Redfern et al., (1981) and Ivbijaro (1986) used plant products (friendly pesticide). In the present investigations treatment of A. mexicana and L. camara inhibited alkaline phosphatase and acid phosphatase enzymes in S. oryzae adults. Many workers reported inhibition of alkaline phosphatase and acid phosphatase enzymes (Inove et al., 1983 and Qureshi et al., 1983, Naqvi, et al., 1992).

Inhibition of proteins by different pesticides were reported by Ahmed and Naqvi (1985), Azmi (1993) and Akhtar et al., (1994).

CONCLUSIONS

It could be concluded that A. mexicana and L. camara extract prove safer alternative pesticides for controlling insect pests. They have low mammalian toxicity, less persistent and do not pollute the environment.

ACKNOWLEDGEMENT

The authors are grateful to Chairman, Dept. of Zoology, Univ. of Karachi for help during this work.

Table 1. Toxicity of Argemone mexicana against Sitophilus oryzae.

Concentration in μg/cm2

Mortality mean %

S.D. (+) S.E. (+) Range at 95% confidence limit

Control - - - -

1060.47 22 4.21 1.33 19.392-24.606

1590.70 32 7.88 2.492 27.116-36.884

2120.90 43 4.830 1.527 40.008-45.992

2651.0 54 5.163 1.632 50.802-57.198

3181.0 61 4.215 1.333 59.406-63.612


Table 2. Toxicity of L. camara against Sitophilus oryzae.

Concentration in

μg/cm2 Mortality mean

% S.D. (+) S.E. (+) Range at 95%

confidence limit Control - - - -

795.30 18.571 3.781 1.424 15.78-21.362

1590.70 27.142 4.898 1.852 23.513-30.771

2386.06 38.571 3.785 1.431 35.767-41.357

3181.42 51.428 3.790 1.432 48.622-54.234

3976.77 55.714 5.349 2.022 51.751-59.677

Table 3.

Alkaline phosphatase inhibition in S. oryzae treated with A. mexicana and L. camara.

Compound Mean of unit (μ/L)

S.D. (+) S.E. (+) Range at 95% confidence

limit

Inhibition %

Control 3.68 1.593 0.919 1.879-5.481 0.0%

A. mexicana 2.76 0.022 0.012 2.736-2.783 50%

L. camara 1.84 1.593 0.919 0.039-3.641 33.333%

Table 4.

Acid phosphatase inhibition in S. oryzae treated with A. mexicana and L. camara.



limit

Inhibition %

Control 1.238 0.429 0.247 0.745-1.722 0.0%

A. mexicana 0.262 0.262 0.151 0.0765-0.666 80%

L. camara 0.247 0.429 0.247 0.237-0.731 80%

Table 5.

Total protein inhibition in S. oryzae treated with A. mexicana and L. camara.



limit

Inhibition %

Control 16.656 14.574 8.414 0.165-33.147 0.0%

A. mexicana 9.89 6.079 3.509 3.003-16.757 40.6%

L. camara 11.59 16.143 9.320 6.677-29.857 65.14%


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AND TABASSUM, R., 1998. Toxicity of cypermethrin and Acorous calamus extract on Sitophilus oryzae. Proc Pad. Congr Zool., 18: 31-36.

AHMAD, I., SHUJA, F., AZMI, M.A., AKHTAR, K. AND RIZVI, S.A. 2001. Comparative toxicological studies of neem leaves extract (NL) and cyhalothrin (pyrethroid) against Sitophilus oryzae and their effects on alkaline phosphatase activity. Proc. Pak. Congr. Zool., 21: 255-261.

AHMED, S.O. AND NAQVI, S.N.H. 1985. Toxicity and effects of dimilin on the protein pattern of Aedes aegypti. Proc. Ent. Soc. Kar., 14-15: 119-132.

AKHTAR, K., NAQVI, S.N.H. AND AZMI, M.A. 1994. Determination of toxicity, fecundity and emergence inhibiting effects of neem factors (NfQ) against Callosobruchus analis. Z. Ang. Zool., 80(2): 141-154.

ATHANASSIOU, C.G., PAPAGREGORIOU, A.S. AND BUCHELOS, C. TH., 2004. Insecticidal and residual effects of three Pyrethroids against Sitophilus oryzae (L.), (Coleoptera: Curculionidae) on stored wheat. J. of stored product research, 40: 289-297.

AZMI, M.A., NAQVI, S.N.H., AKHTAR, K., JAHAN, M., HAIBB, R. AND TABASSUM, R., 1993. Toxicological studies of solfac and neem formulations against Sitophilus oryzae. Nat. Acad. Sci. Letters, 16 (5&6): 187-190.

AZMI, M.A., NAQVI, S.N.H., KHAN, M.F., AKHTAR, K. AND KHAN, F.Y., 1998. Comparative toxicological studies of RB-a (neem extract) and coopex (permethrin+ bioallethrin) against Sitophilus oryzae with reference to their effects on oxygen consumption and GOT, GPT activity. Tr. J. of Zool., 22: 307-310.

GUL-E-RUKHSANA, ASLAM, M., KAZMI, M.A., AHMAD, I., KHAN, M.F. AND NAQVI, S.N.H., 1993. Toxicity of neem oil against Callosobruchus analis in comparison with deltamethrina nd dimethoate. Pakistan J. Entomol. Karachi, 8(2): 15-27.

GUPTA, H.C., BARETH, S.S. AND SHARMA, S.K., 1991. Bioefficacy of edible and non-edible oils against pulse beetle

(Callosobruchus chinensis L.) on stored pulses and their effect of germination. Agric. Biol. Res., 7(2): 101-107.

INOVE, Y., 1983. Termicidal activities of synthetic pyrethroids. Pestic. chem.., Hum. Welfare Environ. Proc. 5th Int. Congr. Pestic. Chem., pp. 113-118.

IVBIJARO, M.F., 1986. Prospects for neem in Nigerian agriculture. Proc. 3rd Int. Neem Conf. (Nairobi, 1986), pp. 525-533.

KHAN, M.F., GUL, S., AHMAD, I., AZMI, M.A., NAQVI, S.N.H. AND AKHTAR, K., 2000. Determination of toxicity of fenpropathrin and citronella oil against Red flour beetle, Tribolium castaneum and their effects on cholinesterase enzyme. Proc. Pak. Congr. Zool., 20: 231-235.

KISHORE, P., RAI, G. AND GODHANI, 1991. Relative susceptibility of pearl millet genotypes to Sitophilus oryzae Linn., Rhizopertha dominica Fab. and Tribolium castaneum Herbst. in storage. J. of Entomological Research (New Delhi), 23(4): 339-342.

NAQVI, S.N.H., AND TABASSUM, R., 1992. Probable and development of resistance against neem extract RB-a and cyfluthrin (solfac 1096) Musca domestica (PCSIR strain). Pak. J. Entomol. Karachi, 7: 9-16.

QURESHI, R.A., QADRI, S., ANWARULLAH, M. AND NAQVI, S.N.H., 1983. Effect of neopesticides (JHA) on the morphology, emergence and sterility of Musca domestica L., (PCSIR strain) and its relation to phosphatase. Z. Angew. Entomol., 95: 304-309.

REDFERN, R.E., WARTHEN, J.D. JR., VEBEL, E.C. AND MILLS, G.V. JR., 1981. The antifeedent and growth disruption effect of azadirachtin on Spodoptera frugipedra and Oncopeltus fasciatus. Proc. 1st Int. Neem Conf. (Rottach-Egern, 1980), pp. 129-136.

RIZWAN, S., AHMAD, I., AKHTAR, K., QURESHI, S.A., NAQVI, S.N.H. AND AZMI, M.A., 2000. Toxicological studies on cypermethrin and cyfluthrin against Sitophilus oryzae and their effects on phosphomonoesterases. Proc. Pak. Congr. Zool., 20: 109-115.

SAHOO, B.K. AND ROUT, G., 1990. Comparative efficacy of some synthetic pyrethroids on organophosphatase against rice weevil


Sitophilus oryzae. Environ. Ecol., 6(3): 721-723.

SHARABY, A., 1991. Some Myrtaceae leaves as protectants of rice against the infestation of Sitophilus oryzae. Pol. Pismo. Entomol., 59(2): 377-382.

SRIDEVI, D. AND DHINGRA, S., 1999. Evaluation of mixtures of non-toxic vegetable oils and deltamethrin against susceptible and resistant strains of Tribolium castaneum Herbst. J. of Entomological Research (New Delhi), 23(4): 323-330.

SU, H.C.F., 1991. Laboratory evaluation of toxicity of calamus oil against four species of stored product insects. J. Entomol. Sci., 26(1): 76-80.

TABASSUM, R., KHAN, M.F., NAQVI, S.N.H., AZMI, M.A. AND AHMAD, I., 1998. Efficacy of neem extract (FNLX, H-27 and CA) against stored grain pest, Callosobruchus analis. Proc. Pak Congr. Zool., 18: 25-29.

TAHIR, S., ANWAR, T. AND NAQVI, S.N.H., 1992. Toxicity and residual effects of novel pesticides against rice weevil, Sitophilus oryzae. Pakistan J. Zool., 24(2): 111-114.


A CLADISTIC ANALYSIS OF THE GENERA OF FAMILY CURCULIONIDAE (COLEOPTERA) FROM SINDH, PAKISTAN

ZUBAIR AHMAD1, SYED ANSER RIZVI2 AND SAIMA NAZ2

1Department of Zoology, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. 2Department of Zoology, University of Karachi, Karachi, Pakistan, 75270.

ABSTRACT

7 genera accommodating 21 species of the family Curculionidae, Latereilla have been cladistically analyzed and a cladogram has been constructed and discussed in the light of apomorphies found in the included genera of family Curculionidae.

KEYWORD INDEX: Cladistic Analysis, Coleoptera, Curculionidae, Genera, Sindh, Pakistan.

INTRODUCTION The super-family Curculionioidea Hopkins, 1915

includes the families Platypodidae, Scolytidae, Anthribidae, Brentidae and Curculionidae. The present work deals with the cladistic analysis of Curculionidae (Curculio, from Latin a weevil, a snout beetle).

The only subsequent attempt to survey the Curculionidae of the world as a whole is contained in Lacordaire’s ‘Genera des Coleopteres’ (vols. vi & vii, 1863 & 1866). In this work he recognized 834 genera, which were divided into 82 “tribes” or subfamilies. It is true that many modifications in his system have been rendered necessary as a result of the enormous increase in the number of described genera and species during the last fifty years, but nevertheless Lacordaire’s classification, on account of its comprehensive character, remain today the standard one for the student who has to deal with the weevils of any fauna other than those of Europe or North America.

Prior to the present studies, the curculionid fauna of Pakistan was very poorly known. Hashmi and Tashfeen (1992) gave only an inventory of the collection housed in different institutions and very little was known from the areas now included in Pakistan.

At the same time no morphological work was ever undertaken to help understand characters of taxonomic importance. Minute to large species of the family Curculionidae are characterized by the prolongation of the head with a snout of variable length, width and shape. Mostly hard, dull-coloured or brilliant metallic beetles; shining, smooth, rugose, sculptured, punctured or striated; scaly or hairy; oval, elongated, cylindrical, robust or long, slender and even ant-like in form. Head prognathous, globose,

slightly or greatly extended, and with mouthparts at the end of the snout. Eyes prominent. Antennae straight, geniculate, moniliform, clavate, 10 to 12 segmented and with three segmented club. Snout short and wider or long and decurved; may be grooved for reception of the antennae. Mouthparts are small, strong. Labrum present or absent. Mandibles flat, pincer-like, toothed on one or both sides. Palpi short and usually concealed. Prothorax variable, narrow or as wide as mesothorax. Legs short or very long, fore and middle coxae rounded, hind pair oval, fore coxal cavities closed behind. Tibiae sometimes armed. Tarsi five segmented, simple or pad-like, fourth segment often very small. Claws usually one pair or may be absent, free or fixed. wings well developed, rudimentary or absent. Elytra usually completely covering abdomen but may expose the pygidium. Abdomen with five sternites, the first two fused.

Many of the authorities have worked on various aspects of different genera of Curculionidae (Marshall, 1916; Ramamurthy and Ghai, 1998; Anderson and Anelia, 2000; Maregalli, 1991; Mihajlova, 1978; Monguzzi, 1999; Pesarini, 1973; Vaurie, 1973; Zarzaga, 1984), but the cladistic analysis of the genera of the family Curculionidae has not yet been done.

CLADISTIC ANALYSIS OF GENERA OF FAMILY CURCULIONIDAE

a0 Head without snout.

a1 Head with snout or rostrum. (Curculionidae).

a2 Rostrum very short, usually less than 1mm. (Burmanicus, Tanymecus and Myllocerus).

a3 Rostrum slightly long, usually less than 3 mm. (Pissodes and Hylobius).

12 Z. Ahmad et. al.

a4 Rostrum very long, more than 3 mm. (Nassophasis).

b0 Rostrum not dilated.

b1 Rostrum dilated at apex (Hylobius).

c0 Base of rostrum laterally smooth.

c1 Base of rostrum laterally punctured (Nassophasis).

d0 Basal joint of antennae usually small.

d1 Basal joint of antennae greatly elongated (Curculionidae).

e0 Antennae not concealed.

e1 Antennae not visible from above (Pissodes).

e2 Antennae visible from above (Hylobius).

f0 Antennae not inserted.

f1 Antennae inserted in between eye and rostrum (Tanymecus).

f2 Antennae inserted at apex of rostrum (Myllocerus).

g0 Club of antennae short.

g1 Club of antennae elongated and mucronate (Tanymecus).

g2 Club of antennae compact (Myllocerus).

h0 Club of antennae unsegmented.

h1 Club of antennae 3 segmented (Myllocerus).

h2 Club of antennae 4 segmented (Tanymecus).

i0 Scape usually smooth.

i1 Scape gradually clavate at apex (Myllocerus).

i2 Scape abruptly calvate at apex (Tanymecus).

j0 1st joint of funicle equal to 2nd.

j1 1st joint of funicle longer than 2nd (Burmanicus).

k0 Palpi longer and open.

k1 Palpi short and usually concealed (Curculionidae).

l0 Body without any vestiture.

l1 Vestitures consists of scales, pubescent, setae etc. (Burmanicus, Hylobius, Myllocerus, Nassophasis, Pissodes, Tanymecus).

l2 Vestitures consists of large punctuation with one long scale (Sitophilus).

m0 Pronotum very short.

m1 Pronotum shorter than elytra (Burmanicus, Hylobius, Myllocerus, Nassophasis, Pissodes, Tanymecus).

m2 Pronotum as wide as elytra (Sitophilus).

n0 Pronotum without any ridge.

n1 Pronotum with black rounded ridges (Hylobius).

o0 No lateral carinae.

o1 Lateral carinae not distinct (Nassophasis).

o2 Lateral carinae distinct (Hylobius and Pissodes).

p0 Elytra smooth.

p1 Elytra with black or white patches of hairs (Hylobius).

p2 Elytra with three or four black lines (Pissodes).

p3 Elytra with yellowish green stripes (Burmanicus).

p4 Elytra without any stripes (Myllocerus and Tanymecus).

q0 Forea or punctation absent.

q1 Numerous forea or punctations on thorax and intervals of elytra (Nassophasis).

r0 Prothorax not constricted.

r1 Prothorax anteriorly constricted (Hylobius).

s0 Tibia without hairy processes.

s1 Prothoracic tibia with fringe of dark brown hairs laterally (Burmanicus).

t0 Precoxae very far to each other.

t1 Precoxae close, never touched each other (Pissodes).

t2 Precoxae contiguous (Hylobius).

u0 Tibia without spurs.

A cladistic analysis of the family Curculionidae 13

u1 Tibia with spurs (Hylobius, Nassophasis and Pissodes).

v0 Fore and middle coxae not rounded.

v1 Fore and middle coxae rounded (Curculionidae).

w0 Legs triangular.

w1 Legs cylindrical or simple (Hylobius, Nassophasis and Pissodes).

w2 Legs denticulate or simple (Burmanicus, Myllocerus and Tanymecus).

x0 Femora without toothed.

x1 Femora with one or more toothed (Myllocerus).

y0 Metasternum with median longitudinal lines.

y1 Metasternum without longitudinal lines (Myllocerus and Tanymecus).

y2 Metasternum with lateral longitudinal lines (Burmanicus).

CHARACTERS AND CHARACTERSTATES

Head (a) Head with snout or rostrum in Curculionidae

shows its autapomorphic condition (a1). Rostrum very short, usually less than 1mm in Burmanicus, Tanymecus and Myllocerus shows their synapomorphic condition (a2). In Pissodes and Hylobius, rostrum less long usually less than 3mm long shows their derived synapomorphic condition (a3) while in Nassophasis it is very long exceeding 3 mm long shows its autapomorphic condition (a4).

Dilation of Rostrum (b) In Hylobius rostrum dilated at apex shows its

autapomorphic character (b1).

Punctation of Rostrum (c) Base of rostrum laterally punctured in

Nassophasis shows its autapomorphic character (c1).

Basal joint of Antennae (d) Basal joint of antennae greatly elongated in

Curculionidae shows its autapomorphic character (d1).

Visibility of Antennae (e) Antennae not visible from above in Pissodes

show its autapomorphic character (e1) while in case of Hylobius it is visible from above shows its derived autapomorphic character (e2).

Insertion of Antennae (f) Antennae inserted in between eyes and rostrum

in Tanymecus shows its autapomorphic condition (f1) and in Myllocerus antennae inserted at apex of rostrum shows its derived autapomorphic character (f2).

Club of Antennae (g) Club of antennae elongated and mucronate in

Tanymecus shows its autapomorphic character (g1) and the club of antennae compact in case of Myllocerus shows its derived autapomorphic character (g2).

Segmentation of club of Antennae (h) In Myllocerus club of antennae 3 segmented

shows its autapomorphic condition (h1) while in Tanymecus club of antennae 4 segmented shows its derived autapomorphic condition (h2).

Scape of Antennae (i) Scape of antennae gradually clavate at apex in

Myllocerus shows its autapomorphic condition (i1). In Tanymecus scape abruptly clavate at apex shows its derived autapomorphic condition (i2).

1st and 2nd funicle (j) 1st segment of funicle longer than 2nd in

Burmanicus shows its autapomorphic condition (j1).

Palpi (k) Palpi short and usually concealed in

Curculionidae shows its autapomorphic condition (k1).

Vestiture on body (l) Vestiture consists of scales, pubescent, setae,

etc. in Nassophasis, Hylobius, Pissodes, Burmanicus, Tanymecus and Myllocerus shows their synapomorphic condition (l1). In Sitophilus vestiture consists of only tubercles shows its autapomorphic condition (l2).

Pronotum (m) Pronotum shorter than elytra in Nassophasis,

Hylobius, Pissodes, Burmanicus, Tanymecus and Myllocerus shows their synapomorphic condition (m1), in Sitophilus vestiture only consists of tubercles shows its autapomorphic condition (m2).

Ridges on Pronotum (n) Pronotum with black round ridges in Hylobius

shows its autapomorphic condition (n1).

14 Z. Ahmad et. al.

Lateral Carinae (o) Lateral carinae not distinct in Nassophasis show

its autapomorphic condition (o1). In Hylobius and Pissodes lateral carinae distinct show their synapomorphic condition (o2).

Elytra (p) Elytra with black or white patches of hairs in

Hylobius shows its autapomorphic condition (p1), elytra with three or four black lines in Pissodes shows its derived autapomorphic condition (p2). In Burmanicus it is with yellow green stripes show its more derived autapomorphy (p3). In Myllocerus and Tanymecus, elytra without any stripes shows their synapomorphic condition (p4).

Punctation (q) Numerous forea or punctation on thorax and

intervals of elytra in Nassophasis shows its autapomorphic condition (q1).

Constriction of Prothorax (r) Prothorax anteriorly constricted in Hylobius

shows its autapomorphic condition (r1).

Hairy Process (s) Prothoracic tibiae with fringe of dark brown hairs

laterally in Burmanicus show its autapomorphic condition (s1).

Closeness of Procoxae (t) Procoxae close but never touched each other in

Pissodes show its autapomorphic condition (t1). In Hylobius it is contiguous which shows its derived autapomorphic condition (t2).

Tibial Spurs (u) Tibiae with spur in Nassophasis, Hylobius and

Pissodes show their synapomorphic condition (u1).

Shape of Fore and Middle coxae (v) Fore and Middle coxae rounded in Curculionidae

show its autapomorphic condition (v1).

Denticulation in legs (w) Legs cylindrical or simple in Nassophasis,

Hylobius and Pissodes show their synapomorphic condition (w1). In Burmanicus, Tanymecus and Myllocerus legs denticulate or simple show their derived synapomophic condition (w2).

Toothed in Femora (x) Femora with one or more toothed in Myllocerus

shows its autapomorphic condition (x1).

Longitudinal lines on Metasternum (y) Metasternum without longitudinal lines in

Tanymecus and Myllocerus shows their

synapomorphic condition (y1). In Burmanicus, metasternum with lateral longitudinal lines shows its autapomorphic condition (y2).

DISCUSSION ON CLADOGRAM OF THE GENERA OF FAMILY CURCULIONIDAE

The family Curculionidae Latreille, comprising seven genera viz.: Myllocerus Schoenherr, Tanymecus Germar, Burmanicus Supare, Pissodes Germar, Hylobius Germar, Nassophasis Waterhouse and Sitophilus Linnaeus appears to fall into two groups.

Group I includes only one genus Sitophilus which probably appears most advanced in the entire clade and place out group relationships with other curculionid genera in having vestiture consist of one long scale on large each punctured (l2) and pronotum as wide as elytra (m2).

Group II comprises rest of the above six genera. This group is further divided into two subgroups. Subgroup I comprises Pissodes, Hylobius and Nassophasis in which Nassophasis plays sister group relationships to each other by having lateral carinae distinct (o2), rostrum less long usually less than 3 mm (a3) shows apomorphic character and also plays outgroup relationships with Nassophasis which has lateral carinae (o1) not distinct, rostrum very long more than 3 mm (a4), base of rostrum laterally punctured (c1) and numerous fovea or punctation on thorax and intervals of elytra (q1) shows its apomorphies.

Figure-1. Cladogram, showing the relationship of the genera of family Curculionidae.

A cladistic analysis of the family Curculionidae 15

The subgroup II comprises three genera in which Burmanicus plays outgroup relationships with other genera and shows its apomorphies by having prothoracic tibiae with fringe of dark brown hairs laterally (s1), elytra with yellowish green stripes (p3), metasternum with lateral longitudinal line (y2), 1st joint of funicle longer than 2nd (j1). The rest of the two genera Myllocerus and Tanymecus appear to be most closely related playing sister group relationships to each other by having apomorphies of elytra without any stripes (p4) and metasternum without longitudinal lines (y1).

REFERENCES ANDERSON, R. S. AND ANALIA, A. (2000). New

genera and species of weevil from the Galapagos Islands, Ecuador and Cocos Island, Costa Rica (Coleoptera: Curculionidae: Entiminae: Entimini). American Museum Novitates, (3299) 28: 1-15.

HASHMI, A. A. AND TASHFEEN, A. (1992). Coleoptera of Pakistan. Proc. Pak. Congr. Zool., 12: 133-170.

HOPKINS, A. D. (1915) A preliminary classification of the superfamily Scolytoidea. U.S. Dept. agric. Ent. Tech., 17: 165 – 232, 7pls.

MARSHALL, G. A. K. (1916). The Fauna of British India. Coleoptera, Rhynchophora, Curculionidae. London.

MARVALDI, A. F. (1999). Larval morphology in Curculionidae (Insecta: Coleoptera). Acta Zoologica Lilloana, 45 (1): 7-24.

MAREGALLI, M. (1991). Notes of some Iberian Otiorhynchus of the Otiorhynchus stricticollis Fairmaire group with description of four new taxa. Eos. Rev. Esp. Entomol., 67(Nov.): 85-101.

MIHAJLOVA, B. (1978). Snout beetles (Coleoptera: Curculionidae) of Macedonia, Yuguslavia. Fragm. Balc. Mus. Macedonici. Sci. Nat., 10(14): 123-133.

MONGUZZI, R. (1999). A new Otiorhynchus (Troglorhynchus) of “baldensis group” and notes about species of the some group (Coleoptera: Curculionidae: Polydrusinae). Bollethino della Societa Entomologica Italiana, 131 (3): 233-238.

PESARINI, C. (1973). [Weevils new for Lombardy: Contribution to the knowledge of Coleoptera: Curculionidae]. Boll. Soc. Entomol. Ital., 105 (1-3): 36-38.

RAMAMURTHY, V. V. AND GHAI, S. (1988). A study of the genus Myllocerus (Coleoptera: Curculionidae), Oriental Insects, 22: 377-500.

VAURIE, P. (1973). The weevil genera Homalinotus and Ozopherus of the neotropical cholinae (Coleoptera: Curculionidae). Bull. Am. Mus. Nat. Hist., 152 (1): 1-49.

ZARAZAGA, M. A. A. (1984). The Curculionidae (Coleoptera): 3. New species of Otiorhynchinae of Iberia and taxonomic comments on some genera of Otiorhynchinae and Brachyderinae. Bol. Asoc. Esp. Entomol., 8: 207-218.


1

EFFECT OF SOME SPRING HOSTS ON THE LIFE CYCLE OF WHITEFLY, BEMISIA TABACI (GENN.)

ABDUL GHANI LANJAR AND HAKIM ALI SAHITO Sindh Agriculture University, Tando Jam

ABSTRACT

The experiment on "effect of some spring host, on life cycle of Bemisia tabaci" was carried out in the laboratory, Department of Entomology, Faculty of Crop Protection, Sindh Agriculture University, Tandojam. Three sets of experiment were conducted, which started from 15-02-2005, 28-02-2005, 10.03.2005 respectively, on each experimental date, the branches/leaves of okra, cabbage, mustard and brinjal were brought in the laboratory. One male and two females from the culture (already maintained in the lab) were released on them to record crop wise life spent by the fly.

Whitefly reared from 15-02-05 spent much time on sesame and mustard less time on okra. It spent 9.00 ± 0.70, 6.80 ± 0.37, 6.40 ± 0.24, 6.40 ± 0.40, 9.00 ± 0.44, 7.20 ± 0.73, 10.20 ± 6.73, 44.80 ± 2.88, 47.80 ± 2.80 days as egg, 1st instar, 2nd instar, 3rd instar, 4th instar male adult female adult, total life male and total life female respectively, whereas, total life of male adult on brinjal, cabbage and okra was recorded 40.00 ± 00, 43.50 ± 3.12 and 28.40 ± 2.86 respectively. During same period the total life of female was 44.60 ± 3.49, 45.80 ± 3.24 and 40.80 ± 3.08 days, respectively.

The shorter life span was recorded when reared from 10-02-05. The total maximum life span of female adult flies was 29.00 ± 2.09 on mustard and minimum 28.2 ± 2.29 day on okra for 27.4 ± 2.30 and 25.8 ± 2.56 day respectively. The quick life span of various life stages after 10-03-05 was recorded on okra. Egg, 1st, 2nd, 3rd, 4th, adult male and female live for 4.80 ± 0.37, 3.80 ± 0.37, 3.40 ± 0.40, 3.00 ± 0.54, 5.80 ± 0.31, 7.40 ± 0.28 days, respectively.

Analysis of variance showed significant difference in the development of whitefly on crops, dates, life stages was recorded on okra.

KEY WORDS: Bemisia tabacci, Brinjal, Cabage, life cycle, mustard, and okra.

INTRODUCTION

Whitefly, Bemisia tabaci stands out as the most important member of the family aleyrodidae: Homoptera (Basu, 1995) for its grave impact on tropical and sub-tropical agriculture. The whitefly causes direct damage by feeding, soil the plant by producing honeydew and more alarming. (Singh et al. 1994) inflects severe crop losses by transmitting fairly large number of viral diseases (Safdar, 2005). It is a polyphagus pest and has been recorded on very wide range of cultivated and wild plants (Greathead, 1986). However, the magnitude of infestation and the nature of extent of injury vary with host plants, seasons and localities. Similar variation has been recorded with regards to its biology. The local duration of the immature stages of B. tabaci reportedly varies widely and correlated with climate and host plants conditions (Basu, 1995). Mohanty and Basu (1995) reported that one-year’s continuous rearing of B.tabaci on four species of plants revealed variation due to seasons as well as hosts. They

further mentioned that the seasonal factors have pronounced effect while the effect of hosts was not substantial. However, month wise breakup of eleven experiments revealed adequate host wise differences especially during November to a lesser extent during February. Coudriet et al. (1986) showed very marked variation due to host factor, with development on sweet potato, lettuce etc. being much faster than on carrot, broccoli and tomato. The differences between the two extremes, that is between sweet potato and carrot, work out to as much as 40%. Host correlation variations were pronounced to less than 20% between the extremes (Lopez-Avila, 1986).

The variation in biology of B.tabaci on various host plants prompted to work on impact of host plant on the biology of B.tabaci in laboratory conditions.


The experiment on impact of some spring hosts on life cycle of Bemisia tabaci was carried


2

out in the laboratory, Department of Entomology, Faculty of Crop Protection, Sindh Agriculture University, Tando Jam. Three sets of experiment were conducted i.e. Ist experiment was started from 15-02-2005, 2nd from 28-02-2005 and 3rd from 10.03.2005.

Culture development of Whitefly: The branches/leaves of okra, cabbage,

mustard and brinjal were brought in the laboratory. After stripping off all leaves except two, which had sufficient number of crawlers, were kept in a chimney glass for culture developed of whitefly. The basal end of branch/petiole of leaf was kept in a beaker containing water to keep the branches alive and fresh for longer period. The beaker had a lid on it from which petiole was passed in the beaker. The immature of whiteflies were regularly examined until adult stage.

Observations of Life-cycle:

At the each experimental date the branches/leaves of okra, mustard, cabbage and brinjal were brought in the laboratory. After removing all the leaves except one, the branch was then kept in the chimney glass for life cycle study. In this way five chimneys were kept for each crop (5 replicates). Case was taken that none of the life stage was already presented on the leaf, if found was removed.

A pair of whitefly (female and male) was released separately on leaves kept in chimneys of each crop. After 24 hours the adult whiteflies were removed and the number of eggs laid by female was counted (if any pair of whitefly was failed to oviposit, it was replaced with new ones). After counting the eggs the egg incubation period and development period of subsequent life stages were recorded for each crop separately. The observations on changes in the life-cycle were recorded twice a day in the morning and in the evening.

Meteorological record was also maintained during study period. The data thus obtained were subjected to analysis of variance for variation in time interval and development period of each life stage. Least significant difference was also observed by applying LSD test.

RESULTS

The whitefly had under gone the following life stages i-e. egg, 1st instar crawler, 2nd instar, 3rd instar nymph, 4th instar nymph + pupal duration

and adult longevity (male + female). The duration of all developmental stages was recorded on different host plants.

The data on life span on host plants, i.e. mustard, brinjal, cabbage and okra at different time intervals is furnished in Tables 1–3. The results of effects on different life stages are presented as under.

a. Development of Egg:

The egg incubation period of the whitefly reared on various dates of spring hosts indicated that maximum incubation period was recorded when reared from 15-02-2005 and minimum period was observed when reared from 10-03-2005. However, incubation period on 15-02-2005 revealed that maximum period 9.00 ± 0.70 days was recorded when reared on mustard followed by cabbage (8.40 ± 0.40), okra (8.00 ± .070) and brinjal (7.40 ± 0.67). Incubation period of the whitefly was shortened when whitefly reared 15 days later i.e. 28-02-2005. During this period the incubation 6.20 ± 0.37 days was recorded on mustard and cabbage and 6.00 ± 0.44 for brinjal and okra respectively. Incubation period still shortened when reared on 10-03-2005. Minimum incubation period 4.40 ± 0.40 days was recorded on mustard followed by brinjal (4.80 ± 0.66) okra (4.80 ± 0.37) cabbage (5.00 ± 0.54) respectively.

b. Nymphal Development:

1st instar nymph

First instar nymph spent 6.50 ± 0.37 days on mustard when reared on 15-02-2005. However, minimum time spent 5.80±0.73 days was recorded on okra. The 1st instar nymphs spent lesser time when reared from 28-02-2005 as compared to 15-02-2005. The 1st instar nymphs life cycle of 28-02-2005 spent lesser time i.e. 4.60 ± 0.24 days on cabbage and much time on brinjal and mustard that was 5.00 ± 0.44 and 5.00 ± 0.31 days, respectively. Still lesser time spent by 1st instar when reared from 10-03-2005. The crop wise time spent indicated that faster development 3.80 ± 0.20 and 3.80 ± 0.24 days was on brinjal and cabbages, respectively, than mustard (4.00 ± 0.31) and cabbages (4.40 ± 0.24).

2nd Instar

The development period of 2nd instar nymph 6.80 ± 0.37, 6.60 ± 0.50, 6.20 ± 0.58 and 5.80 ± 2.27 days was recorded on mustard, brinjal, cabbage and okra, respectively, when reared from 15-02-2005. A bit faster development was recorded when the whitefly was reared from 28-02-


3

2005. During this period, it spent 4.40 ± 0.40 days on mustard and cabbage 4.60 ± 0.50 day on okra and brinjal, respectively. However, faster development was recorded when reared from 10-03-2005. During this period the 2nd instar spent 3.40 ± 0.24 days on mustard and brinjal 3.80 ± 0.37 and 3.40 ± 0.40 days on cabbage and okra respectively.

3rd Instar

The development of 3rd instar was also slower when reared from 15-02-2005 than reared from 10-03-2005. The 3rd instar spent maximum time 6.40 ± 0.40 days on mustard and cabbage when reared from 15-02-2005. The shortest time period 3.00 ± 0.54 days was recorded when reared from 10-02-2005.

4th Instar nymph + pupa

The fourth instar nymph spent more days when reared from 15-02-2005. it lived for 9.00 ± 0.44 days on mustard followed by 8.20 ± 0.66 days on cabbage, 7.80 ± 0.86 on brinjal and 6.80 ± 0.57 days on okra. Fourth instar spent 7.60 ± 0.50, 7.80 ± 0.58, 8.00 ± 0.44 and 7.40 ± 0.40 days on mustard, brinjal, cabbage and okra respectively when reared from 28-02-2005. Minimum time spent was recorded when reared from 10-03-2005. The development of 4th instar completed with in 6 days. However, crop wise days spent indicated that 4th instar lived for 6.60 ± 0.40, 6.80 ± 0.58, 6.20 ± 0.58 and 5.80 ± 0.37 days on mustard, brinjal, cabbage and okra respectively.

Adult Development:

Male Whitefly

Male fly longevity 7.20 ± 0.73, 6.40 ± 0.74, 7.80 ± 0.58, 6.60 ± 0.24 days was recorded on mustard, brinjal, cabbage and okra respectively when reared from 15-02-2005. The male lived for 6.40 ± 0.50, 7.00 ± 0.70, 6.40 ± 0.74 and 5.80 ± 0.37 days on mustard, brinjal, cabbage and okra when reared from 28-02-2005. The male adult lived shorter life when reared from 10-03-2005. The days spent by male adult whitefly after 10-03-2005 was 5.80 ± 0.58, 6.00 ± 0.54, 5.40 ± 0.24 and 5.00 ± 0.31 day on mustard, brinjal, cabbage and okra, respectively.

Female Whitefly

The female of B. tabaci lived longer than male flies during all life cycle development. However, the life of the females reared from 15-02-2005 lived longer then males, which were reared from 10-03-2005. The life spent 10.20 ± 0.73, 11.00 ±

0.54, 10.00 ± 0.70 and 9.0 ± 0.44 days was recorded on mustard, brinjal, cabbage and okra, respectively, when reared from 15.02.2005. The female reared from 28-02-2005 spent 9.80 ± 0.37, 9.60 ± 0.24, 9.20 ± 0.37 and 9.00 days on mustard, brinjal, cabbage and okra, respectively. The female lived for 7.80 ± 0.37, 7.60 ± 0.24, 8.00 ± 0.31 and 7.40 ± 0.24 days on mustard, brinjal, cabbage and okra, respectively, when reared from 10-03-2003.

The life spent

Tables 1-3 reveal the overall life spent of B. tabaci on different host plants at different dates of spring season. The whitefly male spent lesser time to complete its life cycle when reared from mid March to early April. It spent 25.8 ± 2.36 day on okra followed by mustard (27.4 ± 2.30), brinjal (28.00 ± 2.29) and cabbage (28.20 ± 2.28). It completed it life cycle in 33.00 ± 2.68, 33.60 ± 2.52, 33.60 ± 2.63 and 35.20 ± 3.03 days on okra, mustard, cabbage and brinjal, respectively, when reared from end of February to the end of the March.

The male spent maximum days when it reared from mid February to March during this period it completed its life cycle in 38.40 ± 2.88, 40.00 ± 3.69, 43.50 ± 3.12 and 44.80 ± 2.88 days on okra, brinjal, cabbage and mustard, respectively.

The female spent more days than males. The female longevity was more on brinjal which was recorded 11.00 ± 0.54 days followed by cabbage 10.00 ± 0.70, mustard 10.20 ± 0.73 and okra 9.00 ± 0.44 when reared from 15-02-2005. Female took lesser time when reared from 28-02-2005. During this period female spent much time 9.80 ± 0.77 days on mustard, 9.00 ± 0.24 on brinjal, 9.20 ± 0.37 on cabbage and 9.00 days on okra. Female spent still lesser days when reared from 10-03-2005. It took 7.60 ± 0.24, days on okra, 7.80 ± 0.37 on mustard and 8.00 ± 0.31 days on cabbage. The life spent of female from egg to adult 47.80 ± 2.88, 45.80 ± 3.24, 44.60 ± 3.49 and 40.8 ± 3.08 days was recorded on mustard, cabbage, brinjal and okra, respectively, when reared from 15.02.2005. The total life cycle of female reared from 28-02-2005 took 37.80 ± 2.39, 37.80 ± 2.57, 36.20 ± 2.75 and 36.4 ± 2.26 days on mustard, brinjal, cabbage and okra, respectively.

It was recorded that the females reared from 10-03-2005 lived for 29.00 ± 2.09, 29.60 ± 2.27, 30.80 ± 2.28 and 28.2 ± 2.29 days on mustard, brinjal, cabbage and okra respectively.


4

Temperature and Relative Humidity

Temperatures were recorded between 19.15 ± 0.70 to 27.44 ± 1.01 and RH% 42.74 ± 1.42 to 62.38 ± 1.46 for life cycle reared from 15-02-2005. The range of temperature 20.43 ± 0.44 to 30.17 ± 7.32 and relative humidity 42.7 ± 1.62 to 53.13 ± 1.92 was recorded for life cycle reared from 28-02-2005. The temperature and humidity for the life cycle reared from 10-03-2005 was ranged between 24.44 ± 1.07 to 30.17 ± 1.32 and 42.7 ± 1.62 to 45.44 ± 1.35, respectively. It was observed that increase in temperature shortened the life of the whitefly. However, RH had little or no effect on life cycle development.

Statistical Analysis

Analysis of variance of life cycle reared from 15-02-2005 showed that the period of development of the life stages (D.F= 6, F=27, P<0.01) and development with response to crop (D.F= 3, F=5.24, P<0.01) was highly significant. However, LSD test showed that maximum time 10.05 days was spent by female in it development and survival. LSD further indicated that no significant difference was recorded in the development period of egg and 4th instar, 2nd and 3rd instar nymph.

Life cycle development with response to crop showed no significant difference between mustard

and cabbage. Analysis of variance for the life cycle reared from 28-02-2005 showed no significant difference between crops (F.D= 3, F=1.15, P<0.33). LSD test has well supported it by showing highly significant difference in the development period of the stages. LSD test further showed the same period for male longevity, egg incubation period 1st instar, 2nd instar and 3rd instar. Life period on mustard and brinjal, cabbage and okra had no significant difference. Life period after 10-03-2005 had reduced as compared to life period of 15, Feb and 28 Feb. Analysis of variance showed non significant difference in life period on crops. (D.F= 3, F=120, P<0.31). LSD showed non-significant difference in the life period on all crops.

The overall analysis of variance for all season showed significant difference in the development of the whitefly on crops (D.F= 3, F=5.18, P<0.01), dates (D.F= 2, F=169.81, P<0.01) and life stages (D.F= 6, F=131.49, P<0.01). However LSD showed non-significant difference in development period of egg and male longevity. But the crop wise development indicated that faster development was recorded on okra only with regards to dates significant difference was recorded in life cycle development.

Table 1. Total live span (x ± S.E) of B. tabaci on various host plants reared from 15-02-2005 onwards.

Life Stage

Mustard Brinjal Cabbage Okra

Egg Incubation Period

9.00 ± 0.70

7.40 ± 0.67

8.40 ± 0.40

8.00 ± 0.70

1st Instar

6.80 ± 0.37

6.6 ± 0.50

6.20 ± 0.58

5.8 ± 0.73

2nd Instar

6.40 ± 0.24

6.00 ± 0.44

6.60 ± 0.50

5.60 ± 0.60

3rd Instar

6.40 ± 0.40

5.80 ± 0.48

6.40 ± 0.40

5.60 ± 0.24

4th Instar

9.00 ± 0.44

7.80 ± 0.86

8.20 ± 0.66

6.80 ± 0.37

Male (Adult)

7.20 ± 0.73

6.40 ± 0.74

7.80 ± 0.58

6.60 ± 0.24

Female (Adult)

10.20 ± 0.73

11.00 ± 0.54

10.00 ± 0.70

9.00 ± 0.44

Male

44.80 ± 2.88

40.00 ± 3.69

43.50 ± 3.12

28.40 ± 2.86

Female

47.80 ±2.88

a

44.60 ± 3.49

ab

45.80 ± 3.24

a

40.8 ± 3.08

b


5


Life Stage



6.20 ± 0.37

6.00 ± 0.44

6.40 ± 0.37

6.00 ± 0.44

1st Instar

5.00 ± 0.31

5.00 ± 0.44

4.60 ± 0.24

4.80 ± 0.37

2nd Instar

4.40 ± 0.40

4.60 ± 0.50

4.40 ± 0.40

4.60 ± 0.50

3rd Instar

4.00 ± 0.44

4.80 ± 0.37

4.00 ± 0.44

4.40 ± 0.60

4th Instar

7.60 ± 0.50

7.80 ± 0.58

8.00 ± 0.44

7.40 ± 0.40

Male (Adult)

6.40 ± 0.50

7.00 ± 0.70

6.40 ± 0.74

5.8 ± 0.37

Female (Adult)

9.80 ± 0.37

9.60 ± 0.24

9.20 ± 0.37

9.00 ± 0.44

Male

33.6 ± 2.52

35.20 ± 3.03

33.60 ± 2.63

33.00 ± 2.68

Female

37.00 ± 2.39

a

37.80 ± 2.57

a

36.4 ± 2.26

ab

36.20 ± 2.75

ab


Life Stage



4.40 ± 0.40

4.80 ± 0.66

5.00 ± 0.54

4.80 ± 0.37

1st Instar

4.00 ± 0.31

3.80 ± 0.20

4.40 ± 0.24

3.80 ± 0.37

2nd Instar

3.40 ± 0.24

3.40 ± 0.24

3.80 ± 0.37

3.40 ± 0.40

3rd Instar

3.20 ± 0.37

3.20 ± 0.37

3.40 ± 0.24

3.00 ± 0.54

4th Instar

6.60 ± 0.40

6.80 ± 0.58

6.20 ± 0.58

5.80 ± 0.37

Male (Adult)

5.80 ± 0.58

6.00 ± 0.54

5.40 ± 0..24

5.00 ± 0.31

Female (Adult)

7.80 ± 0.37

7.60 ± 0.24

8.00 ± 0.31

7.4 ± 0.24

Male

2.74 ± 2.30

28.00 ± 2.09

28.20 ± 2.21

25.8 ± 2.56

Female

29.00 ± 2.09

a

29.60 ± 2.29

a

30.80 ± 2.28

a

28.2 ± 2.29

a


6

CONCLUSIONS

The results of present study revealed that B. tabaci completed its life cycle on all four host plants i.e. mustard, brinjal, cabbage and okra. El-Khayat, et al. (1994) included okra as one of the hosts of whitefly. Balaji and Veeravel (1994) mentioned the ovipostional preference of B. tabaci on brinjal due to its hairing leaves. Singh et al. (1994) reported that the mustard crop harbouring B. tabaci in rabi season. Aslam and Gebara (1995) reported that among four hosts the whitefly showed its maximum preference to okra. Monhanty et al. (1996). The life cycle started in 2nd week of March had taken shorter time as compared to the life cycle started in 2nd week of February. Bosco and Caciagli (1998) reported that development period varied with temperature lower the temperature higher the duration of life cycle. Chaudhary et al. (2001) reported that developmental period was longer in duration during December to January and Singh et al. (2003) reported brinjal is one of the most preferred hosts of B. tabaci. Xuwei Hong et al. (2003) tested 7 host plants for feeding and development including cabbage. Li et al. (2003) maintained that the developmental periods from egg to adult varied from 48.7 days at 17°C to 13.9 days at 29°C. In present study the whitefly took shorter time to complete it life cycle when reared on okra as compared to mustard, brinjal and cabbage. Al-Zyoud and Sengonca (2004) reported that development of life stages is much faster on cotton than lobicco at 30°C. Cotton and okra both belong to Melvin Family. In present study the period of female longevity was higher than male. The same is reported by Abdullah and Singh (2004) and Al-Zyoud Sengonca (2004). Safdar et al. (2005) reported okra variety subzpari as most susceptible to whitefly attack. The same variety was used in present study. The development period varied with regard to host plants as investigated in present study Demichelis et al. (2005) reported that development time and some morphological character also vary on different host plant. Fekrat and Bor (2005) also reported the same variation in development time on different cultivars of aubergine.

On the behalf of the result achieved it is concluded that:

1. Whitefly spent much time during cooler days to complete its life cycle.

2. Female live longer than male whitefly.

3. Faster life cycle was recorded on okra plants in all three life cycles.

REFERENCES

ABDULLAH, N. M. M. AND J. SINGH (2004).

Biology of whitefly, Bemisia tabaci (Gennadius) on cotton under Punjab conditions. Status Manage. and Eco. Zoo. 12 (1): 1-6.

AL-ZYOUD, F. AND SENGONCA, C. (2004). Development, longevity and fecundity of Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) on different host plants at two temperatures. Mitteilungen der Deut. G. Fur. Al. and A. Ento.14 (1-6): 373-376.

ASLAM, M. AND F. GEBARA. (1995). Host plant preference of vegetables by cotton whitefly, Bemisia tabaci (Genn.). Pak. Jr. of Zoo. 27 (3): 269-272.

BASU, A.N. (1995). Bemesia tabaci (Gennedius) crop pest and principle whitefly vector of plant viruses.West view press, Boulder. San Francisco, Oxford. Pp. 183.

BOSCO, D. AND P. CACIAGLI (1998). Bionomics and ecology of Bemisia tabaci (Sternorrhyncha: Aleyrodidae) in Italy. Eur. Jr. of Ento. 95 (4): 519-527.

CHAUDHURI, N., D.C. DEB AND S.K. SENAPATI (2001). Biology and fluctuation of white fly (Bemisia tabaci Genn.) population on tomato as influenced by abiotic factors under Terai region of West Bengal. Ind. Jr. of Agri. Res. 35 (3): 155-160.

COUDRIET, D.L., PRABHAKER, N., KISHABA, A.N. AND MEYERDRIK, D.E. (1986). Variation in development rate on different hosts and over wintering of the sweet potato whitefly, Bemisia tabaci (Homoptera: Alerodidae). Env. Ento. 14: 516-519.

DEMICHELIS, S., C. ARNÒ, D. BOSCO, D. MARIAN AND P. CACIAGLI (2005). Characterization of biotype T of Bemisia tabaci associated with Euphorbia characias in Sicily. Phy. 33 (2): 196-208.

EL-KHAYAT, E.F., M. EL-SAYED, F.F. SHALABY AND S.A. HADY (1994). Infestation rates with Bemisia tabaci (Genn.) to different summer and winter vegetable crop plants. An. of Agri. Sci., Mosh. 32(1): 577-594.


7

FEKRAT, L. AND P. S. BOR (2005). A study of biology of cotton whitefly Bemisia tabaci (Gennadius) on three cultivars of eggplant in laboratory conditions. Ir. Jr. of Agri. Sci. 36 (1): 137-143.

GREATHEAD, D. J. AND F. D. BENNETT (1986). Possibilities for the use of biotic agents in the control of the white fly, Bemisia tabac. Bio. News and Inf. 2 (1): 7-13.

LI, Q.B., XIANG, R.S. MANDOUR, N.S. AND LI, L. (2003). Effect of temperature on the development and reproduction of Bemisia tabaci, B. biotype (Homoptera: Aleyrodidae), Ento. Sin. 10 (1): 43-49.

LOPEZ A. (1986). Taxonomy and biology in Bemisia tabaci. A literature survey of International Institute of Biological Control. U.K. Pp.27-36.

MOHANTY, A.K., A.K. KAR, P.N. SETHI AND A. DHAL (1996). Brinjal varieties as sources of rearing host plants for Bemisia tabaci Genn. Cr. Res. (Hisar). 11(3): 386-387.

SAFDAR, A.M., A.KHAN, A. HABIB, S. RASHED AND Y. IFTIKHAR (2005). Correlation of

environmental conditions with okra yellow vein mosaic virus and Bemisia tabaci population density. Int. Jr. of Agri. and Bio. 7(1): 142 – 144.

SINGH, J., A. S. SOHI, H. S. MANN AND S. P. KAPUR (1994). Studies on whitefly, Bemisia tabaci (Genn.) transmitted cotton leaf curl disease in Punjab. Jr. of Inse. Sci. 7(2): 194-198.

SINGH, D., R.S. JAGLAN AND R. CHAUHAN. (2003). Field studies on the efficacy of insecticides against brinjal whitefly (Bemisia tabacci. Genn.). An. of Bio. 19 (1): 109-112.

XU WEIHONG, ZHU GUOREN, LI GUILAN, XU BAOYUN, ZHANG YOUJUN, WU QINGJUN (2003-a). Influence of temperature on the biology of Encarsia formosa parasitizing the whitefly Bemisia tabaci. Chi. Jr. of Bio. Con.19 (3): 103-106.

XU WEIHONG, ZHU GUOREN, ZHANG YOUJUN, WU QINGJUN, XU BAOYUN, LI GUILAN (2003-b). An analysis of the life table parameters of Bemisia tabaci feeding on seven species of host plants. Ento. Know. 40 (5): 453-455.


HYDROPHILUS PICEUS L., (COLEOPTERA: POLYPHAGA: HYDROPHILIDAE) RECORDED FIRST TIME AND DESCRIBED

IN DETAIL WITH ITS CLADISTIC RELATIONSHIP

TABINDA ATTIQUE AND SYED KAMALUDDIN Federal Urdu University of Arts, Science and Technology

Gulshan-e-Iqbal Campus, Karachi.

ABSTRACT

Hydrophilus piceus L., the great scavenger and predacious water beetle is described in detail with special reference to its genitalia. The cladistic relationship is also briefly discussed using apomorphic characters. Key Words: Hydrophilus piceus, Coleoptera, Hydrophilidae, Cladistic relationships.

INTRODUCTION

Cox (1874) has keyed out the different genera of Hydrophilidae. Miall (1895) has described life Cycle of Hydrophilus as well as general characteristics of H. piceus. Lefroy and Hewlett (1909) has discussed the five sub families and the life cycle of Hydrophilus as well as general characters and also illustrated H. olivaceus and H. piceus.

Folsom (1923) described the locomotion of Hydrophilus triangularis. Browne (1925) considered the difference between Hydrophilus (Great silver beetle) and the (Hydroous or Hydrocharis) lesser silver beetle with Dytiscus.

Femald and Shepard (1942) has described the characters of family Hydrophilidae and illustrated the diagram of H. triangularis Say. Kloet and Hincks (1945) have listed the only single species of Hydrophilus in their Catalogue. Comstook (1950) described habitat, mutation and characters of different species and genera of the family Hydrophilidae. Jeques (1951) keyed out the different genera and species of Hydrophilidae except H. piceus. Swain (1952) has described the adults, young and their importance of the Hydrophilidae.


The specimens were collected from the fresh water pools in Sindh and on light. For the study of female genitalia, the abdomen was excised at base and warmed in 10% KOH solution on a burner for about 2 to 3 minutes. It was then washed with tap water and inflated under Leitz binocular microscope in the same medium, the examination of various structures were made and their diagrams were drawn by placing them on the

cotton threads immersed in glycerine with the help of eye piece graticule and were later preserved in microvials with a drop of glycerine later pinned with the specimen.

HydrophUusficeus L. (Figs. 1-3)

Hydrophilus piceus L. 1758, S.N., ed. X (I): 411; Alien, 1956, Ent. Mon. Mag. 92: 153; Leech, 1975, Ent. Rec. J. 87(5): 146; Knill-Jones 1985, Ent. Gaz. 35. (2): 112.

General body:

Body broadly ovate.

Colouration:

Body generally dark browinish except yellow, labral margin fine punctures on elytra, with three doted lines and some punctures on ronotum.

Head:

Head more than 2X longer than broad, anteocular distance 3mm, posterior of head including eyes 3mm, width of head including eyes 4mm, eyes prominent, somewhat triangular, basal segments of antennae longest, 2nd- 3rd- 4th and 5th segment equal to each other, 6th segment somewhat triangular, 9th segment club shape, maxillary palpi with basal segment longest, labial palpi two segmented, basal segment longest.

Thorax:

Pronotum broader than long, length of pronotum 5mm, width 6mm, lateral margins of pronotum slightly convex, anterior angles sub rounded, humeral angles rounded, posterior margin sinuated, scutellum not covered, distinctly broader than long, triangular, apex pointed, length of scutellum 3mm, width 4mm, punctures on elytra


with four rows of dots, lateral margins of elytra slightly convex, apical margins sub-rounded, length of elytra 25mm, width 10mm, meso-thoracio spine reaching 5th abdominal segment.

Female genitalia (Fig. 3):

Genital plate somewhat quadrangular-shaped, 1st valvuale large elongated, 2nd valvuale shorter than 1st, medially bulbus-like, distally narrowed with acute apex, uterus large broad, ductus bursae narrowed convoluted with a bulb like accessory gland, corpus bursae elongated and broad.

Abdomen:

Abdomen convex beneath, unarmed, pygidium (Fig. 2) covered, total length 36mm.

Comparative note:

This species is most closely related to H. triangularis (Say) in having last joint of maxillary palpi shorter than the preceding but it can easily be separated from the same in having posterior margin of pronotium sinuated and the abdominal segments have no triangular yellow spots at the side in contrast posterior margin of pronotum concave and the abdominal segments have distinct triangular yellow spots at the side in H. triangularis and by the other characters as noted in the description.

DISCUSSION The representatives of the genus Hydrophilus

Geoff., are distributed all over the world. This genus plays sister group relationship with Hydrous Brull by their synapomorphies like posterior tarsi strongly compressed, oar-like, prosternum and mesosternum riged and out group relation by its autapomorphies like posterior tarsi scarcely compressed, prosternuna smooth.

The species H. piceus L., recorded from Sindh, Pakistan plays sister group group relationship with H. triaagularis Say recorded from Pakistan by their synapomorphies like last joint of maxillary palpi shorter than the preceding and out group relationships by its autapomorhies, the abdominal segments have no more or less distinct triangular, yellow spots at the side.

REFERENCES BROWNE F.B. (1925). Concerning the habits of

insects. Cambridge University press London: Fetter Lane: 112-137.

COMSTOOK, J.H. (1950). An introduction to entomology. Coleoptera. Comstock publishing company, INC.

COX, H.E. (1874). Ahand book. Coleoptera or Beetles of Great Britain and Ireland. E.W. Janson, 28. Museum Street, London: 95 — 124.

FERNALD, H.T AND SHEPARD, H. H. (1942). Applied entomology. An introductory text book of insects in their relations to man. McGraw-Hill book Company, INC. New York and London.

FOLSOM, J.W. (1923). Entomology with special reference to its ecological aspects, John murray, Albemarle street, W.London: 166-167.

JAQUES H.E. (1951). How to know the beetles. W.M.C, Brown Company Dubuque, Lowa: 78-82.

KLOET G.S. AND HINCKS W.D. (1945). A Check list of British insects. T. Buncle & Co.Ltd. Kloet & Hincks, 322 Wellington road north, Heaton Chapel, Tockport.

LEFROY, H.M. AND HEWLETT. P.M. (1909). Indian Insects Life, Tacker and Co., 2 Creed Lane, London.

MIALL, L.C. (1895). The natural history of aquatic insects. Macmillian and Co. New York. London, 395 PP.

SWAIN R.B. (1952). The insects guide order and major families of north American insects. Doubledaj and Company Inc. Garden city, New York: 120-121.

Illustration of Figures Figs. 1-3. Hydrophilus piceus L. 1. entire,

dorsal view; 2. Pygidium, dorsal view; 3.. female genitalia, ventral view.

Key to the letterings ac. gl. (accessory gland), o. br. (corpus

bursae), d.br. (ductus bursae), pyg. (pygidium). ut. (uterus), ist. val. (first valvulae), 2nd val. (second valvulae), 8th. sg. (eight segment), 9th. sg. (ninth segment).


1

PANCREATIC GLUCAGON IN CERTAIN UNGULATES: COMPARATIVE STUDY OF EXTRACTION AND BIOASSAY

M. AHMED AZMI1*, S.N.H. NAQVI2, REHANA PERVEEN3,

M. USMAN4, M. ALI SHERAZ5 AND SOFIA AHMED6

1* Corresponding Author: Muhammad Ahmed Azmi, Ph.D. Baqai Medical University, 51 Deh Tor Gadap Road, Near Toll Plaza, Super Highway, P.O. Box 2407, Karachi–74600, Pakistan.

Tel: +9221-4410293-8 Ext: 221, Dir: +9221-4410434 Fax: +9221-4410439 Email: [email protected] 2,3,5,6, Institute of Pharmaceutical Sciences, Baqai Medical University, Karachi. 4, Institute of Hematology, Baqai Medical University, Karachi.

ABSTRACT

Pacreatic pieces upto 20 gm of four ungulates i.e., buffalo, cow, sheep and goat have been subjected to isolation and extraction of glucagon like material which have been tested by an in vivo qualitative bioassay. The presence of glucagon like material had been interpreted from the increase in protein content after alcohol other precipitation from the sources under investigation and confirmed by the hyperglycemic activity of each extracted material when physiologically tested. The present study demonstrates the comparative presence of glucagon like material in the pancreases of four ungulates required for immunological characterization of glucagon, as a part of this work.

Key words: Pancreatic glucagon, Ungulates, Comparative Study, Extraction, Bioassay

INTRODUCTION

Glucagon, a hormone secreted by the alpha cells of the pancreatic islets of Langerhans when the blood glucose concentration falls. This hormone has multiple functions that are diametrically opposed to those of insulin. One of the important of one is to increase the blood glucose concentration, an effect which is exactly opposite to that of insulin. On injection of purified glucagon into an animal, a profound hyperglycemic effect occurs. Only 1 μg/kg of glucagon can elevate the blood glucose concentration approximately to 20 mg/dl of blood in about 20 minutes. For this reason glucagon is also called the hyperglycemic hormone (Guyton, 1971). Several other researchers also reported about the biological activities of glucagon such as Unson et al. (1989) described that hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucose in the blood.

They also reported that glucagon analog, des-His 1 [Gluaa] glucagon amide has been designed and synthesized as well as considering considered as a useful tool for investigating the mechanisms of glucagon action. In addition they also provide

the evidence that glucagon is a contributing factor in the pathogenesis of diabetes.

Azizeh et al. (1997) also reported that five new glucagon analogues have been designed, synthesized and characterized determining their biological activities. Similarly Pospisilik et al. (2001) reported that glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis.

It was therefore worth wile to plan the present study that is concerned with isolation and extraction of glucagon followed by its biological, molecular and immunological characterization in pancreas and GIT of lower vertebrates in comparison to higher vertebrates to elucidate the regulation of carbohydrate metabolism involving this hormone as a hyperglycemic-glycogenolytic factor.


1. Animals:

The pancreas of four different animal species (Ungulates i.e. buffalo, sheep, cow and goat) were


2

selected for the processes of extraction and purification of pancreatic glucagon during the present study.

2. Collection of samples:

The samples of pancreas such as buffalo, sheep, cow and goat were obtained from the slaughter house, Korangi, Karachi and were immediately brought to the laboratory in thermos flask. The pancreas samples were then trimmed off the surrounding tissues and stored at −20°C. One pancreas from each animal species was used for every extraction.

3. Crude Extraction of Glucagon:

Extraction of glucagon like material from pancreas was followed by the method of Kenny (1955) and this involves three steps:

a. Preparation of acid ethanol extract before the extraction process was performed. The extract prepared labeled as “Extract-A”.

b. The protein fraction containing glucagon was precipitated by the addition of absolute alcohol and ether solvent (2:1) to the extract-A”. The precipitate was dried in vacuum for overnight and then stored at −20°C, until the re-extraction process for column chromatography was performed.

c. The dried powder obtained in the second step was added in chloride phosphate buffer (0.5 ml/gm tissue) and centrifuged at 2000 rpm for 15 minutes. The supernatant obtained was dialyzed for 72 hours against the same buffer.

The dialyzed sample was then dried in a rotatory evaporator. About 1 ml of the buffer solution was added to the residue. The sample was then designated as “Extract-B”, which is stored at −20°C for further purification and subsequent analysis.

4. Protein Estimation:

Samples obtained (0.1 − 1.0 ml) of both “Extract-A” and “Extract-B” were taken simultaneously for protein estimation.

5. Bioassay:

Presence of glucagon in the test sample of animals was recorded by determining the increase in blood sugar level of rabbit following administration of test sample. The rabbit was fasted for 18 hours prior to the experiment. Samples were injected intravenously in volume of 0.1 cc per animal. The blood sugar determination was carried out by the method of Folin-Wu after

collected the samples at 0, 15 and 30 minutes respectively.

6. Estimation of blood sugar:

Blood sugar estimation was carried out by Folin-Wu method. This method is based on the principle that when glucose or other sugars are heated in an alkaline solution of cupric ions which is reduced to cuprous oxide. This compound is then heated and reacted with phosphomolybydic acid to form a complex molybedenuim, the blue color formed is then read colorimetrically at 440 mμ.

RESULTS AND DISCUSSION

In the present study, the pieces of pancreas from a number of ungulates have been subjected to crude extraction for obtaining material demonstrating hyper-glycemic activity. Homogenates were extracted and purified to obtain both micro and macro methods of extraction. The precipitation of glucagon like material was checked in the extracted portion by determining the increase in protein content after glucagon precipitation with alcohol and ether (2:1). The record obtained for protein estimation and increase in protein content from various samples for buffalo, sheep, goat and cow is presented collectively in table 1.

The protein content determined colorimetrically is compared with normal standard curve (Fig. 1). It is assumed from this table that protein content in all the samples of each species is increased after the purification of glucagon which can be interpreted as the part of glucagon like material, since glucagon is a polypeptide in its chemical nature. Although all the samples showed protein concentration before the addition of alcohol and ether. However, it is increased tremendously after precipitation. The increase in protein (μg/ml) content of the samples of homogenates ranged from 20-75 in buffalo, 16-94 in sheep, 12-49 in goat and 14-76 in case of cow. It may be presumed that by this method maximum precipitation of glucagon-like material has been obtained. The increase in protein content have been expressed as percentage of the original protein content before glucagon precipitation as shown in Fig. 2, 3, 4, and 5, respectively. It has been observed that about 100-1300% increase in protein occurred in all except cow’s sample which showed relatively less increase in percentage (542%). Further more, in all the samples of all species except those of cow increase was always


3

more than 100%. One sample of cow however in protein content by showed 63% increase (Fig. 5).

In the present study, all the extracted samples have been tested for the presence of glucagon like material. Bioassay procedure was normally carried out on glucagon are either quantitative or qualitative as mentioned by (Kawarski et al. 1984; Foster et al. 1993). The literature shows that such a qualitative bioassay on pancreatic extraction of dog for the presence of glucagon was interpreted (Fodden and Reid, 1954; Conlon et al., 2002). Glucose concentration recorded for each extracted sample for various species indicate that there is an abrupt increase in the glucose content of plasma both at Omin, 15 and 30 minutes in all the samples of all the species has been presented collectively in table 2.

It has also been observed that the samples extracted from pancreatic tissue of various species do contain the polypeptide which displays the hyperglycemic activity. This activity has been denoted in literature as glycogenolytic / hyperglycemic factor or glucagon (Hendrick et al., 1990).

It becomes obvious from this preliminary attempt that it is possible to extract glucagon-like material utilizing as a commercially adopted method for tissues weighing far less in quantity than normally used (gm instead of Kg) and that the extracted material in all cases is nothing but glucagon. These samples in due course will be subjected to chromatographic separation so as to further purify it and then glucagon content will be determined radio-immunologically (Angelova and Dimov, 1996). Previously glucagon from pancreas and intestine of duck has been shown in two molecular forms (mol. wt. 5500 and 7000) and the one having smaller mol. wt. have been shown to exhibit glycogenolytic activity in dog (Rigopoulon et al., 1970).

The present investigation is the part of complete program of characterization of different molecular species of glucagon with their specificity towards glycogenolytic / hyperglycemic activity showing towards the successful isolation, extraction of partially purified hyperglycemic factors from pancreas of four ungulates.


4

Fig. 1: Std. curve for protein estimation

Fig. 5: Percent increase in protein

content of beef pancreas

Fig. 2: Percent increase in protein content of buffalo pancreas

Fig. 3: Percent increase in protein content of sheep pancreas

Fig. 4: Percent increase in protein


5

REFERENCES

ANGELOVA, M. AND DIMOV, N. 1996. Effect of mobile phase composition on selectivity in preparative hydrophobic interaction chromatographic purification of glucagon. Biomed. Chromatog. 10(5): 251-255.

AZIZEH, B. Y., VAN TINE, B. A., TRIVEDI, D. AND HRUBY, V. J. 1997. Pure glucagon antagonists: Biological activities and cAMP accumulation using phosphodiesterase inhibitors. Peptides. 18(5): 633-641.

CONLON, J. M., KIM, J. B., JOHANSSON, A. AND KIKUYAMA, S. 2002. Comparative peptidomics of the endocrine pancreas: islet hormones from the clawed frog Xenopus laevis and the red-bellied newt Cynops pyrrhogaster. J. Endocrinol 175(3): 769-777.

FODDEN, J. H. AND REID, W.O. 1954. J. Endocrinol. 54: 303-307.

FOSTER, C.M., BORONDY, M., PADMANABHAN, V., SCHWARTZ, J., KLETTER, G. B., HOPWOOD, N. J. AND BEITINS, I. Z. 1993. Bioactivity of human growth hormone in serum: Validation of an in vitro bioassay. Endocrinology. 132(5): 2073-2082.

GUYTON, A. C. 1975. Glucagon: Its structure and function. Text Book of Medical Physiology. 3rd Ed: 862-863.

HENDRICK, G. K., FRZZELL, R. T., WILLIAMS, P.E. AND CHERRINGTON, A. D. 1990. Effect

of hyperglucagonemia on hepatic glycogenolysis and gluconeogenesis after a prolonged fast. Am. J. Physiol. 258(5): 841-849.

KENNY, J. 1955. Extractable glucagon of the human pancreas. J. Clin. Endocrin. 15: 1089-1105.

KOWARSKI, C. R., LIAOU, M. Y., KOWARSKI, D., WEIZER, J., BOYNES, D. AND KOWARSKI, A. A. 1984. Improved bioassay for glucagon by continuous glucose monitoring. J. Pharm. Sci. 73(9): 1298-1299.

LOWRY, O.H., ROSEBROUGH, A. L., FARR, R. J. AND RANDALL, A. 1951. J. Biol. Chem. 193: 265-267.

POSPISILIK, J. A., HINKE, S. A., PEDERSON, R. A., HOFFMANN, T., ROSCHE, F., SCHLENZIG, D., GLUND, K., HEISER, U., MCINTOSH, C. H. AND DEMUTH, H. 2001. Metabolism of glucagon by dipeptidyl peptidase IV (CD26). Regul. Pept. 96(3): 133-141.

RIGOPULOUS, D. T., VALVUDE, J., MARCO, G., FALCOANA, R. H. 1970. Large glucagon immuno-reactivity in the extract of pancreas. J. Biol. Chem. 245: 496-501.

UNSON, C. G., GURZENDA, E. M. AND MERRIFIELD, R. B 1989. Biological activities of des-His 1 [Glu9] glucagon amide, a glucagon antagonist. Peptides. 10(6): 1171-1177.


6

Table 1: Protein estimation in samples during extraction and purification procedures for glucagon in the pancreases of ungulates

Optical density against standard Conc. Of protein against Std (μg/ml)

Ungulates Samples Before precipitation After precipitation

Before glucagon

precipitation samples

In glucagon precipitation samples

Increase in protein content ((μg/ml)

A 0.048 0.097 20 54 34

B 0.041 0.284 08 44 58

C 0.027 0.108 18 84 66

D 0.071 0.244 08 28 20

E 0.051 0.178 28 103 75

F 0.051 0.367 07 57 50

G 0.061 0.456 06 54 40

H 0.051 0.608 05 73 68

I 0.041 0.237 07 36 29

BUFFALO

J 0.061 0.244 9.5 38 28.5

A 0.065 0.085 49 65 16

B 0.061 0.081 46 62 16

C 0.056 0.171 30 88 58

D 0.038 0.181 18 104 86

E 0.032 0.187 16 110 94

F 0.027 0.284 4 44 40

G 0.032 0.155 16 90 74

SHEEP

H 0.032 0.168 16 98 82

A 0.051 0.347 8 55 47

B 0.061 0.081 9 22 13

C 0.076 0.171 12 34 12

D 0.072 0.181 11 60 49

E 0.027 0.187 4 27 21

F 0.032 0.284 4 47 43

G 0.041 0.155 6 44 38

GOAT

H 0.041 0.168 6 45 39

A 0.032 0.071 23 56 33

B 0.041 0.222 6 34 28

C 0.061 0.097 22 36 14

D 0.061 0.114 14 90 76

E 0.041 0.145 22 80 58

COW

F 0.092 0.284 14 44 50


7

Table 2: Hyperglycemic activity in the extract pancreas samples of different species of ungulates

Optical density of samples taken Glucose conc. (mg%) against std. curve Ungulates Samples

0 min 15 mins 30 mins 0 min 15 mins 30 mins

A 0.022 0.041 0.051 80 168 208

B 0.027 0.061 0.065 104 212 272

C 0.022 0.036 0.041 80 148 170

D 0.022 0.032 0.041 80 124 164

E 0.027 0.038 0.046 104 142 184

F 0.027 0.061 0.071 104 256 296

G 0.027 0.056 0.061 104 240 260

H 0.022 0.038 0.041 80 138 160

I 0.027 0.051 0.066 104 208 276

BUFFALO

J 0.027 0.051 0.056 104 200 232

A 0.027 0.046 0.081 104 192 256

B 0.027 0.051 0.071 104 216 280

C 0.027 0.041 0.051 104 168 208 SHEEP

D 0.027 0.45 0.086 104 184 248

A 0.027 0.041 0.051 106 168 212

B 0.027 0.046 0.061 106 192 252

C 0.027 0.036 0.041 106 148 170 GOAT

D 0.027 0.051 0.066 106 208 278

A 0.027 0.056 0.051 104 148 214

B 0.027 0.051 0.066 104 208 260

C 0.027 0.056 0.046 104 140 186

D 0.027 0.046 0.056 104 190 228

E 0.027 0.036 0.046 104 144 192

COW

F 0.027 0.032 0.038 104 120 144


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A REVIEW COMPARISON OF NEEM FRUIT SEED EXTRACT (RB-a) AND NEEM FRUIT COAT EXTRACT

(RB-b) AGAINST VARIOUS INSECTS

R.M. TARIQ Department of Zoology, University of Karachi, Karachi-75270 (RMT)

ABSTRACT

A lot of research work have been done on the activity of neem seed kernel (NSK). The main active ingredient of neem fresh fruit seed (FFS), shed dried seeds (SDS), Ripe Berries part-a, (RB-a), Neem seed kernel extract (NSK), Neem oil (NO) has been found, a well known pure compound azadirachtin, but very little work has been done on neem fresh fruit coat (FFC), Shed dried coats, (SDC), Ripe Berries part-b, (RB-b), which is mainly from Pakistan. It was reported by Naqvi and Schmidt (1993), that RB-b was more effective than RB-a (Neem seed kernel) against M. domestica for the first time. Since then, several researchers carried out experiments on RB-b against various insects. Most of them found RB-b more effective than RB-a, while some of them also reported less effectiveness of RB-b than RB-a. It has been concluded in this paper that complete neem fruit (RB-a + RB-b) is better to use because it was reported that RB-b (neem fruit coat) is devoid of azadirachtin and contains meliacinin, and azadironic acid, having 13 and 4.5ppm LC50 against An. stephensi and others as well, such as azadiradione and apoxyazadiradione, Limocin-A, Limocin-B and desfuranoepoxyazadiradione (Siddiqui et al., 2000). Where as RB-a contains azadirachtin which is main active ingredient of neem tree. Therefore whole fruit will give better results as compared to neem seed alone and the probability of resistance in insects/pests will also decreased.

INTRODUCTION The neem tree is a par excellence tree which

has provided hundreds of pure compounds from different parts of tree. Neem fruit (Ripe Berries) consists of two parts, the inner seed or kernel coded as RB-a (Ripe Berries part-a). The outer coat of the fruit having pericarp + mesocarp coded as RB-b (Ripe Berries part-b). Beside this several other codes to RB-a & RB-b were also used by the researchers working on neem. It may be shown as follows, Ripe Berries part-a (RB-a), Fresh fruit seed (FFS), Shed dried seed (SDS) Neem seed kernel (NSK) Neem seed kernel extract (NSKE), Neem oil (NO), Ripe Berries under ultra violet (RBU-9), Neem fraction of part-a (N-6a). Where as Ripe Berries part-b (RB-b), Fresh fruit coat (FFC), Shed dried coat (SDC), Neem fraction of coat (NfC), Neem fraction-6 of part-b (N-6b). The above codes were developed and used by Siddiqui et al. 1991a, 1991b, 1992, 2000 and 2004.

Many workers have carried a lot of research on different parts of the neem tree (Pruthi 1937, Siddiqui 1942). The pure compound azadirechtin (Lavie et al. 1971, Nakanishi, 1975) has been isolated from the fruit seed extract (Neem oil)

which has been used to control more than 300 species of the insects. The Margosan OTM, NSKE, Neem azal, neemex, Azatech, Neemark, Neemgold, Biosal, Bioaman, Nimbokil and other neem products have mainly azadirachtin as active ingredient. But in 1993 Prof. Dr. S.N.H. Naqvi and his College in Germany, Prof. Dr. G.H. Schmidt published a report on Musca domestica L. (Common house fly), in which Neem fruit coat extract (RB-b) was reported more effective than neem fruit seed extract (RB-a) and equivalent effective to Margosan OTM, a commercial neem product registered in USA. Since than to date several workers in Pakistan, has taken into consideration the work on the comparison of fruit seed (RB-a) and fruit coat (RB-b). The comparison has been given in form of Table. In which mostly the researchers have concluded the RB-b more effective than RB-a while only some has concluded RB-a more effective than RB-b. Totally fourteen publications are taken into consideration in this respect, among which ten are positive (+ve) in favour of RB-b, whereas four are negative (-ve) in favour of RB-a, which may be concluded from the given Table below.


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Table: Showing more effectiveness of RB-b and RB-a with reference of author and year.

Authors with

year Against the insect/pest

Less effective RB-a Neem fruit seed

extract

More effective RB-b Neem fruit coat

extract 1. Naqvi & Schmidt

(1993)

Musca domestica (House fly)

195 ppm (RB-a) 1.1 ppm (RB-b)

2. Khan et al., 1993 M. domestica (House fly)

IGR effects cyfluthrin less

IGR effects RB-b

3. Tariq et al. (1994)

Aedes aegypti (Dengue mosq.)

NfA on 2.25x104, 10% activity

NfB on 0.049x104, 50% activity

4. Tabassum et al. (1996)

M. domestica (House fly)

N-6a 18.0mg/g N-6b 3.8mg/g

5. Naqvi et al. (1996)

Dacus culurbitae (Fruit fly)

0.64 μg/mg (RB-a) 0.60μg/mg (RB-b)

6. Naqvi & Aslam (1996)

Eyprepoenemis plorans (Grass hopper)

SDS = 20.14 + 2.5% Margosan OTM 27.30 + 9.61%

SDC = 17.28 + 7.58%

7. Ahmed et al. (1996)

Piezodorus hybneri (Legume bug)

3.6% (RB-a) 1.6% (RB-b)

8. Ahmed et al. (1998)

Coccinella spp. (Lady bird beetle)

7.8% (RB-a) 7.4% (RB-b)

9. Tariq et al. (2001) Ae. aegypti (D.F. mosq.) 446ppm (RB-a) 319ppm (RB-b)

10. Tariq et al. (2002) An. stephensi (Malaria mosq.)

784ppm (RB-a) 290ppm (RB-b)

More effective RB-a

Less effective RB-b

1. Naqvi et al. (1994) Ae. aegypti

(Dengue mosq.)

380ppm (RBU-9) Margosan OTM 340

490ppm (RB-b)

2. Naqvi et al. (1995) Cx. Fatiganst (Dirty water mosq.)

502.5ppm (RBU-9) Margosan OTM 154.5

545.0ppm (RB-b)

3. Ahmed et al. (1995) Dysdercus koenigii (Cotton Red stainer)

2.80% (RB-a) 3.02% (RB-b)

4. Khan et al. (1995) Ae. aegypti (PCSIR, OT, GT-strains)

250,450, 600ppm (RB-a)

560, 580, 780ppm (RB-b)


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MATERIALS AND METHODS Literature was studied, the results were

obtained and were described in the form of table as shown in results.

RESULTS AND DISCUSSION Siddiqui et al., 1991a, 1991b and 1992

reported Tetracyclic triterpenoids and terpenoids from the fruit coats of neem fruit but these were not tested against insects/pest.

Naqvi and Schmidt (1993) reported for the 1st time that Neem fruit coat (RB-b) was more effective than neem fruit seed (RB-a) and about equivalent to Margosan-OTM against Musca domestica L. The LC50 of RB-a was found 195ppm, whereas of RB-b was 1.1ppm, and Margosan-OTM was 1.0ppm, supporting more effectiveness of RB-b than RB-a.

Khan et al. (1993) reported the toxicological studies of neem extract RB-b and cyfluthrin against 2nd instar larvae of common house fly Musca domestica L. The RB-b was found better IGR than cyfluthrin as RB-b produced reduction in weight and deformation in larvae, pupae and adults more than cyfluthrin because cyfluthrin had very little activity in these spheres. This report is also in line of above reports and was found in favour RB-b.

Tariq et al. (1994) reported the toxicity of NfA giving low toxicity (10%) on the concentration 2.25x10-4ppm, whereas NfB gave 50% activity at very low concentration 0.049x10-4ppm against dengue fever vector mosquito, Aedes aegypti L. (PCSIR-strain), supporting more effectiveness of NfB than NfA.

Naqvi et al. (1994) reported the toxic and IGR effects of RBU-9, RB-b and Margosan-OTM againstthe 4th instar larvae of Aedes aegypti L. (PCSIR strain). The LC50 was found to be 380, 490 and 340ppm respectively. Here in this report RBU-9 is the neem fruit seed extract (RB-a) having azadirachtin as main active ingredient while Margosan-OTM is a registered neem pesticide in USA. Whose active ingredient is also azadirachtin. Both RBU-9 and Margosan-OTM proved more effective than neem fruit coat (RB-b).

Naqvi et al. (1995) reported the toxic effects of RBU-9, RB-b and Margosan-OTM against the larvae of common house mosquito Culex fatigans (K.U. strain) by W.H.O method. The LC50 was found to be 154.5, 502.5 and 545.0ppm for

Margosan-OTM, RBU-9 and RB-b respectively. This report supports the less effectiveness of neem fruit coat (RB-b).

Ahmed et al. (1995) reported efficacy of neem fruit seed extract (RB-a) and neem fruit coat extract (RB-b) against cotton stainer bug Dysdercus koenigii (F.) by using injection method. The LC50 of RB-a was found to be 2.8% while that of RB-b it was 3.02%. This report supports the less effectiveness of RB-b.

Khan et al. (1995) reported toxicity of RB-a, RB-b against three strains of Aedes aegypti L. The LC50 of RB-a against three strains was found to be 250, 450 and 600ppm for PCSIR, Orangi Town and Gulshan-e-Iqbal strain respectively. The LC50 of RB-b against these strains was found to be 560, 580 and 780 for PCSIR, O.T., and G.I. strain respectively. This report also support less effectiveness of RB-b.

Tabassum et al. (1996) reported the comparative toxicity of neem fruit coat extract N-6a = RB-a giving LC50 = 18.0mg/g and N-6b = RB-b giving LC50 = 3.6mg/g against M. domestica, supporting more effectiveness of N-6b than N-6a. In the publication by Tabassum et al., have reported N-6a = fruit coat and N-6b = fruit seed, which is possibly mistaken by author.

Naqvi et al. (1996) reported toxicity of RB-a, RB-b and azodrin (Organophosphate). The LC50 were 0.60, 0.64 and 0.96 μg/mg of media against Dacus cucurbitae, the fruit fly respectively for RB-b, RB-a and Azodrin. In this report RB-b was found more effective than RB-a and azodrin as well. The reason may be due to the presence of meliacinin, azadironic acid etc., which were found more effective than azadirachtin, the well known pure compound of neem fruit seed and active ingredient of almost all neem pesticide e.g., Margosan-OTM, Neemix, Neemazal, Nimbokil, Biosal, Bioaman etc.

Naqvi and Aslam (1996) reported the phagodeterrent effect of SDC, SDS and Margosan-OTM against a pair of, grass hopper, Eyeprepocnemis plorans by using one inch diameter discs of Cabbage leaf, painted with 0.6ml of 0.1% concentration each of neem extract and Margosan-OTM. The consumed area by grass hoppers was calculated by using computer and data analyzed statistically. The area consumed in control was 92.89 + 4.35, SDC 17.28 + 7.58, in SDS 20.14 + 2.49 and in Margosan-OTM 27.30 + 9.61%. The neem extract SDC which is neem fruit coat proved more effective not only than SDS


- 4 -

(which is neem fruit seed) but also than Margosan-OTM (Whose active ingredient is azadirachtin).

Ahmed et al. (1996) reported the efficacy of RB-a and RB-b against legume bug Piezodorus hybneri (GMELIN). The LC50 of RB-a was 3.6% and for RB-b as 1.6%. This report also confirms the more effectiveness of neem fruit coat than neem fruit seed.

Ahmed et al. (1998) reported the efficacy of RB-a and RB-b against Lady bird beetle, Coccinella sp. by filter paper impregnation method. The LC50 of RB-a was 7.8% and RB-b was 7.4%. This report also confirms the more effectiveness of neem fruit coat than neem fruit seed.

Khan et al. (1998) reported the effect of neem cake, neem seed coat and carbafuran on the population density of four nematodes including Helicotylenchus indicus, Merlinius brevidens, Aphelenchus avenae and Meloidogyne sp. Larvae associated with garlic was studied. The population densities with the exception of H. indicus by neem cake, were significantly reduced by the application of neem products and carbafuran. In all the treatments garlic yield was significantly increased over the control. Carbafuran was most effective against all nematodes. Whereas the neem seed coat powder (RB-a) was less effective than carbafuran but more effective against nematodes as compared to neem cake (P< 0.01). The present work also support the previous work.

Siddiqui et al. (2000) reported two pure new compounds from fruit coat extract meliacinin having 13ppm and azadironic acid having 4.5ppm against An. stephensi the malaria vector mosquito. Beside these two new compounds five known pure compounds were also reported in this report. They are azadiradione (LC50 = 15ppm), epoxyazadiradione (LC50 = 18ppm), Limocin A & B (LC50 = 19ppm) and desfuranoazadiradione (LC50 = 37ppm) against An. stephensi. This report confirmed that fruit coat is devoid of azadirachtin.

Tariq et al. (2001) reported comparative toxicity and insect growth regulator (IGR) effects of neem fresh fruit seed (FFS, RB-a) extract and neem fresh fruit coat (FFC, RB-b) extract against 4th instar larvae of Dengue fever vector mosquito, the Aedes aegypti L. (Orangi Town wild strain) by W.H.O method. The LC50 of RB-a was found to be 446 while that of RB-b was 319ppm. The RB-a contains azadirachtin whereas RB-b (fruit coat) is devoid of azadirachtin. RB-b was also proved to be a better IGR than RB-a (fruit seed). This report

also confirms the more effectiveness of fruit coat than fruit seed.

Tariq et al. (2002) reported the toxic effects of neem fruit seed (RB-a) and neem fruit coat (RB-b) against 4th instar larvae of malaria vector mosquito, Anopheles stephensi Liston (Orangi Town wild strain) by W.H.O method. The LC50 of RB-a (FFS) was found to b3 784 and that of RB-b (FFC) it was 290ppm. This report also confirms the more effectiveness of fruit coat than fruit seed.

Among the 14 reports presented 10 reports support the more effectiveness of the fruit coat (RB-b) whereas only 4 reports were found negative to more effectiveness of RB-b which means 71% results are in favour whereas only 29% results are negative.

Siddiqui et al. (2004) reported 27 pure compounds from neem fruit coat extract (RB-b). Among these 23 were reported for the 1st time and 15 were not reported from any part of the neem tree.

Tariq et al. (2004) reported the toxicity of 16 pure compounds from the fruit coats alone of indigenous (Pakistani) neem tree (Azadirachta indica (L.) A. Juss.) against the 4th-instar larvae of malaria vector mosquito, Anopheles stephensi Liston (Orangi Town wild-strain). The LC50 value of azadiradione, nimbocinol, 17β-hydroxynimbocinol, azadirone, deoxygedunin, gedunin, α-nimolactone, β-nimolactone, 14,15-epoxyazadiradione, desfuranoazadiradione, meliacinin, azadironic acid, methyl ester of 12, limocin-A, limocin-B, desfurano-6α-hydroxyazadiradione and 23,23-dihydronimocinol, (1-16) in sequence was found to be: 15, 30, 15, 10, 150, 120, 60, 45, 18, 37, 13, 4.5, 2.8 (12a), 19, 19, 43 and 60 ppm respectively. The # 12a was the methyl ester derivative of azadironic acid, and that of standard, permethrin (25 EC) LC50 was 0.120 ppm.

The presence of these pure and new compounds in the extract of neem fruit coat (RB-b) was the reason of more effectiveness of RB-b than neem fruit seed extract (RB-a) having well known active ingredient azadirachtin.

CONCLUSION

Neem fruit consists of two parts neem fruit seed (RB-a) and Neem fruit coat (RB-b). Salimuzzaman Siddiqui was the 1st scientist who isolated three principal compounds from neem seed oil (RB-a), namely, Nimbin, Nimbidin and Salanin. Siddiqui (1942). Then Lavie et al. (1971) in Melia azedarach L. reported azadiradione and


- 5 -

epoxyazadiradione found. But more emphasis was given on neem seed only and to some extent to leaves, due to which a lot of work has been reported on seed and leaves and many compounds have been reported from various parts of the tree especially the seed. But very few work has been reported on fruit coat. Whereas fruit coat contains more effective pure compounds than azadirachtin. Therefore it will be more beneficial and more effective if the whole neem fruit will be used as pesticide. Researcher are developing pesticides from neem leaves, neem oil (from neem seed) having azadirachtin as active ingredient but as neem fruit coat have more compounds so the use of whole fruit will also decrease the resistance probability in the insects/pests, and more quantity of pesticide will be obtained by using both parts instead of only fruit seeds.

REFERENCES

AHMAD, I., CHANGEZI, A., AHMAD, A., KHAN, Z., NAQVI, S.N.H. AND MOHAMMAD, F.A. (1996). Efficacy of RB-a and RB-b, the two neem fractions in comparison with Nerium indicum and cypermethrin against legume bug, Piezodorus hybneri (Gmelin). Proc. 16th Congr. Zool. (Islamabad) 16: 259-263.

AHMAD, I., NAQVI, S.N.H., TABASSUM, R., AZMI, M.A., HIDAYAT, Y. AND ANJUM, A. (1998). Efficacy of neem fractions (RB-a, RB-b) in comparison with Melia fraction (BB) against Coccinella sp. J. Exp. Zool. India 1 (2): 85-89.

AHMED, I., AHMED, W., NAQVI, S.N.H. AND AZMI, M.A. (1995). Efficacy of RB-a and RB-b, the two neem fractions in comparison with cypermethrin against cotton stainer, Dysdercus koenigii (F.) (Hemiptera: Pyrrhocoridae). 1st International Congress of Entomology, 178-183.

KHAN, A., QAMAR, F. AND SHAUKAT, S.S. (1998). Relative nematicidal activity of neem seed coat and neem cake against nematodes associated with garlic. Bull. Pure & Appl. Sci. 17A (1): 507.

KHAN, A.H., AZMI, M.A., AHMED, I., NAQVI, S.N.H., KHAN, M.F. AND AKHTAR, K. (1993). Toxicological studies of neem extract RB-b in comparison with cyfluthrin against 2nd instar larvae of Musca domestica L. Pakistan J. Zool., Vol.25 (1): 75-78.

KHAN, M.Z., NAQVI, S.N.H., TABASSUM, R., AZMI, M.A. AND RANI, S. (1995). Determination of tolerance in Aedes aegypti L.

against cypermethrin and neem fractions. Pakistan j. entomol. Karachi 10 (1 & 2): 25-30.

LAVIE, D., LEVY, E.C., JAIN, M.K. (1971). Limnoids of biogenetic interest from Melia azedarach L. Tetrahedron 27, 3927-3939.

NAKANISHI, K. (1975). Structure of the insect antifeedant azadirechtin. Recent Adv. Phytochem. 5: 283-298.

NAQVI, S.N.H. AND ASLAM. M. (1996). Phagodeterrent effect of neem preparations against grasshopper, Eyeprepocnemis plorans. Proc. Pakistan Congr. Zool. Vol.16: 33-36.

NAQVI, S.N.H. AND SCHMIDT, G.H. (1993). Comparative effect of three neem fractions (Tetranortriterpenoids) on Musca domestica L. larvae. Pakistan J. Entomol. Karachi 8(2): 5-14.

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