Eduved International Journal of Interdisciplinary Research

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1 Eduved International Journal of Interdisciplinary Research Journal Webpage : ISSN (Online) (Print) EFFICACY OF ESSENTIAL OIL OF LEAF OF Tagetes erecta ON THE FECUNDITY OF Drosophila melanogaster Vishnu Kumar Upadhayay 1, Prabhu N. Saxena 1, Harendra N. Sharma 1 and Nishi Saxena 1 1 Toxicology Laboratory, Department of Zoology, School of Life Sciences, Khandari Campus, Dr. B.R. Ambedkar University, Agra (U.P.), India INTRODUCTION Indian economics mainly depends on agriculture and for which fertilizers and high yielding seeds have continuously been used. Agriculture is an important environmental quality driver and its effect is not likely to diminish in the future. Furthermore, it has been realized that life on earth depends on the proper functioning of several large scale ecological processes. Many of which provide humanity with irreplaceable benefits, turned ecosystem services. The term pesticides encompasses all chemical compounds used for the control of plant and animal pests. However, no control procedure has brought about 100 percent reduction in questioned pest population. Though the synthetic organic pesticides are in vogue yet the plant origin pesticides and their analogs have an upper hand. It has been used for over 50 years and has provided fast, economical and effective pest control. Nowadays in the chemical control of pests, synthetic organic pesticides dominate the scene. As early as 1982, a synthetic organic pesticide dinitrocresol was used as insecticides in Germany and by 1932 several thiocynates became commercially available. During world war II (1940), several highly toxic insecticides came into practice. A series of synthetic pesticides came into the market, Thiocynates in 1932, organophosphate in 1938, organochloride in1939, pyrethroids in 1949, benzenehexachloride in 1952 and carbonate in The number of synthetic organic compounds have increased at a very rapid pace and at present more than one thousand active pesticidal chemicals are being manufactured throughout the world. Currently, there are 165 pesticides registered for use in India. There is a sequential rise in the production and consumption of pesticides in India during the last three decades. However, the consumption pattern of these chemicals in India differs with rest of the world. The domestic demand in India accounts for about 76% of the total pesticides used in the country against 44% globally. lupinine, the alkaloids extracted from Russian weed, Anabasis aphylla, rotenone Derris eleptica and pyrethrums from Chrysanthemum cinerariaefolium flower have been used as natural insecticides. Insecticidal properties of the oil extracted from root of Tagetes minuta are well known but the significance of the essential oil obtained from shoot for the repelling vector mosquitoes has never been investigated. The plant products can be obtained from the whole plants or specific part by extraction with different types of solvent such as aqueous, methanol, chloroform, and acetone etc. depending on the polarity of the phytochemicals. Studies carried out so for have shown that some phytochemicals act as general toxicant both against adults as well as larval stages of mosquitoes. Initial studies pertain to chemical and biochemical constituents of the plant and plant chemistry and biology in general. Recently, interest has been in the bioactivity of various plant extracts and their isolated products. Among the more promising candidate are Phytochemicals extracted from Tagetes species in the Asteraceae. Tagetes erecta leaf oil affects on higher spore population and Mycorrhizal colonization of Meladogyne incognita. Tagetes erecta oil has been used in the present investigation to assess the sub-lethal effect on wild Drosophila melanogaster. MATERIALS AND METHODS Experiments were conducted on the wild form of Drosophila melanogaster (Meign), which belongs to family Drosophilidae and order Diptera and has been selected as a model insect. Wild flies were 3-4mm long, had radish oval eyes, smooth wings and brownish yellow / brownish black body. The male flies were smaller than the female flies. A pure culture of the flies was maintained in Toxicology Laboratory, Department of Zoology, School of Life Sciences, Khandari Campus, Dr. B.R. Ambedkar University, Agra. Some herbal plant products are those products which are obtained by natural entities. The herbal plant species have shown insecticidal and growth inhibition property at a very high concentration and thus may be of much laboratory importance. The herbal products such as nicotine obtained from Tobacco leaves, Nicotiana tabacum, anabasine and Experimental plant : Common name : Marigold Botanical name : Tagetes erecta Family : Compositae Plant type : Annual Colour : Green 13 Vol. 3 issue 9 September 2016

2 Experimental protocol: Experimental flies were categorized into four groups to assess the sub lethal effect of most active fraction of leaf of Tagetes erecta on the wild Drosophila melanogaster. Biological studies were done in control set (without treatment) and treated set (with Tagetes erecta oil) in triplicate as given below :- i. TM and UTF (Treated males and untreated females) ii. TF and TM (Treated females and treated males) iii. TF and UTM (Treated females and untreated males) iv. UTF and UTM (Untreated females and untreated males) Fecundity: Drosophila flies laid their eggs on upper surface of culture media food. Eggs were collected by Delcour method (1969) and counted under microscope to determine the fecundity. Statistical calculations : The statistics that has been applied to generate data was followed after Fisher and Yates (1963), Finney (1971) and Gad (1999). RESULTS AND DISCUSSION ANOVA has been employed to find out whether the four independent estimates of toxicity have a significant variance. The null hypothesis that is employed in this case include: H0 : (µ1) = (µ2) = (µ3)= (µ4) considering all four samples mean to be equal. To analyze the variance; the sum of squares and mean square within and between the samples have been calculated. The variation between samples takes into account the random variation from observation to number of observations in each group. This has been called the sum of square between group for the calculation of variance between the samples, the total of the square of the deviation of the means of various samples from the grand average are divided by the degree of freedom. Secondly, the sums of square within sample have been calculated. This is done by taking the total of the sum of squares and dividing it by the total degree of freedom. The F ratio is calculated by the formula between column variation divided by within column variation. The calculated F ratio 4.45, has been compared to the tabulated value for 3 and 8 degree of freedom at p<0.01 and p<0.05. The value obtained are 4.07 and 7.59 respectively. Both the values are less than the calculated value. Hence, the null hypothesis has been rejected and can be inferred that there exists variation in the toxicities of the most active fraction of Tagetes erecta leaf oil based on the fecundity of wild Drosophila melanogaster between various cross combinations. Further, for considering variance in various groups of cross combinations of wild Drosophila melanogaster. Post-hoc test has also been employed and for which difference between means of all the four groups of cross combinations of wild Drosophila melanogaster have been calculated. To compare the group toxicity Duncan s multiple range test has been employed. Two sets of calculations have been carried out, first the determination of the difference between the means of pairs of group, second the preparation of a probability rate against which each difference in mean has been compared. The mean square of within group is taken form the ANOVA and ranked in increasing order (X1-X2). The groups have been taken in pairs and difference between means expressed as positive numbers have been calculated. The standard error of the Table- 1 Variance analysis of most active (IInd) fraction of Tagetes erecta leaf oil on fecundity of wild Drosophila melanogaster Replicates UTMxUTF C UTFxTM T1 UTMxTF T2 TMxTF T3 C 2 T1 2 T2 2 T Total ΣC = 393 ΣT1 = 337 ΣT2 = 286 ΣT3 = 255 ΣC 2 = ΣT1 2 = 8005 ΣT2 2 = 7636 ΣT3 2 = X = C=131 T1 = T2 = T3 = 85 observation, while that within samples takes into account the variability around mean of each group. In order to analyse the variance, firstly, the variance between samples has been calculated as the difference between the sample mean of each group and the overall mean weights by the mean has been calculated by taking the square root of the quotient of the mean square within group and number of observations. 14 Vol. 3 issue 9 September 2016

3 A probability table has been established (of p<0.01 and p<0.05) by multiplying the S.Em. and the series of values. The error degree of freedom is the number of degree of freedom within the group. The mean square within group from the ANOVA table is 272. Therefore the calculated S.Em. value is The error of degree of freedom within group was 8 so the following values are used. It is observed that the difference in the mean of 2 Vs1, 3 Vs 2, 4 Vs3 and group to the most active IInd fraction of Tagetes erecta leaf oil is non-significance at p>0.01 and p>0.05, while all other groups of cross combination reveal a significance in the means at p<0.01 and p<0.05 level. Table-2 ANOVA table for fecundity of wild Drosophila melanogaster after treatment with most active (IInd) fraction of Tagetes erecta leaf oil Sum of Variation df Sum of squares Mean of squares F Total Between group Within group Table-3 Multiple comparison using Duncan s Multiple Range Test for fecundity of wild Drosophila melanogaster after treatment with most active (IInd) fraction of Tagetes erecta leaf oil 1. TMxTF ( X =85) 2. UTMxTF ( X =95.33) 3. TMxUTF ( X =112.33) 4. UTMxUTF ( X =131) Group X 1- X 2 No. of Mean apart Probability Significant/non significant 2 Vs P>0.05 Non significant (UTMxTF) Vs (TMxTF) 3 Vs P>0.05 Non significant (TMxUTF) Vs (UTMxTF) 4 Vs P>0.05 Non significant (UTMxUTF)Vs (UTFxTM) 3 Vs P>0.05 Non Significant (TMxUTF) Vs (TMxTF) 4 Vs P<0.05 Highly Significant (UTMxUTF)Vs (UTMxTF) 4 Vs 1 (UTMxUTF) Vs (TMxTF) 46 4 P<0.05 Highly Significant The df** between group is 8 and the values obtained from the table are multiplied with S. Em. 15 Vol. 3 issue 9 September 2016

4 Table obtained by multiplying S.Em. with standardized arrange from multiple rank test (Bliss, 1970) No. of Mean apart **df wg = Degree of freedom within group In various cross combinations, fecundity was decreased after most active fraction of leaf oil of Tagetes erecta intoxication. However, better reduction in eggs laying have been observed in those cross combinations where both sexes were treated as compared to control set. Similar observations have earlier been reported by Saxena et al. (1992) who tested 5 plant extracts (Tagetes erecta, Eichhornia crassipes, Ageratum conyzoids, Cleome icosondra and Tridex procumbens) on Culex quinquefasciatus. Almost similar result have been reported by Saxena et al. (1993) and Emara and Ryan (1997) who tested Annona squamosa and two plant extracts of (leaf of Clerodendron inerme and arial part of Pituranthos tortusus,) on Anopheles stephensi and red flour beetle, Tribolium castaneum respectively. The reduction in number of eggs may possibly be due to inhibitory effect of IInd fraction of leaf oil of Tagetes erecta on gonodial development. The abnormalities have also been observed in eggs which include incomplete covering like chorion and vetelline membrane and absence of spiracles in treated set as compared to control set. The size of the eggs was found to be smaller as compared to control sets. The colouration of the eggs were changed after treatment with active fraction (flavonoids). It appears that disruption in oviposition may be a consequence of flavonoids present in IInd fraction of leaf oil of Tagetes erecta on the hormones responsible for the regulation of reproduction. Probably IInd fraction of leaf oil of Tagetes erecta interfere with morphogenesis during egg laying and development and thereby poor egg formation in the ovarioles takes place which were supported by Saxena et al. (1992). Reduction in fecundity is more in treated sets, where both males and females were treated. The reduction in number of eggs may possibly be due to the impaired vitellogenesis and oviposition. Volatiles have been seen to impair sensory activities related to oviposition. Flavonoids (IInd fraction) of Tagetes erecta leaf oil is also volatile which probably has impaired the sensory activities related to oviposition. Reduced fecundity and complete inhibition of oviposition in Anopheles stephensi and Anopheles culicifascies after treatment with neem and other plant extracts has earlier been observed to Dhar et al. (1996). quadricincta in desert locust, Schistocerca gregaria (Assad et al. 1997), similar explanation could hold true in the present investigation too. Further, reduced number of eggs have also been observed by Chakraborti and Chattarjee (2000), Singh and Arya (2001), Patil and Goud (2003) using four neem formulations (Azadirachtin, Azadirachtina indica - iodine, neem seed kernel extract and oil), mustard seed extract and ten plant extracts against rice moth corcyra cephalonica, Lipaphis erysimi and Plutella xylostella (backdiamond moth) respectively. Again, reduction in number of eggs may possibly be due to easy penetration of extract through delicate covering like chorion and vetelline membrane of eggs and gain support by Dwivedi and Garg (2003). who also observed reduced fecundity in Corcyra ciphalonica after treatment with flower extract of Lantana camara. Present findings are in close conformity with Mitchell et al (2004) who tested neem extract on Clavigralla scutellaris and its parasitoids (Gryon fulviventre) and reported reduced fecundity possibly due to failure of complete egg formation. Reduction in fecundity has also been found by Saber et al. (2004), Bhattacharya and Kalita (2005), Meena and Bhargava (2005), Fakhar-Hammad and Mc Auslane (2006) and Nathan et al. (2006) who used Azadirichtin / neem Azal, few plant (Negela sativa, Mentha spicata and Ocimum basilicum), thirteen plants, Melia azadarach extract and some plant (wheat, rice, millet, and sorghum) lectin on Tricogramma cacoecia, stored grain products (Callosobruchs chinensis, Sitophilus oryzae and Tribolium castaneum), Corcyra cephalonica, Bemisia argentifolii and its bio control agent Eretmocirus ruii and Corcyra cephelonica and Trichogrmma chilonis respectively. The decrease in number of eggs may possibly be due to the adverse effect of leaf extract on the gonadotropic cycle, incomplete vitellogenesis and impaired sensory activities related to oviposion following intoxication with leaf and flower extract s Tagetes erecta on Drosophila melanogaster (Saxena et al. 2006). Present finding gains support by the investigations of Adedowal and Adedire (2006) who tested Jatropha curcas seed oil on Callosobruchus maculatus and discovered reduction in number of eggs possibly due to inhibition in oviposion and egg development at a lower concentration. Reduction in number of eggs could have been due to the unequal size of ovaries, inhibition of oogenesis and vitellogenesis in Aedes aegypti following treatment with seeds of Pangamia pinnata (Padmaja et al. 2007). Further, wheat germ, jacalin, pea and soyabean lactin on Spodoptera litura also provided reduced fecundity due to disturbance in protein metabolism in egg formation (Gupta et al. 2007). Almost similar reduction has been reported by Sujatha et al. (2007) who used Azadiracta, ocimum, vitex and some Eucalyptus based formulations against Callosobruchus respectively. The reduction in number of eggs may possibly be due to Reduction in number of eggs may possibly be due to the shorter first gonadotrophic cycle and gonadotrophic period disturbance in reproductive parameters, length of ovarian in immature females under treatment with Commiphora follicle, total protein content of ovary and incomplete 16 Vol. 3 issue 9 September 2016

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