Chapter-1 EFFECT OF A FEW ANTIDEPRESSANTS ON SEXUAL BEHAVIOR OF DROSOPHILA MELANOGASTER

Transcription

1 Chapter-1 EFFECT OF A FEW ANTIDEPRESSANTS ON SEXUAL BEHAVIOR OF DROSOPHILA MELANOGASTER

2 Introduction All animals exhibit innate behaviors that are specified during their development. Sexual behavior is one such innate behavior performed by the male towards the female. Courtship and mating are part of this sexual behavior and these two activities are important events in the life of the sexually reproducing organisms. A few workers have used sexual behavior as one of the parameters in understanding the evolutionary relationship among closely related organisms. One of the advantages of using behavior as a means for studying the evolution of a group of closely related organism is that the investigators performance must study living organisms, and by inclination often checks his laboratory findings against field studies, thus acquiring a healthy respect for the all pervading power of selection (Tinbergen 1963). The sexual behavior is the action of structure responding to stimuli, either internal or external, that have been received, perceived, and processed by the organism. Drosophila although is a tiny insect, displays an elaborate courtship which could be easily observed and recorded (Spieth 1966). The sexual behavior of Drosophila is a sequential stereotyped stepwise display which exhibits an interesting series of courtship elements consisting of various behavioral displays followed by an interchange of different sensory stimuli (visual, acoustic, olfactory and tactile). Courtship is the behavior that accounts for most of their social life,it is rich in content, complex in structure and robust in execution. As an essential piece in the fly s evolutionary repertoire, it is not surprising that reproductive behavior should be reinforced and buffered against deleterious events, whether developmental or experimental, that might subvert it. Sexual behavior is likely to be the most contextdependent of all phenotypes and thus subject to many influences that are external to the individual (Greenspan 2000). 1

3 Although primarily behavior has been used in understanding the evolutionary pathway of different groups of organisms, it has also been used for investigation of many other aspects of biology. The developmental patterns, mechanisms that underlie in gene expression, complex physiological mechanisms of many metabolic activities have been understood using different behavioral activities. Unlike morphology, behavior has the disadvantage that it is a transient activity performed by the creatures being studied and therefore cannot be preserved for later review. It is, indeed, imperative that the behavioral elements of the organisms be perceived as they occur and recorded by the observer for future reference. Behavior involves a series of complex activities of the organism that are serially expressed. A behavioral act is a reflection of co-ordinated function of many morphological and/or physiological traits. Realizing this, a few workers have used the sexual behavior as a parameter for evaluating the effects of a few antiepileptic and antidepressant drugs, carcinogens, mutagens and other toxic substances. For example, Mos et al (1999) studied the effect of a few selective serotonin reuptake inhibitors (SSRIs) on the sexual behavior of male rats. Nazari (2004) studied the effect of two antidepressant drugs, fluoxetine and amitryptyline both belonging to SSRI on sexual behavior of Drosophila melanogaster. In the light of this the present study has also been carried out on the effect of all four classes of antidepressant drugs on the sexual behavior of Drosophila. 2

4 Review of literature A review of literature reveals that there are several investigation on the effect of drugs, antibiotics, pesticides and other toxic chemicals on Drosophila. Nazir et al (2001) suggests that certain larval tissues of Drosophila, a non target organism are vulnerable to chlorpyrifos. Further, the adverse effect of the toxicant reflected on various stages of development of the fly including reproductive performance. The genotoxicity of acrolein in D. melanogaster was investigated using 2 different SMART assays, the eye spot and wing spot tests, and 2 germinal tests, the sex-linked recessive lethal (SLRLT) and sex chromosome loss (SCLT) tests (Sierra et al 1994). Three well known chemical oxidative stress inducers: hydrogen peroxide (H 2 O 2 ), cadmium chloride (CdCl 2 ) and copper sulfate (CuSO 4 ) showed that the modified Alkaline Comet Assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals (Shukla et al 2011). David et al (1997) tested for mutagenic effects of cryopreservation in D. melanogaster embryos with an X- linked, recessive lethal assay. The action of several metabolism modifiers, namely phenobarbital, an inducer of xenobiotic metabolism, phenylimidazole and iproniazid, inhibitors of oxidative activities of cytochrome P450, and diethyl maleate, a glutathione-depleting agent, have been assayed using the sex-linked recessive lethal (SLRL) test on Drosophila (Barros et al 2011). Depression is a common psychiatric disorder and leading cause of disability worldwide. It is associated with increased mortality, especially from suicide. Heritability is estimated around 40% suggesting that genotyping is a promising field for research in to the development of depression (Harris and Barraclough 1998; 3

5 Schneider et al 2001). According to the dopamine theory of affective disorder, a deficiency in neurotransmission may play a major role in the disorder; specific polymorphism in genes that affect neurotransmission could increase susceptibility (Opmeer et al 2009) Antidepressants are drugs of long term use administered to number of patients. In the evaluation of the benefit /risk ratio it should be considered that among the various adverse reactions that these drugs might cause the occurrence of effect on behavior cannot be excluded. A growing body of evidence has suggested that antidepressant effects can be extended from depressive symptomatology also to personality characteristics that do not fall within the pathological realm. Kramer (1993) has found that subjects with antidepressants showed changes both in symptomatology and also in psychological traits. Medication in most cases may produce health effects of variable degree, which in some cases may be a serious human health hazard. This potential damage is of particular concern with respect to compounds used for long periods. The antidepressants studied are medicaments that may be continuously consumed for 6 months or longer, with a possible repetition of the treatment (Frommer and Kulig 1987). Also, there have been reports showing collateral health effects, mainly on the cardiovascular system (Fasoli and Glauser 1981; Burrows and Vohra 1986). Other types of alterations have been described as well: for eg, myoclonous, sexual dysfunction and hyponatremia (Black and Kilzich 1994; Colgate 1993). Most antidepressant drugs have adverse effects on sexual function, but accurate identification of the incidence of treatment-emergent dysfunction has proved troublesome. Growing awareness of the adverse effects of many antidepressants on 4

6 sexual dysfunction has led to attempts to resolve dysfunction through adjuvant or substitution treatment approaches. Yet many of these attempts are unsuccessful in solving these problems. Therefore there is a need for further studies on the effects of antidepressants on sexual function. In the past, most of the studies on the genetic effects of anticancer drugs have been concentrated on cytogenetic damage (Clements et al 1990). However, it is obviously important to learn more about the different types of mutagenic lesions induced by anticancer drugs. Also, Tiburi et al (2002) reported that vincristine (VCR), vinblastine induced genetic toxicity causing increments in the incidence of mutational events, as well as in somatic recombination in D. melanogaster. The effect of antipsychotics fluphenazine showed sex linked recessive lethal mutations in D. melanogaster (Schaik and Graf 1993) and also the result obtained on work carried by Gocke (1996) revealed positive result of trifluperazine on sex linked recessive lethal mutation on Drosophila. Chlorpromazine also affected on somatic mutation in Drosophila. Although antidepressants of different class were assessed clomipramine, desipramine and imipramine showed somatic mutation and recombination on Drosophila (Balbi et al 1980) where as lofepramine, maprotiline and mianserin are assessed by (Schaik and Graf 1993). Schaik and Graf (1991) also showed effect of protryptyline on somatic mutation and recombination in Drosophila. Tricyclic antidepressants also ricochet off other sites in the central as well as peripheral nervous system. Depending on dosage, TCAs may disrupt brain regions that control heart rate, appetite, muscle tension and sexual function to the point where a person is severely debilitated. The anticholinergic properties of TCAs are responsible for their many undesirable side effects, such as dry mouth and eyes, 5

7 constipation etc. TCAs can also cause loss of sex drive. The most dangerous side effects of it are hypertension, tachycardia or suffer from seizure (Mitchell 2004). Although antidepressants are given to reduce depression, some of them are believed to have side effects, they also carry a risk for increased hostility, agitation and anxiety, and antidepressants may raise a heart patient's risk of dying in 3 years (Harrar and Gould, 2006). Antidepressants deplete neurotransmitter level specifically serotonin, norepinephrine and dopamine are prevalent in the areas of brain like the limbic system and upper brain stem which controls mood and emotion. The depletion of neurotransmitters are believed to influence sexual dysfunction (Hinz 2011) with some common side effects like nausea, agitation, constipation, abdominal pain, tremor, sweating, decreased libido, sexual dysfunction is most common. It is reported that some of the antidepressant drugs cause sexual dysfunction. Although a few studies on the effect of antidepressants on sexual behavior are made they have certain experimental flaw. Only few studies followed a recognized measure (Hamilton rating scale) for sexual dysfunction, the others relying on patients reporting adverse events (Baldwin 2001). In spite of this the antidepressants are being prescribed because in disease treatment, the effect of the administered drug on sexual activity is not taken seriously. Whether all the antidepressants have such effects on sexual activity is also not known. If the antidepressants affect the sexual behavior, and sexual behavior being a fitness character, continuous administration of it could affect the reproductive potentiality. Such long term effects of antidepressants have also not been analyzed. This prompted the author to take up the present studies on the effect of these antidepressant drugs on the sexual behavior of Drosophila. The effect has been studied in flies with each of the four different classes of antidepressant and their progenies. 6

8 Materials and methods Materials: 1. Model organism: Standard Oregon K (OK) strain Drosophila melanogater obtained from Drosophila stock centre, Department of Zoology, University of Mysore, Mysore Karnataka, India was used. 2. Antidepressant drugs: For treatment of depression, four different classes of antidepressants are commercially available. Therefore in the present study the effect of one antidepressant drug from each class was studied. The drugs selected for the study were as follows, Sertraline: Sertraline is a drug belonging to selective serotonin reuptake inhibitors (SSRI) which inhibit the inactivation of serotonin (5-hydroxytryptamine or 5-HT) by blocking its absorption in the central nervous system. The hydrochloride of sertraline is a white crystalline powder; slightly soluble in water and alcohols (ethanol, isopropyl alcohol). Its melting point is C; and molecular weight is The chemical designation is (1S-cis)-4-(3,4-dichlorophenyl)-1,2,3,4- tetrahydro-n-methyl-1-naphthalenamine hydrochloride. Sertraline is absorbed readily through the gastrointestinal tract. Its absolute bioavailability has not been determined in humans. Maximum plasma concentrations of following doses of 50 and 200 mg are µg/l (ng/ml). Serum levels at steady state are ng/ml of sertraline and its desmethyl metabolites. Plasma protein binding is extensive (approx 98%) to both albumin and alpha1-acid glycoprotein. Distribution following oral administration of sertraline is biphasic with a prolonged absorption phase. The elimination phase begins hr following the administration. The volume of distribution has not been determined in humans but is more than 20 L/kg in rats and dogs. Both sertraline 7

9 and its metabolites exhibit extensive distribution into tissues outside the blood. The elimination half-life (beta) of sertraline in humans is hr. The clinically active desmethyl metabolite is eliminated more slowly than the parent drug with a half-life of approx 66 hr. Unchanged sertraline is not detected in the urine. Bioavailability and absorption rate are increased if sertraline is taken with food. In the present study Sertra-100, a drug manufactured by M/S Unichem Laboratories Ltd, India was used. Duloxetine: Duloxetine belonging to Serotonin Noradrenergic Reuptake Inhibitors (SNRIs) with a Molecular weight of and Melting Point of C, freely soluble in water, methanol, ethanol, chloroform and methylene chloride and sparingly soluble in diethyl ether. The absorption of duloxetine is rapid and at least 90% is absorbed. Elimination of duloxetine and its metabolites is predominantly through the urine; greater than 90% of a dose is recovered in urine and about 5% of a dose is recovered in the feces over 96 hours. In the present study Duxela-60, a derivative of duloxetine hydrochloride manufactured from M/S Sun Pharmaceuticals Private Limited was used. Doxepin : Doxepin hydrochloride belongs to Tricyclic antidepressants. It has histamine receptor blocking actions, the exact mechanism by which doxepin exerts its antipyritic effect is unknown. Plasma doxepin concentrations level is 47 ng/ml from percutaneous absorption. Plasma levels from topical application of doxepin can result in CNS and other systemic side effects. Doxepin hydrochloride has an empirical formula of C19H21NO HCl and a molecular weight of 316 and its melting point ºC. Desmethyldoxepin has a half-life that ranges from 28 to 52 hours and is not affected by multiple dosing. Renal disease, genetic factors, age, and other medications affect the metabolism and subsequent elimination of doxepin. Spectra-25 8

10 a derivative of doxepin manufactured by M/S Rexcin Pharmaceuticals Private Limited was used here. Nortryptyline: Nortriptyline belongs to the category Monoamine oxidase inhibitors It has poor long-term efficacy with a high relapse rate in individuals being for depression, possibly due to a toxic metabolite 10-hydroxynortriptyline, being produced. Thus it is significantly inferior to other and older antidepressants. Nortriptyline is metabolised in the liver by hepatic enzyme CYP2D6. Blood levels of nortriptyline should be obtained during long term treatment to avoid toxicity and optimise response. Nortryptyline is insoluble in water but soluble in organic solvents, such as pentane. At room temperature it is a colorless crystalline solid. It has a molecular weight and Melting point of 189 to193ºc. Sensival, a derivative of nortryptyline manufactured by M/S Wallace Pharmaceutical Private Limited was used in present study. Toxicity measurement: The toxicity of the above four drugs were measured by feeding appropriate doses. Then the LC 50 was estimated on the basis of the concentration tested in the toxicity analysis. Based on the probit analysis, sublethal concentrations of each drug was chosen for analysis of the effect of these drugs on the sexual behavior and the reproductive fitness of Drosophila melanogaster. A. Preparation of standard medium: The flies of Oregon-K strain of Drosophila melanogaster were acclimatized to laboratory condition for five to six generation at a temperature 22 ± 2ºC in a standard soji agar medium prepared as described by (Hegde et al 2001). To prepare one litre of the standard medium, 120 gms of jaggery was dissolved in 1000 ml of boiling water. 100gm of soji powder was added to this 9

11 solution while boiling. Gentle stirring is needed to avoid the formation of lumps. 10 gms of agar agar was added to this mixture and then 7.5ml of propionic acid was added. The medium was cooked well, distributed to either 100 quarter pint (250 ml) milk bottles or 1000 glass vials (3"X1" dia) and allowed to cool. Next day, 5-10 yeast granules were added and flies were transferred and maintained in the standard laboratory condition. Table 1. Showing log-dose/probit of antidepressants for estimating their LC50. Antidepressants LC50 Sublethal concentration Regression equation Sertraline 1.25mg/100ml 0.2%; 0.4%; 0.6% Y= Duloxetine 15mg/100ml 3%; 6%; 12% Y= Doxepin 1mg/100ml 0.125%; 0.25%; 0.5% Y= Nortryptyline 2mg/100ml 0.25%; 0.5%; 1.0% Y= B. Preparation of media: The Drosophila food medium was prepared as mentioned above. When once all ingredients were added and the medium is cooked sufficiently, it was removed from the flame and then the appropriate concentration of the drug was added to it (The concentration of each drug used in the present study is shown in table 1). The media was stirred well and then distributed to glass vials and then allowed to cool. C. Study of sexual behavior of antidepressant flies: Freshly emerged virgin females and naïve males were sorted by sex and separately maintained in the glass vials with a normal media for three days. Three day old flies were starved to 8hrs and fed in media supplemented by respective concentration of drugs. These flies were then allowed to feed this food supplemented with the drug for two days. Thus 5days old drug fed male and drug fed female were aspirated out and placed in an mating chamber and allowed to undergo mating where as flies fed in normal media 10

12 were assessed as control or un. The sexual behavior such as courtship latency, mating latency and copulation duration was studied following the procedure described by Hegde and Krishna (1997). While making observation of sexual behavior following combination of pairs were made. Group-I Male un X Female un (Control) Group-II Female X Male un (Only female ) Group-III Male X Female un (Only male ) Group-IV Female X Male (Both ). After completion of mating each pair was transferred to vials containing normal food medium. The pairs were allowed to lay eggs, and produce the progeny. The virgin females and naïve males emerged from each pair were separated, aged for five days and their behavior was also studied. A total of 20 pairs were observed for each drug in all the above four combinations. The pairs after mating were kept separately and allowed to produce the progeny. The sexual behavior of flies obtained in the progeny was also studied. The data obtained were pooled together, means and standard errors were calculated. Student t-test was applied to know the effect of the drug on the above behavioral traits. 11

13 Results Table 2. Showing mean duration in minutes, standard error and student t-test of courtship latency of sertraline and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Parent F1 t-values Treatment Means ±S.E Means ±S.E t-values Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 3.18* 3.91* 3.53 ± * 3.57* 4.21* Female 0.2% 5.80 ± 0.74 X ± 0.42 X 2.83* 3.03* 0.4% 4.01 ± 0.07 X X ± 0.52 X X 3.23* 0.6% 7.50 ± 0.91 X X X 2.20 ± 0.27 X X X Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 4.23* 3.16* 3.43 ± * 3.72* Male 0.2% 4.43 ± 0.10 X * 4.50 ± 0.24 X % 4.41 ± 0.21 X X ± 0.42 X X % 2.75 ± 1.03 X X X 2.13 ±0.1 X X X Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 3.18* 3.91* 3.50 ± * 2.21* 2.41* Both sex 0.2% ± 0.31 X ± 0.65 X 1.21* 2.26* 0.4% 7.40 ± 0.98 X X ± 1.74 X X 2.20* 0.6% 4.70 ± 0.64 X X X 3.40 ± 0.57 X X X *indicates P-value significant at 0.05 level 12

14 Table 3. Showing mean duration in minutes, standard error and student t-test of courtship latency of duloxetine and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Female Male Both sex Treatment Parent Means ± S.E t-values F1 Means ± S.E t-values Parent 3% 6% 12% F1 3% 6% 12% Control ± * 4.02* 4.50* 13.53± * 4.10* 2.67* 3% ± 0.74 X 2.91* 3.38* 17.65± 1.87 X * 6% ± 0.07 X X 3.54* ± 0.56 X X 5.02* 12% ± 0.91 X X X ± 1.05 X X X Parent 3% 6% 12% F1 3% 6% 12% Control ± * 4.90* 4.37* 13.43± * 2.93* 3.72* 3% ± 0.10 X 2.40* ± 1.87 X * 6% ± 0.21 X X ± 0.56 X X 2.83* 12% ± 1.03 X X X ± 0.86 X X X Parent 3% 6% 12% F1 3% 6% 12% Control 12.45± * 2.48* 2.02* ± * 9.72* 2.89* 3% ± 2.18 X ± 1.65 X 4.21* 3.38* 6% ± 0.98 X X ± 0.74 X X 5.66* 12% ± 0.92 X X X 4.70 ± 0.57 X X X *indicates P-value significant at 0.05 level 13

15 Table 4. Showing mean duration in minutes, standard error and student t-test of courtship latency of doxepin and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Female Male Both sex Treatment Parent F1 t-values Means ±S.E Means ± S.E t-values Parent 0.125% 0.25% 0.5% F % 0.25% 0.5% Control 5.13 ± * 9.13* 7.90* 4.82 ± * 6.57* 6.66* 0.125% 7.22 ± 0.28 X ± 0.80 X 11.69* 10.04* 0.25% 3.17 ± 0.66 X X 3.06* 1.83 ± 0.62 X X % 1.70 ± 0.43 X X X 4.05 ± 0.83 X X X Parent 0.125% 0.25% 0.5% F % 0.25% 0.5% Control 2.61 ± * ± * 8.53* 4.04* 0.125% 7.10 ± 1.09 X 4.42* ± 0.66 X 6.60* 9.06* 0.25% 4.14 ± 0.78 X X 3.82* 2.64± 0.32 X X 9.95* 0.5% 1.83 ± 0.45 X X X 2.05 ±0.04 X X X Parent 0.125% 0.25% 0.5% F % 0.25% 0.5% Control 3.38± * 2.14* ± % ± 0.38 X 3.70* 2.84* 2.20 ± 0.10 X 3.61* % 4.76 ± 1.00 X X ± 0.79 X X 6.32* 0.5% 5.82 ± 0.85 X X X 2.70 ± 0.85 X X X *indicates P-value significant at 0.05 level 14

16 Table 5. Showing mean duration in minutes, standard error and student t-test of courtship latency of nortryptyline and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Female Male Both sex Treatment Parent Means + S.E t-values F1 Means + S.E t-values Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 2.50 ± * 4.48* 6.95* 2.40 ± * % 1.85 ± 0.17 X 3.63* 2.84* 2.00± 0.66 X % 2.05 ± 0.29 X X 2.74* 2.58 ± 1.26 X X % 7.78 ± 0.74 X X X 2.90 ± 0.33 X X X Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 2.00 ± * 2.40* 2.05* 2.60 ± * 0.25% 7.63 ± 0.62 X * 1.88 ± 0.42 X 6.38* 2.08* 0.5% 5.18 ± 1.02 X X 2.30* 2.85 ± 0.54 X X 4.74* 1.0% 3.52 ± 0.88 X X X 1.90 ± 0.46 X X X Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 2.36± * ± * 7.70* 3.16* 0.25% 3.50 ± 0.79 X * 2.75 ± 0.05 X 4.70* 7.14* 0.5% 3.60 ± 0.13 X X 2.84* 5.35± 0.21 X X 3.19* 1.0% 5.25 ± 0.34 X X X 2.45 ± 0.70 X X X *indicates P-value significant at 0.05 level 15

17 Table 6. Showing mean duration in minutes, standard error and student t-test of mating latency of sertraline and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Female Male Both sex Treatment Parent Means ± S.E *indicates P-value *indicates P-value significant at 0.05 level t-values F1 Means ± S.E t-values Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 3.39* ± * 4.27* 3.91* 0.2% 7.93 ± 0.95 X ± 0.72 X % 6.72 ± 1.35 X X ± 0.73 X X % ± 2.98 X X X 16.50±3.39 X X X Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 4.97* 5.01* 9.62 ± * 0.2% 6.37 ± 0.16 X 3.90* ± 1.27 X % ± 3.12 X X 3.87* ±1.68 X X % 6.56 ± 0.90 X X X 6.60 ± 0.47 X X X Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * * 10.7 ± * 4.23* 3.71* 0.2% ± 3.14 X * 5.50 ± 0.71 X % ± 0.79 X X ±2.28 X X % ± 2.48 X X X 5.20 ± 0.56 X X X 16

18 Table 7. Showing mean duration in minutes, standard error and student t-test of mating latency of duloxetine and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Treatment Parent F1 t-values Means + S.E Means + S.E t-values Parent 3% 6% 12% F1 3% 6% 12% Control 16.6 ± * 2.60* ± * 4.27* 3.91* Female 3% ± 0.95 X ± 0.84 X % ± 0.35 X X ± 0.73 X X % ± 1.23 X X X ± 0.39 X X X Parent 3% 6% 12% F1 3% 6% 12% Control ± * 3.29* 4.02* 9.62± * 2.81* 3.19* Male 3% ± 0.85 X 3.90* ± 0.27 X % ± 0.98 X X ± 0.68 X X % ± 0.76 X X X ± 0.88 X X X Parent 3% 6% 12% F1 3% 6% 12% Both sex Control 16.80± * 4.50* 4.24* ± * 4.23* 3.71* 3% ± 0.54 X 3.12* 3.88* ± 0.35 X % 14.10± 014 X X 4.96* 17.50± 0.52 X X 2.31* 12% ± 0.82 X X X ± 1.15 X X X *indicates P-value significant at 0.05 level 17

19 Table 8. Showing mean duration in minutes, standard error and student t-test of mating latency of doxepin and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Treatment Parent Means + S.E t-values F1 Means + S.E t-values Female Male Both sex Parent 0.125% 0.25% 0.5% F % 0.25% 0.5% Control ± * ± % 9.22 ± 1.02 X ± 1.61 X % ± 1.66 X X ± 0.83 X X % 2.75 ± 0.53 X X X 1.41 ± 0.67 X X X Parent 0.125% 0.25% 0.5% F % 0.25% 0.5% Control 4.33 ± ± * 2.93* % 9.16 ± 0.59 X ± 0.98 X * 0.25% ± 0.53 X X ± 1.36 X X 2.22* 0.5% 6.16 ± 0.93 X X X 4.00 ±0.30 X X X Parent 0.125% 0.25% 0.5% F % 0.25% 0.5% Control 7.83± * 2.05* 3.79* 2.27 ± * % ± 1.14 X 6.54* 2.62* 3.50 ± 0.97 X 5.79* % 8.50 ± 2.09 X X ± 0.65 X X % ± 0.60 X X X 4.60 ± 1.06 X X X *indicates P-value significant at 0.05 level 18

20 Table 9. Showing mean duration in minutes, standard error and student t-test of mating latency of nortryptyline and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Female Male Both sex Treatment Parent F1 t-values Means + S.E Means + S.E t-values Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 3.73 ± * 5.05* 3.47* 5.65 ± * * 0.25% ± 1.84 X 5.37* 4.42* 4.05± 1.20 X 5.68* 5.27* 0.5% 5.25 ± 0.23 X X 4.07* 6.35 ± 1.52 X X 3.09* 1.0% 4.05 ± 0.48 X X X 4.95 ± 1.47 X X X Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 4.35 ± * 1.84* 5.31± * 6.06* % ± 0.90 X 4.10* 3.79* 4.00 ± 0.72 X * 0.5% 9.00 ± 0.58 X X ± 0.83 X X % ± 1.19 X X X 5.31 ± 0.76 X X X Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 7.84± * * 5.15 ± * % 14.60±0.88 X 3.47* ± 1.20 X 5.05* 2.21* 0.5% 8.92 ± 0.67 X X 2.21* 10.09± 0.44 X X 5.78* 1.0% ± 1.21 X X X 4.90 ± 1.35 X X X *indicates P-value significant at 0.05 level 19

21 Table 10. Showing mean duration in minutes, standard error and student t-test of copulation duration of sertraline and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Treatment Parent Mean ± S.E t-values F1 Mean ± S.E t-values Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 3.15* 3.78* ± * 4.76* 3.98* Female 0.2% ± 1.56 X 2.67* 3.56* ± 1.21 X 2.61* 3.19* 0.4% ± 1.75 X X 3.84* ± 0.78 X X % ± 1.22 X X X ± 1.20 X X X Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 2.92* 3.10* ± * 4.30* 3.89* Male 0.2% ± 0.14 X * ± 1.31 X % ± 0.90 X X 2.86* ± 1.25 X X % ± 0.69 X X X ± 0.68 X X X Parent 0.2% 0.4% 0.6% F1 0.2% 0.4% 0.6% Control ± * 4.30* 3.61* ± * 4.20* 3.19* Both sex 0.2% ± 0.52 X ± 1.10 X % 15.0 ± 1.15 X X ± 1.11 X X % ± 0.87 X X X 17.0 ± 0.77 X X X *indicates P-value significant at 0.05 level 20

22 Table 11. Showing mean duration in minutes, standard error and student t-test of copulation duration of duloxetine and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Treatment Parent Means + S.E t-values F1 Means + S.E t-values Parent 3% 6% 12% F1 3% 6% 12% Control ± * 3.18* 3.78* ± * 4.76* 3.98* Female 3% ± 0.60 X ± 0.36 X 2.61* 3.19* 6% ± 0.60 X X ± 1.68 X X 2.09* 12% ± 1.22 X X X ± 1.02 X X X Male Both sex Parent 3% 6% 12% F1 3% 6% 12% Control ± * 2.67* ± * 4.30* 3.89* 3% ± 1.18 X 3.70* ± 0.24 X % ± 1.25 X X 4.79* ± 0.25 X X % ± 0.97 X X X ± 1.04 X X X Parent 3% 6% 12% F1 3% 6% 12% Control ± * 2.09* 2.70* ± * 4.20* 3.19* 3% ± 0.30 X ± 0.22 X % ± 1.15 X X 2.28* ± 1.39 X X % ± 1.01 X X X ± 0.88 X X X *indicates P-value significant at 0.05 level 21

23 Table 12. Showing mean duration in minutes, standard error and student t-test of copulation duration of doxepin and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Treatment Parent F1 t-values Means + S.E Means + S.E t-values Parent 0.5% 0.25% 0.125% F1 0.5% 0.25% 0.125% Control ± * * ± * Female 0.5% ± 2.91 X 4.21* 4.63* ± 1.46 X 6.76* 7.01* 0.25% 24.7 ± 1.67 X X 2.57* ± 1.44 X X 6.76* 0.125% ± 2.75 X X X ± 0.10 X X X Parent 0.5% 0.25% 0.125% F1 0.5% 0.25% 0.125% Control ± * 9.33* 3.28* ± * 5.44* 3.34* Male 0.5% ± 1.07 X 5.15* ± 3.70 X 3.92* 3.63* 0.25% ± 2.32 X X 2.86* ± 1.65 X X 3.54* 0.125% ± 1.33 X X X ±1.49 X X X Parent 0.5% 0.25% 0.125% F1 0.5% 0.25% 0.125% Control ± * ± * Both sex 0.5% ± 1.25 X * ± 3.92 X 5.79* 5.47* 0.25% ± 2.24 X X 6.92* ± 3.46 X X % ± 1.89 X X X ± 2.65 X X X *indicates P-value significant at 0.05 level 22

24 Table 13. Showing mean duration in minutes, standard error and student t-test of copulation duration of nortryptyline and un groups of D.melanogaster flies and their progeny (The values after the decimal point is seconds). Crosses Female Male Both sex Parent F1 t-values Treatment Means + S.E Means + S.E t-values Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control ± ± * % ± 0.84 X 5.86* ± 0.72 X * 0.5% ± 3.85 X X 5.98* ± 1.27 X X 6.40* 1.0% ± 0.93 X X X ± 0.83 X X X Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control ± * 19.30± * * 0.25% ± 0.92 X * ± 0.97 X 5.60* 6.18* 0.5% ± 1.04 X X 3.60* ± 1.45 X X 3.60* 1.0% ± 1.03 X X X ± 0.76 X X X Parent 0.25% 0.5% 1.0% F1 0.25% 0.5% 1.0% Control 20.31± * ± * 2.86* 6.02* 0.25% ± 1.21 X 2.25* 2.18* ± 1.29 X 2.28* % ± 2.99 X X 2.67* ± 0.87 X X 2.38* 1.0% ± 1.70 X X X ± 1.10 X X X *indicates P-value significant at 0.05 level 23

25 Adult Progeny Figure 1: Showing courtship latency of D.melanogaster flies with sertraline. Adult Progeny Figure 2: Showing courtship latency of D.melanogaster flies with duloxetine. 24

26 Adult Progeny Figure 3: Showing courtship latency of D.melanogaster flies with doxepin. Adult Progeny Figure 4: Showing courtship latency of D.melanogaster flies with nortryptyline. 25

27 Adult Progeny Figure 5: Showing mating latency of D.melanogaster flies with sertraline. Adult Progeny Figure 6: Showing mating latency of D.melanogaster flies with duloxetine. 26

28 Adult Progeny Figure 7: Showing mating latency of D.melanogaster flies with doxepin. Adult Progeny Figure 8: Showing mating latency of D.melanogaster flies with nortryptyline. 27

29 Adult Progeny Figure 9: Showing copulation duration of D.melanogaster flies with sertraline. Adult Progeny Figure 10: Showing copulation duration of D.melanogaster flies with duloxetine. 28

30 Adult Progeny Figure 11: Showing copulation duration of D.melanogaster flies with doxepin. Adult Progeny Figure 12: Showing copulation duration of D.melanogaster flies with nortryptyline. 29

31 Results The courtship latency of D.melanogaster flies with different concentrations of sertraline is provided in table 2 and figures 1. The effect of sertraline on the progeny of these flies is also given here. The scrutiny of the table shows that the courtship latency (13.82 ± 0.31) of flies with 0.2% concentration increased significantly when compared control and other concentrations. The effect was found in all combination of crosses. When compared to control, the treatment affected the courtship latency of flies with different concentrations of sertraline. However the difference among different concentrations was insignificant. The treatment appears to have an effect on the progeny also. However, the effect was limited only to the two lower concentrations (i.e.0.2% and 0.4%) and in 0.6% the courtship latency had decreased. Of course the differences in the courtship latency of progeny were also significant. Table 3 and figures 2 shows the effect of duloxetine on courtship latency of D.melanogaster flies. Here the courtship latency was highest in male group with a value of (18.41±0.21). The courtship latency decreased in certain treatments while it had increased in certain other concentrations. For example in both female and male groups, the courtship latency had decreased (10.90±2.18) in 3% concentration while increased in 6% and 12% concentrations (17.19±0.98 and 14.12±0.92 respectively). The differences in the courtship latency of most of the combinations were significant. In contrast to the adult flies, their progeny showed distinct difference in the effect of duloxetine on courtship latency. For example, in both female and male group, the courtship latency had decreased when compared to control while in the male or female groups, the courtship 30

32 latency had increased. The increase in all these combinations was statistically significant. Table 4 and figure 3 shows the effect of doxepin on the courtship latency of Drosophila melanogaster flies with the antidepressant drug doxepin. The scrutiny of the table shows that in both female and male group the courtship latency had increased in all the three concentrations when compared to control. Except 0.5% concentration, the increase was statistically significant. In the female and male groups, the courtship latency was increased at 0.125% concentration and then it was decreased in higher concentrations. Here also the difference in the courtship latency was significant when compared to control. At the highest concentration of 0.5% concentration the courtship latency was lower than that of control. In contrast to this, in the progeny, the treatment lead to an increase in the courtship latency of both female and male group at all concentrations of which 0.25% and 0.5% concentrations the values were significant. The courtship latency of female group had decreased compared to control while male groups showed a decrease in the courtship latency. The effect of the treatment on courtship latency was significant in most of the treatments. Table 5 and figure 4 shows the effect of nortryptyline on courtship latency of D.melanogaster flies with three different concentrations. The treatment has lead to an increase of courtship latency in male and both male and female groups. In the female group, the courtship latency was decreased at 0.25% and 0.5% concentrations when compared to control (2.50± 0.71 and 2.05 ± 0.29). The courtship latency was increased only in 1.0% concentration 31

33 which was the highest concentration used. The increase or the decrease in the courtship latency was statistically significant in most of the cases. In the progeny also the courtship latency was increased in all the three concentrations of both female and male groups, while in the male and female groups the courtship latency had decreased. Here also the differences in the courtship latency between control and was statistically significant. The mean mating latency of the D.melanogaster flies with different concentrations of sertraline is shown in table 6 and figure 5. The table shows that the treatment of sertraline has lead to a decrease in the mating latency in most of the treatments. For example in the female groups the mating latency of control was (16.86 ± 1.32) while in 0.2% and 0.4% concentrations mating latency was (7.93 ± 0.95) and (6.72 ± 1.35) respectively. Only in 0.6% concentration, the mating latency had increased. Similar result was observed in the male and both male and female group also mating latency had decreased when compared to control. The difference in the mating latency in most of these treatments was also statistically significant. Similar results were found in the progenies of these groups showing that the sertraline treatment decreases the mating latency of the courting flies. The effect of treatment of duloxetine on the mean mating latency of the D.melanogaster flies is shown in table 7 and figure 6. The careful observation of the table shows that like sertraline treatment, the treatment of duloxetine also leads to a decrease in the mating latency in most of the treatments. For example in the female groups the mating latency of control was (16.6 ± 1.32) while in 3% it was (12.93 ± 0.95) minutes. Although there was a slight increase in the mating latency at 32

34 6% concentration when compared to control, the increase was not significant. In the male and both male and female groups the mating latency was reduced in all concentrations when compared to control. Interestingly in the progeny of the groups the mating latency had increased in all the treatments and in all the concentrations. More over in most of the cases the increase in the mating latency was significant when compared to control and also when different treatments are compared. Here it is evident that the treatment of duloxetine although has no immediate effect, the progeny of the groups are affected by the treatments. The effect of doxepin treatment on the mean mating latency of the D.melanogaster flies is depicted in table 8 and figure 7. This table shows that the effect of doxepin on the mating latency is different from those of sertraline or duloxetine. In the female group, the mating latency was significantly decreased in 0.125% and 0.5% concentrations while there was no change in the mating latency in 0.25% concentration. The mating latency of flies in this group at 0.25% concentration was (13.00 ± 1.66) which when compared to control was insignificant. In contrast to this, the mating latency in the male and both male and female groups had increased due to the treatment. However, among these two groups, the mating latency of males with different concentrations showed insignificant differences when compared to control. On the other hand the difference in the mating latency between control and various concentrations of doxepin was significant. In the progeny of both groups also the mating latency was increased significantly while in other groups the progeny flies showed inconsistent differences compared to control. 33

35 Table 9 and figure 8 shows the effect of nortryptyline on mean mating latency of control and the group of D.melanogaster. It is observed that the nortryptyline treatment affects the mating latency of all groups. In the female group, the mating latency of control was (3.73 ± 0.61) whereas in 0.25% concentration mating latency was (15.28 ± 1.84). The lowest mating latency in the female group was (4.05 ± 0.48) which was the lowest mating latency of all groups. Furthermore, this value was higher than that of control. Except for the mating latency between 0.5% and 1.0% in the male group and 0.25% and 1.0% concentrations of both male and female groups, all other combinations showed significant difference in the mating latency between the control and groups. In some combinations of the progeny of nortryptyline groups the mating latency had decreased while in others it had increased. The mating latency of flies with 0.25% and 1.0% in the female groups, 0.25% in the male group and 1.0% in the both male and female groups, the mating latency had decreased when compared to control while in others the mating latency had increased. Whether it is decrease or increase the difference between the control and was significant in most of the cases. Table 10 and figure 9 shows that the sertraline treatment to the D.melanogaster flies decreases the copulation duration. The copulation duration of un flies in the female group was (24.18 ± 0.18), while in the groups it was (22.80 ± 1.56); (22.01 ± 1.75); (19.90 ± 1.22) respectively in 0.2%, 0.4% and 0.6% concentrations respectively. Similarly in the male group, the copulation duration of control flies was (24.16 ± 0.21) and the copulation duration of the three concentrations were (20.56 ± 0.14), (21.41 ± 0.90) and (16.62 ± 0.69) respectively. 34

36 In the both sex groups also the copulation duration had decreased when compared to control. However, the decrease was not statistically significant in male groups and in some combinations of female and both sex groups. When the copulation duration of the progeny of these flies were analyzed, it showed that in some combinations the treatment decreased the copulation duration while in others it increased the copulation duration. In all the three groups, the difference between the control and was significant. However, the differences among different concentrations are compared, the values were insignificant. In table 11 and figure 10 the effect of duloxetine on copulation duration on the D.melanogaster flies is shown. The copulation duration of the flies was decreased in both female and male groups when compared to control. The decrease was significant particularly when the copulation duration of groups was compared with control and between different treatments the increase was nonsignificant. In contrast to this, in both female and male group the copulation duration had decreased compared to control. The copulation duration was (18.75± 0.68) minutes in control while it was (13.90 ± 0.30), (12.55 ± 1.15), and (15.55 ± 1.01) minutes in 3%, 6% and 12% concentrations respectively. When compared to control here also the decrease was significant. In the progeny of these groups, the copulation duration had increased in 12% of female group (18.40 ± 1.02) and both female and male (both ) group while in all other concentrations, the copulation duration had decreased. Here also the differences in the copulation duration of control and were statistically significant while between different treatments the differences were insignificant. 35

37 Table 12 and figure 11 shows the effect of doxepin on the copulation duration of D.melanogaster flies with different concentration of the antidepressant drug. The scrutiny of the table shows that only 0.25% concentration of doxepin increases the copulation duration of the flies in the female group (24.70 ± 1.67). The increase when compared to control was statistically significant. The copulation duration was decreased in most of the concentrations of male as well as both female and male groups. In the progeny, the copulation duration had increased in 0.25% of female, and in all the concentrations of male groups. In both female and male group the copulation duration had decreased when compared to control. The effect of different concentrations of nortryptyline on copulation duration of D.melanogaster is shown in table 13 and figure 12. Compared to control, the copulation duration of the flies had increased in some treatments and decreased in others. For example, in the female group, copulation duration was decreased in 0.25% while increased in 0.5% and 1.0% concentrations. Moreover, except between 0.5% and 1.0% concentration the increase was not significant. Similar result was obtained in the male groups. Here 1% concentration showed significant difference when compared with other concentrations. In both female and male group, the copulation duration had decreased in 0.25 and 1.0% concentration while it was increased in 0.5% concentration. The progeny of these flies also showed no distinct trend with regard to copulation duration. In some concentrations or treatments, the duration had increased while in others it had decreased; of course the differences were significant when compared to control or with other concentrations. 36

38 Discussion: Sexuality is one of the important events in the life time of sexually reproducing organisms. During this process the two sexes alternately produce stimuli to which each of them responds. Through this well knit stimulus-response mechanism they produce the offspring which allows the perpetuation of the species. This stimulus-response mechanism is affected by various factors both intrinsic and extrinsic. The intrinsic factors include the physiological condition of both male and female, including hormones and pheromones. The extrinsic factors include both normal and artificial environments. The sexual behavior could be affected even by the extreme conditions such as treatments of drugs or antibiotics. In the present studies the author has noticed the reduction in courtship latency of D.melanogaster flies when they are fed with the antidepressant drug sertraline (table 2). Sertraline belongs to (SSRI). Treatment of other antidepressants namely, duloxetine (SNRI), doxepin (TCA) and nortryptyline (MAOI) had affected the courtship latency but the results were inconsistent (tables 3-5). In some concentration the courtship latency was increased while in others it was decreased. Of course the differences between the control and the were statistically significant. It is evident that the antidepressants affect the courtship latency. The results of the author also show that different classes of antidepressants have different type of effects on the sexual behavior. Courtship latency is one of the parameters which indicate the vigor of male D.melanogaster (Eastwood and Burnett 1977). It represents the time between introduction of male and female flies in to the observation chamber and initiation of courtship (Markow and Kerr 1978, 1985, Markow and Ankey 1988; Hegde and 37

39 Krishna 1997). A male with high vigor reacts quickly in the presence of female while the one with less vigor reacts slowly (Markow and Kerr 1978). The present studies of the author thus show that the antidepressant treatment affects the courtship latency and in particular, sertraline increases the vigor of males. Selective serotonin reuptake inhibitors (SSRIs) are known to induce delayed orgasm and ejaculation, while their effect on other aspects of sexual function, such as sexual motivation, arousal, and erectile function are unclear. In humans Sukoff et al (2008) showed that antidepressive long-term treatment (citalopram and fluoxetine) delays ejaculation but did not affect sexual desire. In addition, several evidences observed that, in male rats, acute administration of fluoxetine results in an inhibition of sexual behavior evidenced by prolonged ejaculation latency and/or by increased number of mounts and/or intromissions exhibited prior to ejaculation (Baum and Starr 1980; Yells et al 1992, 1994, 1995). Taylor et al (1983) showed that the effects of administration of fluoxetine did not modify the propensity of the male rat however; the copulatory pattern was altered in those rats, which exhibited longer latencies of intromission. Serotonin reuptake inhibitors, despite marked differences in chemical structure, induce similar sexual side effects; it is likely that these effects are, indeed, attributable to inhibition of the serotonin transporter and to a subsequent increase in the synaptic concentrations of serotonin (Matuszczyk et al 1998). The author's observation further confirms that the sertraline treatment can cause secondary side effects. Mating latency is time introduction of female and males in to mating chamber until initiation of copulation (Lucic and Kekic 2009). In the present study of the author has noticed significant decrease in the mating latency of flies with sertraline (table 6). Although 0.6% concentration has lead to a slight increase in the 38