Journal of Genetics, Vol. 97, No. 5, December 2018, pp. 1253–1261 © Indian Academy of Scienceshttps://doi.org/10.1007/s12041-018-1045-2

RESEARCH ARTICLE

Complex effects of Ayurvedic formulation: Guduchi andMadhuyashtion different components of life history may elude the elixir effect

SURABHI SINGH1, BODHISATTA NANDY2∗ and MADHU G. TAPADIA1∗

1Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India2Indian Institute of Science Education and Research Berhampur, Berhampur Transit Campus, Government IndustrialTraining Institute (ITI) Building, Engineering School Road, NH 59, Ganjam 760 010, India*For correspondence. E-mail: Madhu G. Tapadia, madhu@bhu.ac.in; Bodhisatta Nandy, nandy@iiserbpr.ac.in.

Received 13 March 2018; revised 29 April 2018; accepted 8 May 2018; published online 3 December 2018

Abstract. Formulations from the traditional Indian medicine, Ayurveda, have long been considered to have potent life-style-enhancing effects, possibly by their effect(s) on key life-history attributes. Although several studies have reported beneficial effectsof these formulations on different components of life history, few have investigated their concurrent influence on various life-historytraits. Here, we report the results of an investigation showing the effect of two well-known Ayurvedic formulations, Guduchi andMadhuyashti, on fecundity and longevity ofDrosophila melanogaster.Flies were either grown (i.e., larval exposure) and/ormaintained(i.e., adult exposure) on standard food supplemented with 0.5% Guduchi or 0.5% Madhuyashti. It was observed that the longevityof adult flies of both sexes was not affected on feeding Guduchi food, but fecundity of the females was greatly enhanced. Fecunditywas also found to be affected by the adult food and whether their mates were grown on Guduchi or normal food. Madhuyashti, onthe other hand, significantly reduced mean longevity and had a stimulatory effect on female fecundity. This fecundity enhancingeffect however seemed to be mediated through its effect on the males. Interestingly, much of these effects interacted with age in acomplex way, making it difficult to generalize the overall effect of these formulations on the reproductive output of the flies. Ourstudy underlines the importance of evaluating the interacting effects of these (and similar) formulations on a range of life-historytraits in a holistic way to understand their utility better.

Keywords. Guduchi; Madhuyashti; life-history traits; fecundity; longevity; Ayurveda.

Introduction

Ayurveda—‘Science of Life’ is an ancient healingscience which originated 5000 years ago in Indian sub-continent (Singh 2010; Patwardhan et al. 2015). VariousAyurvedic formulations are thought to promote generalhealth of the individuals by improving various aspects oflife, such as reproductive output, longevity and diseasecuring/preventing abilities and hence, they have receivedsubstantial attention from a broad range of biologistsover the past couple of decades (Patwardhan 2014).Many studies claimed to have found sufficient evidenceof the beneficial effects of these formulations on a rangeof traits, from fecundity to longevity, by individually

Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12041-018-1045-2) contains supplemen-tary material, which is available to authorized users.

assessing each of these traits. For example, feedingDrosophila melanogaster larvae with rose extract obtainedfrom Rosa damascena resulted in a significant decrease inthe mortality rate in adults (Jafari et al. 2008). Similareffects were seen with water or ethanol extract of Stachyslavandulifolia Vahl. var. lavandulifolia (Altun et al. 2010);Aloe vera extract (Chandrashekara and Shakarad 2011);curcumin powder (Chandrashekara et al. 2011). FeedingCaenorhabditis elegans with Withania somnifera tends toincrease their lifespan (Kumar et al. 2013). Since most ofthese studies have limited themselves to studying only oneor few components of life-history traits, one fundamen-tal challenge at the heart is to conclusively show theiroverall beneficial effect on most (if not all) life-history

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traits, such that the sum total of the effect on survivaland reproduction remains beneficial. Importantly, noneof the above mentioned studies shed any light on repro-ductive output, while emphasizing the effect on lifespan.Since trade-off between reproduction and longevity iswell known (Flatt 2011; Flatt and Heyland 2011), it isalmost impossible to assess if the observed lifespan exten-sion is due to active lowering of rate of ageing by theformulation or a by-product of reduced reproduction. Thisline of reasoning canbe extended to other life-history traitsaswell,making the investigation of the concurrent effect ofthe formulation on multiple life-history traits important.Life history, the scheduling of reproduction and mor-

tality through the life time of an organism (Stearns 1992;Roff 1993), encompasses a range of crucial traits, suchas the rate of ageing, fecundity, development time, ageat reproductive maturity, age at cessation of reproduc-tion and reproductive output at various stages of life. Inaddition, there are traits, such as immunity to pathogens,cost of reproduction, that indirectly affect life historyand therefore referred to as life-history-related traits. Alllife-history traits share a common underlying physiology,which is manifested as life-history trade-offs. In addition,since every organism is adapted to a particular life history(Partridge andFowler 1992;Reznick et al. 1996),most life-history traits are usually optimized by several generationsof adaptive evolution. The utility of a life-style-enhancingformulation is likely to be constrained by various intercon-nected life-history traits. Hence, understanding how theinterconnected life-history traits respond to the Ayurvedicformulations is of prime importance, an idea which hasonly recently received empirical attention.Results fromsomeof the relatively recent studies empha-

size our criticism. Investigating the effect of Ayurvedicformulations, Amalaki Rasayana and Ras Sindoor on var-ious life-history traits usingD. melanogaster, Dwivedi andLakhotia (2016) reported longevity, stress resistance andfecundity. Not only did they find interesting concurrenteffects, but also observed that some traits were either unaf-fected or adversely affected. Zou et al. (2010) reportedincrease in longevity without affecting the fecundity ofMexican fruit flies (Anastrepha ludens) upon feeding adultswith amixture of oregano and cranberry. Feeding larvae ofD. melanogaster, with food supplemented with the extractof fruits of Emblica officinalis (Indian gooseberry) nec-tarine (Prunus persica) (Boyd et al. 2011; Pathak et al.2011) and pomegranate (Balasubramani et al. 2014) sig-nificantly increased both lifespan and reproduction of theadults. Feeding a blueberry extract was found to extendthe lifespan and increase thermotolerance in C. elegans(Wilson et al. 2006). In addition, as evident in the resultsof Dwivedi and Lakhotia (2016), the time and manner ofexposure (larval development or adult feeding) might sig-nificantly alter the final outcome. If a formulation affectslife-history traits by primarily affecting resource acquisi-tion then larval exposure is likely to be more potent than

adult exposure. However, that depends on the sex as well,as sexes may differ substantially in terms of when theyacquire resources and how they allocate them. Therefore,the outcomes may also depend on the sex under investiga-tion, indeedmany life-history traits are sex specific in theirexpression (Nylin and Gotthard 1998; Vieira et al. 2000).

Here, we have investigated the effect of two well-knownAyurvedic formulations:Guduchi andMadhuyashti on twofundamental life-history traits, fecundity and longevityusing D. melanogaster as a model system. We specif-ically investigated the effect of time of exposure, sexspecific effects and concurrent expression of both repro-duction and lifespan. Guduchi, also known as Giloy orAmrita (Tinospora cordifolia) belongs to family Menisper-maceae and the stem of the plant is used for its medicinalproperties. Madhuyashti (Glycirrhiza glabra) belongs tothe family Fabaceae and its root is claimed to possessmedicinal properties. Both the formulations have been cat-egorized under Medha Kamya Rasayana as documentedin the Ayurvedic literature. Guduchi or Tinospora cordifo-lia comprises of many well-documented constituents suchas berberine, palmatine (Qudrat-I-Khuda et al. 1964; Bis-set and Nwaiwu 1983; Kumar et al. 2000; Singh et al.2003), tinocordiside (Maurya et al. 1995; Ghosal andVishwakarma 1997), tinocordifolioside (Wazir et al. 1995;Maurya et al. 1997), cordifolioside A, cordifolioside B(Gangan et al.1994;Maurya et al.1996) and tinocordifolin(Maurya and Handa 1998). Similarly, Madhuyashti hasglycyrrhizin, glycyrrhizic acid or glycyrrhizinic acid as itsconstituents (Damle 2014). These components are thoughtto be responsible for anti-inflammatory, antiulcer, laxa-tive, antimicrobial, antiviral, immune booster, antipyretic,antioxidant, emollient and immunomodulator propertiesof the formulations, thus promoting disease-free long life(Patel et al. 2009; Gupta and Sharma 2011; Damle 2014;Goel et al. 2014; Mittal et al. 2014). We grew/held flies onfood supplemented with (i) Guduchi and (ii) Madhuyashtiand investigated the effect of the exposure on fecundityoutput for the first 20 days of their adult life and on adultlongevity. We analysed the effect of assay food (AF), malegrowth food (MGF), female growth food (FGF) and ageon fecundity in a full factorial design.We also analysed theeffect of exposure to the formulations on adult survivor-ship and mean longevity.

Material and methods

All the experiments were carried out using OregonR+strain of D. melanogaster. The strain is maintained onstandard sugar–cornmeal–yeast medium at 23±1◦C. Forthe purpose of the experiment, flies/eggs were derived fromthe stockand then transferred tomedia containing the sup-plement depending on the specific experiment. To preparethe supplemented food, 100 mL of fresh normal sugar–cornmeal–yeast food was taken and allowed to cool to

Complex effects of Ayurvedic formulation 1255

39–40◦C. To this, 0.5 g powder of Guduchi (for Guduchisupplemented food, hereafter referred to as G-food) orMadhuyashti (for Madhuyashti supplemented food, here-after referred to as M-food) was added and thoroughlymixed. The powders were made from the root of the plantGlycyrrhiza glabra (Madhuyashti) and the stemof the plantT. cordifolia (Guduchi) which was commercially procuredfrom Arya Vaidya Sala, Kottakal, Kerala. After mixing,the warm food was poured into bottles, plates or vialsas per the procedure of the particular assay. The above-mentioned concentration of the supplement (i.e. 0.5%)waschosen after careful screening of a range of concentrations.Both the formulations were tested for their effects on lar-val to adult survivorship and development time at variousconcentrations ranging from0.5% to 5%.Complete detailsof this calibration study can be found in the figure 1 inelectronic supplementary material at http://www.ias.ac.in/jgenet/.

Fecundity assay

To assess the effect of the supplements on the reproductiveoutput, flieswere first grownon formulation supplementedand normal food. To this effect, 100-synchronized firstinstar larvae were collected from normal food and trans-ferred to experimental bottles having 20 mL of food(normal or supplemented). Adults were collected within1 h of eclosion as virgins under ether-anaesthesia. Virginmales and females were held in separate vials for threedays at the density of 20 individuals per vial. Thus, we col-lected two types of males: normal and supplemented; andtwo types of females: normal and supplemented. On the3rd day, sexes were combined to set up crosses in assaycages (volume of the cage is 261,562 mm3). The combina-tion was done in a full 2×2 factorial design, by combiningone vial of females and one vial of males. The assay cagesthus contained 20 males and 20 females. Ten assay cageswere set up for each combination, five in normal and fivein supplemented food. Thus, the design of the experimentinvolved a full factorial set up assessing the effect of growthfood (food on which the larvae were grown) and AF (foodon which the adults were combined, the assay cages). Aschematic diagram of the full factorial design is given infigure 2 in electronic supplementary material. For every 24h fresh food was provided in these cages and the numberof eggs in the old food plate was counted. This was donefor 20 days, i.e., till 23 days post eclosion. Separate experi-ments were conducted to investigate the effects ofGuduchiand Madhuyashti.

Longevity assay

First instar larvae were collected and grown in normaland supplemented food. One-hundred synchronized lar-vae were cultured in 20 mL of food in a bottle. The adults

(both males and females) were collected as virgins (<1 hpost eclosion) and were held in cages with 20 males and 20females in a cage. Adults collected from each food regimewere held in a matched food regime. Five replicate cageswere set up for each treatment. Flies were transferred tofresh food every alternate day and mortality was recordedevery day. The assay continued till all the flies were dead.A single assay was done with three treatments: control,G-food and M-food.

Data analyses

The results of the fecundity assay were analysed usingrepeated measure analysis of variance (ANOVA) in fullfactorial design. AF, MGF and FGF were taken as fixedfactors, while age was modelled as repeated measure.Multiple comparisons were done using Tukey honestlysignificant difference (HSD). The mortality data wereanalysed in two approaches: (i) using Kaplan–Meier sur-vivorship analysis. Here, data from the two sexes wereanalysed separately. The two formulations were comparedwith the controls in separate analyses and (ii) two-factorANOVAon themean longevity, using treatment and sex asfixed factors. The three treatmentswere considered as threelevels, making this a three-sample ANOVA. However, M-food and G-food are two completely different treatmentsand the analysis did not aim at comparing M versus G.Specifically, the multiple comparisons were done betweenM versus C andG versus C using a Student’s t-test. All theanalyses were done at α = 0.5 level of significance usingStatistica (Statsoft v. 13.3) for Windows.

Results

Analyses of the fecundity data from the Guduchi assayindicated a significant effect of all main effects and inter-actions, except the three-way assay, i.e. food×MGF×FGFinteraction (table 1). Clearly,G-food inwhich the fliesweregrown and were held as adults (AF) had significant effecton the fecundity of the females. On an average, femaleshad ∼17.5% higher fecundity when the AF was Guduchisupplemented (figure 3 in electronic supplementary mate-rial), but the relative difference in fecundity was also foundto depend on the type of food the females grew in (figure4 in electronic supplementary material), the type of foodtheir mates grew in (figure 5 in electronic supplementarymaterial), and the age at which fecundity was being mea-sured (figure 1). Females grown on G-food on an averagehad 7.3% higher fecundity (figure 1, c&d) compared withfemales grown on control food (figure 1, a&b). Similarly,males grown on supplemented foodmade females produce∼3%more eggs (figure 1, a&c) comparedwithmales grownon control food (figure 1, b&d). On an average, fecundityshowed two distinct peaks, one around 2 days of age, whilethe other one at around 13 days of age (figure 1, a–c) and

1256 Surabhi Singh et al.

Table 1. Summary of the results of the analysis of fecundity data fromthe Guduchi assay using a full factorial repeated measure ANOVA, takingAF, MGF and FGF as fixed factors and age as the repeated measure.

Effect SS DF MS F P

AF 510.02 1 510.02 179.85 <0.001MGF 17.27 1 17.27 6.09 0.019FGF 105.53 1 105.53 37.21 <0.001AF×MGF 281.01 1 281.01 99.09 <0.001AF×FGF 19.12 1 19.12 6.74 0.014MGF×FGF 25.62 1 25.62 9.03 0.005AF×MGF×FGF 0.01 1 0.01 0.00 0.952Age 7000.91 19 368.47 631.49 <0.001AF×age 1085.34 19 57.12 97.90 <0.001MGF×age 86.45 19 4.55 7.80 <0.001FGF×age 108.58 19 5.71 9.79 <0.001AF×MGF×age 34.75 19 1.83 3.13 <0.001AF×FGF×Age 55.58 19 2.93 5.01 <0.001MGF×FGF×age 202.84 19 10.68 18.30 <0.001AF×MGF×FGF×age 82.30 19 4.33 7.42 <0.001

SignificantP values at α = 0.05 are in bold. SS, sumof squares;DF, degreeof freedom; MS, mean square.

(figure 6 in electronic supplementary materials). This gen-eral pattern of age-specific fecundity is however completelyviolated if females were grown in G-food and the assaywas done in the same food as well, while the males weregrown in control food (figure 1d).Here, after the first peak,fecundity consistently declined with age till the next 20days. Though the beneficial effect of Guduchi on the earlylife fecundity was not much significant, later life (post 8day) fecundity is significantly elevated in the presence ofGuduchi supplement either in AF or in growth food (maleor female). Higher fecundity of females underGuduchiAFcompared to control AF, especially post 8 day, was not evi-dent when both males and females were grown on G-food(figure 1c; for comparison of mean fecundity, see figure 7in electronic supplementary material).Analyses of the fecundity data from the Madhuyashti

assay indicated significant effects of assay condition andMGF (table 2) (figures 8 and 9 in electronic supplemen-tary material). Fecundity was almost 20% higher whenadult flies were held in M-food. Females mated to malesthat grew inM-food on an average had∼4% higher fecun-dity. Interestingly, the effect of FGF was not significant,neither were any of the two-way interactions involvingassay condition and either of the growth foods (table 2)(figures 10 and 11 in electronic supplementary mate-rial). However, the three-way interaction between assaycondition×MGF×FGF was found to have a significanteffect, indicating that relative advantage of the assay condi-tion supplement depends on the growth foodof bothmalesand females (figure 12 in electronic supplementary mate-rial).This is unlikewhatwasobserved earlierwhereG-fooddid not provide relative advantage.Although age had a sig-nificant main effect, the rest of the interactions involving

age also had significant effects (table 2). Figure 2 describesthe age-specific schedule of fecundity output observed inthe assay females. Although the scheduling is roughly sim-ilar across the treatments, mean per capita fecundity ofthe females grown on control food (figure 2a) or M-food(figure 2b) was clearly higher when they were held in (i.e.,AF) supplemented food compared to the unsupplementedcontrol food in most of the later age classes.Analyses of the mortality data revealed that the two for-

mulations differentially affected survivorship (i) Guduchireduced female survivorship, especially at the later partof the life, although the effect was only marginally sig-nificant (log-rank test, χ2 = 3.745, df = 1, P = 0.05,figure 3a). It did not significantly affect male survivorship(log-rank test,χ2 = 0.704, df = 1,P = 0.4). Analysis usingtwo-factor ANOVA revealed a significant effect of thetreatment onmean longevity of the flies (table 3; figure 3b).However, multiple comparison did not show any signifi-cant difference between theG-treatment andC-treatments(Student’s t-test, t = 0.289, df = 24,P = 0.77).(ii) Madhuyashti drastically reduced male survivorship(log-rank test, χ2 = 11.954, df = 1,P = 0.0005, fig-ure 3a),while it didnot affect female survivorship (fromthegraph it looks like that the females are affected too) (log-rank test, χ2 = 0.298, df = 1,P = 0.6, figure 3b). Multi-ple comparisons on the mean longevity results revealed asignificant difference between M-treatment andC-treatments (Student’s t-test, t = −2.14, df = 24,P =0.04, figure 3b). On an average, a 12% reduction inmean longevity was observed in the presence of Mad-huyashti. This effectwasmore pronounced inmales (∼16%reduction). Mean longevity results also validated the well-known effect of sex (table 3), where females were found to

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Figure 1. Effect of AF×MGF×FGF× age interaction onmean per capita fecundity of the females. Per capita fecundity of femalesgrown in control food (FC, a, b) and G-food (FG c, d), mated to males grown in either control food (MC, b, d) or G-food (MG, a,c), when held in either control food (C) or G-food (G) at various ages (1–20 days). Error bars represent standard error of means.

Table 2. Summary of the results of the analysis of fecundity data from theMadhuyashti assay using a full factorial repeatedmeasureANOVA, takingAF, MGF and FGF as fixed factors and age as the repeated measure.

Effect SS DF MS F P

AF 631.14 1 631.14 87.83 <0.001MGF 36.26 1 36.26 5.05 0.032FGF 1.04 1 1.04 0.15 0.706AF×MGF 3.80 1 3.80 0.53 0.472AF×FGF 10.98 1 10.98 1.53 0.226MGF×FGF 1.56 1 1.56 0.22 0.645AF×MGF×FGF 33.80 1 33.80 4.70 0.038Age 4642.62 19 244.35 107.09 <0.001AF×age 301.03 19 15.84 6.94 <0.001MGF×age 212.84 19 11.20 4.91 <0.001FGF×age 130.15 19 6.85 3.00 <0.001AF×MGF×age 158.50 19 8.34 3.66 <0.001AF×FGF×age 153.61 19 8.08 3.54 <0.001MGF×FGF×age 110.60 19 5.82 2.55 <0.001AF×MGF×FGF×age 104.94 19 5.52 2.42 0.001

Significant P values at α = 0.05 are in bold.

1258 Surabhi Singh et al.

Figure 2. Effect of AF×MGF×FGF× age interaction onmean per capita fecundity of the females. Per capita fecundity of femalesgrown in (a, b) control food (FC) and (c, d) M-food (FM), mated to males grown in either control food (MC, b & d) or M-food(MM, a and c), when held in either control food (C) or M-food (M) at various ages (1–20 days). Error bars represent standard errorof means.

have almost 14% shorted longevity on an average, irrespec-tive of the treatment. The interaction between treatment× sex was not significant.

According to us, in figure 3A, a&b do not match and weobserve that M does not affect longevity of females, but infigure 3B we observe that compared between C GM,M isless than G in comparison to C.

Discussion

Ayurvedic formulations are widely known to have ‘qualityof life’ enhancing properties. We subjected the conjec-tured beneficial effect of two suchAyurvedic formulations,Guduchi and Madhuyashti, to experimental verificationusing a fruit fly model system. We quantified the effect ofthese formulations on two fundamental life-history traits,fecundity and longevity. The results suggest that depend-ing on the context, both the formulations have beneficialand detrimental effects on the two traits. These effectsare (i) expressed in a complex way often showing age-specific pattern of cost and benefits, (ii) sex specific in

nature and (iii) dependent on the time of exposure to theformulation marginally detrimental, sex specific, effects.While Guduchi was found to be marginally detrimental tofemales, increasing mortality in the later part of their lifes-pan, Madhuyashti was highly detrimental to the males.

When adults were held on formulation supplementedfood (either Guduchi or Madhuyashti), reproductive out-put was generally higher indicating that both formulationssomehow stimulated reproduction. The mechanism ofsuch stimulation may happen through involvement ofneurogenic genes such as Notch and Delta (Ruoholaet al. 1991) and segment polarity genes such as hedge-hog (Forbes et al. 1996), ovo (Mkvel-Ninio et al. 1994),Bicaudal-D (Suter et al. 1989). The observation thatGuduchi supplemented FGF significantly increases thefecundity of the females indicates that Guduchi may beinvolved in increasing the ovarian reserve (Bouletreau-Merle et al. 1982; R’kha et al. 1997; Klepsatel et al.2013; Mendes and Mirth 2016; Lobell et al. 2017), eitherby increasing the size of the ovary and/or by increasingthe number of developing oocytes a female is born with.

Complex effects of Ayurvedic formulation 1259

Figure 3. Survivorship curves of (a) females and males grown and held in the three food types, control, Guduchi supplemented andMadhuyashti supplemented. (b) Mean longevity of the adult females (grey bars) and males (black bars) grown and held in the threefood types, control, Guduchi andMadhuyashti supplemented. The error bars represent standard error of means.

Madhuyashti supplement in the FGF was found to haveno effect on fecundity. Such effect of juvenile growth envi-ronment on adult phenotypes is not unheard of in D.melanogaster, various environmental conditions, such as,dietary restriction, crowding, heat shock, etc. have pro-found consequences on a range adult traits (reviewed inPrasad and Joshi 2003).A similar effect of the MGF, where females produced

significantly higher number of eggs when mated/heldwith males grown in supplemented food, is relatively dif-ficult to explain because that would require males tostimulate females for increased egg production and/oroviposition. Both the formulations were found to havethis effect. Although the increase in fecundity was onlymarginal (3–4%) compared to the enormous effect of thesupplement in the adult food (which is also accessible to the

Table 3. Summary of the results of two-factor ANOVA onmean longevity data using treatment (control,M-food,G-food)and sex as fixed factors.

Treatment DF SS F P

Treatment 2 135.96 3.51 0.045Sex 1 185.16 9.57 0.005Treatment × sex 2 59.64 1.54 0.235

Significant P values at α = 0.05 are in bold.

females), the effectwas nonetheless statistically significant.Further experiments are needed to assess the mecha-nism of such effects. One additional observation howevershed some light on this issue. In Madhuyashti experi-ment, we found males which grew in supplemented food

1260 Surabhi Singh et al.

showed significantly higher mortality as compared withG-food. Among many things that may affect longevity ofmale fruit flies, reproductive effort is one of the strongdeterminants of male longevity. Several previous studieshave reported the physiological trade-off between malereproductive effort and somatic maintenance, leading tomore reproductively active males dying faster (Prowseand Partridge 1997). Therefore it is possible that malesthat were grown on M-food invested more in reproduc-tion (behaviour and/or physiology) and therefore hadreduced longevity. This theory is also in line with theobservation that MGF, i.e. Madhuyashti, had a greatereffect on female fecundity (4% increase) thanGuduchi (3%increase). As fruit fly males are known to possess abilityto modulate their mate’s oviposition through the seminalfluid-mediated effects (Wolfner 2009), one possible mech-anism of such effect is that Madhuyashti males producequantitatively and/or qualitatively different seminal fluidthat cause both increased fecundity in their mates andincreased mortality in them as a cost of producing richerseminal fluid (Wigby et al. 2009; Bretman et al. 2013).

One of the most important finding in our study is themyriad significant interaction terms in fecundity analyses.Most of the two-way, three-way and four-way interac-tion terms were found to be statistically significant in thefecundity experiments with both the formulations. Thesegenerally indicate the complex nature of the effects of eachof the factors we have discussed so far and the cautionthat we should take in emphasizing on the effect of anyof such formulations. For example, Guduchi supplementin the assay (adult) food generally caused a significantincrease in female fecundity, however the increase in fecun-dity was restricted to middle and later part of life (after 8days of adult life). In addition, the increase in fecunditywas also found to depend on the nature of female andMGF. This effect on late life fecundity was much lowerwhen bothmales and females were grown in supplementedfood (figure 1b). Similarly, the advantage of Madhuyashtisupplement in the adult food was found to depend on age,male and FGF. Such complex nature of the effect of expo-sure to the formulations is not surprising given that thelife-history traits, including lifespan, fecundity, reproduc-tive effort etc. are usually physiologically interconnected.Such interconnection has been repeatedly shown in a largenumber of studies (Rose et al. 2004) and is considered asa general attribute of life history.In conclusion, our studyprovides ample evidence to sug-

gest that Madhuyashti and Guduchi affect fecundity andlongevity in fruit flies. These effects are expressed in anage-specific manner and appear to depend on the timeof exposure. At least some of the effects of Madhuyashtiare sex specific in nature. The overall effect represents thecomplexity of the physiology underlying the life-historytraits. To the best of our knowledge this is the first studyattempting to understand the effect of Ayurvedic formu-lations on the life history of a model organism in the

context of the complexity of the underlying physiologicalconnections between traits.

Acknowledgements

This work was supported by grants from the Department of Sci-ence and Technology, Science and Engineering Research Board(SERB), Government of India, New Delhi, to Madhu G. Tapa-dia and JRF to Surabhi Singh. The funders had no role in studydesign, data collection andanalysis, decision topublish, or prepa-rationof themanuscript.Wealso acknowledgeAryaVaidyaSala,Kotakkal, Kerela for providing the Ayurvedic formulations. Wealso thank the editor and anonymous reviewers for their valuablecomments which has improved the clarity of the manuscript to agreat extent.

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Corresponding editor: H. A. Ranganath