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INTRODUCTIONMany species of social insects such as ants, bees, wasps and termiteslive in colonies showing high levels of cooperation and division oflabour. These features are achieved by the differentiation of colonymembers into one or a small number of fertile queens (or kings)and a large number of sterile (and therefore defined as altruistic)workers (Wilson, 1971). The evolution by natural selection of suchaltruistic behaviour on the part of the workers remains a majorunsolved problem in insect sociobiology. Inclusive fitness theory(also often referred to as kin selection or Hamilton’s rule) providesa powerful theoretical framework for exploring the evolution ofaltruistic behaviour. Hamilton’s rule (rb>c) states that an altruistictrait can spread by natural selection if the benefit of the altruisticact to the recipient (b), devalued by the genetic relatedness betweenactor and recipient (r), is greater than the cost to the altruist (c)(Hamilton, 1964a; Hamilton, 1964b). Most studies attempting totest Hamilton’s rule have measured genetic relatedness betweenaltruists and recipients but relatedness values alone can tell us littleabout the validity or otherwise of Hamilton’s rule (Evans, 1977;Gadagkar, 1990; West-Eberhard, 1978). Attention should thereforenow focus on the cost and benefit terms in Hamilton’s rule, whichso far remain poorly studied (Choe and Crespi, 1997; Gadagkar,2001; West-Eberhard, 1975). One way in which Hamilton’s rulecan be relatively easily satisfied is for the altruist to pay only a smallcost, and this could happen if the altruist has a lower fertilitycompared with other individuals in the population. This idea hasbeen proposed repeatedly in such forms as the ‘sub-fertilityhypothesis’, ‘parental manipulation’, ‘pre-imaginal caste bias’, etc.(Alexander, 1974; Craig, 1983; Field and Foster, 1999; Gadagkar

et al., 1988; Gadagkar et al., 1990; Gadagkar et al., 1991; West-Eberhard, 1975).

These ideas are best tested in so-called primitively eusocialspecies where many workers can be reproductively totipotent andcapable of full ovarian growth, unlike highly eusocial species whereworkers have permanently reduced or atrophied ovaries even at thetime of eclosion (Oertel, 1930; Snodgrass, 1956). Reproductivetotipotency makes it possible for workers in primitively eusocialspecies to transform themselves from sterile to fertile individualsand become replacement queens upon the loss or death of the originalqueen (Chandrashekara and Gadagkar, 1992; Monnin and Peeters,1999; Smith et al., 2009; Tibbetts and Huang, 2010). In many socialinsect species such as honey bees, sweat bees, bumble bees,polistine wasps and vespine wasps, ovarian development isinfluenced by physiological, social and environmental factors suchas body size, larval and adult nutrition, presence or absence of queenpheromones, social interactions and time of year, leading toconsiderable heterogeneity in the extent of ovarian developmentamong colony members (Backx et al., 2012; Bloch et al., 2002;Duchateau and Velthuis, 1989; Hoover et al., 2006; Kapheim et al.,2012; Motro et al., 1979; Smith et al., 2008; Smith et al., 2009;Tibbetts et al., 2011; Toth et al., 2009; Wheeler, 1986; Wheeler,1996; Wheeler et al., 1999). In the primitively eusocial waspRopalidia marginata, there is a clear pre-imaginal caste bias. Onlyabout 50% of the eclosing individuals build nests and lay eggs whenisolated into individual holding boxes in the laboratory and providedwith ad libitum food and building material. The other half die withoutever laying eggs in spite of living, on average, as long as or longerthan the egg layers (Gadagkar et al., 1988; Gadagkar et al., 1990;

SUMMARYSocial insects are characterized by reproductive caste differentiation of colony members into one or a small number of fertilequeens and a large number of sterile workers. The evolutionary origin and maintenance of such sterile workers remains anenduring puzzle in insect sociobiology. Here, we studied ovarian development in over 600 freshly eclosed, isolated, virgin femaleRopalidia marginata wasps, maintained in the laboratory. The wasps differed greatly both in the time taken to develop theirovaries and in the magnitude of ovarian development despite having similar access to resources. All females started with noovarian development at day zero, and the percentage of individuals with at least one oocyte at any stage of developmentincreased gradually across age, reached 100% at 100days and decreased slightly thereafter. Approximately 40% of the femalesfailed to develop ovaries within the average ecological lifespan of the species. Age, body size and adult feeding rate, whenconsidered together, were the most important factors governing ovarian development. We suggest that such flexibility andvariation in the potential and timing of reproductive development may physiologically predispose females to accept worker rolesand thus provide a gateway to worker ontogeny and the evolution of sociality.

Key words: ovarian development, social wasp, caste differentiation, eusociality.

Received 2 April 2012; Accepted 29 August 2012

The Journal of Experimental Biology 216, 181-187© 2013. Published by The Company of Biologists Ltddoi:10.1242/jeb.073148

RESEARCH ARTICLE

Ovarian developmental variation in the primitively eusocial wasp Ropalidia marginatasuggests a gateway to worker ontogeny and the evolution of sociality

Shantanu Shukla1, Swarnalatha Chandran1 and Raghavendra Gadagkar1,2,*1Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, India and 2Evolutionary and Organismal

Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India*Author for correspondence (ragh@ces.iisc.ernet.in)

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Gadagkar et al., 1991). The advantage of experimenting with freshlyeclosed female wasps maintained in isolation in the laboratory isthat we may rule out or minimize the influence of such factors associal interactions, dominance, queen pheromone, etc., and attributethe observed inter-individual variation in reproductive potential tointrinsic properties of the eclosing wasps, including any effects ofthe larval environment. However, data on ovarian development atdifferent ages could not be collected in the studies mentioned above,in which all females were kept alive until they laid at least one eggor died naturally. In the present study, we therefore measured ovariandevelopment at different ages by similarly maintaining freshlyeclosed, virgin female wasps under isolation in the laboratory andfound as much or more inter-individual variation in ovariandevelopment as we found in egg-laying ability in the previous studiesmentioned above.

To begin with, we provide a brief overview of the natural historyof R. marginata in order to help place the results of our laboratorystudies in the ecological context of natural colonies. Ropalidiamarginata is a tropical, old-world, primitively eusocial paper waspwidely distributed in peninsular India. New colonies may befounded throughout the year, by one or a small group of females.In single-foundress nests, the foundress not only lays eggs but alsoperforms nest building, foraging, brood rearing and all other tasks,until the eclosion of her daughters. In multiple-foundress nests, onlyone female wasp lays eggs (queen) while all non-reproductive taskssuch as nest building, brood care and foraging are performed by theremaining females, who function as sterile workers. However, thequeen is replaced from time to time and one of the workers becomesthe new queen, and she may in turn be replaced after some time.Because of the tropical climate, the nesting cycle of these wasps isperennial and the nests may be very long lived. The wasps (at least,the females) are also quite long lived. Males stay on their natal nestsfor about a week, after which they leave, to lead a nomadic life.Female wasps may also leave at various times to found new single-or multiple-foundress nests or to join other newly founded nests, or

may stay in their natal nests for their entire life, with or withoutbecoming queens. Thus, there is no real distinction between ‘queens’and ‘replacement queens’. The mean duration of residence on theirnatal nests for female wasps is 27days (s.d. 23days) (Gadagkar etal., 1982), the mean age at which they become queens (if they do),is 35days (s.d. 20days), and they then remain as queens up to amean age of 103days (s.d. 65days). Thus, at least some femalesare known to remain on their natal nests for over 100days, takeover as queens as late as 78days after their eclosion and continueto remain as queens for as long as 262days after eclosion (Gadagkaret al., 1993). Most queens have already mated, although someindividuals might mate after assuming the role of queen. Someworkers are mated and some are not. Mating is neither necessarynor sufficient for an individual to develop her ovaries, suppressovarian development in other females, including mated ones, andbecome the sole egg layer of a colony (Chandrashekara andGadagkar, 1991). While queens always have well-developed ovaries,and a few workers may have ovaries at an intermediate stage ofdevelopment, most workers have completely undeveloped ovaries.However, workers can rapidly develop their ovaries and lay eggswithin a week or less after they become queens (Gadagkar, 2001).

MATERIALS AND METHODSStudy animals

Thirty-seven nests of Ropalidia marginata (Lepeletier 1836) werecollected in and around Bangalore, India, and brought to thelaboratory, where they were cleared of all existing adult wasps andlarvae. The nests containing the pupae were then maintained inaerated plastic boxes, and monitored daily for eclosing females. Six-hundred and thirty-two freshly eclosed females were removed fromtheir natal nest on the day of their eclosion, immediately transferredto transparent, ventilated plastic boxes (22×11×11cm), one femaleper box, and provided with ad libitum food (Corcyra cephalonicalarvae, Lepidoptera: Pyralidae), honey, water and a soft woodenblock as building material. Each female was randomly allotted to

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Fig.1. Ovarian development in Ropalidia marginatafemales. (A) Well-developed ovaries with several oocytesat various stages. (B) Undeveloped ovaries.

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one of the following 22 age classes: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 40, 50, 60, 80, 100, 150 and 200days. At the stipulatedage, the females were collected, dissected and their ovarianmeasurements (length of largest oocyte, width of largest oocyte,mean length of proximal oocyte, mean width of proximal oocyte,number of mature oocytes, total number of oocytes and number ofoocytes with yolk) were subjected to principal components analysis.Principal component 1 from this analysis was used as the ovarianindex (OI) for each female. Similarly, 27 different bodymeasurements (see Gadagkar, 2001) were subjected to principalcomponents analysis and the first principal component was used asan index of body size. Adult feeding was determined by noting thenumber of C. cephalonica larvae consumed during each day of theexperiment and was then averaged across all days.

Data analysisPrincipal component scores were calculated using StatistiXL, version1.8 (www.statistixl.com). All statistical analyses were done in R,version 2.11.1 (R Development Core Team, 2010). One-hundred andninety-three females from all age classes showing at least one oocyte(of any stage of development) were used to fit four functionsdescribing the relationship between age and ovarian index (bothvariables square-root transformed) to explore trends in the pattern ofovarian development with age: (1) linear increase, (2) saturation(Michaelis–Menten function), (3) unimodal skewed distribution(Ricker function) and (4) quadratic function. An information-theoreticapproach (minimum AIC) was used to select the best-fit function,and each function was fitted using lm or nls commands in R, withOI as the response variable and age as the predictor variable. Toidentify important variables affecting ovarian development, weconstructed seven models using unique combinations of age, bodysize index and feeding. Two-hundred and six females (including thosethat did not have a single oocyte at any stage of development) at agemore than or equal to 20days were included in this analysis.Individuals below 20days of age were excluded from this analysisbecause most of them showed no ovarian development at all. Modelselection was performed using information-theoretic criteria (Burnhamand Anderson, 2002), viz. second-order Akaike information criterion(AICc) values, Akaike’s weights and AIC differences using theAICcmodavg package (Mazerolle, 2010) in R.

RESULTSA total of 632 freshly eclosed, isolated female wasps were studiedby sampling them at predetermined ages ranging from 0 to 200daysand measuring their body size, ovarian development and feedingrate. The wasps showed large variation in ovarian development, evenamong individuals of similar ages, despite being kept under similarconditions and with access to similar resources (Fig.1, Fig.2A,Table1). All eclosing females started with no ovarian developmentat day0, and the number of individuals showing some ovariandevelopment (i.e. with at least one oocyte at any stage ofdevelopment) increased gradually with age, reached a peak at 100%at 100days, and decreased slightly thereafter (Fig.3). The extent ofinter-individual variation is dramatically reflected in the fact thatthe maximum ovarian index in each age class rose very sharplywith age, peaking at a value of 8.4 at 80days, while the mean OIof each age class rose more gradually and peaked at a value of only4.4 at 100days (Fig.2B).

Considering only those wasps that had developed ovaries (196individuals), we found that ovarian development increased gradually,peaked at a value of 4.8 at 156days and then decreased slightly, asbest described by a Ricker function (Fig.2C). Thus, a large number

of individuals failed to develop their ovaries even up to 80days ofage and a few individuals resorbed their ovaries beyond 100daysof age. Moreover, developed ovaries did not necessarily guaranteeegg laying. One-hundred and twenty-seven females that did not layany eggs had better-developed or similar ovaries compared with the23 females that laid eggs. Among the 100day old females, all ofwhich had developed ovaries, eggs layers and non-egg layers didnot differ significantly in their ovarian indices (unpaired Wilcoxonrank sum test, W=15, n1=3, n2=16, P>0.05). Age, body size and

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Fig.2. (A) Ovarian index (OI) values across age varied greatly, showingthat for each age class, females had a wide range of OI values. The plussigns indicate females with at least one oocyte in any stage ofdevelopment and the open circles indicate females without a single oocytein any stage of development. (B) Maximum OI (open squares) and meanOI (filled squares with standard deviation) for each age class. (C) Theplotted Ricker function (AIC=237.6) was the best-fit function for OI valuesacross age, with the quadratic function (AIC=239.8) ranking second. Basedon minimum AIC values, we ruled out the linear function (AIC=266.7) and(saturating) Michaelis–Menten function (AIC=243.5). The Ricker functiony=axe(–bx), where a=0.48±0.02 and b=0.08±0.00 (means ± s.e.m.),modelled OI values as a unimodal distribution with a positive skew,indicating gradual ovarian resorption after 156days of age.

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adult feeding rate positively correlated with ovarian development.When taken together, these three variables had higher explanatorypower than when they were considered one at a time or in pairs(Tables2, 3). The age of the wasps studied was varied as part ofthe experimental design from 0days (day of eclosion) to 200days.Addition of other variables to the model, such as the proportion ofempty cells, the number of females present on the nests from whichthese individuals eclosed (before nest collection), the date ofeclosion and nest of eclosion, did not add any additional explanatorypower (data not shown).

We confirmed that body size was normally distributed (Fig.4).Feeding rate varied in a highly skewed manner, with most waspsfeeding very little. Feeding rate was zero for wasps in the firstquartile and was 0.12 C. cephalonica larvae per day for wasps inthe third quartile (Fig.5). It should be emphasized that these feedingrates were observed in spite of the fact that all wasps were providedwith ad libitum access to food, although it is not the absolute feedingrates but the highly skewed inter-individual feeding rates that areof interest here. There was a weak but statistically significant positivecorrelation between body size and adult feeding rate (Fig.6).

DISCUSSIONRopalidia marginata females showed a very high level of variationin the time taken to develop their ovaries and also in the extent ofovarian development, despite having access to similar resources.The average lifespan of R. marginata workers (duration of residence)on their nest is 27±23days (mean ± s.d.) (Gadagkar et al., 1982).In the present study, about 40% of females failed to show any ovariandevelopment within their lifespan. Because R. marginata nests canbe initiated by solitary females, and because mating is not necessaryfor ovarian development, we consider the possibility that thevariation in ovarian development we have observed here may alsooccur in nature among solitary nest foundresses (which also haveno social stimuli, like the isolated wasps in our laboratoryexperiment). The lack of ovarian development that we observed in40% or more of the females is thus expected to facilitate the ontogenyof worker behaviour, as the cost of foregoing direct reproductionwould be relatively small for many females, i.e. Hamilton’s rule(Hamilton, 1964a; Hamilton, 1964b) would be more easily satisfiedfor some females than for others (Gadagkar, 2001; West-Eberhard,1975). The fact that it took as many as 100days for all females toshow at least one oocyte suggests considerable delay in reproductive

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Fig.3. Histogram of females without a single oocyte at any developmentalstage (white bars) showing that several females failed to develop ovariesuntil as late as 80days. Females with at least one oocyte at any stage(black bars) showed a negatively skewed distribution, indicating that asmall number of females developed ovaries much earlier than others, butthe frequency rose steadily and decreased after 100days.

Table1. Summary of ovarian development for Ropalidia marginata females used in the study

Age No. of Wasps with Wasps with OI No. of femalesSerial no. (days) wasps1 OD+ OD– Median Minimum Maximum that laid eggs2

1 0 35 0 35 −1.44 −1.44 −1.44 02 1 32 1 31 −1.44 −1.44 0.62 03 2 35 0 35 −1.44 −1.44 −1.44 04 3 34 0 34 −1.44 −1.44 −1.44 05 4 37 3 34 −1.44 −1.44 0.29 06 5 33 2 31 −1.44 −1.44 1.56 07 6 32 2 30 −1.44 −1.44 0.80 08 7 36 4 32 −1.44 −1.44 4.98 09 8 33 3 30 −1.44 −1.44 2.08 010 9 29 5 24 −1.44 −1.44 2.50 011 10 33 8 25 −1.44 −1.44 3.01 012 15 29 6 23 −1.44 −1.44 2.25 013 20 25 12 13 −1.44 −1.44 5.29 114 25 25 14 11 1.63 −1.44 5.77 015 30 28 17 11 1.61 −1.44 6.96 116 40 29 19 10 1.80 −1.44 7.38 017 50 24 19 5 2.70 −1.44 7.04 318 60 18 15 3 2.59 −1.44 8.28 319 80 20 19 1 3.69 −1.44 8.41 320 100 19 19 0 3.62 2.33 7.62 521 150 19 16 3 4.46 −1.44 7.19 922 200 14 12 2 2.83 −1.44 6.22 3

OI, ovarian index.OD+, data for females with at least one oocyte at any stage of development.OD–, data for females with no oocyte at any stage of development.1Number of females for which data on ovarian development were collected. In total, 19 females were excluded.2Data obtained before females were collected for ovarian and body size measurements.

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maturation in many females. Such delayed reproductive maturationcan facilitate the commonly observed strategy of female wasps tofirst function as sterile workers and later become future queens oftheir colonies (Gadagkar, 1991). It is of course possible thatvariation in ovarian development evolved after the origin ofeusociality, although we do not know which came first. Nevertheless,variation in ovarian development makes it easier for females withpoor ovarian development to satisfy Hamilton’s rule because theywould be paying a smaller cost by staying back in their natal nestas sterile workers. This advantage for some females may havefacilitated either the origin of eusociality (if variation in ovariandevelopment preceded the origin of eusociality) or the maintenanceof eusociality (if the origin of eusociality preceded variation inovarian development). In short, females with poor potential forovarian development should find it easier to forego nest foundingand become sterile workers. Thus, our study provides furtherevidence in support of models for the evolution of eusocialitydiscussed in the literature under the theme of ‘sub-fertility’(Alexander, 1974; Craig, 1983; Gadagkar, 1991; West-Eberhard,1975) but it should be emphasized that these models in turn showhow Hamilton’s rule can be satisfied.

Our results also show that some females fail to initiate nests andlay eggs even though their ovaries are as well developed as thoseof females that do initiate nests and lay eggs. Such females may beincapable of becoming or reluctant to become solitary foundressesbut may be capable of becoming replacement queens upon the lossof the original queen or of usurping colonies with weak queens.Thus, our study also provides evidence for direct fitness models forthe evolution of sociality discussed in the literature as the ‘sit andwait’ strategy (Nonacs and Reeve, 1993; Starks, 1998). We see no

reason why indirect fitness models such as Hamilton’s rule and directfitness models such as ‘sit-and-wait’ should be mutually exclusive;wasps may be expected to gain fitness by any route available tothem.

We have shown that age, body size and adult feeding rate are allcorrelated with an individual’s ovarian development. That age is oneof the variables important in ovarian development is not surprising.However, age is only one of the variables important in ovariandevelopment, so that, depending on the values of other factors suchas body size and feeding rate, individuals of the same age have verydifferent levels of ovarian development and, conversely, individualsof very different ages may have similar ovarian development. Wehypothesize that it is such plasticity in ovarian development that allowsindividuals to adopt different strategies such as nest founding, joining,staying back in the natal nest, usurping, etc., depending on theirinternal physiological state and the prevailing external environmentalconditions. Individuals that resorb their ovaries after a certain agecould also contribute to the variable effect of age on ovariandevelopment. Ovarian resorption, also known in ants and bees(Billen, 1982; Duchateau and Velthuis, 1989), could be a strategy toavoid ovipositioning in unfavourable conditions or be aimed atconserving nutrients (Sundberg et al., 2001). As females had ad libitumfood available to them throughout their lifetime, starvation may not

Table2. Results of model selection using the AICc approach for ovarian development in females as a function of age, body size and feeding

Model ID Model K AICc ΔAICc wi log likelihood

Model 1 Age + age 2 4 960.61 19.18 0.00 −476.20Model 2 Feeding 3 1001.42 59.99 0.00 −497.65Model 3 Body size 3 990.76 49.33 0.00 −492.38Model 4 Age + age2 + feeding 5 957.58 16.16 0.00 −473.64Model 5 Age + age2 + body size 5 945.24 3.81 0.13 −467.47Model 6 Body size + feeding 4 986.75 45.33 0.00 −489.28Model 7 Age + age2 + body size + feeding 6 941.43 0.00 0.87 −464.50

K, number of estimated parameters included in the model; AICc, second-order Akaike information criterion; ΔAICc, difference in AICc values between a modeland that with least AICc model; wi=Akaike weights.

Model 7 (containing age, age2, body size and feeding) had the lowest AICc value and was therefore the best fit model to explain ovarian development. Model 5(with age, age2 and body size) had a ΔAICc of 3.81 and was ranked second amongst the models. All other models had a very high ΔAICc values and can beconsidered unlikely. Evidence ratio for model 7 and model 5 was 6.72. The global model (model 7) satisfied assumptions of normality of residuals(Shapiro–Wilk test, P>0.05) and constant variance (score test for non-constant error variance, P>0.05). Missing values were deleted from the data set toavoid incorrect AICc results.

Table3. Model-averaged parameter estimates for the variablesused in model selection

Model-averaged 95% confidenceSerial no. Variable estimate interval

1 Age 0.08 0.06, 0.112 Age2 –0.0003 –0.0004, −0.00013 Body size 0.22 0.12, 0.324 Feeding 3.29 0.63, 5.95

Age, age2, body size and feeding had 95% unconditional confidenceintervals excluding zero, indicating a strong effect of these variables onovarian development different from zero.

Age, body size and feeding positively affected ovarian development, withfeeding having the largest model-averaged estimate.

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be the cause of ovarian resorption in our study. However, the metabolicand immunological costs of ageing (Bell and Bohm, 1975; Collier,1995) may lead to a greater demand by somatic tissue, andsurvivability may be achieved by compromising reproduction(Ohgushi, 1996). Resorption may also occur because of age and alack of nesting sites (Bell and Bohm, 1975; Lopez-Guerrero, 1996),and this may be the case here, as many females that developed ovariesfailed to initiate nests.

Body size was the second factor that was important in ovariandevelopment. Body size, in turn, is generally influenced by larvalnutrition (Edgar, 2006; Karsai and Hunt, 2002; Plowright and Jay,1977; Richards and Packer, 1996; Roulston and Cane, 2002).Similarly, body size is known to be correlated with foodconsumption in many insects (Reichle, 1968). As there was a positivecorrelation between body size and adult feeding rate even when allindividuals had access to unlimited food, it may be argued that well-fed larvae developed into larger sized individuals predisposed toconsume more food, which then consumed more food and developedbetter ovaries. Conversely, poorly fed larvae developed into smallersized individuals predisposed to consume less food, which thereforeconsumed less food and had poorly developed ovaries (Gadagkaret al., 1988; Gadagkar et al., 1991). Variation in larval nutrition isthus expected to be the starting point of this cascade of events leadingto variation in ovarian development that ultimately facilitates theevolution of a worker caste. Such variation in larval nutrition maybe brought about by seasonal and other variation in food availabilityto the foragers, the number of foragers available and perhaps someactive differential feeding of larvae to channel them preferentiallyinto queen or worker roles during specific periods in the colonycycle (Gadagkar, 1996; Gadagkar, 2001; Gadagkar et al., 1988;Hunt, 2007; Hunt and Nalepa, 1994; Kapheim et al., 2011).

Adult nutrition was the third important predictor of ovariandevelopment. Oocyte development is essentially a nutrient-drivenprocess requiring large protein and fat resources. Therefore, it isnot surprising that females with a higher average caterpillarconsumption rate also had better developed ovaries. Adult nutritionhas been shown to affect reproductive potential in wasps (Gadagkaret al., 1988; Hunt, 2007; Tibbetts et al., 2011; Toth et al., 2009).Variation in adult nutrition in natural colonies may be brought about

by the unequal exchange of food between foragers and intranidalworkers as well as being due to unequal trophallaxis among theworkers and between workers and larvae. In addition to differentialfeeding, the actual nutritional status of an individual may dependon its social role in the colony and how much energy is required toperform that role. Energetically expensive behaviours such asforaging may reduce resource allocation to reproduction(Markiewicz and O’Donnell, 2001; West-Eberhard, 1969). It shouldbe noted that it was not possible to measure honey consumption inthis study, though sugar consumption is known to affect ovariandevelopment (Nilsen et al., 2011; Page and Amdam, 2007);therefore, our interpretation of nutrition affecting female ovaries islargely based on protein consumption through Corcyra larvae.

We have shown that pre-imaginal factors such as larval nutritionand body size and post-imaginal factors such as age and feedingrate can act together to produce variability in adult reproductivepotential, which can in turn facilitate the ontogeny of a worker caste.Our experiments using female wasps in isolation show that theresulting variation in reproductive potential can be very large in R.marginata, thus providing an explanation for how Hamilton’s rulecould be satisfied even when intra-colony relatedness is quite low(Gadagkar, 2001). Thus, the observed variation in reproductivepotential can by itself provide a powerful gateway for workerontogeny and the evolution of sociality.

ACKNOWLEDGEMENTSR.G. designed the study, S.C. performed the experiments, S.S. analyzed the data,and R.G. and S.S. co-wrote the paper. Our experiments comply with regulationsof animal care in India. We thank James Hunt, William Wcislo and an anonymousreferee for their helpful comments.

FUNDINGThis work was supported by grants from the Department of Science andTechnology, the Department of Biotechnology, the Council of Scientific andIndustrial Research, and the Ministry of Environment and Forests, Government ofIndia.

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Fig.5. Histogram of mean feeding rate for adult wasps, showing a highlyskewed distribution, with median 0.03, range 0.00–0.88. Most femalesconsumed very little food, and only a few females consumed a large meannumber of Corcyra caterpillars per day despite ad libitum availability.

Fig.6. There was a weak but positive statistically significant correlationbetween body size of a female and her adult rate of feeding (Pearsonʼsproduct-moment correlation, d.f.=594, P<0.01, R=0.16).

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Billen, J. (1982). Ovariole development in workers of Formica sanguinea Latr.(Hymenoptera: Formicidae). Insectes Soc. 29, 86-94.

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