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

RESEARCH ARTICLE Ovarian developmental variation in the primitively eusocial wasp marginata suggests a gateway to worker ontogeny and the evolution of

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 ([email protected])

SUMMARY Social are characterized by reproductive caste differentiation of colony members into one or a small number of fertile queens and a large number of sterile workers. The evolutionary origin and maintenance of such sterile workers remains an enduring puzzle in . Here, we studied ovarian development in over 600 freshly eclosed, isolated, virgin female wasps, maintained in the laboratory. The wasps differed greatly both in the time taken to develop their ovaries and in the magnitude of ovarian development despite having similar access to resources. All females started with no ovarian development at day zero, and the percentage of individuals with at least one oocyte at any stage of development increased gradually across age, reached 100% at 100days and decreased slightly thereafter. Approximately 40% of the females failed to develop ovaries within the average ecological lifespan of the . Age, body size and adult feeding rate, when considered together, were the most important factors governing ovarian development. We suggest that such flexibility and variation in the potential and timing of reproductive development may physiologically predispose females to accept worker roles and thus provide a gateway to worker ontogeny and the evolution of sociality. Key words: ovarian development, social wasp, caste differentiation, . Received 2 April 2012; Accepted 29 August 2012

INTRODUCTION et al., 1988; Gadagkar et al., 1990; Gadagkar et al., 1991; West- Many species of social insects such as ants, bees, wasps and termites Eberhard, 1975). live in colonies showing high levels of cooperation and division of These ideas are best tested in so-called primitively eusocial labour. These features are achieved by the differentiation of colony species where many workers can be reproductively totipotent and members into one or a small number of fertile queens (or kings) capable of full ovarian growth, unlike highly eusocial species where and a large number of sterile (and therefore defined as altruistic) workers have permanently reduced or atrophied ovaries even at the workers (Wilson, 1971). The evolution by natural selection of such time of eclosion (Oertel, 1930; Snodgrass, 1956). Reproductive altruistic behaviour on the part of the workers remains a major totipotency makes it possible for workers in primitively eusocial unsolved problem in insect sociobiology. Inclusive fitness theory species to transform themselves from sterile to fertile individuals (also often referred to as or Hamilton’s rule) provides and become replacement queens upon the loss or death of the original a powerful theoretical framework for exploring the evolution of queen (Chandrashekara and Gadagkar, 1992; Monnin and Peeters, altruistic behaviour. Hamilton’s rule (rb>c) states that an altruistic 1999; Smith et al., 2009; Tibbetts and Huang, 2010). In many social trait can spread by natural selection if the benefit of the altruistic insect species such as honey bees, sweat bees, bumble bees, act to the recipient (b), devalued by the genetic relatedness between polistine wasps and vespine wasps, ovarian development is actor and recipient (r), is greater than the cost to the altruist (c) influenced by physiological, social and environmental factors such (Hamilton, 1964a; Hamilton, 1964b). Most studies attempting to as body size, larval and adult nutrition, presence or absence of queen test Hamilton’s rule have measured genetic relatedness between , social interactions and time of year, leading to altruists and recipients but relatedness values alone can tell us little considerable heterogeneity in the extent of ovarian development about the validity or otherwise of Hamilton’s rule (Evans, 1977; among colony members (Backx et al., 2012; Bloch et al., 2002; Gadagkar, 1990; West-Eberhard, 1978). Attention should therefore Duchateau and Velthuis, 1989; Hoover et al., 2006; Kapheim et al., now focus on the cost and benefit terms in Hamilton’s rule, which 2012; Motro et al., 1979; Smith et al., 2008; Smith et al., 2009; so far remain poorly studied (Choe and Crespi, 1997; Gadagkar, Tibbetts et al., 2011; Toth et al., 2009; Wheeler, 1986; Wheeler, 2001; West-Eberhard, 1975). One way in which Hamilton’s rule 1996; Wheeler et al., 1999). In the primitively eusocial wasp can be relatively easily satisfied is for the altruist to pay only a small Ropalidia marginata, there is a clear pre-imaginal caste bias. Only cost, and this could happen if the altruist has a lower fertility about 50% of the eclosing individuals build nests and lay eggs when compared with other individuals in the population. This idea has isolated into individual holding boxes in the laboratory and provided been proposed repeatedly in such forms as the ‘sub-fertility with ad libitum food and building material. The other half die without hypothesis’, ‘parental manipulation’, ‘pre-imaginal caste bias’, etc. ever laying eggs in spite of living, on average, as long as or longer (Alexander, 1974; Craig, 1983; Field and Foster, 1999; Gadagkar than the egg layers (Gadagkar et al., 1988; Gadagkar et al., 1990;

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Fig.1. Ovarian development in Ropalidia marginata females. (A) Well-developed ovaries with several oocytes at various stages. (B) Undeveloped ovaries.

Gadagkar et al., 1991). The advantage of experimenting with freshly may stay in their natal nests for their entire life, with or without eclosed female wasps maintained in isolation in the laboratory is becoming queens. Thus, there is no real distinction between ‘queens’ that we may rule out or minimize the influence of such factors as and ‘replacement queens’. The mean duration of residence on their social interactions, dominance, queen , etc., and attribute natal nests for female wasps is 27days (s.d. 23days) (Gadagkar et the observed inter-individual variation in reproductive potential to al., 1982), the mean age at which they become queens (if they do), intrinsic properties of the eclosing wasps, including any effects of is 35days (s.d. 20days), and they then remain as queens up to a the larval environment. However, data on ovarian development at mean age of 103days (s.d. 65days). Thus, at least some females different ages could not be collected in the studies mentioned above, are known to remain on their natal nests for over 100days, take in which all females were kept alive until they laid at least one egg over as queens as late as 78days after their eclosion and continue or died naturally. In the present study, we therefore measured ovarian to remain as queens for as long as 262days after eclosion (Gadagkar development at different ages by similarly maintaining freshly et al., 1993). Most queens have already mated, although some eclosed, virgin female wasps under isolation in the laboratory and individuals might mate after assuming the role of queen. Some found as much or more inter-individual variation in ovarian workers are mated and some are not. Mating is neither necessary development as we found in egg-laying ability in the previous studies nor sufficient for an individual to develop her ovaries, suppress mentioned above. ovarian development in other females, including mated ones, and To begin with, we provide a brief overview of the natural history become the sole egg layer of a colony (Chandrashekara and of R. marginata in order to help place the results of our laboratory Gadagkar, 1991). While queens always have well-developed ovaries, studies in the ecological context of natural colonies. Ropalidia and a few workers may have ovaries at an intermediate stage of marginata is a tropical, old-world, primitively eusocial development, most workers have completely undeveloped ovaries. widely distributed in peninsular India. New colonies may be However, workers can rapidly develop their ovaries and lay eggs founded throughout the year, by one or a small group of females. within a week or less after they become queens (Gadagkar, 2001). In single-foundress nests, the foundress not only lays eggs but also performs nest building, foraging, brood rearing and all other tasks, MATERIALS AND METHODS until the eclosion of her daughters. In multiple-foundress nests, only Study one female wasp lays eggs (queen) while all non-reproductive tasks Thirty-seven nests of Ropalidia marginata (Lepeletier 1836) were such as nest building, brood care and foraging are performed by the collected in and around Bangalore, India, and brought to the remaining females, who function as sterile workers. However, the laboratory, where they were cleared of all existing adult wasps and queen is replaced from time to time and one of the workers becomes larvae. The nests containing the pupae were then maintained in the new queen, and she may in turn be replaced after some time. aerated plastic boxes, and monitored daily for eclosing females. Six- Because of the tropical climate, the nesting cycle of these wasps is hundred and thirty-two freshly eclosed females were removed from perennial and the nests may be very long lived. The wasps (at least, their natal nest on the day of their eclosion, immediately transferred the females) are also quite long lived. Males stay on their natal nests to transparent, ventilated plastic boxes (22×11×11cm), one female for about a week, after which they leave, to lead a nomadic life. per box, and provided with ad libitum food (Corcyra cephalonica Female wasps may also leave at various times to found new single- larvae, Lepidoptera: Pyralidae), honey, water and a soft wooden or multiple-foundress nests or to join other newly founded nests, or block as building material. Each female was randomly allotted to

THE JOURNAL OF EXPERIMENTAL BIOLOGY Ovarian development in a social wasp 183 one of the following 22 age classes: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, A 15, 20, 25, 30, 40, 50, 60, 80, 100, 150 and 200days. At the stipulated 8 age, the females were collected, dissected and their ovarian measurements (length of largest oocyte, width of largest oocyte, 6 mean length of proximal oocyte, mean width of proximal oocyte, number of mature oocytes, total number of oocytes and number of 4 oocytes with yolk) were subjected to principal components analysis. Principal component 1 from this analysis was used as the ovarian 2 index (OI) for each female. Similarly, 27 different body measurements (see Gadagkar, 2001) were subjected to principal 0 components analysis and the first principal component was used as an index of body size. Adult feeding was determined by noting the –2 number of C. cephalonica larvae consumed during each day of the experiment and was then averaged across all days. 8 B

Data analysis 6 Principal component scores were calculated using StatistiXL, version 1.8 (www.statistixl.com). All statistical analyses were done in R, 4 version 2.11.1 (R Development Core Team, 2010). One-hundred and ninety-three females from all age classes showing at least one oocyte 2 (of any stage of development) were used to fit four functions Ovarian index describing the relationship between age and ovarian index (both 0 variables square-root transformed) to explore trends in the pattern of ovarian development with age: (1) linear increase, (2) saturation –2 (Michaelis–Menten function), (3) unimodal skewed distribution (Ricker function) and (4) quadratic function. An information-theoretic C approach (minimum AIC) was used to select the best-fit function, 8 and each function was fitted using lm or nls commands in R, with OI as the response variable and age as the predictor variable. To 6 identify important variables affecting ovarian development, we constructed seven models using unique combinations of age, body 4 size index and feeding. Two-hundred and six females (including those that did not have a single oocyte at any stage of development) at age 2 more than or equal to 20days were included in this analysis. Individuals below 20days of age were excluded from this analysis 0 because most of them showed no ovarian development at all. Model selection was performed using information-theoretic criteria (Burnham –2 and Anderson, 2002), viz. second-order Akaike information criterion 0 50 100 150 200 250 (AICc) values, Akaike’s weights and AIC differences using the Age (days) AICcmodavg package (Mazerolle, 2010) in R. Fig.2. (A) Ovarian index (OI) values across age varied greatly, showing RESULTS that for each age class, females had a wide range of OI values. The plus A total of 632 freshly eclosed, isolated female wasps were studied signs indicate females with at least one oocyte in any stage of by sampling them at predetermined ages ranging from 0 to 200days development and the open circles indicate females without a single oocyte and measuring their body size, ovarian development and feeding in any stage of development. (B) Maximum OI (open squares) and mean OI (filled squares with standard deviation) for each age class. (C) The rate. The wasps showed large variation in ovarian development, even plotted Ricker function (AIC=237.6) was the best-fit function for OI values among individuals of similar ages, despite being kept under similar across age, with the quadratic function (AIC=239.8) ranking second. Based conditions and with access to similar resources (Fig.1, Fig.2A, on minimum AIC values, we ruled out the linear function (AIC=266.7) and Table1). All eclosing females started with no ovarian development (saturating) Michaelis–Menten function (AIC=243.5). The Ricker function at day0, and the number of individuals showing some ovarian y=axe(–bx), where a=0.48±0.02 and b=0.08±0.00 (means ± s.e.m.), development (i.e. with at least one oocyte at any stage of modelled OI values as a unimodal distribution with a positive skew, development) increased gradually with age, reached a peak at 100% indicating gradual ovarian resorption after 156days of age. at 100days, and decreased slightly thereafter (Fig.3). The extent of inter-individual variation is dramatically reflected in the fact that of individuals failed to develop their ovaries even up to 80days of the maximum ovarian index in each age class rose very sharply age and a few individuals resorbed their ovaries beyond 100days with age, peaking at a value of 8.4 at 80days, while the mean OI of age. Moreover, developed ovaries did not necessarily guarantee of each age class rose more gradually and peaked at a value of only egg laying. One-hundred and twenty-seven females that did not lay 4.4 at 100days (Fig.2B). any eggs had better-developed or similar ovaries compared with the Considering only those wasps that had developed ovaries (196 23 females that laid eggs. Among the 100day old females, all of individuals), we found that ovarian development increased gradually, which had developed ovaries, eggs layers and non-egg layers did peaked at a value of 4.8 at 156days and then decreased slightly, as not differ significantly in their ovarian indices (unpaired Wilcoxon best described by a Ricker function (Fig.2C). Thus, a large number rank sum test, W=15, n1=3, n2=16, P>0.05). Age, body size and

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Table1. Summary of ovarian development for Ropalidia marginata females used in the study OI Age No. of Wasps with Wasps with No. of females Serial no. (days) wasps1 OD+ OD– Median Minimum Maximum that laid eggs2 1035035−1.44 −1.44 −1.44 0 2132131−1.44 −1.44 0.62 0 3235035−1.44 −1.44 −1.44 0 4334034−1.44 −1.44 −1.44 0 5437334−1.44 −1.44 0.29 0 6533231−1.44 −1.44 1.56 0 7632230−1.44 −1.44 0.80 0 8736432−1.44 −1.44 4.98 0 9833330−1.44 −1.44 2.08 0 10 9 29 5 24 −1.44 −1.44 2.50 0 11 10 33 8 25 −1.44 −1.44 3.01 0 12 15 29 6 23 −1.44 −1.44 2.25 0 13 20 25 12 13 −1.44 −1.44 5.29 1 14 25 25 14 11 1.63 −1.44 5.77 0 15 30 28 17 11 1.61 −1.44 6.96 1 16 40 29 19 10 1.80 −1.44 7.38 0 17 50 24 19 5 2.70 −1.44 7.04 3 18 60 18 15 3 2.59 −1.44 8.28 3 19 80 20 19 1 3.69 −1.44 8.41 3 20 100 19 19 0 3.62 2.33 7.62 5 21 150 19 16 3 4.46 −1.44 7.19 9 22 200 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. adult feeding rate positively correlated with ovarian development. We confirmed that body size was normally distributed (Fig.4). When taken together, these three variables had higher explanatory Feeding rate varied in a highly skewed manner, with most wasps power than when they were considered one at a time or in pairs feeding very little. Feeding rate was zero for wasps in the first (Tables2, 3). The age of the wasps studied was varied as part of quartile and was 0.12 C. cephalonica larvae per day for wasps in the experimental design from 0days (day of eclosion) to 200days. the third quartile (Fig.5). It should be emphasized that these feeding Addition of other variables to the model, such as the proportion of rates were observed in spite of the fact that all wasps were provided empty cells, the number of females present on the nests from which with ad libitum access to food, although it is not the absolute feeding these individuals eclosed (before nest collection), the date of rates but the highly skewed inter-individual feeding rates that are eclosion and nest of eclosion, did not add any additional explanatory of interest here. There was a weak but statistically significant positive power (data not shown). correlation between body size and adult feeding rate (Fig.6).

40 DISCUSSION Ropalidia marginata females showed a very high level of variation 35 in the time taken to develop their ovaries and also in the extent of

30 ovarian development, despite having access to similar resources. The average lifespan of R. marginata workers (duration of residence) 25 on their nest is 27±23days (mean ± s.d.) (Gadagkar et al., 1982). 20 In the present study, about 40% of females failed to show any ovarian development within their lifespan. Because R. marginata nests can 15 be initiated by solitary females, and because mating is not necessary No. of females 10 for ovarian development, we consider the possibility that the variation in ovarian development we have observed here may also 5 occur in nature among solitary nest foundresses (which also have

0 no social stimuli, like the isolated wasps in our laboratory 0 1 2 3 4 5 6 7 8 9 10 15 20 25 30 40 50 60 80 100 150 200 experiment). The lack of ovarian development that we observed in Age (days) 40% or more of the females is thus expected to facilitate the ontogeny of worker behaviour, as the cost of foregoing direct reproduction Fig.3. Histogram of females without a single oocyte at any developmental would be relatively small for many females, i.e. Hamilton’s rule stage (white bars) showing that several females failed to develop ovaries (Hamilton, 1964a; Hamilton, 1964b) would be more easily satisfied until as late as 80days. Females with at least one oocyte at any stage (black bars) showed a negatively skewed distribution, indicating that a for some females than for others (Gadagkar, 2001; West-Eberhard, small number of females developed ovaries much earlier than others, but 1975). The fact that it took as many as 100days for all females to the frequency rose steadily and decreased after 100days. show at least one oocyte suggests considerable delay in reproductive

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Table2. 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 −476.20 Model 2 Feeding 3 1001.42 59.99 0.00 −497.65 Model 3 Body size 3 990.76 49.33 0.00 −492.38 Model 4 Age + age2 + feeding 5 957.58 16.16 0.00 −473.64 Model 5 Age + age2 + body size 5 945.24 3.81 0.13 −467.47 Model 6 Body size + feeding 4 986.75 45.33 0.00 −489.28 Model 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 model and 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 be considered 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 to avoid incorrect AICc results. maturation in many females. Such delayed reproductive maturation reason why indirect fitness models such as Hamilton’s rule and direct can facilitate the commonly observed strategy of female wasps to fitness models such as ‘sit-and-wait’ should be mutually exclusive; first function as sterile workers and later become future queens of wasps may be expected to gain fitness by any route available to their colonies (Gadagkar, 1991). It is of course possible that them. variation in ovarian development evolved after the origin of We have shown that age, body size and adult feeding rate are all eusociality, although we do not know which came first. Nevertheless, correlated with an individual’s ovarian development. That age is one variation in ovarian development makes it easier for females with of the variables important in ovarian development is not surprising. poor ovarian development to satisfy Hamilton’s rule because they However, age is only one of the variables important in ovarian would be paying a smaller cost by staying back in their natal nest development, so that, depending on the values of other factors such as sterile workers. This advantage for some females may have as body size and feeding rate, individuals of the same age have very facilitated either the origin of eusociality (if variation in ovarian different levels of ovarian development and, conversely, individuals development preceded the origin of eusociality) or the maintenance of very different ages may have similar ovarian development. We of eusociality (if the origin of eusociality preceded variation in hypothesize that it is such plasticity in ovarian development that allows ovarian development). In short, females with poor potential for individuals to adopt different strategies such as nest founding, joining, ovarian development should find it easier to forego nest founding staying back in the natal nest, usurping, etc., depending on their and become sterile workers. Thus, our study provides further internal physiological state and the prevailing external environmental evidence in support of models for the conditions. Individuals that resorb their ovaries after a certain age discussed in the literature under the theme of ‘sub-fertility’ could also contribute to the variable effect of age on ovarian (Alexander, 1974; Craig, 1983; Gadagkar, 1991; West-Eberhard, development. Ovarian resorption, also known in ants and bees 1975) but it should be emphasized that these models in turn show (Billen, 1982; Duchateau and Velthuis, 1989), could be a strategy to how Hamilton’s rule can be satisfied. avoid ovipositioning in unfavourable conditions or be aimed at Our results also show that some females fail to initiate nests and conserving nutrients (Sundberg et al., 2001). As females had ad libitum lay eggs even though their ovaries are as well developed as those food available to them throughout their lifetime, starvation may not of females that do initiate nests and lay eggs. Such females may be incapable of becoming or reluctant to become solitary foundresses but may be capable of becoming replacement queens upon the loss of the original queen or of usurping colonies with weak queens. 150 Thus, our study also provides evidence for direct fitness models for the evolution of sociality discussed in the literature as the ‘sit and wait’ strategy (Nonacs and Reeve, 1993; Starks, 1998). We see no 100

Table3. Model-averaged parameter estimates for the variables used in model selection

Model-averaged 95% confidence No. of females 50 Serial no. Variable estimate interval 1 Age 0.08 0.06, 0.11 2Age2 –0.0003 –0.0004, −0.0001 3 Body size 0.22 0.12, 0.32 0 4 Feeding 3.29 0.63, 5.95 –10 –6 –2 2 6 10 2 Age, age , body size and feeding had 95% unconditional confidence Body size index intervals excluding zero, indicating a strong effect of these variables on ovarian development different from zero. Fig.4. Histogram of body size index for females, showing a normal Age, body size and feeding positively affected ovarian development, with distribution (Shapiro–Wilk normality test, W=0.99, P=0.66), with mean feeding having the largest model-averaged estimate. –0.07±3.29, range –8.90 to 11.07.

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400 0.8

300 0.6

200

0.4 No. of females 100 Adult feeding rate Adult 0.2 0

0 0.2 0.4 0.6 0.8 1.0 0 Feeding rate (no. day–1) –10 –5 0510 Fig.5. Histogram of mean feeding rate for adult wasps, showing a highly Body size index skewed distribution, with median 0.03, range 0.00–0.88. Most females consumed very little food, and only a few females consumed a large mean Fig.6. There was a weak but positive statistically significant correlation number of Corcyra caterpillars per day despite ad libitum availability. between body size of a female and her adult rate of feeding (Pearsonʼs product-moment correlation, d.f.=594, P<0.01, R=0.16). be the cause of ovarian resorption in our study. However, the metabolic by the unequal exchange of food between foragers and intranidal and immunological costs of ageing (Bell and Bohm, 1975; Collier, workers as well as being due to unequal trophallaxis among the 1995) may lead to a greater demand by somatic tissue, and workers and between workers and larvae. In addition to differential survivability may be achieved by compromising reproduction feeding, the actual nutritional status of an individual may depend (Ohgushi, 1996). Resorption may also occur because of age and a on its social role in the colony and how much energy is required to lack of nesting sites (Bell and Bohm, 1975; Lopez-Guerrero, 1996), perform that role. Energetically expensive behaviours such as and this may be the case here, as many females that developed ovaries foraging may reduce resource allocation to reproduction failed to initiate nests. (Markiewicz and O’Donnell, 2001; West-Eberhard, 1969). It should Body size was the second factor that was important in ovarian be noted that it was not possible to measure honey consumption in development. Body size, in turn, is generally influenced by larval this study, though sugar consumption is known to affect ovarian nutrition (Edgar, 2006; Karsai and Hunt, 2002; Plowright and Jay, development (Nilsen et al., 2011; Page and Amdam, 2007); 1977; Richards and Packer, 1996; Roulston and Cane, 2002). therefore, our interpretation of nutrition affecting female ovaries is Similarly, body size is known to be correlated with food largely based on protein consumption through Corcyra larvae. consumption in many insects (Reichle, 1968). As there was a positive We have shown that pre-imaginal factors such as larval nutrition correlation between body size and adult feeding rate even when all and body size and post-imaginal factors such as age and feeding individuals had access to unlimited food, it may be argued that well- rate can act together to produce variability in adult reproductive fed larvae developed into larger sized individuals predisposed to potential, which can in turn facilitate the ontogeny of a worker caste. consume more food, which then consumed more food and developed Our experiments using female wasps in isolation show that the better ovaries. Conversely, poorly fed larvae developed into smaller resulting variation in reproductive potential can be very large in R. sized individuals predisposed to consume less food, which therefore marginata, thus providing an explanation for how Hamilton’s rule consumed less food and had poorly developed ovaries (Gadagkar could be satisfied even when intra-colony relatedness is quite low et al., 1988; Gadagkar et al., 1991). Variation in larval nutrition is (Gadagkar, 2001). Thus, the observed variation in reproductive thus expected to be the starting point of this cascade of events leading potential can by itself provide a powerful gateway for worker to variation in ovarian development that ultimately facilitates the ontogeny and the evolution of sociality. evolution of a worker caste. Such variation in larval nutrition may be brought about by seasonal and other variation in food availability ACKNOWLEDGEMENTS to the foragers, the number of foragers available and perhaps some R.G. designed the study, S.C. performed the experiments, S.S. analyzed the data, active differential feeding of larvae to channel them preferentially and R.G. and S.S. co-wrote the paper. Our experiments comply with regulations of care in India. We thank James Hunt, William Wcislo and an anonymous into queen or worker roles during specific periods in the colony referee for their helpful comments. cycle (Gadagkar, 1996; Gadagkar, 2001; Gadagkar et al., 1988; Hunt, 2007; Hunt and Nalepa, 1994; Kapheim et al., 2011). FUNDING Adult nutrition was the third important predictor of ovarian This work was supported by grants from the Department of Science and development. 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