A striking feature of hymenopterans societies is the absence of male workers. Foraging, nest building, brood care and all other activities required for the functioning of the colony are carried out by the females. These behaviours of the females and the implied cooperation and altruism have led social insect researchers to focus almost exclusively on the female members of hymenopterans societies. As a consequence, males have remained relatively neglected. The imbalance in the attention paid by researchers to females and males has been even more striking in the case of the extensively studied primitively eusocial wasp Rosalinda marginata. I have therefore focussed most of my attention to male R. marginata but of course, wherever possible and appropriate, I have compared the males with the females. Since almost nothing was known about R. marginata males, I began by obtaining basic information on the natural history and behaviour of the males. Male R. marginata are smaller in size and are different in the shape of the head as compared to the females. By conducting a three year survey, I found that just as nests of this species are found throughout the year, so are the males. The presence of males throughout the year has implications for the evolution of sociality by making available the opportunity to mate and found new nests, for females eclosing at any time of the year. Unlike the females who spend all their life on their nests, males stay on their natal nests only for one to twelve days (mean ± sd: 6.0 ± 2.6, N = 55) and leave their natal nest once and for all, to lead a nomadic life. It is difficult to determine how long males live after leaving their natal nests. However, I maintained males in the laboratory with ad libitum food and found that under these conditions they can live up to 140 days (mean physiological life span ± SD: 61.3 ± 28.0, N = 106). Like all eusocial hymenopterans males, R. marginata males also do not take part in any colony maintenance activities. They however, occasionally perform the following behaviours: solicit food, antennae nest, antennae another wasp, feed self, snatch food (from females), fan wings, body jerk and wing jerk, dominance and subordinate behaviours. Females of course perform all of these behaviours and many more. But there are no behaviours which are restricted to the males. Borrowing methods used by ecologists to measure species richness and diversity, I have computed the behavioral richness and diversity of male and female R. marginata. As expected, female behaviour is richer and more diverse compared to males. Comparing what the males did during their short stay on the nest and what females did during their long stay on the nest, I found that males did not forage or feed larvae (although I recorded one male feeding larvae thrice on one occasion when there seemed to be excess food on the nest). Males showed, dominance and subordinate behaviours and being solicited behaviour, significantly less often than females. On the other hand, males showed higher frequencies of feeding self and soliciting behaviour. However, these comparisons may not be fair because what males do in the first few days of their life is being compared with what females do throughout their life. Hence I truncated the female data at six days to make it comparable to the average age of the males on the nest. Even after doing so I found similar differences except that the males show similar rates of feed self and higher rates of subordinate behaviour compared to the young females. As mentioned in the beginning, absence of male workers is a striking feature of social Hymenoptera. I therefore naturally turned my attention to the possible reasons for this. As there has been much speculation on the ultimate, evolutionary explanations for why males do not work, I decided to investigate possible proximate explanations. To make my goal experimentally tractable, I decided to focus on the behaviour of feeding larvae as an example of work. In spite of the fact that R. marginata has been studied for over two decades, male R. marginata had never been seen to feed larvae, before my study. I advanced three hypotheses for why males do not feed larvae. (1) Males are incapable of feeding larvae. (2) Males do not feed larvae because they have insufficient access to food to satisfy themselves and to feed the larvae. (3) Males do not feed larvae because females perform the same task very efficiently. In a series of experiments designed to test these hypotheses, I showed that males are indeed capable of feeding larvae; they do so at small frequencies when given access to additional food and do so at impressive frequencies when deprived of females, provided with excess food and confronted with hungry larvae. Nevertheless, their ‘feed larva’ behaviour is less sophisticated and relatively inefficient compared to females. Thus I have shown male wasps can work, given an opportunity. In addition to being very satisfying, this result negates the preadaptation hypothesis which argues that male social Hymenoptera do not work because their solitary ancestors did not. In spite of what I have shown above, mating remains the main roleof the males. Therefore, I next turned my attention to a study of mating behaviour. It is well known that mating never takes place on the nest of R. marginata. Although some sporadic attempts had been made before, mating behaviour had never been observed in laboratory conditions before my study. In a series of trial-and-error pilot experiments, varying the age, cage size, number of wasps per cage, period of isolation from other wasps, lighting conditions etc., and with considerable help from enthusiastic volunteers among my lab-mates, I succeeded in observing mating behaviour in the laboratory. In the final, standardized experimental set up, we introduced a single male and a single female wasp, both isolated from their nests and other wasps for at least five days, into an aerated transparent plastic box and made observations for one hour. Using such an experimental set up, we first made a detailed qualitative description of mating behaviour. All behavioral interactions were initiated by the males. Males often attempted to mount the females but sometimes the females flew away, making the attempt to mount unsuccessful. On other occasions males successfully mounted the females, which involved climbing on the female and drumming and rubbing his antennae and abdomen on the corresponding parts of the female body. On some occasions mounting led to interlocking of the abdominal tips, a process we refer to as ‘conjugation’. Sometimes the conjugation lasted less than five seconds and during which the male remained on the back of the female; this was referred to as short conjugation (SC). But at other times the conjugation lasted for more than 20 seconds, and the male flipped on its back; this was Referred to as long conjugation (LC). I dissected all females, involved ineither LC or SC or both. Of the 47 pairs, 21 pairs mated successfully as judged by the transfer of sperm into the spermatheca. With the goal of developing a strategy to obtain live mated females for any future experiments, we attempted to determine the behavioral correlate/s of successful sperm transfer. It turns out that SC is inadequate for sperm transfer and LC is a good predictor of sperm transfer. Five to twenty days of age is optimal for mating for both males and females. There was a significant reduction in the probability of mating when one or both partners were younger or older than 5-20 days. We did not find any evidence for nestmate discrimination in the context of mating. This is not surprising because mating takes place away from the nest and active nestmate discrimination may therefore be unnecessary to avoid inbreeding. Under these experimental conditions neither body size of males and females nor ovarian conditions of the females appeared to influence mating success. I hope that my studies will enhance attention of future researchers to the males and will also facilitate experiments requiring mated wasps and permit the study of mate choice and other reproductive behaviours in this otherwise well- studied species.

Many animals live in societies of varying degrees of organization. Some individuals in these societies seem to sacrifice their own fitness to increase the fitness of some others. Understanding the forces that mould the evolution of such altruistic behaviour has become a dominant theme in modern evolutionary biology. Primitively eusocial polistine wasps provide excellent model systems to study the evolution of altruism as they show high degrees of plasticity in their behaviour. Different individuals in the same population pursue different social strategies such as nesting alpne or nesting in groups. When wasps nest in groups, usually only one individual becomes the egg layer, while die rest assume the role of sterile workers. Why do the workers not become solitary foundresses and rear their own offspring instead of working to rear the brood of another individual? Here I have used the tropical primitively eusocial wasp Ropalidia marginata to explore some factors that might potentially favour the worker strategy over the solitary founding strategy. Workers in multiple foundress nests may benefit by rearing brood more closely related to them than their own offspring would be. However, from previous work on this species it is known that relatedness between sisters is rather low and that workers therefore rear quite distantly related brood. Therefore, I have concentrated on factors other than genetic relatedness that might potentially favour the worker strategy. A total of 145 naturally initiated nests with different numbers of foundresses was monitored over a period of 16 months, and their productivities were compared. Although the total colony productivity increased, the per capita productivity did not increase with increasing foundress numbers. Colonies with larger foundress numbers did not produce significantly heavier progeny and did not produce them significantly faster than colonies with fewer individuals. The conspecific usurpers preferred to usurp single foundress colonies more often than multiple foundress colonies. Therefore, protection from conspecific usurpers might be an advantage of multiple foundress associations. About 10% of the multiple foundress nests experienced queen turnovers. This provides a finite chance to reproduce and gain some individual fitness for workers, at some future point of time. Wasps may not be similar in their reproductive abilities and those who are less fertile might be joining others who are more fertile. Testing such a hypothesis would require that individuals who have chosen to be subordinate cofoundresses in multiple foundress associations are forced to nest alone. During this study a total of 77 nests was monitored. Cofoundresses forced to nest alone had significantly lower productivity than natural solitary foundresses and also queens of multiple foundress nests who were forced to nest alone. This suggested that wasps are not similar either in their reproductive ability or brood rearing ability or both. To ascertain which of the factors was responsible for lower productivity in cofoundresses, productivity of wasps isolated into laboratory cages was compared. There was no significant difference in the productivity of isolated cofoundresses and isolated queens. This suggests that wasps are not subfertile per se but probably differ in their foraging and brood rearing abilities. The certainty with which resources are brought into the nest and therefore, the certainty with which the mean per capita productivity is attained, provides an automatic benefit of group living according to the central limit theorem. This prediction was also tested. The coefficient of variation of mean per capita productivity decreased significantly with increasing foundress numbers. Behavioural observations on another 36 colonies, with different number of adults, showed that the coefficient of variation of food brought to the nest and the rate at which larvae were fed, decreased significantly with increasing number of adults. A computer simulation was used to find out the effect of group size on the variance in feed larva. Assuming that larvae cannot be starved for too long and cannot utilize more than a certain amount of food at a time, the fitness of larvae was found to increase with an increase in the number of adults attending the nest. Previous work on R. marginata has been largely confined to postemergence colonies. An attempt was made to look at and compare social organization in preemergence colonies with that of postemergence colonies. It was found that the egg layer was not the most dominant animal in the well-established preemergence colonies. There were no detectable differences in the social organization of the preemergence colonies (of this study) with that of postemergence colonies of the earlier studies. Perhaps my conclusions drawn from studying preemergence colonies are therefore applicable more widely to the species. It can be concluded that the apparent increased fitness of the worker strategy over solitary foundress strategy does not come from any increase in per capita productivity, but comes instead from (i) the greater predictability with which the mean per capita productivity is attained in larger colonies, (ii) the lower probabilities of usurpation of larger colonies, (iii) queen turnovers that provide opportunities for workers in multiple foundress colonies to gain some direct individual fitness and (iv) the lower brood rearing abilities of workers in multiple foundress nests that make the worker strategy the best of a bad job.

Ropalidia marginata is characterized as a primitively eusocial wasp due to the absence of morphological differentiation between the queen and worker castes. Unlike other primitively eusocial wasps, however, the queen in this species is a docile individual, who does not use aggression to regulate worker reproduction, and does not act as the central pacemaker of her colony. However, if the queen dies or is experimentally removed, one of the workers steps up her aggression immensely within minutes, and if the queen is not replaced, she develops her ovaries, reduces aggression and takes over as the new queen of the colony. We call her the potential queen (PQ). When I started my work on R. marginata, two very intriguing questions were demanding to be answered, which had developed from work done by my immediate seniors in the lab. I decided to pursue both of these for my thesis. My work has been enriched by inputs from several collaborators and colleagues - I couldn’t have done all of it by myself. So, henceforth, I will be using the word “we”, instead of the first person singular to describe the work that has gone into this thesis. Question 1: Is there a designated successor to the queen in R. marginata? My senior Sujata P. Kardile has shown in her thesis, that in R. cyathiformis, a primitively eusocial wasp very closely related to R. marginata, the queen is always succeeded by the next most aggressive individual in the colony, and so the PQ is easily predictable in the presence of the queen. However, in R. marginata, the PQ appears to be an unspecialized individual, who cannot be predicted in the presence of the queen by using age, ovarian profile or behaviour as the yardsticks. However, the PQ becomes evident within minutes after queen removal. The swiftness with which the PQ is established led us to believe that perhaps the successor to the queen in R. marginata is known to the wasps, though we cannot identify her in the presence of the queen. We designed an experiment to check for the presence of such a ”cryptic successor” in R. marginata. Our experiments involved splitting a normal, queen-right nest into two halves separated by a wire mesh partition, so that the wasps could not move across the mesh. Earlier we had used this set-up to demonstrate that a PQ always establishes herself on the queen-less fragment of the nest. So, to test if there is a cryptic successor, we allowed a PQ to establish herself on the queen-less fragment, and then exchanged the queen and the PQ (designated as PQ1) between the two sides. There is a 50% probability that the cryptic successor, if present, would be on the queen-less side in the beginning. Then, upon exchange, she should be able to hold her position on the other side easily. On the other hand, if the cryptic successor is first on the queen-right side, then, upon exchange, she should take over as the PQ (PQ2), and PQ1 should not be able to hold her status. The cryptic successor hypothesis had two predictions: (i) the PQ1 would lose to a PQ2 in about half the cases, and (ii) there would never be a PQ3. We obtained a PQ2 in 5 out of 8 cases, and we never had a PQ3. So we could conclude that there is indeed one individual who is the designated successor to the queen in R. marginata. Since we could not identify her in the presence of the queen, we call her the cryptic successor. The cryptic successor did not receive even a single act of aggression from the PQ1, or from any other individual in the colony. Thus we conclude that she is acceptable to all the wasps in the colony. We next used the more sophisticated and rigorous method of network analysis to check if the PQ could be predicted due to some unique position she might be holding in the social network on her colony. Since this was a first study in a primitively eusocial insect using network tools, we began by characterizing the social networks of queen-right and queen-less colonies of R. marginata, and compared them with the R. cyathiformis networks to see how different the R. marginata society is from a typical primitively eusocial one. The R. marginata social networks based on dominant-subordinate interactions were low in their centrality measure as compared to the R. cyathiformis networks. However, in both the species, the queen-less networks were highly centralized, star-shaped networks with the PQs at the centre. Neither the queens, nor the PQs were key individuals in the queen-right colonies, but it is interesting to note that the removal of an insignificant node, the queen, resulted in a major change in the network architecture, converting the de-centralized queen-right network into a highly centralized one. Such centralized star-shaped networks are unique, and to our knowledge, the first ever described, in any social system. When we removed the queen from the data set (in silico removal), the resulting network was similar in centrality to the queen-right networks. We then did a comparative analysis of the positional importance of the PQs of the two species, and tried to see if we could use this as a tool to predict the PQ in the queen-right network. In R. cyathiformis, the PQs had consistently high ranks (mostly rank 2) in the network based on the degree index, while the PQs in R. marginata had random ranks in the hierarchy. However, since the PQs are known not to have unique ranks in the dominance hierarchies, we repeated the analysis using data on all interactions from the Q-PQ exchange experiments described above. Neither the cryptic successors nor the losers occupied any unique ranks in the all interactions networks. Thus the successors in R. marginata are truly cryptic, even in their social networks. Since R. marginata is known to be more evolved than typical primitively eusocial species, it is likely that the queen’s successor is identified by the wasps through some subtle cue like smell, and so we cannot identify her using the methods that are adequate for the identification of the PQ in a typical primitively eusocial species like R. cyathiformis. Question 2: How does the queen signal her presence and reproductive status to her workers or, how do the workers perceive the presence of their queen? The fact that in spite of her docility, the queen in R. marginata manages to maintain complete reproductive monopoly in her colony, gives rise to the obvious question of how she suppresses worker reproduction. The most attractive hypothesis is that she uses a pheromone like queens of highly eusocial species. My senior A. Sumana had shown that the queen pheromone, if present, is not a volatile substance. She also showed that the queen interacts at a very low rate with her workers, and so they cannot possibly perceive her by means of direct interactions. Since the PQ steps up her aggression within minutes of queen removal, we used her as a proxy to know how soon the queen’s absence is felt in the colony. We built a model to delineate the relationship between the decay time of the pheromone (td), the average age of the queen’s signal present with the PQ (ta), and the average realization time (tr); where tr = td − ta. Using Dijkstra’s algorithm, we showed that the queen could interact faster with the PQ by using relay interactions. Then using experimental data from 50 colonies, we obtained a ta of 102.9 minutes. The td was 340 minutes, and so we obtained a tr of 237.1 minutes; which meant that the PQ should not perceive the queen’s absence within 237 minutes of queen removal, if the queen pheromone is transmitted by a relay mechanism. However, from our experimental data, we had obtained a tr of 30 minutes. So we concluded that physical interactions, both direct and indirect were inadequate for the workers to perceive their queen. As we had ruled out physical interactions, we then wanted to check if it is possible that the queen applies her pheromone to the nest material, from where it is perceived by the workers when they walk or sit on the nest, or antennate the nest surface. The “rub abdomen behaviour (RA)” has been observed to be quite typical of R. marginata queens, and is not very common in the workers of the species. RA involves rubbing the ventral side of the tip of the abdomen or dragging it on the nest surface while walking. We thought that the queen might be using this behaviour to apply her pheromone on the nest material. So we characterized this behaviour using focal behaviour sampling, and found that the queen rubs her abdomen on the nest once in every 23 minutes. Since the observed tr is 30 minutes, it is quite likely that the queen uses the rub abdomen behaviour to apply her pheromone on the nest. The next step was to check for the source of the queen pheromone. We looked for glands that open near the base of the sting, and the Dufour’s gland was a good choice, as it is known to be involved in the recognition of egg-laying workers in the honeybees. We performed a bioassay in the blind using the crude extract of the Dufour’s gland (prepared in Ringer’s solution) from the queen. The Dufour’s gland extract of a randomly chosen worker and the solvent were used as controls. We found that the PQ responds to the queen’s Dufour’s gland extract by lowering her aggression to 65% of what she was showing on queen removal and before the application of the extract. However, the PQ did not change her behaviour significantly when the worker’s extract or Ringer’s solution was applied. The PQ’s reduction of aggression on application of the queen’s extract mimicked the reaction of PQ’s when the queen is re-introduced on the nest some time after removal. So we hypothesize that the Dufour’s gland is the source of the queen pheromone (signal) in R. marginata. This thesis has opened up newer questions pertaining to the power of the queen and the intricacies of the succession to power in R. marginata. For example, we need to pursue chemical analyses of the Dufour’s gland extract of R. marginata to have conclusive proof of it’s being the source of the queen pheromone. But that is perhaps suitable topic for my juniors in the lab, who can continue the tradition of beginning with questions opened up by their seniors!

Altruism is defined as a trait in an individual that increases some other individual’s fitness at the expense of her own. Therefore, existence of such traits in a population is an evolutionary paradox, as natural selection should eliminate such a trait. Extreme altruism in the form of eusociality where individuals relinquish their own reproduction to help raise other’s offspring has been an enigma in evolutionary biology since Darwin. Primitively eusocial organisms provide one with a unique system to study the evolution and maintenance of altruism as in these kind of species most of the individuals are capable of developing their reproductive organs, although at a certain point in time, only one or a few individuals actually reproduce. Ropalidia marginata is a primitively eusocial wasp belonging to the insect order Hymenoptera, Family Vespidae. R. marginata colonies are monogynous, although serial polygyny is observed in a colony’s lifetime. Colony initiation happens either by single founding or multiple founding. Newly founded colonies may accept individuals from other colonies, but mature colonies seldom do. Production of males is irregular, and once eclosed, they generally leave their natal nest within a week. The haplodiploidy of Hymenopteran species, i.e. the males being haploid and the females diploid, make them uniquely genetically predisposed for eusociality to evolve as was shown by William Donald Hamilton in his kin selection theory. Primitvely eusocial Hymenopteran species, being susceptible to experimental manipulation, allows one to test the predictions of this theory. In this thesis I have addressed three aspects of the biology of R. marginata using microsatellite markers, which are the following: 1) Distribution of nestmate genetic relatedness in early founding (pre‐emergence) and mature ( post‐emergence colonies) and their comparison (Chapter 3) 2) Role of relatedness and fertility in predicting the queen’s successor (Chapter 4) 3) Genetic structure of populations (Chapter 5) CHAPTER 1. INTRODUCTION: This chapter gives a brief outline of the field of molecular ecology putting its techniques to the context of insect sociobiology. CHAPTER 2. METHODS: This chapter gives a general outline of the molecular genetic methods involved. In addition, the issue of the mutation process in R. marginata microsatellites has also been addressed. There are two main models of mutation for microsatellite evolution i.e. infinite alleles model (IAM) and the step‐wise mutation model (SMM). To understand the actual process of mutation in R. marginata, sets of alleles with continuous sizes were sequenced and aligned. This was repeated for several of the loci. Seven out of the nine loci genotyped revealed clear step‐like mutation pattern and was binned accordingly. Two loci were dropped as the actual nature of step‐sizes in these two loci was unclear. Therefore, the final dataset consisted of genotype for 7 loci. This chapter also discusses the initial steps in data formatting and analysis. CHAPTER 3. GENETIC RELATEDNESS IN DIFFERENT STAGES OF COLONY DEVELOPMENT: In this chapter I have estimated nestmate genetic relatedness using seven polymorphic microsatellite loci in two different stages of colony development of the primitively eusocial wasp Ropalidiamarginata and compared them. In both kinds of nests the average colony relatedness was observed to be less than 0.75, i.e., what is expected for full sib females in Hymenoptera. Moreover, it was observed that the nestmates at the initial colony founding stage are on average less related to each other than in mature colonies. From this, one may postulate that the indirect component of inclusive fitness plays a relatively minor role than its direct component as individuals chose to leave a higher relatedness background in favour of a lower relatedness background. As newly founded colonies are relatively smaller in size than mature colonies, the probability of an individual wasp becoming the queen in this kind of colony is higher than in mature colonies. CHAPTER 4. TESTING THE ROLE OF RELATEDNESS AND FERTILITY IN PREDICTING THE QUEEN’S SUCCESSORS: R. marginata colonies are headed by docile queens. When this queen dies or is removed, one of the workers becomes extremely aggressive. She is known as the potential queen because within a few days she becomes the new queen of the colony and her aggression comes down. Predicting the successor in the presence of the queen has eluded most of the approaches attempted so far. The probability of an individual becoming the queen has been found to be uncorrelated with her body size, aggression, ovarian status or mating status. The only trend that has been observed till date, is a positive correlation with age, but the pattern is not perfect. However, the workers themselves seem to be perfectly aware of who their immediate successor going to be. In this chapter, I have tested several models of queen succession constructed in an inclusive fitness framework. These models have been tested both using relatedness alone as well as using fertility along with relatedness. Predictions of none of the models actually matched the observed sequence of successors. The wasps do not seem to be choosing their successor to maximize their inclusive fitness. CHAPTER 5. GENETIC STRUCTURE OF NATURAL POPULATIONS: I have also looked at the genetic structure of R. marginata populations in a large part of its natural distribution. I have used both F and R statistics to estimate the level of structuring and compared them. Both Fat as well Rst were found to be significantly larger than 0. Also Fis and Ris both were small and not significant suggesting lack of inbreeding. Rst was observed to be higher than Fst. Permutation test revealed a higher contribution of mutation in this structuring than migration, suggesting Rst to be a better measure of genetic structuring in this case. Similar pattern was observed with Anlysis of MOlecular VAriance. Pairwise Fst/(1‐Fst) values were found to be uncorrelated with distance, whereas barely significant trend was observed with Rst/(1‐Rst). The scatter across the trend line in both the cases suggested lack of migration drift equilibrium, with drift being more relative to migration. Higher level of structuring was observed at the level of the colony. However, colonies were rather outbred as was suggested by high and negative values of Fia and Ria values. This is not at all surprising as nestmates are related to each other. The pattern of isolation by distance at the colony level was similar to that observed in case of the populations. However, there was even higher degree of scattering of the individual points in this case. CHAPTER 6. CONCLUSIONS: Hamilton’s inclusive fitness theory has received a wide attention from and acceptance by sociobiologists, and relatedness have been measured in a wide variety of social insects. In this thesis relatedness in the context of colony founding was measured and compared with mature colonies. Also, several models constructed in an inclusive theory framework were experimentally tested. In both, support for indirect fitness was found wanting. The population genetic structure of R. marginata revealed that the sub populations are small in size and migration among them low. It also suggested significant contribution of colony level structuring on the population genetic structuring. Using more modern molecular genetic and statistical techniques, these and similar other questions can be addressed with higher precision and rigour, and such studies are expected to greatly advance our understanding of the basic premise of this thesis, i.e., how can eusociality evolve and be maintained? We hope that the current work will encourage others to ask such questions in other species.

Social insects are the most dominant terrestrial fauna for the last 50 million years. This tremendous ecological success is accompanied by the fact that sociality has evolved multiple times independently and achieved highest degree of complexity in insect lineages. The remarkable social organization found in insect societies is the result of finely balanced cooperation and conflict among the colony members. A typical hymenopteran colony is characterised by one or a few queens monopolizing reproduction and several sterile workers co-operatively raising brood and performing colony activities. The colonies are often conceptualized as superorganisms where groups of cooperative workers are compared with organs in the body, each of which accomplish a particular task like brood care, foraging and defence. The choice of tasks is often regulated by a systematic age polyethism. As the queens monopolize reproduction, they serve as the sole suppliers of eggs in the colony. Therefore, loss or death of the queen creates a crucial void which exposes the colony to potential reproductive conflict for the position of egg-layer. This crisis is expected to be extreme in monogynous colonies. The situation is rescued only after a new queen is established, and the whole process is known as queen succession. I am interested in this crisis management, and my thesis deals with potential and realized conflicts associated with queen succession and behavioural strategies involved in resolution of these conflicts. The queen can be replaced in two ways - either by a newly eclosed specialized reproductive individual, which happens in highly eusocial hymenopterans, or by an existing member of the colony (worker), as it happens in primitively eusocial hymenopterans. Unlike in highly eusocial species, the workers of primitively eusocial species retain their ancestral capability of mating and activating ovaries to produce both sons and daughters, which makes them suitable for taking up the role of queen in their lifetime. Hence, primitively eusocial species provide a unique situation where loss or death of the queen might result in severe reproductive conflict as the queen can be replaced by any one of the existing workers. Strictly monogynous colonies of the tropical primitively eusocial wasp Ropalidia marginata provide ideal opportunities to study the reproductive conflict and its resolution associated with queen succession because the queen is frequently replaced by one of her nestmates resulting in a serial polygyny. These queens have highly variable tenures of queenship ranging from seven to over 200 days, which, together with the fact that they are replaced by a variety of relatives such as daughters, niece and cousins, suggests a potential reproductive conflict with variable degrees of complexity. I have divided my thesis in three parts which are as follows -Natural queen turnover: Previous works from this lab have tried to characterize the queen succession in R. marginata colonies by experimentally removing the queen from the colony. As this design involves the experimenter intervening at a random point of the colony cycle, the colony might not respond in the similar way as it might have done to a natural succession necessitated by loss or death of the queen. But rarity and unpredictability of natural queen turnovers made them difficult to study. Therefore, in this section, we gathered a dataset of long-term and opportunistic quantitative behavioural observations on eleven natural queen turnovers and compared them with available data on queen removal experiments. All our queen removal experiments resulted in a hyper-aggressive potential queen who gradually reduced her aggression, activated her ovaries and went on to become the unanimously accepted new queen of the colony if the original queen was not returned. Here we found a similar phenomenon in natural colonies where a single un-challenged potential queen took over the colony as new queen after the old queen was lost, died or was driven out of the colony. In some of the natural colonies, the transition was preceded by aggression shown by the potential queens towards their nestmates including the queens, which indicates that they might have pre-empted the transition. The potential queens in natural colonies started laying eggs much faster than in experimental colonies suggesting their physiological readiness for the transition. How does a colony respond to a declining queen?: As we could show that some of the potential queens might perceive the upcoming queen turnover, a fair prediction would be that they sense it through the declining fertility status of the queens. Therefore, we tried to ex-perimentally induce situations where the queen appears to be declining, expecting that it might lead to a queen turnover. The growing evidence suggests that R. marginata queen maintains her status by applying a pheromone on the nest surface by rubbing the tip of her abdomen. We knocked down the nest to deny the queen the surface for applying her pheromone, and argued that the queen would be overthrown as the workers would sense her as infertile. To our surprise, the queen maintained her status in six out of seven colonies by applying her pheromone on the entire surface of the cage. However, the effectively insufficient concentration of pheromone elicited aggression from workers towards the queen, and the queen retaliated back with aggression. These results suggest that the pheromone, being an honest signal of fertility, is extremely important for the queen for maintaining her reproductive monopoly, and the workers are able to perceive the decline of the queen from her pheromone. Queen-successor conflict over access to reproduction: Here we more explicitly looked at the potential reproductive conflict between the queen and her successor over access to direct reproduction. We used the theory of parent-offspring conflict proposed by Robert Trivers (1974) as the conceptual framework and adapted it to unravel the pat-tern of queen-successor conflict in R. marginata colonies. According to this idea, we expected that there should be a pre-conflict zone where the queen and the successor both would agree that the queen should continue to reproduce, followed by a conflict zone where the successor would try to takeover but the queen would hang on, finally followed by post-conflict zone where they both would agree that the successor should reproduce. To test this expectation, we maintained the queen and the potential queen on either side of a wire-mesh partition, each with randomly chosen half of the workers. It allowed the potential queen (successor) to establish herself and then we reintroduced the queen to her side of the mesh daily till the queen gave up. We could behaviourally characterise all three zones which always appeared in the expected sequence. The pre and post-conflict zones had no aggressive interaction between the queen and the potential queen, whereas the conflict zone was characterized by aggressive falling fight between them. This is our first success in experimentally creating overt conflict between the queen and her successor. Overall we can say, that the queens and the potential queens of R. marginata show great behvioural plasticity which might have been shaped by natural selection as an adaptation for conflict resolution. We could show that the potential queens sometimes can predict the upcoming transition and pre-pare themselves accordingly, whereas they can also react to an experimentally created sudden loss of queen by hugely elevating their aggression. The docile queens, on the other hand, maintain their reproductive monopoly by a pheromone, which is essentially a feature of highly eusocial species. But these docile queens have not lost their capability to show aggression and can use that to complement the insufficient concentration of her pheromone. This and the behaviour of potential queens in their establishment phase are strongly reminiscent of typical primitively eusocial species. We conclude that Ropalidia marginata is, perhaps, a particularly advanced primitively eusocial hymenopteran situated on an evolutionary continuum from primitive to highly eusocial species.