SERIES ARTICLE How to Design Experiments in Animal Behaviour∗ 8. How Do Wasps Decide Who Would Be the Queen? Part 2 Raghavendra Gadagkar Continuing to explore the fascinating world of the Indian pa- per wasp Ropalidia marginata, in this article, we will ask how wasps choose their queens in another context. In the previ- ous article in this series, we saw how a simple experiment re- vealed that wasps fight, i.e., indulge in dominance-subordinate interactions, and the winner becomes the queen and the loser becomes the worker. This was in the context of new nestfoundation. But contextmatters. Whenthe same wasps once again have to decide who will be their next queen Raghavendra Gadagkar is if the first one dies or is experimentally removed, the same DST Year of Science Chair Professor at the Centre for rules do not hold. The wasps in a mature colony continue Ecological Sciences, Indian to show dominance-subordinate interactions and can even be Institute of Science, arranged in a dominance hierarchy, but the dominance ranks Bangalore, Honorary of the wasps do not predict who their next queen will be. Professor at JNCASR, and Non-Resident Permanent How they choose their next queen in this context continues Fellow of the to be an enduring mystery. In this article, I will describe four Wissenschaftskolleg (Institute simple experiments that have helped us come close to nail- for Advanced Study), Berlin. ing the culprit, although I must confess that we have not yet During the past 40 years he has established an active found the smoking gun—the chase is on, and we are hot on school of research in the area the trail—please join in! of animal behaviour, ecology and evolution. The origin and In describing the four experiments below, I will use the following evolution of cooperation in pattern. For each experiment, I will begin by asking a question, animals, especially in social provide some necessary background information, outline the ex- insects, such as ants, bees and perimental design, state the predictions, describe the results and wasps, is a major goal of his research. finally, answer the question raised at the beginning. http://ces.iisc.ac.in/hpg/ragh. https://www.researchgate.net/ profile/Raghavendra Gadagkar ∗DOI: https://doi.org/10.1007/s12045-019-0895-0 RESONANCE | November 2019 1287 SERIES ARTICLE Experiment 1 Question Keywords How does the queen signal her presence to her workers? Ropalidia marginata, paper wasp, potential queen, queen Background pheromone, dominance be- We have already seen in the last two articles [1, 2] that Ropa- haviour, cryptic successor. lidia marginata wasps live in colonies consisting of a single fertile queen and several non-reproducing workers. This species is clas- sified as primitively eusocial because the queens and workers can- not be distinguished morphologically (Figure 1). In other prim- itively eusocial species, the queen is usually a very aggressive and highly interactive individual, showing dominance behaviour and bullying her workers. It is believed that by such bullying, queens inhibit their workers from reproducing and ensure that In R. marginata, they work for the colony. In R. marginata, however, the queen however, the queen is a is a remarkably meek and docile individual who seldom shows remarkably meek and any dominance behaviour and rarely interacts with her workers. docile individual who seldom shows any We can only be really sure who the queen is when we see her lay dominance behaviour eggs. This raises many interesting questions. Why do the work- and rarely interacts with ers accept her as their queen? How does she manage to inhibit her workers. We can worker reproduction and maintain reproductive monopoly? How only be really sure who the queen is when we see does she ensure that the workers actually work and do not laze her lay eggs. This raises away? Indeed, how do the workers even know that they still have many interesting a queen and a healthy one at that? In other words, how does the questions. queen signal her presence to her workers, if she is not actually bullying them from time to time? The experiment that I will de- scribe in this section is meant to answer this last question. This was a rather massive and tedious experiment that was conducted by my PhD student Annagiri Sumana, with help from two other PhD students Sujata Deshpande and Anindita Bhadra (Figure 2, upper row). We hypothesized that the queens of R. marginata signal their presence to their workers by means of pheromones. Queens in highly eusocial species are known to do so. A honey- bee queen with tens of thousands of workers does not of course physically interact with them or bully them; instead, she produces volatile pheromones which the workers smell and thus detect the 1288 RESONANCE | November 2019 SERIES ARTICLE Figure 1. A large nest of Ropalidia marginata, show- ing how we uniquely mark the wasps for individual identification, using spot of paint of different colours on different parts of their body [Photo: Souvik Mandal]. presence of their queen, and indeed, her health. Then why not the same in R. marginata? Even though it is a primitively euso- cial species, R. marginata queens may produce pheromones, and the species may be intermediate between primitively and highly eusocial levels of organization. In order to find and study such pheromones, it would be helpful to know whether they are volatile or non-volatile; both kinds of pheromones are known in other an- imal species. We do know that workers in R. marginata are aware of the pres- ence of their queen because they change their behaviour within minutes of the experimental removal of the queen from their nest —one of the workers becomes hyper-aggressive and begins to show very high levels of dominance behaviour toward all the re- maining workers. This hyper-aggressive worker will immediately stop being aggressive and go back to work if the queen is returned. If the original queen is not returned, however, she will reduce her aggression gradually and become the next queen of the colony in about a week. Hence, we have labelled such a hyper-aggressive individual as the potential queen (PQ). We have exploited this very striking response of the workers to the presence or absence of their queen to understand how they might sense her. RESONANCE | November 2019 1289 SERIES ARTICLE Figure 2. Photo gallery of my five PhD students and one post-doc, who con- ducted the four experiments described in this article. Upper row, from left to right: Annagiri Sumana, Su- jata Deshpande and Anin- dita Bhadra; lower row, from left to right: Anirud- dha Mitra, Kannepalli Chan- drasekhar and Alok Bang. Experimental Design Sumana, Sujata and Anindita brought 22 naturally occurring nests of R. marginata to the Vespiary and transplanted each of them into closed, wood and wire mesh cages of dimension 30 X 30 X 30 cm, fed them with Corcyra cephalonica larvae, honey and water, and offered them a piece of soft wood to build their nests (Figure 3). The wasps do well under these conditions, display be- haviour indistinguishable from that in natural nests, and produce both male and female adult offspring. As is our usual practice, they uniquely marked all the wasps with spots of coloured paint (see Figure 1), censused the population and maintained a map of the nest and its brood every day. After the nests had settled well in their cages, the student trio began the experiment, which lasted for three days for each nest. On day 1, they conducted behavioural observations for six hours to record the various behaviours shown by the wasps. Follow- ing methods standardized in the lab, they used a set of unbi- ased sampling methods to record the behaviour of every wasp [3]. Their special interest was to compute the frequency per hour at which each wasp shows dominance behaviour to every other wasp. These observations also allowed them to identify the queen 1290 RESONANCE | November 2019 SERIES ARTICLE Figure 3. Sujata Desh- pande at work using the lab- oratory cage used in experi- ment 1. of the colony. If the queen does not lay an egg and thus give her identity away when we are making observations, we generally remove one of the eggs with a fine forceps, and this usually moti- vates her to lay an egg in that cell. On day 2, they temporarily removed all the adult wasps, placed them in individual glass vials, and cut the nest in two halves, tak- ing care to leave a roughly equal amount of brood in each half. They then inserted a wire-mesh between the two nest halves so that the wasps could not move across the mesh. Next, they rein- troduced all the workers, one by one, on one or the other side of the mesh, by tossing coins and randomly assigning them to the left or the right side. Finally, they similarly tossed a coin Thus they now had two and introduced the queen randomly to one of the two sides. Thus nest fragments, one with they now had two nest fragments, one with half the workers and half the workers and the queen (the queen-right the queen (the queen-right side) and the other with the remaining side) and the other with half of the workers but without the queen (the queen-less side). the remaining half of the Following this procedure, they repeated six hours of observation, workers but without the paying equal attention to all the wasps on both sides of the mesh. queen (the queen-less side). On day 3, they moved the queen from its original side to the op- posite side, leaving the workers undisturbed and repeated another day’s observations as before.