Reproductive division of labor is the most distinctive characteristic of the social Hymenoptera; some individuals reproduce and others forego their own reproduction to raise non-descendant offspring. In species where females are reproductively totipotent and lack morphologically distinct castes, there is potential for reproductive conflict because more than one female in a colony may attempt direct reproduction. I focused my dissertation research on a subtropical population of the primitively eusocial paper wasp, Mischocyttarus mexicanus, to investigate the initiation, establishment, and development of the colony before the emergence of adult offspring. Female M. mexicanus exhibit variation in behavior and task performance, and switch between reproductive and non-reproductive roles. These changes in behavior and reproduction may be influenced by social context. In three studies, I investigated the role of social context on reproduction, behavior, and brain structure. In the first study, I tested the role of body size, reproductive potential, and immediate egg-laying potential on the reproductive tactic employed by females. I found that large females either became solitary foundresses or became part of a group-initiated colony. In contrast, small females left their natal colony and pursued joining other colonies. This joiner tactic is unique to this population and has not been observed in temperate zone populations. I also found that subordinate females had the potential to lay eggs if given the opportunity. This suggests an incentive to remain in a colony for future opportunities of direct reproduction. In the second study, I investigated the effect of three variables on non-nestmate acceptance: non-nestmate age, stage of colony development, and non-nestmate aggressive behavior. I demonstrated that non-nestmate acceptance was context-dependent. Both non-nestmate age and stage of colony development had an effect on the proportion of accepted non-nestmates. Although, non-nestmate aggressive behavior did not affect non-nestmate acceptance, it did trigger an aggressive response from colony nestmates. In the third study, I assessed the relationship of Mushroom Bodies (MB) volume, the brain neuropils associated with learning and memory, to environmental conditions and social interactions. I compared MB volume of newly-established colonies initiated by solitary foundresses to groups of foundresses. In addition, I performed laboratory experiments to differentiate between the effect of environmental conditions and social interactions. I found a positive relationship between MB volume and environmental conditions including light intensity and foraging experience. In contrast to previous studies, I found no association between MB volume and social interactions. Ovary development was positively correlated with MB development. This result suggests that although reproductive dominance is established in newly-initiated colonies, social dominance may not yet be established. In summary, my studies found an effect of social context on behavior, adoption of reproductive tactics and brain structure in colonies of M. mexicanus during the offspring pre-emergence phase.

Altruism in its extreme form is seen in social insects where most individuals give up their own reproduction and work to rear the offspring of their queen. The origin and evolution of such sterile worker castes remains a major unsolved problem in evolutionary biology. Primitively eusocial polistine wasps are an attractive model system for investigating this phenomenon. Ropalidia marginata (Lep.) (Hymenoptera: Vespidae) is one such tropical primitively eusocial wasp, in which new nests are initiated either by a single foundress or by a group of female wasps. Worker behaviour in Ropalidia marginata cannot be satisfactorily explained by the haplodiploidy hypothesis due to the existence of polyandry and serial polygyny which reduce intra-colony genetic relatedness to levels lower than the value expected between a solitary foundress and her offspring. Besides, wasps appear to move frequently between newly initiated nests, perhaps further reducing intracolony genetic relatedness. To study social organization and examine the possibility of kin recognition and task specialization under conditions of low intra-colony relatedness, genetically mixed colonies were created by introducing alien one-day old wasps onto recipient nests. As a first step I have tried to determine the factors that influence the acceptance of foreign wasps onto established colonies. I have introduced wasps between 1 to 20 days of age from donor colonies located at least 10 km away onto 12 different recipient colonies, observed these wasps for a period of 10 hours and later dissected them to examine their ovarian condition. Observations were carried out in the blind i.e. the observer was unaware of the identity of the wasps. Wasps upto 6 days of age were accepted by the alien nests. Older wasps may have been rejected because their relatively better ovarian condition may have been perceived as a reproductive threat to the recipient nest. Alternatively, younger wasps may have been accepted because they may be more easily moulded to the desired roles or due to some other correlate of age per se independent of ovarian condition. Although ovarian condition appeared to influence the probability of acceptance, it was not statistically significant in the presence of age in multiple regression models, making a favourable case for the 'ease of moulding hypothesis' or 'age per se hypothesis' over the 'reproductive threat hypothesis'. In any case these findings gave me a method to create genetically mixed colonies. On 12 different nests Ropalidia marginata, I similarly introduced one-day old wasps and thus created genetically mixed colonies. Such an introduction simulates the eclosion of distantly related individuals which is quite common on nests of R. marginata due to the presence of serial polygyny. About 7 such wasps were introduced per colony and the introductions were so arranged as to matched with natural eclosions on the recipient nest. After 7 days following the last introduction, colonies were observed for 20 hours each. Alien wasps became well integrated and performed most of the behaviours and tasks shown by the natal wasps. There was no evidence of kin recognition or task specialization between natal and introduced wasps. The introduced wasps also sometimes became replacement queens. In an attempt to test the costs in terms of brood rearing efficiency, of living in such genetically variable groups, I created kin and non-kin pairs of wasps in plastic containers. They were provided with ad libitum food, water and building material. The nests initiated were monitored till an adult offspring eclosed. There were no detectable differences in either the productivities or the developmental periods of immature stages in the kin and nonkin pairs suggesting that there is no apparent cost of living with unrelated or distantly related individuals. To compare the extent of cooperation between the two wasps in kin and non-kin pairs, I conducted behavioural observations on 12 pairs each of kin and nonkin wasps. I found no difference in the rates at which the non-egg layers brought food and pulp, fed larvae and built the nest in the kin and nonkin pairs suggesting that cooperative nest building and brood rearing was common to the kin as well as non-kin pairs. The results reported here strengthen the idea that factors other than genetic relatedness must play a prominent role in the maintenance of worker behaviour in Ropalidia marginata.

This thesis has looked at the proximate mechanisms by which eusociality is maintained in colonies of the primitively eusocial wasp Ropalidia marginata. Unlike other typical primitively eusocial species, the R, marginata queen is remarkably docile and non-interactive and hence cannot possibly use aggression to maintain her status. Recent evidence hints at pheromonal queen signalling through the Dufour’s gland. Hence, queen-worker difference in Dufour’s gland composition has been studied in details. Queens and workers differ with respect to overall composition of Dufour’s glands, categories of compounds, and individual compounds as well. The Dufour’s gland compounds may be having a bouquet effect in queen signalling, with individual compounds being less important than the overall composition. The queen pheromone also appears to be an honest signal of fertility, as compounds that differ consistently between queens and workers are correlated with ovarian development of queens, and solitary foundresses and potential queens, who are intermediate between queens and workers in ovarian development, are intermediate in their Dufour’s gland profile as well. When the queen is removed from a colony, one of the workers (potential queen, PQ) shows high aggression, and if the queen is not returned, goes on to become the next queen of the colony. The aggression of PQ comes down as a function of time since queen removal, and correlated with this, the ovaries of PQ increase. Dufour’s gland profile of PQ is similar to workers immediately after queen removal, but comes closer to queens with passage of time. This hints at an interesting transition in maintenance of eusociality from “queen control” by aggression to “queen signal” by pheromone during the queen establishment phase. It has generally been assumed that one set of chemicals can carry multiple information, namely queen signal and colony signal. Initial statistical analysis of chemical composition data showed that perhaps both caste and colony signals can be conveyed by the Dufour’s gland compounds, but detailed analysis cast some doubt on this, as the Dufour’s gland compounds could not be separated into non-overlapping subsets with respect to importance in caste and colony discrimination. A bioassay showed that the wasps do not make colony discrimination from Dufour’s gland compounds. This suggests that the ability to statistically differentiate groups of organisms from their chemical profiles does not guarantee similar discrimination by the organisms themselves, emphasising the need for bioassays to resolve such issues.

abstract: Social animals benefit from the aggregation of knowledge and cognitive processing power. Part of this benefit comes from individual heterogeneity, which provides the basis to group-level strategies, such as division of labor and collective intelligence. In turn, the outcomes of collective choices, as well as the needs of the society at large, influence the behavior of individuals within it. My dissertation research addresses how the feedback between individual and group-level behavior affects individuals and promotes collective change. I study this question in the context of seed selection in the seed harvester ant, Pogonomyrmex californicus. I use both field and laboratory studies to explore questions relating to individual behavior: how forager decision-making is affected through information available in the nest and at the seed pile; how workers interact with seeds in the nest; and how forager preferences diverge from each other’s and the colony’s preference. I also explore the integration between individual and colony behavior, specifically: how interactions between the foraging and processing tasks affect colony collection behavior; how individual behavior changes affect colony preference changes and whether colony preference changes can be considered learning behavior. To answer these questions, I provided colonies with binary choices between seeds of unequal or similar quality, and measured individual, task group, and colony-level behavior. I found that colonies are capable of learning to discriminate between seeds, and learned information lasts at least one month without seed interaction outside of the nest. I also found that colony learning was coordinated by foragers receiving updated information from seeds in the nest to better discriminate and make choices between seed quality during searches for seeds outside of the nest. My results show that seed processing is essential for stimulating collection of novel seeds, and that foraging and processing are conducted by behaviorally and spatially overlapping but distinct groups of workers. Finally, I found that foragers’ preferences are diverse yet flexible, even when colonies are consistent in their preference at the population level. These combined experiments generate a more detailed and complete understanding of the mechanisms behind the flexibility of collective colony choices, how colonies incorporate new information, and how workers individually and collectively make foraging decisions for the colony in a decentralized manner. / Dissertation/Thesis / Doctoral Dissertation Biology 2020

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!

At certain stages of a bumblebee colony life cycle workers lay eggs. Not all workers reproduce, however, since many continue to forage and care for the nest. This leads to questions regarding what differentiates a reproductive worker from a non-reproductive one. It is hypothesized that a form of reproductive competition takes place, where the most behaviourally dominant worker becomes reproductively dominant. The behaviour of orphaned Bombus impatiens pairs was recorded and aggression, social interactions, egg-laying, and ovarian development were identified. Experiment 1 examined the association between aggression and egg-laying. Contrary to the hypothesis, the most aggressive worker did not lay more eggs. When the ovarian development of workers was manipulated and two workers with developed ovaries were paired (Experiment 3), they were more aggressive than pairs with discouraged ovarian development. This provides support for the supposition that aggression and reproduction are related, however, it is only partial support as worker pairs with encouraged ovarian development did not lay more eggs. Since aggression is believed to be only one part of behavioural dominance, Experiment 2 studied the association between social interactions and aggression and reproduction. Results showed that when two socially active bees were paired they were more aggressive than pairs including one or two socially inactive bumblebees. No significant difference in ovarian development between socially active pairs and socially inactive pairs was found. Brood presence was also predicted to affect reproductive control. Experiment 1 found egg-laying and aggression were more likely to co-occur in the absence of brood. Results from Experiment 2 supplemented the first experiment since the absence of brood increased rates of aggression and ovarian development in pairs. Whereas the results confirm aggression has a role in worker reproduction the findings also reveal that behavioural dominance does not equate to reproductive dominance under all conditions. The primary contributions of this thesis were the development of a method to distinguish behavioural dominance from reproductive dominance and determining their relationship under different environments (brood presence) and experimental manipulations (ovarian development). These contributions further define dominance in Bombus impatiens.

Synalpheus shrimp species of the gambarelloides group are the only marine organisms displaying the highest level of social functioning, eusociality. Their social hierarchies are equally complex compared to the reproductive abnormalities that have been recently discovered. For instance, snapping shrimp of the genus Synalpheus were thought to be gonochoric, i.e. developing as independent sexes, until scanning electron microscopy studies revealed intersexed gonopores in several species. This project analyzed both the species composition, and accompanying reproductive structures, of Synalpheus spp. (Caridea: Alpheidae) comprised of densely aggregating communal and pair-living colonies in the Florida Keys, Florida. Colonies of pair-living and communal Synalpheus spp. were observed from hosts Spheciospongia vesparium and Spongia sp. from hard bottom assemblages of the Florida Reef Tract in order to assess differing population structures. Comparisons were made of the measures of overall and relative abundance, frequency by species and sex category, and variation in growth by species and sex category, for each individual colony. We then used scanning electron microscopy (SEM) images to determine the secondary sexual characteristics of three species (Synalpheus brooksi, S. herricki, and S. cf. herricki) which range in social behavior. Species were widely consistent in both host choice and distribution, across all sampling areas. The abundance of communal species Synalpheus brooksi was much greater than expected at all sites, in comparison to previously published work. While Synalpheus longicarpus was reported at higher frequencies in prior studies, our results yielded a much lower frequency of this species, often found in pairs, rather than dense aggregations. Average sizes of ovigerous and non-ovigerous individuals in dense colonies of Synalpheus brooksi and S. pectiniger did not differ significantly. However, total body length of individuals differed within species groups, specifically related to sex and presence of ova. Within colonies of S. brooksi, mid-development, or ‘transitional’ individuals, were discovered in nearly all populations. However, the reproductive and social function of these individuals displaying mixed sexual characteristics could not be determined from this study. Individuals of S. brooksi displaying ‘transitional’ external morphology, i.e. masculine abdominal pleura paired with clutches of eggs, displayed higher incidences of intersex gonopores per colony than did conspecific non-ovigerous and ovigerous individuals. These results suggest that colonies of S. brooksi may be comprised of a subset of helpers, or individuals undergoing a transitional sexual development phase, similar to prior published findings of intersexed helpers among eusocial colonies (Toth and Bauer 2007). In comparison to S. brooksi colonies, nearly all colonies of S. herricki and S. cf. herricki were composed of intersex individuals. In conjunction with previous instances documented in eusocial Synalpheus paraneptunus groups, the data provide substantial evidence of intersexing at all levels of social organization in Synalpheus spp. (pair living, communal, and eusocial). These findings nonetheless provide a clearer picture of how social structure and life history influence adaptation of a particular reproductive strategy. Quantifying features of Spheciospongia vesparium populations and comparing results to neighboring hosts, such as Spongia sp., provided evidence for potential influences of host choice, and variation in growth and reproductive capacity temporally and spatially. These observations of species’ growth patterns and abundances contribute greatly to our understanding of life history of Synalpheus spp., and, furthermore, adaptation of social organization.

At certain stages of a bumblebee colony life cycle workers lay eggs. Not all workers reproduce, however, since many continue to forage and care for the nest. This leads to questions regarding what differentiates a reproductive worker from a non-reproductive one. It is hypothesized that a form of reproductive competition takes place, where the most behaviourally dominant worker becomes reproductively dominant. The behaviour of orphaned Bombus impatiens pairs was recorded and aggression, social interactions, egg-laying, and ovarian development were identified. Experiment 1 examined the association between aggression and egg-laying. Contrary to the hypothesis, the most aggressive worker did not lay more eggs. When the ovarian development of workers was manipulated and two workers with developed ovaries were paired (Experiment 3), they were more aggressive than pairs with discouraged ovarian development. This provides support for the supposition that aggression and reproduction are related, however, it is only partial support as worker pairs with encouraged ovarian development did not lay more eggs. Since aggression is believed to be only one part of behavioural dominance, Experiment 2 studied the association between social interactions and aggression and reproduction. Results showed that when two socially active bees were paired they were more aggressive than pairs including one or two socially inactive bumblebees. No significant difference in ovarian development between socially active pairs and socially inactive pairs was found. Brood presence was also predicted to affect reproductive control. Experiment 1 found egg-laying and aggression were more likely to co-occur in the absence of brood. Results from Experiment 2 supplemented the first experiment since the absence of brood increased rates of aggression and ovarian development in pairs. Whereas the results confirm aggression has a role in worker reproduction the findings also reveal that behavioural dominance does not equate to reproductive dominance under all conditions. The primary contributions of this thesis were the development of a method to distinguish behavioural dominance from reproductive dominance and determining their relationship under different environments (brood presence) and experimental manipulations (ovarian development). These contributions further define dominance in Bombus impatiens.

Diferenças no timing da melanização e esclerotização do exoesqueleto são evidentes quando se compara a morfologia externa de abelhas de hábitos sociais e as solitárias. A esta diferença convencionamos chamar de heterocronia da maturação cuticular, o termo heterocronia significando variações no tempo relativo, ou ritmo, de um evento ontogenético em relação ao ancestral ou entre taxons. Propusemos que as abelhas sociais, que após a ecdise permanecem na colônia por vários dias, alcançariam a maturidade de alguns sistemas orgânicos, entre eles o tegumento, muito mais tarde que as espécies de abelhas solitárias que ao emergir partem imediatamente para atividades extra-nidais. Neste contexto, o objetivo deste trabalho consistiu em testar esta hipótese utilizando o tegumento em maturação das espécies de abelhas sociais, Apis mellifera e Frieseomelitta varia, e da espécie solitária Centris analis, em estudos comparativos de expressão gênica, ultraestrutura e quantificação de hidrocarbonetos cuticulares (CHCs). Para isto utilizamos sequenciamento de mRNA (RNA-seq), microscopia eletrônica de transmissão (MET) e cromatografia de gás e espectrometria de massas (CG/MS). Os perfis de expressão de genes da via de melanização/esclerotização cuticular (ebony e tan) diferenciaram as espécies sociais da solitária, assim como a expressão de genes com função na via de metabolismo de quitina (Cda5, Idgf4 e chitooligosacchariodolytic-domain-like) e de genes codificadores de proteínas estruturais da cutícula (CPR14, CPR17, CPR18, CPR25, CPR23, CPR26, Apd-3 e Apd-like). Genes com função na regulação da maturação cuticular (FTZ-F1, E74, Hr46 e Hr4) se mostraram co-expressos nas espécies sociais e os perfis de expressão destes genes, exceto Hr46, e de outros reguladores (Ethr, Hr38, Rickets e Ptx-1) também diferenciaram as espécies sociais da solitária. Ressaltamos em nossas análises os genes do ciclo circadiano, cuja expressão tem relação com a deposição de quitina cuticular, além de genes de vias de pigmentação não melanínicas. As análises de MET, abrangendo outras três espécies de abelhas (Bombus brasilienses: primitivamente eussocial; Euglossa cordata: facultativamente social; Tetrapedia diversipes: solitária), mostraram diferenças consistentes entre a ultraestrutura e espessura das cutículas das espécies sociais e solitárias, o que reforçou nossos resultados de RNA-seq. A quantificação absoluta dos CHCs diferenciou as abelhas sociais da solitária, consistente com a hipótese de heterocronia da maturação cuticular e com os perfis de expressão de genes envolvidos na biossíntese de CHCs. Assim, além de desvendar transcriptomas de tegumento de três espécies de abelhas, a comparação da expressão gênica aliada à análise de ultraestrutura da cutícula e quantificação de CHCs levaram à caracterização de diferenças no processo de maturação cuticular entre as espécies sociais e solitárias / Differences in the timing of exoskeleton melanization and sclerotization processes are evident when comparing the external morphology of social and solitary bee species. Such differences may constitute a relevant example of cuticular maturation heterochrony, this term referring to a genetic change in timing of an ontogenetic process relative to an ancestor or between taxons. We proposed that social bees, which remain protected inside the colony for many days before initiating outside nest activities, would reach the maturity of some organic systems, such as the integument (epidermis and cuticle), later than solitary bees, which start such activities immediately after ecdysis. We tested this hypothesis in a comparative study of the developing integument of eusocial bees, Apis mellifera and Frieseomelitta varia, and the solitary bee Centris analis. Using RNA-seq, we verified that the expression profiles of genes involved in cuticular melanization and sclerotization (ebony and tan), chitin deposition and organization (Cda5, Idgf4, chitooligosacchariodolytic-domain-like), and cuticle formation (CPR14, CPR17, CPR18, CPR25, CPR23, CPE26, Apd-3, Apd-like) were positively, correlated between the two eusocial species, but not between the eusocial and the solitary species. Some of the genes with roles in regulating exoskeleton maturation (FTZ-F1, E74, Hr46, Hr4) were co-expressed only in the eusocial species. The expression profiles of these genes (except Hr46) and other regulatory genes (Ethr, Hr38, Rickets, Ptx-1) were also positively correlated exclusively in the eusocial bees. We also highlighted the expression of genes involved in non-melanin pigment production and the expression of circadian rhythm genes that could be related to chitin layers deposition. Transmission electron microscopy analysis of the integument of the two eusocial and the solitary bee species, in addition to other three bee species (the primitively eusocial Bombus brasilienses; the facultatively social Euglossa cordata; the solitary bee Tetrapedia diversipes), showed differences in cuticle ultrastructure and thickness, thus supporting the RNA-seq data. In agreement with our hypothesis, CHC quantifications were consistent with the expression levels of genes involved in CHC biosynthesis, thus differentiating the superficial cuticle layer of the eusocial and solitary species. Together, the integument transcriptomes, ultrastructure, and CHC quantification allowed us to characterize differences in the timing of cuticle maturation in social and solitary bees

Diferenças no timing da melanização e esclerotização do exoesqueleto são evidentes quando se compara a morfologia externa de abelhas de hábitos sociais e as solitárias. A esta diferença convencionamos chamar de heterocronia da maturação cuticular, o termo heterocronia significando variações no tempo relativo, ou ritmo, de um evento ontogenético em relação ao ancestral ou entre taxons. Propusemos que as abelhas sociais, que após a ecdise permanecem na colônia por vários dias, alcançariam a maturidade de alguns sistemas orgânicos, entre eles o tegumento, muito mais tarde que as espécies de abelhas solitárias que ao emergir partem imediatamente para atividades extra-nidais. Neste contexto, o objetivo deste trabalho consistiu em testar esta hipótese utilizando o tegumento em maturação das espécies de abelhas sociais, Apis mellifera e Frieseomelitta varia, e da espécie solitária Centris analis, em estudos comparativos de expressão gênica, ultraestrutura e quantificação de hidrocarbonetos cuticulares (CHCs). Para isto utilizamos sequenciamento de mRNA (RNA-seq), microscopia eletrônica de transmissão (MET) e cromatografia de gás e espectrometria de massas (CG/MS). Os perfis de expressão de genes da via de melanização/esclerotização cuticular (ebony e tan) diferenciaram as espécies sociais da solitária, assim como a expressão de genes com função na via de metabolismo de quitina (Cda5, Idgf4 e chitooligosacchariodolytic-domain-like) e de genes codificadores de proteínas estruturais da cutícula (CPR14, CPR17, CPR18, CPR25, CPR23, CPR26, Apd-3 e Apd-like). Genes com função na regulação da maturação cuticular (FTZ-F1, E74, Hr46 e Hr4) se mostraram co-expressos nas espécies sociais e os perfis de expressão destes genes, exceto Hr46, e de outros reguladores (Ethr, Hr38, Rickets e Ptx-1) também diferenciaram as espécies sociais da solitária. Ressaltamos em nossas análises os genes do ciclo circadiano, cuja expressão tem relação com a deposição de quitina cuticular, além de genes de vias de pigmentação não melanínicas. As análises de MET, abrangendo outras três espécies de abelhas (Bombus brasilienses: primitivamente eussocial; Euglossa cordata: facultativamente social; Tetrapedia diversipes: solitária), mostraram diferenças consistentes entre a ultraestrutura e espessura das cutículas das espécies sociais e solitárias, o que reforçou nossos resultados de RNA-seq. A quantificação absoluta dos CHCs diferenciou as abelhas sociais da solitária, consistente com a hipótese de heterocronia da maturação cuticular e com os perfis de expressão de genes envolvidos na biossíntese de CHCs. Assim, além de desvendar transcriptomas de tegumento de três espécies de abelhas, a comparação da expressão gênica aliada à análise de ultraestrutura da cutícula e quantificação de CHCs levaram à caracterização de diferenças no processo de maturação cuticular entre as espécies sociais e solitárias / Differences in the timing of exoskeleton melanization and sclerotization processes are evident when comparing the external morphology of social and solitary bee species. Such differences may constitute a relevant example of cuticular maturation heterochrony, this term referring to a genetic change in timing of an ontogenetic process relative to an ancestor or between taxons. We proposed that social bees, which remain protected inside the colony for many days before initiating outside nest activities, would reach the maturity of some organic systems, such as the integument (epidermis and cuticle), later than solitary bees, which start such activities immediately after ecdysis. We tested this hypothesis in a comparative study of the developing integument of eusocial bees, Apis mellifera and Frieseomelitta varia, and the solitary bee Centris analis. Using RNA-seq, we verified that the expression profiles of genes involved in cuticular melanization and sclerotization (ebony and tan), chitin deposition and organization (Cda5, Idgf4, chitooligosacchariodolytic-domain-like), and cuticle formation (CPR14, CPR17, CPR18, CPR25, CPR23, CPE26, Apd-3, Apd-like) were positively, correlated between the two eusocial species, but not between the eusocial and the solitary species. Some of the genes with roles in regulating exoskeleton maturation (FTZ-F1, E74, Hr46, Hr4) were co-expressed only in the eusocial species. The expression profiles of these genes (except Hr46) and other regulatory genes (Ethr, Hr38, Rickets, Ptx-1) were also positively correlated exclusively in the eusocial bees. We also highlighted the expression of genes involved in non-melanin pigment production and the expression of circadian rhythm genes that could be related to chitin layers deposition. Transmission electron microscopy analysis of the integument of the two eusocial and the solitary bee species, in addition to other three bee species (the primitively eusocial Bombus brasilienses; the facultatively social Euglossa cordata; the solitary bee Tetrapedia diversipes), showed differences in cuticle ultrastructure and thickness, thus supporting the RNA-seq data. In agreement with our hypothesis, CHC quantifications were consistent with the expression levels of genes involved in CHC biosynthesis, thus differentiating the superficial cuticle layer of the eusocial and solitary species. Together, the integument transcriptomes, ultrastructure, and CHC quantification allowed us to characterize differences in the timing of cuticle maturation in social and solitary bees