Conservation in Mammals of Genes Associated with Aggression-Related Behavioral Phenotypes in Honey Bees

The emerging field of sociogenomics explores the relations between social behavior and genome structure and function. An important question is the extent to which associations between social behavior and gene expression are conserved among the Metazoa. Prior experimental work in an invertebrate model of social behavior, the honey bee, revealed distinct brain gene expression patterns in African and European honey bees, and within European honey bees with different behavioral phenotypes. The present work is a computational study of these previous findings in which we analyze, by orthology determination, the extent to which genes that are socially regulated in honey bees are conserved across the Metazoa. We found that the differentially expressed gene sets associated with alarm pheromone response, the difference between old and young bees, and the colony influence on soldier bees, are enriched in widely conserved genes, indicating that these differences have genomic bases shared with many other metazoans. By contrast, the sets of differentially expressed genes associated with the differences between African and European forager and guard bees are depleted in widely conserved genes, indicating that the genomic basis for this social behavior is relatively specific to honey bees. For the alarm pheromone response gene set, we found a particularly high degree of conservation with mammals, even though the alarm pheromone itself is bee-specific. Gene Ontology identification of human orthologs to the strongly conserved honey bee genes associated with the alarm pheromone response shows overrepresentation of protein metabolism, regulation of protein complex formation, and protein folding, perhaps associated with remodeling of critical neural circuits in response to alarm pheromone. We hypothesize that such remodeling may be an adaptation of social animals to process and respond appropriately to the complex patterns of conspecific communication essential for social organization.     

Hormone response to bidirectional selection on social behavior

Behavior is a quantitative trait determined by multiple genes. Some of these genes may have effects from early development and onward by influencing hormonal systems that are active during different life-stages leading to complex associations, or suites, of traits. Honey bees (Apis mellifera) have been used extensively in experiments on the genetic and hormonal control of complex social behavior, but the relationships between their early developmental processes and adult behavioral variation are not well understood. Bidirectional selective breeding on social food-storage behavior produced two honey bee strains, each with several sublines, that differ in an associated suite of anatomical, physiological, and behavioral traits found in unselected wild type bees. Using these genotypes, we document strain-specific changes during larval, pupal, and early adult life-stages for the central insect hormones juvenile hormone (JH) and ecdysteroids. Strain differences correlate with variation in female reproductive anatomy (ovary size), which can be influenced by JH during development, and with secretion rates of ecdysteroid from the ovaries of adults. Ovary size was previously assigned to the suite of traits of honey bee food-storage behavior. Our findings support that bidirectional selection on honey bee social behavior acted on pleiotropic gene networks. These networks may bias a bee's adult phenotype by endocrine effects on early developmental processes that regulate variation in reproductive traits.     

Sequence and Expression Characteristics of Long Noncoding RNAs in Honey Bee Caste Development – Potential Novel Regulators for Transgressive Ovary Size

Division of labor in social insect colonies relies on a strong reproductive bias that favors queens. Although the ecological and evolutionary success attained through caste systems is well sketched out in terms of ultimate causes, the molecular and cellular underpinnings driving the development of caste phenotypes are still far from understood. Recent genomics approaches on honey bee developmental biology revealed a set of genes that are differentially expressed genes in larval ovaries and associated with transgressive ovary size in queens and massive cell death in workers. Amongst these, two contigs called special attention, both being over 200 bp in size and lacking apparent coding potential. Herein, we obtained their full cDNA sequences. These and their secondary structure characteristics placed in evidence that they are bona fide long noncoding RNAs (lncRNA) differentially expressed in larval ovaries, thus named lncov1 and lncov2. Genomically, both map within a previously identified QTL on chromosome 11, associated with transgressive ovary size in honey bee workers. As lncov1 was over-expressed in worker ovaries we focused on this gene. Real-time qPCR analysis on larval worker ovaries evidenced an expression peak coinciding with the onset of autophagic cell death. Cellular localization analysis through fluorescence in situ hybridization revealed perinuclear spots resembling omega speckles known to regulate trafficking of RNA-binding proteins. With only four lncRNAs known so far in honey bees, two expressed in the ovaries, these findings open a novel perspective on regulatory factors acting in the fine tuning of developmental processes underlying phenotypic plasticity related to social life histories.     

Gene expression patterns associated with caste and reproductive status in ants: worker‐specific genes are more derived than queen‐specific ones

Variation in gene expression leads to phenotypic diversity and plays a central role in caste differentiation of eusocial insect species. In social Hymenoptera, females with the same genetic background can develop into queens or workers, which are characterized by divergent morphologies, behaviours and lifespan. Moreover, many social insects exhibit behaviourally distinct worker castes, such as brood‐tenders and foragers. Researchers have just started to explore which genes are differentially expressed to achieve this remarkable phenotypic plasticity. Although the queen is normally the only reproductive individual in the nest, following her removal, young brood‐tending workers often develop ovaries and start to reproduce. Here, we make use of this ability in the ant Temnothorax longispinosus and compare gene expression patterns in the queens and three worker castes along a reproductive gradient. We found the largest expression differences between the queen and the worker castes (~2500 genes) and the smallest differences between infertile brood‐tenders and foragers (~300 genes). The expression profile of fertile workers is more worker‐like, but to a certain extent intermediate between the queen and the infertile worker castes. In contrast to the queen, a high number of differentially expressed genes in the worker castes are of unknown function, pointing to the derived status of hymenopteran workers within insects.     

Differential circular RNAs expression in ovary during oviposition in honey bees

Circular RNAs (circRNAs) are non-coding RNAs newly identified and play important roles in RNA regulation. The mechanism and function of circRNAs have been reported in some species. However, little is known regarding circRNAs in honey bees. In this study, we analyzed circRNAs through bioinformatics, and predicted 12,211 circRNAs in the ovary of honey bee queens. 1340, 175 and 100 circRNAs were differentially expressed in comparisons of egg-laying queens vs virgin queens, egg-laying inhibited queens vs egg-laying queens and egg-laying recovery queens vs egg-laying inhibited queens. Further, functional annotation of differentially expressed circRNAs revealed several pathways that are closely related to ovary activation and oviposition, including insulin secretion and calcium signaling pathways. Moreover, the potential interactions among circRNAs, miRNAs, lncRNAs and mRNAs were investigated. Ame_circ_0005197 and ame_circ_0016640 were observed to sponge several reproductive related miRNAs. These findings demonstrate that circRNAs have potential effects in ovary activation and oviposition of honey bees.     

Sensory response system of social behavior tied to female reproductive traits

Background

Honey bees display a complex set of anatomical, physiological, and behavioral traits that correlate with the colony storage of surplus pollen (pollen hoarding). We hypothesize that the association of these traits is a result of pleiotropy in a gene signaling network that was co-opted by natural selection to function in worker division of labor and foraging specialization. By acting on the gene network, selection can change a suite of traits, including stimulus/response relationships that affect individual foraging behavior and alter the colony level trait of pollen hoarding. The ‘pollen-hoarding syndrome’ of honey bees is the best documented syndrome of insect social organization. It can be exemplified as a link between reproductive anatomy (ovary size), physiology (yolk protein level), and foraging behavior in honey bee strains selected for pollen hoarding, a colony level trait. The syndrome gave rise to the forager-Reproductive Ground Plan Hypothesis (RGPH), which proposes that the regulatory control of foraging onset and foraging preference toward nectar or pollen was derived from a reproductive signaling network. This view was recently challenged. To resolve the controversy, we tested the associations between reproductive anatomy, physiology, and stimulus/response relationships of behavior in wild-type honey bees.

Methodology/Principal Findings

Central to the stimulus/response relationships of honey bee foraging behavior and pollen hoarding is the behavioral trait of sensory sensitivity to sucrose (an important sugar in nectar). To test the linkage of reproductive traits and sensory response systems of social behavior, we measured sucrose responsiveness with the proboscis extension response (PER) assay and quantified ovary size and vitellogenin (yolk precursor) gene expression in 6–7-day-old bees by counting ovarioles (ovary filaments) and by using semiquantitative real time RT-PCR. We show that bees with larger ovaries (more ovarioles) are characterized by higher levels of vitellogenin mRNA expression and are more responsive to sucrose solutions, a trait that is central to division of labor and foraging specialization.

Conclusions/Significance

Our results establish that in wild-type honey bees, ovary size and vitellogenin mRNA level covary with the sucrose sensory response system, an important component of foraging behavior. This finding validates links between reproductive physiology and behavioral-trait associations of the pollen-hoarding syndrome of honey bees, and supports the forager-RGPH. Our data address a current evolutionary debate, and represent the first direct demonstration of the links between reproductive anatomy, physiology, and behavioral response systems that are central to the control of complex social behavior in insects.     

Differential expression of antioxidant system genes in honey bee (Apis mellifera L.) caste development mitigates ROS-mediated oxidative damage in queen larvae

The expression of morphological differences between the castes of social bees is triggered by dietary regimes that differentially activate nutrient-sensing pathways and the endocrine system, resulting in differential gene expression during larval development. In the honey bee, Apis mellifera, mitochondrial activity in the larval fat body has been postulated as a link that integrates nutrient-sensing via hypoxia signaling. To understand regulatory mechanisms in this link, we measured reactive oxygen species (ROS) levels, oxidative damage to proteins, the cellular redox environment, and the expression of genes encoding antioxidant factors in the fat body of queen and worker larvae. Despite higher mean H₂O₂ levels in queens, there were no differences in ROS-mediated protein carboxylation levels between the two castes. This can be explained by their higher expression of antioxidant genes (MnSOD, CuZnSOD, catalase, and Gst1) and the lower ratio between reduced and oxidized glutathione (GSH/GSSG). In worker larvae, the GSG/GSSH ratio is elevated and antioxidant gene expression is delayed. Hence, the higher ROS production resulting from the higher respiratory metabolism in queen larvae is effectively counterbalanced by the up-regulation of antioxidant genes, avoiding oxidative damage. In contrast, the delay in antioxidant gene expression in worker larvae may explain their endogenous hypoxia response.     

Honey bee queen mandibular pheromone inhibits ovary development and fecundity in a fruit fly

A key feature of eusocial insects is their reproductive division of labour. The queen signals her fecundity to her potentially reproductive daughters via a pheromone, which renders them sterile. In contrast, solitary insects lack division in reproductive labour and there is no such social signalling or need for ovary‐regulating pheromones. Nonetheless, females from both non‐social and eusocial lineages are expected to regulate their ovaries to maximize inclusive lifetime reproductive success. It is not known, however, whether the underlying networks that regulate ovary activation are homologous between non‐social and eusocial taxa, especially when these taxa are phylogenetically distant. In this study, we provide evidence that solitary fruit flies may share a conserved ovary‐regulating pathway with a eusocial honey bee, Apis mellifera L. (Hymenoptera: Apidae). Specifically, we demonstrate that honey bee queen mandibular pheromone (QMP) inhibits fly ovaries in much the same way as it suppresses worker ovaries. Drosophila melanogaster Meigen (Diptera: Drosophilidae) exposed to sufficient doses of QMP showed a reduction in ovary size, produced fewer eggs, and generated fewer viable offspring, relative to unexposed controls. Drosophila melanogaster therefore responds to an interspecific social cue to which it would not normally be exposed. Although we cannot strictly rule out an incidental effect, this conspicuous response suggests that these two species may share an underlying mechanism for ovary regulation. Why a non‐social species of fly responds to a highly social bee's pheromone is not clear, but one possibility is that solitary and social insects share pathways associated with female reproduction, as predicted by the ‘groundplan’ hypothesis of social evolution.     

Differential gene expression between adult queens and workers in the ant Lasius niger

Ants and other social insects forming large societies are generally characterized by marked reproductive division of labour. Queens largely monopolize reproduction whereas workers have little reproductive potential. In addition, some social insect species show tremendous lifespan differences between the queen and worker caste. Remarkably, queens and workers are usually genotypically identical, meaning that any phenotypic differences between the two castes arise from caste-specific gene expression. Using a combination of differential display, microarrays and reverse Northern blots, we found 16 genes that were differentially expressed between adult queens and workers in the ant Lasius niger, a species with highly pronounced reproductive division of labour and a several-fold lifespan difference between queens and workers. RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) and gene walking were used to further characterize these genes. On the basis of the molecular function of their nearest homologues, three genes appear to be involved in reproductive division of labour. Another three genes, which were exclusively overexpressed in queens, are possibly involved in the maintenance and repair of the soma, a candidate mechanism for lifespan determination. In-depth functional analyses of these genes are now needed to reveal their exact role.     

Surgically increased ovarian mass in the honey bee confirms link between reproductive physiology and worker behavior

Honey bee (Apis mellifera L.) workers are essentially sterile females that are used to study how complex social behavior develops. Workers perform nest tasks, like nursing larvae, prior to field tasks, like foraging. Despite worker sterility, this behavioral progression correlates with ovary size: workers with larger ovaries (many ovary filaments) start foraging at younger ages on average. It is untested, however, whether the correlation confers a causal relationship between ovary size and behavioral development. Here, we successfully grafted supernumerary ovaries into worker bees to produce an artificial increase in the amount of ovary tissue. We next measured fat body mRNA levels for the yolk precursor gene vitellogenin, which influences honey bee behavioral development and can correlate with ovary size. Vitellogenin was equally expressed in surgical controls and bees with supernumerary ovaries, leading us to predict that these groups would be characterized by equal behavior. Contrary to our prediction, bees with supernumerary ovaries showed accelerated behavioral development compared to surgical controls, which behaved like reference bees that were not treated surgically. To explore this result we monitored fat body expression levels of a putative ecdysteroid-response gene, HR46, which is genetically linked to ovary size in workers. Our data establish that social insect worker behavior can be directly influenced by ovaries, and that HR46 expression changes with ovary size independent of vitellogenin.     

Support for the reproductive ground plan hypothesis of social evolution and major QTL for ovary traits of Africanized worker honey bees (Apis mellifera L.)

Background  

The reproductive ground plan hypothesis of social evolution suggests that reproductive controls of a solitary ancestor have been co-opted during social evolution, facilitating the division of labor among social insect workers. Despite substantial empirical support, the generality of this hypothesis is not universally accepted. Thus, we investigated the prediction of particular genes with pleiotropic effects on ovarian traits and social behavior in worker honey bees as a stringent test of the reproductive ground plan hypothesis. We complemented these tests with a comprehensive genome scan for additional quantitative trait loci (QTL) to gain a better understanding of the genetic architecture of the ovary size of honey bee workers, a morphological trait that is significant for understanding social insect caste evolution and general insect biology.     

Caste-specific gene expression in the stingless bee <Emphasis Type="Italic">Melipona quadrifasciata</Emphasis> – Are there common patterns in highly eusocial bees?

Summary. Caste polyphenism is a multifaceted phenomenon, most evident in the marked differences in reproductive capacity and longevity between queens and workers. The mechanisms underlying caste differentiation and division of labor are mainly addressed in the honey bee, and recently have been studied at the molecular level. Yet, generalizations drawn from studies on this model organism require validation by comparative studies. We choose Melipona quadrifasciata, a sister-group species to honey bees, to investigate differences in gene expression between newly emerged adult queens and workers. RNA extracts were subjected to a differential display protocol (DDRT-PCR). The putative differentially expressed genes, for which annotation was available, were validated by RT-PCR and hybridization. Differential expression was observed for myosin, projectin, kettin, cytochrome P450, Rab11 and Sas10. Except for kettin, all of these were overexpressed in the worker caste. Projectin and kettin could play roles in caste-specific flight muscle organization. The putative Rab11 and Sas10 homolog genes could be involved in fertility-related cell signaling and in longevity-related gene silencing, respectively. Cytochrome P450 overexpression in Melipona workers corroborates similar findings in the honey bee, thus indicating a common function in the social insect caste syndrome.Received 9 January 2003; revised 10 March 2004; accepted 2 April 2004.     

Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis

Phenotypic plasticity is a hallmark of the caste systems of social insects, expressed in their life history and morphological traits. These are best studied in bees. In their co-evolution with angiosperm plants, the females of corbiculate bees have acquired a specialized structure on their hind legs for collecting pollen. In the highly eusocial bees (Apini and Meliponini), this structure is however only present in workers and absent in queens. By means of histological sections and cell proliferation analysis we followed the developmental dynamics of the hind legs of queens and workers in the fourth and fifth larval instars. In parallel, we generated subtractive cDNA libraries for hind leg discs of queen and worker larvae by means of a Representational Difference Analysis (RDA). From the total of 135 unique sequences we selected 19 for RT-qPCR analysis, where six of these were confirmed as differing significantly in their expression between the two castes in the larval spinning stage. The development of complex structures such as the bees’ hind legs, requires diverse patterning mechanisms and signaling modules, as indicated by the set of differentially expressed genes related with cell adhesion and signaling pathways.     

DNA methylation in the termite <Emphasis Type="Italic">Coptotermes lacteus</Emphasis>

Social insects are key examples of organisms that display polyphenism. Their genomes encode instructions for the development of multiple phenotypes, known as castes, which typically have highly divergent morphology, physiology and behaviour. DNA methylation, an epigenetic mechanism associated with modulation of gene expression in various eukaryotes, has recently been shown to provide a key link between environmental cues and caste-specific gene expression in honey bees (Hymenoptera). In termites—a major social insect group phylogenetically distant from Hymenoptera—the existence of DNA methylation has not, to our knowledge, been reported to date. Since genes encoding key DNA methylation enzymes are known to be absent in the genomes of a number of insect species, we sought to test whether termites are able to methylate their DNA, and, if so, whether caste-specific patterns of DNA methylation exist. We performed methylation-specific amplified fragment length polymorphism on the termite Coptotermes lacteus, and found evidence for DNA methylation. However, a comparison of methylation levels in different castes did not reveal any significant differences in methylation levels. The demonstration of DNA methylation in termites sets the stage for future epigenetic studies in these important social insects.     

Gene expression analysis following olfactory learning in Apis mellifera

The honeybee has a strong learning and memory ability, and is recognized as the best model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the gene expression difference following proboscis extension response-based olfactory learning in the A. mellifera using a tag-based digital gene expression (DGE) method. We obtained about 5.71 and 5.65 million clean tags from the trained group and untrained group, respectively. A total of 259 differentially expressed genes were detected between these two samples, with 30 genes up-regulated and 229 genes down-regulated in trained group compared to the untrained group. These results suggest that bees tend to actively suppress some genes instead of activating previously silent genes after olfactory learning. Our DGE data provide comprehensive gene expression information for olfactory learning, which will facilitate our understanding of the molecular mechanism of honey bee learning and memory.