Genetic and environmental effects on morphological traits of social phenotypes in wasps.

Many species exhibit distinct phenotypic classes, such as sexes in dioecious species or castes in social species. The evolution of these classes is affected by the genetic architecture governing traits shared between phenotypes. However, estimates of the genetic and environmental factors contributing to phenotypic variation in distinct classes have rarely been examined. We studied the genetic architecture underlying morphological traits in phenotypic classes in the social wasp Vespula maculifrons. Our data revealed patriline effects on a few traits, indicating weak genetic influences on caste phenotypic variation. Interestingly, traits exhibited higher heritability in queens than workers. This result suggests that genetic variation has a stronger influence on trait variation in the queen caste than the worker caste, which is unexpected because queens typically experience direct selection. Moreover, estimates of heritability for traits were correlated between the castes, indicating that variability in trait size was governed by similar genetic architecture in the two castes. However, we failed to find evidence for a significant relationship between caste dimorphism and caste correlation, as would be expected if trait evolution was constrained by intralocus genetic conflict. Our analyses also uncovered variation in the allometric relationships for traits. These analyses suggested that worker traits were proportionally smaller than queen traits for most traits examined. Overall, our data provide evidence for a strong environmental and moderate genetic basis of trait variation among castes. Moreover, our results suggest that selection previously operated on caste phenotype in this species, and phenotypic variation is now governed primarily by environmental differences.

Gene Duplication in the Honeybee: Patterns of DNA Methylation, Gene Expression, and Genomic Environment.

Gene duplication serves a critical role in evolutionary adaptation by providing genetic raw material to the genome. The evolution of duplicated genes may be influenced by epigenetic processes such as DNA methylation, which affects gene function in some taxa. However, the manner in which DNA methylation affects duplicated genes is not well understood. We studied duplicated genes in the honeybee Apis mellifera, an insect with a highly sophisticated social structure, to investigate whether DNA methylation was associated with gene duplication and genic evolution. We found that levels of gene body methylation were significantly lower in duplicate genes than in single-copy genes, implicating a possible role of DNA methylation in postduplication gene maintenance. Additionally, we discovered associations of gene body methylation with the location, length, and time since divergence of paralogous genes. We also found that divergence in DNA methylation was associated with divergence in gene expression in paralogs, although the relationship was not completely consistent with a direct link between DNA methylation and gene expression. Overall, our results provide further insight into genic methylation and how its association with duplicate genes might facilitate evolutionary processes and adaptation.

Temporal Analysis of Effective Population Size and Mating System in a Social Wasp.

Highly social species are successful because they cooperate in obligately integrated societies. We examined temporal genetic variation in the eusocial wasp Vespula maculifrons to gain a greater understanding of evolution in highly social taxa. First, we wished to test if effective population sizes of eusocial species were relatively low due to the reproductive division of labor that characterizes eusocial taxa. We thus estimated the effective population size of V. maculifrons by examining temporal changes in population allele frequencies. We sampled the genetic composition of a V. maculifrons population at 3 separate timepoints spanning a 13-year period. We found that effective population size ranged in the hundreds of individuals, which is similar to estimates in other, non-eusocial taxa. Second, we estimated levels of polyandry in V. maculifrons in different years to determine if queen mating system varied over time. We found no significant change in the number or skew of males mated to queens. In addition, mating skew was not significant within V. maculifrons colonies. Therefore, our data suggest that queen mate number may be subject to stabilizing selection in this taxon. Overall, our study provides novel insight into the selective processes operating in eusocial species by analyzing temporal genetic changes within populations.

Epigenetics in Insects: Genome Regulation and the Generation of Phenotypic Diversity.

Epigenetic inheritance is fundamentally important to cellular differentiation and developmental plasticity. In this review, we provide an introduction to the field of molecular epigenetics in insects. Epigenetic information is passed across cell divisions through the methylation of DNA, the modification of histone proteins, and the activity of noncoding RNAs. Much of our knowledge of insect epigenetics has been gleaned from a few model species. However, more studies of epigenetic information in traditionally nonmodel taxa will help advance our understanding of the developmental and evolutionary significance of epigenetic inheritance in insects. To this end, we also provide a brief overview of techniques for profiling and perturbing individual facets of the epigenome. Doing so in diverse cellular, developmental, and taxonomic contexts will collectively help shed new light on how genome regulation results in the generation of diversity in insect form and function.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

Genetic diversity and sex ratio of naked mole rat, Heterocephalus glaber, zoo populations.

The naked mole rat, Heterocephalus glaber, is a highly unusual mammal that displays a complex social system similar to that found in eusocial insects. Colonies of H. glaber are commonly maintained in zoo collections because they represent fascinating educational exhibits for the public. However, little is known about the genetic structure or sex ratio of captive populations of H. glaber. In this study, we developed a set of microsatellite markers to examine genetic variation in three captive zoo populations of H. glaber. We also studied sex ratio of these captive populations. Our goal was to determine levels of genetic variation within, and genetic differences between, captive populations of H. glaber. Overall, we found modest levels of genetic variation in zoo populations. We also uncovered little evidence for inbreeding within the captive populations. However, zoo populations did differ genetically, which may reflect the isolation of captive naked mole rat colonies. Finally, we found no evidence of biased sex ratios within colonies. Overall, our study documents levels of genetic variation and sex ratios in a captive eusocial mammalian population. Our results may provide insight into how to manage captive populations of H. glaber.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Climbing, falling, and jamming during ant locomotion in confined environments.

Locomotion emerges from effective interactions of an individual with its environment. Principles of biological terrestrial locomotion have been discovered on unconfined vertical and horizontal substrates. However, a diversity of organisms construct, inhabit, and move within confined spaces. Such animals are faced with locomotor challenges including limited limb range of motion, crowding, and visual sensory deprivation. Little is known about how these organisms accomplish their locomotor tasks, and such environments challenge human-made devices. To gain insight into how animals move within confined spaces, we study the locomotion of the fire ant Solenopsis invicta, which constructs subterranean tunnel networks (nests). Laboratory experiments reveal that ants construct tunnels with diameter, D, comparable to body length, L = 3.5 ± 0.5 mm. Ants can move rapidly (> 9 bodylengths per s) within these environments; their tunnels allow for effective limb, body, and antennae interaction with walls, which facilitate rapid slip-recovery during ascending and descending climbs. To examine the limits of slip-recovery in artificial tunnels, we perform perturbations consisting of rapid downward accelerations of the tunnels, which induce falls. Below a critical tunnel diameter, Ds = 1.31 ± 0.02 L, falls are always arrested through rapid interaction of appendages and antennae with tunnel walls to jam the falls. Ds is comparable to the size of incipient nest tunnels (D = 1.06 ± 0.23 L), supporting our hypothesis that fire ants construct environments that simplify their control task when moving through the nest, likely without need for rapid nervous system intervention.

Behavioral and mechanical determinants of collective subsurface nest excavation.

Collective construction of topologically complex structures is one of the triumphs of social behavior. For example, many ant species construct underground nests composed of networks of tunnels and chambers. Excavation by these 'superorganisms' depends on the biomechanics of substrate manipulation, the interaction of individuals, and media stability and cohesiveness. To discover principles of robust social excavation, we used X-ray computed tomography to monitor the growth in three dimensions of nests built by groups of fire ants (Solenopsis invicta) in laboratory substrates composed of silica particles, manipulating two substrate properties: particle size and gravimetric moisture content. Ants were capable of nest construction in all substrates tested other than completely dry or fully saturated; for a given particle size, nest volume was relatively insensitive to moisture content. Tunnels were deepest at intermediate moisture content and the maximum tunnel depth correlated with measured yield force on small rod-shaped intruders (a proxy for cohesive strength). This implies that increased cohesive strength allowed creation of tunnels that were resistant to perturbation but did not decrease individual excavation ability. Ants used two distinct behaviors to create pellets composed of wetted particles, depending on substrate composition. However, despite the ability to create larger stable pellets in more cohesive substrates, pellet sizes were similar across all conditions. We posit that this pellet size balances the individual's load-carrying ability with the need to carry this pellet through confined crowded tunnels. We conclude that effective excavation of similarly shaped nests can occur in a diversity of substrates through sophisticated digging behaviors by individuals which accommodate both differing substrate properties and the need to work within the collective.

Glass-like dynamics in confined and congested ant traffic.

The collective movement of animal groups often occurs in confined spaces. As animal groups are challenged to move at high density, their mobility dynamics may resemble the flow of densely packed non-living soft materials such as colloids, grains, or polymers. However, unlike inert soft-materials, self-propelled collective living systems often display social interactions whose influence on collective mobility are only now being explored. In this paper, we study the mobility of bi-directional traffic flow in a social insect (the fire ant Solenopsis invicta) as we vary the diameter of confining foraging tunnels. In all tunnel diameters, we observe the emergence of spatially heterogeneous regions of fast and slow traffic that are induced through two phenomena: physical obstruction, arising from the inability of individual ants to interpenetrate, and time-delay resulting from social interaction in which ants stop to briefly antennate. Density correlation functions reveal that the relaxation dynamics of high density traffic fluctuations scale linearly with fluctuation size and are sensitive to tunnel diameter. We separate the roles of physical obstruction and social interactions in traffic flow using cellular automata based simulation. Social interaction between ants is modeled as a dwell time (Tint) over which interacting ants remain stationary in the tunnel. Investigation over a range of densities and Tint reveals that the slowing dynamics of collective motion in social living systems are consistent with dynamics near a fragile glass transition in inert soft-matter systems. In particular, flow is relatively insensitive to density until a critical density is reached. As social interaction affinity is increased (increasing Tint) traffic dynamics change and resemble a strong glass transition. Thus, social interactions play an important role in the mobility of collective living systems at high density. Our experiments and model demonstrate that the concepts of soft-matter physics aid understanding of the mobility of collective living systems, and motivate further inquiry into the dynamics of densely confined social living systems.

Evolution at two levels in fire ants: the relationship between patterns of gene expression and protein sequence evolution.

Variation in protein sequence and gene expression each contribute to phenotypic diversity, and may be subject to similar selective pressures. Eusocial insects are particularly useful for investigating the evolutionary link between protein sequence and condition-dependent patterns of gene expression because gene expression plays a central role in determining differences between eusocial insect sexes and castes. We investigated the relationship between protein coding sequence evolution and gene expression patterns in the fire ants Solenopsis invicta, S. richteri, and their hybrids to gain greater insight into how selection jointly operates on gene expression and coding sequence. We found that genes with high expression variability within castes and sexes were frequently differentially expressed between castes and sexes, as well as between species and hybrids. These results indicate that genes showing high variation in expression in one context also tend to show high variation in expression in other contexts. Our analyses further revealed that variation in both intra- and interspecific gene expression was positively associated with rate of protein sequence evolution in Solenopsis. This suggests that selective constraints on a gene operate both at the level of protein sequence and at the level of gene expression regulation. Overall, our study provides one of the strongest demonstrations that selective constraints mediate both protein sequence evolution and gene expression variability across different biological contexts and timescales.

Epigenetic inheritance and genome regulation: is DNA methylation linked to ploidy in haplodiploid insects?

Organisms show great variation in ploidy level. For example, chromosome copy number varies among cells, individuals and species. One particularly widespread example of ploidy variation is found in haplodiploid taxa, wherein males are typically haploid and females are typically diploid. Despite the prevalence of haplodiploidy, the regulatory consequences of having separate haploid and diploid genomes are poorly understood. In particular, it remains unknown whether epigenetic mechanisms contribute to regulatory compensation for genome dosage. To gain greater insights into the importance of epigenetic information to ploidy compensation, we examined DNA methylation differences among diploid queen, diploid worker, haploid male and diploid male Solenopsis invicta fire ants. Surprisingly, we found that morphologically dissimilar diploid males, queens and workers were more similar to one another in terms of DNA methylation than were morphologically similar haploid and diploid males. Moreover, methylation level was positively associated with gene expression for genes that were differentially methylated in haploid and diploid castes. These data demonstrate that intragenic DNA methylation levels differ among individuals of distinct ploidy and are positively associated with levels of gene expression. Thus, these results suggest that epigenetic information may be linked to ploidy compensation in haplodiploid insects. Overall, this study suggests that epigenetic mechanisms may be important to maintaining appropriate patterns of gene regulation in biological systems that differ in genome copy number.

Relaxed selection is a precursor to the evolution of phenotypic plasticity.

Phenotypic plasticity allows organisms to produce alternative phenotypes under different conditions and represents one of the most important ways by which organisms adaptively respond to the environment. However, the relationship between phenotypic plasticity and molecular evolution remains poorly understood. We addressed this issue by investigating the evolution of genes associated with phenotypically plastic castes, sexes, and developmental stages of the fire ant Solenopsis invicta. We first determined if genes associated with phenotypic plasticity in S. invicta evolved at a rapid rate, as predicted under theoretical models. We found that genes differentially expressed between S. invicta castes, sexes, and developmental stages all exhibited elevated rates of evolution compared with ubiquitously expressed genes. We next investigated the evolutionary history of genes associated with the production of castes. Surprisingly, we found that orthologs of caste-biased genes in S. invicta and the social bee Apis mellifera evolved rapidly in lineages without castes. Thus, in contrast to some theoretical predictions, our results suggest that rapid rates of molecular evolution may not arise primarily as a consequence of phenotypic plasticity. Instead, genes evolving under relaxed purifying selection may more readily adopt new forms of biased expression during the evolution of alternate phenotypes. These results suggest that relaxed selective constraint on protein-coding genes is an important and underappreciated element in the evolutionary origin of phenotypic plasticity.

The genome of the fire ant Solenopsis invicta.

Ants have evolved very complex societies and are key ecosystem members. Some ants, such as the fire ant Solenopsis invicta, are also major pests. Here, we present a draft genome of S. invicta, assembled from Roche 454 and Illumina sequencing reads obtained from a focal haploid male and his brothers. We used comparative genomic methods to obtain insight into the unique features of the S. invicta genome. For example, we found that this genome harbors four adjacent copies of vitellogenin. A phylogenetic analysis revealed that an ancestral vitellogenin gene first underwent a duplication that was followed by possibly independent duplications of each of the daughter vitellogenins. The vitellogenin genes have undergone subfunctionalization with queen- and worker-specific expression, possibly reflecting differential selection acting on the queen and worker castes. Additionally, we identified more than 400 putative olfactory receptors of which at least 297 are intact. This represents the largest repertoire reported so far in insects. S. invicta also harbors an expansion of a specific family of lipid-processing genes, two putative orthologs to the transformer/feminizer sex differentiation gene, a functional DNA methylation system, and a single putative telomerase ortholog. EST data indicate that this S. invicta telomerase ortholog has at least four spliceforms that differ in their use of two sets of mutually exclusive exons. Some of these and other unique aspects of the fire ant genome are likely linked to the complex social behavior of this species.

Novel insights into paternity skew in a polyandrous social wasp.

Females of many species are polyandrous. However, polyandry can give rise to conflict among individuals within families. We examined the level of polyandry and paternity skew in the common eastern yellowjacket wasp, Vespula maculifrons, in order to gain a greater understanding of conflict in social insects. We collected 10 colonies of V. maculifrons and genotyped workers and prereproductive queens at highly variable microsatellite markers to assign each to a patriline. Genotypic data revealed evidence of significant paternity skew among patrilines. In addition, we found that patrilines contributed differentially to caste production (worker vs. queen), suggesting an important role for reproductive conflict not previously discovered. We also investigated if patterns of paternity skew and mate number varied over time. However, we found no evidence of changes in levels of polyandry when compared to historical data dating back almost 40 years. Finally, we measured a suite of morphological traits in individuals from the most common and least common patrilines in each colony to test if males that showed highly skewed reproductive success also produced offspring that differed in phenotype. Our data revealed weak correlation between paternity skew and morphological phenotype of offspring sired by different males, suggesting no evidence of evolutionary tradeoffs at the level investigated. Overall, this study is the first to report significant paternity and caste-associated skew in V. maculifrons, and to investigate the phenotypic consequences of skew in a social wasp. Our results suggest that polyandry can have important consequences on the genetic and social structure of insect societies.

Kin selection, genomics and caste-antagonistic pleiotropy.

Kin selection is a fundamentally important process that affects the evolution of social behaviours. The genomics revolution now provides the opportunity to test kin selection theory using genomic data. In this commentary, we discuss previous studies that explored the link between kin selection and patterns of variation within the genome. We then present a new theory aimed at understanding the evolution of genes involved in the development of social insects. Specifically, we investigate caste-antagonistic pleiotropy, which occurs when the phenotypes of distinct castes are optimized by different genotypes at a single locus. We find that caste-antagonistic pleiotropy leads to narrow regions where polymorphism can be maintained. Furthermore, multiple mating by queens reduces the region in which worker-favoured alleles fix, which suggests that multiple mating impedes worker caste evolution. We conclude by discussing ways to test these and other facets of kin selection using newly emerging genomic data.

Agitated ants: regulation and self-organization of incipient nest excavation via collisional cues.

Ants are millimetres in scale yet collectively create metre-scale nests in diverse substrates. To discover principles by which ant collectives self-organize to excavate crowded, narrow tunnels, we studied incipient excavation in small groups of fire ants in quasi-two-dimensional arenas. Excavation rates displayed three stages: initially excavation occurred at a constant rate, followed by a rapid decay, and finally a slower decay scaling in time as t-1/2. We used a cellular automata model to understand such scaling and motivate how rate modulation emerges without global control. In the model, ants estimated their collision frequency with other ants, but otherwise did not communicate. To capture early excavation rates, we introduced the concept of 'agitation'-a tendency of individuals to avoid rest if collisions are frequent. The model reproduced the observed multi-stage excavation dynamics; analysis revealed how parameters affected features of multi-stage progression. Moreover, a scaling argument without ant-ant interactions captures tunnel growth power-law at long times. Our study demonstrates how individual ants may use local collisional cues to achieve functional global self-organization. Such contact-based decisions could be leveraged by other living and non-living collectives to perform tasks in confined and crowded environments.

DNA methylation is widespread and associated with differential gene expression in castes of the honeybee, Apis mellifera.

The recent, unexpected discovery of a functional DNA methylation system in the genome of the social bee Apis mellifera underscores the potential importance of DNA methylation in invertebrates. The extent of genomic DNA methylation and its role in A. mellifera remain unknown, however. Here we show that genes in A. mellifera can be divided into 2 distinct classes, one with low-CpG dinucleotide content and the other with high-CpG dinucleotide content. This dichotomy is explained by the gradual depletion of CpG dinucleotides, a well-known consequence of DNA methylation. The loss of CpG dinucleotides associated with DNA methylation also may explain the unusual mutational patterns seen in A. mellifera that lead to AT-rich regions of the genome. A detailed investigation of this dichotomy implicates DNA methylation in A. mellifera development. High-CpG genes, which are predicted to be hypomethylated in germlines, are enriched with functions associated with developmental processes, whereas low-CpG genes, predicted to be hypermethylated in germlines, are enriched with functions associated with basic biological processes. Furthermore, genes more highly expressed in one caste than another are overrepresented among high-CpG genes. Our results highlight the potential significance of epigenetic modifications, such as DNA methylation, in developmental processes in social insects. In particular, the pervasiveness of DNA methylation in the genome of A. mellifera provides fertile ground for future studies of phenotypic plasticity and genomic imprinting.

Social structure of perennial Vespula squamosa wasp colonies.

Many social species show variation in their social structure in response to different environmental conditions. For example, colonies of the yellowjacket wasp Vespula squamosa are typically headed by a single reproductive queen and survive for only a single season. However, in warmer climates, V. squamosa colonies sometimes persist for multiple years and can grow to extremely large size. We used genetic markers to understand patterns of reproduction and recruitment within these perennial colonies. We genotyped V. squamosa workers, pre-reproductive queens, and males from perennial colonies in the southeastern United States at 10 polymorphic microsatellite loci and one mitochondrial DNA locus. We found that V. squamosa from perennial nests were produced by multiple reproductives, in contrast to typical annual colonies. Relatedness of nestmates from perennial colonies was significantly lower than relatedness of nestmates from annual colonies. Our analyses of mitochondrial DNA indicated that most V. squamosa perennial colonies represented semiclosed systems whereby all individuals belonged to a single matriline despite the presence of multiple reproductive females. However, new queens recruited into perennial colonies apparently mated with non-nestmate males. Notably, perennial and annual colonies did not show significant genetic differences, supporting the hypothesis that perennial colony formation represents an instance of social plasticity. Overall, our results indicate that perennial V. squamosa colonies show substantial changes to their social biology compared to typical annual colonies and demonstrate variation in social behaviors in highly social species.