Krista K. Ingram

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.

The molecular clockwork of the fire ant Solenopsis invicta.

The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.

Colony life history and lifetime reproductive success of red harvester ant colonies

1. We estimate colony reproductive success, in numbers of offspring colonies arising from a colonys daughter queens, of colonies of the red harvester ant, Pogonomyrmex barbatus. 2. A measure of lifetime reproductive success is essential to understand the relation of ecological factors, phenotype and fitness in a natural population. This was possible for the first time in a natural population of ant colonies using data from long-term study of a population of colonies in south-eastern Arizona, for which ages of all colonies are known from census data collected since 1985. 3. Parentage analyses of microsatellite data from 5 highly polymorphic loci were used to assign offspring colonies to maternal parent colonies in a population of about 265 colonies, ages 1-28 years, sampled in 2010. 4. The estimated population growth rate Ro was 1.69 and generation time was 7.8 years. There was considerable variation among colonies in reproductive success: of 199 possible parent colonies, only 49 (˜ 25%) had offspring colonies on the site. The mean number of offspring colonies per maternal parent colony was 2.94 and ranged from 1 to 8. A parent was identified for the queen of 146 of 247 offspring colonies. There was no evidence for reproductive senescence; fecundity was about the same throughout the 25-30 year lifespan of a colony. 5. There were no trends in the distance or direction of the dispersal of an offspring relative to its maternal parent colony. There was no relationship between the number of gynes produced by a colony in 1 year and the number of offspring colonies subsequently founded by its daughter reproductive females. The results provide the first estimate of a life table for a population of ant colonies and the first estimate of the female component of colony lifetime reproductive success. 6. The results suggest that commonly used measures of reproductive output may not be correlated with realized reproductive success. This is the starting point for future investigation asking whether variation in reproductive success is related to phenotypic variation among colonies in behavioural and ecological traits.

Differential regulation of the foraging gene associated with task behaviors in harvester ants

BackgroundThe division of labor in social insect colonies involves transitions by workers from one task to another and is critical to the organization and ecological success of colonies. The differential regulation of genetic pathways is likely to be a key mechanism involved in plasticity of social insect task behavior. One of the few pathways implicated in social organization involves the cGMP-activated protein kinase gene, foraging, a gene associated with foraging behavior in social insect species. The association of the foraging gene with behavior is conserved across diverse species, but the observed expression patterns and proposed functions of this gene vary across taxa. We compared the protein sequence of foraging across social insects and explored whether the differential regulation of this gene is associated with task behaviors in the harvester ant, Pogonomyrmex occidentalis.ResultsPhylogenetic analysis of the coding region of the foraging gene reveals considerable conservation in protein sequence across insects, particularly among hymenopteran species. The absence of amino acid variation in key active and binding sites suggests that differences in behaviors associated with this gene among species may be the result of changes in gene expression rather than gene divergence. Using real time qPCR analyses with a harvester ant ortholog to foraging (Pofor), we found that the brains of harvester ant foragers have a daily fluctuation in expression of foraging with mRNA levels peaking at midday. In contrast, young workers inside the nest have low levels of Pofor mRNA with no evidence of daily fluctuations in expression. As a result, the association of foraging expression with task behavior within a species changes depending on the time of day the individuals are sampled.ConclusionsThe amino acid protein sequence of foraging is highly conserved across social insects. Differences in foraging behaviors associated with this gene among social insect species are likely due to differences in gene regulation rather than evolutionary changes in the encoded protein. The task-specific expression patterns of foraging are consistent with the task-specific circadian rhythms observed in harvester ants. Whether the molecular clock plays a role in regulating foraging gene expression (or vice versa) remains to be determined. Our results represent the first time series analysis of foraging gene expression and underscore the importance of assaying time-related expression differences in behavioral studies. Understanding how this gene is regulated within species is critical to explaining the mechanism by which foraging influences behavior.

Context-dependent expression of the foraging gene in field colonies of ants: the interacting roles of age, environment and task.

Task allocation among social insect workers is an ideal framework for studying the molecular mechanisms underlying behavioural plasticity because workers of similar genotype adopt different behavioural phenotypes. Elegant laboratory studies have pioneered this effort, but field studies involving the genetic regulation of task allocation are rare. Here, we investigate the expression of the foraging gene in harvester ant workers from five age- and task-related groups in a natural population, and we experimentally test how exposure to light affects foraging expression in brood workers and foragers. Results from our field study show that the regulation of the foraging gene in harvester ants occurs at two time scales: levels of foraging mRNA are associated with ontogenetic changes over weeks in worker age, location and task, and there are significant daily oscillations in foraging expression in foragers. The temporal dissection of foraging expression reveals that gene expression changes in foragers occur across a scale of hours and the level of expression is predicted by activity rhythms: foragers have high levels of foraging mRNA during daylight hours when they are most active outside the nests. In the experimental study, we find complex interactions in foraging expression between task behaviour and light exposure. Oscillations occur in foragers following experimental exposure to 13 L : 11 D (LD) conditions, but not in brood workers under similar conditions. No significant differences were seen in foraging expression over time in either task in 24 h dark (DD) conditions. Interestingly, the expression of foraging in both undisturbed field and experimentally treated foragers is also significantly correlated with the expression of the circadian clock gene, cycle. Our results provide evidence that the regulation of this gene is context-dependent and associated with both ontogenetic and daily behavioural plasticity in field colonies of harvester ants. Our results underscore the importance of assaying temporal patterns in behavioural gene expression and suggest that gene regulation is an integral mechanism associated with behavioural plasticity in harvester ants.

Diurnal Preference Predicts Phase Differences in Expression of Human Peripheral Circadian Clock Genes

Background: Circadian rhythms play an integral role in human behavior, physiology and health. Individual differences in daily rhythms (chronotypes) can affect individual sleep-wake cycles, activity patterns and behavioral choices. Diurnal preference, the tendency towards morningness or eveningness among individuals, has been associated with interpersonal variation in circadian clock-related output measures, including body temperature, melatonin levels and clock gene mRNA in blood, oral mucosa, and dermal fibroblast cell cultures. Methods: Here we report gene expression data from two principal clock genes sampled from hair follicle cells, a peripheral circadian clock. Hair follicle cells from fourteen individuals of extreme morning or evening chronotype were sampled at three time points. RNA was extracted and quantitative PCR assays were used to measure mRNA expression patterns of two clock genes, Per3 and Nr1d2. Results: We found significant differences in clock gene expression over time between chronotype groups, independent of gender or age of participants. Extreme evening chronotypes have a delay in phase of circadian clock gene oscillation relative to extreme morning types. Variation in the molecular clockwork of chronotype groups represents nearly three-hour phase differences (Per3: 2.61 hours; Nr1d2: 3.08 hours, both: 2.86) in circadian oscillations of these clock genes. Conclusions: The measurement of gene expression from hair follicles at three time points allows for a direct, efficient method of estimating phase shifts of a peripheral circadian clock in real-life conditions. The robust phase differences in temporal expression of clock genes associated with diurnal preferences provide the framework for further studies of the molecular mechanisms and gene-by-environment interactions underlying chronotype-specific behavioral phenomena, including social jetlag.

Circadian Clock Model Supports Molecular Link Between PER3 and Human Anxiety

Generalized anxiety and major depression have become increasingly common in the United States, affecting 18.6 percent of the adult population. Mood disorders can be debilitating, and are often correlated with poor general health, life dissatisfaction, and the need for disability benefits due to inability to work. Recent evidence suggests that some mood disorders have a circadian component, and disruptions in circadian rhythms may even trigger the development of these disorders. However, the molecular mechanisms of this interaction are not well understood. Polymorphisms in a circadian clock-related gene, PER3, are associated with behavioral phenotypes (extreme diurnal preference in arousal and activity) and sleep/mood disorders, including seasonal affective disorder (SAD). Here we show that two PER3 mutations, a variable number tandem repeat (VNTR) allele and a single-nucleotide polymorphism (SNP), are associated with diurnal preference and higher Trait-Anxiety scores, supporting a role for PER3 in mood modulation. In addition, we explore a potential mechanism for how PER3 influences mood by utilizing a comprehensive circadian clock model that accurately predicts the changes in circadian period evident in knock-out phenotypes and individuals with PER3-related clock disorders.

Molecular insights into chronotype and time-of-day effects on decision-making

Recent reports highlight that human decision-making is influenced by the time of day and whether one is a morning or evening person (i.e., chronotype). Here, we test whether these behavioral effects are associated with endogenous biological rhythms. We asked participants to complete two well-established decision-making tasks in the morning or evening: the matrix task (an ethical decision task) and the balloon analog risk task (BART; a risk-taking task), and we measured their chronotype in two ways. First, participants completed a self-report measure, the Horne-Östberg Morningness-Eveningness Questionnaire (MEQ). Second, we measured the expression of two circadian clock-regulated genes—Per3 and Nr1d2—from peripheral clock cells in participants’ hair follicle samples. Using a cosinor model, we estimated the phase of the peripheral clock and assigned RNA chronotypes to participants with advanced (larks) or delayed (owls) phases. The behavioral data were analyzed independently for self-reported (MEQ) and RNA-based chronotypes. We find that significant chronotype and/or time-of-day effects between larks and owls in decision-making tasks occur only in RNA-based chronotypes. Our results provide evidence that time-of-day effects on decision-making can be explained by phase differences in oscillating clock genes and suggest that variation in the molecular clockwork may influence inter-individual differences in decision-making behavior.

Modeling Strengthens Molecular Link between Circadian Polymorphisms and Major Mood Disorders

Anxiety and other mood disorders, such as major depressive disorder (MDD) and seasonal affective disorder (SAD), affect nearly one-fifth of the global population and disproportionately affect young adults. Individuals affected by mood disorders are frequently plagued by sleep and circadian problems, and recent genetic studies provide ample support for the association of circadian and sleep syndromes with depression and anxiety. Mathematical modeling has been crucial in understanding some of the essential features of the mammalian circadian clock and is now a vital tool for dissecting how circadian genes regulate the molecular mechanisms that influence mood. Here, we model the effect of five clock gene polymorphisms, previously linked to mood disorders, on circadian gene expression and, ultimately, on the period length and amplitude of the clock, two parameters that dictate the phase, or alignment, of the clock relative to the environment. We then test whether these gene variants are associated with circadian phenotypes (Horne-Ostberg Morningness-Eveningness scores) and well-established measures of depression (Beck Depression Inventory) and anxiety (State-Trait Anxiety Inventory) in a population of undergraduates (n = 546). In this population, we find significant allelic and/or genotypic associations between CRY2 and two PER3 variants and diurnal preference. The PER3 length polymorphism (rs57875989) was significantly associated with depression in this sample, and individuals homozygous for the PER3 single nucleotide polymorphism (SNP) (rs228697) reported significantly higher anxiety. Our simple model satisfies available experimental knockdown conditions as well as existing data on clock polymorphisms associated with mood. In addition, our model enables us to predict circadian phenotypes (e.g., altered period length, amplitude) associated with mood disorders in order to identify critical effects of clock gene mutations on CRY/BMAL binding and to predict that the intronic SNPs studied represent gain-of-function mutations, causing increased transcription rate. Given the user-friendly structure of our model, we anticipate that it will be useful for further study of the relationships among clock polymorphisms, circadian misalignment, and mood disorders.

Sporadic Infection of Wolbachia in a Recently Established Population of Formica fusca

This study examines the distribution and invasion dynamics of Wolbachia in a recently established Formica fusca population. Preliminary data revealed the intermittent infection of Wolbachia across colonies, providing the opportunity to test for ecological factors affecting the acquisition and spread of the parasite. Only 35% of colonies are infected in this population. Both infected and noninfected nests have similar dispersion patterns that approximate a random distribution, suggesting that transmission of Wolbachia between adjacent colonies is not common. There is no difference in the infection rate between workers and brood, indicating that workers are not actively eliminating the infection. Our results show no significant association between Wolbachia infection and nest size; however, infected colonies tend to be larger than noninfected colonies. Finally, Wolbachia infection was not associated with queen number. Overall, our results suggest no large fitness differences between infected and noninfected colonies, although small fitness effects cannot be ruled out for this population.