ASU Scholarship Showcase

Honeybee workers are essentially sterile female helpers that make up the majority of individuals in a colony. Workers display a marked change in physiology when they transition from in-nest tasks to foraging. Recent technological advances have made it possible to unravel the metabolic modifications associated with this transition. Previous studies have revealed extensive remodeling of brain, thorax, and hypopharyngeal gland biochemistry. However, data on changes in the abdomen is scarce. To narrow this gap we investigated the proteomic composition of abdominal tissue in the days typically preceding the onset of foraging in honeybee workers.

In order to get a broader representation of possible protein dynamics, we used workers of two genotypes with differences in the age at which they initiate foraging. This approach was combined with RNA interference-mediated downregulation of an insulin/insulin-like signaling component that is central to foraging behavior, the insulin receptor substrate (irs), and with measurements of glucose and lipid levels.
Our data provide new insight into the molecular underpinnings of phenotypic plasticity in the honeybee, invoke parallels with vertebrate metabolism, and support an integrated and irs-dependent association of carbohydrate and lipid metabolism with the transition from in-nest tasks to foraging.

Background: Mutual policing is an important mechanism for reducing conflict in cooperative groups. In societies of ants, bees, and wasps, mutual policing of worker reproduction can evolve when workers are more closely related to the queen's sons than to the sons of workers or when the costs of worker reproduction lower the inclusive fitness of workers. During colony growth, relatedness within the colony remains the same, but the costs of worker reproduction may change. The costs of worker reproduction are predicted to be greatest in incipient colonies. If the costs associated with worker reproduction outweigh the individual direct benefits to workers, policing mechanisms as found in larger colonies may be absent in incipient colonies.

Results: We investigated policing behavior across colony growth in the ant 'Camponotus floridanus.' In large colonies of this species, worker reproduction is policed by the destruction of worker-laid eggs. We found workers from incipient colonies do not exhibit policing behavior, and instead tolerate all conspecific eggs. The change in policing behavior is consistent with changes in egg surface hydrocarbons, which provide the informational basis for policing; eggs laid by queens from incipient colonies lack the characteristic hydrocarbons on the surface of eggs laid by queens from large colonies, making them chemically indistinguishable from worker-laid eggs. We also tested the response to fertility information in the context of queen tolerance. Workers from incipient colonies attacked foreign queens from large colonies; whereas workers from large colonies tolerated such queens. Workers from both incipient and large colonies attacked foreign queens from incipient colonies.

Conclusions: Our results provide novel insights into the regulation of worker reproduction in social insects at both the proximate and ultimate levels. At the proximate level, our results show that mechanisms of social regulation, such as the response to fertility signals, change dramatically over a colony's life cycle. At the ultimate level, our results emphasize the importance of factors besides relatedness in predicting the level of conflict within a colony. Our results also suggest policing may not be an important regulatory force at every stage of colony development. Changes relating to the life cycle of the colony are sufficient to account for major differences in social regulation in an insect colony. Mechanisms of conflict mediation observed in one phase of a social group's development cannot be generalized to all stages.

Recent advancements in genomics provide new tools for evolutionary ecological research. The paper wasp genus Polistes is a model for social insect evolution and behavioral ecology. We developed RNA interference (RNAi)-mediated gene silencing to explore proposed connections between expression of hexameric storage proteins and worker vs. gyne (potential future foundress) castes in naturally-founded colonies of P. metricus. We extended four fragments of putative hexamerin-encoding P. metricus transcripts acquired from a previous study and fully sequenced a gene that encodes Hexamerin 2, one of two proposed hexameric storage proteins of P. metricus. MALDI-TOF/TOF, LC-MSMS, deglycosylation, and detection of phosphorylation assays showed that the two putative hexamerins diverge in peptide sequence and biochemistry. We targeted the hexamerin 2 gene in 5th (last)-instar larvae by feeding RNAi-inducing double-stranded hexamerin 2 RNA directly to larvae in naturally-founded colonies in the field. Larval development and adult traits were not significantly altered in hexamerin 2 knockdowns, but there were suggestive trends toward increased developmental time and less developed ovaries, which are gyne characteristics. By demonstrating how data acquisition from 454/Roche pyrosequencing can be combined with biochemical and proteomics assays and how RNAi can be deployed successfully in field experiments on Polistes, our results pave the way for functional genomic research that can contribute significantly to learning the interactions of environment, development, and the roles they play in paper wasp evolution and behavioral ecology.

Background: Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life.

Results: Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees.

Conclusion: Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

Background: Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone’s source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. Here we adapt a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system we test potential explanations for how juvenile hormone modulates aging.

Results: A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state.

Conclusions: Reduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. Our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. We identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity.

Background: Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data.

Results: We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation.

Conclusions: This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.

Background: The molecular mechanisms of variations in individual longevity are not well understood, even though longevity can be increased substantially by means of diverse experimental manipulations. One of the factors supposed to be involved in the increase of longevity is a higher stress resistance. To test this hypothesis in a natural system, eusocial insects such as bees or ants are ideally suited. In contrast to most other eusocial insects, ponerine ants show a peculiar life history that comprises the possibility to switch during adult life from a normal worker to a reproductive gamergate, therewith increasing their life expectancy significantly.

Results: We show that increased resistance against major stressors, such as reactive oxygen species and infection accompanies the switch from a life-history trait with normal lifespan to one with a longer life expectancy. A short period of social isolation was sufficient to enhance stress resistance of workers from the ponerine ant species Harpegnathos saltator significantly. All ant groups with increased stress resistances (reproducing gamergates and socially isolated workers) have lower catalase activities and glutathione levels than normal workers. Therewith, these ants resemble the characteristics of the youngest ants in the colony.

Conclusions: Social insects with their specific life history including a switch from normal workers to reproducing gamergates during adult life are well suited for ageing research. The regulation of stress resistance in gamergates seemed to be modified compared to foraging workers in an economic way. Interestingly, a switch towards more stress resistant animals can also be induced by a brief period of social isolation, which may already be associated with a shift to a reproductive trajectory. In Harpegnathos saltator, stress resistances are differently and potentially more economically regulated in reproductive individuals, highlighting the significance of reproduction for an increase in longevity in social insects. As already shown for other organisms with a long lifespan, this trait is not directly coupled to higher levels of enzymatic and non-enzymatic antioxidants.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance.

Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

Epigenetic changes enable genomes to respond to changes in the environment, such as altered nutrition, activity, or social setting. Epigenetic modifications, thereby, provide a source of phenotypic plasticity in many species. The honey bee (Apis mellifera) uses nutritionally sensitive epigenetic control mechanisms in the development of the royal caste (queens) and the workers. The workers are functionally sterile females that can take on a range of distinct physiological and/or behavioral phenotypes in response to environmental changes. Honey bees have a wide repertoire of epigenetic mechanisms which, as in mammals, include cytosine methylation, hydroxymethylated cytosines, together with the enzymatic machinery responsible for these cytosine modifications. Current data suggests that honey bees provide an excellent system for studying the “social repertoire” of the epigenome. In this review, we elucidate what is known so far about the honey bee epigenome and its mechanisms. Our discussion includes what may distinguish honey bees from other model animals, how the epigenome can influence worker behavioral task separation, and how future studies can answer central questions about the role of the epigenome in social behavior.