A role for DNA methylation in bumblebee morphogenesis hints at female-specific developmental erasure.

Epigenetic mechanisms, such as DNA methylation, are crucial factors in animal development. In some mammals, almost all DNA methylation is erased during embryo development and re-established in a sex- and cell-specific manner. This erasure and re-establishment is thought to primarily be a vertebrate-specific trait. Insects are particularly interesting in terms of development as many species often undergo remarkable morphological changes en route to maturity, that is, morphogenesis. However, little is known about the role of epigenetic mechanisms in this process across species. We have used whole-genome bisulfite sequencing to track genome-wide DNA methylation changes through the development of an economically and environmentally important pollinator species, the bumblebee Bombus terrestris (Hymenoptera:Apidae Linnaeus). We find overall levels of DNA methylation vary throughout development, and we find developmentally relevant differentially methylated genes throughout. Intriguingly, we have identified a depletion of DNA methylation in ovaries/eggs and an enrichment of highly methylated genes in sperm. We suggest this could represent a sex-specific DNA methylation erasure event. To our knowledge, this is the first suggestion of possible developmental DNA methylation erasure in an insect species. This study lays the required groundwork for functional experimental work to determine if there is a causal nature to the DNA methylation differences identified. Additionally, the application of single-cell methylation sequencing to this system will enable more accurate identification of if or when DNA methylation is erased during development.

Does the use of chlorantraniliprole during queen development adversely impact health and viability?

The queen is the sole reproductive individual and the maturing brood replenishes the shorter-lived worker bees. Production of many crops relies on both pesticides and bee pollination to improve crop quantity and quality. Despite the certain knowledge on chemical pesticides caused damage to worker bee physiology and behavior, our understanding of the relationship between honeybee queen development and chemical pesticides remains weak. Here, we comprehensive investigate the effects of the widely used insecticide chlorantraniliprole on the growth, hormone levels, and detoxifying enzyme activity of queen larvae. It has been determined that chlorantraniliprole present a chronic toxic effect on queen larvae and also reduced the fitness of queen, and that these effects are positively correlated with pesticide levels. It has been found that queen larvae began to show reduced capping and emergence rates when exposed to 2 ng/larva of chlorantraniliprole. At 20 ng/larva, queen capping and emergence rates were the lowest, and there were significant reductions in larval hormone level. Chlorantraniliprole have an effect on detoxification enzyme activity and hormone levels in queen larvae. In conclusion, chlorantraniliprole can adversely affect the growth and development of queen larvae. Our findings may guide the scientifically sound use of chemical pesticides to reduce potential risks to queen larvae.

Honey bee colony behavior and ontogeny are adversely affected when exposed to a pesticide-contaminated environment.

Honey bees exhibit age polyethism and thus have a predictable sequence of behaviors they express through developmental time. Numerous laboratory studies show exposure to pesticides may impair critical honey bee behaviors (brood care, foraging, egg-laying, etc.) that adversely affect colony productivity and survival. There are fewer studies that examine the impacts of pesticides in natural field settings, especially given the challenges of implementing treatment groups and controlling variables. This study helps address the need for impact studies on pollinators under field conditions to assess the consequences of chemical overuse and dependency in agricultural and urban landscapes. To assess the impact of systemic pesticides in a natural field setting on worker bee behavioral development, observation hives were established to monitor changes in behaviors of similarly aged workers and sister queens within 2 experimental groups: (i) colonies located near point-source systemic pesticide pollution (pesticide contaminated treatment), and (ii) colonies embedded within a typical Midwestern US agricultural environment (control). In this study, worker bees in the contaminated environment exhibited important and biologically significant behavioral differences and accelerated onset of hive tasks (i.e., precocious behavioral development) compared to similarly aged bees at the control site. Queen locomotion was largely unaffected; however, the egg-laying rate was reduced in queens at the contaminated (treated) site. These results show that environmental pesticide exposure can disrupt colony function and adversely affect worker bee behavioral maturation, leading to reduced worker longevity and decreased colony efficiency.

Elevated developmental temperatures impact the size and allometry of morphological traits of the bumblebee Bombus terrestris.

The impact of global warming on wild bee decline threatens the pollination services they provide. Exposure to temperatures above optimal during development is known to reduce adult body size but how it affects the development and scaling of body parts remains unclear. In bees, a reduction in body size and/or a reduction in body parts, such as the antennae, tongue and wings, and how they scale with body size (i.e. their allometry) could severely affect their fitness. To date, it remains unclear how temperature affects body size and the scaling of morphological traits in bees. To address this knowledge gap, we exposed both males and workers of Bombus terrestris to elevated temperature during development and assessed the effects on (i) the size of morphological traits and (ii) the allometry between these traits. Colonies were exposed to optimal (25°C) or stressful (33°C) temperatures. We then measured the body size, wing size, antenna and tongue length, as well as the allometry between these traits. We found that workers were smaller and the antennae of both castes were reduced at the higher temperature. However, tongue length and wing size were not affected by developmental temperature. The allometric scaling of the tongue was also affected by developmental temperature. Smaller body size and antennae could impair both individual and colony fitness, by affecting foraging efficiency and, consequently, colony development. Our results encourage further exploration of how the temperature-induced changes in morphology affect functional traits and pollination efficiency.

Developmental plasticity shapes social traits and selection in a facultatively eusocial bee.

Developmental plasticity generates phenotypic variation, but how it contributes to evolutionary change is unclear. Phenotypes of individuals in caste-based (eusocial) societies are particularly sensitive to developmental processes, and the evolutionary origins of eusociality may be rooted in developmental plasticity of ancestral forms. We used an integrative genomics approach to evaluate the relationships among developmental plasticity, molecular evolution, and social behavior in a bee species (Megalopta genalis) that expresses flexible sociality, and thus provides a window into the factors that may have been important at the evolutionary origins of eusociality. We find that differences in social behavior are derived from genes that also regulate sex differentiation and metamorphosis. Positive selection on social traits is influenced by the function of these genes in development. We further identify evidence that social polyphenisms may become encoded in the genome via genetic changes in regulatory regions, specifically in transcription factor binding sites. Taken together, our results provide evidence that developmental plasticity provides the substrate for evolutionary novelty and shapes the selective landscape for molecular evolution in a major evolutionary innovation: Eusociality.

Multiplex Polymerase Chain Reaction Reveals Unique Trends in Pathogen and Parasitoid Infestations of Alfalfa Leafcutting Brood Cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. M. rotundata is a solitary cavity nesting bee that forms brood nests where its larvae can develop. During the developmental stages of growth, brood can be preyed upon by multiple different fungal pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollination services provided by M. rotundata. Reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. The presence of multiple Ascosphaera species in a single brood cell was observed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species, including multiple predators and pathogenetic species not previously documented in association with M. rotundata brood cells.

Supercooling points of freeze-avoiding bumble bees vary with caste and queen life stage.

Bumble bees thrive in cold climates including high latitude and high altitude regions around the world, yet cold tolerance strategies are largely unknown for most species. To determine bumble bee cold tolerance strategy, we exposed bumble bees to a range of low temperatures and measured survival 72 h post-exposure. All bees that froze died within 72 h while only one bee died without freezing, suggesting that bumble bees are generally freeze-avoiding insects and may be slightly chill susceptible. We then assessed whether temperatures that cause internal ice formation (supercooling points, SCP) varied among bumble bee castes (drones, workers, and queens), or across queen life stages, collection elevation, species, or season. Males froze at significantly lower temperatures than workers or queens. Queens in pre-overwintering or overwintering states froze at significantly lower temperatures than queens stimulated to initiate ovary development by CO2 narcosis (i.e., "spring" queens). We also tested whether the presence of water (i.e., wet or dry) or ramping rate affected SCP. As expected, queens inoculated with water froze at significantly higher temperatures than dry queens. SCP tended to be lower, but not significantly so, at faster ramping rates (0.5 °C/min vs 0.25 °C/min). We also found no differences in SCP between queen bumble bees collected in spring and fall, between queens collected at two sites differing in elevation by 1100 m, or between three field-caught bumble bee species. Bumble bees appear to have relatively high, invariable SCPs, likely making them highly susceptible to freezing across all seasons. As bumble bees are not freeze-tolerant and appear to lack the ability to prevent freezing at temperatures much below 0 °C, they may rely on season- and caste-specific micro-habitat selection to thrive in cold climates.

Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development.

The capacity of the honey bee to produce three phenotypically distinct organisms (two female castes; queens and sterile workers, and haploid male drones) from one genotype represents one of the most remarkable examples of developmental plasticity in any phylum. The queen-worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux acting via epigenetic regulation, in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. We produce the first genome-wide maps of chromatin structure in the honey bee at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. We find extensive genome-wide differences in H3K4me3, H3K27ac, and H3K36me3, many of which correlate with caste-specific transcription. Furthermore, we identify H3K27ac as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. Our results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. We show that at 96 h of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females. In a broader context, our study provides novel data on environmentally regulated organismal plasticity and the molecular foundation of the evolutionary origins of eusociality.

miRNA-34 and miRNA-210 target hexamerin genes enhancing their differential expression during early brain development of honeybee (Apis mellifera) castes.

During the honeybee larval stage, queens develop larger brains than workers, with morphological differentiation appearing at the fourth larval phase (L4), just after a boost in nutritional difference both prospective females experience. The molecular promoters of this caste-specific brain development are already ongoing in previous larval phases. Transcriptomic analyses revealed a set of differentially expressed genes in the L3 brains of queens and workers, which represents the early molecular response to differential feeding females receive during larval development. Three genes of this set, hex70b, hex70c and hex110, are more highly transcribed in the brain of workers than in queens. The microRNAs miR-34, miR-210 and miR-317 are in higher levels in the queens' brain at the same phase of larval development. Here, we tested the hypothesis that the brain of workers expresses higher levels of hexamerins than that of queens during key phases of larval development and that this differential hexamerin genes expression is further enhanced by the repressing activity of miR-34, miR-210 and miR-317. Our transcriptional analyses showed that hex70b, hex70c and hex110 genes are differentially expressed in the brain of L3 and L4 larval phases of honeybee queens and workers. In silico reconstructed miRNA-mRNA interaction networks were validated using luciferase assays, which showed miR-34 and miR-210 negatively regulate hex70b and hex110 genes by directly and redundantly binding their 3'UTR (untranslated region) sequences. Taken together, our results suggest that miR-34 and miR-210 act together promoting differential brain development in honeybee castes by downregulating the expression of the putative antineurogenic hexamerin genes hex70b and hex110.

When a Tritrophic Interaction Goes Wrong to the Third Level: Xanthoxylin From Trees Causes the Honeybee Larval Mortality in Colonies Affected by the River Disease.

The "River Disease" (RD), a disorder impacting honeybee colonies located close to waterways with abundant riparian vegetation (including Sebastiania schottiana, Euphorbiaceae), kills newly hatched larvae. Forager bees from RD-affected colonies collect honeydew excretions from Epormenis cestri (Hemiptera: Flatidae), a planthopper feeding on trees of S. schottiana. First-instar honeybee larvae fed with this honeydew died. Thus, we postulated that the nectars of RD-affected colonies had a natural toxin coming from either E. cestri or S. schottiana. An untargeted metabolomics characterization of fresh nectars extracts from colonies with and without RD allowed to pinpoint xanthoxylin as one of the chemicals present in higher amounts in nectar from RD-affected colonies than in nectars from healthy colonies. Besides, xanthoxylin was also found in the aerial parts of S. schottiana and the honeydew excreted by E. cestri feeding on this tree. A larva feeding assay where xanthoxylin-enriched diets were offered to 1st instar larvae showed that larvae died in the same proportion as larvae did when offered enriched diets with nectars from RD-colonies. These findings demonstrate that a xenobiotic can mimic the RD syndrome in honeybee larvae and provide evidence of an interspecific flow of xanthoxylin among three trophic levels. Further, our results give information that can be considered when implementing measures to control this honeybee disease.

Seed coating with a neonicotinoid insecticide negatively affects wild bees.

Understanding the effects of neonicotinoid insecticides on bees is vital because of reported declines in bee diversity and distribution and the crucial role bees have as pollinators in ecosystems and agriculture. Neonicotinoids are suspected to pose an unacceptable risk to bees, partly because of their systemic uptake in plants, and the European Union has therefore introduced a moratorium on three neonicotinoids as seed coatings in flowering crops that attract bees. The moratorium has been criticized for being based on weak evidence, particularly because effects have mostly been measured on bees that have been artificially fed neonicotinoids. Thus, the key question is how neonicotinoids influence bees, and wild bees in particular, in real-world agricultural landscapes. Here we show that a commonly used insecticide seed coating in a flowering crop can have serious consequences for wild bees. In a study with replicated and matched landscapes, we found that seed coating with Elado, an insecticide containing a combination of the neonicotinoid clothianidin and the non-systemic pyrethroid ß-cyfluthrin, applied to oilseed rape seeds, reduced wild bee density, solitary bee nesting, and bumblebee colony growth and reproduction under field conditions. Hence, such insecticidal use can pose a substantial risk to wild bees in agricultural landscapes, and the contribution of pesticides to the global decline of wild bees may have been underestimated. The lack of a significant response in honeybee colonies suggests that reported pesticide effects on honeybees cannot always be extrapolated to wild bees.

Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees.

Chalkbrood infection caused by the fungus Ascosphaera apis currently has a significant impact on Australia's apicultural industry. We investigated the genetic variation of A. apis and colony and apiary level conditions to determine if an emerging, more virulent strain or specific conditions were responsible for the prevalence of the disease. We identified six genetically distinct strains of A. apis, four have been reported elsewhere and two are unique to Australia. Colonies and individual larvae were found to be infected with multiple strains of A. apis, neither individual strains, combinations of strains, or obvious colony or apiary characteristics were found to be predictive of hive infection levels. These results suggest that host genotype plays an important role in colony level resistance to chalkbrood infection in Australia.

Virulence variations between clonal complexes of Melisococcus plutonius and the possible causes.

Melissococcus plutonius is a pathogenic bacterium that affects honeybee brood triggering colony collapse in severe cases. The bacterium causes a European foulbrood (EFB) disease in the honeybee populations, impacting beekeeping and agricultural industries. The pathogenesis, epidemiology, and variants of M. plutonius have been studied, but the virulence factors involved in larval infection are still unknown. Recently, an in-silico study suggested putative genes that might play a role in the pathogenesis of EFB. However, studies are required to determine their function as virulence factors. In addition, the few studies of clonal complexes (CCs), virulence factors, and variation in the honeybee larvae mortality have interfered with the development of more efficient control methods. The research, development, and differences in virulence between genetic variants (CCs) of M. plutonius and potential virulence factors implicated in honeybee larval mortality are discussed in this review.

Identification of a new P450s gene (AccCYP4AV1) and its roles in abiotic stress resistance in the Apis cerana cerana Fabricius.

Cytochrome P450 monooxygenases (P450s) play significant roles in protecting organisms from abiotic stress damage. Here, we report the sequence and characterization of a P450s gene (AccCYP4AV1), isolated from Apis cerana cerana Fabricius. The open reading frame of AccCYP4AV1 is 1506 base pairs long and encodes a predicted protein of 501 amino acids and 57.84 kDa, with an isoelectric point of 8.67. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis indicated that AccCYP4AV1 is more highly expressed in the midgut than in other tissues. In addition, the highest expression occurs in newly emerged adult workers, followed by the first instar of the larval stage. In addition, the expression of the AccCYP4AV1 was upregulated by low temperature (4 °C), ultraviolet radiation, hydrogen peroxide, paraquat, and dichlorvos treatments. In contrast, AccCYP4AV1 transcription was downregulated by other abiotic stress conditions: exposure to increased temperature (44 °C), deltamethrin, cadmium chloride, and mercury (II) chloride. Moreover, when AccCYP4AV1 was knocked-down by RNA interference, the results suggested that multiple antioxidant genes (AccsHSP22.6, AccSOD2, AccTpx1, and AccTpx4) were downregulated and antioxidant genes AccGSTO1 and AccTrx1 were upregulated. The activity levels of peroxidase and catalase were upregulated in the AccCYP4AV1-knocked-down samples, compared with those in the control groups. These findings suggest that the AccCYP4AV1 protein might be involved in the defense against abiotic stress damage.

Fipronil pesticide as a suspect in historical mass mortalities of honey bees.

Mass mortalities of honey bees occurred in France in the 1990s coincident with the introduction of two agricultural insecticides, imidacloprid and fipronil. Imidacloprid, a neonicotinoid, was widely blamed, but the differential potency of imidacloprid and fipronil has been unclear because of uncertainty over their capacity to bioaccumulate during sustained exposure to trace dietary residues and, thereby, cause time-reinforced toxicity (TRT). We experimentally quantified the toxicity of fipronil and imidacloprid to honey bees and incorporated the observed mortality rates into a demographic simulation of a honey bee colony in an environmentally realistic scenario. Additionally, we evaluated two bioassays from new international guidance for agrochemical regulation, which aim to detect TRT. Finally, we used analytical chemistry (GC-MS) to test for bioaccumulation of fipronil. We found in demographic simulations that only fipronil produced mass mortality in honey bees. In the bioassays, only fipronil caused TRT. GC-MS analysis revealed that virtually all of the fipronil ingested by a honey bee in a single meal was present 6 d later, which suggests that bioaccumulation is the basis of TRT in sustained dietary exposures. We therefore postulate that fipronil, not imidacloprid, caused the mass mortalities of honey bees in France during the 1990s because it is lethal to honey bees in even trace doses due to its capacity to bioaccumulate and generate TRT. Our results provide evidence that recently proposed laboratory bioassays can discriminate harmful bioaccumulative substances and, thereby, address evident shortcomings in a regulatory system that had formerly approved fipronil for agricultural use.

Comparative ecotoxicity of insecticides with different modes of action to Osmia excavata (Hymenoptera: Megachilidae).

Osmia excavata is an important pollinator in commercial fruit orchards. Little information has been published about ecotoxicity to O. excavata, especially the larvae. To clarify the risk of commonly used insecticides with different modes of action to the larvae of O. excavata, six insecticides (clothianidin, acetamiprid, sulfoxaflor, lambda-cyhalothrin, chlorfenapyr and abamectin) were selected for evaluation of their acute lethal toxicity and sublethal effects. Clothianidin and abamectin were the two most toxic insecticides to the larvae of O. excavata with LD50 values of 0.007 (0.006-0.008) and 0.0004 (0.0003-0.0006) µg active ingredient (a.i.) bee-1, respectively. And their ecological risks were high according to the hazard quotient values (HQ > 2500). Sulfoxaflor was identified as the only safe insecticide for O. excavata (HQ < 50) under field conditions. Sublethal toxicity tests showed that larval weight was significantly decreased by ingesting food treated with clothianidin, lambda-cyhalothrin and abamectin (less than the maximum field registered concentrations on fruit trees) due to interference with consumption per larva and reduction of the efficiency of conversion of ingested food. Additionally, above three insecticides significantly prolonged larval developmental duration before cocooning and decreased eclosion rate. Overall, there results suggested that clothianidin and abamectin should not be applied, especially during the flowering phase, the application frequency of lambda-cyhalothrin should be minimized for the purpose of conserving O. excavata. Our results provided important evidences for selecting appropriate insecticides for use in fruit orchards.

Characterization and Developmental Expression Patterns of Four Hexamerin Genes in the Bumble Bee, Bombus terrestris (Hymenoptera: Apidae).

Hexamerins are members of the hemocyanin superfamily and play essential roles in providing amino acids and energy for the nonfeeding stages of insects. In this study, we cloned and analyzed the expression patterns of four hexamerin genes (hex 70a, hex 70b, hex 70c, and hex 110) at different worker development stages and queen diapause statuses in the bumble bee, Bombus terrestris. The results of this study showed that hex 110 has the longest open reading frame (ORF; 3,297 bp) compared to the ORFs of hex 70a (2,034 bp), hex 70b (2,067 bp), and hex 70c (2,055 bp). The putative translation product of Hex 70a, Hex 70b, Hex70c, and Hex 110 has 677, 688, 684, and 1,098aa with predicted molecular mass of 81.13, 79.69, 81.58, and 119 kDa. In the development stages of workers, the expression levels of hex 70a, hex 70b, and hex 70c increased gradually from the larval stage and exhibited high expression levels at the pink eyed and brown eyed pupae stage, whereas hex 110 exhibited the highest expression level at the larval period. Four hexamerin genes were highly expressed at the prediapause status of queen (P < 0.05), and compared to the eclosion queen, the lowest upregulation was 3.7-fold, and the highest upregulation was 1,742-fold. The expression levels of hex 70b, hex 70c, and hex 110 at diapause were significantly higher than those at postdiapause (P < 0.05). In conclusion, hexamerins may play important roles in queen diapause and metamorphosis of larval and pupal stages.

The sublethal effects of ethiprole on the development, defense mechanisms, and immune pathways of honeybees (Apis mellifera L.).

Ethiprole has been widely used in agriculture, but there have been few studies on the adverse effects of ethiprole on nontarget organisms. This study focused on the mechanism of the sublethal effects of ethiprole on the development, antioxidation mechanisms, detoxification mechanisms and immune-related gene expression of honeybees (Apis mellifera L.). Honeybee larvae were found to be more sensitive than pupae to ethiprole. It was found that ethiprole inhibited the pupation and eclosion of bee larvae in a dose-dependent manner, with ethiprole doses of 1 × 10-3 mg/L decreasing pupation and eclosion rates to 50.00 ± 8.84% and 20.83 ± 10.62%, respectively. The activities of antioxidative enzymes (superoxide dismutase and catalase) and detoxification factors (glutathione and glutathione S-transferase) were also significantly increased in ethiprole-exposed honeybees, indicating that a sublethal dose of ethiprole also induced oxidative stress in honeybees. In the 1 × 10-3 mg/L ethiprole-exposure group, the expression of pathogen recognition-related gene PGRP-4300 was upregulated 11.10 ± 0.45-fold, and that of detoxification-related gene CYP4G11 was upregulated 8.84 ± 0.11-fold, indicating that ethiprole induced an immune reaction in honeybees. To the best our knowledge, this study represents the first demonstration that sublethal concentrations of ethiprole inhibit honeybee development and activate honeybee defense and immune systems.

Transcriptome analysis of Apis mellifera under benomyl stress to discriminate the gene expression in response to development and immune systems.

The health and safety of the honeybees are seriously threatened due to the abuse of chemical pesticides in modern agriculture and apiculture. In this study, the RNA Seq approach was used to assess the effects of the honeybees treated with benomyl. The results showed that there were a total of 11,902 differentially expressed genes (DEGs). Among them, 5,759 DEGs were up-regulated and involved in the functions of immunity, detoxification, biological metabolism, and regulation. The DEGs were clustered in the GO terms of epidermal structure and response to external stimuli, and most of the DEGs were enriched in 15 pathways, such as light conduction, MAPK, calcium ion pathway, and so on. Moreover, the pathway of the toll signal transduction was activated. The data investigated that the expression of functional genes involved in the growth, development, foraging, and immunity of honeybees were significantly affected by benomyl stress, which would seriously threaten the health of the honeybees. This study provided a theoretical basis for revealing the response mechanism of honeybees to pesticides stress.

Transcriptome analysis reveals nutrition- and age-related patterns of gene expression in the fat body of pre-overwintering bumble bee queens.

Many diapausing insects undergo a nutrient storage period prior to their entry into diapause. Bumble bee queens diapause as adults in the winter preceding their spring nest initiation period. Before diapause, they sequester glycogen and lipids, which they metabolize during the overwintering period. We used RNA sequencing to examine how age and nectar diet (specifically, the concentration of sucrose in nectar) impact gene expression in the pre-overwintering bumble bee queen fat body, the "liver-like" organ in insects with broad functions related to nutrient storage and metabolism. We found that diet on its own, and in combination with age, impacts the expression of genes involved in detoxification. Age was also a strong driver of gene expression, especially at earlier ages (up to 3 days). In addition to these molecular correlates of diet and age, we also found a putative molecular signature of diapause entry or preparation in adult queens in the oldest age group (12 days) fed the most sucrose-rich diet, based on comparisons between our data set and another transcriptome data set from bumble bee queens. This transcriptomic pattern suggests that preparation for (or entry into) diapause might be in part mediated by nutritional state in bumble bee queens. Collectively, these findings show that there are molecular processes in the fat body that are responsive to sucrose levels in the diet and/or associated with age-related maturational changes. A better understanding of these processes may shed light on important aspects of bumble bee biology, such as queen responses to nutritional and other forms of stress, and the factors that regulate their entrance into diapause.