Transcriptional signature of host shift in the seed beetle Zabrotes subfasciatus.

In phytophagous insects, adaptation to a new host is a dynamic process, in which early and later steps may be underpinned by different features of the insect genome. Here, we tested the hypothesis that early steps of this process are underpinned by a shift in gene expression patterns. We set up a short-term artificial selection experiment (10 generations) for the use of an alternative host (Cicer arietinum) on populations of the bean beetle Zabrotes subfasciatus. Using Illumina sequencing on young adult females, we show the selected populations differ in the expression of genes associated to stimuli, signalling, and developmental processes. Particularly, the "C. arietinum" population shows upregulation of histone methylation genes, which may constitute a strategy for fine-tuning the insect global gene expression network. Using qPCR on body regions, we demonstrated that the "Phaseolus vulgaris" population upregulates the genes polygalacturonase and egalitarian and that the expression of an odorant receptor transcript variant changes over generations. Moreover, in this population we detected the existence of vitellogenin (Vg) variants in both males and females, possibly harbouring canonical reproductive function in females and extracellular unknown functions in males. This study provides the basis for future genomic investigations seeking to shed light on the nature of the proximate mechanisms involved in promoting differential gene expression associated to insect development and adaptation to new hosts.

Immunosenescence in honey bees (Apis mellifera L.) is caused by intrinsic senescence and behavioral physiology.

Young honey bee workers (0 to 2-3 weeks old) perform tasks inside the colony, including brood care (nursing), whereas older workers undergo foraging tasks during the next 3-4 weeks, when an intrinsic senescence program culminates in worker death. We hypothesized that foragers are less able to react to immune system stimulation than nurse bees and that this difference is due to an inefficient immune response in foragers. To test this hypothesis, we used an experimental design that allowed us to uncouple chronological age and behavior status (nursing/foraging). Worker bees from a normal age demography colony (where workers naturally transit from nursing to foraging tasks as they age) and of a single-cohort colony setup (composed of same-aged workers performing nursing or foraging tasks) were tested for survival and capability of activation of the immune system after bacterial injection. Expression of an antimicrobial peptide gene, defensin-1 (def-1), was used to assess immune system activation. We then checked whether the immune response includes changes in the expression of aging- and behavior-related genes, specifically vitellogenin (vg), juvenile hormone esterase (jhe), and insulin-like peptide-1 (ilp-1). We found a significant difference in survival rate between bees of different ages but carrying out the same tasks. Our results thus indicate that the bees' immune response is negatively affected by intrinsic senescence. Additionally, independent of age, foragers had a shorter lifespan than nurses after bacterial infection, although both were able to induce def-1 transcription. In the normal age demography colony, the immune system activation resulted in a reduction in the expression of vg, jhe and ilp-1 genes in foragers, but not in the nurse bees, demonstrating that age and behavior are both important influences on the bees' immune response. By disentangling the effects of age and behavior in the single-cohort colony, we found that vg, jhe and ilp-1 response to immune system stimulation was independent of behavior. Younger bees were able to mount a stronger immune response than older bees, thus highlighting age as an important factor for immunity. Taken together, our results provide new insights into how age and behavior affect the honey bee's immune response.

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs ß bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs ß and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs ß and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera.

Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs.

Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes.

Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes.

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity.

Nuclear Immunolocalization of Hexamerins in the Fat Body of Metamorphosing Honey Bees.

Hexamerins are storage proteins with primordial functions in insect metamorphosis. They are actively secreted by the larval fat body and stored in the hemolymph. During metamorphosis, they return to the fat body to be processed. For decades, these proteins were thought to exclusively function as an amino acid source for tissue reconstruction during the non-feeding pupal and pharate adult stages and, in some species, for egg production. Recently, new findings have linked the hexamerins to caste polyphenism and gonad development in social insects. To explore the roles of hexamerins during the honey bee metamorphosis, we used specific antibodies in expression analysis by western blot, in situ immunolocalization by confocal laser-scanning microscopy and in vivo injections to lower their endogenous levels. Our expression analysis highlighted the changing expression patterns in the fat body and hemolymph during development, which is consistent with the temporal dynamics of hexamerin secretion, storage and depletion. Confocal microscopy showed hexamerin expression in the cytoplasm of both types of fat body cells, trophocytes and oenocytes. Notably, hexamerin foci were also found in the nuclei of these cells, thus confirming our western blot analysis of fat body nuclear-enriched fractions. We also observed that the decrease in soluble hexamerins in antibody-treated pharate adults led to a precocious adult ecdysis, perhaps in response to the lack (or decrease) in hexamerin-derived amino acids. Taken together, these findings indicate that hexamerins have other functions in addition to their well-established role as amino acid sources for development.

A honeybee storage protein gene, hex 70a, expressed in developing gonads and nutritionally regulated in adult fat body.

In preparing for metamorphosis, insect larvae store a huge amount of proteins in hemolymph, mainly hexamerins. Out of the four hexamerins present in the honeybee larvae, one, HEX 70a, exhibited a distinct developmental pattern, especially since it is also present in adults. Here, we report sequence data and experimental evidence suggesting alternative functions for HEX 70a, besides its well-known role as an amino acid resource during metamorphosis. The hex 70a gene consists of 6 exons and encodes a 684 amino acid chain containing the conserved hemocyanin N, M, and C domains. HEX 70a classifies as an arylphorin since it contains more than 15% of aromatic amino acids. In the fat body of adult workers, hex 70a expression turned out to be a nutrient-limited process. However, the fat body is not the only site for hex 70a expression. Both, transcript and protein subunits were also detected in developing gonads from workers, queens and drones, suggesting a role in ovary differentiation and testes maturation and functioning. In its putative reproductive role, HEX 70a however differs from the yolk protein, vitellogenin, since it was not detected in eggs or embryos.