A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera,Apidae)

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera, Apidae). Metamorphosis in holometabolous insects occurs through two subsequent molting cycles: pupation (metamorphic molt) and adult differentiation (imaginal molt). The imaginal molt in Apis mellifera L. was recently investigated in both histological and physiological-molecular approaches. Although the metamorphic molt in this model bee is extremely important to development, it is not well-known yet. In the current study we used this stage as an ontogenetic scenario to investigate the transcriptional profile of the gene Amlac2, which encodes a laccase with an essential role in cuticle differentiation. Amlac2 expression in epidermis was contrasted with the hemolymph titer of ecdysteroid hormones and with the most evident morphological events occurring during cuticle renewal. RT-PCR semiquantitative analyses using integument samples revealed increased levels of Amlac2 transcripts right after apolysis and during the subsequent pharate period, and declining levels near pupal ecdysis. Compared with the expression of a cuticle protein gene, AmelCPR14, these results highlighted the importance of the ecdysteroid-induced apolysis as an ontogenetic marker of gene reactivation in epidermis for cuticle renewal. The obtained results strengthen the comprehension of metamorphosis in Apis mellifera. In addition, we reviewed the literature about the development of A. mellifera, and emphasize the importance of revising the terminology used to describe honey bee molting cycles

Ecdysteroid-dependent expression of the tweedle and peroxidase genes during adult cuticle formation in the honey bee, Apis mellifera.

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut

Effect of HEX 70a depletion on queen ovary growth and worker cuticle formation.

<p>(<b>A</b>) Width of the ovarioles of queens injected with anti-HEX 70a in 0.9% NaCl or saline vehicle only. Measurements were made in two regions of the germarium of 120 ovarioles, 60 of them dissected from 3 anti-HEX 70a injected queens (20 ovarioles per queen), and 60 from 3 control queens. Measurements obtained from bees injected with the antibody, or the antibody vehicle only, were compared using Two-Way ANOVA and the post-hoc Holm-Sidak multiple comparison test (Jandel SigmaStat 3.1 software, Jandel Corporation, San Rafael, CA, USA). (<b>B</b>) Western blot levels of HEX 70a in the hemolymph samples of workers at 4 and 72 h after injection with anti-HEX 70a or saline vehicle only (control). The levels of the ∼200 kDa lipophorin in the same samples were used as loading control. (<b>C</b>) Hind legs of workers injected with anti-HEX 70a in 0.9% NaCl, in comparison to workers injected with mouse IgG in 0,9% NaCl, or those of the 0.9% NaCl injected group.</p

Immunolocalization of HEX 70a in the queen ovariole.

<p>(<b>A</b>) Schematic representation of an ovariole of an egg laying queen (seen at the upper left corner): only the terminal filament, the germarium and early follicles initiating previtellogenic growth in the upper region of the vitellarium are shown in A. Confocal microscopy images: (<b>B</b>) Part of an ovariole showing the middle and lower regions of the vitellarium labeled with rhodamin/phalloidin (green) to highlight F-actin. The arrows and arrowheads show developing nurse cell- and oocyte- chambers, respectively. (<b>C–E</b>) the terminal filament (the lower region is oriented downward) shows HEX 70a foci in the nuclei (D, E) and in cytoplasm (arrows in D, E). (<b>F–H</b>) Nurse cell nuclei in the nurse cell chamber (lower region of the vitellarium as indicated by arrows in B). (<b>I–K</b>) Follicle cell nuclei covering an oocyte at the lower region of the vitellarium (as indicated by arrowheads in B). (<b>C, F, I</b>) DAPI-stained cell nuclei (blue); (<b>D, G, J</b>) anti-HEX 70a/Cy3-staining for HEX 70a detection (red) and (<b>E, H, K</b>) merged images.</p

Detection of HEX 70a in ovarioles of workers at the beginning of the pharate-adult development (∼1 day after pupal ecdysis) (the developmental stage is illustrated at the upper left corner of the figure).

<p>(<b>A</b>) Light microscopy of ovarioles (covered by their respective peritoneal sheath) stained with methylene blue/basic fuchsin. Only the germarium is focused in this figure (the most anterior region of the ovariole, or terminal filament, is not shown). A rosette formed by germline cells (oocyte and nurse cell precursors) is distinguishable (circle) in the germarium. (<b>B, C</b>) Confocal microscopy image of rhodamine/phalloidin labeled F-actin (green) and DAPI-labeled cell nuclei (blue) showing aspects of the structure of the ovarioles (peritoneal sheath removed) at the time they were used for HEX 70A detection. The actin-rich polyfusomes (arrowheads in B) are seen in the center of the cystocyte rosettes in the upper region of the germarium. Ring canals derived from polyfusomes (arrows in B and C) are apparent in the lower region of the germarium shown in B and in higher magnification in C. (<b>D</b>) Confocal microscopy of an ovariole (upper portion of the germarium) stained with DAPI. (<b>E</b>) The same ovariole showing foci of HEX 70a detected with anti-HEX 70a/Cy3 (red). (<b>F</b>) The merged D and E images. The insert in F shows a “control” ovariole (upper portion of the germarium) incubated with the pre-immune serum and subsequently stained with Cy3/DAPI. Arrowheads in D-F show nuclei of germline cells. Arrows in D–F point to nuclei of follicle cell precursors. In all figures, the upper portion of the germarium is oriented upward.</p

Immunolocalization of HEX 70a in the testioles of drone pupae (1 day after pupal ecdysis; developmental stage shown at the upper left corner).

<p>(<b>A</b>) Light microscopy section of a testiole stained with methylene blue/basic fuchsin showing a region containing groups of cystocytes (spermatogonia) (arrows) involved by somatic cells (somatic cell nuclei pointed by arrowheads). (<b>B</b>) Confocal microscopy image showing rhodamine/phalloidin labeled F-actin (green) and DAPI-labeled cell nuclei (blue); somatic cell nuclei are pointed by arrowheads; insert shows a magnified image of a cyst containing cystocytes (cystocyte nuclei pointed by arrows) and ring canals (asterisks). (<b>C–E</b>) Confocal microscopy images of a testiole from a drone taken at the same developmental phase, showing (<b>C</b>) DAPI-stained cell nuclei, (<b>D</b>) foci of HEX 70a detected with anti-HEX 70a/Cy3 (red), and (<b>E</b>) the merged C and D images. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029006#pone-0029006-g001" target="_blank">Figure 1E</a> the thick arrows show germ cell nuclei, the thin arrows show HEX 70a foci in the cytoplasm and the arrowheads show somatic cell nuclei.</p