Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera

The caste-specific regulation of vitellogenin synthesis in the honeybee represents a problem with many yet unresolved details. We carried out experiments to determine when levels of vitellogenin are first detected in hemolymph of female castes of Apis mellifera, and whether juvenile hormone and ecdysteroids modulate this process. Vitellogenin levels were measured in hemolymph using immunological techniques. We show that in both castes the appearance of vitellogenin in the hemolymph occurs during the pupal period, but the timing was different in the queen and worker. Vitellogenin appears in queens during an early phase of cuticle pigmentation approximately 60h before eclosion, while in workers the appearance of vitellogenin is more delayed, initiating in the pharate adult stage, approximately 10h before eclosion. The timing of vitellogenin appearance in both castes coincides with a slight increase in endogenous levels of juvenile hormone that occurs at the end of pupal development. The correlation between these events was corroborated by topical application of juvenile hormone. Exogenous juvenile hormone advanced the timing of vitellogenin appearance in both castes, but caste-specific differences in timing were maintained. Injection of actinomycin D prevented the response to juvenile hormone. In contrast, queen and worker pupae that were treated with ecdysone showed a delay in the appearance of vitellogenin. These data suggest that queens and workers share a common control mechanism for the timing of vitellogenin synthesis, involving an increase in juvenile hormone titers in the presence of low levels of ecdysteroids

A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera

The honeybee Apis mellifera has major ecological and economic importance. We analyze patterns of genetic variation at 8.3 million SNPs, identified by sequencing 140 honeybee genomes from a worldwide sample of 14 populations at a combined total depth of 634×. These data provide insight into the evolutionary history and genetic basis of local adaptation in this species. We find evidence that population sizes have fluctuated greatly, mirroring historical fluctuations in climate, although contemporary populations have high genetic diversity, indicating the absence of domestication bottlenecks. Levels of genetic variation are strongly shaped by natural selection and are highly correlated with patterns of gene expression and DNA methylation. We identify genomic signatures of local adaptation, which are enriched in genes expressed in workers and in immune system– and sperm motility–related genes that might underlie geographic variation in reproduction, dispersal and disease resistance. This study provides a framework for future investigations into responses to pathogens and climate change in honeybees.Swedish Research Council Formas (grant 2010-1295).http://www.nature.comhb201

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xyp type in D. nisus and D. semisquamosus and of the Xy r type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group

A study of the constitutive heterochromatin and nucleolus organizer regions of Isocopris inhiata and Diabroctis mimas (Coleoptera: Scarabaeidae, Scarabaeinae) using C-banding, AgNO3 staining and FISH techniques

Meiotic and mitotic chromosomes of Isocopris inhiata and Diabroctis mimas were studied by standard staining procedures, C-banding, silver nitrate staining and FISH using Apis mellifera 28S rDNA as probe. Isocopris inhiata presented a 2n = 18 (8II+ Xy p) karyotype, composed of meta-submetacentric chromosomes with gradual reduction in size. The karyotype of D. mimas was 2n = 20 (9II+ Xy p), composed of meta-submetacentric (pairs 1, 2, 3, 4 and 7) and acrocentric (pairs 5, 6, 8 and 9) chromosomes, with gradual reduction in size. Analysis of constitutive heterochromatin revealed similar C-banding patterns for both species, showing pericentromeric and telomeric bands and diphasic chromosomes. In addition, the X chromosomes of these species were found to be almost completely heterochromatic. The presence of chromocenters was checked in one or more phases of prophase I of these species. All heterochromatin reacted positively for the silver stain. By FISH analysis we were able to locate the rDNA in medium-size autosome pairs in both species and in the X chromosome of D. mimas

Cell death in ovarioles causes permanent sterility in Frieseomelitta varia worker bees

Frieseomelitta varia worker bees do not lay eggs even when living in queenless colonies, a condition that favors ovary development and oviposition in the majority of highly social bees. The permanent sterility of these worker bees was initially attributed to a failure in ovary morphogenesis and differentiation. Using transmission electron microscopy we found that at the beginning of the pupal phase the ovaries of F. varia workers are formed by four ovarioles, each of them composed of 1) a terminal filament at the apex of the ovarioles, containing juxtaposed and irregularly shaped cells, 2) a germarium with clusters of cystocytes and prefollicular cells showing long cytoplasmic projections that envelop the cystocyte clusters, 3) fusiform interfollicular and basal stalk precursor cells, and 4) globular, irregularly contoured basal cells with large nuclei. However, during the pupal phase an accentuated and progressive process of cell death takes place in the ovarioles. The dying cells are characterized by large membrane bodies, electron-dense apoptotic bodies, vacuoles, vesiculation, secondary lysosomes, enlarged rough endoplasmic reticulum cisternae, swollen mitochondria, pycnotic nuclei, masses of chromatin adjacent to the convoluted nuclear envelope, and nucleoli showing signs of fragmentation. Cell death continues in ovarioles even after the emergence of the workers. Once they become nurse bees, the ovaries have become transformed into a cell mass in which structurally organized ovarioles can no longer be identified. In F. varia workers, ovariole cell death most certainly is part of the program of caste differentiation

Regression of the lateral oviducts during the larval-adult transformation of the reproductive system of Melipona quadrifasciata and Frieseomelitta varia

Toward the end of the larval phase (pre-pupa), the reproductive systems of Melipona quadrifasciata and Frieseomelitta varia workers are anatomically similar. Scanning electron microscopy showed that during this developmental phase the right and left ovaries are fused and form a heart-shaped structure located above the midgut. Each ovary is connected to the genital chamber by a long and slender lateral oviduct. During pupal development, the lateral oviducts of workers from both species become extremely reduced due to a drastic process of cell death, as shown by transmission electron microscopy. During the lateral oviduct shortening, their simple columnar epithelial cells show some signs of apoptosis in addition to necrosis. Cell death was characterized by cytoplasmic vesiculation, peculiar accumulation of glycogen, and dilation of cytoplasmic organelles such as mitochondria and rough endoplasmic reticulum. The nuclei, at first irregularly contoured, became swollen, with chromatin flocculation and various areas of condensed chromatin next to the nuclear envelope. At the end of the pupal phase, deep recesses marked the nuclei. At emergence, worker and queen reproductive systems showed marked differences, although reduction in the lateral oviducts was an event occurring in both castes. However, in queens the ovarioles increased in length and the spermatheca was larger than that of workers. At the external anatomical level, the reproductive system of workers and queens could be distinguished in the white- and pink-eyed pupal phase. The metamorphic function of the death of lateral oviduct cells, with consequent oviduct shortening, is discussed in terms of the anatomical reorganization of the reproductive system and of the ventrolateral positioning of adult worker bee ovaries. (C) 2000 Wiley-Liss, Inc

Induction of heat shock proteins in the larval fat body of Apis mellifera L. bees

Five proteins were expressed in larval honey bee fat bod incubated in vitro in response to heat shock, as shown by SDS-PAGE and fluorography. The large heat shock proteins (82, 70 kDa) were inducible throughout the 5th instar whereas the small ones (29, 26, 16 kDa) were inducible only in certain phases of this instar. The synthesis of these HSPs was accompanied by generalized inhibition of overall protein synthesis and secretion in the culture medium. Fluorograms showed that the 76 and 74 kDa proteins were strongly inhibited by heat treatment. Western blots using a mouse monoclonal antibody against HSP72 and HSC73 permitted the inference that the 70 kDa larval protein accumulated in the honey bee fat body in response to heat shock corresponds to the HSP72 isoform. The Western blots also showed a 70 kDa faint band in fat bodies incubated at the control temperature (34 oC). This protein, also detected in incubation media independently of the temperature used, was interpreted as being the constitutively synthesized and secreted HSC73 isoform.Induction de protéines de choc thermique dans le corps adipeux des larves d' Apis mellifera L. L'expression de protéines en réponse à un choc thermique a été étudiée in vitro dans le corps adipeux de larves d'abeilles domestiques, Apis mellifera L., placées en étuve à 42, 47 et 55 oC durant 24 h en présence de [ $^3$H] L-leucine. Les protéines induites à ces températures ont été étudiées dans le corps adipeux de larves F1 et F2, qui sont aux phases initiales d'alimentation du $\rm 5^e$ stade larvaire, et celui de larves S1, qui ont terminé de s'alimenter et commencé à filer leur cocon (phase filante). Pour chaque expérience et pour les 3 phases larvaires, nous avons procédé à l'incubation in vitro de six corps adipeux pour le groupe témoin et d'autant pour le groupe expérimental. Les expériences ont été répétées au moins trois fois. L'incubation des témoins a été faite à 34 oC, température normalement maintenue au sein de la colonie. Après l'incubation, on a récolté séparément les corps adipeux et leur milieu. Des parties aliquotes d'extraits de corps adipeux et du milieu d'incubation ont été concentrées par précipitation acide. Les protéines radioactives synthétisées de novo par les cellules du corps adipeux et sécrétées dans le milieu d'incubation ont été quantifiées par spectrométrie à scintillation liquide et identifiées par SDS-PAGE et fluorographie. La technique du "Western blotting", utilisant un anticorps monoclonal de souris qui reconnaît deux membres de la famille HSP70, les isoformes HSC73 et HSP72, a été utilisé pour caractériser les protéines de choc thermique chez les larves d'abeilles. Bien qu'on ait observé une diminution significative de la synthèse et de la sécrétion totales de protéines dans les corps adipeux incubés à 42 oC (Figs. 1 et 2), les résultats ont montré que cette température induit la synthèse de cinq protéines dans les corps adipeux des larves. Deux d'entre elles, de 82 et 70 kDa, ont été induites à toutes les phases larvaires (F1, F2 et S1). Les trois autres protéines plus petites n'ont été induites qu'à certaines phases larvaires : la 29 kDa n'est apparue que chez les larves F1, la protéine 26 kDa chez les larves F2 et S1 et la protéine 16 kDa chez les larves F2 (Figs. 3a et 3b). à l'exception de cette dernière, les protéines induites par la chaleur n'ont pas été identifiées dans le milieu d'incubation (Fig. 4a). Ceci montre qu'elles n'ont pas été sécrétées mais sont restées stockées dans les cellules du corps adipeux, protégeant probablement les cellules des effets nocifs des températures élevées. Les protéines de choc thermique 26 et 16 kDa sont régulées par le développement. En comparant l'électrophorèse des corps adipeux des stades F1 , F2 et S1 incubés à 34 oC (Figs. 3a, 3b et 4a) on peut voir que la synthèse de la protéine 26 kDa est plus élevée en F1 qu'en F2 et inférieure en S1 ; la protéine 16 kDa, présente dans le corps adipeux de F1, est pratiquement indécelable dans ceux de F2 et de S1. D'autres protéines, régulées par le développement, ont été également notées : la protéine 180 kDa, bien visible au stade F1 seulement et la protéine 150 kDa bien évidente aux stades F2 et S1. La synthèse de ces deux protéines n'est pas affectée par le choc thermique. Une température élevée, tout en induisant des PCT, réduit simultanément la synthèse et la sécrétion des protéines 76 et 74 kDa (Figs. 3a, 3b et 4a, 4b), ce qui contribue à caractériser la réponse au stress thermique. Un anticorps monoclonal de souris contre l'isoforme HSP70 constitutive bovine (73 kDa) et l'isoforme HSP70 inductible (72 kDa) (Figs. 5 et 6) a reconnu les protéines correspondantes dans le corps adipeux larvaire. Ceci indique des propriétés structurales similaires et une homologie entre les protéines de choc thermique des larves d'abeilles et celles des mammifères

Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR

For obtaining accurate and reliable gene expression results it is essential that quantitative real-time RT-PCR (qRT-PCR) data are normalized with appropriate reference genes. The current exponential increase in postgenomic studies on the honey bee, Apis mellifera, makes the standardization of qRT-PCR results an important task for ongoing community efforts. For this aim we selected four candidate reference genes (actin, ribosomal protein 49, elongation factor 1-alpha, tbp-association factor) and used three software-based approaches (geNorm, BestKeeper and NormFinder) to evaluate the suitability of these genes as endogenous controls. Their expression was examined during honey bee development, in different tissues, and after juvenile hormone exposure. Furthermore, the importance of choosing an appropriate reference gene was investigated for two developmentally regulated target genes. The results led us to consider all four candidate genes as suitable genes for normalization in A. mellifera. However, each condition evaluated in this study revealed a specific set of genes as the most appropriated ones