Comparative analysis of vitellogenin expression in stinglessbee species (Meliponini) that are different on reproductive activity

As operárias de abelhas sem ferrão são peculiares com relação à divisão do trabalho reprodutivo para a manutenção da colônia, visto que podem produzir ovos reprodutivos que originam os machos, além dos ovos tróficos destinados a alimentar a rainha. Desta forma, os estudos de expressão de genes e de proteínas envolvidos na biossíntese de vitelo vitelogênese - e incorporação deste material aos ovócitos contribuem para evidenciar diferenças intra e inter-específicas entre as fêmeas, em relação à fertilidade e comportamento reprodutivo. Os perfis de expressão do gene codificador da vitelogenina e da própria proteína, precursora da principal constituinte do vitelo, foram determinados para várias etapas do desenvolvimento de três espécies de abelhas sem ferrão. Para este estudo foram selecionadas as espécies Frieseomelitta varia, cujas operárias nunca põem ovos, mesmo em condições de orfandade, Scaptotrigona postica e Melipona scutellaris, cujas operárias desenvolvem os ovários e participam ativamente da produção de machos. O RNA total de corpo gorduroso sítio de biossíntese de vitelogenina de operárias destas espécies foi extraído e o cDNA obtido por transcrição reversa semiquantitativa foi amplificado, clonado e seqüenciado utilizando-se primers específicos para a vitelogenina de Apis mellifera. Os resultados revelaram que os cDNA parciais obtidos são bastante conservados entre F. varia, S. postica e M. scutellaris e mostram alta identidade (93-100%) em relação à região 3-terminal do cDNA da vitelogenina de A. mellifera. Entretanto, o perfil de abundância do transcrito difere entre as espécies de meliponíneos e entre estas e as abelhas melíferas. Em F. varia e S. postica a expressão do transcrito mostrou-se constitutiva ao longo dos períodos pupal e adulto, mas M. scutellaris mostrou diminuição da abundância de transcritos nas fases pupais mais avançadas e nas operárias recém-emergidas. Estas espécies diferem de A. mellifera cujas pupas não expressam o gene da vitelogenina. A expressão constitutiva deste gene em F. varia e S. postica mostra que a atividade do gene em questão não é modificada pela variação dos títulos de ecdisteróides e hormônio juvenil descrita para A. mellifera e outros insetos, indicando, portanto, ausência de controle da transcrição de vitelogenina por hormônios nas espécies de meliponíneos estudadas. No entanto, os resultados indicam a existência de controle nutricional da atividade do gene da vitelogenina, dado o aumento de expressão verificado em operárias F. varia alimentadas com dieta rica em proteínas (contendo pólen - a fonte de proteínas para as abelhas - e açúcar) em comparação com aquelas que receberam dieta exclusiva de carboidrato (açúcar). A presença da proteína vitelogenina na hemolinfa de F. varia ocorre concomitantemente com a expressão constitutiva do transcrito. Neste aspecto, difere de S. postica e M. scutellaris que também expressam o transcrito da vitelogenina ao longo do estágio pupal e adulto, mas a proteína correspondente somente é detectada nas operárias destas espécies que estão exercendo a função de nutridoras de crias. Pode-se concluir que o gene da vitelogenina é conservado entre as espécies de abelhas até aqui estudadas, porém sua expressão e possivelmente sua regulação diferem entre meliponíneos e abelhas melíferas, refletindo as diferentes estratégias utilizadas na divisão do trabalho reprodutivo.The stingless bee workers are peculiar with relation to reproductive division of labor to colony maintenance, since they are able to produce reproductive eggs that will develop in males, beyond trophic eggs to feed the queen. In this way, studies about gene and protein expression involved on the yolk biosynthesis vitellogenesis and the incorporation of this material to the oocytes contributes to evidence intra and inter-specific differences between females, in relation to fertility and reproductive behavior. The vitellogenin, the main yolk constituent precursor, gene expression and the protein itself profile were determined to several developmental stages of three stinglessbee species. To this work were selected the species Frieseomelitta varia, whose workers never lay eggs, even in a queenless condition, Scaptotrigona postica and Melipona scutellaris, whose workers develop their ovaries and participate actively to male production. The whole RNA of the fat body the vitellogenin biosynthesis site - of these worker species was extracted and the cDNA obtained by semiquantitative reverse transcription amplified, cloned and sequenced through Apis mellifera vitellogenin specific primers. The results reveal that the obtained partial cDNAs are very conserved among F. varia, S. postica and M. scutellaris and show high identity (93-100%), in relation to 3-end A. mellifera vitellogenin gene. However, transcript abundance profile is different among stinglessbee species and with honeybees. In F. varia and S. postica the transcript expression is constitutive during pupal and adult periods, but M. scutellaris showed transcript reduction in the advanced pupal phases and newly emerged workers. These species are different from A. mellifera pupae that do not express vitellogenin gene. This constitutive gene expression in F. varia and S. postica shows that the gene activity is not modified by the ecdysteroid and juvenile hormone titers as descript to A. mellifera and other insects, indicating absence of vitellogenin transcriptional control by hormones in the studied stingless bee species. However, the results indicate the existence of vitellogenin gene activity nutritional control, given the verified expression increase in F. varia workers and drones fed with a rich protein diet (with pólen the protein bee source and sugar) in comparison with which ones received exclusive sugar diet. The presence of vitellogenin protein on the F. varia hemolimph occurs concomitantly with the constitutive transcript expression. In this aspect, is different of S. postica and M. scutellaris which also express the vitellogenin transcript during pupal and adult stages, but the correspondent protein is only detected on the workers of these species that are exerting nurse tasks. Is possible conclude that vitellogenin gene is conserved among the studied bee species, but its expression and possibly regulation are different among stingless bees and honey bees, reflecting different strategies used on reproductive division of labor

Comparative analysis of vitellogenin expression in stinglessbee species (Meliponini) that are different on reproductive activity

As operárias de abelhas sem ferrão são peculiares com relação à divisão do trabalho reprodutivo para a manutenção da colônia, visto que podem produzir ovos reprodutivos que originam os machos, além dos ovos tróficos destinados a alimentar a rainha. Desta forma, os estudos de expressão de genes e de proteínas envolvidos na biossíntese de vitelo vitelogênese - e incorporação deste material aos ovócitos contribuem para evidenciar diferenças intra e inter-específicas entre as fêmeas, em relação à fertilidade e comportamento reprodutivo. Os perfis de expressão do gene codificador da vitelogenina e da própria proteína, precursora da principal constituinte do vitelo, foram determinados para várias etapas do desenvolvimento de três espécies de abelhas sem ferrão. Para este estudo foram selecionadas as espécies Frieseomelitta varia, cujas operárias nunca põem ovos, mesmo em condições de orfandade, Scaptotrigona postica e Melipona scutellaris, cujas operárias desenvolvem os ovários e participam ativamente da produção de machos. O RNA total de corpo gorduroso sítio de biossíntese de vitelogenina de operárias destas espécies foi extraído e o cDNA obtido por transcrição reversa semiquantitativa foi amplificado, clonado e seqüenciado utilizando-se primers específicos para a vitelogenina de Apis mellifera. Os resultados revelaram que os cDNA parciais obtidos são bastante conservados entre F. varia, S. postica e M. scutellaris e mostram alta identidade (93-100%) em relação à região 3-terminal do cDNA da vitelogenina de A. mellifera. Entretanto, o perfil de abundância do transcrito difere entre as espécies de meliponíneos e entre estas e as abelhas melíferas. Em F. varia e S. postica a expressão do transcrito mostrou-se constitutiva ao longo dos períodos pupal e adulto, mas M. scutellaris mostrou diminuição da abundância de transcritos nas fases pupais mais avançadas e nas operárias recém-emergidas. Estas espécies diferem de A. mellifera cujas pupas não expressam o gene da vitelogenina. A expressão constitutiva deste gene em F. varia e S. postica mostra que a atividade do gene em questão não é modificada pela variação dos títulos de ecdisteróides e hormônio juvenil descrita para A. mellifera e outros insetos, indicando, portanto, ausência de controle da transcrição de vitelogenina por hormônios nas espécies de meliponíneos estudadas. No entanto, os resultados indicam a existência de controle nutricional da atividade do gene da vitelogenina, dado o aumento de expressão verificado em operárias F. varia alimentadas com dieta rica em proteínas (contendo pólen - a fonte de proteínas para as abelhas - e açúcar) em comparação com aquelas que receberam dieta exclusiva de carboidrato (açúcar). A presença da proteína vitelogenina na hemolinfa de F. varia ocorre concomitantemente com a expressão constitutiva do transcrito. Neste aspecto, difere de S. postica e M. scutellaris que também expressam o transcrito da vitelogenina ao longo do estágio pupal e adulto, mas a proteína correspondente somente é detectada nas operárias destas espécies que estão exercendo a função de nutridoras de crias. Pode-se concluir que o gene da vitelogenina é conservado entre as espécies de abelhas até aqui estudadas, porém sua expressão e possivelmente sua regulação diferem entre meliponíneos e abelhas melíferas, refletindo as diferentes estratégias utilizadas na divisão do trabalho reprodutivo.The stingless bee workers are peculiar with relation to reproductive division of labor to colony maintenance, since they are able to produce reproductive eggs that will develop in males, beyond trophic eggs to feed the queen. In this way, studies about gene and protein expression involved on the yolk biosynthesis vitellogenesis and the incorporation of this material to the oocytes contributes to evidence intra and inter-specific differences between females, in relation to fertility and reproductive behavior. The vitellogenin, the main yolk constituent precursor, gene expression and the protein itself profile were determined to several developmental stages of three stinglessbee species. To this work were selected the species Frieseomelitta varia, whose workers never lay eggs, even in a queenless condition, Scaptotrigona postica and Melipona scutellaris, whose workers develop their ovaries and participate actively to male production. The whole RNA of the fat body the vitellogenin biosynthesis site - of these worker species was extracted and the cDNA obtained by semiquantitative reverse transcription amplified, cloned and sequenced through Apis mellifera vitellogenin specific primers. The results reveal that the obtained partial cDNAs are very conserved among F. varia, S. postica and M. scutellaris and show high identity (93-100%), in relation to 3-end A. mellifera vitellogenin gene. However, transcript abundance profile is different among stinglessbee species and with honeybees. In F. varia and S. postica the transcript expression is constitutive during pupal and adult periods, but M. scutellaris showed transcript reduction in the advanced pupal phases and newly emerged workers. These species are different from A. mellifera pupae that do not express vitellogenin gene. This constitutive gene expression in F. varia and S. postica shows that the gene activity is not modified by the ecdysteroid and juvenile hormone titers as descript to A. mellifera and other insects, indicating absence of vitellogenin transcriptional control by hormones in the studied stingless bee species. However, the results indicate the existence of vitellogenin gene activity nutritional control, given the verified expression increase in F. varia workers and drones fed with a rich protein diet (with pólen the protein bee source and sugar) in comparison with which ones received exclusive sugar diet. The presence of vitellogenin protein on the F. varia hemolimph occurs concomitantly with the constitutive transcript expression. In this aspect, is different of S. postica and M. scutellaris which also express the vitellogenin transcript during pupal and adult stages, but the correspondent protein is only detected on the workers of these species that are exerting nurse tasks. Is possible conclude that vitellogenin gene is conserved among the studied bee species, but its expression and possibly regulation are different among stingless bees and honey bees, reflecting different strategies used on reproductive division of labor

Testicular reabsorption in adult males of Melipona bicolor bicolor lepeletier (Hymenoptera, Apidae, Meliponini)

The present paper reports morphological features of the testes reabsorption in adult male of Melipona bicolor bicolor. The ultrastructural features of the degenerating sperm cells and cyst cells are described. It is suggested that the material of the reabsorbed testes may serve as nutritional source for the adult male

Morphology and protein patterns of honey bee drone accessory glands.

We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen

Dimorphic Ovary Differentiation in Honeybee (<i>Apis mellifera</i>) Larvae Involves Caste-Specific Expression of Homologs of <i>Ark</i> and <i>Buffy</i> Cell Death Genes

<div><p>The establishment of the number of repeated structural units, the ovarioles, in the ovaries is one of the critical events that shape caste polyphenism in social insects. In early postembryonic development, honeybee (<i>Apis mellifera</i>) larvae have a pair of ovaries, each one consisting of almost two hundred ovariole primordia. While practically all these ovarioles continue developing in queen-destined larvae, they undergo massive programmed cell death (PCD) in worker-destined larvae. So as to gain insight into the molecular basis of this fundamental process in caste differentiation we used quantitative PCR (qPCR) and fluorescent <i>in situ</i> hybridization (FISH) to investigate the expression of the <i>Amark</i> and <i>Ambuffy</i> genes in the ovaries of the two honeybee castes throughout the fifth larval instar. These are the homologs of <i>ark</i> and <i>buffy Drosophila melanogaster</i> genes, respectively, involved in activating and inhibiting PCD. Caste-specific expression patterns were found during this time-window defining ovariole number. <i>Amark</i> transcript levels were increased when ovariole resorption was intensified in workers, but remained at low levels in queen ovaries. The transcripts were mainly localized at the apical end of all the worker ovarioles, but appeared in only a few queen ovarioles, thus strongly suggesting a function in mediating massive ovariolar cell death in worker larvae. <i>Ambuffy</i> was mainly expressed in the peritoneal sheath cells covering each ovariole. The levels of <i>Ambuffy</i> transcripts increased earlier in the developing ovaries of queens than in workers. Consistent with a protective role against cell death, <i>Ambuffy</i> transcripts were localized in practically all queen ovarioles, but only in few worker ovarioles. The results are indicative of a functional relationship between the expression of evolutionary conserved cell death genes and the morphological events leading to caste-specific ovary differentiation in a social insect.</p></div

<i>Amark</i> transcript localization in worker ovaries at the L₅F phase of the fifth larval instar.

<p>(A) Ovarioles showing DAPI-stained nuclei. (B) The same ovarioles as in A, but labeled with the AlexaFluor555-<i>Amark</i> sense probe (FISH negative control), shows only a reddish background coloration. (C and D) Ovarioles labeled with the AlexaFluor555-<i>Amark</i> antisense probe and DAPI: the dashed line in C highlights an ovariole with large <i>Amark</i> foci (red) in the intermediary region (arrowheads). <i>Amark</i> foci (arrowheads in D) are also concentrated at the apical region of some ovarioles (shown in higher magnification and outlined by dashed lines in D).</p

Honeybee developmental stages.

<p>Developmental phases and ovaries of honeybee workers and queens in the fifth larval instar, which is subdivided into feeding (L₅F), cocoon-spinning (L₅S) and prepupal (PP) phases.</p

<i>Amark</i> transcript localization in worker ovaries at the L₅S and PP phases of the fifth larval instar.

<p>FISH with AlexaFluor555-labeled <i>Amark</i> antisense probe (red foci). Cell nuclei stained with DAPI (blue). (A) An L₅S-phase ovary showing <i>Amark</i> transcripts highly concentrated at the apical end of the ovarioles (arrows). <i>Amark</i> foci are also seen outside the apical region (arrowheads). This pattern of <i>Amark</i> labeling is generalized throughout the worker ovaries. (B and C) At the end of the fifth larval instar (PP phase) the ovary continues to show <i>Amark</i> transcripts concentrated at the apical end of the ovarioles (arrows). (D) Detail showing small-sized degenerating nuclei (arrowheads) at the tip of the ovarioles (PP phase). (E) The same ovary as seen in D, but showing <i>Amark</i> foci (arrowheads) in the region where degenerating nuclei were identified.</p

<i>Ambuffy</i> transcripts localization in the ovaries of queens at the L₅F, L₅S and PP phases of the fifth larval instar.

<p>FISH with AlexaFluor488-labeled <i>Ambuffy</i> antisense probe (green foci). Cell nuclei stained with DAPI (blue). Arrowheads and arrows point to peritoneal sheath cells and ovariole cells, respectively. (A) An L₅S-phase ovary showing <i>Ambuffy</i> foci in the cytoplasm of the peritoneal sheath cells covering all ovarioles. (B) Apical portion of one of the ovarioles shown in A: <i>Ambuffy</i> foci were evident in the peritoneal sheath cells, but barely seen in the interior of the ovariole. (C) Detail of a L₅F-phase ovariole in higher magnification: <i>Ambuffy</i> expression is clearly higher in the peritoneal sheath cells than in the ovariole cells. (D) At the PP phase, <i>Ambuffy</i>-labeling was comparatively less intense than in the previous L₅S phase shown in A. The asterisk marks an ovariole where <i>Ambuffy</i> foci were no longer evident.</p

Gene expression profiles in honeybee ovaries.

<p>Relative quantification (RT-qPCR) of <i>Amark</i> (A) and <i>Ambuffy</i> (B) transcripts in the ovaries of queens and workers in the feeding (L₅F), cocoon-spinning (L₅S) and prepupal (PP) phases of the fifth larval instar. The gene encoding an <i>A. mellifera</i> ribosomal protein (<i>Amrp49</i>) was used for normalization. Each column represents the mean of three independent samples, each composed of five ovary pairs. Different letters indicate significant differences between groups (p≤0.001).</p