A. P. Lourenço

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 realtime 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.ZusammenfassungDie quantitative real-time RT-PCR (qRT-PCR) Methode enwickelt sich zu einer der am häufigsten benutzten Methode zur Quantifizierung von mRNAs. Für exakte und zuverlässig reproduzierbare Ergebnisse ist es allerdings notwendig, dass die jeweiligen qRT-PCR Werte mittels eines geeigneten Referenzgens normalisiert werden. Trotz der Verfügbarkeit von Referenzgen-Studien bei verschiedenen Organismen ist die Bewertung geeigneter Referenzgene für den jeweiligen Organismus, in diesem Fall die Honigbiene Apis mellifera, erforderlich. Genau dies war das Ziel der vorliegenden Untersuchung, und dafür wählten wir folgende Gene als Kandidaten aus: actin, rp49, ef1-alpha and tbp-af (Tab. I). Diese wurden mittels der Computerprogramme geNorm, BestKeeper und Normfinder auf ihre Eignung und Eigenschaften als Referenzgene getestet. Wir untersuchten dies in verschiedenen biologischen Zusammenhängen: in verschiedenen Phasen der postembryonalen Entwicklung, in verschiedenen Geweben und Organen und nach Behandlung mit Juvenilhormon (JH-III).Für Studien zur Postembryonalentwicklung erwiesen sich bei Verwendung von geNorm actin und tbp-af und bei Verwendung von BestKeeper und NormFinder rp49 als die jeweils stabilsten Gene (Tab. II und III). Trotz dieser Unterschiede in der Rangfolge der Eignung zeigten alle vier Referenzgene für das Entwicklungsstadium F3 die höchsten mRNA-Werte an (Abb. 1). Dies weist darauf hin, dass die Regulierung der Genexpression in der Entwicklung in weitem Maße korreliert ist, und dass sie in der Larval- und Pupalentwicklung wahrscheinlich stark von physiologischen Faktoren beeinflusst wird. Ähnliche Unterschiede in der Eignung als Referenzgene waren auch bei den Gewebestudien und nach JH-III-Behandlung zu sehen. Während geNorm in diesen Situationen actin und ef1-alpha als die am besten geeigneten Gene auswies (Tab. II und III), waren dies rp49 und ef1-alpha, wenn BestKeeper und NormFinder als Bewertungsprogramme eingesetzt wurden (Tab. II und III). Obwohl bei den Vergleichen verschiedener Gewebe und Organe die Expressionswerte aller vier Gene signifikant schwankten (Abb. 2), wurden sie bei den BestKeeper- und geNorm-Analysen als stabile Referenz-Gene bewertet. Für Untersuchungen zu Behandlungen mit JH-III waren die stabilsten Gene ef1-alpha und tbp-af (geNorm), tbp-af (Best-Keeper) und actin (NormFinder) (Tab. II and III). Da wir für die Kontrollen (Aceton behandelteArbeiterinnen) und die JH-III behandelten Arbeiterinnen keine Unterschiede in der Expression dieser Gene fanden (Abb. 3), zeigt dies, dass bei solchen Versuchen alle vier Gene zur Normalisierung eingesetzt werden können.In einem weiteren Versuchsansatz untersuchten wir, welche Auswirkungen die Auswahl von Referenzgenen auf die jeweiligen Zielgene hat. Für die Zielgene jhe-like und proPO fanden wir, dass die Normalisierung mittels eines weniger geeigneten Referenzgens eine deutliche Auswirkung auf die Berechnung der relativen Genexpressionswwerte hat, insbesondere, wenn die Proben niedrige mRNA-Werte haben (Abb. 4).Unsere Untersuchung zeigt, dass alle vier Gene im Prinzip als Referenzgene für quantitative Genexpressionsstudien bei A. mellifera eingesetzt werden können. Da jedoch jeder biologische Kontext eine jeweils andere Kombination von Referenzgenen als am besten geeignet auswies, schlagen wir vor, dass jeweils immer zwei dieser Gene für die Normalisierung verwendet werden.

Trade-off between immune stimulation and expression of storage protein genes.

Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.

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.

Areas of endemism in the Atlantic Forest: quantitative biogeography insights from orchid bees (Apidae: Euglossini)

Orchid bees are endemic to the Neotropics and were sampled more intensively in the Atlantic Forest in the last decade than in that of other Brazilian biomes. In this study, we aimed at identifying the main distributional patterns and areas of endemism of Euglossini orchid bee species in the Atlantic Forest using parsimony analysis of endemism and endemicity analysis. The results of these analyses were partly congruent and supported the idea that the distribution of orchid bees is structured into at least five areas of endemism: Pernambuco/coastal Bahia; Espírito Santo/Rio de Janeiro/south of Minas Gerais; north of Minas Gerais/central Bahia; southeast of Minas Gerais/northeast, central and coast of São Paulo/central and coastal Paraná; and central/coast of Santa Catarina-Rio Grande do Sul. Most of these areas were consistent with other groups of organisms and indicate the existence of real areas of endemism in the Atlantic Rain Forest.

Propolis consumption ramps up the immune response in honey bees infected with bacteria

Among their natural defenses against pathogens and parasites, honey bees coat nest cavity surfaces with propolis. Consequently, they are able to economize on immune system activation, lowering energetic costs and improving longevity. However, the mechanisms through which propolis acts to protect bees are unknown. Here we show that 0.1% propolis fed in a pollen substitute diet greatly increases activation of antimicrobial peptide genes (defensin-1, abaecin, hymenoptaecin, and apidaecin) in bees injected with Escherichia coli, compared to infected bees fed the same diets without propolis. This increase was not seen in uninfected bees fed propolis. In addition to its protective role in the hive, propolis stimulates high-level expression of the immune system response in bees challenged with microorganisms. Whether this increase translates into improved disease control will require laboratory and field tests with pathogens.