Functional analysis of 10-hydroxy-2-decenoic acid (10HDA), inhibitor of histone deacetylases and main organic acid in royal jelly, in the differentiation of Apis mellifera L. castes

Author(s)

Giovana Craveiro

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-02-26

Examining board members

Klaus Hartmann Hartfelder; Maria Cristina Arias; Claudia Aparecida Rainho

• Advisor: Klaus Hartmann Hartfelder

Abstract

Bees of the species Apis mellifera are characterized by a highly advanced social organization, in which the queen and the workers are morphologically distinct and perform specific functions in the hive. The queen is responsible for reproduction, while the workers carry out all the maintenance tasks of the hive. Despite the great morphological and functional differences, queens and workers do not differ genetically, but represent a polyphenism. This is triggered mainly by the differential feeding of the larvae. Royal jelly, produced by nurse bees and offered in large quantities to queen larvae, contains compounds that are thought to regulate the development of the larvae, including their epigenetic state. One of these molecules is 10-hydroxy-2-decenoic acid (10HDA), which is present in higher quantities in royal jelly than in worker jelly. 10HDA has been identified as an inhibitor of histone deacetylase (HDAC) enzyme activity in mammalian cells, and based on this result our aim here was to discover its actual mode of action in the honey bee. We re-annotated the genes that encode the HDACs in the honey bee genome and we analyzed their expression pattern in the larvae. The results showed that there are differences between queens and workers with respect to expression levels, mainly in the older larvae, in the L5F2 stage for HDAC4, and in L5S1 for HDAC6. Subsequently we performed an experiment where larvae were reared for 24 h in vitro on a diet enriched in 10HDA, and we found that the highest concentration used was able to affect the HDAC4 transcript levels, which also is the histone deacetylase that is most highly expressed in bees. We also found that this treatment affected the expression of the dnmt3 gene, which encodes a DNA methyl transferase. In addition, we could see that the treatment was able to affect the expression levels of Krüppel-homolog 1 (Kr-h1), an immediate response gene to juvenile hormone (HJ), which plays an important role in caste development, as it promotes the differentiation of the queen ovary phenotype. Regarding the gene expression analyses made with ovarian tissue treated with 10HDA in vitro, we found that some key genes in ovarian differentiation, such as Tudor-SN, lncov2 and ILP-1, had expression levels similar to those seen in queens. Other genes of interest, such as dnmt3 and Egfr, showed an increase in expression in this treatment Finally, in experiments where were larvae received a 10HDA-enriched diet, or where dissected ovaries or nuclear extracts were exposed to 10HDA in vitro, we found that 10HDA did not affect the HDAC enzymatic activity. This was contrary to what we had expected from our initial hypotheses and the expression pattern of the HDAC genes, but it is consistent with the results of the analysis of respective enzyme activity in queen and worker larvae, where we also could not detect a difference. From this we conclude that the different quantities of 10HDA fed by the nurse bees to the queen and worker larvae do not seem to affect the HDACs enzymatic activity, despite the differences seen in the expression levels of the HDAC genes. This indicates that the primary function of 10HDA provided to the larvae in their diets may not be the inhibition of the histone deacetylases, and as such, would not be likely to have a major effect on the larval epigenetic states. (AU)

• FAPESP's process: 18/11271-9 - Functional analysis of 10-hydroxy-2-decenoic acid (10HDA), a histone deacetylase inhibitor and principal organic acid of royal jelly, with respect to the differentiation of the castes of Apis mellifera L.
• Grantee: Giovana Craveiro
• Support Opportunities: Scholarships in Brazil - Master