Transcriptional analysis of candidate genes for honey bees (Apis mellifera L.) caste determination

Author(s)

Marina Carvalho Peruzzolo

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-02-23

Examining board members

Klaus Hartmann Hartfelder; Lívia Maria Rosatto Moda; Francis de Morais Franco Nunes

• Advisor: Klaus Hartmann Hartfelder; Carlos Antônio Mendes Cardoso Júnior

Abstract

Apis mellifera bees are characterized by their advanced social organization, in which queens, workers and drones are morphologically distinct and perform specific functions in the hive. The queen and drone are responsible for reproduction, while the workers carry out the other tasks of colony maintenance, like brood care and foraging. Despite being morphologically and functionally distinct, queens and workers do not differ genetically, but it is the quality of the diet supplied to the larva that promotes the differentiation of their morphologically distinct characteristics. This polyphenism is due, in part, to epigenetic factors, such as DNA methylation and non-coding RNAs, which affect and, consequently, alter the expression of key genes in the processes of cell differentiation and programmed cell death. One of the main morphological characteristics that differs among the castes is the reproductive system: the queens fully develop their ovaries, while a programmed cell death process degrades most of the workers\' ovaries during larval development, thus guaranteeing the queens\' reproductive monopoly. With cell proliferation in the ovaries during the larval stage, the adult queens end up with up to 200 ovarioles per ovary, and they can lay approximately one thousand eggs per day. On the other hand, workers, after the process of cell death in the larval ovaries, have only between 2 and 20 ovarioles left per ovary, and these are facultatively inactive. The aim of this work was to quantify the transcript levels of candidate genes, especially ones encoding epigenetic factors, during the caste-specific ovarian development in queen and worker larvae. The results showed that the transcript levels of the genes lncov1 and Tudor-SN in the larval ovaries of workers coincide with expressive programmed cell death at the L5F3 stage. The lncov2 and fringe genes showed statistically significant expression peaks in the L5F1 stage, which marks the beginning of programmed cell death. However, these genes are expressed with opposite directionality: while lncov2 expression was higher in queens, fringe was significantly higher in workers. Interestingly, lncov2 is hosted in an intron of fringe, indicating a possible repressor action of this lncRNA on fringe. The expression of the Hdac4 gene, the most expressed histone deacetylase in bee larvae, was not different between queens and workers, suggesting that the differential expression observed in whole body samples is likely due to other tissues, whose expression is caste-specific, potentially the brain and/or fat body. Although previous experiments indicated that the knockdown of the DNA methyltransferase 3 (DNMT3) results in the development of queen-like ovaries, the gene was not differentially expressed in the larval ovaries, but like Hdac4 it showed a marked expression peak in the early fifth larval instar. For another gene, Anarchy, which is a key factor for sterility in adult workers, we found that its expression is higher in the ovaries of worker larvae at the stage of programmed cell death (L5F3). In addition to these genes, we also analyzed the Krüppel-homolog 1 (Kr-h1) gene, a transcription factor in the immediate response to juvenile hormone, which plays an important role in caste development. promoting differentiation of ovaries into queens. For Kr-h1 we observed significant expression in the ovaries of workers in the L5F3 stage. Finally, Egfr, another gene of interest, did not show caste-specific ovarian expression. With this work we identified genes that are potentially involved in the differential development of the ovaries, providing a set of data for future functional validation. (AU)

• FAPESP's process: 19/07264-0 - Role of the DNA methyltransferase 3 in the life cycle of adult honey bee workers, Apis mellifera L (Apidae, Hymenoptera)
• Grantee: Marina Carvalho Peruzzolo
• Support Opportunities: Scholarships in Brazil - Master