Positive selection in cytochrome P450 genes is associated with gonad phenotype and mating strategy in social bees.

The honey bee, Apis mellifera differs from all other social bees in its gonad phenotype and mating strategy. Honey bee queens and drones have tremendously enlarged gonads, and virgin queens mate with several males. In contrast, in all the other bees, the male and female gonads are small, and the females mate with only one or very few males, thus, suggesting an evolutionary and developmental link between gonad phenotype and mating strategy. RNA-seq comparisons of A. mellifera larval gonads revealed 870 genes as differentially expressed in queens versus workers and drones. Based on Gene Ontology enrichment we selected 45 genes for comparing the expression levels of their orthologs in the larval gonads of the bumble bee Bombus terrestris and the stingless bee, Melipona quadrifasciata, which revealed 24 genes as differentially represented. An evolutionary analysis of their orthologs in 13 solitary and social bee genomes revealed four genes with evidence of positive selection. Two of these encode cytochrome P450 proteins, and their gene trees indicated a lineage-specific evolution in the genus Apis, indicating that cytochrome P450 genes may be involved in the evolutionary association of polyandry and the exaggerated gonad phenotype in social bees.

Sex- and caste-specific transcriptomes of larval honey bee (Apis mellifera L.) gonads: DMRT A2 and Hsp83 are differentially expressed and regulated by juvenile hormone.

The gonads of honey bee, Apis mellifera, queens and drones are each composed of hundreds of serial units, the ovarioles and testioles, while the ovaries of the adult subfertile workers consist of only few ovarioles. We performed a comparative RNA-seq analysis on early fifth-instar (L5F1) larval gonads, which is a critical stage in gonad development of honey bee larvae. A total of 1834 genes were identified as differentially expressed (Padj < 0.01) among the three sex and caste phenotypes. The Gene Ontology analysis showed significant enrichment for metabolism, protein or ion binding, and oxidoreductase activity, and a KEGG analysis revealed metabolic pathways as enriched. In a principal component analysis for the total transcriptomes and hierarchical clustering of the DEGs, we found higher similarity between the queen and worker ovary transcriptomes compared to the drone testis, despite the onset of programmed cell death in the worker ovaries. Four DEGs were selected for RT-qPCR analyses, including their response to juvenile hormone (JH), which is a critical factor in the caste-specific development of the ovaries. Among these, DMRT A2 and Hsp83 were found upregulated by JH and, thus, emerged as potential molecular markers for sex- and caste-specific gonad development in honey bees.

Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development.

Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the final larval instar, when massive programmed cell death leads to the degeneration of 95-99% of the ovariole anlagen in workers. The higher juvenile hormone (JH) levels in queen larvae protect the ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate and worker sterility, we performed a microarray analysis on fourth and early fifth instar queen and worker ovaries. For the fourth instar we found nine differentially expressed genes (DEGs) with log2FC > 1.0, but this number increased to 56 in early fifth-instar ovaries. We selected 15 DEGs for quantitative PCR (RT-qPCR) analysis. Nine differed significantly by the variables caste and/or development. Interestingly, genes with enzyme functions were higher expressed in workers, while those related to transcription and signaling had higher transcript levels in queens. For the RT-qPCR confirmed genes we analyzed their response to JH. This revealed a significant up-regulation for two genes, a short chain dehydrogenase reductase (sdr) and a heat shock protein 90 (hsp90). Five other genes, including hsp60 and hexamerin 70b (hex70b), were significantly down-regulated by JH. The sdr gene had previously come up as differentially expressed in other transcriptome analyses on honey bee larvae and heat shock proteins are frequently involved in insect hormone responses, this making them interesting candidates for further functional assays.