Transcriptome analysis of brain, intestine and hypopharyngeal glands of Apis mellifera using the Oxford Nanopore third generation sequencing technology

Introduction Apis mellifera plays a crucial role in biodiversity and ecological balance. So far, honey bee transcriptome and its modifications induced by stress or pathogens have been poorly studied. Thus, the characterization of the transcriptional profiles of key organs in response to biotic and abiotic factors could fill these gaps and provide new knowledge. Materials and Methods Honey bees from four apiaries in Northern Italy were collected and immediately frozen in liquid nitrogen. Brain, hypopharyngeal glands, and intestine were excised and total RNA was extracted from four pools of each tissue (1 pool = 3 bees from a single apiary). Twelve tissue-specific cDNA libraries were constructed and sequenced using the Nanopore Mk1C. State-of-the-art software were used for raw data analysis, reads alignment, and splicing isoforms detection. Results About 27 million sequences per tissue were obtained. A total of 55,651 isoforms were identified, of which 21,501 were expressed in at least one of target tissues. Out of these, 6,719 transcripts were annotated, while 10,071 were identified as novel transcripts. The brain showed the highest number of transcripts (14,689), followed by the intestine (8,953) and hypopharyngeal glands (5,227). The analysis of differential expression among the tissues highlighted 2,371 differentially expressed transcripts (DETs), of which 1,236 with known biological function and 715 novel transcripts. The most abundant DETs were those 'activated' exclusively in the brain (some opsins, crucial for vision, and a gene with a fundamental role in longevity) or intestine (genes involved in apoptosis). Several DETs were moderately, but differentially, expressed in all the target organs. As an example, Glutathione S-transferase theta-3 and peroxiredoxin 6 genes, involved in oxidative stress response, were significantly more ‘activated’ in the intestine and hypopharyngeal glands. Discussion and Conclusion The present study, carried out within the RC IZS VE 09/20 project and funded by the Ministry of Health, has improved the knowledge of the transcriptome of three key organs of A. mellifera and investigated its complexity by identifying new transcripts. Therefore, it represents a valuable basis for further molecular investigations into honey bee response to environmental stress.