A Study on the Possibility of Molecular Communication between Honey Bees and Sunflower through the Interspecies Transfer of Small RNAs

Background: Honeybees play a crucial role in nature, primarily through their involvement in pollination, which significantly enhances agricultural productivity and contributes to environmental restoration.It is estimated that nearly one-third of global food production is directly or indirectly reliant on insect pollination, with honeybees being the most prominent among them.Studies indicate that the contribution of honeybees to agricultural output in various countries is 69-143 times greater than their direct production.Over a decade has passed since the initiation of research on the interspecies transfer of dietary microRNAs (miRNAs) and their regulatory roles in host organisms, yet this topic remains vibrant and current.Limited studies have been conducted on the role of dietary miRNAs in honeybees, which are critical pollinators in agricultural industries and natural ecosystems.The potential for gene expression regulation among different species via dietary miRNAs could provide significant benefits in the interactions between plants and their pollinators, allowing plants to modulate the gene expression of pollinating insects through small RNA transfer.In other words, miRNAs can be transferred not only between cells and tissues within an organism but also across different species while maintaining their regulatory functions.Given the vital role of honeybees in plant pollination, this insect may serve as a more suitable model for studying these interactions.Therefore, the present study aims to investigate the molecular relationship between this beneficial social insect and its host plants through the dietary transfer of exogenous miRNAs, potentially illuminating new and extensive dimensions of the beneficial effects of this interaction.Methods: Pollens were collected using pollen traps from honeybee hives located on a sunflower farm in the Shal region of Qazvin, Iran, during thepeak flowering days of the plant.The collected pollens were immediately examined morphologically and transferred to liquid nitrogen tanks to preserve freshness and prevent RNA degradation.For nutritional experiments, four hives were utilized to gather nurse bees under controlled conditions.Approximately 100 young bees from each hive were transferred into specially designed cages.Two cages served as controls (fed with sugar syrup) while the two others were treated with sunflower pollens.To maintain natural hive conditions (queen pheromone, temperature, and humidity) during the experiment, the cages containing nurse bees were placed on the upper level of the hive.To cleanse the digestive system of the bees, all treatments were fed with sugar syrup for 48 h.Subsequently, to encourage further feeding, the bees were kept hungry for about 3 h.In the following 24 h, the control group continued receiving sugar syrup while the treatment group was fed a solution consisting of 30% sugar and 70% sunflower pollen.After completing the nutritional experiments, the bees were anesthetized using cold temperatures, and their midgut tissues were collected, homogenized in Trizol, and stored at -80 C until extraction.RNA from both pollen and honeybee midgut tissues was extracted using a miRNeasy mini Kit from Qiagen, and samples were sent to Novogene in Beijing, China, for sequencing.Following successful Small RNA-Seq sequencing, bioinformatics analyses were conducted to identify sunflower-derived miRNAs and track their presence in bees from different feeding groups.Subsequently, RT-qPCR was employed to validate results obtained from bioinformatics analyses.Results: The results of bioinformatics analysis indicated the identification of 11 plant miRNAs (miR-148a, miR-26a, miR-21-5p, miR-143, miR-27a, miR-203, let-7g, miR-126, miR-30d, miR-101, and miR-103) in honeybees fed with sunflower pollen while no plant miRNAs were found