Long-term inhibition of ferritin2 synthesis in trophocytes and oenocytes by ferritin2 double-stranded RNA ingestion to investigate the mechanisms of magnetoreception in honey bees (Apis mellifera)

Abstract

Behavioral studies indicate that honey bees (Apis mellifera) have a capacity for magnetoreception and superparamagnetic magnetite is suggested to be a magnetoreceptor. The long-term inhibition of magnetite formation can be employed to explore the bee’s magnetoreception. A recent study shows that magnetite formation, ferritin2 messenger RNA (mRNA) expression, and the protein synthesis of ferritin2 in trophocytes and oenocytes were all inhibited by a single injection of ferritin2 double-stranded RNA (dsRNA) into the hemolymph of honey bees but how to maintain this knockdown of ferritin2 for the long-term is unknown. In this study, we injected ferritin2 dsRNA into the hemolymph of worker bees three times every six days to maintain long-term inhibition; however, multi-microinjections accelerated the death of the bees. To overcome this problem, we further reared newly emerged worker bees daily with ferritin2 dsRNA throughout their lives, demonstrating no impact on their lifespans. Follow-up assays showed that the mRNA expression and protein synthesis of ferritin2 were persistently inhibited. These findings verified that daily ferritin2 dsRNA ingestion not only displays the long-term inhibition of mRNA expression and protein synthesis of ferritin2, but also did not damage the bees. This method of long-term inhibition can be used in behavioral studies of magnetoreception in honey bees.

Middle-Aged Worker Bees Express Higher Innate Immunity than Young Worker Bees in the Abdomen without the Digestive Tract of Worker Bees Reared in an Incubator

“…The brood combs containing pupae and a few newly emerged worker bees from different colonies were transferred to an incubator (34 • C, 75% relative humidity) [25]. The newly emerged worker bees from brood combs were randomly collected, labeled with white paint, and put into field hives [26].…”

“…5-day-old or 25-day-old worker bees were collected and anesthetized on ice. Total RNA was extracted from the whole body of individual 5-day-old or individual 25-day-old worker bees reared in field hives, the whole body of individual 5-day-old or individual 25-day-old worker bees reared in a 34 • C incubator, and the abdomen without the digestive tract of individual 5-day-old or individual 25-day-old worker bees reared in a 34 • C incubator using Trizol ® Reagent (15596018; Invitrogen, Carlsbad, CA, USA) [25]. To prepare the abdomen without the digestive tract, the digestive tract was pulled out from the anus of the abdomen by tweezers.…”

“…The qPCR was performed using a CFX connect RT-PCR detection system (Bio-Rad Laboratories), and each reaction contained 0.5 µL of 10 µM of each primer, 12.5 µL of SYBR Green (170-8882; Bio-Rad Laboratories), 1 µL of diluted cDNA, and 10.5 µL of ddH 2 O in a final volume of 25 µL. The β-actin gene was used as a reference gene when measuring gene expression in honey bees [16,25,[28][29][30]. The primers were designed according to GenBank's nucleotide sequences, and primer sequences are shown in Table 1.…”

“…The primers were designed according to GenBank's nucleotide sequences, and primer sequences are shown in Table 1. The PCR program was 95 • C for 3 min, followed by 39 cycles of denaturation at 95 • C for 10 s and annealing at 60 • C for 30 s [25]. All samples were run in quadruplicate [25].…”

“…The PCR program was 95 • C for 3 min, followed by 39 cycles of denaturation at 95 • C for 10 s and annealing at 60 • C for 30 s [25]. All samples were run in quadruplicate [25]. The relative expression levels of genes were calculated using the 2 −∆∆Ct method [31].…”