Honeybee products and edible insect powders improve locomotive and learning abilities of Ubiquilin-knockdown Drosophila

Abstract

Background: Mutations in the human Ubiquilin 2 gene are associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD), the fatal neurodegenerative disease that progressively affected neuronal cells in both brain and spinal cord. There is currently no effective therapy for these diseases. Over the last decade, researchers have focused on the potential use of natural products especially in neurodegenerative studies. Insect products have been used as traditional medicines, however, scientific information is still lacking. Fruit fly is recently used as a model organism to investigate degenerative diseases related to the nervous system because it has a short life span and produces a large number of offspring.

Methods: The present study investigated the effects of honeybee products and edible insect powders on the locomotive and learning abilities, neuromuscular junctions (NMJs) structure, and reactive oxygen species (ROS) in larval brains of Ubiquilin- knockdown Drosophila.

Results: dUbqn knockdown flies showed defects in locomotive and learning abilities accompanied with structural defects in NMJs. The results obtained revealed that the recovery of locomotive defects was significantly greater in dUbqn knockdown flies fed with coffee honey from Apis cerana (1% v/v) or Apis dorsata melittin (0.5 μg/ml) or wasp powder (2 mg/ml) than that of in untreated dUbqn knockdown flies. Furthermore, dUbqn knockdown flies fed with coffee honey showed the partial rescue of structural defects in NMJs, improved learning ability, and reduced the accumulation of ROS caused by dUbqn depletion in the brain over the untreated group.

Conclusion: These results suggest that coffee honey from Apis cerana contains a neuroprotective agent that will contribute to the development of a novel treatment for ALS/FTD.

Keywords: Coffee honey; Edible insect; Honeybee product; Neuroprotective agent; Ubiquilin.

Fig. 1

Effects of honeybee products and edible insect powders on locomotive abilities of pan neuron-specific dUbqn knockdown (w; UAS-dUbqnIR_107–494/+;elav-GAL4). The distance (a, b), length (c, d), and speed (e, f) of dUbqn knockdown larvae covered in one minute in comparison between untreated and treated conditions. The results represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (n = 20 larvae per group). ADM: Apis dorsata melittin; ACM: Apis cerana melittin; AFM: Apis florea melittin; CH: coffee honey; LH: longan honey; TP: tea pollen; FDRJ: freeze-dried fresh royal jelly; Ci: cicada powder; Si: silkworm powder; Ba: bamboo borer powder; Be: honeybee larva powder; Gi: giant water bug power; Cr: crickets powder; Wa: wasp power; Su: subterranean ants powder

Fig. 2

Effects of honeybee products and edible insect powders on learning abilities of pan neuron-specific dUbqn knockdown larvae. The larvae were sequentially exposed to n-amyl acetate (AM) in the presence of reward and then 1-octanol (OCT) in the absence of reward (AM+/OCT). The reciprocal training the larvae was sequentially exposed to OCT in the presence of reward and AM in the absence of reward (OCT+/AM). After training, larvae were tested by exposure to AM and then OCT in the absence of reward to test their preference. Larval AM preference score was shown in (a-e) (score = 1 means all preferred AM; score = − 1 means all preferred OCT). The preference score of control larvae (w; UAS-GFPIR/+;elav-GAL4/+) and dUbqn knockdown larvae (w; UAS-dUbqnIR_107–494/+;elav-GAL4) without treatment was shown in (a, b) whereas the preference score of dUbqn knockdown larvae treated with coffee honey (1% v/v), A. dorsata melittin (0.5 μg/ml), and wasp powder (2 mg/ml) was shown in (c, d, e). Box-and-whisker plots represent the minimum, first quartile, median, third quartile, and maximum. Normalized learning index of all groups was shown in (f). The higher LI means the higher ability of larvae to learn and perform the conditional tasks. The results represent mean ± SEM. * p < 0.05, N.S. not significant. (n = 3 of 6 larvae per group)

Fig. 3

Effects of honeybee products and edible insect powders on synaptic structures. The synaptic structures of control larvae (w; UAS-GFPIR/+;elav-GAL4/+) (a), pan neuron-specific dUbqn knockdown larvae (w; UAS-dUbqnIR_107–494/+;elav-GAL4) without treatment (b), with coffee honey treatment (c), Apis dorsata melittin treatment (d), and wasp powder treatment (e) were shown. HRP and Dlg which are pre- and post-synaptic markers were observed in green and red signals, respectively. The quantification of the main branch length (f), number of boutons (g), number of branches (h), and average terminal boutons size (i) was performed by ImageJ. Quantification results represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S. not significant. Scale bar is 30 μm. (n = 10 larvae per group)

Fig. 4

Reactive oxygen species (ROS) generation in response to coffee honey. The ROS levels of the control larvae (w; UAS-GFPIR/+;elav-GAL4/+), dUbqn knockdown larvae (w; UAS-dUbqnIR_107–494/+;elav-GAL4) without treatment and with coffee honey treatment were measured using fluorescence emitted by dichlorofluorescein (DCF). Quantification results represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. (n = 3 of 20 larvae per group)