Differential gene expression analysis following olfactory learning in honeybee (Apis mellifera L.).

Muhammad Fahad Raza,Ahmed Rady,Pavol Hlaváč,Zhiguo Li,Songkun Su,Muhammad Anwar,Muhmmad Rizwan,Hongyi Nie,Arif Husain,M Ajmal Ali,Khalid Ali Khan

doi:10.1371/journal.pone.0262441

Muhammad Fahad Raza, Ahmed Rady + Show 9 more

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https://doi.org/10.1371/journal.pone.0262441

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Abstract

Insects change their stimulus-response through the perception of associating these stimuli with important survival events such as rewards, threats, and mates. Insects develop strong associations and relate them to their experiences through several behavioral procedures. Among the insects, Apis species, Apis mellifera ligustica are known for their outstanding ability to learn with tremendous economic importance. Apis mellifera ligustica has a strong cognitive ability and promising model species for investigating the neurobiological basis of remarkable olfactory learning abilities. Here we evaluated the olfactory learning ability of A. mellifera by using the proboscis extension reflex (PER) protocol. The brains of the learner and failed-learner bees were examined for comparative transcriptome analysis by RNA-Seq to explain the difference in the learning capacity. In this study, we used an appetitive olfactory learning paradigm in the same age of A. mellifera bees to examine the differential gene expression in the brain of the learner and failed-learner. Bees that respond in 2nd and 3rd trials or only responded to 3rd trials were defined as learned bees, failed-learner individuals were those bees that did not respond in all learning trials The results indicate that the learning ability of learner bees was significantly higher than failed-learner bees for 12 days. We obtained approximately 46.7 and 46.4 million clean reads from the learner bees failed-learner bees, respectively. Gene expression profile between learners’ bees and failed-learners bees identified 74 differentially expressed genes, 57 genes up-regulated in the brains of learners and 17 genes were down-regulated in the brains of the bees that fail to learn. The qRT-PCR validated the differently expressed genes. Transcriptome analyses revealed that specific genes in learner and failed-learner bees either down-regulated or up-regulated play a crucial role in brain development and learning behavior. Our finding suggests that down-regulated genes of the brain involved in the integumentary system, storage proteins, brain development, sensory processing, and neurodegenerative disorder may result in reduced olfactory discrimination and olfactory sensitivity in failed-learner bees. This study aims to contribute to a better understanding of the olfactory learning behavior and gene expression information, which opens the door for understanding of the molecular mechanism of olfactory learning behavior in honeybees.

Highlights

This study aims to contribute to a better understanding of the olfactory learning behavior and gene expression information, which opens the door for understanding of the molecular mechanism of olfactory learning behavior in honeybees
Our result revealed that percent responses were lower on the first conditioning trial and increased on subsequent trials, indicating that the bees learned the odor-sucrose association Fig 1
The study was conducted to identify the gene whose expression differs in the learner and faillearner bees following proboscis extension response, olfactory learning was performed on restrained individuals using the conditioned proboscis extension response

Summary

Introduction

Insects are essential for agriculture, orchards, horticultural crops, and seed production for fiber and forage crops [1]. Insects such as bees, butterflies, wasps, and beetles are increasing the output of the world’s food crop production. Most insects learn about the ecologically related stimuli and modify the behavioral responses to these stimuli to accept the new associations in their ecosystem. Diverse methods can trigger these behavioral changes due to new stimuli [2, 3]. These plasticity types vary how the behavior and stimuli are linked with significant events, including the availability of food, danger, or a mate. The evaluation of behavioral plasticity in any organism involves an experimental level of control throughout several factors, which is not accomplishable in field conditions to conduct behavioral research in natural circumstances, it is necessary to develop a standard conditioning procedure that can be performed under controlled conditions while still being useful [6]

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