Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

Kumi Kaneko,Kenichi Shirai,Tsubomi Ikeda,Takekazu Kunieda,Takayoshi Nakaoka,Tomoko Fujiyuki,Chie Umatani,Rajib Kumar Paul,Atsushi Ugajin,Takeo Kubo,Hideaki Takeuchi,Hiroto Tadano,Mirai Nagai,Sayaka Hori,Hiromu Tanimoto

doi:10.1371/journal.pone.0071732

Abstract

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel ‘middle-type Kenyon cells’ that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large– and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large– and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large– and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a ‘clustering’ of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.

Highlights

The European honeybee (Apis mellifera L) is a social insect that forms a sophisticated society
Subsequent quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that expression of Clone #3 in the optic lobes (OLs) was approximately 2.5 fold higher than in other brain regions (Fig. S1), which was consistent with the results of cDNA microarray analysis
The GB18367 product contained both arrestin-like_N and arrestin-like_C domains (Fig. 1B), which are contained in arrestins [47], neither of these domains had any significant sequence identities with those of Apis mellifera arrestin homolog isoform 1 (GB16006), isoform 2 (GB12766), or b-arrestin (GB13683), three arrestin homologs predicted in the honeybee genome, suggesting that the GB18367 product is structurally not closely related to arrestins

Summary

Introduction

The European honeybee (Apis mellifera L) is a social insect that forms a sophisticated society. Female adult honeybees differentiate into two castes, reproductive queens and sterile workers [1,2]. Queens are engaged in laying eggs, while workers shift their labors in an age-dependent manner from nursing their brood (nurse bees) to foraging for nectar and pollen outside the hives (foragers). Foragers transmit information on the distance and direction of a food source to their nestmates using dance communication [1,2,3]. Despite these highly advanced social behaviors, the honeybee brain is rather compact and simple compared to the mammalian brains [4,5]. The honeybee is an excellent model for studies of the neural and molecular bases of animal social behaviors and higher brain functions [6]

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Results

Conclusion