Gene expression and immunohistochemical analyses of mKast suggest its late pupal and adult-specific functions in the honeybee brain

Abstract

In insect brains, the mushroom bodies (MBs, a higher center) comprise intrinsic neurons, termed Kenyon cells (KCs). We previously showed that the honeybee (Apis mellifera L.) MBs comprise four types of KCs, in addition to the previously known three types of KCs: class I large-type KCs (lKCs), class I small-type KCs (sKCs) and class II KCs, novel class I ‘middle-type’ KCs (mKCs), which are characterized by the preferential expression of a gene, termed mKast. Although mKast was originally discovered during the search for genes whose expression is enriched in the optic lobes (OLs) in the worker brain, subsequent analysis revealed that the gene is expressed in an mKC-preferential manner in the MBs. To gain more insights into the function of mKast in the honeybee brain, we here performed expression analysis of mKast and immunohistochemistry of the mKast protein. Prominent mKast expression was first detected in the brain after the P7 pupal stage. In addition, mKast was expressed almost selectively in the brain, suggesting its late pupal and adult specific functions in the brain. Immunohistochemistry revealed that mKast-like immunoreactivity is detected in several regions in the worker brain: inside and around the MB calyces, at the outer edges of the OL lobula, at the outer surface of and posterior to the antennal lobes (ALs), along the dorsal midline of the anterior brain and at the outer surface of the subesophageal ganglions (SOG). mKast-like immunoreactivities in the MBs, OLs, ALs and SOG were due to the corresponding neurons, while mKast-like immunoreactivities beneath/between the MB calyces were assumed to most likely correspond to the lateral/medial neurosecretory cells.

Kenyon Cell Subtypes/Populations in the Honeybee Mushroom Bodies: Possible Function Based on Their Gene Expression Profiles, Differentiation, Possible Evolution, and Application of Genome Editing

“…Although mKast belongs to the α-arrestin family, which is involved in downregulation of membrane receptors (Kaneko et al, 2013 ), the role of mKast in the honeybee is currently obscure. mKast expression in the brain begins at the late pupal stages and is detectable almost exclusively in the adult brain, suggesting its role in regulating adult honeybee behaviors and/or physiology (Yamane et al, 2017 ).…”

mKast is dispensable for normal development and sexual maturation of the male European honeybee

“…Four microliters of the protein solution was denatured in sample buffer (50 mM Tris-HCl, pH 6.8, containing 10% glycerol, 2% SDS, 0.002% bromophenol blue, and 3% 2-mercaptethanol), and electrophoresed in polyacrylamide gel (5% for stacking, 10% for separation). Immunoblot analysis was performed essentially as described previously 26 , with some modifications, using 0.2 μg/ml anti-mKast antibody 26 or normal guinea pig IgG (Santa Cruz Biotechnology, Germany) as the first antibody, 0.01 μg/ml goat anti-guinea pig IgG-HRP conjugated (A7289, Sigma, Japan) as the second antibody. Signals were detected using ECL select Western Blotting Detection Reagent (GE Healthcare, Japan) and ImageQuant LAS 4000mini (GE Healthcare, Japan).…”

“…Here, we conducted gene knockout using CRISPR/Cas9 methods targeting middle-type Kenyon cell-preferential arrestin-related protein ( mKast ), which is preferentially expressed in the mKCs in honeybee MBs 22 . In situ hybridization analysis revealed that mKast is also expressed in optic lobes (OL, a visual center), antennal lobes (AL, an olfactory center), and subesophageal ganglion (SOG, a center for regulating proboscis function) 22 , 26 . In addition, quantitative reverse transcription- polymerase chain reaction (PCR) analysis revealed its adult brain-specific expression 26 .…”

“…In situ hybridization analysis revealed that mKast is also expressed in optic lobes (OL, a visual center), antennal lobes (AL, an olfactory center), and subesophageal ganglion (SOG, a center for regulating proboscis function) 22 , 26 . In addition, quantitative reverse transcription- polymerase chain reaction (PCR) analysis revealed its adult brain-specific expression 26 . These mKast expression patterns imply that mKast is related to higher-order function of the adult brain by regulating the processing of various types of sensory information.…”

“…These mKast expression patterns imply that mKast is related to higher-order function of the adult brain by regulating the processing of various types of sensory information. In addition, mKast begins to be expressed in the mKCs after the differentiation of lKCs and sKCs ceases in the pupal brain during metamorphosis, raising the possibility that mKast expression is associated with mKC differentiation 22 , 26 . To analyze the function of mKast in the regulation of honeybee social behaviors, it is necessary to produce mKast knockout workers through production of the mKast knockout drones (see Fig.…”