Gustatory Responsiveness of Honey Bees Colonized with a Defined or Conventional Gut Microbiota.

Gut microbes have many beneficial functions for host animals, such as food digestion and development of the immune system. An increasing number of studies report that gut bacteria also affect host neural function and behavior. The sucrose responsiveness of the western honey bee Apis mellifera, which harbors a characteristic gut microbiota, was recently reported to be increased by the presence of gut microbes. However, this responsiveness may vary depending on the experimental design, as animal behavior may be modulated by physiological states and environmental conditions. To evaluate the robustness of the effects of the gut microbiota on host gustatory responsiveness, we herein examined the sucrose responsiveness of honey bees colonized with a defined bacterial community or a conventional gut microbiota extracted from a field-collected bee. Although colonization was experimentally verified, sucrose responsiveness did not significantly differ among treatments after the 2- or 5-h starvation period. We concluded that the sucrose responsiveness of A. mellifera is not always affected by its gut microbiota. Therefore, host physiological conditions and environmental factors need to be considered when evaluating the impact of the gut microbiota on host neural function and behavior.

Basic Structures of Gut Bacterial Communities in Eusocial Insects.

Gut bacterial communities assist host animals with numerous functions such as food digestion, nutritional provision, or immunity. Some social mammals and insects are unique in that their gut microbial communities are stable among individuals. In this review, we focus on the gut bacterial communities of eusocial insects, including bees, ants, and termites, to provide an overview of their community structures and to gain insights into any general aspects of their structural basis. Pseudomonadota and Bacillota are prevalent bacterial phyla commonly detected in those three insect groups, but their compositions are distinct at lower taxonomic levels. Eusocial insects harbor unique gut bacterial communities that are shared within host species, while their stability varies depending on host physiology and ecology. Species with narrow dietary habits, such as eusocial bees, harbor highly stable and intraspecific microbial communities, while generalists, such as most ant species, exhibit relatively diverse community structures. Caste differences could influence the relative abundance of community members without significantly altering the taxonomic composition.

Vast Differences in Strain-Level Diversity in the Gut Microbiota of Two Closely Related Honey Bee Species.

Most bacterial species encompass strains with vastly different gene content. Strain diversity in microbial communities is therefore considered to be of functional importance. Yet little is known about the extent to which related microbial communities differ in diversity at this level and which underlying mechanisms may constrain and maintain strain-level diversity. Here, we used shotgun metagenomics to characterize and compare the gut microbiota of two honey bee species, Apis mellifera and Apis cerana, which diverged about 6 mya. Although the host species are colonized largely by the same bacterial 16S rRNA phylotypes, we find that their communities are host specific when analyzed with genomic resolution. Moreover, despite their similar ecology, A. mellifera displayed a much higher diversity of strains and functional gene content in the microbiota compared to A. cerana, both per colony and per individual bee. In particular, the gene repertoire for polysaccharide degradation was massively expanded in the microbiota of A. mellifera relative to A. cerana. Bee management practices, divergent ecological adaptation, or habitat size may have contributed to the observed differences in microbiota genomic diversity of these key pollinator species. Our results illustrate that the gut microbiota of closely related animal hosts can differ vastly in genomic diversity while displaying similar levels of diversity based on the 16S rRNA gene. Such differences are likely to have consequences for gut microbiota functioning and host-symbiont interactions, highlighting the need for metagenomic studies to understand the ecology and evolution of microbial communities.

Community analysis of gut microbiota in hornets, the largest eusocial wasps, Vespa mandarinia and V. simillima.

Gut microbiota are important for various aspects of host physiology, and its composition is generally influenced by both intrinsic and extrinsic contexts of the host. Social bee gut microbiota composition is simple and highly stable hypothesized to be due to their unique food habit and social interactions. Here, we focused on hornets, the largest of the eusocial wasps - Vespa mandarinia and V. simillima. Unlike the well-studied honey bees, adult hornets are generally herbivorous but also hunt insects for broods, a unique behavior which could influence their gut microbiota. Analysis of the gut microbiome using 16S rRNA gene sequencing revealed that the two species have simple gut microbiota, composed of seven or eight consistently maintained 'core' operational taxonomic units (OTUs). While the two Vespa species shared some OTUs, the structures of their gut communities differed. Phylogenetic analysis indicated association of core OTUs with host diet. Intriguingly, prey honey bee gut microbes were detected in the V. simillima gut (and to a lesser extent in V. mandarinia), suggesting migration of microorganisms from the prey gut. This is the first report uncovering gut microbiome in hornets, giving additional insight into how food habit affects gut microbiota of social insects.

Frischella japonica sp. nov., an anaerobic member of the Orbales in the Gammaproteobacteria, isolated from the gut of the eastern honey bee, Apis cerana japonica Fabricius.

The gut of honey bees is characterized by a stable and relatively simple community of bacteria, consisting of seven to ten phylotypes. Two closely related honey bees, Apis mellifera (western honey bee) and Apis cerana (eastern honey bee), show a largely comparable occurrence of those phylotypes, but a distinct set of bacterial species and strains within each bee species. Here, we describe the isolation and characterization of Ac13T, a new species within the rare proteobacterial genus Frischella from A. cerana japonica Fabricius. Description of Ac13T as a new species is supported by low identity of the 16S rRNA gene sequence (97.2 %), of the average nucleotide identity based on orthologous genes (77.5 %) and digital DNA-DNA hybridization relatedness (24.7 %) to the next but far related type strain Frischella perrara PEB0191T, isolated from A. mellifera. Cells of Ac13T are mesophilic and have a mean length of 2-4 µm and a width of 0.5 µm. Optimal growth was achieved in anoxic conditions, whereas growth was not observed in oxic conditions and strongly reduced in microaerophilic environment. Strain Ac13T shares several features with other members of the Orbaceae, such as the major fatty acid profile, the respiratory quinone type and relatively low DNA G+C content, in accordance with its evolutionary relationship. Unlike F. perrara, strain Ac13T is susceptible to a broad range of antibiotics, which could be indicative for an antibiotic-free A. cerana bee keeping. In conclusion, we propose strain Ac13T as a novel species for which we propose the name Frischella japonica sp. nov. with the type strain Ac13T (=NCIMB 15259=JCM 34075).

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions. We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

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.

Mushroom bodies (MBs), a higher-order center in the honeybee brain, comprise some subtypes/populations of interneurons termed as Kenyon cells (KCs), which are distinguished by their cell body size and location in the MBs, as well as their gene expression profiles. Although the role of MBs in learning ability has been studied extensively in the honeybee, the roles of each KC subtype and their evolution in hymenopteran insects remain mostly unknown. This mini-review describes recent progress in the analysis of gene/protein expression profiles and possible functions of KC subtypes/populations in the honeybee. Especially, the discovery of novel KC subtypes/populations, the "middle-type KCs" and "KC population expressing FoxP," necessitated a redefinition of the KC subtype/population. Analysis of the effects of inhibiting gene function in a KC subtype-preferential manner revealed the function of the gene product as well as of the KC subtype where it is expressed. Genes expressed in a KC subtype/population-preferential manner can be used to trace the differentiation of KC subtypes during the honeybee ontogeny and the possible evolution of KC subtypes in hymenopteran insects. Current findings suggest that the three KC subtypes are unique characteristics to the aculeate hymenopteran insects. Finally, prospects regarding future application of genome editing for the study of KC subtype functions in the honeybee are described. Genes expressed in a KC subtype-preferential manner can be good candidate target genes for genome editing, because they are likely related to highly advanced brain functions and some of them are dispensable for normal development and sexual maturation in honeybees.

Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.).

Although the molecular mechanisms involved in learning and memory in insects have been studied intensively, the intracellular signaling mechanisms involved in early memory formation are not fully understood. We previously demonstrated that phospholipase C epsilon (PLCe), whose product is involved in calcium signaling, is almost selectively expressed in the mushroom bodies, a brain structure important for learning and memory in the honeybee. Here, we pharmacologically examined the role of phospholipase C (PLC) in learning and memory in the honeybee. First, we identified four genes for PLC subtypes in the honeybee genome database. Quantitative reverse transcription-polymerase chain reaction revealed that, among these four genes, three, including PLCe, were expressed higher in the brain than in sensory organs in worker honeybees, suggesting their main roles in the brain. Edelfosine and neomycin, pan-PLC inhibitors, significantly decreased PLC activities in homogenates of the brain tissues. These drugs injected into the head of foragers significantly attenuated memory acquisition in comparison with the control groups, whereas memory retention was not affected. These findings suggest that PLC in the brain is involved in early memory formation in the honeybee. To our knowledge, this is the first report of a role for PLC in learning and memory in an insect.

Production of Knockout Mutants by CRISPR/Cas9 in the European Honeybee, Apis mellifera L.

The European honeybee (Apis mellifera L.) is used as a model organism in studies of the molecular and neural mechanisms underlying social behaviors and/or advanced brain functions. The entire honeybee genome has been sequenced, which has further advanced molecular biologic studies of the honeybee. Functions of genes of interest, however, remain largely to be elucidated in the honeybee due to the lack of effective reverse genetic methods. Moreover, genetically modified honeybees must be maintained under restricted laboratory conditions due to legal restrictions, further complicating the application of reverse genetics to this species. Here we applied CRISPR/Cas9 to the honeybee to develop an effective reverse genetic method. We targeted major royal jelly protein 1 (mrjp1) for genome editing, because this gene is predominantly expressed in adult workers and its mutation is not expected to affect normal development. By injecting sgRNA and Cas9 mRNA into 57 fertilized embryos collected within 3 h after oviposition, we successfully created six queens, one of which produced genome-edited male offspring. Of the 161 males produced, genotyping demonstrated that the genome was edited in 20 males. All of the processes necessary for producing these genome-edited queens and males were performed in the laboratory. Therefore, we developed essential techniques to create knockout honeybees by CRISPR/Cas9. Our findings also suggested that mrjp1 is dispensable for normal male development, at least till the pupal stage. This new technology could pave the way for future functional analyses of candidate genes involved in honeybee social behaviors.

Gene expression profiles and neural activities of Kenyon cell subtypes in the honeybee brain: identification of novel 'middle-type' Kenyon cells.

In the honeybee (Apis mellifera L.), it has long been thought that the mushroom bodies, a higher-order center in the insect brain, comprise three distinct subtypes of intrinsic neurons called Kenyon cells. In class-I large-type Kenyon cells and class-I small-type Kenyon cells, the somata are localized at the edges and in the inner core of the mushroom body calyces, respectively. In class-II Kenyon cells, the somata are localized at the outer surface of the mushroom body calyces. The gene expression profiles of the large- and small-type Kenyon cells are distinct, suggesting that each exhibits distinct cellular characteristics. We recently identified a novel gene, mKast (middle-type Kenyon cell-preferential arrestin-related gene-1), which has a distinctive expression pattern in the Kenyon cells. Detailed expression analyses of mKast led to the discovery of novel 'middle-type' Kenyon cells characterized by their preferential mKast-expression in the mushroom bodies. The somata of the middle-type Kenyon cells are localized between the large- and small-type Kenyon cells, and the size of the middle-type Kenyon cell somata is intermediate between that of large- and small-type Kenyon cells. Middle-type Kenyon cells appear to differentiate from the large- and/or small-type Kenyon cell lineage(s). Neural activity mapping using an immediate early gene, kakusei, suggests that the small-type and some middle-type Kenyon cells are prominently active in the forager brain, suggesting a potential role in processing information during foraging flight. Our findings indicate that honeybee mushroom bodies in fact comprise four types of Kenyon cells with different molecular and cellular characteristics: the previously known class-I large- and small-type Kenyon cells, class-II Kenyon cells, and the newly identified middle-type Kenyon cells described in this review. As the cellular characteristics of the middle-type Kenyon cells are distinct from those of the large- and small-type Kenyon cells, their careful discrimination will be required in future studies of honeybee Kenyon cell subtypes. In this review, we summarize recent progress in analyzing the gene expression profiles and neural activities of the honeybee Kenyon cell subtypes, and discuss possible roles of each Kenyon cell subtype in the honeybee brain.

Analysis of the Differentiation of Kenyon Cell Subtypes Using Three Mushroom Body-Preferential Genes during Metamorphosis in the Honeybee (Apis mellifera L.).

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages.