Tomoko Fujiyuki

Novel Insect Picorna-Like Virus Identified in the Brains of Aggressive Worker Honeybees

ABSTRACT To identify candidate genes involved in the aggressive behavior of worker honeybees, we used the differential display method to search for RNAs exclusively detected in the brains of aggressive workers that had attacked a hornet. We identified a novel, 10,152-nucleotide RNA, termed Kakugo RNA. Kakugo RNA encodes a protein of 2,893 amino acid residues that shares structural features and sequence similarities with various picorna-like virus polyproteins, especially those from sacbrood virus, which infects honeybees. The Kakugo protein contains several domains that correspond to the virion protein, helicase, protease, and RNA-dependent RNA polymerase domains of various picorna-like virus polyproteins. When the worker bee tissue lysate was subjected to sucrose density gradient centrifugation, Kakugo RNA, except for the material at the bottom, was separated into two major peaks. One of the peaks corresponded to the position of Kakugo mRNA, and the other corresponded to the position of the poliovirus virion. These results suggest that the Kakugo RNA exists as an mRNA-like free RNA and virion RNA in the honeybee. Furthermore, injection of the lysate supernatant from the attacker heads into the heads of noninfected bees resulted in a marked increase in Kakugo RNA. These results demonstrate that Kakugo RNA is a plus-strand RNA of a novel picorna-like virus and that the brains of aggressive workers are infected by this novel virus. Kakugo RNA was detected in aggressive workers but not in nurse bees or foragers. In aggressive workers, Kakugo RNA was detected in the brain but not in the thorax or abdomen, indicating a close relation between viral infection in the brain and aggressive worker behaviors.

Carbohydrate metabolism genes and pathways in insects: insights from the honey bee genome.

Carbohydrate‐metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate‐rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate‐metabolizing enzymes and 28 genes encoding lipid‐metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose‐methanol‐choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes.

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) is a socially problematic pathogen that infects healthy individuals, causing severe disease. CA-MRSA is more virulent than hospital associated-MRSA (HA-MRSA). The underlying mechanism for the high virulence of CA-MRSA is not known. The transcription product of the psm-mec gene, located in the mobile genetic element SCCmec of HA-MRSA, but not CA-MRSA, suppresses the expression of phenol-soluble modulin α (PSMα), a cytolytic toxin of S. aureus. Here we report that psm-mec RNA inhibits translation of the agrA gene encoding a positive transcription factor for the PSMα gene via specific binding to agrA mRNA. Furthermore, 25% of 325 clinical MRSA isolates had a mutation in the psm-mec promoter that attenuated transcription, and 9% of the strains had no psm-mec. In most of these psm-mec-mutated or psm-mec-deleted HA-MRSAs, PSMα expression was increased compared with strains carrying intact psm-mec, and some mutated strains produced high amounts of PSMα comparable with that of CA-MRSA. Deletion of psm-mec from HA-MRSA strains carrying intact psm-mec increased the expression of AgrA protein and PSMα, and virulence in mice. Thus, psm-mec RNA suppresses MRSA virulence via inhibition of agrA translation and the absence of psm-mec function in CA-MRSA causes its high virulence property.

Differential expression of HR38 in the mushroom bodies of the honeybee brain depends on the caste and division of labor

We used a cDNA microarray to identify genes expressed in a caste (worker)‐ and division of labor (nurse bees or foragers)‐dependent manner in the honeybee brain. Among the identified genes, one encoded a putative orphan receptor (HR38) homologue that mediates ecdysteroid‐signaling. Real‐time reverse transcription‐polymerase chain reaction indicated that expression of this gene is higher in forager brains, as compared to nurse bees and queens. In the forager brain, expression was concentrated in a subset of the mushroom body neurons, suggesting that ecdysteroid‐signaling in the mushroom bodies might be involved in the division of labor of the workers.

Identification of a novel gene, Mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type Kenyon cells of the honeybee brain

Mushroom bodies (MBs) are considered to be involved in higher‐order sensory processing in the insect brain. To identify the genes involved in the intrinsic function of the honeybee MBs, we searched for genes preferentially expressed therein, using the differential display method. Here we report a novel gene encoding a putative transcription factor (Mblk‐1) expressed preferentially in one of two types of intrinsic MB neurones, the large‐type Kenyon cells, which makes Mblk‐1 a candidate gene involved in the advanced behaviours of honeybees. A putative DNA binding motif of Mblk‐1 had significant sequence homology with those encoded by genes from various animal species, suggesting that the functions of these proteins in neural cells are conserved among the animal kingdom.

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray1

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display‐positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5‐trisphosphate (IP3) 5‐phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large‐type Kenyon cells, suggesting that IP3‐mediated Ca2+ signaling is enhanced in these neurons.

Prevalence and Phylogeny of Kakugo Virus, a Novel Insect Picorna-Like Virus That Infects the Honeybee (Apis mellifera L.), under Various Colony Conditions

ABSTRACT We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), from the brains of aggressive worker honeybees that had counterattacked a giant hornet. To survey the prevalence of KV in worker populations engaged in various labors, we quantified KV genomic RNA. KV was detected specifically from aggressive workers in some colonies, while it was also detected from other worker populations in other colonies where the amount of KV detected in the workers was relatively high, suggesting that KV can infect various worker populations in the honeybee colonies. To investigate whether the KV strains detected were identical, phylogenetic analysis was performed. There was less than a 2% difference in the RNA-dependent RNA polymerase (RdRp) sequences between KV strains from aggressive workers and those from other worker populations, suggesting that all of the viruses detected were virtually the same KV. We also found that some of the KV-infected colonies were parasitized by Varroa mites, and the sequences of the KV strains detected from the mites were the same as those detected from the workers of the same colonies, suggesting that the mites mediate KV prevalence in the honeybee colonies. KV strains had approximately 6% and 15% sequence differences in the RdRp region from deformed wing virus and Varroa destructor virus 1, respectively, suggesting that KV represents a viral strain closely related to, but distinct from, these two viruses.

Identification of proteins whose expression is up- or down-regulated in the mushroom bodies in the honeybee brain using proteomics

To identify protein(s) with different expression patterns in the mushroom bodies (MBs) in the honeybee brain, we compared the protein profiles of MBs and optic lobes (OLs) using proteomics. Two‐dimensional gel electrophoresis revealed that five and three spots were selectively expressed in the MBs or OLs, respectively. Liquid chromatography tandem mass spectrometry analysis identified juvenile hormone diol kinase and glyceraldehyde‐3‐phosphate dehydrogenase as MB‐ and OL‐selective proteins, respectively. In situ hybridization revealed that jhdk expression was upregulated in MB neuron subsets, whereas gapdh expression was downregulated, indicating that MBs have a distinct gene and protein expression profile in the honeybee brain.

Distribution of Kakugo Virus and Its Effects on the Gene Expression Profile in the Brain of the Worker Honeybee Apis mellifera L.

ABSTRACT We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), obtained from the brains of aggressive honeybee worker bees that had counterattacked giant hornets. Here we examined the tissue distribution of KV and alterations of gene expression profiles in the brains of KV-infected worker bees to analyze possible effects of KV infection on honeybee neural and physiological states. By use of in situ hybridization, KV was broadly detected in the brains of the naturally KV-infected worker bees. When inoculated experimentally into bees, KV was detected in restricted parts of the brain at the early infectious stage and was later detected in various brain regions, including the mushroom bodies, optic lobes, and ocellar nerve. KV was detected not only in the brain but also in the hypopharyngeal glands and fat bodies, indicating systemic KV infection. Next, we compared the gene expression profiles in the brains of KV-inoculated and noninoculated bees. The expression of 11 genes examined was not significantly affected in KV-infected worker bees. cDNA microarray analysis, however, identified a novel gene whose expression was induced in the periphery of the brains of KV-infected bees, which was commonly observed in naturally infected and experimentally inoculated bees. The gene encoded a novel hypothetical protein with a leucine zipper motif. A gene encoding a similar protein was found in the parasitic wasp Nasonia genome but not in other insect genomes. These findings suggest that KV infection may affect brain functions and/or physiological states in honeybees.

Evidence of a novel immune responsive protein in the Hymenoptera

Honeybee populations are severely threatened by parasites and diseases. Recent outbreaks of Colony Collapse Disorder (CCD) has caused loss of more than 35% of bee colonies in the USA, and this is thought to at least in part be due to parasites and/or disease. Interestingly, the honeybee possesses of a limited set of immune genes compared to other insects. Non-canonical immune genes of honeybee are of interest because they may provide greater insights into the peculiar nature of the immune system of this social insect. Previous analyses of bee haemolymph upon bacterial challenge identified a novel leucine-rich repeat protein termed IRP30. Here we show that IRP30 behaves as a typical secreted immune protein. It is expressed simultaneously with carboxylesterase upon treatment with bacteria or other elicitors of immune response. Furthermore we characterize the gene and the mRNA encoding this protein and the IRP30 protein itself. Its regulation and evolution reveal that IRP30 belongs to a protein family, distributed broadly among Hymenoptera, suggesting its ancient function in immune response. We document an interesting case of a recent IRP30 loss in the ant Atta cephalotes and hypothesize that a putative IRP30 homolog of Nasonia emerged by convergent evolution rather than diverged from a common ancestor.