Genes associated with hot defensive bee ball in the Japanese honeybee, Apis cerana japonica.

BACKGROUND: The Japanese honeybee, Apis cerana japonica, shows a specific defensive behavior, known as a "hot defensive bee ball," used against the giant hornet, Vespa mandarinia. Hundreds of honeybee workers surround a hornet and make a "bee ball" during this behavior. They maintain the ball for around 30 min, and its core temperature can reach 46. Although various studies have been conducted on the characteristics of this behavior, its molecular mechanism has yet to be elucidated. Here, we performed a comprehensive transcriptomic analysis to detect candidate genes related to balling behavior. RESULTS: The expression levels of differentially expressed genes (DEGs) in the brain, flight muscle, and fat body were evaluated during ball formation and incubation at 46 °C. The DEGs detected during ball formation, but not in response to heat, were considered important for ball formation. The expression of genes related to rhodopsin signaling were increased in all tissues during ball formation. DEGs detected in one or two tissues during ball formation were also identified. CONCLUSIONS: Given that rhodopsin is involved in temperature sensing in Drosophila, the rhodopsin-related DEGs in A. cerana japonica may be involved in temperature sensing specifically during ball formation.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity.

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.

Expression analysis of Egr-1 ortholog in metamorphic brain of honeybee (Apis mellifera L.): Possible evolutionary conservation of roles of Egr in eye development in vertebrates and insects.

Specific genes quickly transcribed after extracellular stimuli without de novo protein synthesis are known as immediate early genes (IEGs) and are thought to contribute to learning and memory processes in the mature nervous system of vertebrates. A recent study revealed that the homolog of Early growth response protein-1 (Egr-1), which is one of the best-characterized vertebrate IEGs, shared similar properties as a neural activity-dependent gene in the adult brain of insects. With regard to the roles of vertebrate Egr-1 in neural development, the contribution to the development and growth of visual systems has been reported. However, in insects, the expression dynamics of the Egr-1 homologous gene during neural development remains poorly understood. Our expression analysis demonstrated that AmEgr, a honeybee homolog of Egr-1, was transiently upregulated in the developing brain during the early to mid pupal stages. In situ hybridization and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry revealed that AmEgr was mainly expressed in post-mitotic cells in optic lobes, the primary visual center of the insect brain. These findings suggest the evolutionarily conserved role of Egr homologs in the development of visual systems in vertebrates and insects.

Expression profile of the sex determination gene doublesex in a gynandromorph of bumblebee, Bombus ignitus.

Gynandromorphy that has both male and female features is known in many insect orders, including Hymenoptera. In most cases, however, only external morphology and behavioral aspects have been studied. We found a gynandromorph of bumblebee, Bombus ignitus, that showed almost bilateral distribution of external sexual traits, with male characters observed on the left side and female characters on the right side. This individual never exhibited sexual behavior toward new queens. The dissection of the head part showed that it had bilaterally dimorphic labial glands, only the left of which was well developed and synthesized male-specific pheromone components. In contrast, the gynandromorph possessed an ovipositor and a pair of ovaries in the abdominal part, suggesting that it had a uniformly female reproductive system. Furthermore, we characterized several internal organs of the gynandromorph by a molecular biological approach. The expression analyses of a sex determination gene, doublesex, in the brain, the fat bodies, the hindgut, and the ovaries of the gynandromorph revealed a male-type expression pattern exclusively in the left brain hemisphere and consistent female-type expression in other tissues. These findings clearly indicate the sexual discordance between external traits and internal organs in the gynandromorph. The results of genetic analyses using microsatellite markers suggested that this individual consisted of both genetically male- and female-type tissues.

CpG methylation in the hexamerin 110 gene in the European honeybee, Apis mellifera.

The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), has a full set of machinery for functional CpG methylation of its genome. A recent study demonstrated that DNA methylation in the honeybee is involved in caste differentiation. In this study, the expression and methylation of the hexamerin 110 gene (Hex110), which encodes a storage protein, was analyzed. High levels of the Hex110 transcript were expressed in both worker and queen larvae. Low levels of this transcript were also detected in adult fat bodies, and the expression level was higher in the queen than in the worker. Bisulfite sequencing revealed that the Hex110 gene is overall methylated at a low level, with a limited number of CpG sites methylated at relatively high levels. These highly methylated sites were exclusively located in the exon regions. The average methylation rate of the Hex110 gene was higher in the adult stage than in the larval stage. Furthermore, several CpG sites were differentially methylated between the worker and queen larvae. These observations suggest that the methylation of the Hex110 gene is regulated at the developmental stage and in a caste-dependent manner.

Differential gene expression in the mandibular glands of queen and worker honeybees, Apis mellifera L.: implications for caste-selective aldehyde and fatty acid metabolism.

In honeybees, queens synthesize the "queen pheromone," whereas workers synthesize fatty acid components of "royal jelly" in their mandibular glands (MGs). To identify candidate proteins involved in the caste-selective MG function, we performed a proteomic analysis and identified three proteins that were expressed selectively in queen MGs (aldehyde dehydrogenase 1 [ALDH1], medium-chain acyl-CoA dehydrogenase [MCAD], and electron transfer flavoprotein alpha [ETFalpha)]), and a protein that was expressed selectively in worker MGs (fatty acid synthase [FAS)]). The quantitative reversed transcription-polymerase chain reaction demonstrated that the level of aldh1 transcription in MGs was significantly higher, whereas that of fas transcription was lower in queens than in workers. Among the eight genes encoding proteins similar to ALDH1 that are registered in the honeybee genome database, aldh6, aldh7, and aldh1 were expressed at significantly higher levels in queen MGs than in worker MGs. In situ hybridization showed that in the queen head, aldh1 was expressed in MG cells, whereas aldh6 and aldh7 were expressed in fat cells attached to the MGs. These results suggest caste- and cell type-selective aldehyde/fatty acid metabolism in honeybee MGs.

Complete WO phage sequences reveal their dynamic evolutionary trajectories and putative functional elements required for integration into the Wolbachia genome.

Wolbachia endosymbionts are ubiquitously found in diverse insects including many medical and hygienic pests, causing a variety of reproductive phenotypes, such as cytoplasmic incompatibility, and thereby efficiently spreading in host insect populations. Recently, Wolbachia-mediated approaches to pest control and management have been proposed, but the application of these approaches has been hindered by the lack of genetic transformation techniques for symbiotic bacteria. Here, we report the genome and structure of active bacteriophages from a Wolbachia endosymbiont. From the Wolbachia strain wCauB infecting the moth Ephestia kuehniella two closely related WO prophages, WOcauB2 of 43,016 bp with 47 open reading frames (ORFs) and WOcauB3 of 45,078 bp with 46 ORFs, were characterized. In each of the prophage genomes, an integrase gene and an attachment site core sequence were identified, which are putatively involved in integration and excision of the mobile genetic elements. The 3' region of the prophages encoded genes with sequence motifs related to bacterial virulence and protein-protein interactions, which might represent effector molecules that affect cellular processes and functions of their host bacterium and/or insect. Database searches and phylogenetic analyses revealed that the prophage genes have experienced dynamic evolutionary trajectories. Genes similar to the prophage genes were found across divergent bacterial phyla, highlighting the active and mobile nature of the genetic elements. We suggest that the active WO prophage genomes and their constituent sequence elements would provide a clue to development of a genetic transformation vector for Wolbachia endosymbionts.

Transinfection reveals the crucial importance of Wolbachia genotypes in determining the type of reproductive alteration in the host.

Wolbachia , a group of endosymbiotic bacteria in arthropods, alter the reproduction of their hosts in various ways. A Wolbachia strain (wSca) naturally infecting the adzuki bean borer moth Ostrinia scapulalis induces male killing, while another strain (wKue) infecting the Mediterranean flour moth Ephestia kuehniella induces cytoplasmic incompatibility (CI) in the resident host. Transinfection of Wolbachia can be a powerful tool to elucidate the relative importance of Wolbachia and the host in determining the type of reproductive alterations. Recently, male killing was shown to occur in E. kuehniella transinfected with w Sca. In the present study, we transferred w Kue to O. scapulalis by embryonic microinjection. In the O. scapulalis transinfected with wKue, CI, but not male killing occurred. Thus, in addition to wSca, wKue was shown to induce the same type of alteration in a foreign host as in its natural host. These results demonstrate the crucial role of the Wolbachia genotype in determining the type of reproductive alteration. However, the present study also revealed the involvement of host factors. First, the degree of incompatibility induced by wKue in O. scapulalis was stronger than that in E. kuehniella , indicating that host factors can affect the level of CI. Second, the vertical transmission rate of wKue in O. scapulalis was generally low, suggesting that the host affects the dynamics of Wolbachia transmission.

Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont.

Wolbachia is a group of obligate symbiotic bacteria found in many insects and other arthropods. The presence of Wolbachia alters reproduction in the host, but the mechanisms are unknown. Molecular biological studies of Wolbachia have delayed significantly, and one of the reasons is the lack of transformation techniques of this bacterium. In the present study, bacteriophage particles were isolated from Wolbachia for the first time. The purified phage had an isometric head that was approximately 40 nm in diameter and contained linear double-stranded DNA of approximately 20 kbp. Partial sequence information (total of 20,484 bp) revealed that there were 24 open reading frames including a structural gene module, and genes for replication and lysogenic conversion. This bacteriophage is the only known mobile genetic element potentially used for transformation of Wolbachia.

Novel insect picorna-like virus identified in the brains of aggressive worker honeybees.

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.

Infection density of Wolbachia and level of cytoplasmic incompatibility in the Mediterranean flour moth, Ephestia kuehniella.

Wolbachia, a causative agent of various reproductive changes in arthropods, induces cytoplasmic incompatibility (CI) in the Mediterranean flour moth, Ephestia kuehniella. Two strains of E. kuehniella, Yokohama and Tsuchiura, harbor closely related Wolbachia, but the Yokohama strain expresses stronger CI than the Tsuchiura strain. A transinfected E. kuehniella strain that harbors the Wolbachia derived from the almond moth Cadra cautella, expresses weak CI at a similar level to the Tsuchiura strain. In the present study, we measured the Wolbachia density in the testis of the three E. kuehniella strains in order to examine the effects of bacterial strain and infection load on the expression of CI. When individuals of the same strain were compared, a correlation of bacterial density to CI level was observed. In addition, the Wolbachia density was higher in the Yokohama strain than the Tsuchiura strain in agreement with the CI levels expressed. However, this relationship did not hold in the comparison between the naturally infected and transinfected strains that carried phylogenetically distant Wolbachia.

Regulation of Wolbachia density in the Mediterranean flour moth, Ephestia kuehniella, and the almond moth, Cadra cautella.

The Mediterranean flour moth, Ephestia kuehniella, is infected with A-group Wolbachia (wKue), and the almond moth, Cadra cautella, is doubly infected with A- and B-group Wolbachia, which are designated as wCauA and wCauB, respectively. In both insects, the Wolbachia populations increased greatly during embryonic and larval stages. The Wolbachia population doubled every 3.6 days on average in E. kuehniella larvae, whereas those of wCauA and wCauB doubled every 2.1 days in C. cautella larvae. The populations of wCauA and wCauB that had been transferred into the E. kuehniella background increased at similar rates to that of wKue in the natural host E. kuehniella, suggesting that the host genetic background influences Wolbachia proliferation. To examine whether the populations of the two Wolbachia variants in double infection is regulated collectively or independently, we measured the infection load in the ovaries of three transfected E. kuehniella lines in different infection states: single infection with wCauA, single infection with wCauB, and double infection. The density of each Wolbachia variant did not differ significantly between the singly and doubly transfected hosts, suggesting independent regulation.

Interspecific transfer of Wolbachia between two lepidopteran insects expressing cytoplasmic incompatibility: a Wolbachia variant naturally infecting Cadra cautella causes male killing in Ephestia kuehniella.

Wolbachia is known as the causative agent of various reproductive alterations in arthropods. The almond moth Cadra cautella is doubly infected with A- and B-group Wolbachia and expresses complete cytoplasmic incompatibility (CI). The Mediterranean flour moth Ephestia kuehniella carries A-group Wolbachia and expresses partial CI. In the present study, the Wolbachia in C. cautella was transferred to E. kuehniella from which the original Wolbachia had been removed. We obtained transfected lines of three different infection states: single infection with A, single infection with B, and double infection with A and B. The doubly transfected lines and those transfected with only A produced exclusively female progeny. Two lines of evidence suggested that the sex ratio distortion was due to male killing. First, reduced egg hatch rate was observed. Second, removal of the Wolbachia from the transfected lines resulted in the recovery of a normal sex ratio of approximately 1:1. The occurrence of male killing following transfection showed that host factors influence the determination of the reproductive phenotype caused by Wolbachia. The transfected E. kuehniella males carrying exclusively B-group Wolbachia expressed partial incompatibility when crossed with the uninfected females. In addition, the transfected lines were bidirectionally incompatible with the naturally infected strain, which was the first demonstration of bidirectional CI in a lepidopteran.