Tetsuhiko Sasaki

Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods.

Abstract.Wolbachia are obligatory intracellular and maternally inherited bacteria, known to infect many species of arthropod. In this study, we discovered a bacteriophage-like genetic element in Wolbachia, which was tentatively named bacteriophage WO. The phylogenetic tree based on phage WO genes of several Wolbachia strains was not congruent with that based on chromosomal genes of the same strains, suggesting that phage WO was active and horizontally transmitted among various Wolbachia strains. All the strains of Wolbachia used in this study were infected with phage WO. Although the phage genome contained genes of diverse origins, the average G+C content and codon usage of these genes were quite similar to those of a chromosomal gene of Wolbachia. These results raised the possibility that phage WO has been associated with Wolbachia for a very long time, conferring some benefit to its hosts. The evolution and possible roles of phage WO in various reproductive alterations of insects caused by Wolbachia are discussed.

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.

Production of essential amino acids from glutamate by mycetocyte symbionts of the pea aphid, Acyrthosiphon pisum

Abstract Glutamine was the most abundant amino acid constituent in the hemolymph of pea aphids, Acyrthosiphon pisum . While mycetocytes isolated from the aphids took up glutamine actively, the intracellular symbionts isolated from the mycetocytes scarcely took up the amino acid, and instead took up glutamic acid actively. [U- 14 C]Glutamine incorporated by mycetocytes was converted into glutamate. When [ϵ- 15 N]glutamine was introduced into mycetocytes, [ 15 N]glutamate was found in the cytosol. These results suggested that glutamine taken up by mycetocytes was hydrolyzed into glutamic acid and ammonia, and at least a portion of ammonia was assimilated into glutamate in the cytosol, probably through reaction with α-ketoglutarate. When isolated symbionts were incubated with [ 15 N]glutamic acid, the following amino acids were found highly labeled: alanine, aspartic acid, glutamine, isoleucine, leucine, phenylalanine, proline and valine.

Nitrogen recycling in the brown planthopper, Nilaparvata lugens: involvement of yeast-like endosymbionts in uric acid metabolism.

Abstract Planthopper harbour yeast-like symbionts in the mycetocyte. A possible role that the symbionts may play for the host insect is nitrogen recycling, i.e. conversion of nitrogenous waste products of the insect into compounds of nutritional value. To examine this possibility, we determined the uric acid content in the honeydew and the whole tissues of control and symbiont-depleted brown planthoppers, Nilaparvata lugens . The symbiont-depleted insects were obtained by exposing newly hatched nymphs to 35 °C for three days. In the honeydew, uric acid was not detected regardless of the presence or absence of symbionts. Nevertheless, a considerable amount of uric acid was found in the insect tissues, and the content was markedly higher in heat-treated insects than in controls. We also determined the uricase activity in the insect whole tissues and in the isolated symbionts. While in control insects uricase was detectable, there was no uricase activity in heat-treated insects. Isolated symbionts had uricase activity 15 times higher than in the whole tissues of control insects. These results suggested that uric acid synthesized by the insect is stored in insect tissues rather than excreted, and that uric acid is recycled in the planthopper with the aid of its endosymbionts.

Growth and reproduction of the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum maintained on artificial diets

Abstract In an effort to study the roles of the intracellular symbionts in the nitrogen metabolism of aphids, the symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum were maintained on their natural host plant and on two kinds of artificial diets. While the amino acid composition of Diet A was designed to mimic that of the plant sap, Diet B was prepared based on the amino acid composition of the total tissue of aphids. Symbiotic aphids grew well and produced progeny on all the three diets. Aposymbiotic aphids, which were sterile on their natural host plant and Diet A, did bear nymphys on Diet B. The capability of amino acid conversion of the aphid was investigated with glutamine labelled with heavy nitrogen. The following amino acids were found labelled exclusively in the symbiotic aphids: arginine, histidine, isoleucine and/or leucine, lysine, phenylalanine, threonine and valine, suggesting that-these so-called essential amino acids were produced with the aid of symbionts.

CpG Methylation in the Hexamerin 110 Gene in the European Honeybee, Apis mellifera

Abstract 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.

Wolbachia variant that induces two distinct reproductive phenotypes in different hosts

Wolbachia is an intracellular endosymbiont that induces a variety of reproductive alterations in diverse arthropods. The almond moth, Cadra cautella, is double infected with two Wolbachia variants, wCauA and wCauB, and expresses complete cytoplasmic incompatibility (CI). The individual contribution of wCauA and wCauB to the expression of CI are unclear, however, because the two variants have not been separated in this host. The effect of wCauA is of particular interest because it induces male killing when transferred into the Mediterranean flour moth, Ephestia kuehniella. In the present study, we generated C. cautella infected with only wCauA by treating double-infected insects with tetracycline. Single-infected C. cautella exhibited strong CI, demonstrating that wCauA induces two distinct reproductive phenotypes in different hosts: CI in C. cautella and male killing in E. kuehniella. CI was also observed in the cross of double-infected males and single-infected females. Comparison of the single- and double-infected insects by real-time quantitative polymerase chain reaction suggested that the wCauA density is not affected much by the presence or absence of wCauB.

Genes for the Type IV Secretion System in an Intracellular Symbiont, Wolbachia, a Causative Agent of Various Sexual Alterations in Arthropods

Wolbachia species are intracellular bacteria known to cause reproductive abnormalities in their hosts. In this study, we identified Wolbachia genes encoding homologs to the type IV secretion system by which many pathogenic bacteria secrete macromolecules. The genes identified encoded most of the essential components of the secretion system and were cotranscribed as an operon.

Amino acid composition of the honeydew of symbiotic and aposymbiotic pea aphids Acyrthosiphon pisum.

Abstract In an attempt to establish the roles played by intracellular symbionts in the nitrogen metabolism of aphids, the level of uric acid, ammonia and amino acids in the honeydew of symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum , were determined. It was shown that the honeydew of neither symbiotic nor aposymbiotic aphids contained any detectable amount of uric acid. A small amount of ammonia was present in the honeydew of both symbiotic and aposymbiotic aphids. The amino acid composition of the honeydew was compared with that in the phloem sap on which the aphids feed. Asparagine and glutamine were the two most abundant amino acids in the phloem sap. It was found that the amino acid composition of the honeydew changed with the age of the aphid. Amounts of asparagine and glutamine were smaller in the honeydew of symbiotic adults (21–23 day) than in that of sterile, aposymbiotic adults at the same age, suggesting that these amino acids were utilized efficiently by symbiotic aphids. In the honeydew from old symbiotic aphids (42–48 day) that no longer produced progeny, asparagine was the only amino acid in appreciable amount. In the honeydew excreted by old aposymbiotic aphids, not only asparagine but also glutamine was abundant.

Wolbachia Infections and Cytoplasmic Incompatibility in the Almond Moth and the Mediterranean Flour Moth

Abstract Wolbachia are a group of inherited bacteria found in a number of arthropods and cause various reproductive alterations in their hosts, including feminization, parthenogenesis and cytoplasmic incompatibility. We examined Wolbachia infection in three species of moths belonging to the sub-family Phycitinae, the Indianmeal moth Plodia interpunctella, the almond moth Ephestia cautella and the Mediterranean flour moth Ephestia kuehniella. We detected infections in E. cautella and two strains of E. kuehniella, one from Tsuchiura city and the other from Yokohama city. Wolbachia was not detected in P. interpunctella. The phylogenetic positions of Wolbachia harbored by E. cautella and E. kuehniella were estimated based on the sequences of the wsp gene which encodes a Wolbachia surface protein. We also performed crossing experiments to examine cytoplasmic incompatibility. It was shown that Wolbachia in E. cautella cause complete cytoplasmic incompatibility: no egg-hatch was observed in the cross between infected males and uninfected females. Both Tsuchiura and Yokohama strains of E. kuehniella showed partial cytoplasmic incompatibility, but the levels were significantly different between the two strains. The rates of egg hatch in the incompatible crosses within Tsuchiura and Yokohama strains were 60.8% and 16.9%, respectively.