Hisato Kuniyoshi

Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins

A Bombyx mori cDNA was cloned that hybridized with Hyalophora cecropia attacin probe and its nucleotide sequence was determined. This cDNA consisted of 846 nucleotides and the deduced amino acid sequence showed that the cDNA encodes an attacin precursor protein. The putative mature protein of B. mori attacin had 70.4, 68.3 and 18.8% identity in amino acid sequences with that of H. cecropia acidic and basic attacins and Sarcophaga peregrina sarcotoxin IIA, respectively. B. mori and H. cecropia attacins and S. peregrina sarcotoxin IIA had two subdomains in each G domain, suggesting that common amino acid residues in the subdomains are conserved during evolution and plays an important role in the activity of the antibacterial proteins. Expression of B. mori attacin gene was rapidly induced by the injection of Escherichia coli cells into B. mori larvae and continued at least for 48 h mainly in fat bodies and hemocytes.

N-terminal modified analogs of C-terminal fragments of PBAN with pheromonotropic activity

Abstract Various analogs corresponding to the C-terminal portion of pheromone biosynthesis activating neuropeptide of silkmoth, Bombyx mori (Bom-PBAN), were synthesized and tested for pheromonotropic activity. Analogs modified at N-terminal amino group, N -acyl, N -alkyl, D -alanyl and pyroglutamyl hexapeptides, exhibited much greater activity than the corresponding peptides with a free N-terminal amino group. Some of them exhibited higher activity than the unoxidized Bom-PBAN. These blocked peptides were stable in diluted haemolymph from B. mori in vitro , while the non-blocked peptide decomposed rapidly. These results suggest that the stability of the blocked peptides in haemolymph might account for their enhancement of biological activity in the in vivo assay.

Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm, Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.

Phylogenetic relationship of two Mola sunfishes (Tetraodontiformes: Molidae) occurring around the coast of Japan, with notes on their geographical distribution and morphological characteristics

Phylogenetic analyses were conducted using complete nucleotide sequences of the D-loop in the mitochondrial genome of Mola specimens, collected mainly in Japanese waters, to clarify the genetic features and distribution patterns of Mola sunfishes. Two significantly distinct groups (designated A and B) were present in the genus, with a considerable net nucleotide sequence divergence between the two (8.4%). The two groups occurred sympatrically around the Japanese coast, as previously suggested by Sagara et al. (2005). Group A occurred mostly on the Pacific coast of eastern Japan, while group B was widely distributed along the Kuroshio Current, strongly suggesting different migration routes for each group. The morphological characteristics of the two group specimens were differentiated via the head bump, body proportions and shape of the clavus. Through the addition of Mola sequence data taken from outside Japan to our phylogenetic analyses, three independent groups, including groups A and B, were found, each with a wide geographical distribution, which suggests the presence of at least three independent species within the genus Mola.

Neural Inactivation of Sex Pheromone Production in Mated Females of the Silkworm Moth, Bombyx mori

Abstract Pheromone content in the pheromone gland of the female moth, Bombyx mori, declines after mating with a time course closely resembling that of decapitated females. The inactivation of pheromone production after mating was prevented when the ventral nerve code or a pair of peripheral nerves (N4) extending from the terminal abdominal ganglion was severed before mating. In contrast, the post-mating inactivation of pheromone production was not prevented when the ventral nerve cord was transected 1 hr after the initiation of mating. Although females produced only a small amount of pheromone when the connection between the brain and the suboesophageal ganglion (SG) was cut at an early pupal stage, mating did not induce a significant decline of pheromone production in these females. The results suggest that inactivation of pheromone production is mediated by a neural signal, originating from a peripheral receptor, that is sent via the ventral nerve cord to the brain-SG complex to suppress activity of the neurosecretory cells responsible for the release of pheromonotropic neuropeptides, such as a pheromone biosynthesis activating neuropeptide (PBAN).

Molecular Cloning and Expression Profile of Sex-Specific Genes, Figla and Dmrt1, in the Protogynous Hermaphroditic Fish, Halichoeres Poecilopterus

The genes folliculogenesis specific basic helix-loop-helix (facor in the germline alpha, Figla) and doublesex and mab-3 related transcription factor 1 (Dmrt1) are female- and male-specific genes that play key roles in sex differentiation. To obtain a better understanding of the molecular mechanisms underlying female-to-male sex change, we cloned the cDNAs of these genes from an ovary and a testis of the protogynus wrasse, Halichoeres poecilopterus. This fish has two isoforms of Dmrt1, Dmrt1a and Dmrt1b, caused by an alternative splicing. The Dmrt1b has an insertion of three nucleotides (CAG) in the open reading frame. Figla and Dmrt1 displayed gonadal-specific expression and abundant in the ovaries and in the testes, respectively. In particular, levels of Figla expression in the ovaries were higher in the spawning season than in the non-spawning season. Once sex change began, Figla mRNA decreased and Dmrt1 mRNA increased with progression of oocyte degeneration and spermatogenesis. These expression levels were maintained until the completion of the sex change. Low Figla and high Dmrt1 were also observed in testes of primary males, which functioned as a gonochoristic male throughout its life span in this wrasse. The results of this study suggest that these genes may regulate the gonadal transition from ovary to testis by the same mechanism as that of formation and maintenance of the primary testis in H. poecilopterus.

Indomethacin induction of metamorphosis from the asexual stage to sexual stage in the moon jellyfish, Aurelia aurita.

We found while screening a chemical library that indomethacin, an inhibitor of prostaglandin biosynthesis, induced strobilation (metamorphosis from the asexual to sexual stage) in the moon jellyfish, Aurelia aurita. Indomethacin initiated strobilation in a dose-dependent manner, but was not involved in the progression of strobilation. Pharmacological experiments suggested that indomethacin could induce strobilation independently of prostaglandin biosynthesis.

Social control of terminal phase transition in primary males of the diandric wrasse, Halichoeres poecilopterus (Pisces: Labridae)

In many diandric fishes, large territorial males with bright body coloration (terminal phase (TP) males) are derived either from initial phase (IP) females that change sex to male or from IP primary males that change color and behavior, but do not change sex. The mechanism controlling the transition of IP primary males into TP males is not well understood. We conducted cohabitation experiments to examine social conditions favoring TP transition by primary males in the diandric wrasse, Halichoeres poecilopterus. IP primary males always started TP-specific sexual behavior in the presence of a smaller subordinate, and subsequently acquired TP body coloration. In contrast, primary males under subordinate conditions often performed female-like sexual behavior. In pairs with similar body sizes, both individuals initiated TP male behavior. The results suggest that TP transition in primary males may be closely related to a dominance relationship (or size order) within social groups, as it is in the case of sex change by females.

Cloning of Full-Length cDNA of Teleost Corticotropin-Releasing Hormone Precursor by Improved Inverse PCR

We designed a new inverse PCR protocol combined with switching mechanism at 5′ end of RNA transcript (SMART) technology, and applied it to the cloning of teleost corticotropin-releasing hormone precursor cDNA. Due to the advantages of both techniques, this method can efficiently amplify the complete 5′- and 3′-ends of cDNA in a single reaction, and might prove to be an alternative to the conventional rapid amplification of cDNA ends (RACE) approaches.

Ten novel polymorphic microsatellite loci of Red-spotted grouper (Epinephelus akaara) revealed from full-sib progeny and unrelated individuals

We isolated 12 candidate microsatellite loci from a small insert genomic DNA library of the Red-spotted grouper, Epinephelus akaara. Evaluation of full-sib progeny and unrelated individuals revealed that two of the 12 loci had distorted segregation states based on lack of adherence to Mendelian laws and Hardy–Weinberg equilibrium. We assessed polymorphism in the 10 loci using 20 unrelated individuals from a single population. The number of alleles per locus ranged from 2 to 19, allelic richness ranged from 2 to 17.6, and the observed heterozygosity ranged from 0.35 to 1.00 with no evidence of linkage disequilibrium.