To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.

We morphologically examined postembryonic compound eye development in Reticulitermes speratus (Kolbe) to understand developmental regulation during caste differentiation. The eye primordia were shown to exist from the larval stage. The number of ommatidia and compound eye size greatly increased over the course of imaginal development. Nymphoids (second-form reproductives) possessed a developed compound eye structure on the surface of the cuticle and thick optic nerves, but individual ommatidia were not clearly discriminated. However, in the line of apterous workers and soldiers, although the outer rim of the eye was observed from second-stage workers, there were few morphological differences among instars, including ergatoids (third-form reproductives). Both nymphoids and ergatoids are slightly physogastric and have highly developed reproductive organs. These results suggest that eye development in the apterous line could be strongly arrested and that there is a weak developmental correlation between the eyes and reproductive organs in R. speratus.

Here we report a novel small protein that is highly conserved across vertebrates. The protein, which we have named TRIQK, has no homology to any previously reported proteins or functional domains, but all vertebrate homologs of this protein share a characteristic triple repeat of the sequence QXXK/R, as well as a hydrophobic C-terminal region. The Xenopus triqk gene (xTriqk) was isolated in an expression screen on the basis of its ability to cause dramatic changes in cell size and nuclear size and morphology in developing embryos. The Xenopus and mouse triqk genes are broadly expressed throughout embryogenesis, and mtriqk is also generally expressed in mouse adult tissues. TRIQK proteins are localized to the endoplasmic reticulum membrane. Depletion of endogenous xTRIQK protein in Xenopus embryos causes no detectable morphological or functional changes in tadpoles.

To identify mammalian carnivore species distributed in the Kushiro Wetland, eastern Hokkaido, Japan, we developed molecular-genetic methods for identification of the species from fecal samples collected from the field. Species-specific primers and PCR programs were established for five native and six alien species of carnivores: Martes zibellina, Mustela nivalis, Mustela erminea, Vulpes vulpes, and Nyctereutes procyonoides as native species, and Neovison vison, Martes melampus, Mustela itatsi, Canis familiaris, Felis catus, and Procyon lotor as alien species in Hokkaido. Touchdown PCR, in which the annealing temperature is decreased 1°C every cycle, was more effective for some species from which fecal DNA was not amplified species-specifically with standard PCR programs. Of 405 fecal samples collected from the Kushiro Wetland, the species of origin of 246 samples were successfully identified: 88 samples for N. vison, 140 for M. zibellina, 13 for V. vulpes, four for C. familiaris and one for F. catus. The results show the particular applicability of this method to monitoring M. zibellina and N. vison. In addition, methods to PCR-amplify DNA from two crayfish species (Pacifastacus leniusculus and Cambaroides japonicus) were developed to determine whether the carnivore fecal samples contained detectable DNA from the prey crayfishes. DNA from P. leniusculus was amplified from feces of N. vison identified in the present study, but no DNA from C. japonicus was detected. This indicates that N. vison preys on the alien species P. leniusculus.

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.

Stanniocalcin 1 (Stc1) was originally identified as an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) associated with the kidney in teleosts. While the stc1 gene is expressed in various tissues in fishes, its role and regulation in extra-CS tissues are unexplored. In the present study, we characterized a cDNA of stc1 in a euryhaline fish, the Japanese flounder (Paralichyhus olivaceus), and examined its expression in peripheral tissues in response to different salinities and Ca² ion concentrations. The Japanese flounder stc1 cDNA (1331 bp) encodes a preprohormone of 251 amino acids (aa), with a signal peptide of 17 aa and a pro-sequence peptide of 15 aa followed by the mature protein of 219 aa. The deduced aa sequence of Japanese flounder stc1 showed highest sequence identity (94.0%) with the European flounder Stc1 among fish and mammalian species, but lower identity to zebrafish, pufferfish, and human STC2 (23.1–25.4%). Lowered environmental salinity resulted in a decrease in stc1 mRNA expression in vivo in the gills, kidney, intestine, and CS glands of the Japanese flounder. Furthermore, we found that extracellular Ca² increased steady-state stc1 mRNA levels in gill and kidney cells as well as in the CS cells. Our findings suggest that Stc1 synthesis in the ionregulatory tissues is responsive to environmental salinity and Ca² level.

In teleosts, prolactin is involved in calcium regulation, but its role in scale/bone metabolism is unclear. Using the in-vitro system with goldfish scales developed recently, we explored the effects of teleost prolactin, growth hormone, and somatolactin on osteoclasts and osteoblasts. Addition of prolactin at concentrations of 0.01–100 ng/ml reduced osteoclastic activity, partly via osteoclast apoptosis, after 6–18 h incubation. Conversely, growth hormone and somatolactin at a concentration of 100 ng/ml increased osteoclastic activity after 18 h incubation, indicating the specificity of the inhibitory effect of prolactin on osteoclastic activity. On the other hand, these three hormones promoted osteoblastic activity at concentrations of 10–100 ng/ml. The results from this study are the first demonstration of direct effects of prolactin on scale/bone metabolism and osteoclastic activity in a teleost.

A transposon-inserted mutant of Drosophila melanogaster was recently identified, and the larvae show no food preference (Ryuda and Hayakawa, 2005). To reveal the genetic mechanism underlying the preference change in this mutant, a large-scale oligo-DNA microarray screening was carried out to identify genes whose expression is different in control and mutant strains. We focused especially on hunger-driven changes in gene expression in the larval central nervous system (CNS) of both strains, because the state of food depletion should promote a feeding response due to changed expression of certain genes in the CNS. We identified 22 genes whose expression changed after starvation in either or both of the two strains. Quantitative RT-PCR analyses confirmed the expression changes in four genes, CG6271, CG6277, CG7953, and new glue 3 (ng3, encoding a putative structural molecule). CG6271 and CG6277 encode triacylglycerol lipase, and CG7953 produces a protein homologous to a juvenile hormone (JH) binding protein. The expression of these two groups of genes was enhanced in control strain larvae with a normal food preference but not in GS1189 strain larvae. Given that these genes contribute to mediating hunger-driven changes in food preference and intake in D. melanogaster larvae, the dysfunction of these key genes could cause the defect in food preference observed in GS1189-strain larvae.

Apoptotic and necrotic changes in the midgut epithelium cells of Allacma fusca (Collembola, Symphypleona) are described at the ultrastructural level. The morphological sign indicating the beginning of the apoptotic process in these cells is their shrinkage and the transformation of their mitochondria. The nucleus assumes a lobular shape and finally undergoes fragmentation. The intercellular junctions between an apoptotic cell and adjacent epithelial cells gradually disappear. Apoptotic cells are discharged into the midgut lumen just beneath the peritrophic membrane, where they are initially distributed singly but ultimately form a single layer. No phagocytosis was observed, so no apoptotic bodies are formed. Only young midgut epithelium shows apoptosis; as cells age, necrosis accompanies apoptosis, and necrosis finally completely replaces apoptosis.

It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.

In resource-based promiscuous mating systems, synchronous spawning of females affects competition among males and variation in the reproductive success of males. We documented the mating behavior of Japanese charr (Salvelinus leucomaenis japonicus) through an annual breeding season to examine the relationship between female spawning synchrony and male mating behavior. Females spawned highly synchronously in the population studied, i.e., approximately half the spawning was finished within the first three days of the entire spawning season (11 days). The daily operational sex ratio (OSR) was nearly 1:1 through the spawning period. The number of males around a spawning female was very small (1.21±0.49 males per female) over the spawning ground and period, suggesting that a competitive male could effectively chase subordinate males away from a spawning female. A few males attempted to sneak near the oviposition site of females (16%; 9 of 57 breeding groups), while some males adopted sneaking tactics in the initial phase of females’ spawning (24%). We did not observe any males to succeed in sneak fertilizations. We conclude that in this Japanese charr population, the synchronous spawning of females was related to the unbiased daily OSR, male aggregation around females, and consequently whether and how efficiently males engaged in sneak mating behavior.

It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3β (GSK3β) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3β are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3β, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3β is a component of the PI3K signal transduction pathway.