Cockroaches have endosymbiotic bacteria in their fat bodies. Recent molecular phylogenetic analyses on both hosts and endosymbionts have revealed that co-evolution has occurred throughout the history of cockroaches and termites. Co-cladogenesis was also shown among closely related taxa (woodroach genus Cryptocercus; Cryptocercidae), and thus endosymbiont data are likely to be useful for biogeographical analyses. To test the possibility of co-cladogenesis among inter- and intraspecific taxa, as well as the utility of endosymbiont data for inferring biogeographical scenarios, we analyzed rRNA genes of endosymbionts of Japanese and Taiwanese Panesthiinae (Salganea and Panesthia; Blaberidae), on which phylogenetic analyses previously had been performed based on the mitochondrial genes. Statistical analyses on the topologies inferred from both endosymbiont and host mitochondria genes showed that co-cladogenesis has occurred. The endosymbiont sequences examined appear to have evolved in a clock-like manner, and their rate of evolution based on the host fossil data showed a major difference in the time of invasion of the two Japanese genera, that is congruent with the recent analyses of their mitochondrial genes.

Phylogeography of the ermine Mustela erminea and the least weasel M. nivalis from Palae-arctic and Nearctic regions were investigated based on mitochondrial DNA control region sequences. Mustela erminea exhibited a very low level of genetic variation, and geographic structures among populations were unclear. This may indicate that M. erminea recently reoccupied a wide territory in Eurasia following the last glacial retreat. In comparison with M. erminea, genetic variations within and among populations of M. nivalis were much greater. Molecular phylogenetic relationships showed that two lineages of M. nivalis occurred in the Holarctic region: one spread from the Eurasian region to North America, and the other occurred in south-eastern Europe, the Caucasus and Central Asia. The results suggest either mitochondrial DNA introgression among populations of south-eastern Europe, the Caucasus and Central Asia, or ancestral polymorphisms remaining in those populations. Contrastive phylogeographic patterns between the two mustelid species could reflect differences of their migration histories in Eurasia after the last glacial age.

Cuticular substances on the body surface of crickets serve as pheromones that elicit a variety of different behaviors in male crickets. Antennal contact between males and females resulted in courtship behavior, and that between two males resulted in aggressive displays. As a first step in elucidating how crickets recognize and discriminate individuals, behavioral responses of male individuals to cuticular substances of conspecific males or females were investigated. The behavioral responses of males to antennal or palpal stimulation with an isolated antenna from a male or a female were recorded. To both antennal and palpal stimulation with female antennae, the majority of males responded with courtship behavior; to stimulation with male antennae, males responded with aggressive displays. To gain insight into the chemical nature of the behaviorally relevant components, isolated antennae were washed in either n-hexane, acetone or ethanol before behavior assays. Washed antennae no longer elicited courtship or aggressive responses in males. Next, polypropylene fibers were smeared with substances from the body surface of females and used for antennal stimulation. This experiment showed that the quality and quantity of cuticular substances appear to be highly age-dependent. Significantly more males responded with courtship behavior to cuticular substances from younger females. Isolated males generally showed higher levels of aggression than males reared in groups. Grouped males also were more likely to display courtship behavior towards antennae from younger females, and aggressive behavior towards antennae from older females. These results suggest that male discrimination of mating partners depends on the nature of female cuticular substances.

Bengalese finches, Lonchura striata, are extremely sexually dimorphic in their singing behavior; males sing complex songs, whereas females do not sing at all. This study describes the developmental differentiation of the brain song system in Bengalese finches. Nissl staining was used to measure the volumes of four telencephalic song nuclei: Area X, HVC, the robust nucleus of the arcopallium (RA), and the lateral portion of the magnocellular nucleus of the anterior nidopallium (LMAN). In juveniles (circa 35 days old), Area X and the HVC were well developed in males, while they were absent or not discernable in females. The RA was much larger in males but barely discernable in females. In males, the volumes of Area X and the RA increased further into adulthood, but that of the HVC remained unchanged. The LMAN volume was greater in juveniles than in adults, and there was no difference in the LMAN volume between the sexes. The overall tendency was similar to that described in zebra finches, except for the volume of the RA, where the degree of sexual dimorphism is larger and the timing of differentiation occurs earlier in Bengalese finches. Motor learning of the song continues until day 90 in zebra finches, but up to day 120 in Bengalese finches. Earlier neural differentiation and a longer learning period in Bengalese finches compared with zebra finches may be related to the more elaborate song structures of Bengalese finches.

The chemotactic response of the nematode Caenorhabditis elegans is known to be affected by the population density on an assay plate, suggesting the existence of interactions between individual animals. To clarify the interactions between individuals during chemotaxis, we investigated the effect of population density at an attractant area on the chemotactic response to water-soluble sodium acetate and odorant diacetyl using wild-type N2 animals and daf-22 (m130) mutants, which have defective pheromone secretion but can sense pheromone. The chemotaxis index of N2 animals at 90 min of the assay negatively correlated with the number of animals on the assay plate regardless of the type of attractant used (p<0.01). On the other hand, there was no significant difference in the chemotaxis indices of daf-22 (m130) mutants for either of the attractants between the low- and high-population groups. When daf-22 (m130) mutants of a high population density were placed at the attractant location in advance, the chemotaxis index of N2 animals was almost the same as that in the control assay in which no animals were placed at the attractant location in advance. When N2 animals of a high population density were placed at the attractant location in advance, the chemotaxis indices of N2 animals and daf-22 (m130) mutants were significantly smaller than those obtained in the control assay (p<0.05). In the absence of an attractant, we observed a decline in the fraction of animals in the neighborhood of N2 animals of a high population density, although the nematodes were not influenced by daf-22 (m130) mutants of a high population density. These results suggest that the attraction of nematodes to chemicals is inhibited by an increase in the concentration of the pheromone generated by N2 animals at the attractant location.

Leydig cells of the adult mouse testis express at a detectable level three distinct glandular (tissue) kallikrein genes: mKlk21, mKlk24, and mKlk27. Recently, the proteins encoded by these genes were characterized using active recombinant proteases, but their roles in the mouse testis remained to be determined. The present study showed that among the proteases, mK24 markedly enhanced the activity of human recombinant single-chain tissue-type plasminogen activator when the two were incubated together. This activation was found to be due to proteolytic conversion of the single-chain enzyme to a two-chain form. The expression of tissue-type plasminogen activator in interstitial Leydig cells was demonstrated by RT-PCR and immunohistochemical analyses. The primary culture medium of adult male testicular Leydig cells contained immunoreactive substances recognized by anti-mK24 antibodies. In addition, the same medium was capable of converting the single-chain plasminogen activator to the two-chain protein. These results suggest that mK24 may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis, due not only to its own activity, but also to that of plasmin produced by the single-chain tissue-type plasminogen activator-converting activity of mK24.

Sponges (phylum Porifera) have remarkable regenerative and reconstitutive abilities and represent evolutionarily the oldest metazoans. To investigate sponge stem cell differentiation, we have focused on the asexual reproductive system in the freshwater sponge Ephydatia fluviatilis. During germination, thousands of stem cells proliferate and differentiate to form a fully functional sponge. As an initial step of our investigation of stem cell (archeocyte) differentiation, we isolated molecular markers for two differentiated cell types: spicule-making sclerocyte cells, and cells involved in innate immunity. Sclerocyte lineage-specific Ef silicatein shares 45% to 62% identity with other sponge silicateins. As in situ hybridization of Ef silicatein specifically detects archeocytes possibly committed to sclerocytes, as well as sclerocytes with an immature or mature spicule, therefore covering all the developmental stages, we conclude that Ef silicatein is a suitable sclerocyte lineage marker. Ef lectin, a marker for the cell type involved in innate immunity, shares 59% to 65% identity with the marine sponge Suberites domuncula galactose-binding protein (Sd GBP) and horseshoe crab Tachypleus tridentatus tachylectin1/lectinL6. Since Sd GBP and tachylectin1 are known to bind to bacterial lipopolysaccharides and inhibit the growth of bacteria, Ef lectin may have a similar function and be expressed in a specialized type of cell involved in defense against invading bacteria. Ef lectin mRNA and protein are not expressed in early stages of development, but are detected in late stages. Therefore, Ef lectin may be specifically expressed in differentiating and/or differentiated cells. We suggest Ef lectin as a marker for cells that assume innate immunity in freshwater sponges.

Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunore-active adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian α-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-β-endorphin II as well as anti-mammalian α-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHβ and salmon LHβ, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate the transcription of multiple thyroid hormone (TH)-responsive genes. Our study aimed to identify TH-responsive genes in an estrogen-responsive chicken hepatoma cell line, LMH. RNA was prepared from cells treated with or without 10⁻⁸ M 3,3′,5-triiodothyronine (T₃) for 24 h and was analyzed by differential display. At least six cDNAs were detected whose transcript increased in the presence of T₃, and four of them were cloned. The four candidate TH-responsive genes that were identified had high similarity (>83%) to known chicken or mammalian genes, which included the ribosomal protein L7 gene; the cytoplasmic dynein heavy chain gene; the scaffold attachment factor A (SAF-A) gene, also known as heterogenous nuclear ribonucleoprotein U (hnRNP U); and a gene for an unknown protein. Real-time PCR confirmed that the transcription of the four genes was responsive to T₃; their transcript levels increased from four to eleven times with the administration of T₃. The amount of TRβ transcript did not change with the administration of T₃. The physiological reasons for the activation of these genes and the utility of this cell line are discussed.

The alien Chinese mussel Anodonta woodiana was first reported in Poland in the system of heated lakes near Konin in 1993. Genetic studies with use of three molecular techniques (isoenzyme electrophoresis, PCR-RFLP and sequence analysis of a COI gene fragment) were carried out on the Polish first populations of A. woodiana. The studies have revealed low genetic variation between the populations (Nei's genetic distance for 12 loci ranged 0.000 to 0.007) as well as their considerable polymorphism. Each population averaged 2.28 alleles per locus, 2.72 alleles per polymorphic locus, and 75% polymorphic loci. Restriction analysis of the COI gene fragment have not revealed variability between the analysed specimens, including males and females. Restriction enzymes, ScrFI, Csp6I, and EcoRI used in the COI gene fragment PCR-RFLP generate distinct restriction patterns, which can be molecular markers for A. woodiana. The sequence obtained for COI fragment was the same in the examined female and male specimens and represents F mitotype (DNA was isolated from somatic tissues). The divergence between A. woodiana F and M mitotypes is high (34%), however it remains within the range of the general character of the DUI (doubly uniparental inheritance) phenomenon in freshwater bivalves (Unionidae).

Red-green color vision in primates is unique in the sense that it is mediated by two photo-receptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 μm in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.

An attempt was made to determine cyclicity in yaks using plasma progesterone during the breeding and non-breeding seasons. Fifteen non-lactating yaks were used in this experiment. During the breeding season (July to November), blood samples were collected from 8 yaks at least twice weekly until estrus was observed and then at 2 days interval for 30 days. During the non-breeding season (February to March), blood samples were collected from the same number of yaks at 2-day interval for 30 days. Progesterone was determined in plasma samples by radioimmunoassay. During the breeding season, plasma progesterone at estrus was basal (≤0.2 ng/ml). Concentrations increased thereafter with a sharp increase during the late luteal phase, typically reaching peak levels around day 15. Concentrations then declined rapidly over the following 4 days, reaching basal levels at estrus. A high proportion (66.7%) of potential estrous periods (based on progesterone concentrations) went undetected, indicating that silent or weak estrus was a prominent problem in yak cows. During the non-breeding season, three animals were found to be cycling as determined by the patterns of plasma progesterone. Yet, behavioral symptoms of estrus were not observed in any of these yak cows. We conclude that peripheral plasma progesterone concentrations can be used to monitor cyclicity in yak cows effectively.

To induce sex reversal of male to female, freshly-fertilized eggs of the S-rR strain medaka (Oryzias latipes) were immersed in saline containing estradiol-17β (E₂) in different concentrations for various durations until hatching. Results of the present experiment showed that the immersion duration in 1 μg/ml E₂ to induce 100% reversal of sex differentiation in the genotypic males was enough only for one day (24 hr) post-fertilization (dpf) and that treatment with E₂ for 1 dpf resulted in a dose-dependent manner with the maximum sex reversal of 100% at 1 μg/ml. To ascertain early developmental periods efficacious for inducing sex reversal, additional brief immersion treatments of eggs with E₂ were further performed individually for four different early developmental periods (Stages 4–9, 10–12, 13–15 and 16–18) within 1 dpf. As a result, induction of sex reversal was observed in all these short immersion periods without any restricted efficacy. Between both experimental and control groups treated with or without E₂ for 1 dpf, differences in the number of germ cells in a gonad were compared in newly-hatched fry. It was found that gonads of the genotypic males (XY) treated with E₂ revealed the female type which contained many germ cells with much dividing activity. These data suggest that a possible switch mechanism that exogenous E₂ could trigger to change the genetic cascades involved in sex determination upon fertilization exists in early developmental stages.