Female pleopods in Procambarus clarkii have a function of holding eggs, and their length shows positive allometry. It is likely that the positive allometry has resulted from directional selection in terms of egg holding ability. Firstly, we examined the allometry of reproductive potential (ovarian egg number). The allometric value of reproductive potential was lower than that of pleopod length, which indicates that the positive allometry of pleopod length is not merely the reflection of the allometry of reproductive potential. Secondly, we examined the relationship between the relative length of pleopod and egg holding ability to test the presence of directional selection on the pleopod length, and then compared the strength of the directional selection between the individuals differing in their body sizes to infer the mechanisms causing the positive allometry for female pleopods. There was a positive relationship between relative pleopod length and relative egg mass remaining to the pleopod, and this relationship was stronger in small individuals. These relationships were not merely the reflection of relationship between pleopod length and reproductive potential. These results indicate that directional selection was acting on the length of female pleopods, and the strength of the directional selection was stronger in smaller individuals. Therefore, it appears that the positive allometry of female pleopods in P. clarkii has been caused by the constraint for possessing longer pleopods in small individuals, although the mechanisms of the constraint remain unknown.

The draft genome sequence and a large quantity of EST and cDNA information are now available for the ascidian Ciona intestinalis. In the present study, genes involved in pigment synthesis pathways were identified in the decoded genome of Ciona, and information about these genes was obtained from available EST and cDNA sequences. It was found that the Ciona genome contains orthologous genes for each enzyme of the melanin, pteridine, ommochrome, papiliochrome, and heme synthesis pathways. Several appear as independent duplications in the Ciona genome. Because cDNA clones for all but two of these genes have already been isolated by the cDNA project, C. intestinalis will provide an experimental system to explore molecular mechanisms underlying color patterns, through future genome-wide studies.

Soluble guanylyl cyclase (soluble GC) is an enzyme consisting of αand β subunits and catalyzes the conversion of GTP to cGMP. The formation of the heterodimer is essential for the activity of soluble GC. Each subunit of soluble GC has been shown to comprize three functionally different parts: a C-terminal catalytic domain, a central dimerization domain, and an N-terminal regulatory domain. The central dimerization domain of the β₁ subunit, which contains an N-terminal binding site (NBS) and a C-terminal binding site (CBS), has been postulated to be responsible for the formation of α/β heterodimer. In this study, we analyzed heterodimerization by the pull-down assay using the affinity between a histidine tag and Ni² Sepharose after co-expression of various N- and C-terminally truncated FLAG-tagged mutants of the α₁ subunit and the histidine-tagged wild type of the β₁ subunit in the vaculovirus/Sf9 system, and demonstrated that the CBS-like sequence of the α₁ subunit is critical for the formation of the heterodimer with the β₁ subunit and the NBS-like sequence of the α₁ subunit is essential for the formation of the enzymatically active heterodimer, although this particular sequence was not involved in heterodimerization. The analysis of the secondary structure of the α₁ subunit predicted the existence of an amphipathic α-helix in residues 431–464. Experiments with site-directed α₁ subunit mutant proteins demonstrated that the amphipathicity of the α-helix is important for the formation of the heterodimer, and Leu⁴⁶³ in the α-helix region plays a critical role in the formation of a properly arranged active center in the dimer.

Mushroom bodies (MB) are insect brain centers involved in learning and other complex behaviors and they are particularly large in ants. We describe the larval and pupal development of the MB in the carpenter ant, Camponotus japonicus. Based on morphological cues, we characterized the stages of preimaginal development of worker ants. We then describe morphological changes and neurogenesis underlying the MB development. Kenyon cells are produced in a proliferation cluster formed by symmetrical division of MB neuroblasts. While the duration of larval instars shows great individual variation, MB neuroblasts increase in number in each successive larval instar. The number of neuroblasts increases further during prepupal stages and peaks during early pupal stages. It decreases rapidly, and then neurogenesis generally ceases during the mid pupal stage (P4). In contrast to the larval period, the MB development of individuals is highly synchronized with physical time throughout metamorphosis. We show that carpenter ants (C. japonicus) have approximately half as many MB neuroblasts than are found in the honey bee Apis mellifera. Mature MBs of carpenter ants and honey bees reportedly comprise almost the same number of neurons. We therefore suggest that the MB neuroblasts in C. japonicus divide more often in order to produce a final number of MB neurons similar to that of honey bees.

Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegfl7. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as Xl-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.

The roles of photoperiod, melatonin, and the pineal gland in regulating the magnitude of compensatory gonadal hypertrophy (CGH) and other reproductive and non-reproductive organ growth during post-weaning development were examined in the marsh rice rat Oryzomys palustris. Juvenile rice rats of both sexes were left gonadally intact (control group) or unilaterally castrated (ULC) and housed on 12L:12D, 14L:10D, or 16L:8D. Within a photoperiod (14L:10D and 16L:8D, but not 12L:12D), growth of the remaining testis, but not the remaining ovary, as well as several additional organs in both sexes were significantly affected, suggesting that the compensatory hypertrophy of the testis is photoperiod-dependent. There was no effect of testis asymmetry on CGH as ULC of either testis in rice rats housed on 14L:10D resulted in a comparable increase of CGH. Melatonin implants in rice rats maintained on 16L:8D had little to no effect (CGH included) on most parameters examined. Both melatonin implants and pinealectomy (separate experiments) in rice rats transferred to 12L:12D prevented short photoperiod-induced effects on CGH, the growth of the reproductive organs and the Harderian glands. Evening melatonin injections had a significant inhibitory effect on the growth of the remaining testis (no CGH was observed) and all other parameters measured. Lastly, ULC did not alter the percentage of males which successfully mated compared to intact animals. Taken together, these data suggest that photoperiod, melatonin, and the pineal gland can affect and regulate reproductive (e.g., CGH in some cases) and non-reproductive growth during postnatal development in the marsh rice rat.

The cabbage armyworm, Mamestra brassicae, has winter- and aestival- diapause pupae (WD- and AD-pupae) showing differences in the strength of diapause. We tried to quantify diapause-strength by measuring the doses of 20-hydroxyecdysone (20-E) required to induce adult development in WD-, AD- and decerebrated non-diapause pupae (ND-pupae). The role of the brain in the regulation of diapause-strength was studied through the decerebration and brain-reimplantation of WD- and AD-pupae. The 20-E doses required for adult development were small within the first 2 days of pupation, and increased thereafter to reach a constant level about 10 days after pupation in AD- and decerebrated ND-pupae. The required 20-E doses in WD-pupae increased for more than 40 days after pupation. When 0-day-old WD- and 0-day-old AD-pupae were decerebrated, required 20-E doses increased after pupation and reached a constant about 10 days later. The required 20-E dose reached a constant level in decerebrated WD-pupae that was smaller than that observed for decerebrated ND- and WD-pupae. Furthermore, the required doses increased when 0-day-old WD-pupal brains were reimplanted into decerebrated WD-and decerebrated ND-pupae. In WD-, AD- and decerebrated ND-pupae, diapause-strength can be represented as the 20-E dose required for adult development. Diapause-strength is weak after pupation, increases thereafter, and reaches a constant about 10 days later in AD- and decerebrated ND-pupae. In WD-pupae, diapause-strength increases for more than 40 days after pupation and reaches a level that is twice that estimated for AD-pupae. Brains of diapausing WD-pupae may secrete a factor that suppresses the 20-E responsiveness of pupal organs, for the purpose of maintaining winter-diapause.

The epithelium of anterior midgut of adult Cenocorixa bifida was examined with light and electron microscopy. The folded epithelium is composed of tall columnar cells extending to the lumen, differentiating dark and light cells with interdigitating apices and regenerative basal cells in the nidi surrounded by villiform ridges that penetrate deeply into the epithelium. The columnar cells display microvilli at their luminal surface. Microvilli lined intercellular spaces and basal plasma membrane infoldings are associated with mitochondria. These ultrastructural features suggest their role in absorption of electrolytes and nutrients from the midgut lumen. The columnar cells contain large oval nuclei with prominent nucleoli. Their cytoplasm is rich in rough endoplasmic reticulum, Golgi complexes and electron-dense secretory granules indicating that they are also engaged in synthesis of digestive enzymes. The presence of secretory granules in close proximity of the apical plasma membrane suggests the release of secretion is by exocytosis. The presence of degenerating cells containing secretory granules at the luminal surface and the occurance of empty vesicles and cell fragments in the lumen are consistent with the holocrine secretion of digestive enzymes. Apical extrusions of columnar cells filled with fine granular material are most likely formed in response to the lack of food in the midgut. The presence of laminated concretions in the cytoplasm is indicative of storageexcretion of surplus minerals. The peritrophic membrane is absent from the midgut of C. bifida.

Sex reversal of XY male to functional females was induced by estrogen treatment during the embryonic period in the medaka Oryzias latipes. The present study aimed to examine whether exogenous estrogen (estradiol-17β; E₂) affects early sex differentiation, paying particular attention to DMY expression and proliferation activity of germ cells in estrogen treated XY individuals. Our results showed that germ cell number was not affected by E₂ treatment at hatching, and that DMY expression was not suppressed under conditions of sex reversal. Therefore, male differentiation of germ cells, which is triggered by the expression of DMY in the supporting cell lineage, proceeds even in E₂ treated XY individuals until hatching, and early sex differentiation is not altered by estrogen. However, sex reversal occurred after hatching probably because of estrogen remaining in the yolk. Interestingly, DMY expression was also detected in the large follicle layer of E₂ treated XY ovary. These results suggested that DMY regulates male determination in early embryonic stage but does not suppress female follicle development.

Coral reef fishes exhibit a diversity of hermaphroditic strategies and comparisons among species with different ecological characteristics will help identify the underlying basis of this complexity. We used manipulative experiments to test the potential for bi-directional sex change in three species of Pseudochromis (Pseudochromidae): P. flavivertex, P. aldabraensis and P. cyanotaenia. The first two species are sexually monochromatic, whereas, P. cyanotaenia is sexually dichromatic. For each species, where two functional females were kept together, one individual in the pair changed sex to male. Where two functional males were kept together, one individual in the pair changed sex to female. In all three species, functional sex change from male to female (52–93 days) took longer than sex change from female to male (18–56 days). In the sexually dichromatic species, P. cyanotaenia, colour change accompanied adult sex change. Females that changed sex to male took on the bright colouration of males and males that changed sex to female took on the drab colouration of females. These results indicate that bi-directional sex change is probably widespread in the family Pseudochromidae and cannot be predicted by the presence or absence of secondary sexual characteristics.

Karyotype of a Japanese small salamander, Hynobius katoi, was first described. All individuals examined had 2n=58 chromosomes, consisting of nine pairs of biarmed macrochromosomes, four pairs of biarmed medium-sized chromosomes, six pairs of biarmed microchromosomes, and 10 pairs of uniarmed microchromosomes, although distinction of the second and the third groups of chromosome pairs was not clear. All pairs appeared homologous and no sexual dimorphism was found. Possession of 2n=58 chromosomes in H. katoi strongly suggests its lotic-breeding habits as was expected from the number and size of eggs and adult morphology. When compared morphology of chromosomes among lotic-breeders with 2n=58 chromosomes, metacentric nature of No. 10 seems to characterize the karyotype of H. katoi.

A new species of torrent-dwelling bufonid frog of the genus Ansonia is described from the Isthmus of Kra, Thailand. Ansonia kraensis is morphologically similar to Malaysian A. malayana, but differs from it in ventral coloration and larval morphology. Occurrence of A. kraensis in this region suggests a heterogeneous nature of the anuran fauna between northern and southern regions of the Malay Peninsula.