The effects of fasting and increased feeding on plasma thyroid hormones, glucose, and total protein levels were investigated in immature greens (Chelonia mydas) and Kemp's ridley (Lepidochelys kempi) sea turtles. Under constant temperature, nutritional status affected plasma thyroid hormones and glucose level, but not protein level in green and Kemp's ridley turtles. Plasma T₄ in green turtles decreased with deprivation of food, but did not do so in Kemp's ridleys. Increased feeding did not affect circulating levels of plasma T₄ in either species. Plasma T₃, in contrast, tended to decrease during fasting and increase with refeeding or increased feeding. Blood glucose level fell with deprivation of food, but remained constant during increased feeding. Sea turtles may use glycogen or lipid during short-term fasting as in endothermic vertebrates.

Overlapping cDNA clones spanning the entire coding region of a Na channel were isolated from newt retina. The coding region predicts a 2,007 amino acid protein, designated nRNaCh (newt retina sodium channel), which is homologous to other Na channels. In situ hybridization indicated that nRNaCh is expressed exclusively in spiking neurons, where a tetrodotoxin (TTX)-resistant Na current has been recorded. Therefore, nRNaCh cDNA is sure to encode the TTX-resistant Na channel of newt retina. Sequence comparisons show that nRNaCh is more homologous to TTX-sensitive Na channels than to TTX-resistant Na channels. The length of the S5-S6 loop of repeat I of nRNaCh is similar to that of TTX-sensitive channels, whereas TTX-resistant Na channels have a deletion. The 3^rd position in the SS2 region of repeat I of nRNaCh is a non-aromatic amino acid (Ala), which is a common feature of TTX-resistant channels. These findings suggest that whether the amino acid at the 3^rd position in the SS2 region of repeat I is aromatic or non-aromatic determines the TTX sensitivity of Na channels, not the overall structure of the channel.

We investigated learned food-avoidance behavior of conditioned slugs in detail, and examined the effect of removing the inferior or superior tentacles (ITs and STs) on the avoidance behavior. The conditioning procedure significantly lowered the preference level of slugs to the conditioned odor, and the decrease of the preference was maintained for at least 48 hr. Video analysis experiments showed that change in the crawling direction during an aversion response occurred consistently accompanied by an average of 1.2 head swings. The first head swing was usually observed within 1 cm from the conditioned odor. Removal of the ITs after conditioning reversed the trend towards a lowered preference level. On the other hand, removal of the STs did not change the level. These results suggest that the IT inputs are needed to induce conditioned behavior. The contribution of STs to the conditioning behavior is indicated by the significant increase in the average number of head swings (3.6 swings) in a trial after their removal. In some trials, the slugs lacking STs avoided the odor source after crossing it. These observations indicate that removal of STs decreases the orientation ability of conditioned slugs.

The phase of locomotor activity of the onion fly, Delia antiqua, in L (400 lux) L_dim (1.0 lux) cycles delayed as compared with that in LD cycles. The free-running period (τ) in constant dim light (L_dimL_dim) was longer than that in constant darkness (DD), suggesting that the phase delay of locomotor activity in LL_dim cycle was caused by the increase in τ. At L_dimD 12:12 in which the light intensity of the photophase was 1.0 lux, the locomotor activity free-ran with the period shorter than 24 hr until about week 2 after eclosion but thereafter entrained to L_dimD in spite of τ might become longer than 24 hr. This suggests that the flies may become more sensitive to light intensity with age.

A 100 kDa protein was identified by a combination of microtubule affinity chromatography and microtubule co-sedimentation from Xenopus egg extracts. The 100 kDa protein was expressed in Stage VI oocytes and early embryos, and then decreased at tailbud stage. The 100 kDa protein was found in adult organs such as brain and liver. Immunofluorescent microscopy revealed that the 100 kDa protein was mainly detected in spinal cord, notochordal sheath, optic cup, lens and cement gland in tailbud embryos. In Xenopus A6 cells, the 100 kDa protein showed filamentous networks in the peripheral cytoplasm and uniform distribution around the nucleus. During cell division, the 100 kDa protein was localized to the mitotic apparatus. The 100 kDa protein may have some roles in microtubule dynamics, organization of the mitotic apparatus and maintenance of cell shape.

In the crayfish Procambarus clarkii, the gastroliths are formed as a paired structure in the stomach during the premolt period, and contain calcium carbonate and a small amount of an organic matrix. In this investigation, a cDNA encoding an insoluble matrix protein was isolated from P. clarkii. The open reading frame encoded 505 amino acid residues including two unique repeated sequences. The N-terminal half of the amino acid sequence, which included 10-amino-acid repeats, exhibited a high degree of similarity to that of involucrin, a protein synthesized in human keratinocytes. Northern blot analysis revealed that mRNA encoding the matrix protein is specifically expressed in the gastrolith discs during the premolt period in which the gastroliths formed. In the gastrolith discs, levels of expression of this mRNA correlated increases in weights of the gastroliths concomitant with their formation. Organ culture of the gastrolith discs suggested that expression of mRNA in the discs is induced by molting hormone, 20-hydroxyecdysone. These results reinforced the relationship between the matrix protein and formation of the gastroliths. Functional analysis showed that the protein inhibits calcium carbonate crystallization in a solution system, suggesting that the protein plays a role in the calcification of the gastroliths.

Two color patterns of the Tuxedo guppy strain commercially cultured in Singapore were subjected to genetic analyses. Gene control of the black caudal-peduncle and red tail color patterns was elucidated by reciprocal crosses between the Tuxedo (TUX) strain and wild-type (WT) stock. F₁ progenies were produced by single-pair crossing between TUX and WT, while the F₂ generation was obtained from full-sib mating between F₁ males and females. F₁ and F₂ data were segregated according to phenotypes and sex, and tested by chi-square analyses. Both color patterns show single gene inheritance, and are dominantly expressed in both sexes, sex-linked and determined by different loci on the X- and Y-chromosomes. Alleles for the black caudal-peduncle (Bcp) and red tail (Rdt) loci, are dominant over that of the wild-type, Bcp and Rdt , which do not display these color patterns. The typical genotypes for TUX guppies are proposed to be X_Bcp,RdtY_Bcp,Rdt for males and X_Bcp,RdtX_Bcp,Rdt for females. Heterozygous TUX males have the X_Bcp,RdtY_Bcp ,_Rdt, X_Bcp,RdtY_Bcp ,_Rdt and X_Bcp,RdtY_Bcp,Rdt genotypes while the females are X_Bcp,RdtX_Bcp ,_Rdt. The segregation and inheritance of the Bcp and Rdt genes are illustrated by genetic models. Map distances estimated from F₁ and F₂ recombinants are approximately 3.4, 5.1 and 2.4 map units for the sex-determining region (SdR)–Rdt, SdR–Bcp and Rdt–Bcp, respectively. The gene map order is hypothesized to be SdR–Rdt–Bcp.

Male Protophormia terraenovae showed a long-day photoperiodic response at 25°C with respect to the induction of mating behavior. Male adults transferred from long-day to short-day conditions at emergence showed no mating behavior. When the corpus allatum (CA) was removed 6–8 hr or 1 day after adult emergence under long-day conditions, about 60% of males showed mating behavior. When the CA was removed 2 days after adult emergence, however, most males showed mating behavior similarly to the intact controls. Application of a juvenile hormone analog increased the proportion of mating behavior from about 60 to 100% in males allatectomized 6–8 hr after adult emergence under long-day conditions, and from 0 to about 30% in intact males transferred to short-day conditions at adult emergence. Thus, the endocrine control of diapause in males is different from that in females in which the CA plays a predominant role. Male adults responded to photoperiod and showed mating behavior even after the CA was removed. The mating behavior is controlled also by a factor or factors other than the CA with respect to diapause.

We have previously demonstrated that the Japanese eel, Anguilla japonica, has two distinct mRNAs for prepro-mammalian gonadotropin-releasing hormone (prepro-mGnRH) and prepro-chicken GnRHII (prepro-cGnRH-II). In the present study, we found a splicing variant for the prepro-mGnRH mRNA, but not for the prepro-cGnRH-II mRNA in the eel. Genomic Southern blot analysis revealed that each of the eel prepro-GnRHs is encoded by a single gene. Isolation and sequencing of the two prepro-GnRH genes revealed that both of them contain four exons (denoted 1, 2, 3 and 4) separated by three introns (denoted A, B and C). Staining with ethidium bromide and Southern blotting following reverse transcription-polymerase chain reaction and electrophoresis revealed that in addition to a mature prepro-mGnRH mRNA, its splicing variant with retention of intron A is present in all tissues tested including the brain, pituitary, eye, olfactory epithelium, ovary, testis, liver, kidney, spleen, heart, gill, intestine, pancreas, muscle, skin, fin and peripheral blood leukocyte. This result differed to that of mammals, the splicing variant of which is present only in the peripheral tissues, but not in the brain. In contrast, only a mature mRNA for the prepro-cGnRH-II was detected in the middle part of dissected brain, pituitary, olfactory epithelium, ovary and testis, indicating that the two prepro-GnRH genes have different splicing processes. These findings suggested that the splicing pattern which produces the intron A retention variant dates from a common ancestor to teleosts and mammals, and is conserved during evolution. Therefore the splicing may be one of the mechanisms regulating prepro-mGnRH gene expression.

Estrogen receptors (ER) in mammals have recently been shown to be encoded by two distinct genes, ERα and ERβ. In this study, cDNAs encoding two tilapia ER subtypes, tER5.1 and tER4.3, were cloned from an ovarian cDNA library of a teleost fish, the tilapia Oreochromis niloticus. The tER5.1 and tER4.3 contain complete open reading frames encoding 585 and 557 amino acid residues, respectively. The two receptors share about 12% homology in the A/B domain, 96% in the DNA binding domain (C domain), 12% in the D domain, 57% in the ligand binding domain (E damain), and 20% in the F domain. Phylogenetic analysis of ER proteins from various vertebrate species indicated that vertebrate ERs consist of two major groups (ERα and ERβ); tER5.1 and tER4.3 belong to ERα and ERβ subtypes, respectively. Thus, we consider tER5.1 and tER4.3 to be the tilapia homologs of ERα (tERα) and ERβ (tERβ), respectively. In transient transfection assays using mammalian COS-7 cells, both tERα and tERβ showed estradiol-17β dependent activation of transcription from the estrogen-responsive ERE-Luc promoter. This is the first report of the presence of ERα and ERβ within a single non-mammalian vertebrate species.

Phylogenetic analyses were carried out for representatives of all eublepharid genera and a few other gekkonoid taxa using sequence data for 879 base pairs of mitochondrial 12S and 16S ribosomal RNA genes. Neighbor-joining (NJ) distance analysis of the data suggested independent great divergences of Coleonyx and Aeluroscalabotes, and monophyly of the remainder within Eublepharidae (bootstrap proportion [BP]=76%). Of the latter, the two African genera, Hemitheconyx and Holodactylus, were almost certainly monophyletic altogether (BP=99%), whereas their sister-group relationship with Eublepharis received a weaker, but still substantial support (BP=68%). Within Goniurosaurus kuroiwae, G. k. splendens first diverged from the remainder (BP=100%), followed by G. k. kuroiwae from the northern part of Okinawajima (BP=100%): G. k. kuroiwae from the southern part of Okinawajima and G. k. orientalis, differing from each other at only seven bases, diverged finally (BP=99%). Parsimony analysis yielded results consistent with those of NJ analysis with respect to the monophyly of the two African genera and relationships within G. kuroiwae, but retained the other relationships within Eublepharidae unresolved. Our results, while showing no serious discrepancies with the relationships among eublepharid genera hypothesized from morphological data, cast a serious doubt to the currently accepted population systematics within G. kuroiwae. Furthermore, results of both analyses suggested a closer affinity of Diplodactylinae (as represented by Rhacodactylus trachyrhynchus) with Eublepharidae, rather than with Gekkoninae. Our study lends a robust support to the Laurasian origin of the family Eublepharidae.

Several variations in larval life histories were described in a salamander Hynobius retardatus living in Hokkaido, Japan, which had been reported to propagate in larval forms in a specific environment of Lake Kuttara like the axolotl. In almost all populations living in lower land Hokkaido, spawning was observed in early spring, and hatched larvae metamorphosed by August or September. In some populations living in the similar ponds but supplied with a mountain stream or spring water, however, larvae could not complete their metamorphosis by late autumn in the first year. All the larvae passed winter season under snow and then metamorphosed between late May and mid June in the second year. In some specific populations in cold, mountainous ponds, larvae could not metamorphose during the first and second years and metamorphosed in the third year. Thus, three age-groups of larvae, which were 2-year- and 1-year-overwintered larvae, and larvae under the age of one, were concurrently observed in one pond. Body size at the completion of the metamorphosis in the 2-year-overwintered larvae was significantly larger than that in the metamorphosing or metamorphosed larvae under the age of one. When hemoglobin (Hb) transition from larval to adult types in each population was examined, adult globin subunits were expressed in the overwintered larvae, even though they had not completed their morphological metamorphosis, suggesting that the expression of some adult phenotypes was independent of morphological metamorphosis.

Karyological and allozymatic characterizations of recently collected samples of the Iberian Leptynia attenuata complex support the occurrence of three genetically differentiated groups, for which parallel morphological observations evidenced only partially diagnostic characters. The groups are: the Portuguese population of Fóia (Serra de Monchique), referred to as the nominal taxon, L. attenuata; the Spanish populations of the Sistema Central, referred to as L. montana; the populations of the Toledo district, referred to as L. caprai. These taxa seem to represent a case of incipient speciation, with chromosomal and genetic differentiation ahead of the morphological one. Chromosome repatternings, affecting autosomes as well as sex chromosomes, appear to go together with the evolutionary events.

Entire nucleotide sequence of the mitochondrial control region was obtained for the red-crowned crane Grus japonensis and two closely related species, the common crane G. grus and the hooded crane G. monacha. Control regions of these crane species could be divided into three subdomains (I, II, III) by incidence of sequence variation. The central domain II was well conserved with C, D and F sequence motifs, while both flanking domains were variable, with greater base substitution in 5′ domain I than 3′ domain III with CSB-1 motif. In addition, a 51 base-pair (bp) sequence was found to be deleted in the 5′ portion of domain I in G. grus and G. monacha compared with the domain I sequence of G. japonensis. Comparison of 418 bp sequence in variable 5′ portion spanning domain I and 5′ part of domain II among 29 individuals of G. japonensis including 14 from the East Asia mainland population and 15 from the Japan's endangered Hokkaido island population revealed seven haplotypes in the former and only two haplotypes in the latter. Small number of haplotypes in the Hokkaido population suggested less genetic variation than in the mainland population, possibly reflecting a “bottleneck” effect. Although the two studied populations shared no common haplotypes, they are likely to belong to a common lineage because of no independent branch of each population on the parsimony network and neighbor-joining tree.

To assess genetic variations of two Japanese species of the genus Martes, the Japanese marten M. melampus and the sable M. zibellina, the whole regions (1,140 base pairs) of the mitochondrial cytochrome b gene were sequenced. Intraspecific variable sites were different between these two species, and most substitutions were transitions resulting in synonymous mutations. Molecular phylogenetic trees exhibited genetic differentiation between the two species. Genetic variations among M. melampus from Honshu, Shikoku, and Kyushu were larger than those among M. zibellina from Hokkaido. Genetic distance between cytochrome b haplotypes did not correlate to geographic distance between sampling localities. This result suggests the introgression of mitochondrial DNA haplotypes between local populations, probably resulting from incomplete geographic isolation, and/or their recent expansion on each island during a short period.