Recent progress in molecular techniques has generated a wealth of information for phylogenetic analysis. Among metazoans all but a single phylum have been incorporated into some sort of molecular analysis. However, the minute and rare species of the phylum Loricifera have remained elusive to molecular systematists. Here we report the first molecular sequence data (nearly complete 18S rRNA) for a member of the phylum Loricifera, Pliciloricus sp. from Korea. The new sequence data were analyzed together with 52 other ecdysozoan sequences, with all other phyla represented by three or more sequences. The data set was analyzed using parsimony as an optimality criterion under direct optimization as well as using a Bayesian approach. The parsimony analysis was also accompanied by a sensitivity analysis. The results of both analyses are largely congruent, finding monophyly of each ecdysozoan phylum, except for Priapulida, in which the coelomate Meiopriapulus is separate from a clade of pseudocoelomate priapulids. The data also suggest a relationship of the pseudocoelomate priapulids to kinorhynchs, and a relationship of nematodes to tardigrades. The Bayesian analysis placed the arthropods as the sister group to a clade that includes tardigrades and nematodes. However, these results were shown to be parameter dependent in the sensitivity analysis. The position of Loricifera was extremely unstable to parameter variation, and support for a relationship of loriciferans to any particular ecdysozoan phylum was not found in the data.

The lesser Japanese mole, Mogera imaizumii, recognized by Motokawa and Abe (1996), occurs in eastern Honshu, western Honshu and Shikoku. Mitochondrial and nuclear DNA were analyzed for mole samples from eastern Honshu to elucidate intraspecific differentiation. Analyses of sequences of the mitochondrial cytochrome b gene (Cytb) and of a restriction fragment length polymorphism of the nuclear 28S ribosomal RNA gene spacer (rDNA-RFLP) revealed two genetic types, partially corresponding to Hutterer's (1993) taxa, M. wogura (= M. imaizumii) and M. minor. Most samples showed either of two combinations of mitochondrial / nuclear gene types. However, two specimens showed a different combination. This incongruent combination of mitochondrial and nuclear genes might have derived, in part, from an introgression event between genetically differentiated populations after secondary contact during the evolutionary history of the lesser Japanese mole in eastern Honshu.

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (Klk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.

Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosinactin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10–50 μM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of α-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2–3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.

The staging of embryonic and larval development, and the germ cell lineage of the loach, Misgurnus anguillicaudatus, are described. Fertilized eggs were obtained by artificial insemination. For the convenience of detailed observation and photography of the external appearance, we use dechorionated embryos. Through a series of operations, these embryos were cultured at 20°C in an incubator. Embryonic and larval development of the loach was divided into five periods: cleavage, blastula, gastrula, segmentation, and hatching. Stages were assigned within each of these periods. Developmental stages were determined and named by morphological features and somite number. The staging series were photographed and tabulated. The germ cell lineage was then elucidated by whole mount in situ hybridization of mRNA expression of the germ-cell-specific marker vasa and histological analysis. Primordial germ cells (PGCs) of the loach derived from the cleavage furrows of 8-cell stage embryos began proliferation in the late blastula period and migrated to the gonadal anlagen through a migration pathway similar to that of the zebrafish. However, it is characteristic of the loach that PGCs migrate a long distance and stay in the posterior part of the yolk-extension region.

This study examined seasonal changes in body weight, hibernation period, and body temperature of the Japanese badger (Meles meles anakuma) from 1997 to 2001. Adult badgers showed seasonal changes in body weight. Between mid-December and February, badger activity almost ceased, as the animals remained in their setts most of the time. Adult male badgers were solitary hibernators; adult females hibernated either alone or with their cubs and/or yearlings. The total hibernation period of Japanese badgers ranged from 42 to 80 days, with a mean length of 60.1 days. Japanese badgers did not always spend the winters in the same sett, although they seldom changed setts during hibernation. I equipped a male cub with an intraperitoneally implanted data logger to record its body temperature between November and April, while the cub hibernated with its mother. Over the winter, the body weight of the cub decreased from 5.3 kg to 3.6 kg, a weight loss of 32.1%, and its body temperature ranged from 32.0 to 39.8°C. The mean monthly body temperature was 35.1°C in December, 34.8°C in January, 35.9°C in February, 37.1°C in March, and 37.4°C in April, so the monthly decrease in body temperature of this cub was not great. The results indicate that during hibernation, when body temperature is low, there is likely considerable economy of energy and a reduced demand for adipose reserves.

In many benthic organisms with a planktonic larval stage, local populations have different morphology. Such difference may arise from some of the following proximate mechanisms. “Local recruitment (LR)”: no larvae move between local populations, and segregated populations possess alleles coding for locally adaptive morphology. “Intragenerational selection (IS)”: larvae move between local populations, and individuals with alleles for locally adaptive morphology survive after recruitment. “Phenotypic plasticity (PP)”: larvae move between local populations and show phenotypic plasticity to adapt to a locality after recruitment. We examined which mechanism explains our finding that a planktonic developer Turbo coronatus coronatus (Gastropoda) had significantly longer spines on its shell on more exposed shores at scales of < 2 km. Experiments at Ishigaki Island, Okinawa, Japan, showed the following results. (a) Shorter- and longer-spined populations occurring within 2 km showed non-significant low ϕst values (−0.0040 to 0.00095) for the mitochondrial DNA COI region. This suggests no segregation of the local populations, supporting the mechanisms IS and PP. (b) T. c. coronatus generated significantly longer spines 70 days after being transplanted to the habitat of a longer-spined population, supporting IS and PP. (c) Individuals caged in the sea for 79 days generated longer spines than individuals in the laboratory, supporting PP. In conclusion, shore-specific morphology of T. c. coronatus arises most likely from phenotypic plasticity and possibly from intragenerational selection.

The house musk shrew (Suncus murinus), or suncus, is a unique experimental animal. We recently showed that this mammal is cold intolerant and hypothesized that its sensitivity to cold is caused by low thermogenic activity in brown adipose tissue (BAT). Thermogenesis in BAT is performed by a unique mitochondrial protein, uncoupling protein 1 (UCP1). Interestingly, only eutherians possess UCP1, and some traits in the suncus resemble those in the Ucp1-ablated mouse, including cold intolerance, histology of BAT, and obesity resistance. In a previous study, we hypothesized that UCP1 may not be present in BAT of the suncus or may be dysfunctional. Therefore, we performed cDNA cloning of suncus Ucp1 and compared it to homologs from other species. The deduced amino acid sequence showed high similarity to other mammalian UCP1. Northern blot analysis revealed mRNA in BAT, as in other mammals. However, a difference in an amino acid residue was observed in an important residue for thermogenesis. Genomic sequence analysis showed that this difference existed in our two genetically distant laboratory colonies. These results suggest that cold intolerance in the suncus is derived from low thermogenic activity of UCP1 and may exist in wild house musk shrews.

C-values, which estimate genome size, have puzzled geneticists for years because they bear no relationship to organismal complexity. Though C-values have been estimated for thousands of species, considerably more data are required in order to better understanding genome evolution. This is particularly true for mammals, in which C-values are known for less than 8% of the total number of mammalian species. Among marine mammals, a C-value has been estimated only for the bottlenose dolphin (Tursiops truncatus). Thus examination of additional species of marine mammals is necessary for comparative purposes. It will enable a better understanding of marine mammal genome evolution, and it is also relevant to conservation, because larger genome size has been linked to increased likelihood of extinction in some plant and animal groups. Our study presents C-values of seven marine mammal species, including five cetacean species that are endangered to varying degrees. Similarly to the results for other groups, our results suggest that larger genome size in cetaceans is related to an increased likelihood of extinction.

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phos-phatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.

In the varied carpet beetle, Anthrenus verbasci, we examined the effects on the circannual pupation rhythm of a short-day or long-day pulse under naturally changing day length at a constant 20°C. A short-day pulse for 4 weeks caused a prominent phase delay or advance under constant long days, but had little or no effect on the phase under naturally changing day length between 4 August and 24 November. A long-day pulse for 4 weeks given under naturally changing day length caused a phase shift in the first pupation group, as under constant short days. A long-day pulse given on 4 August, 1 September, or 29 September caused a phase delay, and a pulse given on 27 October or 24 November caused a phase advance. Pupation was least synchronous just before the transition from delaying to advancing. However, the magnitude of phase delays was much smaller under natural day length than under short days. In the second pupation group, larvae pupated at the same time as in the control experiment without a long-day pulse, and this result can be attributed to entrainment to the geophysical year by long days in spring and summer.

We describe the new genus Pehuencoris to include P. gurguliophagus n. sp. from southern Argentina and Chile (Patagonia). We illustrate the dorsal habitus and male genitalia, provide a complete description of the ostiolar peritreme and its microsculpture, and show a distributional map. We include comments on the biology and habits of the new species, and discuss relationships of the new genus to other genera of Cardiastethini.

The mudskipper Periophthalmus walailakae is recorded from Singapore, where it was previously misidentified as Periophthalmodon schlosseri, with which it is syntopic. Periophthalmus walailakae is distinguished from its congeners by the following combination of characters: pelvic fins completely united and shaped like a disk, and first dorsal fin dark brown or black, with a rounded posterior edge and a white distal margin. This species most closely resembles Pn. schlosseri but has one row of teeth on the upper jaw, scales on the isthmus, and a different upper lip and jaw morphology. Contrary to an earlier report, scales are present on the snout, interorbital, and isthmus of Ps. walailakae. The two species can also be distinguished by size, external morphology, and body color patterns.