The phylum Mollusca is one of the major groups of Lophotrochozoa. Although mollusks exhibit great morphological diversity, only a few comparative embryological studies have been performed on this group. In the present study, to begin understanding the molecular development of the diverse morphology among mollusks, we observed early embryogenesis in a bivalve, the Japanese spiny oyster, Saccostrea kegaki. Although several studies have begun to reveal the genetic machinery for early development in gastropods, very little molecular information is available on bivalve embryogenesis. Thus, as a step toward identifying tissue-specific gene markers, we sequenced about 100 cDNA clones picked randomly from a gastrula-stage cDNA library. This basic information on bivalve embryology will be useful for further studies on the development and evolution of mollusks.

Only some island populations of Podarcis sicula are hyperchromatic. The study of this phenomenon and its relationship with the lizards of the mainland and other islands, exhibiting a “normal” coloration, provides useful hints in our understanding of evolutionary mechanisms that have created the observed morphological variation. We performed a comparative morphological and genetic analysis of a hyperchromatic lizard population from Licosa Island, and compared the data with that obtained from normal-colored lizard populations both from Ustica and Cirella islands in the Tyrrhenian sea and from nearby mainland Italy. Morphological and microsatellite gene differentiation in the hyperchromatic Licosa population appears to have been much more rapid than the molecular evolution of the mtDNA. We discuss herein that the comparison of hyperchromatism and other types of morphological variation with molecular data in island populations of lizards may provide useful hints as to evolutionary mechanisms.

Heart muscles of hagfishes Paramyxine atami and Eptatretus okinoseanus express the B₄ isozyme of lactate dehydrogenase [L-LDH: NAD oxidoreductase, EC 1.1.1.27] (LDH-B₄) whereas their skeletal muscles express LDH-A₄. To examine the relationship of hagfish LDHs to lamprey and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from three hagfishes and compared them with previously published sequences. A phylogenic tree shows that hagfishes diverged just after lampreys. The deduced amino acid sequences showed ten regions common to all vertebrate LDHs examined, i.e., the active site, the pocket recognizing the substrate-coenzyme complex, part of a loop at the surface, and the substrate binding site. The cyclostomate-specific regions (S1, S2) were located in the neighborhood of the active site loop. Three regions, IGS1, IGS2 and IGS3, seem to have altered their structures during the differentiation of LDH isozymes, and the regions remain in LDH-B of vertebrates hitherto examined. IGS2 and IGS3, which are in the neighborhood of the active site, may regulate catalytic activity. There were differences in six amino acid residues (6, 10, 20, 156, 269, and 341) in LDHs of hagfishes. These differences might reflect the tolerance to high pressure and low temperature of LDHs from hagfishes at different habitat depths.

Porifera (sponges) are the most basal phylum of extant metazoans. To gain insight into sponge genome construction, cytogenetic analysis was performed for ten freshwater sponge species of six genera, using conventional Giemsa staining, chromosome banding, and fluorescence in-situ hybridization. The karyotypes were very similar among the ten species, exhibiting a diploid chromosome number of 2n=46 or 48, and usually consisted of microchromosomes with one or two pairs of large chromosomes. The 18S-28S rRNA genes were localized to a single pair of microchromosomes in two Ephydatia species. Hybridization signals of the telomere (TTAGGG)_n sequences were observed at the ends of metaphase chromosomes. The genome sizes of Ephydatia fluviatilis and Ephydatia muelleri were estimated by flow cytometric analysis as about 0.7 pg per diploid complement. These freshwater sponge species appear to represent a fairly homogeneous group with respect to karyotypes.

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally α-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally α-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 μM and 119 μM, respectively), and produced cell death of mammalian COS7 cells (LD₅₀=28 μM).

Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARα and β, and full sequences of two isoforms of PPARγ. This is the first report of reptilian PPARγ mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARα mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARβ mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARγ isoforms, PPARγa was expressed ubiquitously, whereas the expression of PPARγb was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARβ mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR α and γ are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.

We studied by light microscopy the histological development of the olfactory and vomeronasal organ in tadpoles of the Chinese forest frog, Rana chensinensis, from postembryonic periods to the end of metamorphosis. Unlike Bufo americanus, the olfactory epithelium in larval R. chensinensis is not divided into dorsal and ventral branches in the rostral and mid-nasal regions. The olfactory epithelium in the dorsal portion of the buccal cavity in larval R. chensinensis may correspond to the ventral olfactory epithelium of Bufo, which has been argued to provide a chemosensory function in the tadpoles analogous to the role of taste buds in adults. Bowman's glands were present in the olfactory epithelium of R. chensinensis only after the appearance of the forelimbs during metamorphosis. The appearance of Bowman's glands in the olfactory epithelium at this time suggests that the nose first begins to detect odorants in the air, and this is thus also a metamorphic event. The vomeronasal epithelium appeared a little earlier than the vomeronasal gland in R. chensinensis, unlike in toads (bufonids). This study supports Eisthen's hypothesis that the most recent common ancestor to the tetrapods was aquatic and once had a vomeronasal organ, and that this has been lost in various evolutionary lineages.

The microbrain of the silkmoth, Bombyx mori, is a model system for analyzing the neural mechanisms underlying stimulus-driven behavior, and numerous studies using physiological and morphological methods have accumulated. However, one of the limitations of this system is a lack of methodology for labeling specific subsets of neurons. Targeted gene expression with the GAL4/UAS system, which was recently developed, may overcome this disadvantage. To test the GAL4/UAS system in the silkmoth brain, we generated two GAL4 driver lines in which GAL4 expression was under the control of either the bombyxin or prothoracicotropic hormone (PTTH) promoter. Crosses of moths from these lines with a UAS-GFP line showed that green fluorescent protein (GFP) was exclusively expressed in bombyxin or PTTH neurosecretory brain cells. Using these lines, we developed a visually guided method to selectively insert an electrode into and intracellulary stain GFP-expressing cells using fluorescence as a landmark. This work provides a novel method to visualize specific subsets of neurons in the silkmoth brain and to observe detailed structures in a single identified neuron from different individuals.

Crickets respond to air currents with quick avoidance behavior. The terminal abdominal ganglion (TAG) has a neuronal circuit for a wind-detection system to elicit this behavior. We investigated neuronal transmission from cercal sensory afferent neurons to ascending giant interneurons (GIs). Pharmacological treatment with 500 μM acetylcholine (ACh) increased neuronal activities of ascending interneurons with cell bodies located in the TAG. The effects of ACh antagonists on the activities of identified GIs were examined. The muscarinic ACh antagonist atropine at 3-mM concentration had no obvious effect on the activities of GIs 10-3, 10-2, or 9-3. On the other hand, a 3-mM concentration of the nicotinic ACh antagonist mecamylamine decreased spike firing of these interneurons. Immunohistochemistry using a polyclonal anti-conjugated acetylcholine antibody revealed the distribution of cholinergic neurons in the TAG. The cercal sensory afferent neurons running through the cercal nerve root showed cholinergic immunoreactivity, and the cholinergic immunoreactive region in the neuropil overlapped with the terminal arborizations of the cercal sensory afferent neurons. Cell bodies in the median region of the TAG also showed cholinergic immunoreactivity. This indicates that not only sensory afferent neurons but also other neurons that have cell bodies in the TAG could use ACh as a neurotransmitter.

The mesophragmatica group of Drosophila belongs to the virilis-repleta radiation of the Drosophila subgenus. This group comprises 13 Neotropical species that are endemic to the South-American continent and seem to be fundamentally Andean in their distribution. The mesophragmatica-group phylogeny has been inferred previously by other authors based on morphological, cytological, and isozyme analyses. However, the relationships within the group have not yet been completely resolved, although its monophyletic origin has already been confirmed by molecular data. This work attempts to enhance the molecular approach to the relationships among the species of the mesophragmatica group, using both nuclear and mitochondrial markers. Phylogenetic analyses were performed using fragments of the nuclear alcohol dehydrogenase (Adh; 631 bp), alpha-methyldopa (Amd; 1211 bp), dopa-decarboxylase (Ddc; 1105 bp), and hunchback (Hb; 687 bp) genes and the mitochondrial cytochrome oxidase subunit II (COII; 672 bp) gene, and included a total of 4306 bp. The sequences obtained for eight representatives of the mesophragmatica group were analyzed both individually and in combination by distance methods, maximum parsimony, and maximum likelihood. Our results support subdivision of the mesophragmatica group into three main lineages: the first is composed of D. viracochi; the second comprises a clade grouping the sibling species D. pavani and D. gaucha; and the third encompasses D. gasici, D. brncici, and D. mesophragmatica. The best supported scenario suggests that D. viracochi is an early offshoot in the mesophragmatica group, with this and other early branchings occuring in the Pliocene/Pleistocene Epochs, possibly associated with Andean glacial refuges. Also based on the phylogenies obtained, we present a genealogical view of the evolution of previously described characters within the group.

Five new species of the genus Daphnephila (Diptera: Cecidomyiidae: Asphondyliini), D. ornithocephala, D. stenocalia, D. sueyenae, D. taiwanensis, and D. truncicola, all associated with Machilus thunbergii (Lauraceae), are described from Taiwan, and one previously known species, D. machilicola, is redescribed from Japan. Among the five new species, D. truncicola induces stem galls and the other four species induce leaf galls. A molecular phylogenetic analysis based on partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene suggests that in this genus the stem-galling habit is a more ancestral state compared to the leaf-galling habit. Daphnephila seems to be of tropical origin and to have dispersed to Japan through Taiwan.

A new funnel-web spider genus, Acutipetala, gen. nov., is erected to accommodate two new agelenid species known to occur in evergreen forests of northern Thailand: Acutipetala octoginta, sp. nov. (type species,♂♀) and A. donglini, sp. nov. (♂). The genus is established on the basis of the distinctive appearance of the genital structures, in which the median apophysis of the male palp is petal-shaped, sharply pointed, and strongly sclerotized, and the truncate embolus is short, originates subapically, and is provided with a hook-shaped apical portion.

A total of eleven species of tardigrades from Taiwan are reported in this article. They belong to two classes, three orders, four families, and ten genera. Ten species are new records for Taiwan and one is new to science. Doryphoribius taiwanus sp. nov. is similar to Dor. mariae Pilato & Binda, 1990, but differs from it by larger body size, by conspicuous tubercles on the lateral side and dorsal sides of the body, by lacking gibbosities and undulations, by a narrower buccal tube, and by longer claws.