Folic acid (folate; vitamin Bc) is well recognized as essential for the proper metabolism of the essential amino acid methionine as well as for the synthesis of adenine and thymine. A folate deficiency has been implicated in a wide variety of disorders from Alzheimer's disease to depression and neural tube defects. In the cellular slime molds, including Dictyostelium, vegetative growthphase cells are known to chemotactically move toward folate that is secreted by bacterial food sources such as Escherichia coli. Intracellular folate signal transductlon, including G proteins, Ca² channels, and the PIP3 pathway, has been reported in D. discoideum. To our surprise, the genuine chemoattractant(s) of free-living protozoan amoebae have remained to be determined, possibly because of lack of a pertinent method for assaying chemotaxis. We recently isolated a primitive free-living amoeba from the soil of Costa Rica and identified it as a new species of the genus Vahlkampfia belonging to Subclass Gymnamoebia, which includes Entamoeba and Acanthamoeba. The amoebae can grow and multiply quite rapidly, engulfing nearby bacteria such as E. coli. Importantly, we have demonstrated here using a quite simple but finely designed chemotaxis assay that the Vahlkampfia amoebae exhibit chemotaxis toward higher folate concentrations. Riboflavin and cyanocobalamin were also found to serve as positive chemoattractants. Among these chemoattractants, folate is of particular importance because its function seems to be evolutionarily conserved as a potent chemoattractant of amoeboid cells in a wide range of organisms as well as in the Protista and cellular slime molds.

Multicolor fluorescence in-situ hybridization (FISH) and subsequent reprobing of chromosome preparations increase the number of chromosomes and/or anchor loci on the chromosomes simultaneously identified. Reprobing techniques have been widely applied to chromosomes of vertebrates and plants. We have developed a novel reprobing protocol that utilizes multicolor FISH and bacterial artificial chromosome (BAC) probes to examine chromosome preparations in a model lepidopteran species, the silkworm, Bombyx mori. With standard two-color BAC-FISH, routinely used to map genes on B. mori chromosomes, we could localize only two probes on one preparation, whereas our new protocol combining five-color BAC-FISH and preparation reprobing enabled us to simultaneously map 10 probes, as demonstrated with the Bombyx Z chromosome. The improved BAC-FISH technique will facilitate karyotyping and synteny analysis in Lepidoptera.

Depending on fitness consequences, hybridization may rescue inbred populations; generate premating barriers, reproductive interference, or hybrid species; or extinguish a species. However, the fitness of hybrids is unpredictable without direct quantification of their performance in fitness components across multiple generations. The land snails Bradybaena pellucida and B. similaris, which are indigenous and non-indigenous in Japan, respectively, copulate with each other simultaneously and reciprocally. However, only B. pellucida produces hybrids, because it ends mating by removing the penis before transferring a spermatophore, while B. similaris inseminates B. pellucida. To evaluate the strength of an intrinsic postzygotic barrier against the hybrids produced by B. pellucida, we conducted breeding experiments in the laboratory and measured six life-history traits: (1) growth rate, (2) body weight at maturity, (3) number of days to first oviposition after being permitted to mate, (4) clutch size, (5) fecundity, and (6) hatchability. We also calculated the relative intrinsic fitness based on five of these trait values (excluding clutch size). F₁ hybrids exhibited heterosis in growth rate, body weight at maturity and relative intrinsic fitness. F₂ hybrids also showed heterosis in body weight at maturity. Nevertheless, the F₂ hybrids produced significantly fewer progeny than the mid-point value of the parental species. Thus, the F₂ hybrids exhibited weak out-breeding depression in reproduction, offsetting their vigor in body size. These results indicate that only a weak postzygotic barrier, contrasting with strong F₁ heterosis, has evolved during genetic divergence of the two sibling species in allopatry.

Loss of tropical forests is a major cause of biodiversity loss worldwide. Although drastic modification of the habitat has been shown to negatively affect amphibians, we are far from a complete understanding of the response of amphibian communities to deforestation. We studied frog assemblages in a gradient of forest modification in a humid area of Costa Rica, where the primary forest has been partially converted into pasture. The study area is a mosaic of primary palm forest, abandoned pasture covered by secondary forest, and pasture. Species richness was assessed by randomized walk surveys and audio strip transects. We also measured ecological features to evaluate the relationship between landscape alteration and amphibian distribution. The study area hosted a large number of amphibian species. We focused our monitoring on six anurans: Leptodactylus labialis, Eleutherodactylus fitzingeri, E. diastema, Hyla rosenbergi, H. microcephala, and Cochranella granulosa. Three species (L. labialis, H. rosenbergi, and H. microcephala) were most abundant in pasture areas with livestock presence, while E. fitzingeri, E. diastema, and C. granulosa were associated with primary forest. Most of the variation in community structure was explained by the joint effect of forest alteration and presence of livestock. Whereas forest specialists suffer direct negative effect from deforestation, generalist species can take advantage of forest alteration and the presence of farm animals. Species that are able to take advantage of the new environmental characteristics associated with human modifications of landscapes will come to prevail in the new communities.

In vertebrates, gonadotropin-releasing hormone (GnRH), which is synthesized in the brain, is a key peptide involved in gonadal maturation regulated by the brain-pituitary-gonadal axis. GnRH isoforms and their primary structures have recently been determined in two species of non-chordates, the octopus (Octopus vulgaris) and sea hare (Aplysia californica), which are mollusks. Octopus and sea hare GnRHs are dodecapeptides that contain the structural core of chordate GnRH; however, chordate GnRHs, including tunicate GnRH, are decapeptides. In this study, we examined a GnRHlike peptide in the swordtip squid, Loligo edulis, to provide information on the structural evolution of GnRH in non-chordates. We isolated the full-length cDNA of a GnRH-like molecule from the central nervous system (CNS) of the squid. The open reading frame of this cDNA encodes a protein of 90 amino acids, which consists of a putative signal peptide, a GnRH dodecapeptide, a processing site, and a GnRH-associated peptide. This architecture is generally conserved in chordates. Compared to octopus GnRH, Squid GnRH is identical in the deduced amino acid sequence of the peptide, and 80.5% similar in base sequence. In a phylogenetic analysis, prepro-GnRHs of octopus, sea hare, and squid were segregated from all chordate prepro-GnRHs, in a group designated GnRH5. The squid prepro-GnRH mRNA was expressed mainly in the CNS. This study is the first report of GnRH cDNA cloning in squid and the third in non-chordates.

Worldwide decline has been observed in coral populations due to environmental changes at both regional and global levels. Since corals have a typical metapopulation structure, the recovery of severely disturbed local populations depends on larval recruitment from outside the disturbed region. Therefore, the connectivity among local populations may play a key role in the disturbance/ recovery dynamics of coral populations. On the basis of the simulation of surface currents, derived from ocean currents, we hypothesized that the genetic connectivity of coral populations is weak between the northern and southern regions of Sekisei Reef, Japan. To test this hypothesis, the connectivity of the population of the broadcast-spawning coral Acropora digitifera, one of the dominant corals in the Sekisei Reef, was analyzed at six sampling sites that were 5–25 km apart by using six microsatellite markers. The degree of genetic differentiation of A. digitifera was very low among all the sites (F_ST from -0.006 to 0.015), and no evidence was found in support of our hypothesis. The relative lack of genetic differentiation of A. digitifera among the sites may be attributable to the large effect of wind-driven surface currents, which highly vary on an annual basis, on coral larvae or to the time gap between simulation based on ocean current patterns and accumulation of genetic differences. Considering the pattern of the genetic connectivity of A. digitifera in the Sekisei Reef, the sites that are located within less-disturbed areas may act as larval sources and should, therefore, be preserved.

Proper identification of the black flies is facilitated by chromosomal characteristics when morphotaxonomic parameters are insufficient to distinguish a species. Using photo composites, we constructed and describe from the salivary glands of larvae a standard polytene chromosome map of a Himalayan black fly, Simulium (Simulium) dentatum, in the multistriatum group (Diptera: Simuliidae). This species has three metacentric polytene chromosomes (n=3), with homologues more or less intimately paired and having a prominent centromere. The largest chromosome (I) was 40.55% of total complementary length (TCL); chromosomes II and III were 30.14% and 29.31%, respectively. Chromosome I is characterized by the presence of a prominent nucleolar organizer at IS 19. Chromosome II is characterized by the presence of a Balbiani ring at IIS 41B, followed by bulge and a Parabalbiani ring at IIL 64 B. Prominent homogeneously stained centromeres of chromosomes I, II, and III lie at the map positions L21A, L55, and L84, respectively.

Skin plays a key role in the daily survival of amphibians. In the present study, six cDNAs encoding amphibian skin antimicrobial peptide precursors from the Chinese brown frog Rana chensinensis, were cloned and identified as preprobrevinin-1CEa, preprobrevinin-1CEb, preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, and preprochensinin-1. Preprotemporin-1CEa, CEb, and CEc are members of the temporin family, which are usually short, hydrophobic, and C-terminally α-amidated antimicrobial peptides. Preprobrevinin-1CEa and CEb were identified as members of the brevinin-1 family of antimicrobial peptides, because both peptides contain a “Rana box” that is responsible for forming C-terminal Cys-bridged cyclic heptapeptides. The nucleotide and deduced amino acid sequences of preprochensinin-1 were not similar to any known amphibian skin defensive peptides. Four bioactive peptides were chemically synthesized according to the deduced amino acid sequences of six prepropeptides from R. chensinensis skin, and their antimicrobial, cytotoxic, and haemolytic properties were evaluated. All of the synthesized peptides inhibited the growth of Gram-positive bacteria. Brevinin-1CEa showed a broad spectrum of antimicrobial activity. The novel amphibian skin peptide chensinin-1 was active against Bacillus cereus and Streptococcus lactis at a concentration of 11.6 µM, but did not inhibit the growth of MCF-7 and HeLa cells at 200 µM, and had no haemolytic activity at a concentration of 500 µM. Temporin-1CEa exhibited the greatest ability to inhibit the growth of MCF-7 cells. Its antimicrobial and cytotoxic activities may be due to its high degree of α-helical confirmation and amphipathic nature.

The cephalic neuroendocrine system of the larval blow fly, Protophormia terraenovae was studied by backfills using either horseradish peroxidase or NiCl₂, and peptide immunocytochemistry. Backfills through a proximal part of the ring gland mainly revealed three groups of neurons: (1) neurons with somata in the pars intercerebralis (PI) of the protocerebrum, (2) neurons with somata in the pars lateralis (PL) of the protocerebrum, and (3) neurons with somata in the subesophageal ganglion (SEG). Dense arborization was found mainly in the superior protocerebral, tritocerebral and SEG neuropils. Backfills through a distal part of the ring gland exclusively revealed two types of neurons with somata in the PL, viz., those with ipsilateral projections and those with contralateral projections to the ring gland. Antisera against cholecystokinin-8, FMRFamide, and Gryllus bimaculatus pigment-dispersing factor labeled cells in the PI and PL as well as fibers in the ring gland and aorta. Anti-cholecystokinin-8 and anti-FMRFamide antisera also labeled cells in the SEG. These results suggest that cephalic neurons projecting to the ring gland and aorta receive information at restricted regions in the superior protocerebral, tritocerebral and SEG neuropils, and that they release cholecystokinin-8, FMRFamide, and pigment-dispersing factor-like peptides from the ring gland and aorta into the hemolymph as neurohormones, or locally in the ring gland and aorta to regulate the production and/or release of hormones by the gland cells.

High hydrostatic pressure (HHP) can induce physical changes in DNA, proteins, and lipids, causing lethal or sublethal damage to organisms. However, HHP tolerance of animals has not been studied sufficiently. In this study, HHP tolerance of four species of invertebrate anhydrobiotes (the tardigrade Milnesium tardigradum, a nematode species in the family Plectidae, larvae of Polypedilum vanderplanki, and cysts of Artemia franciscana), which have the potential to enter anhydrobiosis upon desiccation, were investigated by exposing them to 1.2 GPa for 20 minutes. This exposure killed the anhydrobiotes in their ordinary hydrated state, but did not affect their survival in the anhydrobiotic state. The results indicated that the hydrated anhydrobiotes were vulnerable to HHP, but that HHP of 1.2 GPa was not sufficient to kill them in anhyrdobiosis.

A new megophryid species, Leptolalax kecil, is described from the Cameron Highlands of Peninsular Malaysia on the basis of acoustic and morphological characteristics. It has an advertisement call dissimilar to that of other, congeneric species, and is the smallest of the known Leptolalax. It is similar to L. pluvialis from Vietnam in small body size, but differs from it in ventral color, and in the size and color of the pectoral gland. The distributional pattern of the new species is discussed.