Zoological Science

In this review, the mechanisms underlying the intracellular processing of peptide hormone precursors, with a focus on proopiomelanocortin (POMC), were discussed on the basis of recent information. POMC as well as other prohormones is processed to active peptides through proteolytic cleavage by prohormone convertases PC1 and/or PC2. However, the cleavage-specificity of PC1 and PC2 in mammals is somewhat different from that in amphibians. From the comparative endocrinological point of view, expression and tissue distribution of PC1 and PC2 were discussed here. In mammals, proteolytic processing of POMC occurs coordinately with the maturation of secretory granules. Studies using immunoelectron microscopy with DAMP (3-[2,4-dinitroanilino]-3′-amino-N-methyldipropylamine) as a pH probe revealed that the acidic pH in the secretory granules, generated by vacular type-H⁺-ATPase, provides a favorable environment for activating PC1 in AtT-20 cells, a mouse corticotrope tumor cell line. Recent data indicate that the 7B2 protein serves as a chaperone in the regulation of PC2 activation and to control the timing for activating the convertase. Together, secretory granules in endocrine and neuroendocrine cells provide proper sites for biosynthesizing hormones in addition to serving as storage sites and vehicles for the transport of peptide hormones.

In vitro synthetic activities of juvenile hormones (JH) were examined using a radiochemical assay in diapause females and reproductive females of the blow fly, Protophormia terraenovae. Thin layer chromatography showed that products of the corpus allatum (CA) comigrated with a synthetic sample of JH III bisepoxide but neither with JH III nor methylfarnesoate. JH synthetic activities increased in females reared under LD 18:6 at 25°C, as the ovaries developed. The synthetic activities remained low in previtellogenic females reared under LD 12:12 at 20°C. Removal of the pars intercerebralis compelely prevented ovaries from development under reproductive conditions, and removal of the pars lateralis caused partial or full development of ovaries under diapause-inducing conditions. In these operated animals, the JH synthetic activities were not significantly different from those of the intact and sham-operated animals. The results indicate that the CA in P. terraenovae produces mainly JH III bisepoxide and a decrease in the JH production rate is a cause of diapause induction. PI neurons and PL neurons in the brain do not directly mediate changes in the JH production rate, but regulate ovarian development cooperatively with some unknown allatostatic and allatotropic factors.

The humoral fluid of Branchiostoma belcheri tsingtauense was examined for the presence of complement-like activity. The humoral fluid showed hemolytic activity for rabbit erythrocytes and those from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogeny of the target erythrocytes and degree of hemolysis. The hemolytic activity was optimally assayed at 20°C, at pH 7.5, and in the presence of 10 mM Mg²⁺. The hemolytic activity was Mg²⁺-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride. In addition, Western blotting and titration by turbidimetric immunoassay (TIA) revealed that amphioxus humoral fluid contained C3 component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway.

Two morphologically distinct blood cell types (hemocytes), Type I and Type II were found coexisting in hemolymph from two kinds of snails, Oncomelania nosophora strain, viz. from the Nirasaki strain (schistosome-resistant snail) and the Kisarazu strain (schistosome-susceptible snail). Ten min after inoculation of SRBC, the majority of Type I cells from Nirasaki strain flattened and spread over the surface of the glass plate by extending pseudopodia. In the Kisarazu strain, Type I cells adhered to the surface of substrate with spike-like filopodia, but did not form spreading lamellipodia. Type I cell from the Nirasaki strain phagocytosed SRBC but that from the Kisarazu strain did not. The starting time of recognition of foreign materials was slightly different in the Type I hemocytes from the two strains. Type II cells from both strains were round and lymphocyte-like. Ten or sixty min after incubation, Type II cells from neither strain adhered to the surface of substrate or SRBC, and did not phagocytose SRBC. Type II cells from the Nirasaki strain were quite similar to those from the Kisarazu strain. We concluded that Type I cells from the schistosome-resistant snail, Nirasaki strain, possessed higher phagocytic activity than those from the susceptible snail, Kisarazu strain, despite the morphological similarities of the hemocytes from both strains.

A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain ∼16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the “Ciona intestinalis Gene Collection Release I”. In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

The ontogenetic development of gonads from embryo to adult was observed histologically in the viviparous teleost, Gambusia affinis. Primordial germ cells (PGCs) appeared in the subendodermal space of the embryo 14 days before birth, and then transferred to the dorsal mesentery to form paired genital ridges 12 days before birth. The PGCs proliferated in the genital ridge, forming gonadal primordia 10 days before birth. All gonadal primordia differentiated to the ovary containing oocytes 2 days before birth, but then redifferentiated to the ovary and testis just after birth. This indicates that the mosquitofish is a juvenile hermaphroditic species. The characteristics of gonadal sex differentiation just after birth were enlargement of the oocytes in females, and invasion of somatic cells from the hilar region to an inner portion of the gonad in males. The paired ovary fused at the basal area 5 days after birth, then on the ventral and dorsal portions, developing into a single ovary 10 days after birth. During this time a single ovarian cavity was formed on the dorsal portion of the ovary. The paired testes fused only at the basal area and became a single testis having two main lobes 10 days after birth. The oocytes gradually developed and began vitellogenesis 100 days after birth, but did not reach maturation until 110 days after birth. Spermatogenic cells formed cysts at 20 days, began meiosis at 70 days, and matured to form sperm balls 90 days after birth. The male fish sexually matured earlier than the female.

Ascidians inhabit both shallow water and the deep sea. The phylogenetic position of deep-sea ascidians has not been sufficiently investigated because of their unusual habitats. The family Octacnemidae is one such enigmatic deep-sea ascidian. In this report, we determined the sequences of the 18SrDNA and a mitochondrial protein gene of Megalodicopia hians belonging to the family Octacnemidae, and we analyzed its phylogenetic relationship with other ascidians. A phylogenetic relationship of this family with the families Cionidae and/or Corellidae has been suspected based on a small number of morphological characteristics. However, our results suggested that M. hians has a close relationship to the family Corellidae and might originate from them. This is the first report of the molecular phylogenetic analysis of a deep-sea ascidian.

A new mayfly species, Dipteromimus flavipterus sp. nov., of the family Dipteromimidae is described on the basis of specimens of males, females and mature nymphs collected from Ofunato, Iwate Prefecture, Japan. This new Dipteromimus species is characterized by a large body size, a yellowish body color (especially the frontal border of the forewings), short penis lobes, a slight elongation backwards of the female 7th abdominal sternum, and a rounded backward projection of the female 9th abdominal sternum, etc., in comparison with another known dipteromimid mayfly Dipteromimus tipuliformis McLachlan, 1875. Full descriptions of males, females, mature nymphs and eggs of this new species have been provided. We also examined and discussed the genetic relationship of two dipteromimid mayflies to settle the taxonomic status, inferred from the partially sequenced large mitochondrial ribosomal subunit (16S rRNA) genes. Consequently, phenetic and molecular phylogenetic analyses agreed in terms of clustering.

Twelve mite species of the genus Macrocheles (Acari: Macrochelidae) were collected from the body surface of dung beetles in Java, Indonesia. Of these, three species, i.e., Macrocheles jabarensis, M. jonggolensis, and M. sukabumiensis, were described as new to science. Female of M. dispar was redescribed. Two species, i.e., M. baliensis and M. sukaramiensis, were recorded from Java for the first time. The occurrence of five species previously recorded from Java, i.e., M. hallidayi, M. kraepelini, M. limue, M. oigru, and M. merdarius, were reconfirmed. Taxonomic status of M. sp. aff. glaber was not settled in the present study, because we could not obtain the male and immatures which are indispensable for exact identification. In total 15 species of the genus Macrocheles, including 3 species already recorded but not collected in this research (M. crispa, M. krantzi and M. subbadius), are known from Java up to date.

Stephensia armata sp. n., a remarkable species is described from Belize (Central America). The external features and genitalia of the male are figured and described in detail. A distribution map for all six Elachistidae species, currently recorded from tropical America, is given: Perittia smaragdophanes (Meyrick), P. punatensis Kaila, Elachista albisquamella Zeller, E. saccharella (Busck), E. vasrana Kaila and Stephensia armata Sruoga, sp. n.

We conducted a polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3–5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCRRFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.

A cDNA clone encoding the soluble guanylyl cyclase α₂ subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-α₂ . The OlGCS-α₂ cDNA was 3192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian α₂ subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, “RETSL”, which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-α₂ was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-α₂ genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-α₂ gene consisted of 9 exons and 8 introns. The 5′-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-α₂ gene was mapped to LG13; this mapping position was different from those for the OlGCS-α₁ and OlGCS-β₁ genes (LG1).

Differences in the nuclear ribosomal DNA (rDNA), mitochondrial DNA (mtDNA), cytochrome b (Cytb), and Y chromosomal Sry genes were used to assess intra- and interspecific relationships in two Japanese red-backed voles, Eothenomys andersoni and E. smithii, focusing on areas where the two species might come into contact. In the Kii Peninsula, southwestern Honshu, which contains an allopatric population of E. andersoni isolated from its main range, the rDNA-RFLP data provide robust evidence of past mutual interspecific gene introgression, while the Cytb and Sry sequences were specific to this population. In central Honshu, where E. andersoni and E. smithii inhabit higher and lower altitudes, respectively, with a narrow sympatric zone, the rDNA-RFLP and Sry variation was specific for each species, while introgression of the mtDNA from E. smithii to E. andersoni was seen. These complex patterns in the gene markers are consistent with our previous notions derived from sex chromosome variation. Our previous and present data strongly suggest that the evolution of these vole species, which are morphologically and cytogenetically distinct, involves complex genetic interactions and the resultant combinations of genes are sometimes peculiar, mainly due to the Cytb haplotypes. However, phylogenetic analysis using a combination of maternal, paternal, and biparental markers has proven useful for understanding the evolutionary history given the complex phylogenetic background.

Paeonocanthus antarcticensis (Hewitt, 1965) is redescribed based on four specimens recovered from a deep-sea smelt, Bathylagus antarcticus Günther, collected in the Antarctic Ocean (65°S, 139° 59.6′E). Studies on the morphological variations of these four specimens plus comparison with the three documented specimens yielded that the sphyriid reported as P. antarcticensis from the goiter blacksmelt, Bathylagus euryops Goode & Bean, taken in the Northwest Atlantic Ocean is a different species. It is renamed Paeonocanthus hogansi n. sp.