Effect of gonadotropin releasing hormone on the expression of luteinizing hormone and estrogen in the nerve ganglia and ovary of a tropical abalone, Haliotis asinina Linnaeus.

Gonadotropin releasing hormone (GnRH) is a peptide brain hormone that is involved in the regulation of reproduction in vertebrates via stimulation of the secretion of the pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in their turn stimulate sexual development and sex steroid hormone secretion by the gonads. The tropical abalone, Haliotis asinina, in common with many other invertebrates contains a peptide with a similar structure to GnRH. This study looks at its possible involvement in reproduction by injecting groups of one-year-old female abalone at the mature phase by injecting them with synthetic H. asinina (Has) GnRH at doses of 0, 250 and 500 ng/g and then measuring the amount of material in nerve ganglia, ovary and hemolymph that cross-reacted with enzyme-linked immunosorbent assays (ELISA) for vertebrate LH and steroid, estradiol. Immunohistochemistry, using antibodies for the same two compounds, was also carried out to examine the location of immunoactivity in the tissues of the animals. There were slight (in some cases statistically significant) increases in LH-immunoactivity and estradiol in the hemolymph and tissues. However, this applied to the lower dose only (i.e the dose-response relationship was non-monotonic). Using immunohistochemistry, LH-immunoreactive cells were observed in types 1 and 2 neurosecretory (NS1 and NS2) cells within the cerebral and pleuropedal ganglia of H. asinina. In addition, LH-immunoreactive nerve fiber bundles were strongly detected in both ganglia. The immunoactivity against the estrogen appeared to be localized in the granulated cells within the connective tissue and trabeculae of the mature ovary. There was no positive staining in the cytoplasm of any stage of the germ cells. The interpretation of these findings is presently hindered by the fact that the homologous gene for vertebrate LH has not yet been identified in the genomes of any mollusks (so the cause of the immunostaining is as yet unknown) and also by the fact that mollusks are known to readily absorb steroids from the environment and store them long-term in the form of fatty acid esters. More work, involving identification of the protein that cross-reacts with the LH antiserum and also exclusion of the possibility that the estradiol is of exogenous origin, will have to be carried out before these findings can be used to manipulate reproduction in this species.

Spermatogenesis in the rock oyster, Saccostrea forskali (Gmelin, 1791).

Morphology of the differentiating spermatogenic cells of the rock oyster Saccostrea forskali (Bivalve: Ostreidae) was investigated by light and transmission electron microscopy. The testis is formed by several branching acini containing developing spermatogenic cells, classified into 7 stages based on nuclear characteristics, patterns of chromatin condensation and cytoplasmic contents. The spermatogonium is characterized by a euchromatic nucleus with a prominent nucleolus. The cytoplasm contains several round granulo-fibrillar dense bodies surrounded by numerous mitochondria. The round nucleus of the primary spermatocyte contains patches of electron-dense heterochromatin, numerous proacrosomal vesicles, ribosomes and mitochondria. The secondary spermatocytes contain a reticulated chromatin pattern and reduced number of proacrosomal vesicles. The early spermatids contain a small amount of euchromatin among dense patches of heterochromatin. A large single acrosomal vesicle is located in the posterior part of the cell. The middle spermatid is characterized by the migration of an acrosomal vesicle to the anterior part of the nucleus. The late spermatids contain highly condensed heterochromatin blocks and the acrosomal vesicle becomes cup-shaped and invaginated at the basal part. The spermatozoon contains a barrel-shaped head covered with the cup-like acrosome. At this stage, the subacrosomal space contains an axial rod in subacrosomal materials. Three to four transverse bands appear at the anterior region of the acrosome. The middle piece consists of spherical mitochondria surrounding the proximal and distal centrioles. The flagellum consists of 9+2 axonemal microtubule doublets surrounded by the plasma membrane. Our electron microscopic study of spermatogenesis in the S. forskali provides important new information on the mechanism of development of spermatogenesis of this species.

Postmating isolation in six species of three genera (Hoplobatrachus, Euphlyctis and Fejervarya) from family Dicroglossidae (anura), with special reference to spontaneous production of allotriploids.

In light of reproductive isolation being a fundamental aspect of the biological species concept, we performed crossing experiments using six species from three genera (Hoplobatrachus, Euphlyctis and Fejervarya) of family Dicroglossidae to explore postmating isolation in dicroglossid frogs. Our results revealed gametic isolation among these genera, although the intergeneric hybrids between female E. cyanophlyctis and male H. chinensis were not viable at the tadpole stage, while the hybrids between female E. cyanophlyctis and male H. tigerinus were inviable at the hatching stage. These results showed complete hybrid inviability between the two genera. Almost all interspecific hybrids between female H. tigerinus and male H. chinensis died of underdevelopment at the tadpole stage, whereas several hybrids developed normally and survived to maturity. Chromosomal observations and mtDNA and allozyme analyses confirmed that these mature hybrids were allotriploid, with two maternal genomes and one paternal genome. The present results suggest that the allotriploids were produced spontaneously, and histological observations confirmed their sex as sterile males. We also investigated the molecular relationships between H. tigerinus, H. chinensis, and the interspecific allotriploids by mitochondrial Cytb, 12S and 16S rRNA gene analyses. The maternal inheritance mode of mitochondrial genomes was retained in the hybrids. Finally, the present results suggest that the degree of postmating isolation reflects phylogenetic relationship. In addition, we speculate that allotriploids may be produced via hybridization among cryptic species.

Taxonomic status of three types of Fejervarya cancrivora from Indonesia and other Asian countries based on morphological observations and crossing experiments.

Although the crab-eating frog Fejervarya cancrivora is one of the most widely distributed species in Asian region, taxonomic relationships among different populations remain unclarified. In this study, we attempted to elucidate the taxonomic status of F. cancrivora from Indonesian and other Asian populations. Five populations of F. cancrivora from Selangor (Malaysia), Cianjur (Java, Indonesia), Trat (Thailand), Khulna (Bangladesh), and Makassar (Sulawesi, Indonesia) were morphologically observed and subjected to crossing experiments. Principal component and clustering analyses revealed that these five populations could be organized into three groups corresponding to three observed morphological types: a Selangor and Cianjur group (large-type), a Trat and Khulna group (mangrove-type), and a Makassar group (Sulawesi-type). The limited crossing experiments revealed that hybrids between Selangor females and Cianjur and Trat males developed normally, whereas hybrids between Selangor females and Khulna males showed incomplete gametic isolation. Histological observations of the testes of mature males revealed the presence of pycnotic nuclei in the hybrids between Selangor females and Khulna males in addition to normal bundles of spermatozoa. In contrast, no pycnotic nuclei were observed in the Selangor controls. Although meiotic metaphases in the controls were normal, those in hybrids showed several abnormalities, such as the appearance of univalents and an increase in rod-shaped bivalents. Based on our findings from the morphological observations and crossing experiments, we conclude that each of three identified types represents a distinct species. We propose that the large-type is F. cancrivora, the mangrove-type is F. moodiei, and the Sulawesi-type represents an undescribed species.

Genetic divergence and evolutionary relationship in Fejervarya cancrivora from Indonesia and other Asian countries inferred from allozyme and MtDNA sequence analyses.

To elucidate genetic divergence and evolutionary relationship in Fejervarya cancrivora from Indonesia and other Asian countries, allozyme and molecular analyses were carried out using 131 frogs collected from 24 populations in Indonesia, Thailand, Bangladesh, Malaysia, and the Philippines. In the allozymic survey, seventeen enzymatic loci were examined for 92 frogs from eight representative localities. The results showed that F. cancrivora is subdivided into two main groups, the mangrove type and the large- plus Pelabuhan ratu types. The average Nel's genetic distance between the two groups was 0.535. Molecular phylogenetic trees based on nucleotide sequences of the 16S rRNA and Cyt b genes and constructed with the ML, MP, NJ, and BI methods also showed that the individuals of F. cancrivora analyzed comprised two clades, the mangrove type and the large plus Pelabuhan ratu / Sulawesi types, the latter further split into two subclades, the large type and the Pelabuhan ratu / Sulawesi type. The geographical distribution of individuals of the three F. cancrivora types was examined. Ten Individuals from Bangladesh, Thailand, and the Philippines represented the mangrove type; 34 Individuals from Malaysia and Indonesia represented the large type; and 11 individuals from Indonesia represented the Pelabuhan ratu / Sulawesi type. Average sequence divergences among the three types were 5.78-10.22% for the 16S and 12.88-16.38% for Cyt b. Our results suggest that each of the three types can be regarded as a distinct species.