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Three chalcone synthase (CHS) genes were isolated from Curcuma longa Linn. using TAIL-PCR. ClCHS1 and ClCHS2 were 1,460 and 1,407 bp in length, respectively, containing 1,191 bp open reading frame (ORF) that encodes 396 amino acids, whereas ClCHS3 was 1,394 bp in length containing 1,170 bp ORF that encodes 389 amino acids. The structure of all three genes comprise two exons and one intron which are consistent with the other CHS gene family. Southern blot analysis using a ClCHS conserved fragment revealed the ClCHS genes belong to a gene family. Phylogenetic analysis showed the three putative ClCHS proteins to be closely related to DCS (diketide-CoA synthase) protein, a product of CHS-like gene in C. longa, which condenses malonyl-CoA with feruloyl-CoA or coumaroyl-CoA as the substrate in curcuminoid synthesis. These results suggest that the interaction of substrate and enzyme between the three putative ClCHS proteins and DCS might be highly similar. Homology modeling and docking analysis were consistent, indicating that the same substrate (coumaroyl-CoA) can be used in the putative ClCHS1 and ClCHS2 proteins. However, the putative ClCHS3 protein seems to have seven amino acids deletion in a loop involved in the binding site formation, suggesting that the binding site with coumaroyl-CoA might be altered.

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution.

The morphological development and the sequence of organogenesis from glochidium to the early juvenile stage of the freshwater pearl mussel, Hyriopsis bialatus, were observed. Larvae of H. bialatus were cultured in standard tissue culture medium (M199) supplemented with common carp (Cyprinus carpio) plasma and they showed transformation within 10 days. Larval samples were collected every 2 days during glochidia development and subjected to histological processing. Three types of cell masses were developed during this period: the ventral plate (the foot rudiment), lateral pits (the gill rudiment), and the oral plate or endodermic sac (the origin of the digestive tract). The ventral plate gave rise to two foot lobes which fused into a single lobe. The gills were developed from the lateral pits next to the ventral plate, forming a pair of gill buds that became elongated and turned into gill bars. The digestive tract began with the formation of mouth by invagination of the oral plate (or endodermic sac) and fo...

The microscopic structure of the gonad at different ages of the cultured mussel Hyriopsis (Limnoscapha) myersiana is described. Male gonadal acini are first observed at 6 months old; they are small and scarce, and contain only a few germ cells at the early stage of spermatogenesis. At 7 and 8 months old, the male acini increase in size and have more numerous germ cells at subsequent developmental stages: i.e., spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa. Male germ cell morulae are also found in the acini. At 9 months old, the male gonad becomes fully developed and occupies two-thirds of the visceral mass. Sperm are found abundantly in the acini and gonoducts, indicating spawning. The female gonad begins to develop at 8 months old with a few small previtellogenic oocytes and numerous nutritive granules in the acini. The female gonad and gonoduct are well developed at 9 months old. Several large vitellogenic oocytes are found in the acini; the cytoplasm...

The butterfly lizard (Leiolepis reevesii rubritaeniata) has the diploid chromosome number of 2n = 36, comprising two distinctive components, macrochromosomes and microchromosomes. To clarify the conserved linkage homology between lizard and snake chromosomes and to delineate the process of karyotypic evolution in Squamata, we constructed a cytogenetic map of L. reevesii rubritaeniata with 54 functional genes and compared it with that of the Japanese four-striped rat snake (E. quadrivirgata, 2n = 36). Six pairs of the lizard macrochromosomes were homologous to eight pairs of the snake macrochromosomes. The lizard chromosomes 1, 2, 4, and 6 corresponded to the snake chromosomes 1, 2, 3, and Z, respectively. LRE3p and LRE3q showed the homology with EQU5 and EQU4, respectively, and LRE5p and LRE5q corresponded to EQU7 and EQU6, respectively. These results suggest that the genetic linkages have been highly conserved between the two species and that their karyotypic difference might be caused by the telomere-to-telomere fusion events followed by inactivation of one of two centromeres on the derived dicentric chromosomes in the lineage of L. reevesii rubritaeniata or the centric fission events of the bi-armed macrochromosomes and subsequent centromere repositioning in the lineage of E. quadrivirgata. The homology with L. reevesii rubritaeniata microchromosomes were also identified in the distal regions of EQU1p and 1q, indicating the occurrence of telomere-to-telomere fusions of microchromosomes to the p and q arms of EQU1.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L4m + L2sm + M2m + S4m + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.

Summary Culture of the freshwater pearl mussel, Hyriopsis (Limnoscapha) myersiana, was carried out in three consecutive steps: (1) culture of glochidia larvae in artificial media, (2) rearing the early juveniles (0–120 days old) in a nursery, and (3) rearing the juveniles (120–360 days old) in an earthen pond. The percentage survival of glochidia in standard tissue culture medium (M199) supplemented with common carp plasma was 95±2.5. All surviving larvae (100%) transformed to juveniles, the duration of transformation being 8 days. The early juveniles (0–60 days old) were fed with a mixture of four selected phytoplankton species (Chlorella sp., Kirchneriella incurvata, Navicula sp. and Coccomyxa sp.). The survival rate of juveniles was 8±0.2%. The average length of these juveniles increased from 0.13±0.01 mm to 1.41±0.16 mm and the average height from 0.16±0.01 mm to 0.98±0.09 mm. Subsequently, 60–120-day juveniles were fed with one of the same four phytoplankton species or a combination of the four. Feed...

Summary In vitro culture of the glochidia of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900, in M199 medium supplemented with common carp plasma resulted in 95 ± 2% survival, of which 97 ± 2% were transformed to juveniles. Transformation occurred within 10 days. After transformation, early juvenile mussels were reared and fed for 15 days with a mixture of four species of phytoplankton (Chlorella sp.2, Kirchneriella incurvata, Navicula sp. and Coccomyxa sp.). Digestive enzyme activities, not detected in glochidia, were induced after feeding in transformed juveniles. Analysis of juvenile crude enzyme extracts after 15 days of feeding revealed the presence of amylase, proteinases, lipase and cellulase with specific activities 7.29 ± 0.12 U mg protein−1, 0.003 ± 0.0001 U mg protein−1, 1.85 ± 0.24 U mg protein−1 and 0.03 ± 0.01 U mg protein−1, respectively, which corresponded to 39%, 25%, 42% and 6%, respectively, of those found in the gastrointestinal tract of adult mussels. In vitr...

Ultrastructural changes of spermatids during spermiogenesis in a freshwater stingray, Himantura signifer, are described. Differentiation of spermatids begins with modification of the nuclear envelope adjacent to the Golgi apparatus, before the attachment of the acrosomal vesicle. A fibrous nuclear sheath extends over the nuclear surface from the site of acrosomal adherence. The conical apical acrosome is formed during nuclear elongation. At the same time, chromatin fibers shift from an initially random arrangement, assume a longitudinal orientation, and become helical before final nuclear condensation. An axial midpiece rod is formed at the posterior end of nucleus and connects to the base of the sperm tail. Numerous spherical mitochondria surround the midpiece axis. The tail originating from the posterior end of the midpiece is composed of the usual 9 + 2 axoneme accompanied by two longitudinal columns, which are equal in size and round in cross section. The two longitudinal columns are absent at the end piece. A distinctive feature of freshwater stingray sperm is its spiral configuration.

The light microscopic structure of the testis and genital duct system of the freshwater stingray Himantura signifer was observed. The testis is composed of lobes having numerous spermatocysts in a dorsoventral zonated arrangement. The germinal papilla at the middorsal surface of the testicular lobe is the origin site of spermatocyst development, where mesenchymal-like cells are predominantly found. The association of a Sertoli cell precursor with a spermatogonium marks the onset of spermatocyst formation and development. The newly formed spermatocysts at the dorsal end of the germinal zone replace the older ones, which are sequentially moved to the ventral side and are termed spermatogonial, spermatocyte, spermatid, spermatozoal, and degenerate zones. In the degenerate zone, the spermatocysts deteriorate after releasing the spermatozoa into the intratesticular duct, where they are further transported through the extratesticular duct system and finally stored at the seminal vesicle. The epithelial lining of the genital duct is a pseudostratified ciliated columnar with no muscular layer underneath; thus, sperm are conveyed through ciliary activity. The interesting features of the present study are the finding of mesenchymal-like cells in the germinal papilla and the nonaggregated formation of sperm in the seminal vesicle.

The enzymatic properties of the main digestive enzymes, amylase and proteinase, from stomach and intestine of adult freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson 1900, were studied at various pH's (1-11) and temperatures (20-80 °C) as well as their enzymatic stability at various pH's. The pH and temperature profiles of the enzymatic activities of amylase and proteinase were similar between male and female mussels. At least seven amylase activities were observed in both stomach and intestine. Four acidic amylases showing pH stability at 2-11 had temperature optima at 35, 45, 55 and 65 °C. Two neutral amylases with pH stability at 6-7 showed temperature optima at 30 and 40 °C, and alkaline amylase(s) with pH stability at 8-10 had temperature optima range at 60-75 °C. Proteinase, on the other hand, showed its optimal activity at pH 5 (acidic proteinase) and at 60-70 °C in the stomach, while at a pH range of 6-8 (alkaline proteinase) and at 35-40 °C in the intestine. A wide pH stability range was observed for both acidic proteinase (pH 2-6) and alkaline proteinase (pH 4-7). Six proteinase activities were differentiated, two acidic forms with pH stability at 2-3 and 6 having respective optimal temperature at 60 and 70 °C, and four alkaline forms with pH stability at 4, 6, 7 and 10 showing respective optimal temperature of 25, 40, 35 and 55-80 °C. Digestive enzymes seemed to be more sensitive in females than in males. Females seemed to prefer a more alkaline condition for optimal food digestion compared to male mussels, as their alkaline proteinases were less stable in acidic conditions. At optimal conditions, both amylase and proteinase enzymes showed higher specific activities in the stomach than in the intestine. At habitat temperature (28-30 °C), amylase specific activities dominate in the stomach, while both amylase and proteinase specific activities dominate in the intestine.

Nuclear DNA was isolated from the blood cells of catfish representing three families (clariidae, pangasiidae and ictaluridae) for analysis by polymerase chain reaction (PCR) and restriction enzymes. Primers specific for the CH 4 exon of the gene encoding the immunoglobulin M heavy chain of channel catfish ( Ictalurus punctatus ) were used. Nuclear DNA amplified with these primers yielded a single band of about 300 base pairs (bp) for Clarias macrocephalus, Pangasius gigas, Pangasius hypophthalmus and the hybrid of P. gigas X P. hypophthalmus . However, the same primers yielded two DNA bands of about 300 and 340 bp in Clarias gariepinus and in the hybrid of C. macrocephalus X C. gariepinus . Nucleotide sequences of the amplified DNA were determined for I. punctatus, C. macrocephalus, P. gigas and P. hypophthalmus . Based on the DNA sequence data, the restriction enzyme Hpa I was used to further characterize the PCR products of P. gigas, P. hypophthalmus and their hybrid. Digestion with this restriction enzyme yielded one DNA band (300 bp) for P. gigas , two bands (100 and 200 bp) for P. hypophthalmus and three bands (100, 200 and 300 bp) for the hybrid. These findings would aid in identifying genetic contributions in hybrid, androgenetic, gynogenetic and polyploid catfish.

Based on molecular phylogeny of available complete mitochondrial DNA (mtDNA) genome sequences reveals that Crocodylus siamensis and C. porosus are closely related species. Yet, the sequence divergence of their mtDNA showed only a few values under conspecific level. In this study, a new haplotype (haplotype2, EF581859) of the complete mtDNA genome of Siamese crocodile (C. siamensis) was determined. The genome organization, which appeared to be highly similar to haplotype1 (DQ353946) mtDNA genome of C. siamensis, was 16,814 bp in length. However, the sequence divergence between the two genomes differed by around 7–10 and 0.7–2.1% for the haplotype1 between C. siamensis and C. porosus (AJ810453). These results were consistent with the phylogenetic relationship among the three genomes, suggesting that C. siamensis haplotype1 mtDNA genome might be the hybrid or the intraspecific variation of C. porosus. On the other hand, our specimen was found to be a true C. siamensis. Simultaneously, the seven species-specific DNA markers designed based on the distinctive site between haplotype2 mtDNA sequences of C. siamensis and haplotype1 mtDNA sequence of C. siamensis–C. porosus were successfully used to distinguish C. siamensis from C. porosus. These effective markers could be used primarily for rapid and accurate species identification in population, ecology and conservation studies.

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed.

We investigated the histology and histochemistry (of carbohydrates and proteins) of the digestive tract of the freshwater stingray Himantura signifier. The alimentary tract consists of a mouth, pharynx, esophagus, stomach (with a descending cardiac and ascending pyloric part), anterior intestine (with an initial portion and a spiral intestine) and posterior intestine, ending in a cloaca. Histologically, three layers--mucosa, muscularis and adventitia/serosa--were defined from the mouth to esophagus and in the posterior intestine, whereas in the stomach and anterior intestine four layers were present, including a submucosa. The epithelial lining of mouth, pharynx and cloaca was of the stratified cuboidal type, whereas that of the esophagus and posterior intestine was stratified columnar. The stomach and anterior intestine were lined by a simple columnar epithelium with microvilli. Goblet cells were observed along the alimentary tract, except in the stomach. In the descending cardiac portion of this organ, gastric glands composed of oxyntic, oxyntic-peptic and peptic cells were observed. The anterior intestine presented a spiral valve with 11 folds, formed by mucosa and submucosa. The posterior intestine was particular in displaying a three-layered muscularis. Mucosubstances secreted along the alimentary tract contained both neutral and acid mucins, but in the stomach only neutral mucins were detected. The stomach presented intense protein content in the epithelial lining of the gastric pits. Enteroendocrine cells were identified in the stomach and intestine. Overall, our data offer a baseline for comparative purposes and future detailed ultrastructural and immunohistochemical studies.