Kannika Chatchavalvanich

Histology of the digestive tract of the freshwater stingray Himantura signifer Compagno and Roberts, 1982 (Elasmobranchii, Dasyatidae)

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.

Scanning electron microscopy of glochidia and juveniles of the freshwater mussel, Hyriopsis myersiana

Summary The ultrastructure of early stages of the mussel, Hyriopsis (Limnoscapha) myersiana (Lea, 1856), was observed by scanning electron microscopy from the glochidial period until the onset of the juvenile stage 10 days later. Further observations were performed for an additional 13 days to assess juvenile development. Glochidia extracted from the brood chambers have a hookless, semi-oval and equivalve calcareous shell with numerous pores in the internal surface, pits in the external surface and cuticular spines in the ventral region. Keratin fibers with a random arrangement in the cuticle of the glochidial shell were also detected. The appearance of the foot within 10 days of in vitro glochidial culture was considered the main feature of metamorphosis to the juvenile stage. Another change during the following 13 days was the formation of a new periostracum exhibiting growth lines under the old glochidial shell. This development occurs mainly in the anterior region and is followed by hardening of the periostracum matrix by calcium deposition. Periostracum growth gradually became apparent in the lateral and posterior regions at the end of this period. The retraction of spines and the alteration of the external surface of the old shell are also described. It is speculated that transcuticular filaments identified in the juvenile stage may have sensory or metabolic exchange functions. The prominent foot, gradually covered by long dense cilia, shows rhythmical movements which suggest a role in feeding. Similarly, cilia present in the mantle may also be involved in the capture of food, while microvilli may facilitate absorption of dissolved materials. Longer cilia, sparsely distributed in the mantle, may function as chemo- or tactile sensors.

Structure of the testis and genital duct of freshwater stingray, Himantura signifer (Elasmobranchii: Myliobatiformes: Dasyatidae)

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.

Histological structure of gonads in the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900

Summary The macroscopic and microscopic structures of male and female gonads of Hyriopsis (Hyriopsis) bialatus are described. The sex of individuals was determined from the presence or absence of sperm and eggs in the gonadal fluid under light microscopy analysis. The gonads of both sexes are composed of numerous acini. Male acini contain germ cells at various developmental stages of typical spermatogenesis, i.e., spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa. A striking feature is the presence of morulae with atypical spermatogenesis, interspersed among typical germ cells. Early stage morulae comprise a few large spermatogonia, those at middle stage contain spermatocytes and those at late stage consist of spermatids and spermatozoa. Female acini include oogonia and five different stages of developing oocytes. Stage 1 oocytes are attached to the acinar wall without yolk vesicles and stalk. Stage 2 oocytes have a stalk connected to the acinar wall. Stage 3 oocytes are larger in size, having yolk vesicles accumulated both at the base of the stalk and at the periphery of nucleus. Stage 4 oocytes have yolk vesicles dispersed throughout the cytoplasm. During this period, the basophilia of cytoplasm is replaced by eosinophilic yolk. Stage 5 oocytes contain both mature and developing oocytes. Immature oocytes are surrounded by a vitelline layer and attached to the acinar wall by a narrow stalk. Mature oocytes, however, are completely detached from the acinar wall, being released into the acinus lumen. The male and female germinal ducts are lined by a simple ciliated columnar epithelium. Several acini are connected to a single germinal duct. Numerous mature germ cells are found in the lumen of the germinal duct. No change was detected in the histological structure of male and female gonads collected at different times of the year. Female gonads collected at different stages of marsupial development are also morphologically similar. These observations strongly suggest that H. (H.) bialatus has no distinct reproductive season and may be suitable for commercial culture.

Morphological development of glochidia in artificial media through early juvenile of freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900

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 formation of a tube underneath the growing foot. Several controversial aspects of organogenesis have been inferred, e.g., de novo formation of the anterior and posterior juvenile adductors, the fate of the mushroom body structure, and foot lobe formation from two separate precursor lobes. A mushroom body protruded into the mantle cavity and remained there throughout the transformation period. Moreover, the evidence of a supporting band (mucoid structure) in the mature glochidium of H. bialatus has never been reported in other freshwater mussel species, and its function and composition need to be further investigated.

Ultrastructure of spermiogenesis in a freshwater stingray, Himantura signifer

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.

Histological observation on gonad development of the freshwater pearl mussel, Hyriopsis (Limnoscapha) myersiana.

Abstract 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 of these oocytes contains eosinophilic yolk vesicles. At this age the marsupia are formed, confirming that the female reproductive system is fully developed and that spawning is taking place. In this study, the gonads of mussels at 10, 11, and 12 months of age were less developed than those of 9-month-old mussels, most likely due to environmental conditions. Several atretic oocytes were found in the acini of 12-month-old mussels. The present observation strongly suggests that both males and females are sexually mature at 9 months old, and that H. (L.) myersiana at this stage can be kept as brood stock for artificial culture.

Effect of progesterone administration on the distribution of oviductal carbohydrates in Rana tigrina

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.