Satit Kovitvadhi

Phylogeny of the most species-rich freshwater bivalve family (Bivalvia: Unionida: Unionidae): Defining modern subfamilies and tribes

Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies.

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.

Optimization of diet and culture environment for larvae and juvenile freshwater pearl mussels, Hyriopsis (Limnoscapha) myersiana Lea, 1856

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. Feeding the juveniles with K. incurvata resulted in the highest survival rate (65±8.32%), with an average length of 3.46±0.04 mm and an average height of 1.94±0.04 mm. Finally, the 120–360-day juveniles were cultured in an earthen pond. There were progressive changes in average weight (0.0037±0.002 g to 11.24±5.02 g), length (3.48±0.39 mm to 54.08±6.21 mm), height (1.97±0.24 mm to 25.09±2.48 mm) and width (0.98±0.06 mm to 12.28±3.21 mm) from 120 to 360 days. The average growth rates per day of these parameters were 0.0497±0.01 g, 0.2414±0.15 mm, 0.0975±0.08 mm and 0.0493±0.03 mm, respectively. H. (L.) myersiana juveniles developed the complete structural composition of the adult by 160 days, and at 360 days, gametogenesis was complete.

Development of digestive enzymes and in vitro digestibility of different species of phytoplankton for culture of early juveniles of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900

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 vitro digestibility of the four species of phytoplankton using juvenile crude enzyme extract resulted in digestion of carbohydrate, protein and lipid content of Chlorella sp.2, K. incurvata, Navicula sp. and Coccomyxa sp., respectively, at the following levels: carbohydrate, 414 ± 46, 234 ± 38, 98 ± 19 and 188 ± 31 μg of reducing sugar per mg phytoplankton; protein, 75 ± 13, 164 ± 19, 63 ± 7 and 120 ± 11 μg of cleaved peptides per mg phytoplankton; lipid, 369 ± 24, 18 ± 3, 56 ± 7 and 80 ± 11 μg of fatty acid per mg phytoplankton. Among different nutrient digestibilities, that of protein is apparently the key factor determining food quality, followed by carbohydrate as the secondary factor in herbivorous species like mussels. The data indicate that a combination of Chlorella sp.2 and K. incurvata is a suitable food formula for juvenile culture.

Diversity, biogeography and conservation of freshwater mussels (Bivalvia: Unionida) in East and Southeast Asia

Recent research efforts have significantly advanced our knowledge on Asian freshwater mussel (Bivalvia: Unionida) diversity and distribution. Here we provide a modern consensus of the diversity, biogeography and conservation of Unionida in the region comprising East and Southeast Asia (excluding Wallacea) and Asian Russia. A data review confirmed the presence of 228 native and 3 non-native Unionida (98% Unionidae, 2% Margaritiferidae), rendering the region a global hotspot of freshwater mussel diversity. Species richness was highest in China (particularly Yangtze basin) in absolute numbers and Cambodia when correcting for country area, and decreased gradually towards the south and steeply towards the north and east. Six of the seven unionid subfamilies are native to the region, with species richness peaking in Southeast Asia for Rectidentinae, Gonideinae, Parreysiinae and Modellnaiinae, China for Anodontinae and Unioninae, and Asian Russia for Margaritiferidae. Conservation status and data collected after 1980 were not available for 61 and 24% of species, respectively. Dams, deforestation and pollution are likely the major threats to mussels in the region, though data in this respect are scarce. The Philippines, Laos, Indonesia, Myanmar and Malaysia are among the countries with the poorest data availability and urgently require research.

Morphological development of the juvenile through to the adult in the freshwater pearl mussel, Hyriopsis (Limnoscapha) myersiana, under artificial culture

Summary The morphological development of the freshwater mussel, Hyriopsis (Limnoscapha) myersiana (Lea, 1856), was observed using light and scanning electron microscopes, from the newly transformed (0 days old) juvenile to the onset of the adult stage (360 days old). As in the glochidium, the early juvenile has a semi-oval and equivalve shell with an equilateral valve. After day 1 the shell develops a larger anterior than posterior region until day 40, after which the posterior region grows larger than the anterior region. The form of the juvenile at 260-day-old resembles that of a fully grown adult. The shell microstructure of 0–20-day-old juveniles shows two differentiated layers, the periostracum and the prismatic layer. By day 30 the prismatic layer lies under a clear columnar structure that has formed a third layer, the nacreous layer. The mantle develops incurrent and excurrent siphons when juveniles are 60 days old. The development of juvenile gills initiates from a pair of gill bars at 0 days old, and formation of the inner demibranch starts from 10 days old and the outer demibranch from 90 days old. From this stage, numerous cilia form the latero-frontal cirri of the inner demibranchs. Additionally, longitudinal and transversal interfilamentous junctions of the inner and outer demibranchs begin to develop when juveniles are 200 and 240 days old and are complete at 230 and 260 days of age, respectively. Interlamellar septa join the inner surface of descending and ascending gill filaments to form water chambers when juveniles are 250 and 280 days old, respectively, and the development of inner and outer demibranchs is complete.

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification - by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose - was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0-11.0 and an optimal temperature of approximately 55-60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis-Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s(-1), respectively. The catalytic efficiency (Kcat/Km) was 238 s(-1) mM(-1).

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.

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.