Zhifeng Gu

Characterization of the Pearl Oyster (Pinctada martensii) Mantle Transcriptome Unravels Biomineralization Genes

Pearl oyster, Pinctada martensii, is a marine bivalve species widely distributed in tropic and subtropic marine coasts. Mantle is the special tissue of P. martensii that secretes biomineralization proteins inducing shell deposition as well as iridescent nacre both in the inner shell and artificial nucleus. The pearl oyster is very efficient for artificial pearl production and is therefore an ideal organism for studies into the processes of biomineralization. However, deficiency of transcriptome information limits the insight into biomineralization mechanisms and pearl formation. In this study, we sequenced and characterized the P. martensii mantle transcriptome using 454 pyrosequencing. A total of 25,723 unique transcripts were assembled from 220,824 quality reads, followed by annotation and Gene Ontology classification analysis. A total of 146 unique transcript segments homologous to 49 reference biomineralization genes were identified, including calcineurin-binding protein, amorphous calcium carbonate binding protein 1, calmodulin, calponin-like protein, carbonic anhydrase 1, glycine-rich shell matrix protein, lysine-rich matrix protein, mantle gene or protein, nacrein, pearlin, PIF, regucalcin, and shematrin. The sequence data enabled the identification of 10,285 potential single nucleotide polymorphism loci and 7,836 putative indels, providing a resource for molecular biomarker, population genetics, and functional genomic studies. A large number of candidate genes for biomineralization were identified, considerably enriching resources for the study of shell formation. These sequence data will notably advance biomineralization and transcriptome study in pearl oyster and other Pinctada species.

Development of Expressed Sequence Tags from the Pearl Oyster, Pinctada martensii Dunker

The pearl oyster, Pinctada martensii, is the primary species used for the aquaculture production of marine pearls in China and Japan. Genetic tools and resources are needed to study the genome of this species and to understand the molecular basis of development, growth, host defense, pearl formation, and other important traits. In this study, we developed a set of expressed sequence tags (ESTs) for P. martensii. We constructed cDNA libraries from adult tissues and sequenced 7,128 ESTs. Clustering analysis identified 788 contigs (covering 5,769 ESTs) and 1,351 singletons, yielding a total of 2,139 unique genes. Of these unique genes, only 935 had significant (E-value ≤ 0.005) hits in GenBank, and the remaining 1,204 (56.3%) were novel. Most of the known genes are related to cellular structure, protein binding, and metabolic processes. Putative host-defense genes (86) were identified including C-type lectin, ferritin, polyubiquitin, proteases, protease inhibitors, scavenger receptors, heat shock proteins, and RAS oncogenes. The EST sequences developed in this study provide a valuable resource for future efforts on gene identification, marker development, and studies on molecular mechanism of host defense in pearl oysters.

Biochemical Composition of Pond-Cultured vs. Wild Gravid Female Mud Crab Scylla paramamosain in Hainan, China: Evaluating the Nutritional Value of Cultured Mud Crab

ABSTRACT Mud crab (MC) aquaculture is rapidly developing fast worldwide, and there is a high consumer demand for Scylla paramamosain. Demand for MCs is especially high in China, where such crabs, especially the gravid females, are regarded a delicacy. In Hainan Island, pond-rearing wild MC juveniles in the presence of local trash fish and shrimps in brackish water has been shown to be successful. The nutritional value of cultured MC, however, has not been evaluated. In this paper, the biochemical composition of pond-cultured and wild gravid female MCs was studied. The cultured MCs had a higher ratio of total edible yield than the wild ones. The contents of crude protein and crude lipid in the cultured MCs were significantly lower than in the wild MCs (P < 0.05). Furthermore, the total amino acid (TAA) and essential amino acid (EAA) contents of gonads in the wild MCs were both significantly higher than those in the cultured MCs (P < 0.05). The TAAs and EAAs of claw and body muscles in cultured MCs, however, were significantly higher (P < 0.05). There were no significant differences in the contents of polyunsaturated fatty acids (PUFAs), n-3 fatty acids, and n-6 fatty acids in the gonad of bothMCgroups, whereas the contents of total saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) were significantly different between the wild and the cultured MCs (P < 0.05). Furthermore, there were no significant differences in the contents of SFAs, MUFAs, and PUFAs in both claw and body muscle between the wild and cultured MCs.

Byssus Growth in Winged Pearl Oyster Pteria penguin (Röding, 1798)

ABSTRACT Relationships between shell morphological traits and byssus dimensions in adult winged pearl oysters Pteria penguin are described to include a more detailed description of byssus growth in juveniles under typical culture conditions. Byssus growth, byssus diameter (BD) relations between the byssus and shell traits [shell height; total wet weight (TW)], byssus occurrence, and the size distributions in samples collected monthly or bimonthly over a 1-y duration were recorded. Pearl oysters exhibited continuous shell growth and weight increase over the experimental period, but byssal measurements exhibited a different pattern. The increase in BD decreased as oysters aged and no further increase was observed after day 267. Moreover, BD/TW ratio exhibited a steadily diminishing trend as P. penguin aged. The relationships between the BD and the shell traits in 68-day-old oysters proved to be insignificant, probably because the oysters were neither heavy nor large enough to exhibit the relationships, but later, when oysters increased in size and weight, the relationships became significant. Byssus diameter was determined to be slightly more closely related to the weight of the animal than the size of the shell, and oysters at day 183 exhibited comparatively higher r2 values than oysters at day 366 of cultivation. The occurrence of oysters without byssus decreased gradually during cultivation from 30% initially to 0 after a year of cultivation.

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster, Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC50), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.

Sex determination and differentiation in Aurelia sp.1: the absence of temperature dependence

Cnidarians, being regarded as ‘basal’ metazoan animals, are considered to have relatively high plasticity in terms of sex reversal. In this study we used an experimental approach to demonstrate sexual differentiation and plasticity in benthic polyps and pelagic medusae of Aurelia sp.1 maintained at different temperatures. Results indicated that in Aurelia sp.1, sex differentiation has been determined at the polyp stage and that all medusae originating from a given polyp are, phenotypically, of the same sex. In addition, the sex of polyps budding from the same clone (either male or female) at different temperatures appears to be the same as that of the parent. The sex of medusae that had originated from a known-sex polyp was observed to remain the same as that of the parent, irrespective of differences in strobilation or rearing temperatures. These results indicate that the mechanism of sex determination of Aurelia sp.1. is not influenced by prevailing temperature regimes. A comparison of variability in terms of sexual plasticity of Aurelia sp.1 with that of Hydrozoa and Anthozoa suggests that species characterized by a free-swimming medusa life stage have a high dispersal potential, which probably results in a lower rate of sex reversal.

Noble scallop, Chlamys nobilis, sperm motility duration in the post-activation phase

Sperm motility in the post-activation phase is important for conducting and assessing species-specific artificial fertilization protocols. This study characterizes spermatozoa movement of the noble scallop, Chlamys nobilis, during the post-activation phase. Sperm samples were diluted and activated by fresh seawater, and subsequently incubated at 26 °C for 4 h. Sperm movement variables including total motile sperm (TM), rapid sperm (RAP), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), lateral head displacement (ALH) and beat-cross frequency (BCF) after sperm activation was recorded and analyzed using the computer assisted semen analyze system. Based on the motility index (MI), the sperm movement was categorized into four groupings (twitching before full activation, full activation, twitching after full activation, and decreasing during the latter portion of the sperm motility pattern). The full activation lasted 21 min with the greatest movement characteristics except BCF, and there was no difference with twitching before full activation except for the RAP. The greatest TM was observed at 24.5 min after activation. The RAP, VCL, VSL, VAP and ALH values in the post-activation phase increased at full activation, followed by a subsequent decrease, while the BCF continued to trend downward throughout the study. This study contributes to the understanding on the sperm property of the noble scallop for gamete management, fertilization and spat production in aquaculture.

Relationships Between Shell Morphological Traits and the Byssus Dimensions in the Winged Pearl Oyster Pteria penguin (Röding, 1798) Cultivated in Sanya, Hainan Island, China

ABSTRACT The relationships of the shell morphological traits, including the byssus dimensions, were recorded and analyzed in sample groups of 18- and 30-mo-old cultured winged pearl oysters, Pteria penguin. All relationships among shell dimensional traits with respect to shell height (SH) in the 18- and 30-mo-old oysters were observed to be positive and significant (P < 0.05). Although allometric growth was observed in both groups, the r2 values in the relationship between SH and total wet weight (TW) were higher in old oysters than in young ones. These findings indicate that weight in young oysters is heterogeneous. Moreover, the TW—SH ratio showed a clearer tendency to increase relative to exterior dimensions in old oysters. The r2 values in the correlation between SH and maximum dorso-ventral length were the highest in both oyster groups; hence, this dimension is a reliable predictor of SH. On the other hand, the relationships between SH and either hinge length or shell thickness were slightly weak in both age groups. Oysters with no byssus were found only in the younger group. It is possible that older oysters are less susceptible to factors that induce byssus shedding. In young oysters, the length and diameter of the byssus was closely related to the weight of the animal rather than the size of the shell. But, as individuals continued gaining weight, no augmentation in the byssus organ diameter was measured, suggesting a threshold for the byssus diameter in heavier oysters. Because it is possible that byssus size may influence the quality of pearls formed in the oyster due to the proximity of the pearl sac to the byssus root, it is important to consider the weight of the oysters selected or cultivated for pearl production, as this may indirectly affect the quality of the finished pearl product.