Freshwater Pearl Mussel Hyriopsis Cumingii Research Articles

Genome-wide association analysis reveals genetic architecture of growth and inner shell color traits in freshwater pearl mussel Hyriopsis cumingii (Lea 1852)

Triangle sail mussel (Hyriopsis cumingii, Lea 1852) is the most important freshwater pearl mussel in China. Growth traits of host mussels and inner shell color traits of donor mussels are critical determinants for pearl size and color. In this study, we performed GWAS analysis to explore the genetic architecture of growth and inner shell color traits. Using genotype by sequencing (GBS) technique, we obtained 80,562 SNPs from a total dataset of 463 mussels, based on these data, we found weak genetic relationship (−0.1–0.4) and rapid LD decay rate among the population. Moreover, GWAS analysis identified 12 growth-related significant SNPs and 29 inner shell color related significant SNPs. Five of the 12 SNPs linked to growth traits were validated to be credible SNPs in a new population. Cyclin, SNF, PMP22, CPED1, F-box and APC/C genes were found to be significantly associated with growth traits, which may influence the growth and development of H. cumingii by regulating cell mitosis, mantle epithelial tissue activity, and Ca2+ absorption and transport capacity. Notably, redness a* showed an obvious aggregation peak in chromosome 17, indicating that this region may be closely related to the inner shell color. Furthermore, three causal genes in chromosome 17, HSP78, MAS and PKS are inferred to be the key genes on color formation of purple inner shell. These results provide some new insights into the genetic basis and physiological regulation mechanism of growth and inner shell color traits in H. cumingii. However, detailed functional identification and regulation mechanism analysis merits more investigation.

Genotype by environment interactions for growth and inner shell color disparity in Hyriopsis cumingii under different light conditions

The light plays a pivotal role in influencing the growth and body color for aquaculture animals. The freshwater pearl mussel Hyriopsis cumingii, inhabiting benthic environments naturally, is predominantly cultivated by hanging methods. This aquaculture method altersmutiple environmental factors, with light being one of the maximum variationfactor. In order to comprehend the impact of light change on the growth and inner shell color of H. cumingii, a total of 14 full-sibling families were established and cultivated separately under natural light and dim light conditions for a period of 13 months in this study. The results revealed that under natural light conditions, juvenile mussel exhibited accelerated growth rates, while dim light conditions expedited purple pigmentation in the inner shell. Genetic analysis indicated general heritability range for the entire experimental population falls between 0.07 and 0.28. heritability estimates for all traits under natural light conditions (NL) ranged from 0.33 to 0.72, indicating consistently high heritability. However, heritability estimates for all traits under dim light conditions (DL) ranged from 0.05 to 0.16. The growth and inner shell color traits showed significant G × E interactions under different light conditions. The anal angle radial rib length (AARRL) trait exhibited a significantly higher correlation with body weight (BW) compared to shell height (SH) and total shell height (TSH). Furthermore, the growth and inner shell color traits of different families were compared and evaluated under the two environmental conditions in this study. Families 1 and 6 excelled in various traits under both conditions, making them promising candidates for genetic improvement breeding. In this study, pronounced influences of light exposure on the growth of pearl mussels were identified, emphasizing the need for tailored breeding strategies for both bottom seeding and suspended cultivation methods.

Gene identification and functional analysis of a D-type cyclin (CCND2) in freshwater pearl mussel (Hyriopsis cumingii).

Cyclin D (CCND) plays an important role in the cell cycle and is a rate-limiting factor that facilitates the G1/S transition. In this study, the full-length cDNA of Hc-CCND2 was isolated from freshwater pearl mussel (Hyriopsis cumingii; Hc) and amplified using the 3´/5´ RACE system. The Hc-CCND2 expression profiles were analysed by quantitative real-time PCR. Functional analysis of the Hc-CCND2 genes was examined by both RNA interference (RNAi) and overexpression in H. cumingii. Hc-CCND2 protein sequences were 295 amino acids long, possessed D-type cyclin signature motifs and contained conserved cyclin box domains. Hc-CCND2 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonad), with the highest expression levels found in the gill (P < 0.05). During the different developmental periods of the embryo, the relative expression of Hc-CCND2 increased with embryonic development, peaking at the blastula stage and decreasing significantly in the gastrula stage. After knockdown of Hc-CCND2 by RNAi, a significant decrease in CDK6 expression levels was found, while the percentage of cells in the G0/G1 phase significantly increased. Overexpression of Hc-CCND2 in mantle cells led to increased proliferation of cultured cells (P < 0.05). Our results demonstrated that Hc-CCND2 may promote cell cycle progression in H. cumingii, and that overexpression of Hc-CCND2 promotes mantle cell proliferation. These findings may provide a novel approach for improving the slow proliferation rate of shellfish cells in in vitro cultures.

Evaluation of Genetic Breeding for the Color of Shell Nacre in the Freshwater Pearl Mussel Hyriopsis cumingii (Lea, 1852) by Raman Spectroscopy

The color of pearl, one of the important quality traits, is determined by the shell nacre color of host mussel; however, the effective method to evaluate the effect of genetic breeding on the purity and uniformity of the nacre color was still limited to the freshwater mussel Hyriopsis cumingii. In this study, the characteristics of the υ1 band of polyene pigments in the colorful shell nacre and pearl samples from different selective generations of H. cumingii were detected by Raman spectroscopy. The results of this study showed that the number of peaks at the υ1 band of polyene pigments decreased gradually from 6–8 in the shell nacre and pearls with the color scheme of purple for wild mussels to 1 in those from the mussels of the F4 generation by genetic breeding. Similar results were also found in the samples with the color scheme of orange. But, no obvious peak of the υ1 band was detected in the samples with white color after four generations of selective breeding. Thus, it could be speculated that the single peak of the υ1 band in the Raman spectroscopy of shell nacre could be used as the sign of color uniformity to reflect the degree of purification in shell nacre color by genetic breeding. Meanwhile, the results showed that the samples with the same color scheme have a similar main peak at the υ1 band (ca. 1,508 cm–1 for the purple color scheme and ca. 1,528 cm–1 for the orange color scheme), suggesting that the polyene pigments in shell nacre and pearl with the same color scheme have the same length of C = C double bond. The length of C = C double bond were 12 and 9 in purple and orange samples, respectively. In summary, the results of this study support the conclusion that Raman spectroscopy is an efficient method to evaluate the genetic breeding for nacre color in H. cumingii.

Production performance of largemouth bass Micropterus salmoides and water quality variation in monoculture, polyculture and integrated culture

A 74‐day experiment was conducted to evaluate the production performance and water quality variation in three types of farming system for largemouth bass Micropterus salmoides. The tested aquaculture models included monoculture of largemouth bass (MC), polyculture of largemouth bass, gibel carp Carassius auratus gibelio and silver carp Hypophthalmichthys molitrix (PC), and integrated culture of largemouth bass, gibel carp, silver carp and freshwater pearl mussel Hyriopsis cumingii (IC). The ratio of largemouth bass, gibel carp and silver carp was 30:2:1 in the PC model, and the ratio of largemouth bass, gibel carp, silver carp and mussel was 30:2:1:5 in the IC model. The largemouth bass were fed with formulated feed twice daily. No significant differences were found in weight gain and yield of largemouth bass, total fish yield, nitrogen (N) and phosphorus (P) utilization efficiencies, N and P wastes, pH, nitrite, nitrate, reactive phosphate, total nitrogen, total phosphorus, total organic carbon, chemical oxygen demand, 5‐day biochemical oxygen demand, chlorophyll a, primary productivity among the MC, PC and IC models. The ammonia was lower, while the dissolved oxygen was higher in the PC tanks than in the MC tanks. These results suggest that the environment situation was better in the PC tanks relative to that in the MC tanks. The present study reveals that the PC model should be a way to optimize the aquaculture model for commercial largemouth bass farming.

Identification and Molecular Characterization of Hc-Upsalin, a Novel Matrix Protein Involved in Nacreous-Layer Biomineralization in Hyriopsis cumingii

Biomacromolecules, including matrix proteins, are important in shell-mineralization processes, such as inducing crystal nucleation, regulating crystal polymorphism and morphology. In this study, we identified the novel matrix protein hc-upsalin from the freshwater pearl mussel Hyriopsis cumingii, associated with its significantly high similarity to the known matrix protein upsalin in the freshwater mussel Unio pictorum. The full-length cDNA of hc-upsalin encodes 119 amino acids, including a potential signal peptide of 17 residues. The mature protein is characterized by high proportions of Gly (13.7%), Cys (11.8%), and Pro (11.8%), with a molecular weight of 11.4 kDa and a theoretical pI of 6.93. Hc-upsalin is specifically expressed in muscular tissues mostly in the mantle, with positive signals detected in dorsal epithelial cells of the mantle by in situ hybridization, and decreased expression of hc-upsalin leaded to random accumulation of tablet, suggesting its involvement in shell nacreous-layer biomineralization. Furthermore, hc-upsalin gene-expression patterns during pearl biomineralization indicated a potential role in the process of pearl nacreous-layer calcification.

Cloning and Analysis of Gene Expression of Two Toll Receptors in Freshwater Pearl Mussel Hyriopsis cumingii

Toll receptors are involved in innate immunity of invertebrates. In this study, we identify and characterize two Toll genes (named HcToll4 and HcToll5) from triangle sail mussel Hyriopsis cumingii. HcToll4 has complete cDNA sequence of 3,162 bp and encodes a protein of 909 amino acids. HcToll5 cDNA is 4,501 bp in length and encodes a protein of 924 amino acids. Both deduced HcToll4 and HcToll5 protein contain signal peptide, extracellular leucine rich repeats (LRRs), and intracellular Toll/interleukin-1 (IL-1) receptor domains. Quantitative real-time PCR analysis revealed that HcToll4 and HcToll5 were largely distributed in the hepatopancreas and could be detected in the gills and mantle. HcToll4 and HcToll5 expression could respond to Staphylococcus aureus, Vibrio parahaemolyticus, White spot syndrome virus (WSSV) or Poly I:C challenge. RNA interference by siRNA results showed that HcToll4 and HcToll5 were involved in the regulation of theromacin (HcThe) and whey acidic protein (HcWAP) expression. Based on these results, HcToll4 and HcToll5 might play pivotal function in H. cumingii innate immune response.

The effect of four microbial products on production performance and water quality in integrated culture of freshwater pearl mussel and fishes

An 80-day mesocosm experiment was conducted to evaluate the effect of four commercial microbial products on production performance and water quality in integrated culture of freshwater pearl mussel Hyriopsis cumingii, grass carp, gibel carp, silver carp and bighead carp. Five treatments were tested. One treatment with non-supplementation of microbial products served as control (C). In the other four treatments, Novozymes Pond Protect (NO), Bio-Form BZT-Water Reform (WR), Bacillus natto (BN) and Effective Microbes (EM) were added at the intervals of two weeks, respectively. Mussel yield declined in the tanks with supplementation of the microbial products. No significant differences were found in fish yield and chemical water quality among the treatments except total nitrogen (TN) was higher in tanks EM than in tanks C. Biomass of phytoplankton and Cyanophyta was higher in tanks NO, WR, BN and EM than in tanks C. Supplementation of the microbial products resulted in change in bacterial community in which Actinobacteria, Chlorobi, Verrucomicrobia and Proteobacteria dominated. Bacterial community in the tanks was significantly affected by TN, total phosphorus, chemical oxygen demand and Cyanophyta biomass. This study reveals that the function of these microbial products as probiotics is limited in H. cumingii farming.

HcToll3 was involved in anti-Vibrio defense in freshwater pearl mussel, Hyriopsis cumingii

Toll-like receptors (TLRs) play an important role in the activation of innate immune response but their functions in bivalves remain largely unknown. In this study, we identified a TLR from the freshwater pearl mussel Hyriopsis cumingii (HcToll3) and investigated its functions in immunity. The full-length cDNA of HcToll3 is 3852 bp and includes an open reading frame (ORF) of 3228 bp that encodes a polypeptide of 1075 amino acids. The predicted HcToll3 protein shares similar structural characteristics with other known Toll family proteins. Quantitative real-time PCR analysis revealed that HcToll3 mRNA is broadly expressed in all of the examined tissues; its transcript level was significantly up-regulated by challenge with gram-negative bacteria Vibrio parahaemolyticus or lipopolysaccharide, but not gram-positive Staphylococcus aureus or peptidoglycan. RNA interference by siRNA results showed that HcToll3 regulated expression of whey acidic protein (HcWAP) and lysozymes (HcLyso1 and HcLyso2) in vivo and knockdown of HcToll3 suppressed the elimination of V. parahaemolyticus. These findings suggest that HcToll3 might be involved in anti-Vibrio defense in H. cumingii.

Analysis of Selective Breeding of Nacre Color in Two Strains ofHyriopsis cumingiiLea Based on the Cielab Colorspace

The nacre color of the pearl mussels is one of the most important traits for the pearl industry. In this study, to evaluate the effectiveness of the nacre color selection of the freshwater pearl mussel Hyriopsis cumingii, the characteristics of nacre color from a purple, white, and a commercial farmed strain were detected, respectively, by measuring the color parameters of CIEL*a*b* (CIELAB) system. The analysis of the variation among the color parameters showed that the uniformity of the nacre color in purple or white strain had been significantly improved over that of the farmed strain. The principal component analysis indicated that the first principal component (PC1) was most affected by color parameter a* where a positive value indicates a hue of red and a negative value indicates a green in the CIELAB color space. The second principal component (PC2) was most affected by color parameter b* where a positive value indicates a hue of yellow and a negative value indicates blue. The contribution rates of these PCs were 63.64% and 23.81%, respectively. The results also showed that the nacre color was obviously different among purple, white, and farmed strain, and the difference of nacre color between purple and white strain was primarily caused by color parameter a*. The discriminant functions for nacre colors were established and the rate of discriminant accuracy (P1 and P2) was 100% for all three strains. The results indicated that purple, white, and farmed strains could be clearly identified and differentiated on the basis of their nacre color, and the traits of nacre color could be purified through selective breeding.

Optimization of fish to mussel stocking ratio: Development of a state-of-art pearl production mode through fish–mussel integration

Integrated aquaculture has been widely used for pearl production in the freshwater pearl mussel Hyriopsis cumingii farming in China, but the production technology has not reached the state of the art. This study explored the optimal stocking ratio of fish to mussel (fish–mussel) through a 90-day experiment conducted in land-based enclosures. The integrated system included pearl mussel, grass carp, gibel carp, silver carp and bighead carp, with four fish–mussel stocking ratios by number: 1:1 (R1), 2:1 (R2), 3:1 (R3) and 4:1 (R4). The pearl yield was higher in the R2 enclosures than in the R1 and R4 enclosures, whereas the fish yield was higher in the R3 and R4 enclosures than in the R1 and R2 enclosures. The phosphorus (P) utilization efficiency was higher in the R2, R3 and R4 enclosures than in the R1 enclosures. The wastes of nitrogen (N) and P enhanced with the increase of fish–mussel ratio. Regression analyses indicated that the fish–mussel ratio was 2.3:1 for the maximal pearl yield, and 3.6:1 for the maximal fish yield, and 1.6–2.3:1 for the minimal N waste, and 1.9–2.9:1 for the minimal P waste. This study indicated that the suitable fish–mussel stocking ratio was 2:1 in the integrated culture of H. cumingii, grass carp, gibel carp, silver carp and bighead carp.

Characterization and functional analysis of a chitin synthase gene (HcCS1) identified from the freshwater pearlmussel Hyriopsis cumingii.

The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.

Characterization of a novel carbonic anhydrase from freshwater pearl mussel Hyriopsis cumingii and the expression profile of its transcript in response to environmental conditions.

Gene encoding for α-carbonic anhydrases (α-CAs) and their functions in fundamental metabolism and biomineralization are widely identified in mollusks. However, the transcriptional regulation of α-CA genes in response to various environmental conditions remains unknown. In the present study, we characterized a cDNA encoding for an α-CA (HcCA) from the freshwater pearl mussel Hyriopsis cumingii. The spatial and temporal expression patterns of HcCA indicate that this gene is mainly expressed in the mantle of juvenile mussels. The expression profile of HcCA under various environmental conditions reveals that the transcription of HcCA is significantly regulated by Ca(2+) concentration, water temperature, pH and air exposure. Our results suggest that HcCA is a crucial target gene by which the external environmental conditions affecting shell growth and pH homeostasis of H. cumingii.

Comparative analysis of total carotenoid content in tissues of purple and white inner-shell color pearl mussel, Hyriopsis cumingii

The freshwater pearl mussel Hyriopsis cumingii is the most important mussel species for freshwater pearl production in China. Mussel shell color is an important indicator of pearl quality. The objective of this study was to assess whether total carotenoid content (TCC) in H. cumingii is related to shell color. TCC of different tissues (gonad, gill, hepatopancreas, kidney, axe foot, mantle, and adductor muscle) of purple and white inner-shell H. cumingii was determined by UV–Vis spectrophotometry. The results revealed that TCC ranged from 12.91 ± 0.78 to 56.30 ± 0.74 μg/g. In general, TCC was higher in the hepatopancreas, followed by the mantle, gonad, gill, kidney, axe foot, and adductor muscle. TCC in gonad, gill, hepatopancreas, kidney, and mantle of purple mussels was significantly higher than that of white mussels. TCC in mussel tissues of H. cumingii was significantly different (P < 0.001) with respect to shell color. There were significant positive correlations between TCC in mussel mantle and shell color intensity. Future studies will assess the biological roles of carotenoids in shell color formation.

Cloning, differential tissue expression of a novel hcApo gene, and its correlation with total carotenoid content in purple and white inner-shell color pearl mussel Hyriopsis cumingii

As a molecular carrier and storage protein, apolipoprotein (Apo) mediates the intracellular uptake of lipids, proteins, vitamins and carotenoids. In this study, we identified a novel Apo gene, designated hcApo, from the freshwater pearl mussel Hyriopsis cumingii. The complete hcApo cDNA consists of 4104 nucleotides with an open reading frame encoding 1155 amino acid residues. The hcApo protein contains a conserved lipoprotein N-terminal domain (LPD-N) that is a characteristic of the large lipid transfer protein (LLTP) superfamily. The hcApo mRNA is constitutively expressed in a wide range of tissues with the highest expression level in the liver. Moreover, differential expression analysis revealed that the hcApo gene is more highly expressed in the liver, kidney, mantle and gill of purple line mussels compared to white line mussels. In situ hybridization investigations of the precise expression site of hcApo mRNA in the mantle showed that hcApo mRNA is specifically expressed in the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold of the mantle, as well as throughout the outer epithelium of the outer fold and ventral mantle. Another very important finding is that significantly positive correlation existed between the hcApo gene expression level and the total carotenoid content in purple line mussels. These findings may provide a better understanding of the roles of hcApo in the molecular mechanisms of shell formation and coloring of H. cumingii.

Molecular characterization and expression pattern of an α-amylase gene (HcAmy) from the freshwater pearl mussel, Hyriopsis cumingii.

The freshwater pearl mussel Hyriopsis cumingii is of commercial importance because it produces the freshwater pearl; however, knowledge about the molecular characterization and regulation mechanisms of α-amylase remains unknown for this species. In this study, the full-length cDNA of the α-amylase gene (HcAmy) was isolated from H. cumingii by the rapid amplification of cDNA ends. Tissue-specific expression analysis showed that HcAmy mRNA was mainly expressed in the hepatopancreas; although, the gene was also expressed in the adductor muscle, intestine, gill, and crystalline style. After 2 weeks starvation, the expression of HcAmy mRNA in the hepatopancreas was upregulated at 24 h after re-feeding or when exposed to algal concentration of 32 μg/L chlorophyll-a, indicating that the HcAmy mRNA expression in H. cumingii is regulated by algal availability. The results of this study confirm that the HcAmy gene is an important component of the carbohydrate metabolism of H. cumingii fed phytoplankton. In addition, this study demonstrates that the modulation of this gene is dependent on environmental food availability, including starvation, re-feeding time following a period of starvation, and algal concentrations during re-feeding.

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.

Characterization of cDNAs for calmodulin and calmodulin-like protein in the freshwater mussel Hyriopsis cumingii: Differential expression in response to environmental Ca2+ and calcium binding of recombinant proteins

Calmodulin and calmodulin-like protein are two crucial calcium regulators in bivalves. However, molecular characteristics and expression patterns of these genes in the freshwater mussel are poorly understood. In this study, two cDNAs encoding novel calmodulin and calmodulin-like protein (HcCaM and HcCaLP) were cloned and characterized from the freshwater pearl mussel Hyriopsis cumingii. The full-length cDNA of HcCaM was 726 bp, including a 118-bp 5'-untranslated region (UTR), a 447-bp open reading frame (ORF), and a 161-bp 3'-UTR. The 1217-bp HcCaLP cDNA comprised of a 51-bp 5'-UTR, a 447-bp ORF, and a 716-bp 3'-UTR. The potential phosphorylation sites of, Arg(80) and Phe(100) in deduced HcCaM were mutated to Thr(80) and Tyr(100) in HcCaLP. Tissue-specific expression analysis revealed that HcCaM mRNA was prominently expressed in the gill, mantle center, and foot. In contrast, HcCaLP mRNA was mainly expressed in the mantle edge. The recombinant HcCaM and HcCaLP proteins expressed in Escherichia coli showed the typical Ca(2+) dependent electrophoretic shift characterization as CaM and differed in the calcium binding affinity. The calcium stimulation test that lasted 5 weeks implied that HcCaM and HcCaLP had differential expression patterns in response to various environmental Ca(2+) concentrations (0.25-1.25 mM). The expression of HcCaM mRNA was up-regulated by low Ca(2+) concentration (0.25 mM), and the highest expression of HcCaLP mRNA occurred under Ca(2+) concentration of 1 mM.

Healing and regeneration of the freshwater pearl mussel Hyriopsis cumingii Lea after donating mantle saibos

After excision of mantle tissue from Hyriopsis cumingii Lea, survival rates, specific growth rate, healing and regeneration were observed and quantified for three months. High survival rates (94.7%, 92%, 95.3% and 94.7%, respectively) of mussels with small (8–12mm×8–12mm) (S-group)excision taken from the front, middle and back of the ventral mantle margin, and control mussels were observed, and no significant difference was observed among the four groups. The specific growth rates, SGR (length), SGR (thickness) and SGR (weight) of H. cumingii with different excision locations did not differ significantly but the SGR (height) of the group with the front excision was significantly lower than that of the other excised groups and controls. Mussels were excised with small, medium (8–12mm×28–32mm) (M-group) or large (8–12mm×48–52mm) (L-group) from the middle ventral mantle margin. The survival rates of mussels with S-, M- and L-group and controls were, respectively, 92%, 90%, 90.7% and 94.7%. The survival rate of the L-group was significantly lower than that of the other three groups. The trends in SGR (length), SGR (height), SGR (thickness) and SGR (weight) among the four groups were similar: controls>S-group>M-group>L-group. Macroscopic observation of mantle tissue showed that the wound area became reduced since 12th d after the excision. To grow to the same level as the surrounding normal tissue, the regenerated tissue needed 40d, 75d and 90d, respectively, in the S-, M-, and L-group. Histological observation of wound tissues showed that haemocytes aggregated at the wound site soon after excision and completely sealed the wound after 12h. Epithelialization sealed the wound completely after 72h. The three mantle lobes began to form at 15d. The regenerated lobes were morphologically different from normal tissues after 25d but were identical to the normal mantle lobes, in both morphological structure and function, by 90d. This is the first report on the survival rate, growth rate, wound healing, and regeneration in the freshwater pearl mussel after excision of its mantle tissue. The study demonstrates that donor mussels, without being killed, may donate mantle pieces (saibos) for evaluating the effect of donor mussel on pearl quality. Donor mussels contributing to pearl quality, this provides support for selective breeding of donor mussels in the future.

Effect of three fertilization programs on the chemical water quality for integrated culture of freshwater pearl mussel and fish

A 155-day experiment was conducted in land-based enclosures to examine the effects of three fertilization programs on chemical water quality for integrated culture of freshwater pearl mussel(Hyriopsis cumingii)and fishes.The pearl mussel was co-cultured with grass carp,gibel carp,silver carp and bighead carp,and the stocking ratio of fish to mussel was 1.5∶ 1.Three fertilization programs,including fertilizing with duck manure(DM),with chemical fertilizer(CF),and with duck manure and chemical fertilizer in combination(DC),were used.In the experiment,the pH was lower in the CF enclosures than that in the DM enclosures during the earlier phase(from May 20 to July 18).The SD,concentration of dissolved oxygen(DO) and Ca2+,and ratio of Ca2+ to Mg2+ decreased,while the concentration of ammonia(TAN),nitrite(NO2-N),total nitrogen(TN),total phosphorus(TP) and chemical oxygen demand(CODMn)increased,with the progress of the experiment.There were no significant differences in the SD and concentration of dissolved oxygen(DO),SO2-4,Cl-,CO2-3,HCO-3,Ca2+,Mg2+,hardness,alkalinity,ammonia(TAN),NO3-N,NO2-N,PO4-P,total nitrogen(TN),total phosphorus(TP)and chemical oxygen demand(CODMn)between the fertilization treatments.The ratio of PO4-P to TP was higher in the CF enclosures than that in the DM enclosures.Our results indicate that using different fertilization programs had no significantly different effects on the major ions,hardness,alkalinity,TAN,NO3-N,NO2-N,PO4-P,TN,TP and CODMn in the enclosures with integrated culture of freshwater pearl mussel with fishes and fed formulated fish feed.The concentration of CODMn was slightly low,but the ratio of PO4-P to TP was higher,in the enclosures fertilized with chemical fertilizer,relative to the enclosures fertilized with duck manure,whereas the concentration of CODMn and ratio of PO4-P to TP were at median levels in the enclosures fertilized with both chemical fertilizers and duck manure.The higher growth rate and nacre secretion of H.cumingii hung in the enclosures supplied with both chemical fertilizers and duck manure suggest this fertilization program is suitable for H.cumingii farming.