Pearl Sac Formation Research Articles

A proteomic approach reveals biomineralization and immune response for mantle to pearl sac in the freshwater pearl mussel (Hyriopsis cumingii)

In the process of production of freshwater pearl, implanted mantle pieces undergo a series of complex physiological and biochemical processes to form pearl sac, which produce pearl. This is a very important site of occurrence due to immune-induced biomineralization, while its molecular regulatory mechanism is still unclear. Here, we use proteomics to identify differentially expressed proteins (DEPs) of the mantle and pearl sac and examine the biomineralization and immune response of the pearl sac formation process in Hyriopsis cumingii. Using iTRAQ technology and bioinformatics analysis, we obtained DEP profiles between the mantle and pearl sac. A total of 1871 proteins were identified. Of these, 74 DEPs were found between the pearl sac and outer mantle, 112 DEPs between the pearl sac and inner mantle, and 124 DEPs between the outer and inner mantles. Bioinformatics analysis revealed that the screened biomineralization-related DEPs were mainly enriched in signaling pathways associated with calcium signaling, regulation of the actin cytoskeleton and protein processing in the endoplasmic reticulum, while the immune-related DEPs were mainly enriched in the Notch, Hippo, nuclear factor kappa-B (NF-κB), and transforming growth factor-β (TGF-β) signaling pathways. In addition, the expression of six biomineralization-related and four immune-related proteins were verified at the transcriptional level using quantitative real-time PCR. Our findings contribute to furthering the understanding of the mechanisms of pearl formation and immune response, and have long-term implications for future studies on the production of high-quality freshwater pearls and development of the freshwater pearl industry.

Cloning and functional characterization of PmΔ5FAD in pearl oyster Pinctada fucata martensii

Pinctada fucata martensii produces high-quality pearls. However, excess immune and inflammatory response after grafting operation will lead to nucleus rejection, pearl sac formation failure, and death of the host pearl oyster. Polyunsaturated fatty acids (PUFAs) are critically important for its regulation function in inflammation and their synthesis process was limited by the related enzymes including fatty acid desaturase (FAD). In this study, FAD gene in the pearl oyster was characterized in P. f. martensii (PmΔ5FAD). We established that the full-length sequence of PmΔ5FAD contained a 1302-bp open reading frame that encoded a sequence of 433 amino acids. PmΔ5FAD domain analysis revealed the presence of a distinctive Cyt-b5 domain and a FA-desaturase domain that were highly similar to the Δ5FAD protein sequences identified in Haliotis discus hannai (69.25%) and Mimachlamys nobilis (62.96%). PmΔ5FAD expressed in all detected tissues. Functional analysis of PmΔ5FAD in recombinant Saccharomyces cerevisiae yeast showed the existence of Δ5-desaturation activity, which towards PUFA substrates and it efficiently desaturated exogenous PUFA C20:3n-6 to C20:4n-6 (ARA) with a desaturation conversion rate of 19.49%. Furthermore, PmΔ5FAD showed a significantly higher expression level on day 1 after the grafting operation, which may upregulate ARA synthesis in response to inflammatory and immune. These results provide important information concerning the function of FADs during pearl oyster PUFA biosynthesis and grafting operation.

Effects of low-salinity and no-feed post-operative care during pearl sac formation on the expression profiles of shell matrix protein genes in the Akoya pearl oyster, Pinctada fucata

Effects of low-salinity and no-feed post-operative care during pearl sac formation on the expression profiles of shell matrix protein genes in the Akoya pearl oyster, <I>Pinctada fucata</I>

Identification of a uroporphyrinogen III synthetase gene and characterization of its role in pearl sac formation in Hyriopsis cumingii

Uroporphyrinogen III synthetase (UROS) is an important enzyme involved in the structural formation of porphyrin, which is responsible for color intensity. A UROS homolog was isolated and characterized from the pearl mussel, Hyriopsis cumingii (HcUros). The HcUros cDNA was 858 bp in length with an open reading frame that encoded a 285 amino acid protein, which contained a conserved HemD domain. HcUros exhibited widespread tissue distribution, displaying particular enrichment in the mantle and haemolymph of purple mussels, compared to white mussels. In situ hybridization of HcUros in mantle tissue demonstrated that it was specifically expressed in the dorsal epithelial cells of the mantle pallia. Additionally, HcUros expression was upregulated following implantation of the pearl sac to produce pearls. The data suggest that HcUros contributes to shell pigmentation, and nacre formation and coloring in H. cumingii.

Histological and Comparative Transcriptome Analyses Provide Insights Into the Immune Response in Pearl Sac Formation of Hyriopsis cumingii

Immune response plays an important role in the pearl sac formation and pearl quality. However, there is little knowledge about the mode and mechanism of the immune responses in the pearl sac formation of H. cumingii, which produced the largest production of non-nucleated freshwater pearl in China. In this study, we monitored the process of pearl sac formation by histology examination for 18 days after grafting. The results showed that a large number of hemocytes aggregated in the pearl sac cavity on day 8 after grafting and provided organic substrate for the deposition of extracellular matrix and CaCO3 crystal and mediated the initiation of pearl forming on day 12. These results revealed the important role of hemocytes on the early pearl sac and pearl formations. In the transcriptome analysis of pearl sac at the key time points of day 0 and day 6, 8, 12 after grafting, the expression profiles of the immune-related genes revealed the immune response pattern of H. cumingii after grafting. The significant up-regulation of the hydrolytic enzymes, acute phase proteins, and antimicrobial peptides of humoral defense factors on day 6 after grafting suggested that these immune-related gene products were the main immune defense during the early wound healing; Meanwhile, the continuous up-regulation of antioxidant enzymes such as Cu-Zn SOD, Se-GPX, and tyrosinase in prophenoloxidase (proPO) system on day 8 and day 12 suggested the important role of these immune effectors in oxidation-reduction on the later stage of pearl sac and pearl formations in H. cumingii. Moreover, the results of KEGG analysis of differentially expressed genes suggested the potential regulatory function of the signaling pathways of proPO system and complement system during pearl sac formation. These results provided valuable new insights into the roles and functions of the immune system in pearl sac and pearl formation.

Characterization of cyclin dependent kinase-7 and its differential response to grafting challenge in the black shell colored selected line of pearl oyster Pinctada fucata martensii

Cyclin dependent kinase-7 (Cdk-7) is a protein kinase associated with regulating the cell cycle, cell differentiation and proliferation, apoptosis and inflammatory response. This study characterized the full cDNA sequence of Cdk-7 in Pinctada fucata martensii (PmCdk-7). A full length sequence of 1473bp with an open reading frame (ORF) of 915bp and encodes a 304aa, 5′-UTR of 58bp and a 3′-UTR of 500bp was obtained. The construed amino acid sequence of PmCdk-7 comprised of a Serine/Threonine protein kinases, catalytic (S_TKc) domain with a protein kinases ATP-binding region signature (14-38aa) and the serine/Threonine protein kinases active-site signature (129-141aa) within the domain. Tissue distribution analysis revealed a high relative mRNA expression of PmCdk-7 within haemocytes. Following the insertion operation (grafting), the relative expression levels of PmCdk-7 in the haemocyte was expressed differentially among the studied groups; the black shell colored selected line (BS) and the control group (CG). High expression was recorded between 12 h and 5d with a peak at 3d suggesting a heightened level of DNA replication and inflammatory response during the pearl-sac formation and this expression was higher in BS than CS showcasing, the heightened immune capacity of BS to grafting operation. Immune stimulation experiment with bacterial endotoxin and a viral mimic revealed PmCdk-7 response to pathogenic stress. The results from our study showed that PmCdk-7 performs a vital function during the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants (pathogens).

Response of pearl oyster Pinctada fucata martensii to allograft-induced stress from lipid metabolism

The pearl oyster, Pinctada fucata martensii, produces high-quality pearls. During pearl production, excess immune and inflammatory response after transplantation will lead to nucleus rejection, pearl sac formation failure, and death of the host pearl oyster. The hemocyte transcriptome and fatty acid (FA) contents in the adductor muscle before and after transplantation were analyzed to investigate the response of pearl oyster P. f. martensii to allograft-induced stress from lipid metabolism. The hemocyte transcriptome analysis detected 193 lipid metabolism-related genes, such as the elongation of very long-chain FA protein 5, acyl-CoA 6-desaturase, cytochrome P450, phospholipase A2, glycerol-3-phosphate O-acyltransferase, and prostaglandin-H2 d-isomerase. Pathway enrichment analyses revealed that these genes were mainly involved in the “biosynthesis of unsaturated FAs,” “FA biosynthesis,” “ARA metabolism,” and “glycerolipid metabolism.” An analysis of FA contents in the adductor muscle indicated no significant difference in the contents of lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, arachidic acid, α-linolenic acid, eicosadienoic acid, docosadienoic acid, and 11,14,17-eicosatrienoic acid. However, ARA, DHA, and EPA in the adductor muscle after transplantation were significantly greater than those processed without grafting surgery. These results suggest that pearl oysters require more polyunsaturated FAs (PUFAs) to regulate their inflammatory and immune response after transplantation. However, their ability to biosynthesize unsaturated FAs is limited. Given these results, the addition of PUFA-containing diets or selection of a line with strong ability to biosynthesize unsaturated FAs may be valuable for pearl oyster recovery after transplantation.

Greft İmplantasyonunda İnci Kesesi Oluşumunun Dört Midye Türünde (Unionidae) Karşılaştırılması

Bu çalışmada, inci kesesi oluşumu için en uygun manto bölümü ve en iyi konakçı midye türü tespit edilmeye çalışılmıştır. Araştırma için dört farklı midye türünden oluşan (Unio terminalis, Potamida littoralis, Leguminaia wheatleyi ve Anodonta pseudodopsis) toplam 400 adet midye kullanılmıştır. Midyelerin ortalama dorso-ventral uzunlukları sırasıyla; 7,89±0,25; 7,28±0,38; 10,68±0,27 ve 11,14±0,34 cm‘dir. Manto dokusunun pallial kenarından 3×3 mm büyüklüğünde elde edilen manto doku dilimleri, greft olarak kullanılmıştır. Biri manto boşluğu diğeri manto dokusu üzerinde açılan kesitler olmak üzere, greft implantasyonu için iki farklı manto bölümü tercih edilmiştir. Üç aylık inci kültürü sonunda, manto boşluğu bölümü en iyi greft implant bölgesi olarak belirlenmiştir. U. terminalis, yaşama oranı ve inci kesesi oluşumu bakımından en başarılı tür olarak tespit edilmiştir. P. littoralis‘in ikinci başarılı tür olduğu, L. wheatleyi‘nin ise türler arasında en düşük sonuç veren tür olduğu belirlenmiştir. Bu araştırma, inci üretiminde önemli görülen sedef kalınlığı ve kalitesi konusunda, uzun süreli inci üretim çalışmaları için bir rehber niteliği taşımaktadır.

Identification of nacre matrix protein genes hic14 and hic19 and their roles in crystal growth and pearl formation in the mussel Hyriopsis cumingii.

Biological mineralization is a highly programmed process in which inorganic minerals reassociate under the strict control of the extracellular matrix to form minerals with special functions and patterns. Shells are biominerals, and their synthesis is finely regulated by organic matrix including matrix proteins, polysaccharides, lipids, pigments, free amino acids, and small peptides. In this study, two matrix protein genes, hic14 and hic19, were isolated from the mantle of the mussel Hyriopsis cumingii. Tissue expression analysis showed that both proteins were expressed mainly in the mantle, and in situ hybridization of mantle tissues showed that they were specifically expressed in the dorsal epithelial cells of mantle pallial. Therefore, hic14 and hic19 were both nacreous layer matrix proteins. In the pearl insertion experiment, hic14 and hic19 kept low expression during pearl sac formation and disordered calcium carbonate deposition, and increased significantly during pearl nacre accumulation, which showed that both proteins participated in the mineralization of pearl nacre. In the RNA interference experiment, shell nacre tablet growth was inhibited after crystal nucleation due to the decreased expression of hic14, and crystal morphology and arrangement of nacre were highly modified after expression of hic19 was inhibited. These results provided further evidence that both hic14 and hic19 participated in nacreous layer biomineralization.

A Kunitz proteinase inhibitor (HcKuPI) participated in antimicrobial process during pearl sac formation and induced the overgrowth of calcium carbonate in Hyriopsis cumingii

Proteinase inhibitors with the ability to inhibit specific proteinases are usually closely connected with the immune system. Interestingly, proteinase inhibitors are also a common ingredient in the organic matrix of mollusk shells. However, the molecular mechanism that underlies the role of proteinase inhibitors in immune system and shell mineralization is poorly known. In this study, a Kunitz serine proteinase inhibitor (HcKuPI) was isolated from the mussel Hyriopsis cumingii. HcKuPI was specifically expressed in dorsal epithelial cells of the mantle pallium and HcKuPI dsRNA injection caused an irregular surface and disordered deposition on the aragonite tablets of the nacreous layer. These results indicated that HcKuPI plays a vital role in shell nacreous layer biomineralization. Moreover, the expression pattern of HcKuPI during LPS challenge and pearl formation indicated its involvement in the antimicrobial process during pearl sac formation and nacre tablets accumulation during pearl formation. In the in vitro calcium carbonate crystallization assay, the addition of GST-HcKuPI increased the precipitation rate of calcium carbonate and induced the crystal overgrowth of calcium carbonate. Taken together, these results indicate that HcKuPI is involved in antimicrobial process during pearl formation, and participates in calcium carbonate deposition acceleration and morphological regulation of the crystals during nacreous layer formation. These findings extend our knowledge of the role of proteinase inhibitors in immune system and shell biomineralization.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

Treatment effects during the post-operative care on the rate of pearl-sac formation in the pearl oyster Pinctada fucata

The pearl culture industry using the Akoya pearl oyster Pinctada fucata in Japan has been suffering from low productivity due to the proportion of low-quality pearls, which are not round in shape and have blemishes on the surface, is fairly high with over 70% of pearls effected. Recent studies indicate that the immersion treatment of oysters in low salinity seawater (25psu) in a tank for 8days just after the implantation of nuclei has a positive effect on the production of high-quality pearls compared to the suspension of oysters in the sea (33psu), so-called the “conventional method”, throughout two weeks of the post-operative care. However, a mechanism to increase the proportion of high-quality pearls in low salinity seawater is not fully known. In this study, we compared the periods from the implantation of nuclei to the completion of pearl-sac formation among implanted pearl oysters nursed in tanks containing low salinity (25psu) or normal salinity (33psu) seawater in tanks and in the sea (33psu). After the immersion of the implanted oysters in 25 or 33psu seawater in the tanks for 8days, animals were suspended from a raft in an inland bay for the remainder of post-operative care period. The implanted oysters treated with the conventional method were all fastened from the raft throughout the post-operative care period. The oysters from the three treatments were 10% formalin-fixed at periodic intervals and thin slices of the nucleus-implanted part of body were prepared for the examination of the process of pearl-sac formation. The oysters suspended from the raft in the sea throughout the post-operative care period showed the shortest interval from the implantation to the pearl-sac formation, followed by those in normal salinity and low salinity treatments, respectively. This trend corresponds well to that in the proportion of high-quality pearls among the three treatments. The results of the present study suggest that the delay of pearl-sac formation is most likely one of the important factors in preventing deformities and blemishes of pearls in future culture production.

Calmodulin Highly Expressed during the Formation of Pearl Sac in Freshwater Pearl Mussel (Hyriopsis schlegelii)

Calmodulin (CaM) is a pivotal multifunction regulator of calcium metabolism in nearly all organisms, and earlier studies revealed its underlying involvement of shell or pearl formation in oysters. Culture pearls are formed within complete pearl sacs. However, whether CaM functions in pearl sac formation or not is not reported. In this study, we prepared a polyclonal antibody against the CaM from the freshwater pearl mussel Hyriopsis schlegelii (HsCaM) and then detected the expression and distribution of HsCaM in pearl sac using the method of immunofluorescence in situ hybridization (IISH). Results revealed that strong fluorescent signals were observed in pearl sac as well as epithelial border of mantle. In addition, quantitative analysis results indicated that the mRNA expression level of HsCaM in pearl sac of H. schlegelii was significantly up-regulated during pearl sac formation. The fact that HsCaM highly expressed is present in pearl sac suggested that it might facilitate pearl sac formation in H. schlegelii.

Identification, structural characterization and expression analysis of a novel carbonic anhydrase from freshwater mussel Hyriopsis cumingii

In this study, an α-carbonic anhydrase (α-CA), HcCA3, from Hyriopsis cumingii was characterized. The full-length cDNA of HcCA3 was 1628bp, including a CA domain and an ORF of 1053bp which encoded 350 amino acids. Its predicted molecular weight was 39.69kDa and the pI was 5.92. qRT-PCR was used to determine the expression of the gene in various tissues at 0h, 3h, 6h, 12h, 24h, 48h, 96h, 7d, 14d, 21d, 28d and 35d after inserting the pearl nucleus. The results showed that the HcCA3 was highly expressed in the mantle, whereas its expression was low in other tissues. Expression in the posterior mantle pallial (pMP) was significantly higher than that in the anterior mantle pallial (aMP) and mantle center (MC). Expression in the aMP, pMP and MC was significantly higher in purple mussels compared with that in white mussels. At the same time, during the formation of pearls, expression in the aMP, pMP and pearl sac (PS) decreased and then increased; whereas expression in the MC increased and then decreased. In-situ hybridization showed that the HcCA3 was expressed in both inside and outside epidermal cells. In protein level, Western blot showed that HcCA3 was mainly expressed in the aMP, pMP and MC. Our results suggest that HcCA3 play a role in the formation of shell and pearl sac formation.

Hic74, a novel alanine and glycine rich matrix protein related to nacreous layer formation in the mollusc Hyriopsis cumingii

Organic matrix proteins play an important role in the biomineralization of mollusc shells. We have identified and characterized a novel matrix protein, namely hic74, isolated from the mantle of the freshwater mussel Hyriopsis cumingii. The hic74 gene encodes a 850 amino acid protein that is rich in alanine (ala, 30.8%), glycine (gly, 25.8%) and serine (ser, 10.6%), with ala mainly existing in poly-ala forms within the ala/gly-rich regions. Quantitative PCR expression analysis demonstrated that hic74 was specifically amplified from the mantle and in situ hybridization showed a strong signal in the epithelial cells in the pallial region of the mantle. Lower levels of expression were detected during the early stages of pearl sac formation but increased and remained constant during nacreous layer formation suggesting that hic74 might be involved in this process.

Characterization of allograft inflammatory factor-1 in Hyriopsis cumingii and its expression in response to immune challenge and pearl sac formation

The allograft inflammatory factor-1 (AIF-1) is one of the key factors associated with inflammatory response and immune defense. In the present study, we report the identification and characterization of AIF-1 from triangle sail mussel Hyriopsis cumingii (HcAIF-1). The full-length cDNA of HcAIF-1 consisted of a 5′-terminal untranslated region (UTR) of 80 bp, a 3′-UTR of 420 bp with a poly (A) tail, and an open reading frame of 444 bp encoding a polypeptide of 147 amino acids with two conserved EF-hand Ca2+-binding motifs. HcAIF-1 mRNA and protein were expressed in all examined tissues and showed higher mRNA expression levels were observed in immune tissues, especially hemocytes and mantle, and the highest protein expression level was in mantle. The expression level of HcAIF-1 mRNA was significantly upregulated in hemocytes 12–48 h after lipopolysaccharide challenge. After mantle tissue implantation, the expression level of this gene in pearl sac decreased significantly at 3–48 h (P < 0.01), and then was significantly upregulated at 96 h (P < 0.05) and recovered to the control level at 21–28 d. There was significant increase HcAIF-1 transcript abundance in hemocytes 96 h (P < 0.05) after mantle tissue implantation. The phagocytosis rate was significantly enhanced in hemocytes 3–24 h (P < 0.01) after the injection of recombinant HcAIF-1 protein. These findings suggest that HcAIF-1 is important in the underlying mechanism of the innate immune responses and pearl sac formation of H. cumingii.

A galectin from Hyriopsis cumingii involved in the innate immune response against to pathogenic microorganism and its expression profiling during pearl sac formation.

Hyriopsis cumingii is the most important freshwater pearl mussel cultured in China. The operation for implantation is one necessary technical step for pearl culture. However, implantation-induced trauma results in a series of immune responses and can enable the invasion of pathogenic microbes. Lectin proteins are found widely in nature and play important roles in innate immunity. Galectins are members of the lectin superfamily and are characterized by one or several carbohydrate recognition domains (CRDs) that produce multiple sugar binding sites on the protein. Here we cloned and characterized the H.cumingii galectin gene HcGal1, which encodes a 312 amino acid galectin protein. The HcGal1 transcript was detected in all tested H.cumingii tissues and showed higher expression specifically in immune tissues. The significant upregulation of HcGal1 expression was observed after challenging the mussel with lipopolysaccharide or Gram-negative and Gram-positive bacteria. After implantation, significant downregulation of the HcGal1 transcript was noted in the mantle, hemocytes, and pearl sac in the acute-stress stage (0-24h) and the stage of wound healing and pearl-sac formation (24h-7d). In addition, significant upregulation of HcGal1 expression was observed in the liver in the stage of wound healing and pearl-sac formation. In the pearl-secretion stage (7-35d), the HcGal1 transcript levels returned to normal in all tested tissues. We also show that recombinantly expressed and purified HcGal1 can agglutinate some Gram-negative and Gram-positive bacteria. In addition, invivo experiments showed that the recombinant protein HcGal1 could promote phagocytosis by hemocytes. Our data suggest that HcGal1 plays a role in innate immune responses involved in pathogen recognition and wound healing.

Cloning and analysis of gene expression of interleukin-17 homolog in triangle-shell pearl mussel, Hyriopsis cumingii, during pearl sac formation.

Successful allograft of mantle tissues in certain bivalve mollusks can form pearl sacs secreting nacre for pearl production. Little was known, however, about the immune consequences in response to the tissue transplantation. In the present study, interleukin (IL)-17, one of the key regulatory genes of alloimmunity, was cloned from the triangle-shell pearl mussel (HcIL-17) Hyriopsis cumingii by high-throughput sequencing of the mantle transcriptome. The sequence of HcIL-17 contains an open reading frame of 567 bp encoding a putative protein of 188 amino acid residues. Analysis of sequence characteristics, multiple sequence alignment and phylogenetic analysis indicated HcIL-17 was a novel member in the mollusk IL-17 family. Expression of the HcIL-17 gene in donor mantle tissues and in hemocytes of recipient mussel was up-regulated dramatically within 7 days in response to the mantle tissue allograft for pearl aquaculture, suggesting remarkable proinflammatory responses during pearl sac formation in triangle-shell pearl mussels. Analysis of the time-course expression of HcIL-17 gene revealed the induction of HcIL-17 was time-dependent, reflecting the different periods of alloimmune events in triangle-shell mussels. The results of this study provide essential background information for further investigation of mollusk alloimmunity.

The Pearl Sac Formation in Male and Female Pinctada maxima Host Oysters Implanted With Allograft Saibo

An experiment was conducted to study the effect of male and female host oysters on the pearl sac formation in Pinctada maxima oyster. One hundred sixty oysters were used in a completely randomized design with 2 × 4 factorial arrangement and 20 replications. The 1st factor was that sex of host oyster consisted of two levels that is males and females. The 2nd factor was week after nucleus implantation with four levels that is 1, 2, 3, and 4 weeks. The parameters observed were the percentage of successful oysters to form the pearl sac, the speed of pearl sac formation, the percentage of nucleus coverage by the pearl sac, histology of the pearl sac growth and development, and haemolymph glucose, calcium and phosphorus concentrations. Our results showed that the percentages of host oysters that succeeded in forming a pearl sac were 80% and 75% in female and male host oysters, respectively. There was no statistical difference in nucleus rejection and mortality in male and female host oysters but the results indicated that male host oysters showed a numerically higher nucleus rejection. The speed of pearl sac growth and the percentage of nucleus coverage by the pearl sac in female host oysters were better than those in male host oysters. Haemolymph calcium, phosphorus and glucose concentrations, oxygen consumption, and histological development of the pearl sac were not different between male and female host oysters. Pearl sac formation in the female host oysters was better than that in male host oysters.

Development and function of pearl-sacs grown from regenerated mantle graft tissue in the black-lip pearl oyster, Pinctada margaritifera (Linnaeus, 1758)

Current pearl grafting techniques were developed in the early 1900s and have changed little since. They involve the sacrifice of donor pearl oysters to provide graft tissue (saibo) that is implanted into host oysters. This study assessed the feasibility of using regenerated graft tissue for pearl production in the ‘black-lip’ pearl oyster, Pinctada margaritifera. Twelve days after grafting with regenerated graft tissue, there was complete encapsulation of the nucleus by the fully developed pearl-sac and the first layer of organic matrix had been secreted. Sixteen days after grafting, the pearl-sac was completely integrated with host tissue. The epithelial cells in the pearl-sac continued to secrete the organic matrix layer but there were no signs of nacre deposition at this stage. However, after three months of culture, nuclei in oysters grafted with regenerated mantle tissue were completely covered with nacre. The average nacre thickness on pearls produced from regenerated (0.547 ± 0.01 mm, n = 8) and normal (0.532 ± 0.01 mm, n = 8) mantle tissue did not differ significantly (p > 0.05). Nacre secretion rates, over the 80 day period subsequent to pearl-sac formation were 6.84 ± 0.1 μm day−1 and 6.66 ± 0.1 μm day−1 for oysters grafted with regenerated and normal mantle tissue, respectively. These means were not significantly different (p = 0.258). Our results clearly show that regenerated mantle tissue can function successfully as saibo for pearl production in P. margaritifera. This finding could provide significant benefits to pearl farmers and a basis for further development of current pearl grafting practices.