A shell matrix protein of Pinctada mazatlanica produces nacre platelets in vitro.

Nacre is the main component of the pearl oyster shells and it is synthesized by specialized soluble and insoluble shell matrix proteins. Insoluble proteins from the decalcification of the shell are the less studied proteins due to the technical problems to isolate them from the organic matrix. In this study, an insoluble shell matrix protein from Pinctada mazatlanica, pearlin (Pmaz-pearlin), was successfully cloned from the mantle tissue, and the native protein isolated from the shell was functionally characterized. The full coding sequence of Pmaz-pearlin mRNA consists of 423 base pairs, which encode to a 16.3 kDa pearlin. Analysis of the deduced amino acid sequence revealed that Pmaz-pearlin contained four acidic regions, an NG repeat domain, and Cys conserved residues, the latter potentially forms four disulfide bridges which might stabilize the protein structure. The isolated protein from the shell is a glycoprotein of ~ 16.74 kDa which can produce aragonite and calcite crystals in vitro. Our results show that Pmaz-pearlin is a well-conserved protein involved in nacre layer growth, which produces calcite crystals in the presence of CaCl2, aragonite crystal polymorphs with a hexagonal structure in the presence of MgCl2, and needle-like crystal structure polymorphs in the presence of CaCO3 The identity of the crystals was confirmed using RAMAN analyses.

Purification and functional analysis of the shell matrix protein N66 from the shell of the pearl oyster Pteria sterna.

Mollusk biomineralization is a process controlled by a complex interplay of proteins, ions and external regulators. In spite of several studies, there is a lack of knowledge of who (molecules involved), how (mechanism) and why (evolution and adaptation) mollusk are designed as we know them. In this study, a shell matrix protein, N66, has been purified and characterized biochemically from the shell of Pteria sterna. Two protein bands with carbohydrates associated were separated with a molecular weight of ~60 and 64 kDa. It has carbonic anhydrase activity and it is able to form crystal polymorphs of calcium carbonate in vitro. The mRNA N66 was obtained from the mantle tissue of Pteria sterna and the deduced amino acid sequence contained a carbonic anhydrase (CA) domain and a Asn/Gly-rich domain (aa243-439). The CA domain contained three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu166-Thr525), being thus similar to the human isoform hCAVII. Also, to test whether the posttranslational modifications present on the native N66 affects the CA activity and its crystallization capability in vitro, a recombinant N66 was overexpressed in Escherichia coli and functionally characterized. Our results show that recombinant N66 has higher CA activity and produce larger size crystals in vitro than the native N66 protein, suggesting that intrinsic properties of the native N66, such as glycosylations and/or phosphorylations, might regulate its activity.

Molecular cloning and characterisation of scavenger receptor class B in pearl oyster Pinctada fuctada martensii

Background: Molluscs can accumulate carotenoids in their body tissues by predominantly feeding on aquatic plant sources. Carotenoid transport and absorption are determined by the regulation of various proteins such as Scavenger receptor class B(SR-BI). We report the identification and characterisation of pearl oyster Pinctada fuctada martensii SR-BI (PmSR-BI). The correlation between total carotenoid content (TCC) and gene expression was also estimated. Results: The full-length cDNA of PmSR-BI was 1828 bp, including an open-reading frame encoding of 1518 bp with a pI value of 5.83. PmSR-BI protein contains a hydrophobic CD36 domain and four centrally clustered cysteine residues for the arrangement of disulphide bridges. The deduced amino acid sequence had an identity of 30% to 60% with the SR-B of other organisms. Reverse transcription polymerase chain reaction analysis showed that mRNA transcripts were expressed in multiple tissues of adult pearl oyster. A higher expression of PmSR-BI gene was observed in the hepatopancreas than in the adductor muscle, gill and mantle. The TCC and gene expression of PmSR-BI were significantly correlated (P b 0.05), with a correlation coefficient of 0.978. Conclusions: The results suggested that PmSR-BI is involved in the absorption of carotenoids in the pearl oyster P. fuctada martensii.

Pcr-rflp for identification of the pearl oyster Pinctada imbricate from Brazil and Venezuela / Pcr-rflp para identificação de ostras perlíferas (Pinctada imbricate) do Brasil e Venezuela

Oysters of the genus Pinctada are of great economic importance due to their extensive use in human feeding and pearl cultivation. It includes four species: Pinctada radiata (Europe), P. imbricata (Western Atlantic)and the P. fucata-martensi complex (Pacific), the latter being a species complex of difficult morphological differentiation. Although this species complex has several molecular studies corroborating each species as valid, there are still doubts about the validity of P. imbricata in the South of Western Atlantic (ie Brazilian Coast). Here we carried out a RFLP study with populations from Ceará, Rio de Janeiro, São Paulo and Venezuela coast. We analyzed mitochondrial (16S) and nuclear genes (partial IGS). This study confirms the Brazilian and Venezuelan stocks as genetically close to the P. imbricata stocks from Caribbean than the P. martensi-fucata complex. This result is important for pearl-oyster farmers, demonstrating that the Brazilian and Venezuelan stocks are not alien species or hybrids of Indo-Pacific species.

As ostras do género Pinctada são de grande importância económica devido ao seu uso extensivo na alimentação humana e cultivo de pérolas. Inclui quatro espécies: Pinctada radiata (Europa), P. imbricata (Atlântico Ocidental) e P. fucatamartensi (Pacífico), sendo esta última uma espécie complexa de difícil diferenciação morfológica. Embora este complexo de espécies tenha vários estudos moleculares corroborando cada espécie como válida, ainda há dúvidas sobre a validade de P. imbricata no Sul do Atlântico Ocidental. Neste trabalho realializou-se um estudo de RFLP com populações do Ceará, Rio de Janeiro, São Paulo e costa da Venezuela. Foram analisados genes mitocondriais (16S) e nucleares (IGS parcial). Este estudo confirma que os espécimes brasileiros e venezuelanos são geneticamente mais próximos às populações de P. imbricata do Caribe do que o complexo P. martensi-fucata. Esse resultado é importante para os produtores de ostras perlíferas, demonstrando que os estoques brasileiro e venezuelano não são espécies exóticas ou híbridas de espécies indo-pacíficas.

Pcr-rflp for identification of the pearl oyster Pinctada imbricate from Brazil and Venezuela / Pcr-rflp para identificação de ostras perlíferas (Pinctada imbricate) do Brasil e Venezuela

Oysters of the genus Pinctada are of great economic importance due to their extensive use in human feeding and pearl cultivation. It includes four species: Pinctada radiata (Europe), P. imbricata (Western Atlantic)and the P. fucata-martensi complex (Pacific), the latter being a species complex of difficult morphological differentiation. Although this species complex has several molecular studies corroborating each species as valid, there are still doubts about the validity of P. imbricata in the South of Western Atlantic (ie Brazilian Coast). Here we carried out a RFLP study with populations from Ceará, Rio de Janeiro, São Paulo and Venezuela coast. We analyzed mitochondrial (16S) and nuclear genes (partial IGS). This study confirms the Brazilian and Venezuelan stocks as genetically close to the P. imbricata stocks from Caribbean than the P. martensi-fucata complex. This result is important for pearl-oyster farmers, demonstrating that the Brazilian and Venezuelan stocks are not alien species or hybrids of Indo-Pacific species.(AU)

As ostras do género Pinctada são de grande importância económica devido ao seu uso extensivo na alimentação humana e cultivo de pérolas. Inclui quatro espécies: Pinctada radiata (Europa), P. imbricata (Atlântico Ocidental) e P. fucatamartensi (Pacífico), sendo esta última uma espécie complexa de difícil diferenciação morfológica. Embora este complexo de espécies tenha vários estudos moleculares corroborando cada espécie como válida, ainda há dúvidas sobre a validade de P. imbricata no Sul do Atlântico Ocidental. Neste trabalho realializou-se um estudo de RFLP com populações do Ceará, Rio de Janeiro, São Paulo e costa da Venezuela. Foram analisados genes mitocondriais (16S) e nucleares (IGS parcial). Este estudo confirma que os espécimes brasileiros e venezuelanos são geneticamente mais próximos às populações de P. imbricata do Caribe do que o complexo P. martensi-fucata. Esse resultado é importante para os produtores de ostras perlíferas, demonstrando que os estoques brasileiro e venezuelano não são espécies exóticas ou híbridas de espécies indo-pacíficas.(AU)

Molecular characterization of CHST11 and its potential role in nacre formation in pearl oyster Pinctada fucata martensii

Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.

Development of SSR marker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii

Background: Pearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation where a mantle tissue graft from a donor oyster and a round nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contributes to the formation of a pearl sac that secretes pearl nacre to form a pearl should be determined. In April 2012, two full-sib families were separately used as donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed using Illumina HiSeq™ 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster, and pearl sac. Results: A total of 3168 microsatellite loci were identified in 39,078 unigenes, and 1977 SSR primers were designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotype pearl sac, donor oyster, and host oyster. Donor and host oysters were both homogenous, and the alleles in pearl sac were identical to those in donor and host oysters. Conclusions: The present results confirmed that the mantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii.

Development of coding single nucleotide polymorphic markers in the pearl oyster Pinctada fucata based on next-generation sequencing and high-resolution melting analysis.

The pearl oyster Pinctada fucata is an important commercial marine shellfish that is cultured for producing saltwater pearls. In this study, 468 single nucleotide polymorphisms (SNPs) were screened from P. fucata transcriptome data, and 119 polymorphic SNPs were successfully isolated by a two-step small-amplicon high-resolution melting assay. Of these, 88 were annotated with BLAST in the Nr database and 90 were in the open reading frame, including 16 non-synonymous SNPs and 74 synonymous SNPs; 12 SNPs were in the 3'-untranslated region (UTR) and 1 was in the 5'-UTR. Twenty-five SNPs were randomly chosen to test the genetic diversity of 40 wild individuals from Liusha Bay, China. All of the loci had two alleles. The observed and expected heterozygosities ranged from 0.0417 to 0.6042 and from 0.2945 to 0.5053, respectively. Minor allele frequencies ranged from 0.1771 to 0.5000, and the polymorphism information content ranged from 0.2516 to 0.3750. These novel SNP markers can contribute to P. fucata genetics and breeding studies.

Identification of 15 novel polymorphic microsatellite loci in pearl oyster (Pinctada fucata).

The pearl oyster Pinctada fucata is a commercially important marine shellfish. As a result, genetic improvement and selective-breeding program have been conducted for this species. Polymorphic microsatellites are effective molecular markers to investigate molecular marker-assisted selection and genetic variance. In this study, microsatellite DNAs were screened and characterized based on the partial genome sequence of P. fucata. We identified 111 microsatellite DNA motifs through mining the published draft genome sequence of P. fucata. Forty-two loci were screened with 8 P. fucata individuals, and 15 were found to be polymorphic and were therefore further evaluated using 40 wild individuals from the Daya Bay, Shenzhen City, Guangdong Province, China. The number of alleles per locus ranged from 3 to 8, with an average of 5.2667 for the 15 polymorphic loci. Observed and expected heterozygosities ranged from 0.1154 to 0.6216 (0.3321 on average) and 0.4950 to 0.8491 (0.6768 on average), respectively. Of the 15 polymorphic loci, 12 loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0033). Polymorphism information content ranged from 0.44 to 0.83 with a mean value of 0.63. The results suggest that the markers isolated in this study can be used for research on molecular marker-assisted selection and genetic variance of P. fucata.

Molecular cloning and expression analysis of heat shock protein 20 (HSP20) from the pearl oyster Pinctada martensii.

Small heat shock proteins (HSPs) are molecular chaperones with ATP-independent properties. They are involved in a variety of physiological and stress processes. In this study, the full-length HSP 20 (HSP20) from Pinctada martensii, designated as PmHSP20, was obtained from hemocytes using rapid amplification of cDNA ends technology. The PmHSP20 cDNA was 952 bp in length, containing an open reading frame of 534 bp that encoded 177-amino acid residues, with an isoelectric point of 5.86 and molecular weight of 20.24 kDa. The sequence of this deduced polypeptide contained typical structure and function domains conserved in the HSP20 family, providing evidence that PmHSP20 belongs to the HSP20 family. The PmHSP20 mRNA expression levels were detected in various tissues of P. martensii and in hemocytes after challenges with the bacteria Vibrio harveyi and lipopolysaccharide (LPS) using quantitative real-time polymerase chain reaction amplification. The results indicated that PmHSP20 is constitutively expressed in all tissues tested and might be involved in the immune response. The upregulation of PmHSP20 after V. harveyi and LPS challenge suggests that PmHSP20 plays an important role in anti-bacterial immunity. Studies on PmHSP20 are a valuable resource to further explore the immune system in pearl oysters and might enhance our knowledge of molluscan innate immunity.

Histoquímica de la glándula digestiva en la ostra perla Pinctada imbricata (Pterioida: Pteriidae) durante su ciclo gametogénico, Venezuela / Histochemistry of the digestive gland of the pearl oyster Pinctada imbricata (Pterioida: Pteriidae) during the gametogenic cycle, Venezuela

ResumenLas técnicas histoquímicas hoy en día permiten seleccionar áreas de tejido y generar información confiable sobre la distribución de reservas energéticas en los moluscos bivalvos durante su ciclo de vida. Mensualmente se examinaron las gónadas y la glándula digestiva (GD) de 15 individuos recolectados entre abril 2012 y febrero 2013 por técnicas histológicas e histoquímicas de microscopia de luz, para relacionar el ciclo gametogénico y la disponibilidad de reservas energéticas con los parámetros ambientales. En el ciclo gametogénico, la proporción mensual de organismos maduros fue mayor en los machos entre agosto (40 %) y noviembre (53 %), mientras que las hembras tienden a presentar un ciclo más corto y sincronizado de liberación de gametos (septiembre 67 % y octubre 60 %). Los períodos intensos de desoves coinciden en ambos sexos (octubre-enero). Entre abril-agosto 2012 y enero-febrero 2013, se observan los valores más altos del IGl (índice de glúcido), mientras que en septiembre disminuyen y alcanzaron valores mínimos entre octubre y diciembre. El IL (índice de lípidos) presentó valores máximos en abril-2012 y febrero-2013, con un valor intermedio en agosto. Los resultados indican que las reservas de la GD presentan un patrón de movilización en relación inversa con la proliferación de los gametos de ambos sexos, vinculado directamente con la disponibilidad de nutrientes como la clorofila a y el seston orgánico.

AbstractHistochemical techniques today allow you to select areas of tissue and generate reliable information on the distribution of energy reserves in bivalve molluscs during their life cycle. The main objective of this study was to describe and relate the gametogenic cycle with the availability of energy reserves and the environmental parameters. For this, we sampled and examined the gonads and digestive glands (DG) of 15 individuals collected monthly during April 2012 and February 2013. We processed and analyzed the samples by standard histological and histochemical light microscopy techniques. Our results showed that for the gametogenic cycle, the monthly proportion of mature organisms was higher for males, between August (40 %) and November (53 %), while the females tend to have a shorter synchronized cycle and release of gametes in September (67 %) and October (60 %). The intense spawning periods in both sexes was the same (October to January). Between the periods April-August 2012 and January-February 2013, we observed the highest values of IGl and glucide index (instead, a decrease was observed in September, reaching minimum values during the period October-December). Besides, the maximum values of IL, lipid index, were observed in April-2012 and February-2013, with an intermediate value in August-2013. The results indicated that the reserves of the GD have a pattern of mobilization inversely related to the proliferation of gametes in both sexes; this was directly linked to the availability of nutrients such as chlorophyll a and the organic seston. Rev. Biol. Trop. 64 (2): 849-858. Epub 2016 June 01.

[Histochemistry of the digestive gland of the pearl oyster Pinctada imbricata (Pterioida: Pteriidae) during the gametogenic cycle, Venezuela]. / Histoquímica de la glándula digestiva en la ostra perla Pinctada imbricate (Pterioida: Pteriidae) durante su ciclo gametogénico, Venezuela.

Histochemical techniques today allow you to select areas of tissue and generate reliable information on the distribution of energy reserves in bivalve molluscs during their life cycle. The main objective of this study was to describe and relate the gametogenic cycle with the availability of energy reserves and the environmental parameters. For this, we sampled and examined the gonads and digestive glands (DG) of 15 individuals collected monthly during April 2012 and February 2013. We processed and analyzed the samples by standard histological and histochemical light microscopy techniques. Our results showed that for the gametogenic cycle, the monthly proportion of mature organisms was higher for males, between August (40 %) and November (53 %), while the females tend to have a shorter synchronized cycle and release of gametes in September (67 %) and October (60 %). The intense spawning periods in both sexes was the same (October to January). Between the periods April-August 2012 and January-February 2013, we observed the highest values of IGl and glucide index (instead, a decrease was observed in September, reaching minimum values during the period October-December). Besides, the maximum values of IL, lipid index, were observed in April-2012 and February-2013, with an intermediate value in August-2013. The results indicated that the reserves of the GD have a pattern of mobilization inversely related to the proliferation of gametes in both sexes; this was directly linked to the availability of nutrients such as chlorophyll a and the organic seston.

Anatomical differences among specimens of Pinctada imbricata Rõding, 1798 from different South American localities / Diferenças anatômicas entre espécimes de Pinctada imbricata Rõding, 1798 de diferentes localidades da América do Sul

In the present study, we compared the internal and external anatomy of pearl oyster (Pinctada imbricata Rõding, 1798) from some South American localities, including Venezuela and several regions of Brazil. The anatomical data shows non-geographical variations characterized by variable outline in adults, different degrees of posterior auricle shell development and mantle papillae ranging from normal to digitiform. The geographical variations can be divided into two major groups: the Venezuelan group presents a more folded mantle; pallial muscle grouped in anterior and median-posterior sets; poorly-developed perpendicular mantle groove; inner lamella not extended into the anterior mantle. Brazilian group specimens present a smoother mantle, with less clear separation between anterior and posterior mantle muscle groups; externally-developed perpendicular groove; inner lamella extension developed in anterior mantle. These differences may represent ecophenotypes or differences among isolated South American populations, which may be elucidated by subsequent phylogeographic studies.

No presente estudo comparou-se a anatomia da ostra perlífera (Pinctada imbricata Rõding, 1798) de algumas localidades da América do Sul, incluindo a Venezuela e várias regiões do Brasil. Foram encontradas variações não geográficas e geográficas. As variações não geográficas foram caracterizadas por adultos com contorno bem variável, diferentes graus de desenvolvimento da aurícula posterior e papilas da lamela interna do manto variando de normal a digitiformes. Variações geográficas podem ser divididas em dois grandes grupos: o grupo venezuelano apresentou um manto mais pregueado; musculatura palial agrupada em dois conjuntos de feixes um anterior e outro mediano-posterior; sulco perpendicular do manto pouco desenvolvido. Espécimes dos grupos brasileiros apresentaram um manto menos pregueado, com separação menos nítida entre os feixes musculares paliais anterior e posterior do manto; sulco perpendicular do manto bem desenvolvido; lamela interna do manto se estendendo externamente na região do manto anterior. Essas diferenças podem ser ecofenótipos ou diferenças entre as populações com certo grau de isolamento da América do Sul, que podem ser elucidados por estudos filogeográficos subsequentes.

Anatomical differences among specimens of Pinctada imbricata Rõding, 1798 from different South American localities / Diferenças anatômicas entre espécimes de Pinctada imbricata Rõding, 1798 de diferentes localidades da América do Sul

In the present study, we compared the internal and external anatomy of pearl oyster (Pinctada imbricata Rõding, 1798) from some South American localities, including Venezuela and several regions of Brazil. The anatomical data shows non-geographical variations characterized by variable outline in adults, different degrees of posterior auricle shell development and mantle papillae ranging from normal to digitiform. The geographical variations can be divided into two major groups: the Venezuelan group presents a more folded mantle; pallial muscle grouped in anterior and median-posterior sets; poorly-developed perpendicular mantle groove; inner lamella not extended into the anterior mantle. Brazilian group specimens present a smoother mantle, with less clear separation between anterior and posterior mantle muscle groups; externally-developed perpendicular groove; inner lamella extension developed in anterior mantle. These differences may represent ecophenotypes or differences among isolated South American populations, which may be elucidated by subsequent phylogeographic studies.(AU)

No presente estudo comparou-se a anatomia da ostra perlífera (Pinctada imbricata Rõding, 1798) de algumas localidades da América do Sul, incluindo a Venezuela e várias regiões do Brasil. Foram encontradas variações não geográficas e geográficas. As variações não geográficas foram caracterizadas por adultos com contorno bem variável, diferentes graus de desenvolvimento da aurícula posterior e papilas da lamela interna do manto variando de normal a digitiformes. Variações geográficas podem ser divididas em dois grandes grupos: o grupo venezuelano apresentou um manto mais pregueado; musculatura palial agrupada em dois conjuntos de feixes um anterior e outro mediano-posterior; sulco perpendicular do manto pouco desenvolvido. Espécimes dos grupos brasileiros apresentaram um manto menos pregueado, com separação menos nítida entre os feixes musculares paliais anterior e posterior do manto; sulco perpendicular do manto bem desenvolvido; lamela interna do manto se estendendo externamente na região do manto anterior. Essas diferenças podem ser ecofenótipos ou diferenças entre as populações com certo grau de isolamento da América do Sul, que podem ser elucidados por estudos filogeográficos subsequentes.(AU)

Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii.

Purple acid phosphatases (PAPs), also known as type 5 acid phosphatases, are widely present in animals, plants, and fungi. In mammal, PAP was reported to participate in immune defense and bone resorption. In this study, the characteristics and potential functions of a PAP gene from pearl oyster Pinctada martensii (pm-PAP) were examined. The Pm-PAP cDNA was found to be 2777 base pairs, containing a 1581-base pair open reading fragment encoding for 526 amino acids with an estimated molecular mass of 60.1 kDa and theoretical isoelectric point of 5.82. One signal peptide and five conserved motifs [GDXX/GDXXY/GNH(D/E)/XXXH/(A/G)HXH] were present in the entire sequence. Tissue expression profile analysis showed that pm-PAP mRNA was constitutively expressed in all tissues studied with abundant mRNA found in mollusk defense system, including hepatopancreas, gill, and hemocytes. After lipopolysaccharide stimulation, the expression of pm-PAP mRNA in hemocytes was dramatically upregulated at 2 h and achieved the highest level at 36 h. Additionally, pm-PAP mRNA expression was significantly increased and achieved the highest level at 2 days after the surgical implantation during pearl production. These results suggest that pm-PAP is a constitutive and inducible protein that may be involved in the immune defense of pearl oyster.

Molecular cloning and expression analysis of a pearl oyster (Pinctada martensii) heat shock protein 90 (HSP90).

Heat shock protein 90 (HSP90) is an important molecular chaperone required for proper folding of cellular proteins, and thus, it plays an essential role in protecting cells from damage during stress. In this study, an HSP90 cDNA designated PmHSP90 was cloned from the mantle tissue of the pearl oyster Pinctada martensii using reverse transcription polymerase chain reaction (RT-PCR) coupled with the rapid amplification of cDNA ends (RACE) approach. PmHSP90 cDNA was 2584 bp in length, including an open reading frame of 2160 bp, which encodes a polypeptide of 719 amino acid residues, with predicted molecular mass and isoelectric point of 83.0 kDa and 4.87, respectively. Multiple-sequence alignment indicated that HSP90 is highly conserved among species, and PmHSP90 showed 89% sequence identity to Crassostrea gigas HSP90. Five conserved amino acid blocks defined as HSP90 protein family signatures were also observed in PmHSP90, indicating that PmHSP90 may be a cytosolic member of the HSP90 family. Expression levels of PmHSP90 were detected in various tissues of P. martensii and in hemocytes under three different stress conditions using quantitative real-time PCR (qPCR). The results demonstrate that PmHSP90 mRNA is constitutively expressed in all the tested tissues and may be involved in the immune response against thermal stress, lipopolysaccharide stimulation, and nucleus insertion operations. Studies on PmHSP90 are a valuable source to further explore the immune system in pearl oysters during the production of pearls, and may enhance our knowledge of molluscan innate immunity.

Molecular characterization of tumor necrosis factor receptor-associated factor 6 (TRAF6) in pearl oyster Pinctada martensii.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key signaling adaptor molecule for tumor necrosis factor receptor superfamily and Toll-like receptor/interleukin-1 receptor family members. It signals the upstream receptors and is involved in a wide range of biological functions, such as immunity and bone metabolism. In this report, the TRAF6 gene from the pearl oyster Pinctada martensii (designated as PmTRAF6) was identified and characterized. The obtained full-length PmTRAF6 cDNA was 2273 bp, containing a 5'-untranslated region (UTR) of 297 bp, a 3'-UTR of 128 bp with a 42-bp poly (A) tail, and an open reading frame of 1848 bp that encoded 616-amino acid residues. The deduced protein sequence of PmTRAF6 contained a conserved TRAF family motif including a RING-type zinc finger, two TRAF-type zinc fingers, and a coiled-coil region followed by one meprin and TRAF homology domain. Multiple-sequence alignment indicated that TRAF6 was highly conserved among species, and PmTRAF6 showed 53% sequence identity to Azumapecten farreri and Mizuhopecten yessoensis. Furthermore, an amino acid sequence containing a low-complexity region was inserted in the TRAF6s from mollusk. Quantitative real-time polymerase chain reaction analysis demonstrated that PmTRAF6 was constitutively expressed in all tissues studied, with the most abundant mRNA expression in hepatopancreas and gill in P. martensii. After lipopolysaccharide stimulation, the expression of PmTRAF6 mRNA was dramatically upregulated. These results suggested that the obtained PmTRAF6 was a member of the TRAF6 family and perhaps involved in the innate immune response of pearl oyster.

Efficacy of calcein and Coomassie Blue dyeing of shell growing-edges and micro growth-bands: ageing juvenile of Pinctada mazatlanica (Pterioida: Pteriidae).

Age validation is the first step to determine shellfish species age determination. This information is vital for different inferential models used in marine ecosystem management activities. In spite that various validation techniques are used for marking carbon calcium structures, the calcein marking technique for oysters had never been used for age validation in Pinctada mazatlanica. Thus the objectives of this study included: the evaluation of calcein to mark a shell growing-edge, and the efficacy of Coomassie Blue staining on posterior shell growth, to produce visible micro growth-bands that would enable age validation of juvenile mother-of-pearl oysters. Oysters were collected and cultivated at The Perlas del Cortez S. de R. L. MI. pearl-farming operation, in Pichilingue, La Paz Bay, Baja California Sur, Mexico; a total of 36 oysters (shell height 11.5-36.4 mm) were injected with calcein (0.125 g/L), and another 50 oysters (shell height 14.8-42.7 mm) were submersed in calcein (0.4 and 0.7 g/L). Shell slices of calcein-marked oysters were posteriourly stained with Coomassie Blue R-25 for micro growth-band recognition. Our results showed that Calcein marking only worked by submersion and produced a concise bright lime-green florescent band along the growing-edge with clear boundaries for both concentrations. However, marks resulted better at the lower calcein concentration (0.4 g/L) with more "perfect" and "good" marks on the growing-edge (p = 0.0012). Commassie Blue staining technique was successful, and allowed to conclude that one micro growth-band was laid down per day, similar to other oyster species. Mean 15-d increment of shell growth height was slightly greater at the lower calcein concentration (= 0.735 mm) than at the higher one (= 0.577 mm) (not significant difference, p = 0.198). Calcein marking of shell growing-edges and Commassie Blue staining of posterior shell growth, as a method for age validation is recommended for shellfish shell growth-band counts. This will allow back-dating for estimation of very precise colonization dates, both spatially and temporally in future work.

Efficacy of calcein and Coomassie Blue dyeing of shell growing-edges and micro growth-bands: Ageing juvenile of Pinctada mazatlanica (Pterioida: Pteriidae) / Eficiencia de la tinción de bandas de crecimiento con calceina y azul de coomassie, para estimar la edad de jóvenes de Pinctada mazatlanica (Pterioida: Pteriidae)

Age validation is the first step to determine shellfish species age determination. This information is vital for different inferential models used in marine ecosystem management activities. In spite that various validation techniques are used for marking carbon calcium structures, the calcein marking technique for oysters had never been used for age validation in Pinctada mazatlanica. Thus the objectives of this study included: the evaluation of calcein to mark a shell growing-edge, and the efficacy of Coomassie Blue staining on posterior shell growth, to produce visible micro growth-bands that would enable age validation of juvenile mother-of- pearl oysters. Oysters were collected and cultivated at The Perlas del Cortez S. de R. L. MI. pearl-farming opera tion, in Pichilingue, La Paz Bay, Baja California Sur, Mexico; a total of 36 oysters (shell height 11.5-36.4mm) were injected with calcein (0.125g/L), and another 50 oysters (shell height 14.8-42.7mm) were submersed in calcein (0.4 and 0.7g/L). Shell slices of calcein-marked oysters were posteriourly stained with Coomassie Blue R-25 for micro growth-band recognition. Our results showed that Calcein marking only worked by submersion and produced a concise bright lime-green florescent band along the growing-edge with clear boundaries for both concentrations. However, marks resulted better at the lower calcein concentration (0.4g/L) with more “perfect” and “good” marks on the growing-edge (p=0.0012). Commassie Blue staining technique was successful, and allowed to conclude that one micro growth-band was laid down per day, similar to other oyster species. Mean 15-d increment of shell growth height was slightly greater at the lower calcein concentration ( =0.735mm) than at the higher one ( =0.577mm) (not significant difference, p=0.198). Calcein marking of shell growing- edges and Commassie Blue staining of posterior shell growth, as a method for age validation is recommended for shellfish shell growth-band counts. This will allow back-dating for estimation of very precise colonization dates, both spatially and temporally in future work.

La validación de la edad es el primer paso para determinar las edades de las especies de moluscos, esta información es de vital importancia para los diferentes modelos de inferencia utilizados en actividades de gestión de los ecosistemas marinos. Diversas técnicas de valida- ción se utilizan para marcar estructuras de carbonato de calcio, aunque la técnica de marcado de calceína en ostras nunca se había utilizado para la validación de la edad de P. mazatlanica. Los objetivos de este estudio fueron: evaluar la calceína como marcador interno de la concha y la eficiencia del azul de Coomassie en la tinción de la matriz proteica de la concha, para facilitar la observación y conteo de micro bandas de crecimiento que permiten validar la edad de las ostras juveniles de madre perla. Las ostras fue- ron recolectadas en la costa de la empresa Perlas del Cortez S. de RL MI., en Pichilingue en Bahía de La Paz, Baja California Sur, México. Se inyectaron 36 ostras (altura de concha 11.5-36.4mm) (0.125g/L de calceína) y otras 50 ostras (altura de la concha 14.8-42.7mm) se sumergieron (0.4 y 0.7g/L de calceína). Secciones de la concha marcadas con calceína fueron teñidos con azul de Coomassie R-25 para el reconocimiento de las micro bandas de crecimiento. El marcado con calceína fue exitoso por inmersión y produjo una banda fluorescente de color verde lima brillante con- cisa a lo largo del crecimiento interno de la concha. Sin embargo, las marcas fueron mejores a una concentración de calceína inferior (0.4g/L), con mayor cantidad de marcas “buenas” y “perfectas” (p=0.0012). La técnica de tinción con azul de Commassie también fue exitosa. Se detectó un crecimiento diario por micro banda, similar a lo encontrado en otras especies de ostras. La diferencia del crecimiento medio en relación a la altura de la concha en un lapso de 15 días, fue ligeramente mayor con una concentración de calceína inferior ( =0.735mm) que con la de mayor concentración ( =0.577mm), pero no significativamente (p=0.198). El marcado de conchas con calceína y tinción de matrices proteicas con azul de Coomassie posterior a su crecimiento, es recomendando como un método para la validación de la edad facilitando el conteo de micro bandas de crecimiento internas de la concha. Además, permitirá estimar edades con el fin de predecir fechas de colonización y ubicación de bancos naturales.

Comparative dermatology: blue nevus.

There are elements in nature that may be compared to some dermatological lesions, such as the black pearl, which is very similar to a cellular blue nevus observed in the gluteus region of a 31-year-old male patient.