Cytoskeletal polarization and cytokinetic signaling drives polar lobe formation in spiralian embryos.

In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division.

A C-type lectin fold gene from Japanese scallop Mizuhopecten yessoensis, involved with immunity and metamorphosis.

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins that are well known for their participation in pathogen recognition and clearance. In this study, a putative C-type lectin fold (MyCLF) gene was identified from the Japanese scallop Mizuhopecten yessoensis. The full-length of MyCLF was 645 bp, encoding a polypeptide of 167 amino acids. MyCLF carried a signal peptide of 20 amino acid residues, and a single carbohydrate recognition domain, having relatively high amino acid sequence conservation with C-type lectins reported for other bivalves. The expression of MyCLF mRNA transcripts in adult tissues, after bacterial challenge and during different developmental stages was determined using real-time quantitative RT-PCR. MyCLF was mainly distributed in the mantle, gill, and kidney. The expression of MyCLF clearly increased 3 h after Vibrio anguillarum challenge, and dropped to a minimum level after 9 h compared to the control group. During embryonic development, the expression level increased in the gastrulae, trochophore and early D-shaped larvae, decreased in D-shaped larvae, and then increased hundreds of times in metamorphosing larvae. The results suggested that MyCLF was involved in an immune response and it may play important roles during the metamorphosis phase of M. yessoensis.

Functional characterization of a Δ5-like fatty acyl desaturase and its expression during early embryogenesis in the noble scallop Chlamys nobilis Reeve.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in lots of important physiological processes, while, many marine species have no or limited ability of endogenous PUFA biosynthesis, normally due to the lack of key enzymes such as fatty acid desaturase (FAD). In this study, we isolated a scallop Chlamys nobilis cDNA with high homology to vertebrate FADs. Functional characterization in recombinant yeast Saccharomyces cerevisiae showed that scallop FAD exhibited Δ5-desaturation activity towards both saturated and PUFA substrates. Thus, it efficiently desaturated exogenously added PUFA C20:4(n - 3) and C20:3(n - 6) to C20:5(n - 3) (EPA) and C20:4(n - 6) (ARA) respectively. It also converted the yeast's endogenous C18:0 into C18:1(n - 13), and participated in the biosynthesis of non-methylene-interrupted FA by introducing a double bond to C20:3(n - 3) and C20:2(n - 6) in the Δ5 carbon. Temporal transcript profile of scallop FAD was studied during early embryonic development. High level of mRNA was found at the beginning of embryogenesis (egg) and noticeable decreases of were observed during larvae development, suggesting maternal FAD mRNA transfer to the embryo. Further, FAD transcripts were detected in all tissues analyzed, with the gonad and hepatopancreas showing the highest expression.

Expression profiling of the Kdm genes in scallop Patinopecten yessoensis suggests involvement of histone demethylation in regulation of early development and gametogenesis.

Histone demethylation modification is an important means of gene expression regulation and is widely involved in biological processes such as animal reproduction and development. Histone lysine demethylases (Kdm) plays an important role in the demethylation of histones. To understand the role of histone demethylation in scallops, we identified the Kdm gene family of the Yesso scallop Patinopecten yessoensis, and analyzed its expression during the gonad and early development. The results showed that the P. yessoensis has a complete Kdm family including seventeen members that belong to sixteen subfamilies (Hif1an, Hspbap1, Jarid2, Jmjd4, Jmjd6, Jmjd7, Jmjd8, Kdm1, Kdm2, Kdm3, Kdm4, Kdm5, Kdm6, Kdm7, Kdm8 and Kdm9). The Kdm genes showed five different expression patterns in the early development of scallop, with some of them (e.g. Jmjd7, Jmjd8 and Kdm8) being highly expressed in only one or two stage and the others (e.g. Kdm1A, Kdm9, Jmjd4 and Jmjd6) in several consecutive stages. During gonadal development, the Kdm genes also display various expression patterns. Some genes (e.g. Kdm2, Kdm4 and Jmjd7) display preferential expression in the testis, and the others have no obvious sex bias but show stage preference (resting, proliferative, growing or maturation stage). These results suggest that various histone demethylation modifications (e.g. H3K4, H3K9 and H3K27) may participate in the regulation of gametogenesis and early development of Yesso scallop. It will facilitate a better understanding of the epigenetic contributions to mollusk development.

Developmental dynamics of myogenesis in Yesso Scallop Patinopecten yessoensis.

The development of bivalves has been extensively studied over the last 150 years. Despite this, the developmental dynamics of myogenesis in bivalves remains largely unknown, particularly at the early developmental stages. In the present study, we investigate the characteristics of muscle development of Yesso scallop Patinopecten yessoensis by phalloidin staining, light, electron and confocal microscopy. Myoblasts containing chaotic myofilaments are initially found in the blastocoel of trochophore, and become more organized during the transformation from trochophore into veliger. This is followed by a well-structured musculature including an anterior adductor, velum retractors and ventral retractors at the early veliger stage. With development into late veliger, larval muscle system is composed of the branched velum retractors and ventral retractors, anterior and posterior adductors. The most striking change for pediveliger is the development of foot retractor and mantle related muscles at this stage. During metamorphosis, the retractor muscles and anterior adductor undergo the irreversible shrink until vanishing completely towards the end of larval life, which coincide with the growth of foot retractor and mantle margin. All retractor muscles are found to be composed of striated fibres, whereas the adductor muscles have both smooth and striated components. The present study provides new evidences on the dynamic pattern of myogenesis during embryonic and larval development in scallops, which will greatly improve our understanding of scallop myogenesis and provide the basis for comparative analysis of muscle development in bivalves.

Dynamics of DNA Methylation and DNMT Expression During Gametogenesis and Early Development of Scallop Patinopecten yessoensis.

DNA methylation reprograms during gametogenesis and embryo development, which is essential for germ cell specification and genomic imprinting in mammals. Corresponding process remains poorly investigated in molluscs. Here, we examined global DNA methylation level in the gonads of scallop Patinopecten yessoensis during gametogenesis and in embryos/larvae at different stages. DNA methylation level fluctuates during gametogenesis and early development, peaking at proliferative stage of ovary, growing stage of testis, and in blastulae. To understand the mechanisms underlying these changes, we conducted genome-wide characterization of DNMT family and investigated their expression profiles based on transcriptomes and in situ hybridization. Three genes were identified, namely PyDNMT1, PyDNMT2, and PyDNMT3. Expression of PyDnmt3 agrees with DNA methylation level during oogenesis and early development, suggesting PyDNMT3 may participate in de novo DNA methylation that occurs mainly at proliferative stage of ovary and testis, and in blastulae and gastrulae. PyDnmt1 expression is positively correlated with DNA methylation level during spermatogenesis, and is higher at maturation stage of ovary and in 2-8 cell embryos than other stages, implying possible involvement of PyDNMT1 in DNA methylation maintenance during meiosis and embryonic development. This study will facilitate better understanding of the developmental epigenetic reprogramming in bivalve molluscs.

Two novel LRR-only proteins in Chlamys farreri: Similar in structure, yet different in expression profile and pattern recognition.

Leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and be involved in the immune response. In the present study, two novel LRR-only proteins, CfLRRop-2 and CfLRRop-3, were identified and characterized from scallop Chlamys farreri. They both contained nine LRR motifs with the consensus signature sequence LxxLxLxxNxL and formed typical horseshoe structure. The CfLRRop-2 and CfLRRop-3 mRNA transcripts were constitutively expressed in haemocytes, muscle, mantle, gill, haepatopancreas and gonad, with the highest expression level in haepatopancreas and gill, respectively. During the ontogenesis of scallop, the mRNA transcripts of CfLRRop-2 were kept at a high level in oocytes and embryos, while those of CfLRRop-3 were expressed at a rather low level from oocytes to blastula. Their mRNA transcripts were significantly increased after the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), and the mRNA expression of CfLRRop-2 rose more intensely than that of CfLRRop-3. After the suppression of CfTLR (previously identified Toll-like receptor in C. farreri) via RNA interference (RNAi), CfLRRop-3 mRNA transcripts increased more intensely and lastingly than those of CfLRRop-2. The rCfLRRop-3 protein could bind LPS, PGN, GLU and poly I:C, while rCfLRRop-2 exhibited no significant binding activity to them. Additionally, rCfLRRop-2 could significantly induce the release of TNF-α from the mixed primary cultured scallop haemocytes, but rCfLRRop-3 failed. These results collectively indicated that CfLRRop-2 might act as an immune effector or pro-inflammatory factor, while CfLRRop-3 would function as a pattern recognition receptor (PRR), suggesting the function of LRR-only protein family has differentiated in scallop.

The arginine kinase in Zhikong scallop Chlamys farreri is involved in immunomodulation.

Arginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallop AK gene was identified from Chlamys farreri with an open reading frame (ORF) of 1101bp encoding for a protein of 366 amino acids (designed as CfAK). An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain (GS domain) and an ATP-gua Ptrans domian which was responsible for binding ATP, were both identified in CfAK. The mRNA transcripts of CfAK were detectable in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in the muscle and the lowest level in the hemocytes. The expression level of CfAK mRNA increased from fertilized eggs to eyebot, and reached the highest in the trochophore stage. The relative expression level of CfAK mRNA in muscle was up-regulated significantly after LPS (0.5mg/mL) stimulation, and reached the peak at 6h (5.2-fold, P<0.05). The activity of inducible nitric oxide synthase (iNOS) in the supernatant of muscle homogenate increased significantly from 3.2U/mg at 0 h to 9.7 U/mg at 12h after LPS stimulation, while the concentration of nitric oxide (NO) in the supernatant of muscle homogenate began to increase at 3h (21.55 µmol/L), and reached the top concentration at 24h (42.27 µmol/L), then recovered to the normal level after 48 h. The recombinant protein of CfAK (rCfAK) expressed in Escherichia coli displayed Arginine kinase activity, and its apparent K(m) was 0.82 ± 0.11 and 1.24 ± 0.13 mM for L-arginine and ATP-Na, respectively. The results indicated that the CfAK was involved in energy production and utilization during the whole life process, and might refer to the immunomodulation process via altering the NO concentration and iNOS activity in scallop Chlamys farreri.