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Scapharca broughtonii is one of the most important Arcidae aquaculture species in the Asia-Pacific region. We aimed to investigate the immune responses of hemocytes from ark shell S. broughtonii hemolymph against pathogens. Hemocyte ultrastructure and immunological activity in response to Vibrio anguillarum challenge were observed by scanning and transmission electron microscopy. Before ultrastructure observation, we used the API ZYM semi-quantitative kit to evaluate the levels of hydrolytic enzymes in the plasma and hemocytes following V. anguillarum infection. An enzyme-linked immunosorbent assay kit was used to investigate the variation in the lysozyme activity and hemocytes following bacterial infection. The results showed that hemocytes were the main defense cells against bacterial infection, whereas plasma played a role in the transport and support of hemocytes. It was presumed that an important function of lysozymes and hydrolytic enzymes in lysosomes was for bacterial digestion. Three major types of hemocytes were observed, namely, red blood cells (RBCs), white blood cells (WBCs), and thrombocytes (TCs). Scanning electron microscopy showed that the normal RBCs appeared pie-shaped with 10 μm diameter and 4 μm central thickness, whereas WBCs were spherical in shape with varying sizes, 4–8 μm diameter, and included small lymphocytes. TCs were long, spindle-shaped, and 12–20 μm in length. The cell membrane surface was smooth and even for all cells before pathogen challenge. Under transmission electron microscopy, RBCs displayed a limited ability to devour and digest bacteria adherent to the cell surface following infection. Many hemoglobin particles were observed in the RBC cytoplasm. WBCs were very active against bacterial invasion and showed a strong ability to digest and decompose infected and wrapped V. anguillarum through phagocytosis and lysosome fusion. Digestive vacuoles rapidly became transparent and were thought to contain increasing quantities of pathogen-induced lysozymes. WBCs that devoured pathogenic bacteria were prone to deformation as well as adhesion to each other. TCs were rich in endoplasmic reticulum (ER) content in their cytoplasm and were widely connected in a net-shaped structure. Mitochondria in TCs formed clusters upon invasion of V. anguillarum in the hemolymph. TCs disintegrated to release the ER into the plasma to form a mesh that facilitated clotting. The ability of circulating hemocytes to quickly modify their morphologies and stainability suggests that S. broughtonii is endowed with highly dynamic hemocyte populations capable of coping with environmental changes and rapidly growing pathogens.

Increasing evidences point to the potential role of microRNAs (miRNAs) in muscle growth and development in animals. However, knowledge on the identity of miRNAs and their targets in molluscs remains largely unknown. Scallops have one large adductor muscle, composed of fast (striated) and slow (smooth) muscle types, which display great differences in muscle fibers, meat quality, cell types and molecular components. In the present study, we performed a comprehensive investigation of miRNA transcriptomes in fast and slow adductor muscles of Yesso scallop Patinopecten yessoensis. As a result, 47 differentially expressed miRNAs representing ten miRNA families were identified between the striated and smooth adductor muscles. The KEGG enrichment analysis of their target genes were mainly associated with amino acid metabolism, energy metabolism and glycan biosynthesis. The target genes of miR-133 and miR-71 were validated by the dual-luciferase reporter assays and miRNA antagomir treatment in vivo. The identification and functional validation of these different miRNAs in scallops will greatly help our understanding of miRNA regulatory mechanism that achieves the unique muscle phenotypes in scallops. The present findings provide the direct evidences for muscle-specific miRNAs involved in muscle growth and differentiation in molluscs.

Many proteins are known to be phosphorylated, affecting important regulatory factors of muscle quality in the aquatic animals. The striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis were used to investigate muscle texture and identify phosphoproteins by histological methods and phosphoproteomic analysis. Our present study reveals that muscle fiber density is in relation to meat texture of the striated and smooth adductor muscles. The phosphoproteomic analysis has identified 764 down-regulated and 569 up-regulated phosphosites on 743 phosphoproteins in the smooth muscle compared to the striated part. The identification of unique phosphorylation sites in glycolytic enzymes may increase the activity of glycolytic enzymes and the rate of glycolysis in the striated adductor muscle. The present findings will provide new evidences on the role of muscle structure and protein phosphorylation in scallop muscle quality and thus help to develop strategies for improving meat quality of scallop products.

Knowledge of early development in bivalves is of great importance to understand the function of animal organ systems and the evolution of phenotypic diversity. Manila clam (Ruditapes philippinarum) is an economically important bivalve living in marine intertidal zones. To determine the pattern of muscle development in the clams, we investigate the characteristics of musculature development utilizing phalloidin staining and confocal microscopy. Myofilaments first appear at the early trochophore stage, and gradually become orderly arranged during the transition from trochophore to veliger. For veliger, larval muscle system is mainly composed of dorsal velum retractors, medio-dorsal velum retractors, ventral velum retractors, ventral larval retractors and anterior and posterior adductor muscles. After metamorphosis, the muscle system of late veliger has been reconstructed, showing the irreversible shrink of velum retractor muscles, the rapid growth of wedge-shaped foot and mantle margins. One of the most striking changes in settled spats is the development of sophisticated architecture of foot musculature, which consists of transverse pedal muscles, anterior foot retractor and posterior foot retractor. These findings will not only provide the basis to understand the dynamic pattern of myogenesis in the burrowing bivalves, but also provide valuable information for comparative analysis of muscle development among bivalves.

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response.

In order to improve the accuracy of a single low-cost attitude observation sensor of the wave glider (WG), a new multi-sensor hierarchical data fusion algorithm is proposed. The bottom-level fusion strategy is the complementary data fusion of the accelerometer and the magnetometer, and the global optimal estimation of the bottom-level fusion result and the gyroscope based on the unscented Kalman filter (UKF) algorithm in the top-level fusion strategy. However, the estimation accuracy of the standard UKF algorithm depends on the accuracy of the noise model. Inaccurate estimation of the covariance matrix of the state noise and the measurement noise will cause the accuracy of the UKF algorithm to decrease or even diverge. By introducing the covariance matching technology, the innovation sequence and the residual sequence are used to realize the real-time estimation of the covariance matrix of state noise and measurement noise. At the same time, in view of the characteristic that the measurement equation is a strongly nonlinear equation, an improved covariance matching adaptive UKF algorithm is proposed. It effectively overcomes the limitations of the standard UKF and improves the adaptability of the multi-sensor hierarchical data fusion algorithm. Numerical simulation and experiment are carried out and the results demonstrate that the multi-sensor hierarchical data fusion algorithm is effective in solving the precision problem of the WG low-cost attitude observation unit.

To effectively improve system autonomy, increase fault-tolerant resilience, solve low payload capacity and short endurance time of unmanned surface vehicles (USVs), there's a trend to deploy multiple USVs as a formation fleet. The formation path planning algorithms are essential to generate optimal trajectories and provide practical collision avoidance maneuvers to efficiently navigate the USV fleet. To ensure the optimality, rationality and path continuity of the formation trajectories, this paper presents a novel deterministic algorithm named multiple sub-target artificial potential field (MTAPF) based on an improved APF. The MTAPF belongs to the local path planning algorithm, which refers to the global optimal path generated by an improved heuristic A* algorithm. and the optimal path is divided by this algorithm into multiple sub-target points to form sub-target point sequence. The MTAPF can greatly reduce the probability that USVs will fall into the local minimum and help USVs to get out of the local minimum by switching target points. As an underactuated system, the USV is restricted by various motion constraints, and the MTAPF is presented to make the generated path compliant with USV's dynamics and orientation restrictions. The proposed algorithm is validated on simulations and proven to work effectively in different environments.

Path following control plays an essential role in improving accuracy of navigation and achieving a smooth motion control. However, the performance will exhibit large lateral errors caused by weak maneuverability, large time-lag, and large ocean current disturbance when the wave glider is gliding with a low and time-varying speed. An adaptive path following control integrated an adaptive line-of-sight (ALOS) algorithm and an improved artificial potential field (IAPF) algorithm was put forward in this paper, which could extremely guarantee path following accuracy. The adaptive control based a LOS was introduced to dynamically adjust a parameter of the LOS so that the output desired heading of the wave glider was on-time updated based on the velocity of the wave glider and path following error. The IAPF was applied to analyze and modify the output of the ALOS guiding the vehicle always convergent to the desired path, which could make a real time self-diagnosing, monitoring and correction in path following control. Following this, a closed-loop control was put forward with a dynamic model of the “Black Pearl” wave glider and the given path following control. The proposed control was verified by simulations and sea trials compared with conventional LOS, which demonstrated its effectiveness and long-term reliability.

Ferritin, the principle cytosolic iron storage protein in the majority of living organisms, has important roles during immune process in invertebrates. Detailed information about ferritin in the ark shell Scapharca broughtonii, however, has been very limited. In this study, full-length ferritin (termed SbFer) was cloned by the rapid amplication of cDNA ends (RACE) method based upon the sequence from the transcriptome library. The cDNA contained a 182 bp 5'-untranslated region, a 519 bp open reading frame encoding a polypeptide of 172 amino acids, a 229 bp 3'-untranslated region, and three introns (902, 373 and 402 bp) embedded in four exons. There was an iron response element (IRE) in the 5'-untranslated region. The deduced amino acid sequence of SbFer possessed many characteristics of vertebrate H type ferritin, shared 63%-91% identity with mollusks and greater identity with vertebrate H type ferritin compared to the L type. The SbFer gene expression pattern examined by quantitative real-time PCR showed ferritin mRNA was expressed in all ark shell tissues examined. The highest levels of expression were found in hemocytes with decreasing levels of expression in foot, mantle, gill, adductor muscle and hepatopancreas. A challenge with Vibrio anguillarum resulted in time-dependent significant upregulation of SbFer mRNA, indicating SbFer participated actively in the bacterial defense process. Further analysis of the antibacterial activity indicated recombinant SbFer could function as an immune antibacterial agent to both Gram-positive and Gram-negative bacteria. Taken together, these results suggested strongly that ferritin of the ark shell is involved in immune defense against microbial infection and it is a constitutive and inducible acute-phase protein.

To understand the molecular mechanism associated with growth variability in bivalves, we employed Illumina RNA-seq technology to analyze the transcriptomic profiles from fast- and slow-growing individuals in one full-sib family of the ark shell, Scapharca kagoshimensis. De novo assembly of S. kagoshimensis transcriptome yielded 276,082,016 raw reads, which were assembled into 98,502 unique transcripts using the Trinity strategy. A total of 6357 differentially expressed genes (DEGs) were obtained between fast- and slow-growing individuals, with 68 up-regulated genes and 846 down-regulated genes. Functional annotation revealed that most of the DEGs were classified to subunits of the large or small ribosomal protein, all of which showed significantly lower expression levels in the fast-growing group than those in the slow-growing group. Gene Ontology (GO) enrichment analysis indicated that most of the DEGs were involved in biological processes, followed by molecular functions and cellular components. The most Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enriched genes were related to energy metabolism, protein synthesis and degradation. These findings presented the potential links between genes expression and contrasting phenotypes in the ark shell, suggesting that the fast-growing phenotype might be attributed to lower energy demands for metabolism maintenance, and more effective protein biosynthesis and degradation in bivalves.

Myostatin (MSTN) belongs to the transforming growth factor β (TGF-β) family, which plays a critical role in negatively regulating muscle growth in vertebrates. However, little is known about its function in muscle growth of scallops that possess a large adductor muscle composed of striated and smooth muscle fibers. In this study, we first indentify and characterize the complete cDNA sequence of the myostatin gene from Yesso scallop P. yessoensis, an economically important species in Asian countries. The C-terminal mature TGF-β peptides are highly conserved in scallops, with sequence identity of >97% among these species. Gene expression at developmental stages revealed by whole-mount in situ hybridization indicates that myostatin is mainly expressed on the profusely ciliated rim of velum in veligers. The significant increase of muscle cellularity induced by RNA interference of myostatin can be explained as a result of a combination of hyperplasia and hypertrophy of MHC II striated myofibers, rather than myofiber growth of the paramyosin-rich smooth muscle. This is also evidenced by the quantification results of muscle gene expression, which indicate the significant increase of MHC expression after the inhibition of myostatin mRNA in the striated muscle, but not in the smooth muscle. For both of the striated and smooth muscles, however, no significant difference between the treatment and control groups is detected by real-time quantitative PCR analysis in other muscle genes. These findings suggest that the cellular targets of myostatin in scallop muscles are probably regulated by signaling directly to striated MHC. The new additional nuclei are potentially supplied by a population of satellite cells or muscle stem cell-like adult muscle precursors that may fuse with existing muscle fibers during muscle hypertrophy. These evidences found in this study highlight that myostatin may not only be involved in the development of larval musculature, but also play an important role in regulating scallop muscle growth.

Single-nucleotide polymorphisms (SNPs) have become markers of choice in genetic studies and may be assayed by a variety of methods. For purposes of confirming pedigrees of experimental and commercial broodstocks of the Pacific oyster Crassostrea gigas , we developed PCR primers for exonic amplicons, ranging in size from 61 to 165 base pairs (average 95 bp) and high-resolution melting (HRM) assays for a set of 53 coding SNPs, which were previously identified from EST sequences and mapped in families. We define as canonical the three HRM profiles corresponding to the two homozygotes and the heterozygote expected at the target SNP (e.g. AA, GG, AG or AA, CC, AC), with variation at no other site in the PCR amplicon. We summarize data on Mendelian inheritance of HRM profiles in four, large, full-sib progenies from wild-caught parents and in 16 families with smaller numbers of progeny, a dataset of 24,065 HRM phenotypes. Not surprisingly, these family analyses confirm Mendelian inheritance of canonical HRM profiles, while showing evidence for non-amplifying null alleles and distortions of transmission ratios that can be ascribed to early viability selection. Unexpectedly, these family analyses reveal evidence for heritable, non-canonical HRM profiles, which we further describe. Non-canonical HRM phenotypes are found at 40 of the 53 markers but comprise only 5.37% of all phenotypes in our dataset (whereas technical artifacts account for only 0.4% of phenotypes and measured genotyping error from replicates is just 0.2%). Cloning and sequencing of 142, non-canonical HRM haplotypes, at 26 loci, reveal polymorphism at up to four sites besides the target nucleotide. Thus, HRM assays reveal extensive haplotype diversity within exons; interpreted correctly, this additional phenotypic variation increases the allelic diversity and power of parentage assignment for these Type-I markers.

To investigate genetic relationships among three color variants (Red, Green and Black) of sea cucumber Apostichopus japonicus, complete mitochondrial (mt) genomes of two Red and one Green individuals of A. japonicus were sequenced and compared with those of other eight individuals (two Red, two Black and four Green) from GenBank. All the eleven A. japonicus mitogenomes share an identical gene complement, consisting of 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes, arranged in the same order. Comparative genomic analyses reveal that non-coding intergenic regions have the highest mutation rate (7.36% of sites were variable), while tRNA genes have the lowest mutation rates (0.99%). Three hundred and eighty-six single-nucleotide polymorphism sites are observed in protein-coding genes across the eleven mt sequences. The overall mean genetic distance for the complete mtDNA of the eleven individuals is 0.76% (p-distance). For all protein-coding genes, the pairwise p-distances among the eleven A. japonicus range from 0.04% to 1.55%. As the consequences of extremely low levels of divergence, they strongly support that the assertion that the three color variants of sea cucumber belong to a single species A. japonicus.