New genes helped acorn barnacles adapt to a sessile lifestyle.

Barnacles are the only sessile lineages among crustaceans, and their sessile life begins with the settlement of swimming larvae (cyprids) and the formation of protective shells. These processes are crucial for adaptation to a sessile lifestyle, but the underlying molecular mechanisms remain poorly understood. While investigating these mechanisms in the acorn barnacle, Amphibalanus amphitrite, we discovered a new gene, bcs-6, which is involved in the energy metabolism of cyprid settlement and originated from a transposon by acquiring the promoter and cis-regulatory element. Unlike mollusks, the barnacle shell comprises alternate layers of chitin and calcite and requires another new gene, bsf, which generates silk-like fibers that efficiently bind chitin and aggregate calcite in the aquatic environment. Our findings highlight the importance of exploring new genes in unique adaptative scenarios, and the results will provide important insights into gene origin and material development.

Impact of ocean acidification on shells of the abalone species Haliotis diversicolor and Haliotis discus hannai.

Ocean acidification (OA) results from the absorption of anthropogenic CO2 emissions by the ocean and threatens the survival of many marine calcareous organisms including molluscs. We studied OA effects on adult shells of the abalone species Haliotis diversicolor and Haliotis discus hannai that were exposed to three pCO2 conditions (ambient, ∼880, and ∼1600 µatm) for 1 year. Shell periostracum corrosion under OA was observed for both species. OA reduced shell hardness and altered the nacre ultrastructure in H. diversicolor, making its shells more vulnerable to crushing force. OA exposure did not reduce the shell hardness of H. discus hannai and did not alter nacre ultrastructure. However, the reduced calcification also decreased its resistance to crushing force. Sr/Ca in the shell increased with rising calcification rate. Mg/Ca increased upon OA exposure could be due to a complimentary mechanism of preventing shell hardness further reduced. The Na/Ca distribution between the aragonite and calcite of abalone shells was also changed by OA. In general, both abalone species are at a greater risk in a more acidified ocean. Their shells may not provide sufficient protection from predators or to transportation stress in aquaculture.

Effects of one-year exposure to ocean acidification on two species of abalone.

Ocean acidification (OA) resulting from the absorption of excess atmospheric CO2 by the ocean threatens the survival of marine calcareous organisms, including mollusks. This study investigated the effects of OA on adults of two abalone species (Haliotis diversicolor, a subtropical species, and Haliotis discus hannai, a temperate species). Abalone were exposed to three pCO2 conditions for 1 year (ambient, ~ 880, and ~ 1600 µatm), and parameters, including mortality, physiology, immune system, biochemistry, and carry-over effects, were measured. Survival decreased significantly at ~ 800 µatm pCO2 for H. diversicolor, while H. discus hannai survival was negatively affected only at a higher OA level (~ 1600 µatm pCO2). H. diversicolor exhibited depressed metabolic and excretion rates and a higher O:N ratio under OA, indicating a shift to lipids as a metabolism substrate, while these physiological parameters in H. discus hannai were robust to OA. Both abalone failed to compensate for the pH decrease of their internal fluids because of the lowered hemolymph pH under OA. However, the reduced hemolymph pH did not affect total hemocyte counts or tested biomarkers. Additionally, H. discus hannai increased its hemolymph protein content under OA, which could indicate enhanced immunity. Larvae produced by adults exposed to the three pCO2 levels were cultured in the same pCO2 conditions and larval deformation and shell length were measured to observe carry-over effects. Enhanced OA tolerance was observed for H. discus hannai exposed under both of the OA treatments, while that was only observed following parental pCO2 ~ 880 µatm exposure for H. diversicolor. Following pCO2 ~ 1600 µatm parental exposure, H. diversicolor offspring exhibited higher deformation and lower shell growth in all pCO2 treatments. In general, H. diversicolor were more susceptible to OA compared with H. discus hannai, suggesting that H. diversicolor could be unable to adapt to acidified oceans in the future.

Proteome of larval metamorphosis induced by epinephrine in the Fujian oyster Crassostrea angulata.

BACKGROUND: The Fujian oyster Crassostrea angulata is an economically important species that has typical settlement and metamorphosis stages. The development of the oyster involves complex morphological and physiological changes, the molecular mechanisms of which are as yet unclear. RESULTS: In this study, changes in proteins were investigated during larval settlement and metamorphosis of Crassostrea angulata using epinephrine induction. Protein abundance and identity were characterized using label-free quantitative proteomics, tandem mass spectrometry (MS/ MS), and Mascot methods. The results showed that more than 50% (764 out of 1471) of the quantified proteins were characterized as differentially expressed. Notably, more than two-thirds of the differentially expressed proteins were down-regulated in epinephrine-induced larvae. The results showed that "metabolic process" was closely related to the development of settlement and metamorphosis; 5 × 10- 4 M epinephrine induced direct metamorphosis of larvae and was non-toxic. Calmodulin and MAPK pathways were involved in the regulation of settlement of the oyster. Expression levels of immune-related proteins increased during metamorphosis. Hepatic lectin-like proteins, cadherins, calmodulin, calreticulin, and cytoskeletal proteins were involved in metamorphosis. The nervous system may be remodeled in larval metamorphosis induced by epinephrine. Expression levels of proteins that were enriched in the epinephrine signaling pathway may reflect the developmental stage of the larvae, that may reflect whether or not larvae were directly involved in metamorphosis when the larvae were treated with epinephrine. CONCLUSION: The study provides insight into proteins that function in energy metabolism, immune responses, settlement and metamorphosis, and shell formation in C. angulata. The results contribute valuable information for further research on larval settlement and metamorphosis.

Aggregation Pheromone for an Invasive Mussel Consists of a Precise Combination of Three Common Purines.

Most marine benthic invertebrates have a pelagic larval phase, after which they settle preferentially on or near conspecific adults, forming aggregations. Although settlement pheromones from conspecific adults have been implicated as critical drivers of aggregation for more than 30 years, surprisingly few have been unambiguously identified. Here we show that in the invasive dreissenid mussel Mytilopsis sallei (an ecological and economic pest), three common purines (adenosine, inosine, and hypoxanthine) released from adults in a synergistic and precise ratio (1:1.125:3.25) serve as an aggregation pheromone by inducing conspecific larval settlement and metamorphosis. Our results demonstrate that simple common metabolites can function as species-specific pheromones when present in precise combinations. This study provides important insights into our understanding of the ecology and communication processes of invasive organisms and indicates that the combination and ratio of purines might be critical for purine-based signaling systems that are fundamental and widespread in nature.

Effects of acidification on the proteome during early development of Babylonia areolata.

Increases in atmospheric CO2 partial pressure have lowered seawater pH in marine ecosystems, a process called ocean acidification (OA). The effects of OA during the critical stages of larval development may have disastrous consequences for some marine species, including Babylonia areolata (Link 1807), a commercially important sea snail in China and South East Asia. To investigate how OA affects the proteome of Babylonia areolata, here we used label-free proteomics to study protein changes in response to acidified (pH 7.6) or ambient seawater (pH 8.1) during three larvae developmental stages of B. areolata, namely, the veliger larvae before attachment (E1), veliger larvae after attachment (E2), and carnivorous juvenile snail (E3). In total, we identified 720 proteins. This result suggested that acidification seriously affects late veliger stage after attachment (E2). Further examination of the roles of differentially expressed proteins, which include glutaredoxin, heat-shock protein 70, thioredoxin, catalase, cytochrome-c-oxidase, peroxiredoxin 6, troponin T, CaM kinase II alpha, proteasome subunit N3 and cathepsin L, will be important for understanding the molecular mechanisms underlying pH reduction.

Differential proteomic profiles and characterizations between hyalinocytes and granulocytes in ivory shell Babylonia areolata.

The haemocytes of the ivory shell, Babylonia areolata are classified by morphologic observation into the following types: hyalinocytes (H) and granulocytes (G). Haemocytes comprise diverse cell types with morphological and functional heterogene and play indispensable roles in immunological homeostasis of invertebrates. In the present study, two types of haemocytes were morphologically identified and separated as H and G by Percoll density gradient centrifugation. The differentially expressed proteins were investigated between H and G using mass spectrometry. The results showed that total quantitative proteins between H and G samples were 1644, the number of up-regulated proteins in G was 215, and the number of down-regulated proteins in G was 378. Among them, cathepsin, p38 MAPK, toll-interacting protein-like and beta-adrenergic receptor kinase 2-like were up-regulated in G; alpha-2-macroglobulin-like protein, C-type lectin, galectin-2-1, galectin-3, ß-1,3-glucan-binding protein, ferritin, mega-hemocyanin, mucin-17-like, mucin-5AC-like and catalytic subunit of protein kinase A were down-regulated in G. The results showed that the most significantly enriched KEGG pathways were the pathways related to ribosome, phagosome, endocytosis, carbon metabolism, protein processing in endoplasmic reticulum and oxidative phosphorylation. For phagosome and endocytosis pathway, the number of down-regulation proteins in G was more than that of up-regulation proteins. For lysosome pathway, the number of up-regulation proteins in G was more than that of down-regulation proteins. These results suggested that two sub-population haemocytes perform the different immune functions in B. areolata.

Bacterial Nucleobases Synergistically Induce Larval Settlement and Metamorphosis in the Invasive Mussel Mytilopsis sallei.

Marine bacterial biofilms have long been recognized as potential inducers of larval settlement and metamorphosis in marine invertebrates, but few chemical cues from bacteria have been identified. Here, we show that larval settlement and metamorphosis of an invasive fouling mussel, Mytilopsis sallei, could be induced by biofilms of bacteria isolated from its adult shells and other substrates from the natural environment. One of the strains isolated, Vibrio owensii MS-9, showed strong inducing activity which was attributed to the release of a mixture of nucleobases including uracil, thymine, xanthine, hypoxanthine, and guanine into seawater. In particular, the synergistic effect of hypoxanthine and guanine was sufficient for the inducing activity of V. owensii MS-9. The presence of two or three other nucleobases could enhance, to some extent, the activity of the mixture of hypoxanthine and guanine. Furthermore, we determined that bacteria producing higher concentrations of nucleobases were more likely to induce larval settlement and metamorphosis of M. sallei than were bacteria producing lower concentrations of nucleobases. The present study demonstrates that bacterial nucleobases play an important role in larval settlement and metamorphosis of marine invertebrates. This provides new insights into our understanding of the role of environmental bacteria in the colonization and aggregation of invasive fouling organisms and of the metabolites used as chemical mediators in cross-kingdom communication within aquatic systems.IMPORTANCE Invasive species are an increasingly serious problem globally. In aquatic ecosystems, invasive dreissenid mussels are well-known ecological and economic pests because they appear to effortlessly invade new environments and foul submerged structures with high-density aggregations. To efficiently control exotic mussel recruitment and colonization, the need to investigate the mechanisms of substrate selection for larval settlement and metamorphosis is apparent. Our work is one of very few to experimentally demonstrate that compounds produced by environmental bacteria play an important role in larval settlement and metamorphosis in marine invertebrates. Additionally, this study demonstrates that bacterial nucleobases can be used as chemical mediators in cross-kingdom communication within aquatic systems, which will enhance our understanding of how microbes induce larval settlement and metamorphosis of dreissenid mussels, and it furthermore may allow the development of new methods for application in antifouling.

Identification and characteristics of muscle growth-related microRNA in the Pacific abalone, Haliotis discus hannai.

BACKGROUND: The Pacific abalone, Haliotis discus hannai, is the most important cultivated abalone in China. Improving abalone muscle growth and increasing the rate of growth are important genetic improvement programs in this industry. MicroRNAs are important small noncoding RNA molecules that regulate post-transcription gene expression. However, no miRNAs have been reported to regulate muscle growth in H. discus hannai. RESULTS: we profiled six small RNA libraries for three large abalone individuals (L_HD group) and three small individuals (S_HD group) using RNA sequencing technology. A total of 205 miRNAs, including 200 novel and 5 known miRNAs, were identified. In the L_HD group, 3 miRNAs were up-regulated and 7 were down-regulated compared to the S_HD specimens. Bioinformatics analysis of miRNA target genes revealed that miRNAs participated in the regulation of cellular metabolic processes, the regulation of biological processes, the Wnt signaling pathway, ECM-receptor interaction, and the MAPK signaling pathway, which are associated with regulating growth. Bone morphogenetic protein 7 (BMP7) was verified as a target gene of hdh-miR-1984 by a luciferase reporter assay and we examined the expression pattern in different developmental stages. CONCLUSION: This is the first study to demonstrate that miRNAs are related to the muscle growth of H. discus hannai. This information could be used to study the mechanisms of abalone muscle growth. These DE-miRNAs may be useful as molecular markers for functional genomics and breeding research in abalone and closely related species.

Expression profiling of lncRNAs and mRNAs reveals regulation of muscle growth in the Pacific abalone, Haliotis discus hannai.

Long non-coding RNAs (lncRNAs) are known to play a major role in the epigenetic regulation of muscle development. Unfortunately there is little understanding of the mechanisms with which they regulate muscle growth in abalone. Therefore, we used RNA-seq to study the muscle transcriptomes of six Haliotis discus hannai specimens: three large (L_HD group) and three small (S_HD group). We identified 2463 lncRNAs in abalone muscle belonging to two subtypes: 160 anti-sense lncRNAs and 2303 intergenic lncRNAs (lincRNAs). In the L_HD group, we identified 204 significantly differentially expressed lncRNAs (55 upregulated and 149 downregulated), and 2268 significantly differentially expressed mRNAs (994 upregulated and 1274 downregulated), as compared to the S_HD group. The bioinformatics analysis indicated that lncRNAs were relate to cell growth, regulation of growth, MAPK signaling pathway, TGF-ß signaling pathway, PI3K-Akt and insulin signaling pathway, which involved in regulating muscle growth. These findings contribute to understanding the possible regulatory mechanisms of muscle growth in Pacific abalone.

Author Correction: Proteomics Studies on the three Larval Stages of Development and Metamorphosis of Babylonia areolata.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

LC-MS/MS-Based Metabolome Analysis of Biochemical Pathways Altered by Food Limitation in Larvae of Ivory Shell, Babylonia areolata.

Ivory shell, Babylonia areolata, is one of the commercially important mariculture species in China and South East Asia. Survival varies in the artificial hatching and larval rearing of B. areolata. Food deprivation may be involved in rearing mortality, and so, a better understanding of how larvae respond and adjust to starvation is needed. In this study, the metabolite profiles of newly hatched larvae with yolk (I), larvae with yolk exhaustion (II), larvae suffering 24 h starvation after yolk exhaustion (III), and larvae fed with exogenous nutrients after yolk exhaustion (IV) were analyzed by LC-MS/MS. Principal component and cluster analyses revealed differential abundance of metabolite profiles across groups. When compared to metabolite levels of the I group, significantly up-regulated metabolites included polyunsaturated fatty acids, phospholipids, nucleotide, amino acids, and their derivatives were found in the II group, indicating that organisms relied predominantly on glycerophospolipid metabolism and protein-based catabolism for energy production during this stage. During starvation after yolk exhaustion, the levels of all energy related metabolites were significantly reduced, but an increase in products of purine and pyrimidine metabolism indicated an insufficient energy supply and an increase in cellular disintegration. Larvae fed exogenous nutrients can have significantly improved metabolism compared to starved larvae. These findings suggest that metabolomics, using LC-MS/MS, can be used to assess the physiological status and food-affected metabolic changes affecting B. areolata larvae.

Proteomics Studies on the three Larval Stages of Development and Metamorphosis of Babylonia areolata.

The ivory shell, Babylonia areolata, is a commercially important aquaculture species in the southeast coast of mainland China. The middle veliger stage, later veliger stage, and juvenile stage are distinct larval stages in B. areolata development. In this study, we used label-free quantification proteomics analysis of the three developmental stages of B. areolata. We identified a total of 5,583 proteins, of which 1,419 proteins expression level showed significant differential expression. The results of gene ontology enrichment analysis showed that the number of proteins involved in metabolic and cellular processes were the most abundant. Those proteins mostly had functions such as binding, catalytic activity and transporter activity. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the number of proteins involved in the ribosome, carbon metabolism, and lysosome pathways were the most abundant, indicating that protein synthesis and the immune response were active during the three stages of development. This is the first study to use proteomics and real-time PCR to study the early developmental stages of B. areolata, which could provide relevant data on gastropod development. Our results provide insights into the novel aspects of protein function in shell formation, body torsion, changes in feeding habits, attachment and metamorphosis, immune-related activities in B. areolata larvae.

Proteomic analysis of trochophore and veliger larvae development in the small abalone Haliotis diversicolor.

BACKGROUND: Haliotis diversicolor is commercially important species. The trochophore and veliger are distinct larval stages in gastropod development. Their development involves complex morphological and physiological changes. We studied protein changes during the embryonic development of H. diversicolor using two dimensional electrophoresis (2-DE) and label-free methods, tandem mass spectrometry (MS/ MS), and Mascot for protein identification. RESULTS: A total of 150 2-DE gel spots were identified. Protein spots showed upregulation of 15 proteins and downregulation of 28 proteins as H. diversicolor developed from trochophore to veliger larvae. Trochophore and veliger larvae were compared using a label-free quantitative proteomic approach. A total of 526 proteins were identified from both samples, and 104 proteins were differentially expressed (> 1.5 fold). Compared with trochophore larvae, veliger larvae had 55 proteins upregulated and 49 proteins downregulated. These differentially expressed proteins were involved in shell formation, energy metabolism, cellular and stress response processes, protein synthesis and folding, cell cycle, and cell fate determination. Compared with the 5 protein (fructose-bisphosphate aldolase, 14-3-3ε, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase. CONCLUSION: Our results provide insight into novel aspects of protein function in shell formation, torsion, and nervous system development, and muscle system differentiation in H. diversicolor larvae. "Quality control" proteins were identified to be involved in abalone larval development.

Transcriptome analysis of the key role of GAT2 gene in the hyper-accumulation of copper in the oyster Crassostrea angulata.

One paradigm of oysters as the hyper-accumulators of many toxic metals is the inter-individual variation of metals, but the molecular mechanisms remain very elusive. A comprehensive analysis of the transcriptome of Crassostrea angulata was conducted to reveal the relationship between gene expression and differential Cu body burden in oysters. Gene ontology analysis for the differentially expressed genes showed that the neurotransmitter transporter might affect the oyster behavior, which in turn led to difference in Cu accumulation. The ATP-binding cassette transporters superfamily played an important role in the maintenance of cell Cu homeostasis, vitellogenin and apolipophorin transport, and elimination of excess Cu. Gill and mantle Cu concentrations were significantly reduced after silencing the GABA transporter 2 (GAT2) gene, but increased after the injection of GABA receptor antagonists, suggesting that the function of GABA transporter 2 gene was strongly related to Cu accumulation. These findings demonstrated that GABA transporter can control the action of transmitter GABA in the nervous system, thereby affecting the Cu accumulation in the gills and mantles.

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea.

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione-S-transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.

Metal accumulation and differentially expressed proteins in gill of oyster (Crassostrea hongkongensis) exposed to long-term heavy metal-contaminated estuary.

Bio-accumulation and bio-transmission of toxic metals and the toxicological responses of organisms exposed to toxic metals have been focused, due to heavy metal contaminations have critically threatened the ecosystem and food security. However, still few investigations focused on the responses of certain organisms exposed to the long term and severe heavy metal contamination in specific environments. In present investigation, the Hong Kong oyster, Crassostrea hongkongensis were obtained from 3 sites which were contaminated by different concentrations of heavy metals (such as zinc, copper, manganese and lead etc.), respectively. Heavy metal concentrations in the sea water samples collected from the 3 sites and the dissected tissues of the oysters with blue visceral mass were determinated to estimate the metal contamination levels in environments and the bio-accumulation ratios of the heavy metals in the different tissues of oysters. Moreover, Proteomic methods were employed to analyze the differentially expressed proteins in the gills of oysters exposed to long-term heavy metal contaminations. Results indicated that the Jiulong River estuary has been severely contaminated by Cu, Zn and slightly with Cr, Ni, Mn, etc, moreover, Zn and Cu were the major metals accumulated by oysters to phenomenally high concentrations (more than 3.0% of Zn and about 2.0% of Cu against what the dry weight of tissues were accumulated), and Cr, Ni, Mn, etc were also significantly accumulated. The differentially expressed proteins in the gills of oysters exposed to heavy metals participate in several cell processes, such as metal binding, transporting and saving, oxidative-reduction balance maintaining, stress response, signal transduction, etc. Significantly up-regulated expression (about 10 folds) of an important metal binding protein, metallothionein (MT) and granular cells was observed in the gills of oysters exposed to long-term and severely heavy-metal-contaminated estuary, it suggested that binding toxic metals with metallothionein-like proteins (MTLP) and storing toxic metals in metal-rich granules (MRG) with insoluble forms were the important strategies of oyster to detoxify the toxic metals and adapt to the high level of metal-contaminated environment. Most of the stress and immunity responsive proteins, such as heat shock proteins (HSP), extracellular superoxide dismutase (ECSOD) and cavortin, and the cellular redox reaction relative proteins such as 20G-Fe (II) oxygenase family oxidoreductase, aldehyde dehydrogenase and retinal dehydrogenase 2, were detected significantly down-regulated in the gills of oysters exposed to long term heavy metal contaminated environments, it indicated that long term exposure different from emergent exposure to heavy metal contamination may significantly suppress the stress and immunity response system of oysters. Moreover, Formin homology 2 domain containing protein (FH2). The only protein domain to directly nucleate actin monomers into unbranched filament polymers, by which will subsequently control gene expression and chromatin remodelling complexes, was also detected greatly up-regulated in the gills of oysters exposed to long-term heavy metal contaminations. It indicated that nuclear activity regulation may also be important for oyster to adapt to the long-term heavy-metal-contaminated environment.