Gene expression changes in response to low temperatures in embryos of the kelp grouper, Epinephelus moara.

The kelp grouper Epinephelus moara has high economic value and is popular in fisheries and aquaculture in China. In the previous study, we treated the embryos at 16-22 somite stage at 4 °C, -25.7 °C, -140 °C and -196 °C, and successfully obtained surviving embryos in each group. To better understand the molecular changes affected by the low temperatures, we conducted a comparative transcriptome analysis among embryos exposed at 4 °C for 30 min, embryos exposed at -25.7 °C for 30 min and the control group. qPCR assays were conducted for the validation. Signal transduction pathways were highly enriched for the differentially expressed genes. c-Fos, c-Jun, JunD, GADD45, involved in MAPK signaling pathway, were upregulated when embryos were treated at low temperatures. As immediate early genes, Egr-1a and b, and IER2, that respond quickly to the environment stress, their expression increased as well. Hsp70 showed similar expression pattern as immediate early genes. Meanwhile, transcription factors Sox, HES, TFIID, muscle movement and protein synthesis-related genes were downregulated. Taken together, our findings suggest that cooling disrupts gene expression patterns in E. moara embryos. The differentially expressed genes may be involved in cellular resistance against low temperatures, possibly through neural activation, apoptosis, proliferation, differentiation, cellular recovery and heat shock regulation. This study also provides transcriptome dataset of E. moara embryos exposed to cold temperatures for future studies focusing on the molecular effects of cryopreservation.

Quantitative proteomics study of larval settlement in the Barnacle Balanus amphitrite.

Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite.

Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite.

Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.

Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans.

The polychaete Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of H. elegans. A full-length CaM cDNA was successfully cloned from H. elegans (He-CaM) and it contained an open reading frame of 450 bp, encoding 149 amino acid residues. It was highly expressed in 12h post-metamorphic juveniles, and remained high in adults. In situ hybridization conducted in competent larvae and juveniles revealed that He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of H. elegans.

Transcriptomic analysis of neuropeptides and peptide hormones in the barnacle Balanus amphitrite: evidence of roles in larval settlement.

The barnacle Balanus amphitrite is a globally distributed marine crustacean and has been used as a model species for intertidal ecology and biofouling studies. Its life cycle consists of seven planktonic larval stages followed by a sessile juvenile/adult stage. The transitional processes between larval stages and juveniles are crucial for barnacle development and recruitment. Although some studies have been conducted on the neuroanatomy and neuroactive substances of the barnacle, a comprehensive understanding of neuropeptides and peptide hormones remains lacking. To better characterize barnacle neuropeptidome and its potential roles in larval settlement, an in silico identification of putative transcripts encoding neuropeptides/peptide hormones was performed, based on transcriptome of the barnacle B. amphitrite that has been recently sequenced. Potential cleavage sites andstructure of mature peptides were predicted through homology search of known arthropod peptides. In total, 16 neuropeptide families/subfamilies were predicted from the barnacle transcriptome, and 14 of them were confirmed as genuine neuropeptides by Rapid Amplification of cDNA Ends. Analysis of peptide precursor structures and mature sequences showed that some neuropeptides of B. amphitrite are novel isoforms and shared similar characteristics with their homologs from insects. The expression profiling of predicted neuropeptide genes revealed that pigment dispersing hormone, SIFamide, calcitonin, and B-type allatostatin had the highest expression level in cypris stage, while tachykinin-related peptide was down regulated in both cyprids and juveniles. Furthermore, an inhibitor of proprotein convertase related to peptide maturation effectively delayed larval metamorphosis. Combination of real-time PCR results and bioassay indicated that certain neuropeptides may play an important role in cypris settlement. Overall, new insight into neuropeptides/peptide hormones characterized in this study shall provide a platform for unraveling peptidergic control of barnacle larval behavior and settlement process.

Toward an understanding of the molecular mechanisms of barnacle larval settlement: a comparative transcriptomic approach.

BACKGROUND: The barnacle Balanus amphitrite is a globally distributed biofouler and a model species in intertidal ecology and larval settlement studies. However, a lack of genomic information has hindered the comprehensive elucidation of the molecular mechanisms coordinating its larval settlement. The pyrosequencing-based transcriptomic approach is thought to be useful to identify key molecular changes during larval settlement. METHODOLOGY AND PRINCIPAL FINDINGS: Using 454 pyrosequencing, we collected totally 630,845 reads including 215,308 from the larval stages and 415,537 from the adults; 23,451 contigs were generated while 77,785 remained as singletons. We annotated 31,720 of the 92,322 predicted open reading frames, which matched hits in the NCBI NR database, and identified 7,954 putative genes that were differentially expressed between the larval and adult stages. Of these, several genes were further characterized with quantitative real-time PCR and in situ hybridization, revealing some key findings: 1) vitellogenin was uniquely expressed in late nauplius stage, suggesting it may be an energy source for the subsequent non-feeding cyprid stage; 2) the locations of mannose receptors suggested they may be involved in the sensory system of cyprids; 3) 20 kDa-cement protein homologues were expressed in the cyprid cement gland and probably function during attachment; and 4) receptor tyrosine kinases were expressed higher in cyprid stage and may be involved in signal perception during larval settlement. CONCLUSIONS: Our results provide not only the basis of several new hypotheses about gene functions during larval settlement, but also the availability of this large transcriptome dataset in B. amphitrite for further exploration of larval settlement and developmental pathways in this important marine species.