Transcriptomic Analysis of Marine Gastropod Hemifusus tuba Provides Novel Insights into Conotoxin Genes

The marine gastropod Hemifusus tuba is served as a luxury food in Asian countries and used in traditional Chinese medicine to treat lumbago and deafness. The lack of genomic data on H. tuba is a barrier to aquaculture development and functional characteristics of potential bioactive molecules are poorly understood. In the present study, we used high-throughput sequencing technologies to generate the first transcriptomic database of H. tuba. A total of 41 unique conopeptides were retrieved from 44 unigenes, containing 6-cysteine frameworks belonging to four superfamilies. Duplication of mature regions and alternative splicing were also found in some of the conopeptides, and the de novo assembly identified a total of 76,306 transcripts with an average length of 824.6 nt, of which including 75,620 (99.1%) were annotated. In addition, simple sequence repeats (SSRs) detection identified 14,000 unigenes containing 20,735 SSRs, among which, 23 polymorphic SSRs were screened. Thirteen of these markers could be amplified in Hemifusus ternatanus and seven in Rapana venosa. This study provides reports of conopeptide genes in Buccinidae for the first time as well as genomic resources for further drug development, gene discovery and population resource studies of this species

Effects of Dietary Cholesterol Levels on the Growth, Molt Performance, and Immunity of Juvenile Swimming Crab, Portunus trituberculatus

The effects of dietary cholesterol levels on growth, molt performance, and immunity of juvenile swimming crab Portunus trituberculatus, were investigated at four cholesterol levels (0.2%-1.4%) of purified diets. Each diet was fed in triplicate to 18 crabs per replicate for 50 days. Crabs fed the diet with 1.0% cholesterol showed significantly higher (P<0.05) specific growth rate (SGR) than the other groups, who suffered from relatively lower molt death syndrome (MDS). Cholesterol content in the serum, whole body, and hepatopancreas increased in relation to dietary cholesterol. Muscle lipid content was significantly higher (P<0.05) in crabs fed the diet with 0.2% cholesterol compared to the other treatments. Crabs fed moderate dietary cholesterol levels showed higher alkaline phosphatase (AKP) or acid phosphatase (ACP) levels than those fed 0.2% or 1.4% cholesterol diets. The present study also showed that dietary cholesterol supplementation generally increased serum superoxide dismutase (SOD) activity. Overall, moderate dietary cholesterol (1.0 %) enhanced the performance of growth, survival, molting, and immunity of juvenile swimming crab P. trituberculatus

Light Spectrum Impacts on Growth, Molting, and Oxidative Stress Response of the Mud Crab Scylla paramamosain

An 8 weeks trial was performed to test the effects of light spectra [full-spectrum, violet (405 nm), blue (470 nm), cyan (500 nm), green (525 nm), yellow (570 nm), and red (625 nm)] on growth performance, molting, antioxidant capacity, stress response and expression of molting, and apoptosis-related genes in Scylla paramamosain. Results showed that spectrum had a significant effect on S. paramamosain physiology. Compared to blue light, crabs exposed to violet light had a significantly lower survival rate (79.5 ± 3.6% vs. 94.9 ± 3.6%), weight gain (49.2 ± 5.4 vs. 67.6 ± 6.7), molt frequency (4.2 ± 0.2 vs. 4.5 ± 0.1), and extended intermolt intervals between instar 1 and 2 stages (C1–C2) (6.3 ± 0.3 vs. 5.0 ± 0.1 days). Expression of the molt-inhibiting hormone (mih) gene was upregulated in crabs reared under violet light. According to the regression analysis, maximum SGR would be at 449.97 nm. Crabs exposed to blue light also had lower melatonin levels than under full-spectrum and lower cortisol levels than violet and yellow groups. Regarding oxidative stress, crabs in full-spectrum had lower H2O2 and MDA contents, however, no significant difference was found in total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and catalase (CAT) in hepatopancreas from crabs under different spectra. Gene expression of hsp40, hsp70, hsp90 were down-regulated in crabs exposed to the full-spectrum light group. Regarding apoptosis-related genes, bcl-2 gene expression in crabs under cyan and the cox IV and caspase 3 in green were upregulated, suggesting cyan light may inhibit, while green light may promote apoptosis. Taken together, these results suggest that blue or cyan light would promote growth performance, while full-spectrum light could reduce stress response in S. paramamosain

The Whole-Genome Sequencing and Hybrid Assembly of Mytilus coruscus

The hard-shelled mussel (Mytilus coruscus) is an economically important shellfish, which has been cultivated for the last decade. Due to over-exploitation, most mussel stocks have dramatically declined. Efforts to study this species' natural distribution, genetics, breeding and cultivation have been hindered by the lack of a high-quality reference genome. To address this, we produced a hybrid high-quality reference genome of M. coruscus using a long-read platform to assemble the genome and short-reads, high-quality technology to accurately correct for sequence errors. The genome was assembled into 10,484 scaffolds, a total length of 1.90 Gb and a scaffold N50 of 898 kb. Ab initio annotation of the M. coruscus genome assembly identified a total of 42,684 genes. This accurate, reference genome of M. coruscus provides an essential resource with the advantage of enabling the genome-scale selective breeding of M. coruscus. More importantly it will also help the deciphering of the speciation and local adaptation of the Mytilus species

Phytoplankton community structure in the Western Subarctic Gyre of the Pacific Ocean during summer determined by a combined approach of HPLC-pigment CHEMTAX and metabarcoding sequencing

The Western Subarctic Gyre (WSG) is a cyclonic upwelling gyre in the northwest subarctic Pacific, which is a region with a high concentration of nutrients but low chlorophyll. We investigated the community structure and spatial distribution of phytoplankton in this area by using HPLC-pigment CHEMTAX (a chemotaxonomy program) and metabarcoding sequencing during the summer of 2021. The phytoplankton community showed significant differences between the two methods. The CHEMTAX analyses identified eight major marine phytoplankton assemblages. Cryptophytes were the major contributors (24.96%) to the total Chl a, followed by pelagophytes, prymnesiophytes, diatoms, and chlorophytes. The eukaryotic phytoplankton OTUs obtained by metabarcoding were categorized into 149 species in 96 genera of 6 major groups (diatoms, prymnesiophytes, pelagophytes, chlorophytes, cryptophytes, and dinoflagellates). Dinoflagellates were the most abundant group, accounting for 44.74% of the total OTUs obtained, followed by cryptophytes and pelagophytes. Sixteen out of the 97 identified species were annotated as harmful algal species, and Heterocapsa rotundata, Karlodinium veneficum, and Aureococcus anophagefferens were assigned to the abundant group (i.e., at least 0.1% of the total reads). Nutrients were more important in shaping the phytoplankton community than temperature and salinity. The 24 stations were divided into southern and northern regions along 44°N according to the k-means method, with the former being dominated by high Chl a and low nutrients. Although different phytoplankton assemblages analyzed by the two methods showed various relationships with environmental factors, a common feature was that the dinoflagellate proportion showed a significantly negative correlation with low nutrients and a positive correlation with Chl a

Mapping and validation of sex-linked SNP markers in the swimming crab Portunus trituberculatus

Portunus trituberculatus is one of the most commercially important marine crustacean species for both aquaculture and fisheries in Southeast and East Asia. Production of monosex female stocks is attractive in commercial production since females are more profitable than their male counterparts. Identification and mapping of the sex-linked locus is an efficient way to elucidate the mechanisms of sex determination in the species and support the development of protocols for monosex female production. In this study, a sex-averaged map and two sex-specific genetic maps were constructed based on 2b-restriction site-associated DNA sequencing. A total of 6349 genetic markers were assigned to 53 linkage groups. Little difference was observed in the pattern of sex-specific recombination between females and males. Association analysis and linkage mapping identified 7 markers strongly associated with sex, four of which were successfully mapped on the extremity of linkage group 22. Females were homozygous and males were heterozygous for those 7 markers strongly suggesting an XX/XY sex determination system in this species. Three Markers were successfully validated in a wild population of P. trituberculatus and exhibited a specificity ranging from 93.3% to 100%. A high-resolution melting based assay was developed for sex genotyping. This study provides new knowledge and tools for sex identification which will help the development of protocols for monosex female production of P. trituberculatus and support future genomic studies

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge

Molecular cloning and characterization of two isoforms of cyclophilin A gene from Venerupis philippinarum

Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein belonging to the immunophilin family, which is recognized as the cell receptor for the potent immunosuppressive drug cyclosporine A. In the present study, two isoforms of cyclophilin A gene (named as VpCypA1 and VpCypA2) were isolated and characterized from Venerupis philippinarum by RACE approaches. Both VpCypA1 and VpCypA2 possessed all conserved features critical for the fundamental structure and function of CypA, indicating that the two isoforms of cyclophilin A should be new members of CypA family. The expression of VpCypA2 mRNA in haemocytes was significantly up-regulated and the highest expression level was detected at 96 h post-infection with 7.7-fold increase compared with that in the blank group. On the contrary, the relative expression level of VpCypA1 mRNA was down-regulated rapidly at 6 h post-infection and reached 0.4-fold of the control group. They exhibited different expression profile and identical effect of immune modulation, which might suggest the two VpCypA isoforms exert their function in a manner of synergy. These results provide valuable information for further exploring the roles of cyclophilin A in the immune responses of V. philippinarum. (C) 2011 Elsevier Ltd. All rights reserved.Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein belonging to the immunophilin family, which is recognized as the cell receptor for the potent immunosuppressive drug cyclosporine A. In the present study, two isoforms of cyclophilin A gene (named as VpCypA1 and VpCypA2) were isolated and characterized from Venerupis philippinarum by RACE approaches. Both VpCypA1 and VpCypA2 possessed all conserved features critical for the fundamental structure and function of CypA, indicating that the two isoforms of cyclophilin A should be new members of CypA family. The expression of VpCypA2 mRNA in haemocytes was significantly up-regulated and the highest expression level was detected at 96 h post-infection with 7.7-fold increase compared with that in the blank group. On the contrary, the relative expression level of VpCypA1 mRNA was down-regulated rapidly at 6 h post-infection and reached 0.4-fold of the control group. They exhibited different expression profile and identical effect of immune modulation, which might suggest the two VpCypA isoforms exert their function in a manner of synergy. These results provide valuable information for further exploring the roles of cyclophilin A in the immune responses of V. philippinarum. (C) 2011 Elsevier Ltd. All rights reserved