Transcriptome Responses to Different Environments in Intertidal Zones in the Peanut Worm Sipunculus nudus.

The peanut worm (Sipunculus nudus) is an important intertidal species worldwide. Species living in the same aquaculture area might suffer different environmental impacts. To increase knowledge of the molecular mechanisms underlying the response to environmental fluctuations, we performed a transcriptome analysis of S. nudus from different intertidal zones using a combination of the SMRT platform and the Illumina sequencing platform. (1) A total of 105,259 unigenes were assembled, and 23,063 unigenes were perfectly annotated. The results of the PacBio Iso-Seq and IIIumina RNA-Seq enriched the genetic database of S. nudus. (2) A total of 830 DEGs were detected in S. nudus from the different groups. In particular, 33 DEGs had differential expression in the top nine KEGG pathways related to pathogens, protein synthesis, and cellular immune response and signaling. The results indicate that S. nudus from different zones experience different environmental stresses. (3) Several DEGs (HSPA1, NFKBIA, eEF1A, etc.) in pathways related to pathogens (influenza A, legionellosis, measles, and toxoplasmosis) had higher expression in groups M and L. HSPA1 was clearly enriched in most of the pathways, followed by NFKBIA. The results show that the peanut worms from the M and L tidal flats might have suffered more severe environmental conditions. (4) Some DEGs (MKP, MRAS, and HSPB1) were upregulated in peanut worms from the H tidal flat, and these DEGs were mainly involved in the MAPK signaling pathway. These results indicate that the MAPK pathway may play a vital role in the immune response of the peanut worm to the effects of different intertidal flats. This study provides a valuable starting point for further studies to elucidate the molecular basis of the response to different environmental stresses in S. nudus.

Genetic Signature of Pinctada fucata Inferred from Population Genomics: Source Tracking of the Invasion in Mischief Reef of Nansha Islands.

Among the anthropogenic stresses that marine ecosystems face, biological invasions are one of the major threats. Recently, as a result of increasingly intense anthropogenic disturbance, numerous marine species have been introduced to their non-native ranges. However, many introduced species have uncertain original sources. This prevents the design and establishment of methods for controlling or preventing these introduced species. In the present study, genomic sequencing and population genetic analysis were performed to detect the geographic origin of the introduced Pinctada fucata population in the Mischief Reef of the South China Sea. The results of population genetic structure analysis showed a close relationship between the Mischief Reef introduced population and the Lingshui population, indicating that Lingshui may be the potential geographical origin. Furthermore, lower heterozygosity and nucleotide diversity were observed in the introduced population in Mischief Reef, indicating lower genetic diversity than in other native populations. We also identified some selected genomic regions and genes of the introduced population, including genes related to temperature and salinity tolerance. These genes may play important roles in the adaptation of the introduced population. Our study will improve our understanding of the invasion history of the P. fucata population. Furthermore, the results of the present study will also facilitate further control and prevention of invasion in Mischief Reef, South China Sea.

The complete mitochondrial genome and phylogenetic analysis of the ocellated angelshark: Squatina tergocellatoides Chen, 1963.

The ocellated angelshark (Squatina tergocellatoides Chen, 1963) is a threatened shark within the family Squatinidae. In the present study, we reported the mitochondrial genome sequence of the ocellated angelshark. The complete mitochondrial genome is 16,683 bp in length and contains 37 mitochondrial genes and a control region as similar to most fishes. In addition, we constructed a maximum-likelihood phylogenetic tree of S. tergocellatoides and its relative species. This work will provide molecular data for further studies on S. tergocellatoides.

Comparative transcriptome sequencing analysis of female and male Decapterus macrosoma.

Sexual growth dimorphism is a common phenomenon in teleost fish and has led to many reproductive strategies. Growth- and sex-related gene research in teleost fish would broaden our understanding of the process. In this study, transcriptome sequencing of shortfin scad Decapterus macrosoma was performed for the first time, and a high-quality reference transcriptome was constructed. After identification and assembly, a total of 58,475 nonredundant unigenes were obtained with an N50 length of 2,266 bp, and 28,174 unigenes were successfully annotated with multiple public databases. BUSCO analysis determined a level of 92.9% completeness for the assembled transcriptome. Gene expression analysis revealed 2,345 differentially expressed genes (DEGs) in the female and male D. macrosoma, 1,150 of which were female-biased DEGs, and 1,195 unigenes were male-biased DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the DEGs were mainly involved in biological processes including protein synthesis, growth, rhythmic processes, immune defense, and vitellogenesis. Then, we identified many growth- and sex-related genes, including Igf, Fabps, EF-hand family genes, Zp3, Zp4 and Vg. In addition, a total of 19,573 simple sequence repeats (SSRs) were screened and identified from the transcriptome sequences. The results of this study can provide valuable information on growth- and sex-related genes and facilitate further exploration of the molecular mechanism of sexual growth dimorphism.

Complete Mitochondrial Genome of Two Ectoparasitic Capsalids (Platyhelminthes: Monogenea: Monopisthocotylea): Gene Content, Composition, and Rearrangement.

The capsalid monogeneans are important pathogens that generally infect marine fishes and have a substantial impact on fish welfare in aquaculture systems worldwide. However, the current mitogenome information on capsalids has received little attention, limiting the understanding of their evolution and phylogenetic relationships with other monogeneans. This paper reports the complete mitochondrial genomes of Capsala katsuwoni and Capsala martinieri for the first time, which we obtained using a next-generation sequencing method. The mitogenomes of C. katsuwoni and C. martinieri are 13,265 and 13,984 bp in length, respectively. Both species contain the typical 12 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. The genome compositions show a moderate A+T bias (66.5% and 63.9% for C. katsuwoni and C. martinieri, respectively) and exhibit a negative AT skew but a positive GC skew in both species. One gene block rearrangement was found in C. katsuwoni in comparison with other capsalid species. Instead of being basal to the Gyrodactylidea and Dactylogyridea or being clustered with Dactylogyridea, all species of Capsalidea are grouped into a monophyletic clade. Our results clarify the gene rearrangement process and evolutionary status of Capsalidae and lay a foundation for further phylogenetic studies of monogeneans.

Spatial Distribution, Potential Risks and Source Identification of Heavy Metals in the Coastal Sediments of the Northern Beibu Gulf, South China Sea.

Thirty samples of surface sediments (0-5 cm) from the northern Beibu Gulf were analyzed to determine the spatial distribution, potential risks and sources of six heavy metals (Cr, Cu, Zn, As, Cd and Pb). The concentrations (mg/kg, dw) of Cr, Cu, Zn, As, Cd and Pb were 15.38 ± 6.06, 6.54 ± 3.23, 41.86 ± 17.03, 6.92 ± 2.75, 0.04 ± 0.02 and 17.13 ± 6.38, respectively. Higher levels of Cr, Cu, Cd and Zn were observed in the western part of the study area. According to the potential ecological risk indexes and sediment quality guidelines, the measured metals were assessed at low contamination levels, with Pb posing the largest ecological risks. The results of positive matrix factorization (PMF) indicated that Cr and Zn mainly originated from natural geological background sources, while Cu, As, Cd and Pb were influenced by anthropogenic sources such as atmospheric deposition and anthropogenic activities. These three sources contributed 60.4%, 28.1% and 11.5% of the heavy metals, respectively. In addition, further research should be conducted focusing on the general relationships between As and various controls in sediments of the northern Beibu Gulf.

Transcriptomic analysis of male three-spot swimming crab (Portunus sanguinolentus) infected with the parasitic barnacle Diplothylacus sinensis.

Diplothylacus sinensis is reported as an intriguing parasitic barnacle that can negatively affect the growth, molting, reproduction in several commercially important portunid crabs. To better understand the molecular mechanisms of host-parasite interactions, we characterized the gene expression profiles from the healthy and D. sinensis infected Portunus sanguinolentus by high-through sequence method. Totally, the transcriptomic analysis generated 52, 266, 600 and 51, 629, 604 high quality reads from the infected and control groups, respectively. The clean reads were assembled to 90,740 and 69,314 unigenes, with the average length of 760 bp and 709 bp, respectively. The expression analysis showed that 18,959 genes were significantly changed by the parasitism of D. sinensis, including 4769 activated genes and 14,190 suppressed genes. The differentially expressed genes were categorized into 258 KEGG pathways and 647 GO terms. The GO analysis mapped 13 DEGs related to immune system process and 32 DEGs related to immune response, respectively, suggesting a potential alteration of transcriptional expression patterns in complement cascades of P. sanguinolentus. Additionally, 4 representative molting-related genes were down-regulated in parasitized group, indicating D. sinensis infection appeared to suppress the producing of ecdysteroid hormones. In conclusion, the present study improves our understanding on parasite-host interaction mechanisms, which focuses the function of Ecdysone receptor, Toll-like receptor and cytokine receptor of crustacean crabs infestation with rhizocephalan parasites.

Efficacy of EGFR-TKI sequential therapy in patients with EGFR exon 19 insertion-positive non-small-cell lung cancer: A case report.

BACKGROUND: Insertions in exon 19 in the epidermal growth factor receptor gene (EGFR) is a rarely seen mutation in non-small cell lung cancer. These patients have been effectively treated with sequential EGFR tyrosine kinase inhibitors (TKIs). CASE SUMMARY: Here, we presented a case of non-small cell lung cancer, stage IIIB, with EGFR exon 19 insertion mutation as detected in the right lower lobe by next-generation sequencing. The patient was sequentially treated with first, second, and third-generation EGFR TKIs after the surgical operation. The overall survival of the patient was 21.3 mo. There was no dynamic analysis of drug resistance mechanisms in targeted therapy. CONCLUSION: This case emphasized the importance of following the guidelines. In patients with EGFR mutations, repeated and dynamic next-generation sequencing monitoring is necessary to prescribe a personalized treatment plan.

Next generation sequencing yields the complete mitochondrial genome of the darkfin hind Cephalopholis urodeta (Serranidae) from the Mischief Reef, South China Sea.

The complete mitochondrial genome of the Cephalopholis urodeta (Serranidae: Epinephelinae) was sequenced by next-generation sequencing method on the basis of one female specimen collected from coral reef areas at the Mischief Reef, South China Sea. The mitogenome is 16,593 bp in length, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and a D-loop region. The overall content of A + T is 55.66%, which is significantly higher than the C + G content (44.34%). Among four bases, C shows the lowest frequency (15.99%). Phylogenetic tree based on the 13 protein-coding genes was constructed for analyzing the position of C. urodeta. The results provide useful insights into further studies on population genetics and phylogenetics of groupers.

The complete mitochondrial genome and phylogenetic analysis of the reticulated swellshark: Cephaloscyllium fasciatum Chan, 1966.

In the present study, the complete mitogenome of Cephaloscyllium fasciatum has been sequenced and assembled. The complete mitochondrial genome is 16,509 bp in length and contains 37 mitochondrial genes and a control region as other fishes. The nucleotide composition of C. fasciatum mitogenome showed an anti-G bias and an excess of AT content. Furthermore, maximum-likelihood phylogenetic tree was constructed based on 13 protein-coding genes of C. fasciatum and other 15 sharks. This work will provide molecular data for studies on phylogeny and evolution in the family Scyliorhinidae.

De novo assembly and annotation of the whole transcriptome of Muraenesox cinereus.

In this study, we generated the transcriptome of Muraenesox cinereus from four combined tissues (muscle, sexual, liver and heart) using high-seq sequencing technology. De novo assembly was performed using Trinity software and a total of 62,125,296 high-quality clean reads were assembled and clustered into 75,862 unigene with an N50 of 2034 nt. After annotation, 43,157 unigenes had significant hit in Nr database. And then, 24,510 unigenes were annotated into three GO categories: biological processes, cellular components, and molecular functions. Moreover, 33,032 unigenes were mapped 25 different clusters of eukaryotic proteins. Furthermore, we predicted the structure of all unigenes using HMMER and MISA software, respectively. The result showed that a total of 33,183 nucleotide sequences of coding regions (direction of the sequences is 5'- > 3') were confirmed to the protein database and a total of 29,487 simple sequence repeats (SSR) were identified. The whole transcriptome is an important foundation for future genomic research on the M. cinereus, it could provide comprehensively understanding and further characterizations of transcriptomes of non-model organisms.

Identifying Genomic Alterations in Small Cell Lung Cancer Using the Liquid Biopsy of Bronchial Washing Fluid.

Objective: With the rapid development of cancer genomics and immunomics, some new treatments of small cell lung cancer (SCLC) are emerging. However, there are limitations to the clinical use of tumor tissue. Our study aimed to evaluate the potential use of bronchial washing fluid (BWF) in the liquid biopsy of SCLC. Methods: Twenty-one extensive SCLC (ES-SCLC) patients were enrolled in this study. For all patients, four sample types, BWF supernatant (BWFs), BWF precipitate (BWFp), plasma and tumor tissue, were collected before receiving chemotherapy, and one type, plasma, was collected after chemotherapy. All samples were conducted to NGS using the 1021-gene panel. The concordance rates of genomic profiling using NGS in the four types of samples were evaluated. Multiple clinical information was analyzed for correlation. Results: We successfully tested 20 BWFs samples, 21 BWFp samples, 21 tumor tissue samples, 20 pre-treatment plasma, and 13 post-treatment plasma of these 21 patients. The detectability of somatic mutations was 100% for BWFs, BWFp, tumor tissues, and post-treatment plasma, and only one pre-treatment plasma was absent with any mutation. Matched tumor tissue, BWFs, BWFp, and pre-treatment plasma samples were subsistent for 19 patients. For these patients, 204 genomic alterations were identified in tissue samples, while 189 (92.6%), 175 (85.5%), and 163 (79.9%) alterations were detected in the matched BWFs, BWFp, and pre-treatment plasma, respectively. Moreover, we found that the three tumor markers associated with SCLC have a lower sensitivity than genomic alterations. The endocrine resistance pathway was found enriched in hyponatremia patients which may be related to the hyponatremia. The TMBs of BWF, BWFp, and pre-treatment plasma samples all had a strong correlation with that of tissue samples. Both the VAF and the MVAF of mutations in post-treatment plasma were less than those in pre-treatment plasma, which was in accordance with the evaluation of curative effect. Conclusions: For ES-SCLC patients, the liquid biopsy of BWF showed a highly potential advantage to identify DNA alterations, which suggested that genomic analysis of BWF liquid biopsy may have clinical value as a supplement for tissue and blood detection. Through the restricted validation, it can be widely used in routine clinical practice.

The complete mitochondrial genome of Heniochus chrysostomus (Perciformes, Chaetodontidae).

We report the first mitochondrial genome sequences for the three band pennant fish, Heniochus chrysostomus. The whole mitogenome of H. chrysostomus was circular in shape and 16,650 bp in length. The mitogenome consists of 13 typical vertebrate protein-coding genes, 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), and 2 putative non-coding regions. Phylogenetic tree analysis revealed that H. chrysostomus was closely related to Heniochus diphreutes. This study will provide useful genetic information for future phylogenetic and taxonomic classification of Chaetodontidae.

The complete mitochondrial genome of the Capsaloides cristatus (Platyhelminthes, Monogenea), a pathogen of the sailfish (Istiophorus platypterus).

This study reports the complete mitochondrial genome of the Capsaloides cristatus (Monogenea: Capsalidae) collected from the gill lamella of Istiophorus platypterus. The total length of the mitogenome was 13,948 bp, containing 12 typical platyhelminthic protein-coding genes, 22 tRNA genes, 2 rRNA genes and a putative non-coding region, with the atp8 gene being absent. The total A + T content was 65.99%, which was significantly higher than that of the C + G content (34.01%). There were two kinds of start codons (ATG and GTG) and three kinds of terminated codons (TAA, TAG and TGA) in the 12 protein-coding genes. Phylogentic analysis revealed close relationships among the genera Capsaloides, Capsala, Benedenia and Neobenedenia with high bootstrap value supported. This study will provide useful molecular data for a better understanding of the species identification and phylogenetic position of C. cristatus.

Heavy metal pollution and stable isotope ratios (δ13C and δ15N) in marine organisms from the Northern Beibu Gulf, South China Sea.

Twenty biota species were collected from the Northern Beibu Gulf to understand the heavy metal pollution status and biomagnification characteristics. Mean concentrations (µg/g) of Mn, Zn, Pb, Cr, Ni, As, Cu, and Cd in the biota species were 0.99, 38.33, 0.14, 0.079, 0.085, 51.10, 7.92, and 0.21, respectively. As levels in most biota species exceeded the corresponding guidelines. Crabs were detected with high metal accumulation ability for each heavy metal except Pb. The ranges of δ15N and δ13C in organisms were from 8.0‰ to 15.6‰ and from -21.4‰ to -15.6‰, respectively. Cr, Mn, Ni, and As showed potential biomagnification trends in the food webs. The EDI values of these elements decreased by the sequence of As > Zn > Cu > Mn > Cd > Pb > Ni > Cr. High THQ and CR values for As indicated a potential health risk by consumption of these aquatic products.

Comparative transcriptomic analysis for identification of candidate sex-related genes and pathways in Crimson seabream (Parargyrops edita).

Teleost fishes display the largest array of sex-determining systems among animals, resulting in various reproductive strategies. Research on sex-related genes in teleosts will broaden our understanding of the process, and provide important insight into the plasticity of the sex determination process in vertebrates in general. Crimson seabream (Parargyrops edita Tanaka, 1916) is one of the most valuable and abundant fish resources throughout Asia. However, little genomic information on P. edita is available. In the present study, the transcriptomes of male and female P. edita were sequenced with RNA-seq technology. A total of 388,683,472 reads were generated from the libraries. After filtering and assembling, a total of 79,775 non redundant unigenes were obtained with an N50 of 2,921 bp. The unigenes were annotated with multiple public databases, including NT (53,556, 67.13%), NR (54,092, 67.81%), Swiss-Prot (45,265, 56.74%), KOG (41,274, 51.74%), KEGG (46,302, 58.04%), and GO (11,056, 13.86%) databases. Comparison of the unigenes of different sexes of P. edita revealed that 11,676 unigenes (9,335 in females, 2,341 in males) were differentially expressed between males and females. Of these, 5,463 were specifically expressed in females, and 1,134 were specifically expressed in males. In addition, the expression levels of ten unigenes were confirmed to validate the transcriptomic data by qRT-PCR. Moreover, 34,473 simple sequence repeats (SSRs) were identified in SSR-containing sequences, and 50 loci were randomly selected for primer development. Of these, 36 loci were successfully amplified, and 19 loci were polymorphic. Finally, our comparative analysis identified many sex-related genes (zps, amh, gsdf, sox4, cyp19a, etc.) and pathways (MAPK signaling pathway, p53 signaling pathway, etc.) of P. edita. This informative transcriptomic analysis provides valuable data to increase genomic resources of P. edita. The results will be useful for clarifying the molecular mechanism of sex determination and for future functional analyses of sex-associated genes.

Heavy metal concentrations and associated health risks in edible tissues of marine nekton from the outer Pearl River Estuary, South China Sea.

Seven heavy metals including Hg, Cu, Pb, Cd, Zn, Cr, and As were examined in seventeen marine nekton species from the outer Pearl River Estuary (PRE), South China Sea. On the wet weight basis, the metal concentration ranges were 0.016-0.157 µg/g for Hg, 0.18-14.3 µg/g for Cu, 0.26-1.48 µg/g for Pb, 0.021-0.873 µg/g for Cd, 1.35-57.15 µg/g for Zn, 0.15-0.53 µg/g for Cr, and 0.42-7.83 µg/g for As, respectively. The levels of tested metals except for Pb in crustaceans were found to be higher than those in fish and cephalopods, suggesting that the diet and habitat played important roles on heavy metal accumulation ability of marine organism. Except for Cd in Champsodon capensis, Calappa lophos, and Portunus argentatus, all the left metal concentrations of investigated nekton species were below their permissible upper limits, indicating that consumption of examined marine nekton should be considered as safe for human health. The values of single target hazard quotient (THQ) and total THQ were all less than 1 and also suggested that there was no health risk for consumption. Even so, the local people should control their daily intake of crustacean foods from the outer PRE, since there might be potential As and Cd cumulative risks.

The complete mitochondrial genome of milk shark, Rhizoprionodon acutus (Ruppell 1837).

The complete mitogenome sequence of the milk shark, of which Rhizoprionodon acutus was determined using long PCR. The genome was 16,693 bp in length and it contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 D-loop region. The overall base composition of the heavy strand is A (31.63%), C (23.91%), T (31.38%), and G (13.08%). However, the base composition of A (31.38%), C (13.08%), T (31.63%), and G (23.91%) was in the light strand. Except for that COXI began with GTG, all the protein initiation codons were ATG. What's more, all the protein termination codons were TAA, in addition to ND2, ND3, and Cytb ended with TAG, COXII and ND4 finished only with T, and ND6 with AGG. Moreover, based on thirteen concatenated PCGs nucleotide datasets, the phylogenetic analysis showed high value support for the following sister clade among the two genera (Rhizoprionodon acutus, Rhizoprionodon; Scoliodon macrorhynchos and Scoliodon laticaudus, Scoliodon).

Genetic effects of released swimming crab (Portunus trituberculatus) on wild populations inferred from mitochondrial control region sequences.

Along the coast of Shandong Province in China, an extensive hatchery-release programme has been conducted for more than 10 years. However, no information has been reported concerning the long-term effects on the wild population resulting from the release of large numbers of juveniles in Portunus trituberculatus. In this study, sequence variation of swimming crab P. trituberculatus based on the mitochondrial control region was investigated for 946 swimming crabs at the releasing sites from 2012 to 2014. The result showed that the wild P. trituberculatus was characterized by high genetic diversity indices, and not significantly different from the previous study. Low FST values were estimated among the groups of different years and different sites, which suggested no genetic differentiation found in the wild population after the stock enhancement programme. Hence, the long-term extensive hatchery release programme has not affected the genetic structure of wild P. trituberculatus populations along the coast of Shandong Province.

Synchronous multiple carcinoma with small intestine and pulmonary neuroendocrine involvement: A case report.

RATIONALE: In clinical work, neuroendocrine synchronous multiplicity carcinoma was relatively rare. Most were confirmed by the pathological diagnosis of a certain part of the body combined with the imaging of the whole body, while cases that had both pathological and immunohistochemistry diagnosis were few. PATIENT CONCERNS: A patient who presented with abdominal pain visited our hospital, and was diagnosed with lesions in both the small intestine and lung. DIAGNOSES: Both were considered primary tumors by imaging, and diagnosed as neuroendocrine carcinomas by pathology. INTERVENTIONS: The intestinal lesion was surgically resected, and the lung tumor treated by chemoradiotherapy. OUTCOMES: The survival time of this patient exceeded 24 months. LESSONS: The diagnosis relied on clinical, imaging, pathological, and immunohistochemical features, which confirmed a synchronous multiple carcinoma. Treatment was based on the pathological types. Through this case report, the clinical and pathological data of neuroendocrine synchronous multiplicity carcinoma could be enriched.