Seasonal Dynamics of Antibiotic Resistance Genes and Mobile Genetic Elements in a Subtropical Coastal Ecosystem: Implications for Environmental Health Risks.

Antibiotic resistance poses a considerable global public health concern, leading to heightened rates of illness and mortality. However, the impact of seasonal variations and environmental factors on the health risks associated with antibiotic resistance genes (ARGs) and their assembly mechanisms is not fully understood. Based on metagenomic sequencing, this study investigated the antibiotic resistome, mobile genetic elements (MGEs), and microbiomes in a subtropical coastal ecosystem of the Beibu Gulf, China, over autumn and winter, and explored the factors influencing seasonal changes in ARG and MGE abundance and diversity. Results indicated that ARG abundance and diversity were higher in winter than in autumn, with beta-lactam and multidrug resistance genes being the most diverse and abundant, respectively. Similarly, MGE abundance and diversity increased in winter and were strongly correlated with ARGs. In contrast, more pronounced associations between microbial communities, especially archaea, and the antibiotic resistome were observed in autumn than in winter. The co-occurrence network identified multiple interactions between MGEs and various multidrug efflux pumps in winter, suggesting a potential for ARG dissemination. Multivariate correlation analyses and path modeling indicated that environmental factors driving microbial community changes predominantly influenced antibiotic resistome assembly in autumn, while the relative importance of MGEs increased significantly in winter. These findings suggest an elevated health risk associated with antimicrobial resistance in the Beibu Gulf during winter, attributed to the dissemination of ARGs by horizontal gene transfer. The observed seasonal variations highlight the dynamic nature of antibiotic resistance dissemination in coastal ecosystems, emphasizing the need for comprehensive surveillance and management measures to address the growing threat of antimicrobial resistance in vulnerable environments.

miRNA-seq provides novel insight into the response to hyper- and hypo- salinity acclimation in Crassostrea hongkongensis.

The Hong Kong oyster, Crassostrea hongkongensis, is a significant bivalve species with economic importance. It primarily inhabits the estuarine intertidal zones in southern China, making it susceptible to salinity fluctuations. Consequently, investigating the molecular mechanisms governing salinity regulation in C. hongkongensis is essential. In this study, we conducted miRNA-seq on C. hongkongensis to compare miRNA expression differences under varying salinities (5‰, 25‰, and 35‰). The miRNA sequencing revealed 51 known miRNAs and 95 novel miRNAs across nine small RNA libraries (S5, S25, and S35). Among these miRNAs, we identified 6 down-regulated differentially expressed (DE) miRNAs in response to hypo-salinity stress (5‰), while 1 up-regulated DE miRNA and 5 down-regulated DE miRNAs were associated with hyper-salinity stress (35‰). Additionally, we predicted 931 and 768 potential target genes for hypo- and hyper-salinity stress, respectively. Functional gene annotation indicated that the target genes under hypo-salinity stress were linked to vesicle-mediated transport and metal ion binding. Conversely, those under hyper-salinity stress were primarily involved in signal transduction and metabolic processes. These findings have provided insights into the regulatory role of miRNAs, their potential target genes and associated pathways in oyster hypo- and hyper-salinity stress, which establish a foundation for future studies on the roles of miRNAs in salinity acclimation mechanisms in C. hongkongensis.

Transcriptional responses of Crassostrea hongkongensis under high and low salinity stress.

Salinity, a key limiting factor, affects the distribution and survival of marine species. The Hong Kong oyster (Crassostrea hongkongensis), a euryhaline species found along the coast of the South China Sea, has become a major aquaculture bivalve species. To determine the molecular mechanism by which oysters respond to coastal waters with varying salinity levels, we used RNA-seq to sequence the gill samples of oysters exposed to normal (25 ‰, S25), low (5 ‰, S5) and high (35 ‰, S35) salinity conditions for one month. The results revealed different expression transcriptome levels among oysters living under low and high salinity conditions. Using high-throughput sequencing, we identified 811 up-regulated genes and 769 down-regulated genes. As determined by KEGG pathway mapping, the differentially expressed genes (DEGs) were significantly enriched in the prion diseases, histidine metabolism, arginine and proline metabolism, and beta-alanine metabolism pathways in both the S5 vs. S25 and S35 vs. S25 group comparison. Several DEGs including heat shock 70 kDa protein 12B-like, poly (ADP-ribose) polymerase (PARP), and tripartite motif-containing protein 2 (TRIM2), and low-density lipoprotein receptor-like, as well as KEGG pathways, including arginine and proline metabolism, apoptosis, PPAR signaling pathway, the thyroid hormone signaling pathway, were concerning response to salinity stress. Additionally, eight DEGs involved in salinity adaptation were selected for RT-qPCR validation, and the results confirmed the credibility of the transcriptome sequencing data. Overall, we designed a one-month, medium-term experiment to examine the responses of C. hongkongensis exposed to different levels of salinity stress and performed transcriptome analysis using high-throughput sequencing. Our results enhance current understanding of the molecular mechanisms of salinity stress responses in C. hongkongensis and provided insights into the osmotic biology of oysters.

Whole genome sequencing of Crassostrea ariakensis (Mollusca: Ostreidae) and C. hongkongensis expands understandings of stress resistance in sessile oysters.

To understand the environmental adaptations among sessile bivalves lacking adaptive immunity, a series of analyses were conducted, with special emphasis on the widely distributed C. ariakensis. Employing Pacbio sequencing and Hi-C technologies, whole genome for each of a C. ariakensis (southern China) and C. hongkongensis individual was generated, with the contig N50 reaching 6.2 and 13.0 Mb, respectively. Each genome harbored over 30,000 protein-coding genes, with approximately half of each genome consisting of repeats. Genome alignment suggested possible introgression between C. gigas and C. ariakensis (northern China), and re-sequencing data corroborated this result and indicated significant gene flow between C. gigas and C. ariakensis. These introgressed candidates, well-represented by genes related to immunity and osmotic pressure, may be associated with environmental stresses. Gene family dynamics modeling suggested immune-related genes were well represented among the expanded genes in C. ariakensis. These outcomes could be attributed to the spread of C. ariakensis.

The male and female genomes of golden pompano (Trachinotus ovatus) provide insights into the sex chromosome evolution and rapid growth.

INTRODUCTION: Golden pompano (Trachinotus ovatus) is economically significant important for offshore cage aquaculture in China and Southeast Asian countries. Lack of high-quality genomic data and accurate gene annotations greatly restricts its genetic breeding progress. OBJECTIVES: To decode the mechanisms of sex determination and rapid growth in golden pompano and facilitate the sex- and growth-aimed genetic breeding. METHODS: Genome assemblies of male and female golden pompano were generated using Illumina, PacBio, BioNano, genetic maps and Hi-C sequencing data. Genomic comparisons, whole genome re-sequencing of 202 F1 individuals, QTL mapping and gonadal transcriptomes were used to analyze the sex determining region, sex chromosome evolution, SNP loci, and growth candidate genes. Zebrafish model was used to investigate the functions of growth candidate gene. RESULTS: Female (644.45 Mb) and male (652.12 Mb) genomes of golden pompano were assembled and annotated at the chromosome level. Both genomes are highly conserved and no new or highly differentiated sex chromosomes occur. A 3.5 Mb sex determining region on LG15 was identified, where Hsd17b1, Micall2 and Lmx1a were putative candidates for sex determination. Three SNP loci significantly linked to growth were pinpointed, and a growth-linked gene gpsstr1 was identified by locus BSNP1369 (G â†’ C, 17489695, Chr23). Loss of sstr1a (homologue of gpsstr1) in zebrafish caused growth retardation. CONCLUSION: This study provides insights into sex chromosome evolution, sex determination and rapid growth of golden pompano.

Candidate sex-associated gene identification in Trachinotus ovatus (Carangidae) using an integrated SLAF-seq and bulked segregant analysis approach.

It is difficult to distinguish the sexes of Trachinotus ovatus based on appearance, and little data about sex-determining genes are available for this species. Here, we generated 200 F2 individuals using the parents R404 and R403. DNA samples were collected from 50 individuals of each sex and aggregated into sex-specific DNA pools. Specific-locus amplified fragment sequencing was integrated with bulked segregant analysis to detect candidate sex-associated genes. Approximately 3,153,153 high-quality single-nucleotide polymorphism (SNP) markers and 135,363 high-quality insertion-deletion (Indel) markers were generated. Six candidate regions within chromosome 14, encompassing 132 candidate genes, were identified as closely related to sex. Based on annotations, six genes (EVM0019817, EVM0004192, EVM0001445, EVM0005260, EVM0014734, and EVM0009626) were predicted to be closely associated with sex. These results present an efficient genetic mapping approach that lays a foundation for molecular sex discrimination in T. ovatus.

Transcriptomic analysis of Litopenaeus vannamei hepatopancreas under cold stress in cold-tolerant and cold-sensitive cultivars.

Litopenaeus vannamei (L. vannamei) is one of the most widely cultured shrimp species in the world. The species often suffers from cold stress. To understand the molecular mechanism of cold tolerance, we performed transcriptomic analysis on two contrasting cultivars of L. vannamei, namely, cold-tolerant Guihai 2 (GH2) and cold-sensitive Guihai1 (GH1), under a control temperature (28 °C), cold stress (16 °C), and recovery to 28 °C. A total of 84.5 Gb of sequences were generated from 12 L. vannamei hepatopancreas libraries. The de-novo assembly generated a total of 143,029 unigenes with a mean size of 1,052 bp and an N50 of 2,604 bp, of which 34.08% were annotated in the Nr database. We analyzed the differentially expressed genes (DEGs) between nine comparison groups and detected a total of 21,026 DEGs. KEGG pathways, including lysosome, sphingolipid metabolism and nitrogen metabolism, were significantly enriched by DEGs between different temperatures in GH2. Furthermore, eight of the most significantly DEGs under cold stress from the transcriptomic analysis were selected for quantitative real-time PCR (qPCR) validation. Overall, we compared gene expression changes under cold stress in cold-tolerant and cold-sensitive L. vannamei for the first time. The results may further extend our understanding of the cold stress-response mechanism in L. vannamei.

Chromosome-level analysis of the Crassostrea hongkongensis genome reveals extensive duplication of immune-related genes in bivalves.

Crassostrea hongkongensis is a popular and important native oyster species that is cultured mainly along the coast of the South China Sea. However, the absence of a reference genome has restricted genetic studies and the development of molecular breeding schemes for this species. Here, we combined PacBio and 10 × Genomics technologies to create a C. hongkongensis genome assembly, which has a size of 610 Mb, and is close to that estimated by flow cytometry (~650 Mb). Contig and scaffold N50 are 2.57 and 4.99 Mb, respectively, and BUSCO analysis indicates that 95.8% of metazoan conserved genes are completely represented. Using a high-density linkage map of its closest related species, C. gigas, a total of 521 Mb (85.4%) was anchored to 10 haploid chromosomes. Comparative genomic analyses with other molluscs reveal that several immune- or stress response-related genes extensively expanded in bivalves by tandem duplication, including C1q, Toll-like receptors and Hsp70, which are associated with their adaptation to filter-feeding and sessile lifestyles in shallow sea and/or deep-sea ecosystems. Through transcriptome sequencing, potential genes and pathways related to sex determination and gonad development were identified. The genome and transcriptome of C. hongkongensis provide valuable resources for future molecular studies, genetic improvement and genome-assisted breeding of oysters.

Identification and characterization of microRNAs in the gonads of Crassostrea hongkongensis using high-throughput sequencing.

Crassostrea hongkongensis is one of the three most-commonly cultivated oyster species in China. Although microRNAs (miRNAs) expression in the gonads have been widely investigated, few studies of miRNAs in mollusk gonads are available, particularly in oyster. In the present study, we analyzed the miRNAs expressed in the ovaries and testes of C. hongkongensis. We obtained 14,166,409 and 15,133,900 raw reads from the ovaries and testes, respectively, yielding 13,634,997 (ovarian) and 14,494,149 (testicular) 18-35-nt sequences. We mapped these sequences to the C. hongkongensis genome reference sequence, and identified 8,771,717 (ovarian) and 9,926,014 (testicular) sequences corresponding to miRNAs in the Rfam database. After blasting the miRNA sequences against the miRBase database, we identified 50 known mature miRNAs and 53 novel miRNAs. Of these, 27 miRNAs were significantly upregulated in ovaries as compared to the testes, and 43 miRNAs were significantly upregulated in the testes as compared to the ovaries. To validate the differential expression results generated by Illumina sequencing, we used RT-real-time quantitative PCR (RT-qPCR) to characterize the expression patterns of the six most differently expressed miRNAs (lgi-miR-1990, lgi-miR-1986, lgi-miR-263b, lgi-miR-279, lgi-miR-1992, and novel_98) as well as two miRNAs associated with gonad development (lgi-miR-29 and lgi-miR-8). Most of the RT-qPCR miRNA expression patterns were similar to those recovered by high-throughput sequencing with the exceptions of novel_98 and lgi-miR-1992. Gene Ontology (GO) annotations indicated that the multi-organism cellular process GO category was enriched with the target genes of the differentially expressed miRNAs. Additionally, the target genes were enriched in several KEGG pathways, including the ECM-receptor interaction, galactose metabolism, phagosome, and notch signaling pathway. These pathways are involved in gonadal differentiation and the maintenance of gonad function. This identification and characterization of the miRNAs differentially expressed between the ovaries and testes of C. hongkongensis will increase our understanding of the role of miRNAs in gonad differentiation in the oyster.

Identification and characterization of microRNAs in the gonad of Trachinotus ovatus using Solexa sequencing.

The Trachinotus ovatus (T. ovatus) reach sexual maturity as late as 4-5 years, and there are no obvious morphology differences between males and females, even at maturity, making the selection of male and female parents for selective breeding difficult. To examine the potential regulatory mechanism of sexual differentiation, we conducted a microRNAs (miRNAs) analysis on the ovaries and testes of T. ovatus. A total of 13,423,691 and 11,734,953 raw reads, representing 91,883,908 and 86,879,726 unique sequences of 18-35 nt length obtained from the ovaries and testes, respectively. After mapping to the T. ovatus transcriptome reference sequence (unpublished data), and comparing the miRNA sequences with the miRBase database, 179 known mature miRNAs and 100 novel miRNAs were identified. Expression analysis showed that, 165 miRNAs were differentially expressed between the ovary and testis. To validate the Illumina results, the expression patterns of the nine most differently expressed miRNAs (dre-miR-7a, dre-miR-7b, dre-miR-153a-3p, dre-miR-144-3p, dre-miR-301a, dre-miR-92a-3p, dre-miR-727-5p, Novel 124, and Novel 190, as well as those of five miRNAs that may relate to gonad development (dre-miR-143, dre-miR-101a, dre-miR-202-5p, dre-let-7c-5p, and dre-miR-181a-5p), were assayed with RT-qPCR. The expression trends for all the miRNAs validated with RT-qPCR were consistent with the next-generation sequencing data. The identification and characterization of the miRNAs differentially expressed between the ovary and the testis of the T. ovatus will increase our understanding of the role of miRNAs in gonad differentiation. These data also facilitate studies on miRNA regulation of teleost reproduction.

mRNA and microRNA transcriptomics analyses in intermuscular bones of two carp species, rice flower carp (Cyprinus carpio var. Quanzhounensis) and Jian carp (Cyprinus carpio var. Jian).

The rice flower carp (Cyprinus carpio var. Quanzhounensis) is a bony fish (superclass Osteichthyes), with very soft bones that is a significant feature unlike the other carp species. In this study, we analyzed the mRNA and microRNA (miRNA) transcriptomes in the intermuscular bones of rice flower carp and Jian carp (Cyprinus carpio var. Jian) (a typical member of common carp in China), using Illumina RNA sequencing. We identified 55,340 genes (including 47,541 known genes and 8231 predicted new genes) and 662 miRNAs (including 595 known miRNAs and 67 novel miRNAs) in the two species. By comparing the transcriptomes of the two species, we identified 1523 differentially expressed genes (DEGs) (including 576 up - and 947 downregulated DEGs) and 352 differentially expressed miRNAs (DEMs) (including 85 up- and 267 downregulated DEMs). According to the Gene Ontology (GO) annotation, 7 DEGs and 12 DEMs were found to be involved in the regulation of bone mineralization. The results of this study improve our understanding of the mRNA and miRNA profiles of carp bones, particularly those of the rice flower carp.

Single-molecule long-read sequencing facilitates shrimp transcriptome research.

Although shrimp are of great economic importance, few full-length shrimp transcriptomes are available. Here, we used Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology to generate transcripts from the Pacific white shrimp (Litopenaeus vannamei). We obtained 322,600 full-length non-chimeric reads, from which we generated 51,367 high-quality unique full-length transcripts. We corrected errors in the SMRT sequences by comparison with Illumina-produced short reads. We successfully annotated 81.72% of all unique SMRT transcripts against the NCBI non-redundant database, 58.63% against Swiss-Prot, 45.38% against Gene Ontology, 32.57% against Clusters of Orthologous Groups of proteins (COG), and 47.83% against Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Across all transcripts, we identified 3,958 long non-coding RNAs (lncRNAs) and 80,650 simple sequence repeats (SSRs). Our study provides a rich set of full-length cDNA sequences for L. vannamei, which will greatly facilitate shrimp transcriptome research.

Proteomic Responses Under Cold Stress Reveal Unique Cold Tolerance Mechanisms in the Pacific White Shrimp (Litopenaeus vannamei).

The Pacific white shrimp (Litopenaeus vannamei), one of the most widely cultured shrimp species in the world, often suffers from cold stress. To understand the molecular mechanism of cold tolerance in Pacific white shrimp, we conducted a proteomic analysis on two contrasting shrimp cultivars, namely, cold-tolerant Guihai2 (GH2) and cold-sensitive Guihai1 (GH1), under normal temperature (28°C), under cold stress (16°C), and during recovery to 28°C. In total, 3,349 proteins were identified, among which 2,736 proteins were quantified. Based on gene ontology annotations, differentially expressed proteins largely belonged to biological processes, cellular components, and molecular functions. KEGG pathway annotations indicated that the main changes were observed in the lysosome, ribosomes, and oxidative phosphorylation. Subcellular localization analysis showed a significant increase in proteins present in cytosol, extracellular regions, and mitochondria. Combining enrichment-based clustering analysis and qRT-PCR analysis, we found that glutathione S-transferase, zinc proteinase, m7GpppX diphosphatase, AP2 transcription complex, and zinc-finger transcription factors played a major role in the cold stress response in Pacific white shrimp. Moreover, structure proteins, including different types of lectin and DAPPUDRAFT, were indispensable for cold stress tolerance of the Pacific white shrimp. Results indicate the molecular mechanisms of the Pacific white shrimp in response to cold stress and provide new insight into breeding new cultivars with increased cold tolerance.

Identification of microRNAs involved in cold adaptation of Litopenaeus vannamei by high-throughput sequencing.

The Litopenaeus vannamei (L. vannamei) is one of the most widely cultured shrimp species in the world, with low temperature being one of the most serious threats to its growth and survival. To examine the potential regulatory mechanism of cold adaptation, we conducted a microRNAs (miRNAs) analysis on the hepatopancreas of L. vannamei under normal temperature 28 °C (M28), cold acclimation 16 °C for 6 days (M16), and recovered under normal temperature (MR). In total 14,754,823, 14,945,246 and 15,880,093 raw reads representing 10,690,259, 8,587,144, and 11,512,941 unique sequences of 18-32 nt length were obtained from the M28, M16 and MR libraries, respectively. After comparing the miRNA sequences with the miRBase database, 68 known mature miRNAs and 47 novel miRNAs were identified. Expression analysis showed that 34 miRNAs were significantly differential expressed in response to cold adaptation. Compared to the M28 library, 21 miRNAs were upregulated and 13 miRNAs were downregulated significantly in the M16 library. After recovery to normal temperature, there are 16 miRNAs upregulated and 15 miRNAs downregulated significantly compared to M28 library. Then, five significantly differential expressed miRNAs under cold acclimation including three known miRNAs (mja-miR-6491, mja-miR-6494, and Bta-miR-2478) and two newly-identified miRNAs (novel_68 and novel_5) were selected for validation by RT-qPCR in the hepatopancreas and muscle tissues of cold treated shrimps. The expression trend of most the miRNAs from RT-qPCR were consistent with the next-generation sequencing data. Further, the Gene Ontology (GO) annotation showed that the metabolic process GO term was significantly enriched with target genes of the differentially expressed miRNAs. Additionally, KEGG pathway analysis suggested that the fatty acid degradation and glycerolipid metabolism pathways etc. are significantly enriched with the target genes. These findings may contribute to a better understanding of the molecular mechanisms governing the responses to low temperature in L. vannamei.

Identification of cold responsive genes in Pacific white shrimp (Litopenaeus vannamei) by suppression subtractive hybridization.

The Pacific white shrimp (Litopenaeus vannamei) is one of the most widely cultured shrimp species in the world. Despite L. vannamei having tropical origins, it is being reared subtropically, with low temperature stress being one of the most severe threats to its growth, survival and distribution. To unravel the molecular basis of cold tolerance in L. vannamei, the suppression subtractive hybridization (SSH) platform was employed to identify cold responsive genes in the hepatopancreas of L. vannamei. Both forward and reverse cDNA libraries were constructed, followed by dot blot hybridization, cloning, sequence analysis and quantitative real-time PCR. These approaches identified 92 cold induced and 48 cold inhibited ESTs to give a total of 37 cold induced and 17 cold inhibited contigs. Some of the identified genes related to stress response or cell defense, such as tetraspanins (TSPANs), DEAD-box helicase, heat shock proteins (HSPs) and metallothionein (MT), which were more abundant in the forward SSH library than in the reverse SSH library. The most abundant Est was a tetraspanin-8 (TSPAN8) homolog dubbed LvTSPAN8. A multiple sequence alignment and transmembrane domain prediction was also performed for LvTSPAN8. LvTSPAN8 expression was also examined in the gills, muscle, heart and hepatopancreas following cold exposure and showed the highest expression levels in the hepatopancreas. Overall, this study was able to identify several known genes and novel genes via SSH that appear to be associated with cold stress and will help to provide further insights into the molecular mechanisms regulating cold tolerance in L. vannamei.

Gonadal transcriptomic analysis and differentially expressed genes in the testis and ovary of the Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: The Pacific white shrimp (Litopenaeus vannamei) is the world's most prevalent cultured crustacean species. However, the supply of high-quality broodstocks is limited and baseline information related to its reproductive activity and molecular issues related to gonad development are scarce. In this study, we performed transcriptome sequencing on the gonads of adult male and female L. vannamei to identify sex-related genes. RESULTS: A total of 25.16 gigabases (Gb) of sequences were generated from four L. vannamei gonadal tissue libraries. After quality control, 24.11 Gb of clean reads were selected from the gonadal libraries. De-novo assembly of all the clean reads generated a total of 65,218 unigenes with a mean size of 1021 bp and a N50 of 2000 bp. A search of all-unigene against Nr, SwissProt, KEGG, COG and NT databases resulted in 26,482, 23,062, 20,659, 11,935 and 14,626 annotations, respectively, providing a total of 30,304 annotated unigenes. Among annotated unigenes, 12,320 unigenes were assigned to gene ontology categories and 20,659 unigenes were mapped to 258 KEGG pathways. By comparing the ovary and testis libraries, 19,279 testicular up-regulated and 3,529 ovarian up-regulated unigenes were identified. Enrichment analysis of differentially expressed unigenes resulted in 1060 significantly enriched GO terms and 34 significantly enriched KEGG pathways. Nine ovary-specific, 6 testis-specific, 45 testicular up-regulated and 39 ovarian up-regulated unigenes were then confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, using all-unigenes as a reference, a total of 13,233 simple sequence repeats (SSRs) were identified in 10,411 unigene sequences. CONCLUSIONS: The present study depicts the first large-scale RNA sequencing of shrimp gonads. We have identified many important sex-related functional genes, GO terms and pathways, all of which will facilitate future research into the reproductive biology of shrimp. We expect that the SSRs detected in this study can then be used as genetic markers for germplasm evaluation of breeding and imported populations.