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.

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.

First report of black-heart disease in Kumamoto oyster Crassostrea sikamea spat caused by Polydora lingshuiensis in China.

A black-heart disease caused by polydorid infestation is reported for the first time in Kumamoto oyster Crassostrea sikamea Amemiya, 1928 spat in a pond at Beihai city, Guangxi province, China, with a prevalence of 100% and a cumulative mortality rate of 50% within 2 mo. In heavily infected oyster spat, blisters extended toward the center of the inner shell surface, around the adductor muscle scar area to form a large black area occupying approximately 50% of the area of the inner shell surface. Morphological analysis identified the pathogen as Polydora lingshuiensis Ye et al., 2015, which was reconfirmed by comparison of its corresponding 18S rRNA and mitochondrial CO1 gene sequences with those in the GenBank database. The mean abundance of mud blisters was significantly higher in live spat than in dead spat, suggesting that P. lingshuiensis preferentially infests live oyster spat. Additionally, P. lingshuiensis larvae were detected in the inlet near the dam, which suggests that the source of P. lingshuiensis larvae infecting the spat may be larvae entering the ponds through the water current from the sea.