Tissue damage, antioxidant capacity, transcriptional and metabolic regulation of red drum Sciaenops ocellatus in response to nanoplastics exposure and subsequent recovery.

Nanoplastics are recognized as emerging contaminants that can cause severe toxicity to marine fishes. However, limited researches were focusing on the toxic effects of nanoplastics on marine fish, especially the post-exposure resilience. In this study, red drum (Sciaenops ocellatus) were exposed to 5 mg/L polystyrene nanoplastics (100 nm, PS-NPs) for a 7-day exposure experiment, and a 14-day recovery experiment that followed. The aim was to evaluate the dynamic alterations in hepatic and branchial tissue damage, hepatic antioxidant capacity, as well as hepatic transcriptional and metabolic regulation in the red drum during exposure and post-exposure to PS-NPs. Histopathological observation found that PS-NPs primarily triggered hepatic lipid droplets and branchial epithelial liftings, a phenomenon persistently discernible up to the 14 days of recovery. Although antioxidant capacity partially recovered during recovery periods, PS-NPs resulted in a sustained reduction in hepatic antioxidant activity, causing oxidative damage throughout the entire exposure and recovery phases, as evidenced by decreased total superoxide dismutase activities and increased malondialdehyde content. At the transcriptional and metabolic level, PS-NPs primarily induced lipid metabolism disorders, DNA damage, biofilm disruption, and mitochondrial dysfunction. In the gene-metabolite correlation interaction network, numerous CcO (cytochrome c oxidase) family genes and lipid metabolites were identified as key regulatory genes and metabolites in detoxification processes. Among them, the red drum possesses one additional CcO6B in comparison to human and zebrafish, which potentially contributes to its enhanced capacity for maintaining a stable and positive regulatory function in detoxification. This study revealed that nanoplastics cause severe biotoxicity to red drum, which may be detrimental to the survival of wild populations and affect the economics of farmed populations.

Genomic Insights and Synthetic Biology Applications of Marine Actinomycete Streptomyces griseoincarnatus HNS054.

The marine bacterium Streptomyces sp. HNS054 shows promise as a platform for producing natural products. Isolated from a marine sponge, HNS054 possesses several desirable traits for bioengineering: rapid growth, salt tolerance, and compatibility with genetic tools. Its genome contains 21 potential biosynthetic gene clusters, offering a rich source of natural products. We successfully engineered HNS054 to increase the production of aborycin and actinorhodin by 4.5-fold and 1.2-fold, respectively, compared to S. coelicolor M1346 counterparts. With its unique features and amenability to genetic manipulation, HNS054 emerges as a promising candidate for developing novel marine-derived drugs and other valuable compounds.

Transcriptome Profiling and Expression Localization of Key Sex-Related Genes in a Socially-Controlled Hermaphroditic Clownfish, Amphiprion clarkii.

Clownfish can be an excellent research model for investigating the socially-controlled sexual development of sequential hermaphrodite teleosts. However, the molecular cascades underlying the social cues that orchestrate the sexual development process remain poorly understood. Here, we performed a comparative transcriptomic analysis of gonads from females, males, and nonbreeders of Amphiprion clarkii, which constitute a complete social group, allowing us to investigate the molecular regulatory network under social control. Our analysis highlighted that the gonads of nonbreeders and males exhibited high similarities but were far from females, both in global transcriptomic profiles and histological characteristics, and identified numerous candidate genes involved in sexual development, some well-known and some novel. Significant upregulation of cyp19a1a, foxl2, nr5a1a, wnt4a, hsd3b7, and pgr in females provides strong evidence for the importance of steroidogenesis in ovarian development and maintenance, with cyp19a1a playing a central role. Amh and sox8 are two potential key factors that may regulate testicular tissue development in early and late stages, respectively, as they are expressed at higher levels in males than in females, but with slightly different expression timings. Unlike previous descriptions in other fishes, the unique expression pattern of dmrt1 in A. clarkii implied its potential function in both male and female gonads, and we speculated that it might play promoting roles in the early development of both testicular and ovarian tissues.

Balancing selection maintains ancient polymorphisms at conserved enhancers for the olfactory receptor genes of a Chinese marine fish.

The study of balancing selection, as a selective force maintaining adaptive genetic variation in gene pools longer than expected by drift, is currently experiencing renewed interest due to the increased availability of new data, methods of analysis, and case studies. In this investigation, evidence of balancing selection operating on conserved enhancers of the olfactory receptor (OR) genes is presented for the Chinese sleeper (Bostrychus sinensis), a coastal marine fish that is emerging as a model species for evolutionary studies in the Northwest Pacific marginal seas. Coupled with tests for Gene Ontology enrichment and transcription factor binding, population genomic data allow for the identification of an OR cluster in the sleeper with a downstream flanking region containing three enhancers that are conserved with human and other fish species. Phylogenetic and population genetic analyses indicate that the enhancers are under balancing selection as evidenced by their translineage polymorphisms, excess common alleles, and increased within-group diversities. Age comparisons between the translineage polymorphisms and most recent common ancestors of neutral genealogies substantiate that the former are old, and thus, due to ancient balancing selection. The survival and reproduction of vertebrates depend on their sense of smell, and thereby, on their ORs. In addition to locus duplication and allelic variation of structural genes, this study highlights a third mechanism by which receptor diversity can be achieved for detecting and responding to the huge variety of environmental odorants (i.e., by balancing selection acting on OR gene expression through their enhancer variability).

Correction : Characterization of hybridization within a secondary contact region of the inshore fish, Bostrychus sinensis, in the East China Sea.

Following the publication of this article, the authors have requested that the Acknowledgements section be amended to thank Weidi Yang for his assistance with their Bostrychus sinensis photograph that was chosen for the front cover of the January 2018 issue of the journal. This error has been corrected in both the PDF and HTML versions of the paper. Also, the legends for Supplementary Figures 1 and 2 were not posted online. This error has been corrected in the HTML version of the paper.

Diversity and Dynamics of "Candidatus Endobugula" and Other Symbiotic Bacteria in Chinese Populations of the Bryozoan, Bugula neritina.

Bugula neritina is a common invasive cosmopolitan bryozoan that harbors (like many sessile marine invertebrates) a symbiotic bacterial (SB) community. Among the SB of B. neritina, "Candidatus Endobugula sertula" continues to receive the greatest attention, because it is the source of bryostatins. The bryostatins are potent bioactive polyketides, which have been investigated for their therapeutic potential to treat various cancers, Alzheimer's disease, and AIDS. In this study, we compare the metagenomics sequences for the 16S ribosomal RNA gene of the SB communities from different geographic and life cycle samples of Chinese B. neritina. Using a variety of approaches for estimating alpha/beta diversity and taxonomic abundance, we find that the SB communities vary geographically with invertebrate and fish mariculture and with latitude and environmental temperature. During the B. neritina life cycle, we find that the diversity and taxonomic abundances of the SB communities change with the onset of host metamorphosis, filter feeding, colony formation, reproduction, and increased bryostatin production. "Ca. Endobugula sertula" is confirmed as the symbiont of the Chinese "Ca. Endobugula"/B. neritina symbiosis. Our study extends our knowledge about B. neritina symbiosis from the New to the Old World and offers new insights into the environmental and life cycle factors that can influence its SB communities, "Ca. Endobugula," and bryostatins more globally.

Analysis of enzyme activity, antibacterial activity, antiparasitic activity and physico-chemical stability of skin mucus derived from Amphiprion clarkii.

Recently, mucosal surfaces, especially fish skin and its secreted mucus, have attracted significant interest from immunologists. Amphiprion clarkii, a member of the family Pomacentridae, lives symbiosis with sea anemones and has a good resistance to common seawater bacterial diseases and parasites owing to the protection from its abundant skin mucus. In the present work, the activity of immune-related enzymes (lysozyme, protease, antiprotease, cathepsin B, alkaline phosphatase and peroxidase), the antibacterial activity against two Gram-positive bacteria and five Gram-negative bacteria, the antiparasitic activity against the pathogen of marine white spot disease (Cryptocaryon irritans theronts) and the physico-chemical stability (to pH and heat) of the skin mucus of A. clarkii were analysed. The results showed that the levels of lysozyme and peroxidase were very similar (from 2 to 4 U mg-1 protein). However, cathepsin B was detected of 63.32 U mg-1 protein and alkaline phosphatase was only 0.12 U mg-1 protein. Moreover, protease showed a higher percentage of activity than antiprotease. A. clarkii skin mucus showed a strong antibacterial activity against Gram-negative bacteria, particularly against Aeromonas hydrophila and Vibrio parahaemolyticus but showed no effect on Gram-positive bacteria at the tested concentrations. The bactericidal activity functioned within a short time in a distinct time- and dose-dependent manner. SEM showed that after treated with A. clarkii skin mucus, the V. parahaemolyticus cells distorted and piled together, and the filaments appeared and became into cotton-shaped or quasi-honeycomb texture to adhere cells. Meanwhile, A. clarkii skin mucus showed an apparent antiparasitic activity against C. irritans theronts with a distinct dose- and time-dependent relationship. LM and SEM observation showed that after treated with skin mucus, the theronts quickly stopped their swimming and cilia movement, cells became rounded, cilia shed, small bubbles formed on the surface, cell nucleolus enlarged, cytoskeleton deformed, cell membranes ruptured and cell content leaked out. Antibacterial activity was not affected by 30-90 °C heat treatment but was slightly suppressed by 100 °C. In the pH treatment groups, antibacterial activity was not affected by the moderate pH treatment of 5.0-8.0, but slightly suppressed by weak acid and weak base. Therefore, we speculated that the skin mucus of A. clarkii might be a potential source of novel antibacterial and antiparasitic components for fish or human health-related applications. This study broadened our understanding of the role of skin mucus in the innate immune system and provided a basis for the further isolation and purification of active substances.

Genetic diversity within grouper species and a method for interspecific hybrid identification using DNA barcoding and RYR3 marker.

Groupers (family Epinephelidae) are an assemblage of coral reef fishes comprising more than 160 species in 16 genera, many of which are both environmentally and economically valuable. Because of their similar morphology, variable color patterns, and tendency for interspecies hybridization, morphological identification of groupers usually leads to taxonomic confusion. To find an effective method for identifying different grouper species and hybrids, evaluate genetic diversity and uncover any synonymous or cryptic species, we sampled a total of 221 specimens representing 57 species in 9 genera in the China Seas. Both mitochondrial (mt) cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 2 (ND2) were found to be effective barcoding genes. We also developed an efficient protocol for identifying hybrid groupers using mt markers and the nuclear RYR3 gene and found the first record of wide interspecies hybridization in genus Epinephelus. This barcoding study revealed high genetic divergence in many widespread species and possible synonyms. In addition to providing a molecular method for identifying grouper species, this study offers important resources for the further study of grouper conservation genetics, speciation, hybridization and other evolutionary traits.

Characterization of hybridization within a secondary contact region of the inshore fish, Bostrychus sinensis, in the East China Sea.

The northwest Pacific marginal seas are a primary center of phylogeographic and evolutionary research, because of their dynamic geographic history of falling and rising sea levels during the glaciations and interglaciations of the last one million years. Here we present new molecular and morphological data for geographic samples of the four-eyed sleeper (Bostrychus sinensis), which reinforce the evidence for secondary contact and hybridization between two phylogeographic lineages in the East China Sea. Specifically, we find that the secondary contact region is characterized by a low frequency of hybridization, where mitochondrial DNA introgression is relatively common, whereas F1 hybrids are correspondingly scarce. Furthermore, the adult standard lengths of the two phylogeographic lineages vary geographically in a manner that is consistent with reproductive character displacement. Collectively, the molecular and morphological data document that sleeper hybridization conforms to the classic "tension zone" model, where alleles are lost via reduced hybrid viability and/or positive assortative mating but are then replenished by dispersal from south of the secondary contact region. They also indicate that the two phylogeographic lineages are at an incipient stage of the speciation process. These results and conclusions for the four-eyed sleeper are presented as a case study for future research on the vicariance, secondary contact, and hybridization of marine groups in the northwest Pacific marginal seas.

Phylogeography of the inshore fish, Bostrychus sinensis, along the Pacific coastline of China.

This study assesses the phylogeography of the Chinese four-eyed sleeper (Bostrychus sinensis) with one mitochondrial and one nuclear genes and two morphological characters. Phylogenetic and population genetic analyses of the sequences reveals two phylogeographic lineages from the East and South China Seas, which are corroborated by the morphological data. The vicariance of the two lineages is attributed to the Pleistocene Ice Age exposure of the Taiwan Strait and consequent connection of Taiwan to the mainland, which thereby introduced an ecological barrier to gene flow between populations in the East and South China Seas. The distributions of the two lineages now overlap in the East China Sea and this secondary contact is attributed to biased northward migration along the two main currents of the Taiwan Strait following its interglacial re-flooding. In conclusion, this study reinforces the importance of "vicariance, then secondary contact" due to Late Pliocene and Pleistocene sea-level changes to the phylogeography of marine species. Specifically, it corroborates the importance of Pleistocene sea-level changes in the Taiwan Strait to the phylogeography of Chinese inshore species.

Simultaneous Quantitation of Free Amino Acids, Nucleosides and Nucleobases in Sipunculus nudus by Ultra-High Performance Liquid Chromatography with Triple Quadrupole Mass Spectrometry.

To evaluate the nutritional and functional value of Sipunculus nudus, a rapid, simple and sensitive analytical method was developed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 25 free amino acids and 16 nucleosides and nucleobases in S. nudus within 20 min, which was confirmed to be reproducible and accurate. The limits of detection (LODs) and quantification (LOQs) were between 0.003-0.229 µg/mL and 0.008-0.763 µg/mL for the 41 analytes, respectively. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order: (highest) coelomic fluid > body wall > intestine (lowest). The method is simple and accurate, and could serve as a technical support for establishing quality control of S. nudus and other functional seafoods. Moreover, the research results also laid foundation for further exploitation and development of S. nudus.

Transcriptome analysis revealed changes of multiple genes involved in immunity in Cynoglossus semilaevis during Vibrio anguillarum infection.

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most valuable marine aquatic species in Northern China. Given to the rapid development of aquaculture industry, the C. semilaevis was subjected to disease-causing bacteria Vibrio anguillarum. It therefore is indispensable and urgent to understand the mechanism of C. semilaevis host defense against V. anguillarum infection. In the present study, the extensively analysis at the transcriptome level for V. Anguillarum disease in tongue sole was carried out. In total, 94,716 high quality contigs were generated from 75,884,572 clean reads in three libraries (HOSG, NOSG, and CG). 22,746 unigenes were identified when compared with SwissProt, an NR protein database and NT nucleotide database. 954 genes exhibiting the differentially expression at least one pair of comparison in all three libraries were identified. GO enrichment for these genes revealed gene response to biotic stimulus, immune system regulation, and immune response and cytokine production. Further, the pathways such as complement and coagulation cascades and Vibrio cholerae infection pathways were enriched in defensing of pathogen. Besides, 13,428 SSRs and 118,239 SNPs were detected in tongue sole, providing further support for genetic variation and marker-assisted selection in future. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of development of therapeutic regimens and lays the foundation for selecting resistant lines of C. semilaevis against V. anguillarum.

Isolation and diversity of natural product biosynthetic genes of cultivable bacteria associated with marine sponge Mycale sp. from the coast of Fujian, China.

The marine sponge Mycale sp., a potential source of natural bioactive products, is widely distributed along the coast of Fujian, China. The cultivable bacterial community associated with Mycale sp., the antibacterial activities, and the PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetase) gene diversity of these bacteria were investigated. Phylogenetic analysis of the 16S rRNA gene showed that the 51 isolates from Mycale sp. belonged to Actinobacteria, Bacteroidetes, Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Among them, some bacteria were first isolated from marine sponge. The 20 isolates with antimicrobial activities were primarily clustered within the groups Actinobacteria, Gammaproteobacteria, and Bacillus. Strain HNS054, which showed 99% similarity to Streptomyces labedae, exhibited the strongest antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus MTCC 1430, Bacillus subtilis MTCC 441) and Vibrio species. The screening of natural product biosynthetic genes revealed that 8 Actinobacteria species with antimicrobial activities possessed PKS-KS (ketosynthase) or NRPS-A domains, and the Nocardiopsis species contained a hybrid or mixed PKS-NRPS system. The phylogenetic analysis of the amino acid sequences indicated that the identified KS domains clustered with those from diverse bacterial groups, including Actinobacteria, Alphaproteobacteria, Cyanobacteria, and Firmicutes. Most KS domain sequences had high homology (>80%) to type I KSs, but the KS domain of Nocardiopsis sp. strain HNS048 had 77% similarity to the type II KS domain of Burkholderia gladioli. The NRPS-A domains of the 8 isolates were grouped into the Gammaproteobacteria, Actinobacteria, and Firmicutes groups. The NRPS-A gene of strain HNS052, identified as Nocardiopsis cyriacigeorgica, showed only 54% similarity to Rhodococcus opacus. All results suggested that Mycale sp. harboured diverse bacteria that could contribute to the production of novel bioactive substances in the future.

Phylogeography of the Chinese beard eel, Cirrhimuraena chinensis Kaup, inferred from mitochondrial DNA: a range expansion after the last glacial maximum.

The Chinese beard eel (Cirrhimuraena chinensis Kaup) is an intertidal fish and a model organism for the study of impacts caused by topological fluctuations during the Pleistocene and current intricate hydrological conditions on fauna living in the coastal areas of China. In this study, we examined the phylogeographical pattern, population genetic profile and demographical history of C. chinensis using mitochondrial DNA (cytochrome b (cyt b) and control region (CR)) from 266 individuals sampled in seven localities across the coastal area of southeastern China. The combined data indicated high levels of haplotype diversity and low levels of nucleotide diversity. Analyses of molecular variance (AMOVA) and FST statistics suggested the absence of a significant population structure across the Chinese coast. Neutrality tests, mismatch distributions and Bayesian skyline plots uniformly indicated a recent population expansion. The phylogeographic structure of C. chinensis may be attributed to past population expansion and long-distance pelagic larval dispersal facilitated by present-day ocean currents.

Important Role of the Ihh Signaling Pathway in Initiating Early Cranial Remodeling and Morphological Specialization in Cromileptes altivelis.

In this study, we identified the important contribution of frontal bone remodeling in shaping the 'sunken head and humpback' appearance in C. altivelis. Our investigation identified a developmental milestone at a total length of 5-6 cm, making the onset of its morphologic specialization in this species. A comparative analysis with closely related species reveals heightened activity in the frontal osteoblasts of the humpback grouper, potentially providing a physiological basis for its remodeling. Furthermore, our findings highlight that a significant upregulation in the expression levels of Ihhb, Ptch1, and Gli2a genes was seen in C. altivelis within the specified developmental stage, indicating an important involvement of the Ihhb-Ptch1-Gli2a signaling pathway in initiating the morphological specialization. We hypothesized that Ihh signaling could be attributed to shifts in mechanical stress, resulting from muscle traction on the frontal bone due to changes in swimming patterns during development. This study not only offers significant insights into unraveling the molecular mechanisms that govern phenotypic specialization and ecological adaptations in the humpback grouper but also serves as a valuable reference for studies on fishes with a controversial morphology and molecular phylogeny.

Genomic organization, promoter characterization and expression analysis of the leukocyte cell-derived chemotaxin-2 gene in Epinephelus akaraa.

Leucocyte cell-derived chemotaxin 2 (LECT2) was first identified as a chemotactic factor and has been subsequently proven to be a multifunctional protein that mediates the regulation of liver regeneration, carcinogenesis and Natural killer T (NKT) cell homeostasis in mammals. In fish, it has been recently found to be critical for the inflammatory response to stimuli. However, the in vivo function of LECT2 in fish remains obscure. Base on the full-length cDNA of the Epinephelus akaraa LECT2 (EaLECT2) gene we previously isolated, we sought to analyze its genomic structure and context. The genomic DNA of the EaLECT2 gene spans 2866bp from the transcription start site to the termination codon. As in most LECT2 genes in other vertebrates, the EaLECT2 genomic DNA contains four exons and three introns. An analysis of the promoter region revealed the presence of a TATA box and several putative transcription factor-binding sites. And transcriptional activity analysis suggested that most basal DNA regulatory elements required for EaLECT2 transcriptional activity might be contained within the 581bp region upstream of the transcription start codon. A real-time PCR analysis showed that the EaLECT2 expression levels were slightly increased in the head kidney, liver, gill and brain by bacterial challenge with Vibrio harveyi. Furthermore, the transcriptional level of the EaLECT2 gene in the liver was significantly up-regulated within 1h and reached its peak level at 12h post-stimulation. Higher levels of LECT2 expression were also observed in head kidney in challenged individuals.The expression pattern demonstrates the role of EaLECT2 in the immune response and its functions under other conditions. Additionally, we found that the recombinant EaLECT2 could be expressed as a soluble protein using a prokaryotic expression system with the expression vector pET32a(+) and the soluble protein was further proved to be the recombinant EaLECT2 with the rat antiserum against EaLECT2 we obtained. This work provides a unique basis for substantial work in future projects.

Chromosome-Level Genome Assembly of the Speckled Blue Grouper (Epinephelus cyanopodus) Provides Insight into Its Adaptive Evolution.

Epinephelus cyanopodus is a coral reef-dwelling grouper with important economic and ecological value and is widely distributed in the western Pacific Ocean. The lack of genomic resources for E. cyanopodus hinders its adaptive evolution and phylogeny research. We constructed the first high-quality genome of E. cyanopodus based on DNBSEQ, PacBio, and Hic sequencing technologies, with a genome size of 998.82 Mb, contig N50 of 5.855 Mb, and scaffold N50 of 41.98 Mb. More than 99.7% of contigs were anchored to 24 pseudochromosomes, and 94.2% of BUSCO genes were found in the E. cyanopodus genome, indicating a high genome assembly completeness. A total of 26,337 protein-coding genes were predicted, of which 98.77% were functionally annotated. Phylogenetic analysis showed that E. cyanopodus separated from its closely related species Epinephelus akaara about 11.5-26.5 million years ago, and the uplift of the Indo-Australian archipelago may have provided an opportunity for its rapid radiation. Moreover, several gene families associated with innate and adaptive immunity were significantly expanded in speckled blue grouper compared to other teleost genomes. Additionally, we identified several genes associated with immunity, growth and reproduction that are under positive selection in E. cyanopodus compared to other groupers, suggesting that E. cyanopodus has evolved broad adaptability in response to complex survival environment, which may provide the genetic basis for its rapid radiation. In brief, the high-quality reference genome of the speckled blue grouper provides a foundation for research on its biological traits and adaptive evolution and will be an important genetic tool to guide aquaculture and resolve its taxonomic controversies in future studies.

Epinephelusrankini Whitley, 1945, a valid species of grouper (Teleostei, Perciformes, Epinephelidae) from Western Australia and southeast Indonesia.

Background: The grouper Epinephelusrankini, described from the waters off Western Australia, has long been regarded as a junior synonym of Epinephelusmultinotatus. However, the two species are discernible as distinct species on the basis of their morphological characteristics and genetic differences by the holotype material and non-type of specimens. New information: In this study, Epinephelusrankini is considered as a valid species and re-described based on the examination of the holotype and additional specimens. Epinephelusrankini can be distinguished from the closely-related species E.multinotatus by the following combination of characters: body dark greyish-brown to chocolate with irregular white blotches (vs. body pale brownish-grey with irregular and small white blotches in E.multinotatus), absence of small dark brown spots (vs. numerous small dark brown spots in E.multinotatus). Furthermore, genetic differences between the two species strongly support the validity of both species based on molecular analysis (mtDNA, COI gene). In addition based on the sampling range, E.rankini was observed range from the Abrolhos Islands of Western Australia to south-eastern Indonesia, while E.multinotatus ranges from the Persian Gulf to southern Mozambique.

Development and characterization of 20 microsatellite mrkers for Chinese back sleeper, Bostrychus sinensis.

Twenty microsatellite markers were isolated and characterized from the Chinese black sleeper, Bostrychus sinensis. Loci were screened in 30 individuals from Taiwan. For each locus, the number of alleles varied from 4 to 22 with mean expected and observed heterozygosity of 0.79 and 0.66, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was detected. This set of microsatellites will provide a suitable tool for population genetic studies of Chinese black sleeper.

Reproductive biology and annual reproductive cycles of two sympatric lineages of Bostrychus sinensis with a natural habitat on southeastern coast of China.

The two phylogeographic lineages [South China Sea (SCS) lineage and East China Sea (ESC) lineage] of the four-eyed sleeper (Bostrychus sinensis) occur sympatrically along the southeastern coast of China, where there is a small percentage of hybrids in a natural habitat. To assess the mechanism responsible for the incomplete reproductive isolation between the SCS and ECS lineages of four-eyed sleeper, there was sampling of individuals from the Yueqing Bay from November 2018 to November 2019 to determine whether there are differences in characteristics of the reproductive cycles and reproductive biology. The two lineages varied in reproductive seasonality, body size and shape, and egg number. The SCS lineage spawned annually between May and August, with the absolute fecundity (AF) of the SCS lineage being 9960 to 39,517 eggs per female, and there was positive allometric growth of this species. The ECS lineage spawned annually between June and July, with females producing 4064 to 10,370 eggs per fish and individuals having an isometric growth pattern. Preliminary results indicated that there was partial overlap in the spawning season between the two lineages in the sympatric region, which may not be a primary factor in the incomplete reproductive isolation. Furthermore, the lineage differences in fecundity and body shape and size may lead to variation in mate choice and reproductive strategy. The results from this study provide insight into the biology, reproductive strategies, and speciation of the four-eyed sleepers.