Targeting TtVgR via siRNA Knockdown Elicits Ovarian Cell Death in the Tri-spine Horseshoe Crab.

The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.

Pharmaceutical Residues in Edible Oysters along the Coasts of the East and South China Seas and Associated Health Risks to Humans and Wildlife.

The investigation of pharmaceuticals as emerging contaminants in marine biota has been insufficient. In this study, we examined the presence of 51 pharmaceuticals in edible oysters along the coasts of the East and South China Seas. Only nine pharmaceuticals were detected. The mean concentrations of all measured pharmaceuticals in oysters per site ranged from 0.804 to 15.1 ng g-1 of dry weight, with antihistamines being the most common. Brompheniramine and promethazine were identified in biota samples for the first time. Although no significant health risks to humans were identified through consumption of oysters, 100-1000 times higher health risks were observed for wildlife like water birds, seasnails, and starfishes. Specifically, sea snails that primarily feed on oysters were found to be at risk of exposure to ciprofloxacin, brompheniramine, and promethazine. These high risks could be attributed to the monotonous diet habits and relatively limited food sources of these organisms. Furthermore, taking chirality into consideration, chlorpheniramine in the oysters was enriched by the S-enantiomer, with a relative potency 1.1-1.3 times higher when chlorpheniramine was considered as a racemate. Overall, this study highlights the prevalence of antihistamines in seafood and underscores the importance of studying enantioselectivities of pharmaceuticals in health risk assessments.

Effects of bivalve aquaculture on plankton and benthic community.

Global human population has increased dramatically over the past 50 years. As a result, marine fisheries and finfish aquaculture have become increasingly unsustainable, driving bivalve aquaculture to become an important food industry for the production of marine animal protein to support the growing market demand for animal protein. It is projected that the rate of bivalve aquaculture expansion will be greatly accelerated in the near future as the human population continues to increase. Although it is generally believed that unfed bivalve aquaculture has less impact on the environment than finfish aquaculture, the rapid expansion of bivalve aquaculture has raised concerns about its potential negative impact, especially on plankton and benthic community. Therefore, there is an urgent need to update the potential effects of bivalve aquaculture on plankton and benthic community. This article reviews the present state of knowledge on environmental issues related to bivalve aquaculture, and discusses potential mitigation measures for the environmental impacts induced by expansion of bivalve aquaculture. This review provides guidance for scientists and farm managers to clarify the current state of research and identify priority research needs for future bivalve aquaculture research. Therefore, specific management strategies can be formulated for the sustainable development and expansion of bivalve aquaculture.

Regulation of PhoB on biofilm formation and hemolysin gene hlyA and ciaR of Streptococcus agalactiae.

PhoB is a response regulator protein that plays a key role in the PhoBR two-component signal transduction system. In this study, we used transcriptome and proteomics techniques to evaluate the detect the gene network regulated by PhoB of Streptococcus agalactiae. The results showed that expression of biofilm formation and virulence-related genes were changed after phoB deficiency. Crystal violet and CLSM assay confirmed that the deletion of the phoB increased the thickness of S. agalactiae biofilm. The results of lacZ reporter and the bacterial one-hybridization method showed that PhoB could directly bind to the promoter regions of hemolysin A and ciaR genes but not to the promoter regions of cylE and hemolysin III. Through the construction of an 18-base pair deoxyribose nucleic acid (DNA) random fragment library and the bacterial one-hybridization system, it was found that the conservative sequence of PhoB binding was TTGGAGAA(G/T). Our research has uncovered the virulence potential of the PhoBR two-component system of S. agalactiae. The findings of this study provide the theoretical foundation for in-depth research on the pathogenic mechanism of S. agalactiae.

siRNA Silencing of FpVtg Induces Ovarian Cell Apoptosis in Redtail Prawn, Fenneropenaeus penicillatus.

Inadequate gonadal maturation and poor spawning performance increasingly threaten the sustainability of shrimp aquaculture. Unraveling the mechanisms regulating ovarian development and maturation hence is critical to address industry challenges. Vitellogenin (Vtg), a precursor of yolk protein found in the hepatopancreas and ovary of shrimp, plays a key role in facilitating shrimp's oocyte maturation and embryonic development after oviposition. This study found that FpVtg was specifically expressed in F. penicillatus hepatopancreas and ovary. FpVtg was localized predominantly in the oocyte cytoplasm and distributed uniformly in the hepatopancreas tissue. Silencing FpVtg led to apoptosis in both hepatopancreas and ovary tissues. Furthermore, FpVtg depletion upregulated the expression of ovarian peritrophin 1, ovarian peritrophin 2, serine proteinase inhibitor 6, and juvenile hormone esterase-like carboxylesterase 1, while downregulated that of vitellogenin, delta-9 desaturase, and insulin-like receptor. KEGG pathway analysis implicated such as PI3K-AKT signaling, RNA transport, ECM-receptor interaction, hippo signaling, oocyte meiosis, and apoptosis were enriched and involved in ovarian development. These findings have provided insights into the FpVtg's reproductive role and the associated regulatory genes and pathways in F. penicillatus. This knowledge can contribute to establishing strategies to improve the breeding and aquaculture production of F. penicillatus by elucidating its vitellogenesis regulation in redtail prawn and other penaeid species. Further characterization of the implicated pathways and genes will clarify the intricacies underlying ovarian maturation.

Molecular characterization of complement regulatory factor CD46 in Trachinotus ovatus and its role in the antimicrobial immune responses and complement regulation.

CD46, as a cofactor of complement I factor, not only regulates the complement system but also functions as a pathogen receptor and is involved in controlling early pathogen infection through autophagy. In this study, a new CD46 gene (ToCD46) was identified from golden pompano (Trachinotus ovatus), which showed higher sequence homology with other teleosts CD46. Homology comparison showed that ToCD46 had higher sequence homology (46.95-52.85%) with other teleosts CD46 and lower homology with mammal. Tissue expression profile analysis showed that ToCD46 was generally expressed in all tissues with the highest expression level in liver, followed by head kidney, and showed different patterns of up-regulation in immune-related tissues after stimulation by Streptococcus agalactiae and Vibrio alginolyticus. The hemolytic activity analysis and apoptosis assay showed that rToCD46 decreased the hemolytic activity of serum of golden pompano and effectively inhibited the damage of A549 cells, suggesting that ToCD46 might be involved in the regulation of complement activation of golden pompano. In vitro antibacterial experiments showed that rToCD46 had antibacterial activity against gram negative bacteria V. alginolyticus but no effect on positive bacteria S. agalactiae. These results suggest that ToCD46 may be involved in the immune response of golden pompano to pathogens, which will provide important basic information for elucidating the evolutionary history of the complement system of golden pompano.

Silencing of novel TtVtg6-like induced ovarian cell apoptosis in ancient chelicerate Tachypleus tridentatus.

Vitellogenin (Vtg) serves as the precursor of yolk protein and exhibits widespread distribution in tissues, including in the ovary of both vertebrates and invertebrates. Vtg plays a critical role in facilitating oocyte maturation and embryonic development following oviposition. In this study, we have successfully elucidated the complete transcript sequence of TtVtg6-like from an ancient chelicerate Tachypleus tridentatus. The TtVtg6-like transcript encompassed a length of 4887 bp and encoded 1629 amino acids residues. Notably, TtVtg6-like was found to contain 25 exons. Furthermore, the molecular weight and isoelectric point of TtVtg6-like were determined to be 191.6 KDa and 6.73, respectively. Subsequent mRNA expression analysis demonstrated the specific expression of TtVtg6-like in ovary and yellow connective tissue. In addition, TtVtg6-like was located and distributed in both ovary and yellow connective tissue. Intriguingly, employing an siRNA approach to silence TtVtg6-like resulted in a decrease in TtVtg6-like transcription levels. Concomitantly, TtVtg6-like silencing led to increase production of ROS, ultimately resulting in DNA damage and cell apoptosis within the ovarian primary cell. The induction of apoptosis ovarian primary cells due to TtVtg6-like silencing was further corroborated through TUNEL assay and flow cytometry analysis. Overall, our findings underscore the significance of TtVtg6-like in ovarian cell development, revealing its potential association with ovarian cell apoptosis. Consequently, the insights gained from this study contribute to the future exploration of vitellogenesis and ovarian development in T. tridentatus.

A review of natural and anthropogenic radionuclide pollution in marine bivalves.

Radionuclide contamination in food is a public health issue. Bivalves are known to accumulate relatively high levels of radionuclides. Despite many relevant reports, this information is poorly organized. Therefore, in this study, we conducted a comprehensive scientific review of radionuclides in marine bivalves. In general, the accumulation of radionuclides in bivalves is highly species and tissue-specific, which may be due to the different biological half-life of radionuclides in different species and tissues. The trophic pathway is the main pathway for the accumulation of most radionuclides in bivalves, with polonium-210 (210Po) and lead-210 (210Pb) potentially selectively accumulating in the digestive glands, while 134Cs and 137Cs selectively accumulating in the adductor muscle and mantle. Some other radionuclides (radium-226 (226Ra) and strontium-90 (90Sr)) are absorbed along with other minerals (e.g. Calcium) and selectively accumulate in bivalve shells. The information in this study can provide an overview of radionuclide contamination in marine bivalves.

Identification and Functional Analysis of ToBPI1/LBP and ToBPI2/LBP in Anti-Bacterial Infection of Trachinotus ovatus.

Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are a group of antibacterial proteins that play an important role in the host's innate immune defense against pathogen infection. In this study, two BPI/LBPs, named ToBPI1/LBP (1434 bp in length, 478 amino acids) and ToBPI2/LBP (1422 bp in length, 474 amino acids), were identified from the golden pompano. ToBPI1/LBP and ToBPI2/LBP were significantly expressed in immune-related tissues after being challenged with Streptococcus agalactiae and Vibrio alginolyticus. The two BPI/LBPs showed significant antibacterial activity against Gram-negative Escherichia coli and Gram-positive S. agalactiae and Streptococcus iniae. In contrast, the antibacterial activity against Staphylococcus aureus, Corynebacterium glutamicum, Vibrio parahaemolyticus, V. alginolyticus and Vibrio harveyi was low and decreased with time. The membrane permeability of bacteria treated with recombinant ToBPI1/LBP and ToBPI2/LBP was significantly enhanced. These results suggest that ToBPI1/LBP and ToBPI2/LBP may play important immunological roles in the immune response of the golden pompano to bacteria. This study will provide basic information and new insights into the immune response mechanism of the golden pompano to bacteria and the function of BPI/LBP.

Adverse impacts of high-density microplastics on juvenile growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus.

The impacts of high-density microplastics, namely polyamine 6,6 (nylon), polymethyl methacrylate (PMMA) and polyethylene terephthalate (PET), on growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus were investigated for 100 days. Negative changes in wet weight and prosomal width of the juveniles were observed in all treatments of microplastics, but significant difference was only detected in prosomal width between control and PMMA. T. tridentatus became significantly less active upon exposure to nylon and PET. The extent of burrowing by T. tridentatus did not significantly differ among the treatments but was overall significantly reduced towards day 100. T. tridentatus exposed to PET significantly showed the lowest survival probability (30 %), compared to the other treatments (70-90 %). In conclusion, high-density microplastics compromised growth and behaviour of juvenile horseshoe crabs. Among the polymers that were tested, PET was considered more harmful and associated with higher mortality.

Intestinal response of mussels to nano-TiO2 and pentachlorophenol in the presence of predator.

With the development of industry, agriculture and intensification of human activities, a large amount of nano-TiO2 dioxide and pentachlorophenol have entered aquatic environment, causing potential impacts on the health of aquatic animals and ecosystems. We investigated the effects of predators, pentachlorophenol (PCP) and nano titanium dioxide (nano-TiO2) on the gut health (microbiota and digestive enzymes) of the thick-shelled mussel Mytilus coruscus. Nano-TiO2, as the photocatalyst for PCP, enhanced to toxic effects of PCP on the intestinal health of mussels, and they made the mussels more vulnerable to the stress from predators. Nano-TiO2 particles with smaller size exerted a larger negative effect on digestive enzymes, whereas the size effect on gut bacteria was insignificant. The presence of every two of the three factors significantly affected the population richness and diversity of gut microbiota. Our findings revealed that the presence of predators, PCP, and nano-TiO2 promoted the proliferation of pathogenic bacteria and inhibited digestive enzyme activity. This research investigated the combined stress on marine mussels caused by nanoparticles and pesticides in the presence of predators and established a theoretical framework for explaining the adaptive mechanisms in gut microbes and the link between digestive enzymes and gut microbiota.

Chemically mediated rheotaxis of endangered tri-spine horseshoe crab: potential dispersing mechanism to vegetated nursery habitats along the coast.

Background: An enhanced understanding of larval ecology is fundamental to improve the management of locally depleted horseshoe crab populations in Asia. Recent studies in the northern Beibu Gulf, China demonstrated that nesting sites of Asian horseshoe crabs are typically close to their nursery beaches with high-density juveniles distributed around mangrove, seagrass and other structured habitats. Methods: A laboratory Y-maze chamber was used to test whether the dispersal of early-stage juvenile tri-spine horseshoe crab Tachypleus tridentatus is facilitated by chemical cues to approach suitable nursery habitats. The juvenile orientation to either side of the chamber containing controlled seawater or another with various vegetation cues, as well as their movement time, the largest distance and displacement were recorded. Results: The juveniles preferred to orient toward seagrass Halophila beccarii cues when the concentration reached 0.5 g l-1, but ceased at 2 g l-1. The results can be interpreted as a shelter-seeking process to get closer to the preferred settlement habitats. However, the juveniles exhibited avoidance behaviors in the presence of mangrove Avicennia marina and invasive saltmarsh cordgrass Spartina alterniflora at 2 g l-1. The juveniles also spent less time moving in the presence of the A. marina cue, as well as reduced displacement in water containing the S. alterniflora cue at 1 and 2 g l-1. These results may explain the absence of juvenile T. tridentatus within densely vegetated areas, which have generally higher organic matter and hydrogen sulfide. Conclusion: Early-stage juvenile T. tridentatus are capable of detecting and responding to habitat chemical cues, which can help guide them to high-quality settlement habitats. Preserving and restoring seagrass beds in the intertidal areas should be prioritized when formulating habitat conservation and management initiatives for the declining horseshoe crab populations.

Tetrodotoxin in Asian horseshoe crabs Carcinoscorpius rotundicauda and Tachypleus tridentatus across different life stages from northern Beibu Gulf, China.

Horseshoe crabs (HSCs) are a group of ancient chelicerates with great ecological and biomedical importance. Food poisonings caused by the consumption of Asian HSCs have significant impacts on public health and safety. This study measured tetrodotoxin (TTX) concentrations in two HSC species across various life stages in May 2020 from the northern Beibu Gulf, their most important spawning and nursery habitats in China. The average TTX contents in both Carcinoscorpius rotundicauda and Tachypleus tridentatus ranged 6.2-8.0 µg/kg and 3.8-8.4 µg/kg, respectively. While sampling location, growth and molt stages have little influence on TTX distribution in both species, significantly higher levels of TTX were detected in hemolymph, but lower in pooled tissues of early-instar juvenile T. tridentatus. These results provide a regional view of TTX occurrence and distribution in HSCs during their spawning season, which are critical for future studies to enhance understanding of TTX dynamics and formation in HSCs.

Community structure of benthic macroinvertebrates in native and introduced mangroves of northern Beibu Gulf, China: Implication for restoring mangrove ecosystems.

Introduced mangroves are widely used to restore mangrove ecosystems in South China. Results of potential impacts on indicative benthic macroinvertebrates are divergent. We explored the community structure of benthic macroinvertebrates in the mangrove ecosystem of northern Beibu Gulf, China across four habitats: native Avicennia marina mangrove, introduced Laguncularia racemosa mangrove, native-introduced mixed mangrove, and unvegetated intertidal flat. Based on the Hill number, community structure was estimated from the dimensions of estimated species richness, diversity, evenness, and species composition similarity. Benthic macroinvertebrates in the unvegetated flat significantly differed from the other three assemblages in mangroves; introduced L. racemosa mangrove had relatively distinct benthic macroinvertebrate assemblage from the native A. marina and the mixed mangroves, with lower species richness and similarity but higher diversity and evenness. Considering the lack of unanimous conclusion of potential impact on benthic macroinvertebrates under complex species interactions, native mangroves should be of top priority in ecosystem restoration.

Spatiotemporal Distribution of Asian Horseshoe Crab Eggs Are Highly Intermingled with Anthropogenic Structures in Northern Beibu Gulf, China.

Identification, protection and restoration of spawning habitats are vital for protecting the depleted species. Asian horseshoe crabs are ecologically important macroinvertebrates in coastal and estuarine ecosystems. However, their spawning habitat studies were limited to several reports in tropical regions, possibly due to the lack of modified survey methods, particularly in habitats with a lower density of spawning adults, and/or intermingled with anthropogenic structures. In this study, the year-round egg distribution and spawning habitat baselines of Carcinoscorpius rotundicauda and Tachypleus tridentatus were determined in the northern Beibu Gulf, China. Our findings demonstrated that the peak spawning occurred in June-July and ceased in November-January when the average water temperature dropped below 20 °C. Egg aggregations were found < 10 cm beneath the sediment surface with regular tidal inundation, regardless of seasonal changes, in the vicinity of natural and artificial structures with elevated, mildly sloping substratum within the high tide zones. The nests were characterized by medium-sized sediment grains (0.5-0.9 mm), high temperatures (31-34°C), low water contents (0.8%-0.9%), and total organic carbon contents (0.5%-0.7%), which might maximize the hatching success. The identified nesting beaches were close to nursery habitats for juveniles, and tidal creeks were present as the possible corridor connecting these two important habitats through the dominant mangrove forests. The findings provide valuable insights in the scope of spawning behavior and nest-site selection of Asian horseshoe crabs under a mixture of natural and artificial structures, which could benefit future management efforts for the exploited spawning populations.

Ontogenetic resource use and trophic dynamics of endangered juvenile Tachypleus tridentatus among diversified nursery habitats in the northern Beibu Gulf, China.

Horseshoe crabs, the most well-known example of "living fossils", are iconic and ecologically important macroinvertebrates in coastal and estuarine ecosystems. Their blood is a crucial resource for manufacturing Limulus or Tachypleus amebocyte lysate to detect bacterial endotoxins or fungal contamination in drug and medical devices. An enhanced understanding of their ecological roles and trophic interactions in the food webs is fundamental to facilitate resource management for the declining populations in Asia. Foraging information of the Asian species, however, is mainly derived from preliminary, scattered reports from a limited number of study locations. In this study, resource utilization, trophic niche dynamics, and trophic interaction of the juvenile tri-spine horseshoe crab, Tachypleus tridentatus (instars 1-12, approximately 0.5-8 years old) across ontogeny was assessed in diversified nursery habitats along the northern Beibu Gulf, China, using carbon and nitrogen stable isotopes. Our results suggest that: (i) T. tridentatus are ecological generalists given the vast range of carbon isotopic values and trophic niche width estimates exhibited between multiple instar groups; (ii) juvenile T. tridentatus across most habitat types predominantly assimilated energy from a variety of basal production sources in the food web, but primarily depended on sedimentary organic matter and seagrass resource pools; (iii) ontogenetic shifts in juvenile dietary proportions were evident, with decreased reliance on sedimentary organic matter, coupled with increased reliance on benthic macroinvertebrate grazers, detritivores, and omnivores with age; and (iv) nearly all juvenile instars occupied similar trophic positions in the food web with slight shifts in trophic position present with increasing size. Our findings indicate that resource availability and ontogenetic diet shifts strongly influence horseshoe crab trophic dynamics, and age should be accounted when formulating habitat conservation measures based on resource use for Asian horseshoe crabs.

Effects of rubble zones from oyster cultivation on habitat utilization and foraging behaviour of the endangered tri-spine horseshoe crab: An implication for intertidal oyster cultivation practices.

Estuarine structured habitats, including benthic cultivation structure, are widely cited as substantial nursery and feeding grounds for fish and macroinvertebrates. Activities such as locomotion and foraging behaviour of sediment-dwelling species, however, may be hindered in the presence of the obstacles. In this study, collected field evidence demonstrated that habitat utilization of the juvenile tri-spine horseshoe crab Tachypleus tridentatus, an iconic and endangered macroinvertebrate, was adversely affected by high-density intertidal oyster rubble zones. Despite the fact that the utilization area of individual juvenile horseshoe crabs was independent of the presence of these obstacles, the abundance and number of positional fixes of tagged juveniles in these areas were lower. The effects of these obstacles and their associated topographic changes on the movement of the juvenile horseshoe crabs was investigated in a simulated laboratory experiment. Both the simulated oyster cultches and hummocks formed between the cultches reduced the total distance and displacement of the feeding trail, and less juveniles preferred to forage on elevated, dry hummocks (dry at low tide) formed between the cultches. Our findings suggested that oyster cultches and rubbles reduced the habitat quality for juvenile horseshoe crabs. On-bottom oyster cultivation activities seeking to conserve horseshoe cabs by avoiding nursery habitats should instead consider off-bottom or offshore cultivation methods.

Author Correction: Draft genomic and transcriptome resources for marine chelicerate Tachypleus tridentatus.

In the original version of this Data Descriptor the word "Gulf" was incorrectly spelled in the affiliation "Ocean College, Beibu Gulf University, Qinzhou, 535011, Guangxi, China". This has now been corrected in both the HTML and PDF versions.

Draft genomic and transcriptome resources for marine chelicerate Tachypleus tridentatus.

Chinese horseshoe crabs (Tachypleus tridentatus), ancient marine arthropods dating back to the mid-Palaeozoic Era, have provided valuable resources for the detection of bacterial or fungal contamination. However, excessive exploitation for the amoebocyte lysate of Tachypleus has dramatically decreased the population of the Chinese horseshoe crabs. Thus, we present sequencing, assembly and annotation of T. tridentatus, with the hope of understanding the genomic feature of the living fossil and assisting scientists with the protection of this endangered species. The final genome contained a total size of 1.943 Gb, covering 90.23% of the estimated genome size. The transcriptome of three larval stages was constructed to investigate the candidate gene involved in the larval development and validate annotation. The completeness of the genome and gene models was estimated by BUSCO, reaching 96.2% and 95.4%, respectively. The synonymous substitution distribution of paralogues revealed that T. tridentatus had undergone two rounds of whole-genome duplication. All genomic and transcriptome data have been deposited in public databases, ready to be used by researchers working on horseshoe crabs.