Tachypleus Tridentatus Research Articles

A novel antimicrobial peptide CpAMP identified from Chinese horseshoe crab, Tachypleus tridentatus.

Antibacterial peptide (AMP) is a crucial component of the innate immune system in most organism, play an important role in host defense processes. Many of these peptides have broad antimicrobial activity against Gram-negative, -positive bacteria and fungi. In the present study, a novel AMP named CpAMP was identified using transcriptome analysis in Chinese horseshoe crab. The preprotein of CpAMP consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. And the putative mature peptide CpAMP was 4.95 kDa with a theoretical isoelectric point (pI) of 8.73. The mature CpAMP showed α-helix and irregularly curled structure in the cys-stabilized region. CpAMP exhibited a broad spectrum of antimicrobial activity against Gram-negative, -positive bacteria and antibiotic-resistant strains. In addition, CpAMP can significantly increase the survival rate of zebrafish infected with bacteria. Due to its broad-spectrum antibacterial activity and the sensitivity of drug-resistant strains, CpAMP could be used as a new type of drug for inhibition of pathogenic microorganisms or as an immune enhancer in animals.

An effective way to monitor the population of juvenile horseshoe crabs in the Beibu Gulf of China

Population conservation of horseshoe crab is urgently required in China as their population is declining dramatically. However, conservation efforts are severely constrained by the lack of information on the population dynamics of the species. Here, a citizen science-based investigation was conducted to continuously monitor the population dynamics of juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda in the Beibu Gulf of China over five years. A low abundance of horseshoe crabs was observed at all stations. T. tridentatus was primarily observed in the eastern of Beibu Gulf, with an abundance (0.179 ind/100 m2) larger than that of C. rotundicauda (0.007 ind/100 m2). The prosomal width range of T. tridentatus was between 9.0 and 138.0 mm while that of C. rotundicauda was between 20.0 and 111.0 mm, representing their different age distribution patterns. A continuous distribution pattern from 3rd to 14th instar was observed in the T. tridentatus population, whereas a discontinuous distribution pattern from 6th to 16th instars was observed in the C. rotundicauda population. This instar distribution pattern implies that these areas are crucial nursery habitats for the two species. No horseshoe crab within the 1st to 2nd instars (for T. tridentatus) or before 6th instar (for C. rotundicauda) were observed within five years, indicating a declining age structure with few natural births. It was concluded that the declining population would exacerbate the risk of endangered horseshoe crab. There is an urgent need to strengthen the protection of horseshoe crab in the Beibu Gulf, particularly to protect their nursery habitats. The value of long-term citizen science based-monitoring was highlighted, which greatly increased public awareness of horseshoe crab protection.

Morphometric Character of Horseshoe Crab (Tachypleus) at Mempawah Mangrove Park Coastal Area, West Kalimantan, Indonesia

Abstract In Mempawah Mangrove Park (MMP) coastal area, fishers are still often catch horseshoe crabs as a bycatch. One of its genera is Tachypleus. This research aimed to assess the morphometric character of Tachypleus from MMP Coastal area, West Kalimantan. A total of 14 parameters were measured from samples that were collected using a trammel net. The sampling frequency was three times from July to September 2022. Physical and chemical parameters of the waters are also studied to support the ecological information. Two species of Tachypleus were found in these waters, i.e. Tachypleus gigas (8 males and 4 females) and Tachypleus tridentatus (6 males and 11 females). In terms of size, females were larger than males in both species. Based on morphometric comparison between females and males of T. gigas, there was a significant difference in CL/PW ratio (p<0.05). On the other hand, for T. tridentatus, the ratio of PL/OL differed significantly (p<0.05).

Cloning and functionally characterization of TtVtg2-like in horseshoe crab Tachypleus tridentatus: A special focus on ovarian development

Horseshoe crabs are living fossils. In recent decades, the population of horseshoe crabs, especially the tri-spine horseshoe crab Tachypleus tridentatus, has decreased significantly and was listed as an ‘endangered species’ under the IUCN Red List in 2019. In order to improve the reproduction of T. tridentatus to facilitate stock enhancement, it is important to understand their ovarian development. In this study, a novel TtVtg2-like gene from T. tridentatus was cloned and functionally characterized. The total legth of TtVtg2-like was 5469 bp, encoding a protein consisting of 1822 amino acid with a pI value of 6.51 and a molecular weight of 208.68 KDa. The TtVtg2-like was highly expressed in the ovary and yellow connective tissues, mainly localized in cytoplasm and endoplasmic reticulum vesicles of oocytes and yellow connective tissues, respectively. RNA interference of TtVtg2-like caused the accumulation of ROS, DNA damage, and apoptosis of ovarian primary cells. The results of this study provide useful baseline information for future studies on ovarian development in horseshoe crabs.

The development of novel genome-SSRs, multiplex PCR panels, and allelic ladders for parentage identification in Tachypleus tridentatus

Tachypleus tridentatus, one of the oldest animal species on Earth, with significant biological and ecological value, its population sizes have declined due to overfishing. Stock enhancement technology has been used to protect T. tridentatus, however, its efficiency needs to be addressed. In the present study, a parentage identification system was developed. We identified 235,336 SSRs and 182,394 primer pairs were designed. Out of 321 synthesized primers, 14 highly polymorphic primers were selected to reveal the genetic diversity of 110 T. tridentatus. SSRs were amplified with the 14 developed primers, which were fluorescently labeled, and the resulting PCR products were analyzed using the ABI DNA Analyzer 3730xl. The number of alleles per locus, the polymorphism information content, the observed heterozygosity, and the expected heterozygosity ranged from 7 to 48, 0.581 to 0.963, 0.636 to 0.909, and 0.645 to 0.969, respectively. The cumulative exclusion probability with both unknown parents, with only unknown parents, or with both known parents was >99.9999% for the 14 loci. Furthermore, to facilitate data exchange across laboratories and platforms, multiplex PCR detection, SSR fragment size ladders, and parentage identification were employed. Out of the 14 primers, 6 optimal primers were selected for multiplex PCR validation, and two sets of triplex systems were established. Three thousand clones for 160 alleles of the six primers were selected for plasmid extraction and sequencing, and 600 correct plasmids were obtained for new T. tridentatus SSR fragment size ladder construction. Two pairs of parents with 12 offspring each were used for parentage identification by the multiplex PCR systems and ladders, the cumulative average exclusion probability of these 6 primers reached 99.9999% among parents and their offsprings, and was 0% in nonparent-offspring group. The parentage identification system constructed in this study can serve as a tool for monitoring the survival rates of T. tridentatus in stock enhancement programs.

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 1956bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26kDa 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.

Hypoxia impairs cellular energy allocation in the juvenile horseshoe crab Tachypleus tridentatus

Although hypoxia is a serious environmental concern for marine ecosystems globally, its biological effects on the benthic biota remain mostly unclear for some endangered species. To provide an deep understanding of the possible effects of hypoxia on the tri-spine horseshoe crab Tachypleus tridentatus, the cellular energy allocation (CEA) approach was utilized to examine the cellular responses and adaption potential of horseshoe crabs. We examined the energetic responses of T. tridentatus under low dissolved oxygen level (2 mg O2/L). The horseshoe crabs first experienced 14 days of hypoxic stress, and then recovered in a normal dissolved oxygen environment for 7 days. On the 7th and 14th day of hypoxic exposure, the levels of available energy, electron transport system activity, protein, lipids, and carbohydrates were decreased in T. tridentatus (p < 0.05). All measured parameters in the hypoxic group partially or completely recovered after seven days of re‑oxygenation, reaching a level that was significantly up-regulated (p < 0.05) compared with the 14th day and non-significantly different from the 0th day exposure (p ˃ 0.05). In conclusion, hypoxic stress has adverse effects on the energy balance of juvenile T. tridentatus, but these adverse effects can be alleviated in a short recovery period. As a result, our findings provide novel perspectives on the physiology of T. tridentatus under hypoxia acclimation, which is essential information for establishing ideal conditions for the cultivation of this endangered species.

Modeling potential habitats of threatened horseshoe crabs in Indo-Pacific Asia: Suggestions for conservation management

Identifying critical habitats of threatened marine species is key to area-based management measures, while it can be of great challenge for those species that distribute continentally. Asian horseshoe crabs, including tri-spine horseshoe crab Tachypleus tridentatus, coastal horseshoe crab T. gigas, and mangrove horseshoe crab Carcinoscorpius rotundicauda, are globally threatened marine chelicerates, which are largely distributed in developing countries in Indo-Pacific Asia with limited baseline information. Here, we performed the maximum entropy model (MaxEnt), a widely used correlative species distribution model, to explore potential habitats of the Asian horseshoe crabs. Presence-only data of the adult Asian horseshoe crabs and the seven sea benthic environmental predictors, which included temperature, salinity, chlorophyll a concentration, dissolved oxygen, current velocity, bathymetry, and slope, were input into the MaxEnt. The Cloglog output format was adopted to estimate the presence probability of these three species under species-specific regularization multipliers. Our results showed that all three species have potential habitats in Southeast Asia. The range of potential habitats is the most widespread for T. tridentatus, but the most restricted for T. gigas. Bathymetry, temperature, and chlorophyll a concentration are the main contributing environmental predictors for the projected distribution of T. tridentatus, while for T. gigas and C. rotundicauda, temperature and bathymetry have contributed the most. Temperature of the potential habitats is the lowest for T. tridentatus (25.6 ± 4.4 °C), compared to the more tropically distributed T. gigas and C. rotundicauda (around 29 °C). Core habitats of the estuary-bound C. rotundicauda have shallower bathymetry of 5.8 ± 3.5 m in depth. Overall, Asian horseshoe crab species share similar niches, with the common characteristics of shallow bathymetry (<15 m in depth), high temperature (>25 °C), and relatively higher chlorophyll a concentration in coastal waters. We conclude that the projected habitats serve as a critical reference for the conservation management of threatened Asian horseshoe crabs under extensive baseline gaps. Generalized conservation strategies are suggested, depending on the sufficiency of baseline data, to facilitate the formation of specific decision-making.

Impact of hypoxia on glucose metabolism and hypoxia signaling pathways in juvenile horseshoe crabs Tachypleus tridentatus

Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.

Combined effects of norfloxacin and polystyrene nanoparticles on the oxidative stress and gut health of the juvenile horseshoe crab Tachypleus tridentatus

Pollution with anthropogenic contaminants including antibiotics and nanoplastics leads to gradual deterioration of the marine environment, which threatens endangered species such as the horseshoe crab Tachypleus tridentatus. We assessed the potential toxic mechanisms of an antibiotic (norfloxacin, 0, 0.5, 5 μg/L) and polystyrene nanoparticles (104 particles/L) in T. tridentatus using biomarkers of tissue redox status, molting, and gut microbiota. Exposure to single and combined pollutants led to disturbance of redox balance during short-term (7 days) exposure indicated by elevated level of a lipid peroxidation product, malondialdehyde (MDA). After prolonged (14–21 days) exposure, compensatory upregulation of antioxidants (catalase and glutathione but not superoxide dismutase) was observed, and MDA levels returned to the baseline in most experimental exposures. Transcript levels of molting-related genes (ecdysone receptor, retinoic acid X alpha receptor and calmodulin A) and a molecular chaperone (cognate heat shock protein 70) showed weak evidence of response to polystyrene nanoparticles and norfloxacin. The gut microbiota T. tridentatus was altered by exposures to norfloxacin and polystyrene nanoparticles shown by elevated relative abundance of Bacteroidetes. At the functional level, evidence of suppression by norfloxacin and polystyrene nanoparticles was found in multiple intestinal microbiome pathways related to the genetic information processing, metabolism, organismal systems, and environmental information processing. Future studies are needed to assess the physiological and health consequences of microbiome dysbiosis caused by norfloxacin and polystyrene nanoparticles and assist the environmental risk assessment of these pollutants in the wild populations of the horseshoe crabs.

Revitalizing the Population of Tachypleus tridentatus: A Comprehensive Study Tachypleus tridentatus Conservation in China

Tachypleus tridentatus is an ancient marine arthropod. Currently, the population of Tachypleus tridentatus has decreased rapidly, in 2019, the IUCN Red List officially updated the endangered status of Tachypleus tridentatus to Endangered (EN), indicating a global decline in the resources of this species. However, the lack of data, such as population baseline data, acts as a barrier for this nearshore habitat and medically important species. The results of this paper suggest that the main causes of the decline in the population of the Tachypleus tridentatus darter are overfishing and habitat destruction. In addition, the discussion focuses on three techniques presently employed in China to revive the population of Tachypleus tridentatus, including fisheries enhancement, habitat designation for conservation, and educational programs. The study also examines the advantages and disadvantages of conservation methods implemented in China, and while certain methods, such as moratoriums on aquaculture, show significant advantages, others, such as the establishment of protected habitats, have limitations due to incomplete findings. These methods provide a solid foundation for the conservation of the Tachypleus tridentatus, and also point to possible future directions, such as the implementation of polyculture. However, there is a need to improve the efficiency of existing methods through further development and additional data collection studies. To address the acknowledged limitations and gaps in this study, additional exploration and discourse are imperative to advance the preservation of Tachypleus tridentatus.

Integrated transcriptomics and metabolomics reveal the toxic mechanisms of mercury exposure to an endangered species Tachypleus tridentatus

Mercury (Hg) pollution is threatening the health of endangered Tachypleus tridentatus whereas the toxic mechanism is still unclear. This study combined transcriptomic and metabolomics technology to reveal the toxic mechanisms of mercury (Hg 2+, 0.025 mg/L) exposing to T. tridentatus larvae for 15 days. Mercury induced cellular toxicity and cardiovascular dysfunction by dysregulating the genes related to endocrine system, such as polyubiquitin-A, cathepsin B, atrial natriuretic peptide, etc. Mercury induced lipid metabolic disorder with the abnormal increase of lysoPC, leukotriene D4, and prostaglandin E2. Cytochrome P450 pathway was activated to produce anti-inflammatory substances to reconstruct the homeostasis. Mercury also inhibited arginine generation, which may affect the development of T. tridentatus by disrupting the crucial signaling pathway. The mercury methylation caused enhancement of S-adenosylmethionine to meet the need of methyl donor. The mechanisms described in present study provide new insight into the risk assessment of mercury exposure to T. tridentatus.

A new strategy for optimizing marine protection networks by considering functional connectivity: An example of Tachypleus tridentatus

The concept of creating a conservation network through the establishment of multiple protected areas to protect marine fauna is gaining momentum. To effectively establish a network that yields favorable outcomes, it is imperative to identify suitable areas that foster optimal connectivity. Given the swift development of coastal areas, it has become necessary to develop cost-effective measures that yield maximum conservation benefits. However, exploration of these ideas for application to marine conservation networks remains scarce. In this study, we used the horseshoe crab (Tachypleus tridentatus) in the Beibu Gulf as an example. The species and environmental data were collected through field sampling and remote sensing. The species distribution model, the least-cost path model, and circuit theory were employed to establish a marine conservation network. The functional connectivity metrics were employed to optimize both the interconnection and economic efficiency of the network. The results indicated a low presence of adult horseshoe crabs in the Beibu Gulf, with average densities of 1.47 and 1.43 ind./km2 among the 38 surveyed stations in 2018 and 2019, respectively. The species distribution model accurately predicted juvenile presence in mudflats and adult presence offshore, and it identified 43 potential areas that are suitable for the establishment of marine protected areas. The marine protected areas that ranked higher in importance contributed significantly to maintaining the integrity of regional connectivity. The network connectivity shows a law of diminishing returns as the number of marine protected areas increases. The framework developed in this study will provide decision support for building and optimizing marine conservation networks.

Reconstitution of (1→3)-β-D-glucans measurement system using recombinant Limulus polyphemus Factor G.

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-β-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and β from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for β. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and β subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and β subunits for BDG-dependent activation.

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.

Response of moulting genes and gut microbiome to nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus

Nanoplastics (NPs) and heavy metals are typical marine pollutants, affecting the gut microbiota composition and molting rate of marine organisms. Currently, there is a lack of research on the toxicological effects of combined exposure to horseshoe crabs. In this study, we investigated the effects of NPs and copper on the expression of molt-related genes and gut microbiome in juvenile tri-spine horseshoe crabs Tachypleus tridentatus by exposing them to NPs (100 nm, 104 particles L−1) and/or Cu2+ (10 μgL−1) in seawater for 21 days. Compared with the control group, the relative mRNA expression of ecdysone receptor (EcR), retinoid x receptor (RXR), calmodulin-A-like isoform X1 (CaM X1), and heat shock 70 kDa protein (Hsp70) were significantly increased under the combined stress of NPs and Cu2+. There were no significant differences in the diversity and abundance indices of the gut microbial population of horseshoe crabs between the NPs and/or Cu2+ groups and the control group. According to linear discriminant analysis, Oleobacillus was the most abundant microorganism in the NPs and Cu2+ stress groups. These results indicate that exposure to either NPs stress alone or combined NPs and Cu2+ stress can promote the expression levels of juvenile molting genes. NPs exposure has a greater impact on the gut microbial community structure of juvenile horseshoe crabs compared to Cu2+ exposure. This study is helpful for predicting the growth and development of horseshoe crabs under complex environmental pollution.

Morphometry of Limulidae (Oscar E. Brown) in Leidong, Sei Berombang, and Tanjung Tiram, North Sumatera

Limulidae (Horseshoe crab), or Mimi in the local name, are known; there are four species, namely Limulus polyphemus in North America and Asia, namely Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. In Indonesia, these species are protected by the Decree of the Minister of Forestry Number 12/KPTS-II/1987; meanwhile, illegal fishing still occurs. This study aims to identify Limulidae species and analyze Mimi's morphometric characteristics and distribution in North Sumatra to conserve the species. This research was conducted using a quantitative descriptive method in Leidong, Sei Berombang, Tanjung Tiram - North Sumatera, from September to December 2021. Biometric measurements were performed on 98 Mimi, and the data were statistically analyzed using the Kruskal Wallis test, Mann-Whitney test, and Multinomial logistics. The results showed that Mimi in the study area was identified as Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. The most commonly caught was T. tridentatus at the Tanjung Tiram. Based on the maximum width of the prosoma and body length, the largest to most petite sizes are T. tridentatus at Leidong (10.31 cm), T. gigas at Leidong, and Sei Berombang (10 cm) and C. rotundicauda at Sei Berombang (7.56 cm) respectively. The cluster analysis based on 27 morphometric characters showed that T. tridentatus had almost the same characteristics as T. gigas, while C. rotundicauda had its features. It can be concluded that the largest to most petite sizes of Mimi in the study area were T. tridentatus, T. gigas, and C. rotundicauda, respectively.

The use of physiological and transcriptional analyses to examine molting regulatory mechanisms in juvenile horseshoe crab Tachypleus tridentatus

Molting is a necessary developmental process in crustaceans, and is holistically regulated by a range of hormones and associated genes. In order to investigate physiological changes occurring at the molecular level during the molting cycle of juvenile Tachypleus tridentatus, chitinase and N-acetyl-β-D-glucosaminidase (β-NAGase) activities were firstly measured by absorbance spectrophotometry. In addition, ecdysone content was quantified by ELISA, and changes in expression levels of genes relevant to ecdysis were examined based on quantitative real-time PCR (qRT-PCR) and RNA-sequencing (RNA-seq). Results showed that chitinase, β-NAGase activity, and ecdysone content increased at the intermolt stages, peaked at premolt stages, and decreased to a lower level at molt and postmolt stages. Chitinase and β-NAGase were positively correlated with ecdysone. RNA-seq revealed that a total of 6482 differentially expressed genes (DEGs) in the four different molting stages, and 5763 DEGs had higher expression levels in the premolt stage than in the intermolt stage. A total of 59 shared DEGs were identified in all molting stages, and the highly expressed DEGs were mainly related to chitin binding, stratum corneum structural composition and neuropeptide hormone activity signaling pathways. KEGG pathway analysis showed that at the premolt stage, digestion, transport, and catabolic signaling pathways were significantly enriched and innate immune response pathways were significantly enriched as molting progressed. These results suggested that the molting of juvenile horseshoe crabs may be directly regulated by ecdysone, and chitinase and β-NAGase play a synergistic role with ecdysone during the molting cycle. The key biological processes and signaling pathways involved in hormone regulation, epidermal structural proteins, catabolism, apoptosis, and immune response potentially regulate the molting process of horseshoe crab juveniles. This study provides a theoretical basis for enhancing understanding of the molting and growth processes in marine arthropods, and a strong theoretical support for the developmenting of captive rearing technologies for Chinese horseshoe crabs.

COI Gene Analysis of Asian Horseshoe Crab in Banyuasin Estuarine Waters, Sumatra, Indonesia

There is limited study on DNA barcoding of horseshoe crabs in South Sumatra, Indonesia. The present study was the first to record horseshoe crabs' COI DNA barcoding in three localities of Banyasin estuarine waters. The nucleotide composition was in a strong adenine thymine bias (AT = 63.90%) with high haplotypic diversity (Hd = 0.858) relative to low nucleotide diversity (π = 0.0534). A total of 16 haplotypes were recorded, of which two were in Carat Cape, 4 in Makati Jaya, and 12 in the Banyuasin river estuary (BRE), and Hap 1 was a dominant type (62.5%). The population differentiation (FST) value was varied, and only in the comparison of BRE-Carat Cape was significant (FST = 0.637), while the gene flow (Nm) value in Makati Jaya-BRE was high (6.563). The neutrality test, Tajima's D (0.4142), and Fu's Fs values were negative (-0.492), suggesting these populations have experienced a current bottleneck or multiplication. Analysis of molecular variation (AMOVA) suggested that 68% was distributed within populations. The haplotype network, PCoA, and phylogenetic of COI DNA sequences clustered in three groups correspond to Carcinoscorpius rotundicauda, Tachypleus gigas, and Tachypleus tridentatus. This finding is a matter of concern for managing and conserving horseshoe crab species in Banyuasin estuarine waters.

Effects of Oncolytic Vaccinia Viruses Harboring Different Marine Lectins on Hepatocellular Carcinoma Cells.

Oncolytic viruses are being developed as novel strategies for cancer therapy. Our previous studies have shown that vaccinia viruses armed with marine lectins improved the antitumor efficacy in diverse cancer types. The objective of this study was to assess the cytotoxic effects of oncoVV harboring Tachypleus tridentatus lectin (oncoVV-TTL), Aphrocallistes vastus lectin (oncoVV-AVL), white-spotted charr lectin (oncoVV-WCL), and Asterina pectinifera lectin (oncoVV-APL) on HCC. Our data revealed that the effects of recombinant viruses on Hep-3B cells were oncoVV-AVL > oncoVV-APL > oncoVV-TTL > oncoVV-WCL; oncoVV-AVL showed stronger cytotoxicity than oncoVV-APL, while oncoVV-TTL/WCL had no effect on cell killing in Huh7 cells, and PLC/PRF/5 cells exhibited sensitivity to oncoVV-AVL/TTL but not to oncoVV-APL/WCL. The cytotoxicity of oncoVV-lectins could be enhanced by apoptosis and replication in a cell-type-dependent manner. Further research revealed that AVL may mediate various pathways, including MAPK, Hippo, PI3K, lipid metabolism, and androgen pathways through AMPK crosstalk, to promote oncoVV replication in HCC in a cell-dependent manner. OncoVV-APL replication could be affected by AMPK/Hippo/lipid metabolism pathways in Hep-3B cells, AMPK/Hippo/PI3K/androgen pathways in Huh7 cells, and AMPK/Hippo pathways in PLC/PRF/5 cells. OncoVV-WCL replication was also multi-mechanistic, which could be affected by AMPK/JNK/lipid metabolism pathways in Hep-3B cells, AMPK/Hippo/androgen pathways in Huh7 cells, and AMPK/JNK/Hippo pathways in PLC/PRF/5 cells. In addition, AMPK and lipid metabolism pathways may play critical roles in oncoVV-TTL replication in Hep-3B cells, and oncoVV-TTL replication in Huh7 cells may depend on AMPK/PI3K/androgen pathways. This study provides evidence for the application of oncolytic vaccinia viruses in hepatocellular carcinoma.