Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Novel MIP gene mutation causes autosomal-dominant congenital cataract.

AIM: To identify disease-causative mutations in families with congenital cataract. METHODS: Two Chinese families with autosomal-dominant congenital cataract (ADCC) were recruited and underwent comprehensive eye examinations. Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family. Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis. The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis. Major intrinsic protein (MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos. Zebrafish embryonic lens phenotypes were screened using confocal microscopy. RESULTS: A novel heterozygous mutation (c.85G>A; p.G29R) in the MIP gene was identified in the proband of one family. A known heterozygous mutation (c.97C>T; p.R33C; rs864309693) in MIP was found in the proband of another family. In-silico prediction indicated that the novel mutation might affect the MIP protein function. Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP. CONCLUSION: Two missense mutations in the MIP gene in Chinese cataract families are identified, and one of which is novel. These findings expand the genetic spectrum of MIP mutations associated with cataracts. The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

A Frequent Diagnostic Pitfall: Dermatological Clinical Features and Pathological Results in Diagnosing Pagetoid Squamous Cell Carcinoma in situ (Case Series).

Squamous cell carcinoma (SCC) in situ can occur on any skin or mucus surface and is more commonly found in elderly patients on areas of skin that have been sunburnt. Most previous case reports are from dermatologists, with few published reports from pathologists. In this study, three patients underwent pathological routine and auxiliary immunohistochemical (IHC) examination and were ultimately diagnosed with pagetoid SCC in situ - a different diagnosis from the initial clinical assessment. All three patients received a complete resection of the skin mass. After follow-up, as of June 2023, the patients had no tumour recurrence or metastasis. Pagetoid SCC in situ is a particular type of SCC in situ that has no specific features in clinical manifestations, gross diagnosis or histopathological sections. The final diagnosis depends on IHC staining. Pagetoid SCC in situ expresses EMA, CK5/6 and p63 but not CEA, CK8 or S-100, which are expressed in extramammary Paget's disease. Pagetoid SCC in situ is usually only locally invasive, and the main treatment is complete surgical resection. The prognosis is related to human papillomavirus infection, surgical margin closure, disease location, tumour thickness and other factors.

Sparse Generalized Canonical Correlation Analysis: Distributed Alternating Iteration-Based Approach.

Sparse canonical correlation analysis (CCA) is a useful statistical tool to detect latent information with sparse structures. However, sparse CCA, where the sparsity could be considered as a Laplace prior on the canonical variates, works only for two data sets, that is, there are only two views or two distinct objects. To overcome this limitation, we propose a sparse generalized canonical correlation analysis (GCCA), which could detect the latent relations of multiview data with sparse structures. Specifically, we convert the GCCA into a linear system of equations and impose $\ell _1$ minimization penalty to pursue sparsity. This results in a nonconvex problem on the Stiefel manifold. Based on consensus optimization, a distributed alternating iteration approach is developed, and consistency is investigated elaborately under mild conditions. Experiments on several synthetic and real-world data sets demonstrate the effectiveness of the proposed algorithm.

Cisplatin-Based Combination Therapy for Enhanced Cancer Treatment.

Cisplatin, a primary chemotherapeutic drug, is of great value in the realm of tumor treatment. However, its clinical efficacy is strictly hindered by issues, such as drug resistance, relapse, poor prognosis, and toxicity to normal tissue. Cisplatin-based combination therapy has garnered increasing attention in both preclinical and clinical cancer research for its ability to overcome resistance, reduce toxicity, and enhance anticancer effects. This review examines three primary co-administration strategies of cisplatin-based drug combinations and their respective advantages and disadvantages. Additionally, seven types of combination therapies involving cisplatin are discussed, focusing on their main therapeutic effects, mechanisms in preclinical research, and clinical applications. This review also discusses future prospects and challenges, aiming to offer guidance for the development of optimal cisplatin-based combination therapy regimens for improved cancer treatment.

LettuceDB: an integrated multi-omics database for cultivated lettuce.

Crop genomics has advanced rapidly during the past decade, which generated a great abundance of omics data from multi-omics studies. How to utilize the accumulating data becomes a critical and urgent demand in crop science. As an attempt to integrate multi-omics data, we developed a database, LettuceDB (https://db.cngb.org/lettuce/), aiming to assemble multidimensional data for cultivated and wild lettuce germplasm. The database includes genome, variome, phenome, microbiome and spatial transcriptome. By integrating user-friendly bioinformatics tools, LettuceDB will serve as a one-stop platform for lettuce research and breeding in the future. Database URL: https://db.cngb.org/lettuce/.

Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) on bitterling fishes (Acheilognathinae) from China.

Three new species of Gyrodactylus are described from three species of bitterling in Donghu Lake, China: Gyrodactylus ocellorhodei n. sp. from Rhodeus ocellatus; G. sinenorhodei n. sp. from Rhodeus sinensis; and G. acheilorhodei n. sp. from Acheilognathus macropterus. All the three new species showed similar opisthaptor morphology, especially the marginal hooks: all had a slender and perpendicular sickle shaft, and flat sickle base with distinct heel and inner arch which was different from the G. rhodei-group species parasitic on bitterling. Multivariate analyses based on hamulus and marginal hooks suggested that these three new species cannot be completely distinguished, despite some morphology divergence observed in certain less reliable morphometric features, such as hamulus root length, ventral bar total length and process shape. These three new species shared an identical 18S ribosomal RNA gene sequence, while the variation in the Internal Transcribed Spacers (ITS1-ITS2) sequence among them (8.4-11.2%, K2P) far exceeded the 1% ITS sequence difference that had been suggested as a threshold for species delimitation of Gyrodactylus. Phylogenetic analysis based on ITS1-ITS2 showed that all these sequenced Gyrodactylus spp. parasitic on the subfamily Acheilognathinae host formed a monophyletic group. However, a clear differentiation (18.9-20.9%, K2P of ITS1-ITS2) could be found between the subgroup from China (G. ocellorhodei n. sp., G. sinenorhodei n. sp. and G. acheilorhodei n. sp.) and that from Europe (G. rhodei).

Effects of loneliness and isolation on cardiovascular diseases: a two sample Mendelian Randomization Study.

BACKGROUND: Loneliness and isolation are associated with multiple cardiovascular diseases (CVDs), but there is a lack of research on whether they were causally linked. We conducted a Mendelian Randomization (MR) study to explore causal relationships between loneliness and isolation and multiple CVDs. METHODS: Single nucleotide polymorphisms associated with loneliness and isolation were identified from a genome-wide association study (GWAS) of 455,364 individuals of European ancestry in the IEU GWAS database. Summary data for 15 CVDs were also obtained from the IEU GWAS database. We used three MR methods including inverse variance weighting, MR-Egger, and weighted median estimation to assess the causal effect of exposure on outcomes. Cochran's Q test and MR-Egger intercept test were used to evaluate the heterogeneity and pleiotropy. RESULTS: MR analysis showed that loneliness and isolation were significantly associated with essential hypertension (OR = 1.07, 95% CI: 1.03-1.12), atherosclerotic heart disease (OR = 1.04; 95% CI: 1.02-1.06), myocardial infarction (OR = 1.02; 95% CI: 1-1.04) and angina (OR = 1.04; 95% CI =1.02-1.06). No heterogeneity and pleiotropy effects were found in this study. CONCLUSIONS: Causal relationship of loneliness and isolation with CVDs were found in this study.

Investigating the impact of remnant cholesterol on new-onset stroke across diverse inflammation levels: Insights from the China Health and Retirement Longitudinal Study (CHARLS).

BACKGROUND: Prior research underscores the significant impact of remnant cholesterol (RC) on stroke occurrence due to its proatherogenic and proinflammatory traits. This study aims to explore diverse risks of new-onset stroke associated with RC, considering distinct inflammation levels in the middle-aged and senior population in China. METHODS: We analyzed 6509 participants from the China Health and Retirement Longitudinal Study (CHARLS) across four waves (2011-2018). We employed a multivariable Cox proportional hazards regression model, incorporated restricted cubic spline techniques, and conducted sensitivity analyses to evaluate the association among RC, high-sensitivity C-reactive protein (hsCRP), and the risk of new-onset stroke. RESULTS: Over 7 years, 540 new-onset strokes occurred. Individuals in the highest quartile of RC levels exhibited a heightened risk of new-onset stroke, with a multivariable-adjusted hazard ratio (HR) peaking at 1.50 (95% confidence interval 1.12-2.00, P for trend = 0.021), showing a non-linear correlation (P nonlinearity = 0.049). High hsCRP alone had an adjusted HR of 1.10 (95% CI 0.87-1.39), compared to 1.40 (95% CI 1.00-1.96) for high RC alone. Additionally, concurrent high RC and hsCRP showed an adjusted HR of 1.43 (95% CI 1.05-1.96). Consistency persisted across various hsCRP thresholds, after adjusting for additional parameters, or excluding chronic diseases in the primary model, reinforcing result robustness. CONCLUSION: Our findings reveal a substantial and non-linear association between higher baseline RC levels and an elevated risk of new-onset stroke. Moreover, elevated levels of both RC and hsCRP jointly pose the highest risk for new-onset stroke, surpassing the risk associated with each factor individually.

Association between PEMT rs7946 and blood pressure levels in Chinese adolescents.

OBJECTIVES: This study was to explore blood pressure levels in Chinese adolescents with different genotypes of phosphatidylethanolamine N-methyltransferase (PEMT) gene (PEMT) rs7946, as well as effects of dietary intake on blood pressure levels with different genders and different genotypes of PEMT rs7946. METHODS: PEMT rs7946 genotypes were identified by PCR-restriction fragment length polymorphism and verified by DNA sequencing. Blood pressure was measured using a standard mercury sphygmomanometer. Dietary intakes were analyzed based on a 3-day diet diary, and dietary components were calculated using computer software. RESULTS: A total of 721 high school students (314 males and 407 females) at the age of 16.86 ±â€…0.59 years were included. The A allele carriers of PEMT rs7946 had increased levels of SBP, DBP, mean arterial pressure (MAP) and pulse pressure (PP) than the GG homozygotes in the female subjects. There were significant interactions between PEMT rs7946 and gender on SBP and MAP levels, regardless of whether an unadjusted or adjusted model was used. When dietary intake was taken into account, fat intake was positively associated with SBP and PP in the male GG homozygotes, while protein intake was positively associated with PP in the female A allele carriers of PEMT rs7946. CONCLUSION: This study suggests that PEMT rs7946 is significantly associated with blood pressure levels in human being. There might be interactions among PEMT rs7946, gender, and dietary intake on blood pressure levels in the adolescent population.

Stratifying ICIs-responsive tumor microenvironment in HCC: from parsing out immune-hypoxic crosstalk to clinically applicable MRI-radiomics models.

BACKGROUND: We aimed to redefine Immune checkpoint inhibitors (ICIs)-responsive "hot" TME and develop a corresponding stratification model to maximize ICIs-efficacy in Hepatocellular Carcinoma (HCC). METHODS: Hypoxic scores were designed, and the relevance to immunotherapy responses were validated in pan-cancers through single cell analysis. Multi-omics analysis using the hypoxic scores and immune infiltrate abundance was performed to redefine the ICIs-responsive TME subtype in HCC patients from TCGA (n = 363) and HCCDB database (n = 228). The immune hypoxic stress index (IHSI) was constructed to stratify the ICIs-responsive TME subtype, with exploring biological mechanism in vitro and in vivo. MRI-radiomics models were built for clinical applicability. RESULTS: The hypoxic scores were lower in the dominant cell-subclusters of responders in pan-cancers. The higher immune infiltrate-normoxic (HIN) subtype was redefined as the ICIs-responsive TME. Stratification of the HIN subtype using IHSI effectively identified ICIs-responders in Melanoma (n = 122) and urological cancer (n = 22). TRAF3IP3, the constituent gene of IHSI, was implicated in ICIs-relevant "immune-hypoxic" crosstalk by stimulating MAVS/IFN-I pathway under normoxic condition. MRI-radiomics models assessing TRAF3IP3 with HIF1A expression (AUC > 0.80) screened ICIs-Responders in HCC cohort (n = 75). CONCLUSION: The hypoxic-immune stratification redefined ICIs-responsive TME and provided MRI-Radiomics models for initial ICIs-responders screening, with IHSI facilitating further identification.

A strategy of novel molecular hydrogen-producing antioxidative auxiliary system improves virus production in cell bioreactor.

In the increasing demand for virus vaccines, large-scale production of safe, efficient, and economical viral antigens has become a significant challenge. High-cell-density manufacturing processes are the most commonly used to produce vaccine antigens and protein drugs. However, the cellular stress response in large-scale cell culture may directly affect host cell growth and metabolism, reducing antigen production and increasing production costs. This study provided a novel strategy of the antioxidant auxiliary system (AAS) to supply molecular hydrogen (H2) into the cell culture media via proton exchange membrane (PEM) electrolysis. Integrated with a high-density cell bioreactor, the AAS aims to alleviate cellular stress response and increase viral vaccine production. In the results, the AAS stably maintained H2 concentration in media even in the high-air exposure tiding cell bioreactor. H2 treatment was shown safe to cell culture and effectively alleviated oxidative stress. In two established virus cultures models, bovine epidemic fever virus (BEFV) and porcine circovirus virus type 2 (PCV-2), were employed to verify the efficacy of AAS. The virus yield was increased by 3.7 and 2.5 folds in BEFV and PCV-2 respectively. In conclusion, the AAS-connected bioreactor effectively alleviated cellular oxidative stress and enhanced virus production in high-density cell culture.

Proteomic and metabolomic features in patients with HCC responding to lenvatinib and anti-PD1 therapy.

Combination therapy (lenvatinib/programmed death-1 inhibitor) is effective for treating unresectable hepatocellular carcinoma (uHCC). We reveal that responders have better overall and progression-free survival, as well as high tumor mutation burden and special somatic variants. We analyze the proteome and metabolome of 82 plasma samples from patients with hepatocellular carcinoma (HCC; n = 51) and normal controls (n = 15), revealing that individual differences outweigh treatment differences. Responders exhibit enhanced activity in the alternative/lectin complement pathway and higher levels of lysophosphatidylcholines (LysoPCs), predicting a favorable prognosis. Non-responders are enriched for immunoglobulins, predicting worse outcomes. Compared to normal controls, HCC plasma proteins show acute inflammatory response and platelet activation, while LysoPCs decrease. Combination therapy increases LysoPCs/phosphocholines in responders. Logistic regression/random forest models using metabolomic features achieve good performance in the prediction of responders. Proteomic analysis of cancer tissues unveils molecular features that are associated with side effects in responders receiving combination therapy. In conclusion, our analysis identifies plasma features associated with uHCC responders to combination therapy.

Discovery of a nitroaromatic nannocystin with potent in vivo anticancer activity against colorectal cancer by targeting AKT1.

The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.

Megafossils of Betulaceae from the Oligocene of Qaidam Basin and their paleoenvironmental and phytogeographic implications.

Understanding the paleoenvironment and phytogeographical history of the Tibetan Plateau, China relies on discovering new plant fossils. The Qaidam Basin has long been regarded as an ideal 'field laboratory' to investigate the paleoclimate and paleobiological evolution of the northern Tibetan Plateau. However, fossil angiosperms from the Qaidam Basin are rare, and our knowledge of its paleovegetation is poor. Here, we report fossil leaves and fruits of Betulaceae found from the Oligocene Shangganchaigou Formation of northwestern Qaidam Basin (Huatugou area). Comparative morphological analysis led us to assign the fruits to the Betula subgenus Betula and the leaves to Carpinus grandis. These findings, together with other reported fossil plants from the same locality, reveal a close floristic linkage between the Qaidam Basin and Europe during the Oligocene. The northern pathway of this floristic exchange may have crossed through the Qaidam Basin during the late Paleogene. This floristic linkage may have been facilitated by the continuous narrowing of the Turgai Strait and stronger westerlies, which transported moisture and provided favorable climatic conditions. Indeed, fossil plants collected from the Qaidam Basin suggest that during the Oligocene this region had warm and humid deciduous broad-leaf forest, which differs from the region's modern vegetation and indicates that the Qaidam Basin may have been a suitable region for these plants to flourish and spread during the Oligocene.

Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection.

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-ß, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Borneol-modified docetaxel plus tetrandrine micelles for treatment of drug-resistant brain glioma.

OBJECTIVE: Glioma is the most common and deadly primary malignant tumor in adults. Treatment outcomes are ungratified due to the presence of blood-brain barrier (BBB), glioma stem cells (GSCs) and multidrug resistance (MDR). Docetaxel (DTX) is considered as a potential drug for the treatment of brain tumor, but its effectiveness is limited by its low bioavailability and drug resistance. Tetrandrine (TET) reverses the resistance of tumor cells to chemotherapy drugs. Borneol (BO) modified in micelles has been shown to promote DTX plus TET to cross the BBB, allowing the drug to better act on tumors. Therefore, we constructed BO-modified DTX plus TET micelles to inhibit chemotherapeutic drug resistance. SIGNIFICANCE: Provide a new treatment method for drug-resistant brain gliomas. METHODS: In this study, BO-modified DTX plus TET micelles were prepared by thin film dispersion method, their physicochemical properties were characterized. Its targeting ability was investigated. The therapeutic effect on GSCs was investigated by in vivo and in vitro experiments. RESULTS: The BO-modified DTX plus TET micelles were successfully constructed by thin film dispersion method, and the micelles showed good stability. The results showed that targeting micelles increased bEnd.3 uptake and helped drugs cross the BBB in vitro. And we also found that targeting micelles could inhibit cell proliferation, promote cell apoptosis and inhibit the expression of drug-resistant protein, thus provide a new treatment method for GSCs in vitro and in vivo. CONCLUSIONS: BO-modified DTX plus TET micelles may provide a new treatment method for drug-resistant brain gliomas.

Immune response and protective efficacy of Streptococcus agalactiae vaccine coated with chitosan oligosaccharide for different immunization strategy in nile tilapia (Oreochromis niloticus).

In the past decade, the outbreak of Streptococcus agalactiae has caused significant economic losses in tilapia farming. Vaccine immunization methods and strategies have gradually evolved from single-mode to multi-mode overall prevention and control strategies. In this study, an inactivated vaccine of S. agalactiae with a chitosan oligosaccharide (COS) adjuvant was constructed using different administration methods: intraperitoneal injection (Ip), immersion combined with intraperitoneal injection (Im + Ip), immersion combined with oral administration (Im + Or), and oral administration (Or). Safety analysis revealed no adverse effects on tilapia, and the vaccine significantly promoted fish growth and development when administered through Im + Or or Or immunization. Following vaccination, innate immunity parameters including SOD, ACP and CAT activities were all significantly enhanced. Additionally, specific serum IgM antibodies reached their highest level at the 6th week post vaccination. Skin and intestinal mucus IgT antibodies reached peaked at the 6th and 7th week post vaccination, respectively. The relative peak expression values for IL-8, IL-12, MHC-I, MHC-II, IgM, IgT, CD4, CD8, TNFα, IFNγ from Im + Ip group were significantly higher than those in Ip group, Im + Or group and Or group in most cases (p < 0.05). Importantly, the relative protection survival of Im + Ip group was the highest (78.6%), followed by the Ip group (71.4%), the Or group (64.3%) and the Im + Or group (57.1%). In summary, this study encourages further research on multi-channel immunization strategies of other kinds of vaccines in other aquatic economic animals to improve their disease resistance.