Next Article in Journal
Neo-X-Linked Chromosome Polymorphism: Cytogenetic Insights from Passalites nemorivagus (Mammalia, Cervidae)
Previous Article in Journal
Spatiotemporal Patterns of Crested Ibis (Nipponia nippon) Movement
Previous Article in Special Issue
Toxic Effects of Povidone-Iodine on Macrobrachium rosenbergii: Concentration-Dependent Responses in Oxidative Stress, Immunosuppression, and Recovery Potential
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 15
Issue 17
10.3390/ani15172556
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Transcriptomic Analysis of Tachypleus tridentatus Larval Response to Vibrio parahaemolyticus Infection

by
Lei Yan
1,2,†,
Jinxia Liu
1,†,
Boyu Chen
1,2,
Fanxi Gao
1,2,
Zizhuo Liu
1,2,
Zhenwen Zhang
1,2,
Shimiao Li
1,2,
Yan Zhang
1,2,
Jiuman Jia
1,2,
Peng Zhu
1,2,3,* and
Yongyan Liao
1,2,*
1
College of Ocean, Beibu Gulf University, Qinzhou 535011, China
2
Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Qinzhou 535011, China
3
Beibu Gulf Marine Ecological Environment Field Observation and Research Station of Guangxi, Qinzhou 535011, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Animals 2025, 15(17), 2556; https://doi.org/10.3390/ani15172556
Submission received: 25 July 2025 / Revised: 22 August 2025 / Accepted: 27 August 2025 / Published: 30 August 2025
(This article belongs to the Special Issue Ecotoxicology in Aquatic Animals: 2nd Edition)
Browse Figures
Versions Notes

Simple Summary

Tachypleus tridentatus is an economically important but endangered marine organism. Although Vibrio parahaemolyticus is a well-known pathogen affecting a broad range of aquatic species, the molecular immune response of T. tridentatus larvae to V. parahaemolyticus infection has not been systematically investigated. This study aimed to elucidate the immune response mechanisms of T. tridentatus larvae to V. parahaemolyticus infection by integrating acute toxicity assays, histopathological analysis, and transcriptome profiling.

Abstract

Tachypleus tridentatus is a rare and endangered marine organism with considerable scientific and economic value. It has existed on Earth for about 450 million years and its continuation to the present day may be related to its unique immune system. Owing to its drastic population decline, diverse technical approaches are required for its recovery, and the development and growth of its larvae are crucial in this context. Vibrio parahaemolyticus is a common marine pathogen that impairs the healthy growth of marine organisms. The peak period of V. parahaemolyticus occurrence is from May to November, which significantly overlaps with the T. tridentatus spawning period from April to September. However, the response mechanisms of juvenile T. tridentatus to V. parahaemolyticus stress remain unknown. Hence, in this study, we aimed to investigate these response mechanisms through acute toxicity assays, histological observations, and transcriptome analysis. The results showed that the 48 h LD50 of V. parahaemolyticus-infected T. tridentatus larvae was determined to be 1.31 × 108 CFU/g. Histological analysis showed that V. parahaemolyticus damaged the larval tissue. In addition, RNA sequencing (RNA-Seq) identified 2347 differentially expressed genes (DEGs; 1440 upregulated and 907 downregulated genes) and 243 enriched signaling pathways. Functional enrichment analysis revealed the enrichment of immunoregulatory pathways, including the Wnt signaling pathway, ECM-receptor interaction, aminoacyl-tRNA biosynthesis, and Toll and Imd signaling pathways. Seventeen DEGs were randomly selected for real-time RT-PCR (RT-qPCR) validation, and their expression patterns were consistent with those obtained via RNA-Seq. The study of the response mechanism of T. tridentatus larvae to V. parahaemolyticus stress provides scientific references for the protection of T. tridentatus habitats and the recovery of its population size.

1. Introduction

Horseshoe crabs, belonging to the phylum Arthropoda, class Merostomata, and order Xiphosura, have existed on Earth for approximately 450 million years [1]. Their forms have changed little over time, and they are recognized as “living fossils” [2]. Tachypleus tridentatus is one of the four remaining horseshoe crab species and is widely distributed along the west coast of the Pacific Ocean, including Japan, Korea, China, Vietnam, the Philippines, Kalimantan, Sumatra, Indonesia, and the Javanese coast [3]. The population of T. tridentatus has declined significantly because of historical factors such as habitat destruction and overfishing [4]. To protect this rare species, various conservation measures have been implemented both domestically and internationally. T. tridentatus was listed as an Endangered Species in China’s Red List in 2004 [5], as Endangered in the IUCN Red List in 2019 [6], and as a National GradSAe II Protected Animal in China in 2021 [7]. The development and enforcement of relevant laws and regulations provide legal protection for the conservation of T. tridentatus. Additionally, to preserve T. tridentatus habitats, many local governments and environmental organizations have carried out ecological restoration and environmental protection projects [8]. Currently, the Beibu Gulf area supports the highest density of T. tridentatus populations in China and is considered an ideal site for marine protected area research [9,10,11,12].
Vibrio parahaemolyticus is a common Gram-negative bacterial pathogen widely present in marine, estuarine, and coastal environments. Its highly virulent strains (carrying the plasmid-encoded PirA/PirB toxin genes) have spread to China, Vietnam, Thailand, Malaysia, Singapore, Mexico, and other countries [13]. It infects aquatic organisms and causes significant losses to the aquaculture industry [14,15,16,17]. It has been detected in fish [18], shellfish [19], shrimp [20], crabs [21], and other aquatic organisms. It also causes foodborne diseases [22,23], resulting in gastrointestinal symptoms such as diarrhea, nausea, vomiting, and abdominal cramps in humans, and may also lead to wound infections [24].
In recent years, the recovery of T. tridentatus populations has been facilitated through two primary strategies: restocking initiatives and habitat protection. In Guangxi’s Beibu Gulf, for example, conservationists have conducted large-scale restocking programs, but their ecological benefits remain difficult to quantify due to insufficient tracking technologies (e.g., acoustic telemetry) and standardized assessment protocols [25]. The peak period of V. parahaemolyticus occurrence is from May to November, which significantly overlaps with the spawning period of T. tridentatus from April to September. The Beibu Gulf is an essential spawning habitat for T. tridentatus, and V. parahaemolyticus has been isolated from both seawater and aquatic products in this area [26,27]. These findings indicate that diseases induced by V. parahaemolyticus pose a serious threat to marine biodiversity restoration and may hinder the recovery of T. tridentatus populations. As an invertebrate, T. tridentatus lacks immunoglobulins and lymphocytes involved in adaptive immune responses in humans [28]. Natural immune effector proteins should be extracted directly from T. tridentatus hemolymph or obtained from inactive immune receptors using recombinant expression techniques. However, the newly hatched T. tridentatus larvae were less than 1 cm in length, making it difficult to meet the criteria for the experiment. Consequently, V. parahaemolyticus infection may disrupt the physiological functions of T. tridentatus larvae and may even result in mortality.
The effects of V. parahaemolyticus on juvenile T. tridentatus remain poorly understood. Therefore, investigating the response mechanisms of juvenile T. tridentatus to V. parahaemolyticus stress is essential for protecting their habitats and evaluating population recovery. This study aimed to provide foundational data for future research on T. tridentatus conservation strategies using ecotoxicology, histopathology, and molecular biology approaches.

2. Materials and Methods

2.1. Ethics Statement

This study was conducted in accordance with the standards of the Chinese Academy of Sciences. Approvals and documentation were obtained from Beibu Gulf University, the Department of Agriculture and Rural Affairs of the Guangxi Zhuang Autonomous Region, and the Bureau of Agriculture and Rural Affairs of Qinnan District, Qinzhou City (approval No. 2023-0702001).

2.2. Experimental Animals

T. tridentatus larvae (wet weight: 18.33 ± 1.53 mg) used in this experiment were authorized by Beibu Gulf University and provided by Guangxi Lan Gui Aquatic Co., Guangxi, China. A cohort of 360 healthy juveniles, 7 days post-hatching, were selected and acclimated in laboratory culture tanks (48 × 35 × 25 cm, 30 L capacity) for 7 days. The experimental water was obtained from natural seawater at the Sandun Pier in Qinzhou City, Guangxi Province. Seawater quality was monitored daily using a YSI EXO2 multiparameter water quality analyzer (YSI Incorporated, Yellow Springs, OH, USA). The rearing conditions were aerated seawater (salinity 20, pH 7.4, temperature 27 ± 1 °C), with no water changes or feeding during the experimental period.

2.3. Bacterial Preparation and Acute Toxicity Tests

V. parahaemolyticus strains were obtained from the Guangxi Beibu Gulf Key Laboratory of Marine Biodiversity Conservation, China. V. parahaemolyticus grows at a high rate at 37 °C and reaches late growth or pre-stabilization after 12 h of incubation [29]. Therefore, the bacteria were cultured in LB liquid medium at 37 °C and 200 rpm for 12 h and isolated by ultracentrifugation at 4 °C and 8000 rpm for 3 min. The supernatant was discarded, and the precipitate was washed twice with an equal volume of 1× PBS solution. The corresponding optical density (OD) values at 600 nm were measured using a spectrophotometer, and bacterial concentrations were adjusted based on a pre-established standard curve. Final bacterial concentrations were set at 1.0 × 109, 5.3 × 108, 2.5 × 108, 1.3 × 108, and 6.0 × 107 CFU/mL.
The experimental groups were 1.0 × 109, 5.3 × 108, 2.5 × 108, 1.3 × 108, and 6.0 × 107 CFU/mL of bacterial suspension. The control group was 1× PBS solution. A total of 180 T. tridentatus larvae were injected into each group of 30 individuals using a microsyringe. Each larva in the experimental group received a 5 μL injection of bacterial suspension at the heart, and the control group received a 5 μL injection of 1× PBS solution. Clinical symptoms and mortality were monitored for 96 h. Survival curves were analyzed using GraphPad 8.0, and the data were processed using SPSS 26.0 [30].

2.4. Sample Collection

A total of 180 T. tridentatus larvae were randomly selected for further experiments, 90 in each of the experimental and control groups. Each larva in the experimental group received a 5 μL injection of bacterial suspension at a dose of 1.31 × 108 CFU/g (48 h LD50), whereas the control group received a 5 μL injection of 1× PBS solution. Between 17 and 18 h post-injection, three surviving individuals were randomly selected from the control group, and three surviving individuals were randomly selected from the experimental group. Samples were fixed in 10% formalin for paraffin embedding and immuno-TUNEL staining. Additionally, three randomly selected survivors from each group were frozen in liquid nitrogen and stored at −80 °C for subsequent analysis.

2.5. Transcriptome Sequencing, Assembly, and Annotation

Three samples from each group were used for total RNA extraction with TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA), following the manufacturer’s instructions. RNA quality and integrity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA), and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) [31].
Poly(A)+ mRNA was enriched using oligo(dT) magnetic beads, and double-stranded cDNA was synthesized and purified. The cDNA was end-repaired, A-tailed, and ligated with Illumina TruSeq paired-end adapters (Illumina, San Diego, CA, USA). Libraries were constructed using magnetic bead-mediated fragment screening and multiplex PCR amplification.
Six RNA sequencing libraries were constructed on the Illumina NovaSeq 6000 platform using the reference genome (accession number: CNA0000821). Raw data were processed by splice trimming and quality filtering. Reads were aligned to the reference genome, and valid sequences contained at least 90% Q30 bases. Quality assessment was performed using the GO, COG, KEGG, KOG, NR, Pfam, Swiss-Prot, TrEMBL, and eggNOG bioinformatics databases.

2.6. Analysis of Differentially Expressed Genes (DEGs)

Differential gene expression analysis and correction of dispersion and mean relationships were performed using DESeq2 (v1.38.3) [32]. The screening thresholds were set at fold change (FC) of 1.5 and false discovery rate (FDR) of 0.01. FC indicates the ratio of transcript abundance between the experimental and control groups. FDR was adjusted using the Benjamini–Hochberg method to control error accumulation. FC values were log2-transformed to satisfy the log-normal distribution of transcriptome data. A larger absolute log2 FC and smaller FDR indicate more notable gene expression differences.

2.7. RT-qPCR Validation

T. tridentatus β-actin was used as the reference gene [33]. To validate the transcriptome sequencing results, ten DEGs (five upregulated and five downregulated) were randomly selected for RT-qPCR. Additionally, seven DEGs (five upregulated and two downregulated) associated with the Wnt signaling pathway, which was significantly enriched in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were selected for re-validation (Table 1).
Each 10 μL RT-qPCR reaction contained 5 μL of 2 × PerfectStart Green qPCR SuperMix (TransGen Biotech, Haidian District, BJ, China), 1 μL of cDNA template, 0.2 μL each of forward and reverse primers, and 3.6 μL of enzyme-free water. Reactions were performed on a Bio-Rad real-time PCR system (Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows: initial activation at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s and annealing at 60 °C for 30 s. Each sample set was analyzed with three biological and three technical replicates. The results were calculated using the 2−ΔΔCT relative quantification method [34].

3. Results

3.1. Statistical Analysis of Survival

The mean survival times for T. tridentatus larvae at bacterial concentrations of 1.0 × 109 CFU/mL, 5.3 × 108 CFU/mL, 2.5 × 108 CFU/mL, 1.3 × 108 CFU/mL, and 6.0 × 107 CFU/mL, as well as for the 1× PBS control group, were 8 h, 28.8 h, 51.2 h, 64 h, 89.6 h, and 96 h, respectively. The survival time of T. tridentatus larvae was inversely proportional to the V. parahaemolyticus concentration. Survival curves were constructed using the log-rank test to assess differences between the V. parahaemolyticus-infected and control groups. The chi-square value was 80, with a p value < 0.0001 (Figure 1). The calculated 48 h LD50 was 1.31 × 108 CFU/g.

3.2. Histopathological Analysis

Paraffin sections of fixed T. tridentatus larvae were transected and observed under a microscope at 100 μM magnification (Figure 2A). In the control group, the chitin shell, basement membrane, and connective tissues were structurally intact and neatly arranged. In the experimental group, tissue morphology was deformed. Chelicerae in the control group were structurally intact, and muscle fibers were neatly arranged. In the experimental group, muscle fiber morphology was deformed.
Processed samples were stained using TUNEL, DAPI, and MEGE, emitting green, blue, and blue-green fluorescence, respectively, where green fluorescence indicated apoptotic cell nuclei (Figure 2B). The apoptosis rates were 1.86% and 2.95% in the control and infected groups, respectively (Figure 2C). The results showed that the apoptosis rate of positive cells increased in T. tridentatus larvae infected with V. parahaemolyticus.

3.3. Sequencing Data and Quality Assessment

cDNA libraries from six T. tridentatus larvae were successfully constructed. A total of 35.91 Gb of clean data was obtained after quality control. As shown in Table 2, the experimental group yielded 19,933,296; 19,902,312; and 19,926,271 clean reads, whereas the control group yielded 20,062,119; 19,967,925; and 20,304,542 clean reads. The total number of bases in the clean data exceeded 5,951,331,518 reads.
Data quality assessment showed that GC content was no less than 36.73%, Q30 base percentage exceeded 91.08%, and alignment efficiency with the reference genome ranged from 93.07% to 94.08%.

3.4. Functional Annotation and Expression Analysis of Genes

Gene expression levels were quantified using the transcripts per million (TPM) method to assess differential expression between the control and V. parahaemolyticus-infected larvae. The distribution of gene expression densities showed that most genes in all samples had TPM values ranging −2.5–2.5 (Figure 3A). Box plots illustrated that gene expression levels were relatively consistent within groups but significantly different between the control and infected groups (Figure 3B). These results confirmed the reliability of the experimental design, sample selection, and processed data.

3.5. Identification and Analysis of DEGs

The Pearson correlation among samples was more than 0.74 (Figure 4A). RNA-Seq identified 2347 DEGs, including 1440 upregulated and 907 downregulated genes (Figure 4B). Cluster analysis was performed to visualize the distinct gene expression patterns between the control and experimental groups (Figure 4C). Principal component analysis (PCA) further confirmed significant differences between the groups (Figure 4D).

3.6. GO, COG, KEGG Enrichment Analysis of DEGs

GO classification categorized 1707 DEGs into three main categories: biological processes (BP), cellular components (CC), and molecular functions (MF), covering 34 subcategories (Figure 5A). In the BP category, most DEGs were associated with “cellular processes” (954 DEGs), “biological regulation” (657 DEGs), and “metabolic processes” (608 DEGs). In the CC category, most DEGs were linked to “cellular anatomical entity” (1051 DEGs), “intracellular” (518 DEGs), and “protein-containing complex” (143 DEGs). In the MF category, most DEGs were related to “binding” (1052 DEGs) and “catalytic activity” (579 DEGs).
The COG database was used to annotate homologous proteins. COG enrichment analysis categorized 559 DEGs into 26 functional groups (Figure 5B). The largest groups included “post-translational modifications, protein turnover, and chaperones” (85 DEGs), “general function prediction only” (75 DEGs), and “signal transduction mechanisms” (72 DEGs). Two categories, “chromatin structure and dynamics” and “nuclear structure,” contained no DEGs.
KEGG pathway analysis assigned 1580 DEGs to 243 pathways across six categories (Figure 5C). The most enriched pathways included “Wnt signaling pathway” (ko04310, 40 DEGs), “endocytosis” (ko04144, 33 DEGs), “ECM-receptor interaction” (ko04512, 33 DEGs), and “protein processing in the endoplasmic reticulum” (ko04141, 33 DEGs). Additional enriched pathways included “lysine degradation” (ko00310, 31 DEGs), “mTOR signaling pathway” (ko04150, 27 DEGs), “herpes simplex virus 1 infection” (ko05168, 26 DEGs), and “ubiquitin-mediated proteolysis” (ko04120, 24 DEGs).
Among the top 20 DEG-enriched KEGG pathways with the lowest q-values (Figure 5D), the “Toll and Imd signaling pathway” (16 DEGs) was most significantly enriched, followed by “protein processing in the endoplasmic reticulum” (33 DEGs), “aminoacyl-tRNA biosynthesis” (16 DEGs), and the “Wnt signaling pathway” (40 DEGs). The generated bubble plots annotated the up- and down-regulation of each DEGs in the KEGG pathway, which provided a clear visualization basis for subsequent analysis (Figure 5E). The Wnt signaling pathway showed the greatest response in T. tridentatus larvae infected with V. parahaemolyticus. Red boxes in Figure 5F indicate upregulated genes, green boxes indicate downregulated genes, and blue boxes indicate both upregulated and downregulated genes. In the Wnt/PCP (planar cell polarity) pathway, the expression of key effector molecules Daam1, Rac1, and RhoA was upregulated, while non-classical ligands Wnt11 and Wnt11b also showed an upward trend. The classical Wnt pathway receptors Fzd2 and Fzd4 were significantly downregulated.

3.7. Validation by RT-qPCR

Ten DEGs (five upregulated and five downregulated) were randomly selected for RT-qPCR validation. As shown in Figure 6A, the expression patterns from RNA-Seq were consistent with the RT-qPCR results, confirming the reliability of the RNA-Seq data (Table S1). Seven other DEGs (five upregulated and two downregulated) associated with the Wnt signaling pathway were selected for further validation, and the results remained consistent (Figure 6B).

4. Discussion

In the present study, we demonstrated that V. parahaemolyticus is highly pathogenic to T. tridentatus larvae. Injection of 5 μL of V. parahaemolyticus solution into each 20 mg juvenile resulted in reduced vitality or death over time. At the time of death, the larvae exhibited bulging gills and did not respond to abdominal limb contact. The mean survival times of the 1.0 × 109 CFU/mL, 5.3 × 108 CFU/mL, 2.5 × 108 CFU/mL, 1.3 × 108 CFU/mL, 6.0 × 107 CFU/mL experimental groups and 1× PBS control group were 8 h, 28.8 h, 51.2 h, 64 h, 89.6 h, and 96 h, respectively. The 48 h LD50 for the larvae was 1.31 × 108 CFU/g, and the histopathological damage was significantly greater in the experimental individuals than in the dead individuals. In addition, high-density microplastics, chemicals, heavy metals and other contaminants can affect the development and growth of T. tridentatus larvae, and even cause death [35,36].
Scylla paramamosain infected with V. parahaemolyticus had an LD50 of 3.18 × 104 CFU/g and showed black gills and yellowing of the hepatopancreas [37]. Litopenaeus vannamei infected with V. parahaemolyticus had an LC50 of 4.79 × 105 CFU/mL and exhibited loss of appetite and liver and gill lesions [38]. Larimichthys crocea infected with V. parahaemolyticus had an LD50 of 1.0 × 107 CFU/mL, presenting hepatosplenomegaly and ascites [39]. Infected Amphiprion sebae had an LD50 of 1.0 × 105 CFU/tail and showed reduced viability and hepatic and gill lesions [40]. Infected Danio rerio had an LD50 of 5.0 × 105 CFU/tail and showed fusion of the gills and digestive glands [41]. These results highlight the significant danger posed by V. parahaemolyticus to aquatic animals and highlight the need for aquatic disease prevention and control measures. At present, no systematic studies have reported the analytical response mechanism of T. tridentatus larvae infected with V. parahaemolyticus, indicating an important area for future research.
The transcriptome, the central molecular label characterizing the functional state of an organism, is the collection of all RNAs transcribed from a particular cell or tissue at a particular time or state [42]. Advances in RNA-seq technology have provided a fast and reliable platform for the systematic study of transcriptomics at a holistic level owing to its high sensitivity and digital quantification [43]. RNA-Seq technology is not only widely used to analyze key biological processes, such as developmental regulation and environmental stress response, but has also been applied in aquatic animal research to analyze growth and reproduction, protein translation, and immune responses in a multidimensional way [44,45,46]. As a highly efficient tool for analyzing immune responses and associated regulatory mechanisms, RNA-Seq has aided in the transcriptomic analyses of V. parahaemolyticus-infected aquatic animals [47]. In this study, cDNA libraries of six T. tridentatus larvae were constructed using RNA-Seq. A total of 2347 DEGs (1440 upregulated genes and 907 downregulated genes) and 243 enriched signaling pathways were identified. Functional enrichment analysis revealed the enrichment of immune-related pathways, including the Wnt signaling pathway, ECM-receptor interaction, aminoacyl-tRNA biosynthesis, and Toll and Imd signaling pathways. In addition, several DEGs provided valuable insights into the molecular response mechanisms of T. tridentatus larvae to V. parahaemolyticus infection.
Similarly to other invertebrates, T. tridentatus lacks an adaptive immune system and relies on the innate immune system for defense. The innate immune system includes cellular and humoral immunity that work synergistically against environmental stress and pathogen invasion. The Wnt signaling pathway is a core regulatory hub of immune homeostasis and regulates the dynamic balance of immunomodulatory cytokines (e.g., IL-10, TGF-β, and IFN-γ) in both directions through β-catenin-dependent and nondependent mechanisms. It plays a key role in physiological processes, such as tissue regeneration, inflammation abatement and autoimmune tolerance [48,49]. These pathways are dependent on the specific binding of Wnt ligands to Frizzled receptors and LRP5/6 co-receptors, which in turn triggers the activation of downstream signaling cascades [50,51,52]. The relative expression of the Wnt11 gene was significantly upregulated after infection with V. parahaemolyticus in Li. vannamei, and silencing of the gene significantly increased Li. vannamei mortality rate, indicating an important regulatory role for Wnt11 in the antimicrobial immune response of the organism [53]. However, the relative expression of Wnt5b was significantly downregulated after Li. vannamei was infected with V. parahaemolyticus and WSSV. Furthermore, silencing of the gene significantly increased the expression of several antimicrobial peptides (AMPs), indicating an inhibitory role for Wnt5b in the antimicrobial-versus-antiviral response of Li. vannamei [54,55]. The relative expression of β-catenin gene was significantly upregulated in Marsupenaeus japonicus after infection with bacteria and viruses, and silencing of the gene resulted in impaired bacterial clearance and increased viral replication in vivo, suggesting that β-catenin is involved in M. japonicus immune responses [56]. The relative expression of the Rac1 gene was significantly upregulated after La. crocea was infected with V. parahaemolyticus, and silencing of the gene reduced phagocytosis in the immune response of La. crocea, implying the crucial role of Rac1 in the innate immunity of La. crocea [57]. These results are highly consistent with the results obtained in our study, providing a key scientific basis for elucidating the immune mechanism of T. tridentatus larvae infected with V. parahaemolyticus.
Cells have evolved a variety of mechanisms to regulate signaling pathways to resist pathogen invasion, with the Toll and Imd pathways being core immune response pathways in invertebrate immune defense. In invertebrates, the Wnt gene family activates the Toll and Imd pathways by mediating the specific recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Activation of these two pathways induces translocation of nuclear factor-κB (NF-κB) family transcription factors to the nucleus and subsequent alteration of AMP expression [58]. The transcript levels of three AMPs were significantly upregulated in V. parahaemolyticus-infected Macrobrachium nipponense when tested in vivo [59], suggesting the central role of AMPs in crustacean defense mechanism against invading pathogens [60]. In addition, the Toll and Imd pathways were important for the antimicrobial response of S. paramamosain during V. parahaemolyticus infection [61]. Therefore, a systematic study of the immune responses and association regulation mechanisms in T. tridentatus larvae infected with V. parahaemolyticus is vital for the development of effective strategies for the protection and recovery of this species.

5. Conclusions

In this study, we explored the response mechanism of T. tridentatus larvae infected with V. parahaemolyticus via acute toxicity experiments. The 48 h LD50 of the infected larvae was 1.31 × 108 CFU/g. Histopathological analysis and fluorescence staining revealed extensive tissue damage and heightened apoptosis. RNA-Seq of the larvae before and after V. parahaemolyticus infection revealed 2347 DEGs (including 1440 upregulated and 907 downregulated genes), with enrichment in 243 signaling pathways. Functional enrichment analysis indicated significant involvement of immunoregulatory pathways, including the Wnt signaling pathway, ECM-receptor interaction, aminoacyl-tRNA biosynthesis, and Toll and Imd signaling pathways. RT-qPCR of 17 DEGs validated the reliability of RNA-Seq results with consistent gene expression patterns. This study enhances our understanding of the immune defense mechanisms of T. tridentatus larvae and provides a scientific foundation for developing disease prevention and control strategies in invertebrates. However, current technologies have not been able to successfully extract natural immune effector proteins from newly hatched T. tridentatus larvae and validate their biological function. It is expected that future research will develop effective methods to systematically assess the immune function of T. tridentatus larvae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani15172556/s1, Table S1: Differential gene expression levels among the comparison groups.

Author Contributions

L.Y.: Conceptualization, methodology, and writing—original draft preparation. J.L.: Data curation, and investigation. B.C.: Data curation, and visualization. F.G.: Data curation, and validation. Z.L.: Data curation, and validation. Z.Z.: Data curation, and formal analysis. S.L.: Data curation, and software. Y.Z.: Data curation, and project administration. J.J.: Methodology, and funding acquisition. P.Z.: Conceptualization, and funding acquisition. Y.L.: Conceptualization, supervision, and funding acquisition. All authors participate in the writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (NSFC32260918), the Qinzhou Scientific Research and Development Program Project (20242405), the Counterpart Aid for Discipline Construction Project of Guangxi University (2023B03), and the Key Research and Development Program of Guangxi (Guike AB24010084).

Institutional Review Board Statement

This study was conducted in accordance with the standards of the Chinese Academy of Sciences. Approvals and documentation were obtained from Beibu Gulf University, the Department of Agriculture and Rural Affairs of the Guangxi Zhuang Autonomous Region, and the Bureau of Agriculture and Rural Affairs of Qinnan District, Qinzhou City (approval No. 2023-0702001).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors thank the Guangxi Lan Gui Aquatic Co. for providing experimental animals and sites, and Peng Zhu from the Guangxi Beibu Gulf Key Laboratory of Marine Biodiversity Conservation, China for providing the V. parahaemolyticus strain.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Xu, Z.; Wang, Y.J.; Gul, Y.; Li, Q.Z.; Song, J.; Hu, M.H. Effects of copper supplement on the immune function and blood-chemistry in adult Chinese horseshoe crab Tachypleus tridentatus. Aquaculture 2020, 515, 734576. [Google Scholar] [CrossRef]
  2. Ma, X.W.; Zhang, X.Z.; Qiao, Y.; Zhong, S.P.; Xing, Y.Z.; Chen, X.Y. Weighted gene co-expression network analysis of embryos and first instar larvae of the horseshoe crab Tachypleus tridentatus uncovers development gene networks. Comp. Biochem. Phys. D 2022, 42, 100980. [Google Scholar] [CrossRef]
  3. Chen, X.H.; Gu, Y.G.; Xie, X.Y.; Ying, Z.W.; Luo, Z.M.; Zhang, W.L.; Xie, M.J.; Fan, J. A new strategy for optimizing marine protection networks by considering functional connectivity: An example of Tachypleus tridentatus. Sci. Total Environ. 2024, 911, 168763. [Google Scholar] [CrossRef]
  4. Pan, Y.T.; Qian, J.; Ma, X.W.; Huang, W.; Fang, J.K.-H.; Arif, L.; Shang, Y.Y.; Hu, M.H. Response of moulting genes and gut microbiome to nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus. Mar. Environ. Res. 2023, 191, 106128. [Google Scholar] [CrossRef]
  5. Chen, B.Y.; Long, J.; Liu, J.X.; Wang, P.L.; Ma, Z.H.; Lan, Z.Y.; Liang, Z.W.; Fu, Q.N.; Zhang, Z.N.; Zhang, Y.; et al. The development of novel genome-SSRs, multiplex PCR panels, and allelic ladders for parentage identification in Tachypleus tridentatus. Aquaculture 2024, 592, 741262. [Google Scholar] [CrossRef]
  6. Laurie, K.; Chen, C.-P.; Cheung, S.G.; Do, V.; Hsieh, H.; John, A.; Mohamad, F.; Seino, S.; Nishida, S.; Shin, P.; et al. Tachypleus tridentatus (errata version published in 2019). IUCN Red List Threat. Species™ 2019, e.T21309A149768986. [Google Scholar] [CrossRef]
  7. Liu, X.M.; Liu, J.N.; Xiong, K.; Zhang, C.Q.; Fang, J.K.-H.; Song, J.; Tai, Z.G.; Zhu, Q.G.; Hu, M.H.; Wang, Y.J. Effects of Ocean Acidification on Molting, Oxidative Stress, and Gut Microbiota in Juvenile Horseshoe Crab Tachypleus tridentatus. Front. Physiol. 2022, 12, 813582. [Google Scholar] [CrossRef] [PubMed]
  8. Wang, C.-C.; Kwan, K.Y.; Chen, R.F. Seafood harvesting practices in critical nursery habitat of Asian horseshoe crabs in northern Beibu Gulf, China: Suggestions for conservation management. Ocean Coast. Manag. 2025, 263, 107606. [Google Scholar] [CrossRef]
  9. Xie, X.Y.; Wu, Z.; Wang, C.-C.; Fu, Y.J.; Wang, X.P.; Xu, P.; Huang, X.; Liao, Y.Y.; Huang, S.-L.; Kwan, K.Y. Nursery habitat for Asian horseshoe crabs along the northern Beibu Gulf, China: Implications for conservation management under baseline gaps. Aquat. Conserv. 2020, 30, 260–272. [Google Scholar] [CrossRef]
  10. Liao, Y.Y.; Hsieh, H.L.; Xu, S.P.; Zhong, Q.P.; Lei, J.; Liang, M.Z.; Fang, H.Y.; Xu, L.L.; Lin, W.Y.; Xiao, X.B.; et al. Wisdom of Crowds reveals decline of Asian horseshoe crabs in Beibu Gulf, China. Oryx 2019, 53, 222–229. [Google Scholar] [CrossRef]
  11. Sekiguchi, K.; Shuster, C.N. Limits on the Global Distribution of Horseshoe Crabs (Limulacea): Lessons Learned from Two Lifetimes of Observations: Asia and America. In Biology and Conservation of Horseshoe Crabs, 1st ed.; Springer: Boston, MA, USA, 2009; pp. 5–24. [Google Scholar] [CrossRef]
  12. Xie, M.J.; Bao, Y.Y.; Xie, X.Y.; Lin, W.Y.; Chen, X.H.; Zhao, X.Y.; Xiao, X.B. An effective way to monitor the population of juvenile horseshoe crabs in the Beibu Gulf of China. Reg. Stud. Mar. Sci. 2024, 79, 103831. [Google Scholar] [CrossRef]
  13. Dong, X.; Bi, D.X.; Wang, H.L.; Zou, P.Z.; Xie, G.S.; Wan, X.Y.; Yang, Q.; Zhu, Y.P.; Chen, M.M.; Guo, C.C.; et al. pirABvp-Bearing Vibrio parahaemolyticus and Vibrio campbellii Pathogens Isolated from the Same AHPND-Affected Pond Possess Highly Similar Pathogenic Plasmids. Front. Microbiol. 2017, 8, 1859. [Google Scholar] [CrossRef]
  14. Sony, M.; Sumithra, T.G.; Anusree, V.N.; Amala, P.V.; Reshma, K.J.; Alex, S.; Sanil, N.K. Antimicrobial resistance and virulence characteristics of Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi from natural disease outbreaks of marine/estuarine fishes. Aquaculture 2021, 539, 736608. [Google Scholar] [CrossRef]
  15. Bai, Y.; Yang, Q.P.; Sun, Y.N.; Li, F.Q.; Sun, J.L.; Yang, S.R.; Yang, D.J.; Peng, Z.X.; Yang, B.W.; Xu, J.; et al. Antimicrobial susceptibility and genomic characterization of Vibrio parahaemolyticus isolated from aquatic foods in 15 provinces, China, 2020. Int. J. Food Microbiol. 2024, 418, 110737. [Google Scholar] [CrossRef] [PubMed]
  16. Pan, Y.T.; Yang, Z.Y.; Zhao, W.X.; Fang, J.K.-H.; Shi, J.H.; Li, D.J.; Hu, M.H.; Wang, Y.J. Combined effects of polyamide microplastics and the pathogenic bacterium Vibrio parahaemolyticus on the immune parameters of Mytilus coruscus. Mar. Pollut. Bull. 2025, 218, 118204. [Google Scholar] [CrossRef] [PubMed]
  17. Lv, X.R.; Cao, W.W.; Zhang, H.; Zhang, Y.L.; Shi, L.; Ye, L. CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood. Foods 2022, 11, 1681. [Google Scholar] [CrossRef] [PubMed]
  18. Liang, H.Y.; Liang, R.C.; Wang, H.J.; Zhang, X.L.; Yan, X.J.; Shen, W. Apelin participates in host defense against bacterial infection and promotes bacterial clearance in large yellow croaker (Larimichthys crocea). Aquaculture 2022, 549, 737803. [Google Scholar] [CrossRef]
  19. Wang, Y.H.; Li, D.D.; Zhang, H.; Chen, J.Y.; Tang, Y.M.; Nie, H.T.; Huo, Z.M.; Yan, X.W. Transcriptome analysis of Manila clam (Ruditapes philippinarum) under Vibrio parahaemolyticus challenge in a low-salinity environment provides molecular insights into immune response. Aquaculture 2024, 584, 740681. [Google Scholar] [CrossRef]
  20. Zhao, X.L.; Liu, Y.; Yan, F.; Lin, Z.Y.; Zhao, Y.Z.; Chen, X.L.; Zhang, Y.L. OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus. Fish Shellfish Immunol. 2024, 146, 109409. [Google Scholar] [CrossRef]
  21. Li, Y.K.; Lv, J.J.; Sun, D.F.; Guo, J.Y.; Liu, P.; Gao, B.Q. Characterization of a pseudohemocyanin gene (PtPhc1) and its immunity function in response to Vibrio parahaemolyticus infection in the swimming crab Portunus trituberculatus. Fish Shellfish Immunol. 2024, 147, 109435. [Google Scholar] [CrossRef]
  22. Yilmaz, S.; Yilmaz, E.; Dawood, M.A.O.; Ringø, E.; Ahmadifar, E.; Abdel-Latif, H.M.R. Probiotics, prebiotics, and synbiotics used to control vibriosis in fish: A review. Aquaculture 2022, 547, 737514. [Google Scholar] [CrossRef]
  23. Wangman, P.; Surasilp, T.; Pengsuk, C.; Sithigorngul, P.; Longyant, S. Development of a species-specific monoclonal antibody for rapid detection and identification of foodborne pathogen Vibrio vulnificus. J. Food Saf. 2021, 41, e12939. [Google Scholar] [CrossRef]
  24. Hou, Y.C.; Liu, X.P.; Wang, Y.N.; Guo, L.; Wu, L.Y.; Xia, W.R.; Zhao, Y.Q.; Xing, W.W.; Chen, J.; Chen, C.G. Establishment and application of a rapid visualization method for detecting Vibrio parahaemolyticus nucleic acid. Infect. Med. 2024, 3, 100111. [Google Scholar] [CrossRef]
  25. Chen, X.H.; Xie, M.J.; Feng, J.; Zou, J.X.; Shi, J.; Zhuang, L.F.; Deng, J.M.; Xie, X.Y. A rapid bycatch assessment framework for small-scale fisheries: A case study on horseshoe crab (Tachypleus tridentatus) bycatch. Ocean Coast. Manag. 2025, 266, 107701. [Google Scholar] [CrossRef]
  26. Huang, B.Y.; Peng, S.; Liu, S.F.; Zhang, Y.T.; Wei, Y.X.; Xu, X.Y.; Gao, C.H.; Liu, Y.H.; Luo, X.W. Isolation, Screening, and Active Metabolites Identification of Anti-Vibrio Fungal Strains Derived From the Beibu Gulf Coral. Front. Microbiol. 2022, 13, 930981. [Google Scholar] [CrossRef]
  27. Xu, Q.S.; Wang, P.B.; Huangleng, J.H.; Su, H.Q.; Chen, P.Y.; Chen, X.; Zhao, H.X.; Kang, Z.J.; Tang, J.L.; Jiang, G.L.X.; et al. Co-occurrence of chromophytic phytoplankton and the Vibrio community during Phaeocystis globosa blooms in the Beibu Gulf. Sci. Total Environ. 2022, 805, 150303. [Google Scholar] [CrossRef] [PubMed]
  28. Liu, X.M.; Xu, Z.; Chang, X.Q.; Fang, J.K.-H.; Song, J.; He, J.F.; Tai, Z.G.; Zhu, Q.G.; Hu, M.H. Enhanced immunity and hemocytes proliferation by three immunostimulants in tri-spine horseshoe crab Tachypleus tridentatus. Fish Shellfish Immunol. 2021, 115, 112–123. [Google Scholar] [CrossRef]
  29. Liu, B.X.; Liu, H.Q.; Pan, Y.J.; Xie, J.; Zhao, Y. Comparison of the Effects of Environmental Parameters on the Growth Variability of Vibrio parahaemolyticus Coupled with Strain Sources and Genotypes Analyses. Front. Microbiol. 2016, 7, 994. [Google Scholar] [CrossRef] [PubMed]
  30. Lan, Z.Y.; Mao, M.Q.; Liang, Q.; Ma, Z.H.; Zhang, Z.N.; Liang, Z.W.; Chen, B.Y.; Fu, Q.N.; Zhou, Q.L.; Zhang, Y.; et al. Cloning and Characterization of Two Inhibitors of Apoptosis (IAP) from Eriocheir hepuensis and Their Expression Pattern in the Immune Organs under Azadirachtin Stress. Aquac. Res. 2023, 2023, 2507844. [Google Scholar] [CrossRef]
  31. Fu, Q.N.; Liu, J.X.; Ren, T.J.; Zhang, Z.N.; Ma, Z.H.; Lan, Z.Y.; Duan, Y.T.; Liang, Z.W.; Chen, B.Y.; Zhang, Y.; et al. Cloning of Two HSP Genes of Eriocheir hepuensis and Their Expression under Vibrio parahaemolyticus Stress. Fishes 2022, 7, 372. [Google Scholar] [CrossRef]
  32. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef]
  33. Li, W.X.; Huang, J.R.; Ye, H.H.; Huang, H.Y.; Li, S.J. Cloning and tissue expression of β-actin gene in the Chinese horseshoe crab (Tachypleus tridentatus). Xiamen Daxue Xuebao Ziran Kexueban 2015, 54, 188–193. [Google Scholar] [CrossRef]
  34. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT. Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  35. Fang, J.K.-H.; Tse, T.W.; Maboloc, E.A.; Leung, R.K.-L.; Leung, M.M.-K.; Wong, M.W.-T.; Chui, A.D.-Y.; Wang, Y.J.; Hu, M.H.; Kwan, K.Y.; et al. Adverse impacts of high-density microplastics on juvenile growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus. Mar. Pollut. Bull. 2023, 187, 114535. [Google Scholar] [CrossRef]
  36. Kwan, K.Y.; Chan, A.K.Y.; Cheung, S.G.; Shin, P.K.S. Responses of growth and hemolymph quality in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) to sublethal tributyltin and cadmium. Ecotoxicology 2015, 24, 1180–1895. [Google Scholar] [CrossRef]
  37. Chen, X.L.; Cheng, C.H.; Deng, Y.Q.; Ma, H.L.; Su, Y.L.; Feng, J.; Guo, Z.X. Isolation, identification and drug sensitivity of pathogenic Vibrio parahemolyticus of mud crab (Scylla paramamosain). Nanfang Nongye Xuebao 2020, 51, 2846–2855. [Google Scholar] [CrossRef]
  38. Wei, Z.; Chen, X.Y.; Wang, S.; Ye, X.L.; Huang, L.P.; Wei, Y.Y.; Liu, M.; Hu, T.J.; Yu, M.L. Protective effect of Chinese herbal compound preparations on hepatopancreas necrosis of Penaeus vannamei induced by Vibrio parahaemolyticus. Heilongjiang Xumu Shouyi 2022, 16, 108–115+139–140. [Google Scholar] [CrossRef]
  39. Xu, B.F.; Lin, N.F.; Yang, J.X.; Yu, F.S.; Dong, C.F.; Lin, T.L. Isolation, identification and pathogenicity analysis of Vibrio parahaemolyticus from Pseudosciaena crocea. Fujian Nongye Xuebao 2002, 17, 174–177. [Google Scholar] [CrossRef]
  40. Sun, Y.X.; Chang, H.J.; Yang, X.X.; Wu, M.W.; Li, J. Vibriosis caused by Vibrio parahaemolyticus in aquatic animals and bacteriophage therapy: A review. Weishengwuxue Tongbao 2023, 50, 3620–3634. [Google Scholar] [CrossRef]
  41. Li, Y.; Li, P.H.; Liu, C.H.; Ma, Q.J. Characterization of pathogenicity and drug resistance in Vibrio parahaemolyticus D3112. Haiyang Kexue 2019, 43, 25–33. [Google Scholar] [CrossRef]
  42. Wang, Z.; Gerstein, M.; Snyder, M. RNA-Seq: A revolutionary tool for transcriptomics. Nat. Rev. Genet. 2009, 10, 57–63. [Google Scholar] [CrossRef]
  43. Zhang, X.Y. Highly effective batch effect correction method for RNA-seq count data. Comput. Struct. Biotechnol. J. 2025, 27, 58–64. [Google Scholar] [CrossRef]
  44. Ma, B.; Liu, Y.; Zhang, X.; Chen, T.; Zhang, L.P.; Hu, C.J.; Yu, S.Z.; Chen, G.Q.; Liu, L.Y.; Zhu, J.X.; et al. Genome-wide QTL mapping and RNA-seq reveal genetic mechanisms behind discrepant growth traits in Pacific whiteleg shrimp, Litopenaeus vannamei. Aquaculture 2025, 599, 742084. [Google Scholar] [CrossRef]
  45. Li, X.; Wang, W.W.; Wang, X.D.; Wang, H.L. Differential immunotoxicity effects of triclosan and triclocarban on larval zebrafish based on RNA-Seq and bioinformatics analysis. Aquat. Toxicol. 2023, 262, 106665. [Google Scholar] [CrossRef]
  46. Wang, J.W.; Yu, X.F.; Song, S.Q.; Zhang, Y.Q.; Cao, J.L.; Jin, C.F.; Bao, Z.M.; Wang, B.; Hu, J.J. Transcriptome analysis reveals the immune responses of leopard coral grouper to nervous necrosis virus infection. Fish Shellfish Immunol. 2025, 161, 110290. [Google Scholar] [CrossRef] [PubMed]
  47. Liu, Y.J.; Xing, K.F.; Yan, C.C.; Zhou, Y.Z.; Xu, X.M.; Sun, Y.Y.; Zhang, J.Q. Transcriptome analysis of Neocaridina denticulate sinensis challenged by Vibrio parahemolyticus. Fish Shellfish Immunol. 2022, 121, 31–38. [Google Scholar] [CrossRef]
  48. Liu, F.S.; Hu, H.M.; Chen, G.L.; Lin, Y.Q.; Li, W.; Liu, Z.Y.; Chen, C.; Li, X.; Sun, S.J.; Zhang, L.; et al. Pexidartinib hydrochloride exposure induces developmental toxicity and immunotoxicity in zebrafish embryos via activation of Wnt signaling. Fish Shellfish Immunol. 2023, 138, 108849. [Google Scholar] [CrossRef] [PubMed]
  49. Wu, W.H.; Zou, C.C.; Shi, H.; Wang, C.Q.; Lin, W.Y.; Zhang, Y.L.; He, Y.B.; Ruan, L.W. Phosphorylation regulation of Lv-β-catenin of Litopenaeus vannamei by an immediate early protein WSV083 to reduce cell adhesion. Aquaculture 2024, 579, 740244. [Google Scholar] [CrossRef]
  50. Zhang, W.Q.; Zhang, K.X.; Ma, Y.H.; Song, Y.X.; Qi, T.B.; Xiong, G.J.; Zhang, Y.Z.; Kan, C.X.; Zhang, J.W.; Han, F.; et al. Secreted frizzled-related proteins: A promising therapeutic target for cancer therapy through Wnt signaling inhibition. Biomed. Pharmacother. 2023, 166, 115344. [Google Scholar] [CrossRef]
  51. Hua, Y.; Yang, Y.L.; Li, Q.; He, X.Y.; Zhu, W.; Wang, J.Y.; Gan, X.Q. Oligomerization of Frizzled and LRP5/6 protein initiates intracellular signaling for the canonical WNT/β-catenin pathway. J. Biol. Chem. 2018, 293, 19710–19724. [Google Scholar] [CrossRef]
  52. Lee, H.; Lee, S.J.; Kim, G.-H.; Yeo, I.; Han, J.-K. PLD1 regulates Xenopus convergent extension movements by mediating Frizzled7 endocytosis for Wnt/PCP signal activation. Dev. Biol. 2016, 411, 38–49. [Google Scholar] [CrossRef]
  53. Wang, Z.-A.; Zheng, J.F.; Yang, L.W.; Zuo, H.L.; Niu, S.W.; Weng, S.P.; He, J.G.; Xu, X.P. Wnt11 positively regulates immune defense against Vibrio parahaemolyticus but promotes white spot syndrome virus infection in Litopenaeus vannamei. Aquaculture 2021, 542, 736910. [Google Scholar] [CrossRef]
  54. Yang, L.W.; Wang, Z.-A.; Zuo, H.L.; Guo, Z.X.; Weng, S.P.; He, J.G.; Xu, X.P. Wnt5b plays a negative role in antibacterial response in Pacific white shrimp Penaeus vannamei. Dev. Comp. Immunol. 2022, 133, 104411. [Google Scholar] [CrossRef] [PubMed]
  55. Wang, C.Q.; Ruan, L.W.; Shi, H.; Xu, X. Wnt5b regulates apoptosis in Litopenaeus vannamei against white spot syndrome virus. Fish Shellfish Immunol. 2018, 74, 318–324. [Google Scholar] [CrossRef]
  56. Xie, Y.; Ding, D.; Wang, H.M.; Kang, C.J. A homologue gene of β-catenin participates in the development of shrimps and immune response to bacteria and viruses. Fish Shellfish Immunol. 2015, 47, 147–156. [Google Scholar] [CrossRef]
  57. Han, F.; Li, W.B.; Liu, X.D.; Zhang, D.L.; Liu, L.P.; Wang, Z.Y. Rac1 GTPase is a critical factor in phagocytosis in the large yellow croaker Larimichthys crocea by interacting with tropomyosin. Fish Shellfish Immunol. 2019, 91, 148–158. [Google Scholar] [CrossRef]
  58. Li, C.Z.; Wang, S.; He, J.G. The Two NF-κB Pathways Regulating Bacterial and WSSV Infection of Shrimp. Front. Immunol. 2019, 10, 1785. [Google Scholar] [CrossRef]
  59. Wang, K.Q.; Dai, X.L.; Zhang, C.; Cao, X.Y.; Zhang, R.D.; Zhang, Z.X.; Huang, X.; Ren, Q. Two Wnt genes regulate the expression levels of antimicrobial peptides during Vibrio infection in Macrobrachium nipponense. Fish Shellfish Immunol. 2020, 101, 225–233. [Google Scholar] [CrossRef]
  60. Destoumieux-Garzón, D.; Rosa, R.D.; Schmitt, P.; Barreto, C.; Vidal-Dupiol, J.; Mitta, G.; Gueguen, Y.; Bachere, E. Antimicrobial peptides in marine invertebrate health and disease. Philos. Trans. R. Soc. B 2016, 371, 20150300. [Google Scholar] [CrossRef] [PubMed]
  61. Cheng, C.H.; Ma, H.L.; Deng, Y.Q.; Feng, J.; Jie, Y.K.; Guo, Z.X. Effects of Vibrio parahaemolyticus infection on physiological response, histopathology and transcriptome changes in the mud crab (Scylla paramamosain). Fish Shellfish Immunol. 2020, 106, 197–204. [Google Scholar] [CrossRef]
Animals 15 02556 g001
Figure 1. Survival rate of T. tridentatus larvae infected by V. parahaemolyticus.
Figure 1. Survival rate of T. tridentatus larvae infected by V. parahaemolyticus.
Animals 15 02556 g001
Animals 15 02556 g002
Figure 2. Histopathological analysis of T. tridentatus larvae by V. parahaemolyticus. (A) Paraffin sections of fixed T. tridentatus larvae; (B) TUNEL fluorescence staining; (C) Apoptosis rate of positive cells. Different letters represent significant differences (** p < 0.01).
Figure 2. Histopathological analysis of T. tridentatus larvae by V. parahaemolyticus. (A) Paraffin sections of fixed T. tridentatus larvae; (B) TUNEL fluorescence staining; (C) Apoptosis rate of positive cells. Different letters represent significant differences (** p < 0.01).
Animals 15 02556 g002
Animals 15 02556 g003
Figure 3. Expression analysis of genes. (A) The distribution of gene expression densities; (B) The box plots of gene expression.
Figure 3. Expression analysis of genes. (A) The distribution of gene expression densities; (B) The box plots of gene expression.
Animals 15 02556 g003
Animals 15 02556 g004
Figure 4. Transcriptome profiling of the effects of T. tridentatus larvae infected by V. parahaemolyticus. (A) The Pearson correlation among samples; (B) Volcanic map analysis of DEGs; (C) Heat map analysis of DEGs; (D) Principal component analysis of the RNA-Seq data.
Figure 4. Transcriptome profiling of the effects of T. tridentatus larvae infected by V. parahaemolyticus. (A) The Pearson correlation among samples; (B) Volcanic map analysis of DEGs; (C) Heat map analysis of DEGs; (D) Principal component analysis of the RNA-Seq data.
Animals 15 02556 g004
Animals 15 02556 g005
Figure 5. Enrichment analysis of DEGs. (A) GO classification of DEGs; (B) COG classification of DEGs; (C) KEGG Classification on DEGs; (D) The top 20 DEG-enriched KEGG pathways with the lowest q-values; (E) KEGG pathway enrichment on DEGs-Bubble chart; (F) DEGs analysis of the Wnt signaling pathway.
Figure 5. Enrichment analysis of DEGs. (A) GO classification of DEGs; (B) COG classification of DEGs; (C) KEGG Classification on DEGs; (D) The top 20 DEG-enriched KEGG pathways with the lowest q-values; (E) KEGG pathway enrichment on DEGs-Bubble chart; (F) DEGs analysis of the Wnt signaling pathway.
Animals 15 02556 g005
Animals 15 02556 g006
Figure 6. RNA-Seq verification by RT-qPCR. (A) Expression of randomly selected DEGs; (B) Expression of Wnt signaling pathway DEGs.
Figure 6. RNA-Seq verification by RT-qPCR. (A) Expression of randomly selected DEGs; (B) Expression of Wnt signaling pathway DEGs.
Animals 15 02556 g006
Table 1. Transcriptome RT-qPCR validation primer table.
Table 1. Transcriptome RT-qPCR validation primer table.
Primer NamePrimer SequencePrimer Purpose
β-actin-FAGAGCGTGGTTACAGCTTCACRT-qPCR
β-actin-RCAGCTCCTTACGGATGTCAATRT-qPCR
evm.TU.Hic_chr_12.393-FCTCCACCTCACCTTCGTCACTRT-qPCR
evm.TU.Hic_chr_12.393-RCCTGGCTGTTTGAAGGCGTATRT-qPCR
evm.TU.Hic_chr_4.1095-FCCACTTCGTAACCTCAGCAACRT-qPCR
evm.TU.Hic_chr_4.1095-RAAGCTCTCGTGTCGTGGATAGRT-qPCR
evm.TU.Hic_chr_9.1728-FACCTGTCTTCCCCTTCCCCTART-qPCR
evm.TU.Hic_chr_9.1728-RAGGCGTTTCTGGTTGATCCGART-qPCR
evm.TU.Hic_chr_10.1010-FATTATCCGCCACCCCAACCTCRT-qPCR
evm.TU.Hic_chr_10.1010-RTCACACGGTCGAAGTAACGGGRT-qPCR
NewGene_3691-FTGGATGATTCGGGACGGACAART-qPCR
NewGene_3691-RTGGCGGTCAATCTACAGAGGTRT-qPCR
evm.TU.Hic_chr_3.480-FTTGCTGATGCCTGACACTGCTRT-qPCR
evm.TU.Hic_chr_3.480-RTGGTGAAGCTCCTGGCGAAAART-qPCR
evm.TU.Hic_chr_13.3889-FGTCCAAGCAAGTTCACCCCGART-qPCR
evm.TU.Hic_chr_13.3889-RTCAACAGTAGCCAGAGCCTGCRT-qPCR
evm.TU.Hic_chr_3.1525-FCTTGAACGAGTCCACCTTGCCRT-qPCR
evm.TU.Hic_chr_3.1525-RTCACCACAGACCTCCAGTTCGRT-qPCR
evm.TU.Hic_chr_6.2481-FTCGTCAGCCTCGTCATTCCTTRT-qPCR
evm.TU.Hic_chr_6.2481-RCTGTCGCACTTTCCGTCGTTART-qPCR
evm.TU.Hic_chr_6.357-FGCCCTTCTCCCTTCAGCCTAGRT-qPCR
evm.TU.Hic_chr_6.357-RCGTCCCATGCCATTCCCGAGTRT-qPCR
Wnt11-FTCCCGTTCGTTTTCTGATGGTCCTRT-qPCR
Wnt11-RATCAAATTAGCACGCAACGCCCTCRT-qPCR
Wnt11b-FGCTTACCAAATCCAAAGGTCGGTTRT-qPCR
Wnt11b-RTAAGTCCTATAACCTCGTCCGCAGRT-qPCR
Daam1-FAGGTACAGCGTAGCAAGAGGTTAART-qPCR
Daam1-RTAAGACTAACCAGACGGAAACCCART-qPCR
Rac1-FACCATTGCCCAAACACACCCATTART-qPCR
Rac1-RATGGGGGCAAGTTTACGGTCTTTCRT-qPCR
RhoA-FGGGTCGAACAATGGCAGAAAAAATRT-qPCR
RhoA-RTTTCAAAAACTTCCCTCACCCCATRT-qPCR
Fzd2-FCTTGGCACCCCTGTTTTTCTATTTRT-qPCR
Fzd2-RTATCTGTCTTCGTTCCATCATGCTRT-qPCR
Fzd4-FGTTGTTGGGAGCCTTGTTTCTGATRT-qPCR
Fzd4-RTTTTGTTCGGATTTGTCGGTGGAGRT-qPCR
Table 2. Sequencing data table.
Table 2. Sequencing data table.
SampleClean ReadsClean BasesGC ContentQ30Mapped Reads
PBS-120,062,1196,001,896,47936.82%92.24%94.08%
PBS-219,967,9255,972,790,91836.73%91.87%93.69%
PBS-320,304,5426,064,975,42236.90%91.72%93.52%
FC-119,933,2965,965,013,45636.83%91.22%93.07%
FC-219,902,3125,951,331,51836.80%91.54%93.31%
FC-319,926,2715,955,285,11237.17%91.08%93.10%
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yan, L.; Liu, J.; Chen, B.; Gao, F.; Liu, Z.; Zhang, Z.; Li, S.; Zhang, Y.; Jia, J.; Zhu, P.; et al. Transcriptomic Analysis of Tachypleus tridentatus Larval Response to Vibrio parahaemolyticus Infection. Animals 2025, 15, 2556. https://doi.org/10.3390/ani15172556

AMA Style

Yan L, Liu J, Chen B, Gao F, Liu Z, Zhang Z, Li S, Zhang Y, Jia J, Zhu P, et al. Transcriptomic Analysis of Tachypleus tridentatus Larval Response to Vibrio parahaemolyticus Infection. Animals. 2025; 15(17):2556. https://doi.org/10.3390/ani15172556

Chicago/Turabian Style

Yan, Lei, Jinxia Liu, Boyu Chen, Fanxi Gao, Zizhuo Liu, Zhenwen Zhang, Shimiao Li, Yan Zhang, Jiuman Jia, Peng Zhu, and et al. 2025. "Transcriptomic Analysis of Tachypleus tridentatus Larval Response to Vibrio parahaemolyticus Infection" Animals 15, no. 17: 2556. https://doi.org/10.3390/ani15172556

APA Style

Yan, L., Liu, J., Chen, B., Gao, F., Liu, Z., Zhang, Z., Li, S., Zhang, Y., Jia, J., Zhu, P., & Liao, Y. (2025). Transcriptomic Analysis of Tachypleus tridentatus Larval Response to Vibrio parahaemolyticus Infection. Animals, 15(17), 2556. https://doi.org/10.3390/ani15172556

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop