RNA-seq data of healthy and fungal infected Tachypleus tridentatus embryos

Abstract

The “living fossil” Tachypleus tridentatus holds significant medical and economic value but is currently experiencing a severe decline in germplasm resources. The extended incubation period of T. tridentatus eggs make them susceptible to invasion by pathogenic microorganisms, with fungal infections posing a major threat to embryonic development. However, the molecular immune mechanisms underlying embryonic immunity in T. tridentatus remain poorly understood. We collected T. tridentatus embryos at stages 18–20 that were naturally infected with Aspergillus candidus under aquaculture conditions, and conducted RNA sequencing to analyze transcriptomic response to the fungal infection.

Specifications Table

Subject	Biology
Specific subject area	Marine Biology
Type of data	Table, Figure, Raw, Analyzed
Data collection	Embryos were collected from an aquaculture farm. Total RNA was extracted using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA). RNA sequencing of eighteen libraries was performed by Gene Denovo Biotechnology Co (Guangzhou, China). Differential expression analysis was conducted using DESeq2 and edgeR. Functional enrichment of the differentially expressed genes was conducted based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Gene set enrichment analysis was performed using software GSEA and MSigDB.
Data source location	Fourth institute of Oceanography, Ministry of Natural Resources, Beihai, China
Data accessibility	Repository name: CNGBdb Data identification number: CNP0008445 Direct URL to data: https://db.cngb.org/data_resources/project/CNP0008445
Related research article	none

1. Value of the Data

• •This dataset presents the first transcriptomic resource for Tachypleus tridentatus embryos under fungal infection, a major cause of embryonic mortality in aquaculture.
• •The data may provide a basis for exploring the immune mechanisms induced by fungal infection in T. tridentatus embryos, facilitating the study of the regulatory roles of specific genes or signaling pathways in this process.
• •Utilizing this data to further elucidate the immunological characteristics of T. tridentatus embryos could contribute to disease prevention and the development of artificial breeding techniques. These advances would ultimately support the restoration and sustainable management of this species.

2. Background

As a living fossil species, T. tridentatus holds significant economic and medicinal value. Its blood is used to produce the “Limulus Amebocyte Lysate (LAL) test” for detecting bacterial endotoxins, which is widely applied in fields such as food safety and medical diagnostics [1]. However, overharvesting and habitat loss have led to a severe decline in its germplasm resources [2]. Additionally, during its embryonic development stage, the horseshoe crab is susceptible to infections by various pathogenic microorganisms [[3], [4], [5]], thereby reducing its hatching success rate. While the adult T. tridentatus immune system is well-studied, embryonic immune responses remain poorly characterized at the molecular level.

During the artificial breeding of T. tridentatus, we observed diseased embryos exhibiting a white appearance and the causative fungus, Aspergillus candidus were isolated from them. Based on Sekiguchi's observation, the embryonic development of the horseshoe crab comprises 21 periods, with the earliest molting event initially observed during stages 18–20, also referred to as the tissue differentiation phase, during which multiple key organs are formed [6]. Therefore, we selected embryos at these three developmental stages for our data collection. This dataset was generated to capture transcriptomic changes in T. tridentatus embryos following fungal infection, providing a resource for understanding embryonic defense mechanisms and potential vulnerabilities during early development.

3. Data Description

This article describes a transcriptomic dataset of T. tridentatus embryos at stages 18–20, covering healthy and fungal-infected conditions, along with comparative data obtained through differential expression analysis and bioinformatics analysis.

Raw sequencing reads (FASTQ format) are available under CNSA accession CNP0008445 (Three replicates per sample, totaling 18 sequencing files). Sequencing statistics for all samples are listed in Table 1.

Table 1.

Transcriptome statistics.

Developmental stage	Description	Sample	Raw reads	Clean reads	Mapping rate	Unique gene
Stage 18	tissue differentiation stage I	C-V1–1	60,702,556	60,664,272 (99.94 %)	57,265,630 (95.39 %)	32,257
		C-V1–2	49,080,468	49,050,152 (99.94 %)	46,471,780 (96.13 %)
		C-V1–3	47,800,076	47,768,878 (99.93 %)	44,981,012 (96.05 %)
		T-V1–1	37,917,990	37,678,948 (99.37 %)	35,258,302 (94.24 %)
		T-V1–2	37,976,754	37,765,148 (99.44 %)	35,561,810 (94.85 %)
		T-V1–3	38,248,076	38,002,868 (99.36 %)	35,741,124 (94.69 %)
Stage 19	tissue differentiation stage II	C-V2–1	48,175,650	48,149,750 (99.95 %)	45,609,191 (96.33 %)
		C-V2–2	48,235,324	48,196,378 (99.92 %)	44,590,641 (95.80 %)
		C-V2–3	47,231,014	47,201,060 (99.94 %)	44,025,460 (95.87 %)
		T-V2–1	37,964,708	37,746,422 (99.43 %)	35,491,086 (94.61 %)
		T-V2–2	38,262,088	38,041,302 (99.42 %)	35,505,775 (93.99 %)
		T-V2–3	38,493,226	38,250,718 (99.37 %)	35,311,283 (94.27 %)
Stage 20	tissue differentiation stage III	C-V3–1	54,092,094	54,053,214 (99.93 %)	49,123,621 (95.57 %)
		C-V3–2	46,592,324	46,564,720 (99.94 %)	42,361,181 (96.15 %)
		C-V3–3	47,366,076	47,327,164 (99.92 %)	43,599,112 (95.25 %)
		T-V3–1	38,117,924	37,893,722 (99.41 %)	35,581,390 (94.49 %)
		T-V3–2	38,167,278	37,928,086 (99.37 %)	35,489,247 (94.30 %)
		T-V3–3	38,981,552	38,737,210 (99.37 %)	36,236,689 (94.09 %)

The number of differentially expressed genes (DEGs) and their regulation (up- or down-regulated) between the control and infected groups at each embryonic stage are shown in Fig. 1. We additionally generated a Venn diagram to illustrate the intersecting states of these genes across different stages, highlighting several potential key genes (Fig. 2). These genes were screened based on GO and KEGG functional annotations. Significantly up-regulated immune-related genes identified included: HSP83, techylectin-like, Relish, limulus factor D, antifungal protein-like, etc. The enrichment results from GO annotation and KEGG pathway analysis show marked differences among the three developmental stages (Figs. 3 and 4). Notably, embryos at both stages 18 and 19 exhibited enrichment in the “oxidative phosphorylation” pathway, which is associated with inflammatory responses and the activity of polymorphonuclear phagocytosis [7,8]; For embryos at stage 20, the significantly enriched GO terms and KEGG pathway were primarily involved in the cell cycle. DEGs annotated to immune-related GO terms and KEGG pathways were identified as immune-related genes, with a total of 278 genes being screened, and an expression heatmap was generated (Fig. 5). Weakly activated immune-related pathways were identified via GSEA-KEGG analysis, with results summarized in Table 2.

Fig. 1.

Summary of differential gene expression.

Fig. 2.

Venn diagram of differentially expressed genes and expression patterns of key genes.

Fig. 3.

GO annotation classification of differentially expressed genes. A: stage 18; B: stage 19; C: stage 20. First circle: Top 20 enriched GO terms; Second circle: Number of genes associated with the GO term in the background of differentially expressed genes and the corresponding Q-value; Third circle: Bar plot showing the proportion of up- and down-regulated differentially expressed genes; Fourth circle: Rich Factor value for each GO term.

Fig. 4.

KEGG pathway enrichment analysis of differentially expressed genes. A: stage 18; B: stage 19; C: stage 20. Figures were generated using the top 20 pathways with the smallest Q-values. The bubble size corresponds to the number of genes, and the color intensity indicates the Q-value (with redder shades denoting smaller Q-values).

Fig. 5.

Heatmap of immune-related differentially expressed genes. Red represents up-regulated expression, blue represents down-regulated expression, and the color intensity corresponds to the magnitude of the |Log₂FC| value (−6<Log₂FC<4) .

Table 2.

Immune-related pathways based on GSEA-KEGG analysis.

Group	KEGG pathway	NES	P-value	FDR
C-V1-vs-T-V1	NOD-like receptor signaling pathway	1.42	0.001	0.17
	Toll-like receptor signaling pathway	1.38	0.029	0.24
	C-type lectin receptor signaling pathway	1.37	0.016	0.24
C-V2-vs-T-V2	/	/	/	/
C-V3-vs-T-V3	NOD-like receptor signaling pathway	1.24	0.018	0.18
	C-type lectin receptor signaling pathway	1.22	0.037	0.20

In summary, the data presented in this study may advance our understanding of the defense mechanisms against fungal infection in Tachypleus tridentatus embryos at the molecular level.

4. Experimental Design, Materials and Methods

4.1. Sample collection

All T. tridentatus embryos were obtained from adults in the aquaculture farm. The collected embryos were maintained in seawater tanks under controlled temperature and light-proof conditions. After a period of natural development, embryos at stages 18, 19 and 20, both healthy and fungal infected, were selected as experimental subjects, with three biological replicates each.

4.2. RNA extraction

Total RNA extraction was performed using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Quality assessment was conducted on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), with integrity confirmation carried out by RNase-free agarose gel electrophoresis.

4.3. Library construction and sequencing

Following enrichment with Oligo (dT), the mRNA was fragmented and reverse transcribed into cDNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB#7530, New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments were first processed for end repair and A-base addition, followed by ligation of the products with Illumina sequencing adapters. AMPure XP Beads (1.0X) were used to purify the ligation reaction products. Following size selection through agarose gel electrophoresis, ligated fragments were amplified by polymerase chain reaction (PCR). The prepared cDNA libraries were sequenced on the Illumina Novaseq6000 platform by Gene Denovo Biotechnology Co (Guangzhou, China). The 6 groups of libraries corresponded to the infected and control groups at each of the three embryonic developmental stages, and triplicate for each group.

4.4. Bioinformatics analysis

We performed RNAs differential expression analysis using DESeq2 [9] for group comparisons and edgeR [10] for sample comparisons. We subsequently functionally annotated the resulting DEGs via the Gene Ontology (GO) database. (http://www.geneontology.org/). Based on the number of genes per term, we defined significantly enriched GO terms in DEGs relative to the genome background by hypergeometric test. Additionally, we conducted pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

4.5. Gene set enrichment analysis (GSEA)

To identify significantly different gene sets from specific GO terms and KEGG pathways between two groups, we employed gene set enrichment analysis using software GSEA [11] and MSigDB [11]. The brief analysis procedure involved inputting the gene expression matrix and ranking the genes by Signal-to-Noise normalization method. Enrichment scores and p-values were derived with the default parameters.

Limitations

The embryos were obtained from a single aquaculture population. This sampling strategy may limit the genetic diversity represented in the study; consequently, the findings might not fully capture the potential variation in immune responses across different wild populations or geographic strains of T. tridentatus. The fungal infection occurred naturally under aquaculture conditions rather than through a controlled laboratory challenge. Consequently, the precise infectious dose, the exact timing of infection onset, and the progression of infection could not be standardized or quantified across individual embryos. This variability may contribute to background noise in the transcriptomic data.

Ethics Statement

We are in lawful possession of the People's Republic of China Aquatic Wildlife Artificial Breeding Permit for T. tridentatus, and throughout the breeding and cultivation processes, no acts of cruelty or harm have been inflicted upon individual animals. Embryos were obtained from an aquaculture facility under standard rearing conditions.

CRediT Author Statement

Yunfan Huang: Data curation, Writing, Original draft preparation. Ying Qiao: Investigation, Data curation, Writing. Xiaowan Ma: Conceptualization, Writing- Reviewing and Editing. Ruifang Chen, Lanfang Dong and Haijuan Liu: Provide facilities and research seedlings. Theerakamol Pengsakul: Software, Validation.

Data Availability

• CNGBdbTranscriptome data of Tachypleus tridentatus embryos infected by fungi (Original data).