Identification of genes regulated by 20-Hydroxyecdysone in Macrobrachium nipponense using comparative transcriptomic analysis

Abstract

Background: Macrobrachium nipponense is a freshwater prawn of economic importance in China. Its reproductive molt is crucial for seedling rearing and directly impacts the industry's economic efficiency. 20-hydroxyecdysone (20E) controls various physiological behaviors in crustaceans, among which is the initiation of molt. Previous studies have shown that 20E plays a vital role in regulating molt and oviposition in M. nipponense. However, research on the molecular mechanisms underlying the reproductive molt and role of 20E in M. nipponense is still limited.

Results: A total of 240.24 Gb of data was obtained from 18 tissue samples by transcriptome sequencing, with > 6 Gb of clean reads per sample. The efficiency of comparison with the reference transcriptome ranged from 87.05 to 92.48%. A total of 2532 differentially expressed genes (DEGs) were identified. Eighty-seven DEGs associated with molt or 20E were screened in the transcriptomes of the different tissues sampled in both the experimental and control groups. The reliability of the RNA sequencing data was confirmed using Quantitative Real-Time PCR. The expression levels of the eight strong candidate genes showed significant variation at the different stages of molt.

Conclusion: This study established the first transcriptome library for the different tissues of M. nipponense in response to 20E and demonstrated the dominant role of 20E in the molting process of this species. The discovery of a large number of 20E-regulated strong candidate DEGs further confirms the extensive regulatory role of 20E and provides a foundation for the deeper understanding of its molecular regulatory mechanisms.

Keywords: 20-hydroxyecdysone; Macrobrachium nipponense; Molting; Transcriptome.

Fig. 1

Volcano plots and count charts of the DEGS. Each point in the volcano plots (A–C) represents a DEG. The x-axis represents the logarithm of the fold difference in expression of a gene in the two experimental groups, the y-axis represents the negative logarithm of the FDR. Green dots represent down-regulated DEGs, red dots represent up-regulated DEGs, and grey dots represent non-DEGs. A ConG vs. ExpG; B ConM vs. ExpM; C ConO vs. ExpO. D The number of DEGs in the two experimental groups, with the x-axis indicating the number of DEGs and the y-axis showing the comparison between groups

Fig. 2

Statistical plot of the GO classification of the DEGs. The horizontal coordinate shows the GO classification and the vertical coordinate shows the number of genes. A ConG vs. ExpG; B ConM vs. ExpM; C ConO vs. ExpO

Fig. 3

KEGG classification of all DEGs. The left and right y-axes indicate the names of the primary and secondary metabolic pathways, respectively. The x-axis indicates the number and proportion of genes annotated for each pathway. A ConG vs. ExpG; B ConM vs. ExpM; C ConO vs. ExpO

Fig. 4

Heatmaps showing the levels of expression of the genes related to molt. The x-axis indicates the grouping of samples, while the y-axes represent the clustering patterns of both the genes and their expression levels. Each row corresponds to a unique gene and the color gradients within the row indicate the relative expression level of that gene. The levels of gene expression are expressed as FPKM (fragments per kilobase million). A ConG vs. ExpG; B ConM vs. ExpM; C ConO vs. ExpO

Fig. 5

qRT-PCR validation of the RNA sequencing results. A C-type lectin 4. B CYP18A1. C Glutahione S-transferase. D Hemocyanin. E Cuticular protein 34. F Forkhead box protein O3. G Glutamine synthetase. H Metallothionein 1/2. I Vitellogenin. J Glutamine synthetase. K Groucho. L Cathepsin L

Fig. 6

The qRT-PCR results of strong candidate DEGs regulated by 20E during the different stages of molt. The data are presented as the mean ± SD (n = 6). Bars with different letters indicate statistically significant differences (P < 0.05). Prm, pre-molt; M, mid-molt; Pom, post-molt