Differences in alternative splicing events in the adaptive strategies of two Daphnia galeata genotypes induced by fish kairomones

Abstract

Background: Daphnia galeata is a suitable model organism for investigating predator-induced defense. Genes and pathways exhibiting differential expression between fish kairomone-treated and untreated groups in D. galeata have been identified. However, understanding of the significance of alternative splicing, a crucial process of the regulation of gene expression in eukaryotes, to this mechanism remains limited. This study measured life-history traits and conducted short-read RNA sequencing and long-read isoform sequencing of two Korean D. galeata genotypes (KB1 and KE2) to uncover the genetic mechanism underlying their phenotypic plasticity under predation stress.

Results: KB1 exhibited strategies to enhance fertility and decrease body length when exposed to fish kairomones, while KE2 deployed an adaptive strategy to increase body length. Full-length transcriptomes from KB1 and KE2 yielded 65,736 and 57,437 transcripts, respectively, of which 32 differentially expressed transcripts (DETs) were shared under predation stress across both genotypes. Prominent DETs common to both genotypes were related to energy metabolism and the immune system. Additionally, differential alternative splicing (DAS) events were detected in both genotypes in response to fish kairomones. DAS genes shared between both genotypes may indicate their significant role in the post-transcriptional stress response to fish predation. Calpain-3, involved in digestion and nutrient absorption, was identified as a DAS gene in both genotypes when exposed to fish kairomones. In addition, the gene encoding thymosin beta, which is related to growth, was found to be a statistically significant DAS only in KB1, while that encoding ultraspiracle protein, also associated with growth, was only identified in KE2. Moreover, transcripts encoding proteins such as EGF-like domain-containing protein, vitellogenin fused with superoxide dismutase, and others were identified overlapping between DAS events and DETs and potentially elucidating their association with the observed phenotypic variation in each genotype.

Conclusions: Our findings highlight the crucial role of alternative splicing in modulating transcriptome landscape under predation stress in D. galeata, emphasizing the requirement for integrating gene expression and splicing analyses in evolutionary adaptation studies.

Keywords: Daphnia galeata; Alternative splicing; Full-length transcriptome; Fish kairomones; Phenotypic plasticity; Predator-induced response.

Fig. 1

A schematic diagram showing the different stages of this study including sample collection, experimental setup, bioinformatic analysis of the data, and expected outcomes

Fig. 2

Effects of fish kairomone exposure (21 days) on the life-history traits of two D. galeata genotypes. KB1_con is the control group for genotype KB1 without fish kairomones, while KB1_fish is the group exposed to fish kairomones. Similarly, KE2_con is the control group for genotype KE2 without fish kairomones, and KE2_fish is the group exposed to fish kairomones. Statistically significant differences were indicated by ***(P < 0.001), **(P < 0.01), *(P < 0.05), and N.S. (no significance)

Fig. 3

Principal component analysis (PCA) plots and volcano plots of RNA-seq data in D. galeata. PCA plot of gene expression in genotypes KB1 A and KE2 B. The red and cyan indicate the control (without fish kairomones) and experimental groups (fish kairomone-exposed). Volcano plots illustrate the differences in gene expression fold change between control (without fish kairomones) and experimental groups (fish kairomone-exposed) across genotypes KB1 C and KE2 D. Red and blue dots indicate up- and down-regulated DETs, respectively. Gray dots indicate no statistically significant changes in transcript expression

Fig. 4

Sashimi plot display the coverage of RNA-seq reads mapped to the reference genome, including A Calpain-3 in KB1, B Calpain-3 in KE2, C Thymosin beta in KB1, and D Ultraspiracle in KE2, all of which exhibit statistically significant alternative splicing events. The exons are represented on the x-axis, and the read coverage of RNA-seq is represented on the y-axis. The splice junctions are depicted in the form of arcs, with the numerical values on each arc representing the junction-spanning read counts for each sample. KB1/KE2_con and KB1/KE2_fish indicate the control and experimental groups of genotypes KB1/KE2, respectively. Abbreviations: AF—alternative first exon, A3—alternative 3′ splice site, A5—alternative 5′ splice site

Fig. 5

Venn diagram showing the number of DETs and DAS genes identified in each D. galeata genotype