Full-Length Transcriptome of Testis and Ovary Provides Insights into Alternative Splicing During Gonadal Development in Litopenaeus vannamei

Abstract

The Pacific white shrimp, Litopenaeus vannamei (L. vannamei), is an important aquaculture species, yet the molecular mechanisms underlying its sex differentiation and gonadal development remain poorly understood. A deeper understanding of these processes is critical for advancing broodstock quality and enabling unisex breeding strategies. While previous studies have focused on gene expression differences between females and males, structural differences in transcriptomic regulation between sexes have been largely overlooked. Here, we present a comprehensive full-length transcriptome analysis of L. vannamei testis and ovary, identifying 830 and 690 novel genes, respectively, and over 6000 new isoforms. Notably, we discovered extensive alternative splicing (AS) events, with the cartilage oligomeric matrix protein-like gene exhibiting over 300 AS isoforms in the ovary compared to only 2 in the testis, suggesting a potential role in ovarian development. Furthermore, sex-determining genes such as Fem-1a, Fem-1c, and Sxl were found to produce AS isoforms exclusively in ovarian tissue. We also identified three germ cell development-associated genes-MAD2-like, RAD51-like, and Su(dx)-like-that undergo distinct AS events in gonadal tissues, leading to sex-specific structural domain alterations. These findings highlight the complexity of AS-mediated post-transcriptional regulation in L. vannamei and provide novel insights into the molecular mechanisms governing sex differentiation and gonadal development.

Keywords: AS events; PacBio Iso-Seq; gonads; sexual differentiation; shrimp.

Figure 1

Summary of Iso-seq data. (A,B) Length distribution of subreads; the horizontal coordinate indicates the length of the subreads, the vertical coordinate is the number of subreads, (A) denotes the length distribution of subreads for testis, (B) denotes the length distribution of subreads for ovary. (C,D) FLNC length distribution map; the x-axis indicates the length of the FLNC reads, the left Y-axis is a bar graph coordinate indicating the number of reads in a range of lengths (X-axis), the Y-axis on the right is the plot coordinates indicating the number of reads whose length is greater than a certain X-axis value, (C) denotes the FLNC length profile of testis, and (D) denotes the ovary. (E,F) The percentage of high-quality isoforms mapped to the reference genome; (E) denotes the result for testis and (F) denotes the result for ovary.

Figure 2

Function annotation and classification of isoforms. (A) Venn diagrams of annotated isoforms in testis. (B) Venn diagrams of annotated isoforms in ovary. (C) Annotation of GO functional classification of isoforms in testis. (D) Annotation of GO functional classification of isoforms in ovary. (E) KEGG pathway enriched for genes in testis. (F) KEGG pathway enriched for genes in ovary.

Figure 3

Gene statistics and KEGG enrichment in testis and ovary. (A) Venn diagrams for genes in the testis and ovary. (B) KEGG pathways enriched for genes specific to the testis. (C) KEGG pathways enriched for genes specific to the ovary.

Figure 4

Alternative splicing analysis. (A) Classification of alternative splicing events. (B) Distribution of the number of alternative splicing events. (C) Expression pattern of the gene LOC113825377 (NCBI Gene ID: 113825377) in different tissues of adult shrimp L. vannamei. Adult different tissues: Bra (brain), Epi (epidermis), Es (eye stalk), Gill (gill), Hp (hepatopancreas), Ht (heart), In (intestine), Ms (muscle), Oka (lymphoid organ), St (stomach), Tg (thoracic ganglion), Vn (ventral nerve), Hc (hemocyte), Ov (ovary), Te (testis). *** indicates p < 0.001.

Figure 5

Alternative splicing isoform analysis. (A) Structural domains of different transcripts of MAD2-like, RAD51-like, and Su(dx)-like. (B) Alternative splicing analysis of known sex-determining and differential genes.

Figure 6

Validation of selective splicing (AS) events. Gene names and transcript IDs are shown sequentially on the left side of the gene model. The exons of the different transcripts are indicated by yellow or green squares, respectively, and red arrows indicate the position of the PCR primers (F for forward and R for reverse). Introns are indicated by lines. In agarose gel images, arrows indicate PCR products generated from different transcripts.