Osaka University discovers sex differences in Daphnia magna gene isoforms — Selection according to the environment

Daphnia magna, a zooplankton widely found in freshwater and brackish water, usually produces genetically identical clones but adapts to its surroundings by producing diverse phenotypes in response to the environment. One such adaptation is sex switching. Under favorable environmental conditions, Daphnia magna produces only females. However, under a shortened photoperiod, food shortages, or increased population density, males that are genetically identical to females are produced to produce individuals with a new genetic composition. Previously, the doublesex1 (Dsx1) gene, which is highly expressed in males, has been reported as a male determinant gene, and short-read sequencing has identified a group of genes differing in expression levels between males and females.

A research group led by Associate Professor Yasuhiko Kato and Professor Hajime Watanabe of the Graduate School of Engineering, Osaka University, in collaboration with Project Professor Atsushi Toyoda of the National Institute of Genetics, and Project Research Associate Joel Nitta (currently Associate Professor of the Graduate School of Global and Transdisciplinary Studies, Chiba University) and Professor Wataru Iwasaki of the Graduate School of Frontier Sciences at the University of Tokyo has analyzed the transcripts of Daphnia magna, which produces males or females according to the environment using the long-read sequencing method. The research group revealed the diversity of isoforms of each gene and their sex differences. The study was published in Scientific Reports.

In eukaryotes, multiple isoforms with partially different sequences are often synthesized from a single gene. Sequence differences are caused by skipping over specific exons, retaining introns, or other events during the splicing process. As a result, it has been reported that various proteins with differing activity and subcellular localization can be synthesized from the same gene and that the same gene can produce different phenotypes. However, it is difficult to detect different isoforms separately through conventional short-read sequencing, and no studies have examined the expression of gene isoforms in Daphnia magna in detail.

Using the long-read sequencing method, the research group determined the full-length sequence of each transcript synthesized during sex switching in response to the environment and identified a total of 25,654 transcripts derived from 9,710 genes. Of these, 14,924 transcripts were previously unidentified isoforms, and 5,713 genes produced two or more isoforms. Furthermore, based on the information on these transcripts, they analyzed the sex differences in expression levels of respective isoforms using short-read sequencing. Among the 5317 genes from which multiple isoforms were synthesized, they identified 824 genes that produce isoforms showing sex differences in expression levels.

Of these, 723 genes were found to have been overlooked in previous analyses because no sex differences in expression were detected at the gene level. Moreover, this number was found to be greater than the number of genes showing sex differences in expression at the gene level (613 genes). These results highlight the need for gene isoform analysis in the analysis of gene expression in Daphnia magna. Meanwhile, they found that the CREB-regulated transcription coactivator (CRTC) gene, a major regulator of carbohydrate metabolism in animals, was among the genes that produce isoforms showing sex differences.

They found a correlation between the CRTC gene isoform expression pattern and the differential expression of carbohydrate metabolic genes between the sexes, shedding light on how the sex difference is constructed. The study on the sex of the crustacean Daphnia magna could be applied to commercially important shrimp and crabs and may contribute to developing all-male or all-female monosex culture techniques required in crustacean aquaculture.

Kato said, "Through joint research with other institutions, we could obtain information on the expression of gene isoforms in the Daphnia magna females and males, which had been overlooked. Moving forward, functional analysis of the identified isoforms using genome editing and other techniques is expected to provide information necessary to understand a whole picture of the environment-dependent sex determination mechanism."

Journal Information
Publication: Scientific Reports
Title: Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna
DOI: 10.1038/s41598-024-59774-1