Transcriptome analysis of the Japanese eel (Anguilla japonica) during larval metamorphosis

Abstract

Background: Anguillid eels spend their larval period as leptocephalus larvae that have a unique and specialized body form with leaf-like and transparent features, and they undergo drastic metamorphosis to juvenile glass eels. Less is known about the transition of leptocephali to the glass eel stage, because it is difficult to catch the metamorphosing larvae in the open ocean. However, recent advances in rearing techniques for the Japanese eel have made it possible to study the larval metamorphosis of anguillid eels. In the present study, we investigated the dynamics of gene expression during the metamorphosis of Japanese eel leptocephali using RNA sequencing.

Results: During metamorphosis, Japanese eels were classified into 7 developmental stages according to their morphological characteristics, and RNA sequencing was used to collect gene expression data from each stage. A total of 354.8 million clean reads were generated from the body and 365.5 million from the head, after the processing of raw reads. For filtering of genes that characterize developmental stages, a classification model created by a Random Forest algorithm was built. Using the importance of explanatory variables feature obtained from the created model, we identified 46 genes selected in the body and 169 genes selected in the head that were defined as the "most characteristic genes" during eel metamorphosis. Next, network analysis and subsequently gene clustering were conducted using the most characteristic genes and their correlated genes, and then 6 clusters in the body and 5 clusters in the head were constructed. Then, the characteristics of the clusters were revealed by Gene Ontology (GO) enrichment analysis. The expression patterns and GO terms of each stage were consistent with previous observations and experiments during the larval metamorphosis of the Japanese eel.

Conclusion: Genome and transcriptome resources have been generated for metamorphosing Japanese eels. Genes that characterized metamorphosis of the Japanese eel were identified through statistical modeling by a Random Forest algorithm. The functions of these genes were consistent with previous observations and experiments during the metamorphosis of anguillid eels.

Keywords: Anguilla japonica; Eel genome; Leptocephali; Metamorphosis; Transcriptome.

Fig. 1

Photographs of the developmental stages of artificially reared Japanese eels (Anguilla japonica) from the leptocephalus stage to the yellow eel stage, including 3 metamorphosing stages (M1-M3), the non-feeding glass eel stage and the elver stage when feeding begins

Fig. 2

Heat map of Pearson Correlation coefficients (PCC) obtained from the transcriptome datasets based on RPM values for samples from the body (left) and head (right). The PCC values between the samples of the same stages are marked by yellow squares

Fig. 3

Classification modeling for RNA-seq datasets to filter the genes that characterize the developmental stages. A: The feature importance (gini importance) of genes, obtained from each created model. B: Cross-validation using the leave-one-out approach. C: Principal component analysis of the most characteristic genes during metamorphosis

Fig. 4

Correlations from network analysis using the most characteristic genes and their co-expression genes. A: Body Clusters. B: Head Clusters. C: Average expression values of each cluster during metamorphosis