De Novo Assembly of the Whole Transcriptome of the Wild Embryo, Preleptocephalus, Leptocephalus, and Glass Eel of Anguilla japonica and Deciphering the Digestive and Absorptive Capacities during Early Development

Abstract

Natural stocks of Japanese eel (Anguilla japonica) have decreased drastically because of overfishing, habitat destruction, and changes in the ocean environment over the past few decades. However, to date, artificial mass production of glass eels is far from reality because of the lack of appropriate feed for the eel larvae. In this study, wild glass eel, leptocephali, preleptocephali, and embryos were collected to conduct RNA-seq. Approximately 279 million reads were generated and assembled into 224,043 transcripts. The transcript levels of genes coding for digestive enzymes and nutrient transporters were investigated to estimate the capacities for nutrient digestion and absorption during early development. The results showed that the transcript levels of protein digestion enzymes were higher than those of carbohydrate and lipid digestion enzymes in the preleptocephali and leptocephali, and the transcript levels of amino acid transporters were also higher than those of glucose and fructose transporters and the cholesterol transporter. In addition, the transcript levels of glucose and fructose transporters were significantly raising in the leptocephali. Moreover, the transcript levels of protein, carbohydrate, and lipid digestion enzymes were balanced in the glass eel, but the transcript levels of amino acid transporters were higher than those of glucose and cholesterol transporters. These findings implied that preleptocephali and leptocephali prefer high-protein food, and the nutritional requirements of monosaccharides and lipids for the eel larvae vary with growth. An online database (http://molas.iis.sinica.edu.tw/jpeel/) that will provide the sequences and the annotated results of assembled transcripts was established for the eel research community.

Fig 1. Frequency diagram of contig length

This diagram presents an overview of the contigs. In all, approximately 40% of the contigs were <500 bp and 33% of the contigs were >1000 bp. The bar and the solid line individually represent the number of assembled contigs in a specific length interval and the cumulative percentage over a certain length.

Fig 2. Relative abundance of the main taxonomic groups among the BLAST hits using a simplified Tree of Life diagram

Eukaryotes accounted for 99.8% of all positive hits, and Chordata accounted for 98.9%. Among Chordata, Actinopterygii accounted for 90.2% of all positive hits, followed by Mammalia (4.6%). The top-hit species distribution of Actinopterygii was analyzed further: the most heavily represented fish species being Lepisosteus oculatus (33%), followed by Danio rerio (25%), and Oreochromis niloticus (7%).

Fig 3. Functional annotation of assembled contigs associated with GO terms

(A) In biological processes, 21,852 (25.2%) protein coding transcripts participated in cellular processes, 16,925 (19.5%) transcripts were related to metabolic processes, and 15,075 (17.4%) transcripts were associated with single-organism processes. (B) In the cellular component, 12,603 (26.3%) protein-coding transcripts belonged to cell parts, 7,455 (15.5%) transcripts pertained to membrane parts, and 6,682 (13.9%) protein coding transcripts were part of the membrane. (C) Of those related to molecular functions, 32,004 (49.6%) protein coding transcripts were related to binding, 17,955 (27.8%) were believed to display catalytic activity, and 3,788 (5.9%) transcripts showed transporter activity.

Fig 4. Expressional percentages of different categories of digestive enzymes and nutrient transporters at different stages

(A) In preleptocephali, transcript levels of protein digestion enzymes accounted for 98.27% of the total, followed by lipid digestion enzymes (1.48%) and carbohydrate digestion enzymes (0.25%). In leptocephali, the transcript levels of protein digestion enzymes accounted for 90.47% of the total, followed by lipid digestion enzymes (8.77%) and carbohydrate digestion enzymes (0.76%). In glass eels, transcript levels of lipid digestion enzymes accounted for 36.36%, followed by protein digestion enzymes (33.33%) and carbohydrate digestion enzymes (30.3%). (B) In preleptocephali, transcript levels of amino acid transporters accounted for 82.6%, followed by glucose and fructose transporters (9.04%) and cholesterol transporters (8.36%). In leptocephali, transcript levels of amino acid transporters accounted for 52.61%, followed by glucose and fructose transporters (35.34%) and cholesterol transporters (12.05%). In glass eels, transcript levels of amino acid transporters accounted for 89.32%, followed by glucose and fructose transporters (10.26%) and cholesterol transporters (0.42%).

Fig 5. Relative mRNA expression levels of digestive enzymes and nutrient transporters in glass eel

The mRNA expression levels were presented as mean ± SD (n = 6). (A) For the digestive enzymes, the mRNA expression level of pepsinogen (pep) was approximately six times higher than that of α-amylase (amy), and four times higher than that of triglyceride lipase (lip). (B) For the nutrient transporters, the mRNA expression level of large neutral amino acids transporter small subunit 2 (slc7a8) was approximately four point five times higher than that of sodium/glucose co-transporter member 1 (sglt1), and five times higher than that of Niemann-Pick C1-like 1 (npc1l1). Means with the same letter were not significantly different at the 5% level, as determined by Tukey’s HSD test.