A new approach using NGS to identify massive mixed eggs as species level for fish spawning ecology study

Abstract

Distribution of eggs is important information to explore the species-specific spawning times and grounds for fish. However, it has never been widely used to study the spawning ecology of fish because of difficulties in morphological identification of eggs. To understand the egg distribution-based fish spawning ecology, next-generation sequencing (NGS) was applied to the analysis for species identification a large number of mixed eggs. Compared to the list of species already we know the sequences obtained from mixed egg using NGS, species were decided by the OTU method and tree analysis, as a standard for species identification. The species list determined by the phylotype-based OTU was not different from those by the cluster-based OTU one (P>0.05). The number of OTU made by phylotype was 16 times less than those by cluster. The detective ability of NGS was 81%. The correlation of the percentage between the number of egg and the number of sequences was high (r2=0.91, n=21). 17 species of fish eggs were identified using phylotype-based OTU method from the four field samples using NGS. The occurrence timing and composition of these species changed, depending on every two week during summer. NGS was possible to identify a large number of mixed fish eggs to species level. This new approach using NGS will contribute to figuring out the species-specific spawning ecology of fish based on egg distribution.ication of eggs. To understand the egg distribution-based fish spawning ecology, next-generation sequencing (NGS) was applied to the analysis for species identification a large number of mixed eggs. Compared to the list of species already we know the sequences obtained from mixed egg using NGS, species were decided by the OTU method and tree analysis, as a standard for species identification. The species list determined by the phylotype-based OTU was not different from those by the cluster-based OTU one (P>0.05). The number of OTU made by phylotype was 16 times less than those by cluster. The detective ability of NGS was 81%. The correlation of the percentage between the number of egg and the number of sequences was high (r2=0.91, n=21). 17 species of fish eggs were identified using phylotype-based OTU method from the four field samples using NGS. The occurrence timing and composition of these species changed, depending on every two week during summer. NGS was possible to identify a large number of mixed fish eggs to species level. This new approach using NGS will contribute to figuring out the species-specific spawning ecology of fish based on egg distribution.