High‐throughput molecular identification of fish eggs using multiplex suspension bead arrays

Abstract

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.

Molecular prey identification in Central European piscivores

“…Fragments of the mitochondrial 16S rRNA (16S) gene and cytochrome c oxidase subunit I (COI) gene were amplified for all target species (Table S1, Supporting information) using the forward primer 16Sar plus the reverse primer 16Sbr for 16S (Gleason & Burton ) and the forward primer FishF1 plus the reverse primer FishR1 for COI (Ward et al . ), respectively.…”

“…Fragments of the mitochondrial 16S rRNA (16S) gene and cytochrome c oxidase subunit I (COI) gene were amplified for all target species (Table S1, Supporting information) using the forward primer 16Sar plus the reverse primer 16Sbr for 16S (Gleason & Burton 2012) and the forward primer FishF1 plus the reverse primer FishR1 for COI (Ward et al 2005), respectively. Purified PCR products were sequenced by Eurofins MWG Operon (Munich, Germany); the sequences were edited using BioEdit (Hall 1999) and their identity checked by BLAST (NCBI website http://blast.ncbi.nlm.nih.gov/Blas t.cgi).…”

Species composition and assemblages of ichthyoplankton during summer in the East China Sea

“…In the past, unfertilized or early-developed fish eggs with special characteristics on their membranes, such as the reticular structure of Lizardfishes (Mito, 1961;Shao et al, 2001) and the three-feather root structures of Lanternfishes (Shao et al, 2001), allowed their identification to family level. Fish eggs without clear morphological characteristics or with shared morphologies have proven more difficult to classify (Shao et al, 2002;Gleason and Burton, 2012;Harada et al, 2015). However, using DNA barcoding, the eggs from our collection that were morphologically indistinguishable (accounting for 52% of the sample) were easily classified into 30 taxa.…”

Testing and validating a modified CTAB DNA extraction method to enable molecular parentage analysis of fertilized eggs and larvae of an emerging South African aquaculture species, the dusky kob Argyrosomus japonicus

“…japonicus (Archangi et al , ; Mirimin et al , ; Barnes et al , ), which reflect the recent increasing interest of the aquaculture sector in the cultivation of these fast‐growing marine teleosts. Isolation of genomic DNA and amplification of target DNA fragments can be inhibited by the protein‐rich content of fish eggs (Tsamis et al , ); however, an increasing number of methods have been described but may vary among species and type of application (Cary, ; Aranishi, ; Bayha et al , ; Lelièvre et al , ; Gleason & Burton, ). This study describes the successful implementation of a DNA extraction method capable of isolating genomic DNA from A .…”