Using in situ hybridization to expand the daily egg production method to new fish species

Abstract

The capacity to reliably identify fish eggs is critical in the application of the daily egg production method (DEPM) to estimate biomass of commercially important species. This application has largely been confined to species that have easily identifiable eggs. Various molecular strategies have been used to extend the DEPM to a broader range of species, with recent approaches like in situ hybridization (ISH) that preserves the integrity of whole eggs, embryos or larvae recommended as a suitable alternative over destructive procedures like PCR. Here, we designed and validated an ISH approach for the identification of whole eggs and larvae from Snapper (Chrysophrys auratus) from environmental samples using the mitochondrial 16S rRNA gene as a target for specific horseradish peroxidase (HRP)-conjugated oligonucleotide probes. This colorimetric assay allowed the highly specific detection of positive hybridization signals from intact C. auratus larvae and eggs from mixed-species samples comprising closely related taxa. Furthermore, evaluation of whole eggs across a range of developmental stages revealed the sensitivity of the approach for discerning early stages, thereby guiding staging and the identification of otherwise indistinguishable eggs from environmental samples. This approach represents a major advance from current molecular-based strategies as it is nondestructive and allows for the simultaneous identification and staging of fish eggs (and larvae). The resultant 100% egg identification certainty we have achieved allows the DEPM to be applied to a wider array of fish species and is particularly applicable to species in areas where morphologically similar eggs are being spawned at the same time.

Discriminating Natal Source Populations of a Temperate Marine Fish Using Larval Otolith Chemistry

“…Morphological identifications were verified using an in situ hybridization (ISH) molecular technique. This technique uses a horseradish peroxidase (HRP) enzyme conjugated oligonucleotide probe that binds specifically to mitochondrial 16S ribosomal RNA of King George whiting and generates a blue color through oxidization with a HRP reactive substrate (Figure 3; Oxley et al, 2017). The ISH probe was applied to a tissue sample from each larva after the head had been removed to prevent potential contamination of the otoliths.…”

Using a biophysical model to investigate connectivity between spawning grounds and nursery areas of King George whiting (Sillaginodes punctatus: Perciformes) in South Australia’s gulfs

“…Morphological identifications were verified using an in situ hybridization (ISH) molecular technique. This technique uses a horseradish peroxidase (HRP) enzyme‐conjugated oligonucleotide probe that binds specifically to mitochondrial 16S ribosomal RNA of King George whiting and generates a blue colour through oxidization with a HRP reactive substrate (Figure 2) (Oxley et al., 2017). The chorion of each "possible" egg was mechanically pierced to expose the embryonic tissue to the molecular probe.…”

Standing out from the crowd: Spotting your targets in a mixed plankton sample

“…In this issue of Molecular Ecology Resources, Oxley et al. () describe an innovative in situ hybridization ( ISH ) approach that successfully solves this important problem and opens an exciting new avenue to ichthyoplankton analysis that may be widely adopted by both fish ecologists and fisheries managers.…”

“…The new study by Oxley et al. () presents an elegant solution to this problem. Because Snapper eggs and larvae, like those of many species, can't reliably be identified morphologically, previous DEPM studies relied on morphological identification of eggs that has now been shown to be unreliable (Dias et al., ).…”