Genetic evidence of killer whale predation on white sharks in Australia

Killer whales (Orcinus orca) have been documented to prey on white sharks (Carcharodon carcharias), in some cases causing localised shark displacement and triggering ecological cascades. Notably, a series of such predation events have been reported from South Africa over the last decade, with killer whales specifically targeting shark’s liver. However, observations of these interactions are rare and knowledge of their frequency across the world’s oceans remain limited. In October 2023, a 4.7 m (total length) white shark carcass washed ashore in south-eastern Australia, coinciding with reports from citizen scientists of killer whales hunting a large, unidentified prey item in the area. Visual inspection of the carcass revealed that the liver, digestive, and reproductive organs were missing, and the presence of four distinctive bite wounds, one of which was characteristic of killer whale liver extraction as seen in South Africa. Genomic analyses performed on swabs taken from the bite wounds confirmed the presence of killer whale DNA in the major bite area, while the other bites were embedded with genetic material from the scavenging broadnose sevengill shark (Notorynchus cepedianus). These results provide confirmed evidence of killer whale predation on white sharks in Australia, and the likely selective consumption of the liver, suggesting predations of this nature are more globally prevalent than currently assumed.

Methods
We collected genetic material from bite wounds on a white shark carcass by swabbing internal and external wound surfaces with a flocked cotton swab and preserving each in ATL buffer (QIAGEN). We collected a total of 15 swabs four distinct bite wounds, with each swab taken from a different area of each wound site. Samples were subsequently transported to the laboratory on ice and stored at 4oC for 24 h prior to genetic analysis. Total genomic DNA was extracted from the swabs using a QIAGEN DNeasy Blood and Tissue Kit, following the manufacturer's protocol with minor modifications as specified by Clark et al. (2023). We performed four negative extraction controls in parallel (all extraction steps performed but without a swab sample) to control for potential cross-sample contamination.

Polymerase chain reaction (PCR) targeting the mitochondrial 12S rRNA gene (106bp) was performed using a universal vertebrate PCR assay (Riaz et al., 2011; Table 1) following the laboratory workflow outlined by Clark et al. (2023). Briefly, PCR primer combinations were modified to include Illumina adapter tails at their 5′ ends (‘TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG’ and ‘GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G’ for forward and reverse primers, respectively) to enable the addition of Illumina dual-index barcodes. Additionally, blocking primers were used to suppress human and white shark DNA amplification.  We performed all PCR reactions in duplicate for each sample with PCR reaction matrices and thermal cycling conditions being consistent with those described by Clark et al. (2023). A total of eight negative template PCR controls (no DNA extract added) and four negative DNA controls (extract from negative DNA extractions) were included in each step of the metabarcoding process through to sequencing to control for laboratory contamination.

Index PCR reaction matrices and thermal cycling conditions were again performed following Clark et al. (2023). Forward and reverse index primers provided dual indices in unique combinations, allowing demultiplexing of pooled products and Illumina sequencing adapters. PCR products were purified using 0.8× volume of AmpureBead XP (Beckman Coulter) and quantified using a Qubit dsDNA BR Assay Kit (Invitrogen). Indexed PCR products were subsequently normalised and pooled to create a single pooled library for sequencing. The resulting library was finally denatured and sequenced on the Illumina iSeq® platform using the iSeq® Version 2 kit (300 bp paired end), allowing for average read depth of 5 × 104 DNA sequence reads per sample.