Determining the diet of larvae of western rock lobster (Panulirus cygnus) using high-throughput DNA sequencing techniques

Abstract

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

Figure 1. Source of sequence reads among individual larvae.

Relative distribution of combined reads for each sample to ascertain the proportion of potential prey reads against other kinds of reads. Samples 1, 2, 3, 5 and 11 had very little potential prey DNA, whereas samples 4, 7, 8, 10, 12, 13, 15 and 17 contained over 50%. Fungal DNA could originate from laboratory contamination, but along with algae, it is just as likely to originate from the gut and is not relevant to the current study. Contamination was either mammalian (probably human) or plant material and sequencing artefacts were mostly very short reads.

Figure 2. Sampling saturation of (a) v7 and (b) v9 loci.

Rarefaction curves representing the number of OTUs detected in pooled samples for the (a) v7 and (b) v9 loci. OTUs are defined at 93%, 95% and 97% respectively and as the percentage threshold increases so does the number of OTUs detected. However, the estimate of OTU richness for each OTU threshold tends toward an asymptote indicating that there is sufficient sequencing coverage to detect most taxa.

Figure 3. Rank abundance of potential prey reads after standardisation.

Rank abundances for the (a) v7 and (b) v9 loci. The x-axis shows the prey classes that constituted more than 0.5% of the sequencing reads. Samples were standardised into ratios of prey per predator prior to combining them into rank abundance.