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.

The use of DNA barcodes in food web construction—terrestrial and aquatic ecologists unite!

“…An increasing number of studies using next-generation sequencing (including DNA metabarcoding) has offered particular insights into the broad range of taxa consumed by aquatic organisms (e.g., Deagle et al 2009Deagle et al , 2010Murray et al 2011;O'Rorke et al 2012bO'Rorke et al , 2013O'Rorke et al , 2014Leray et al 2013; Barcoding a coral reef food web). However, their main focus has been on the dietary analysis of larger taxa such as seals, penguins, and fish and in marine over freshwater systems.…”

Feeding selectivity of bivalve larvae on natural plankton assemblages in the Western English Channel

“…As part of this study, we performed molecular analysis of gut content of bivalve larvae following the bottle incubations, and where there were sufficient larvae, on the gut content of larvae taken directly from the field. Recent studies have used DNA-based techniques for dietary analysis including PCR amplification of 18S genes from the gut content of zooplankton (Nejstgaard et al 2003;Troedsson et al 2007;Nejstgaard et al 2008;Troedsson et al 2009;Durbin et al 2012), including meroplanktonic larvae (Maloy et al 2009;Riemann et al 2010;Fileman et al 2014). If the gut content of the predator can first be removed to reduce coamplification of host DNA, then general or universal primers designed to conserved regions (Holland et al 1991) targeting the 18S gene can be used.…”

“…Next generation sequencing (NGS) allows sequencing of several thousands of sequences simultaneously, increasing the efficiency and decreasing the cost and time associated with more traditional amplification, cloning and sequencing techniques. DNA metabarcoding using NGS has the potential to reveal many consumed species simultaneously (O'Rorke et al 2012;Pompanon et al 2012;Lindeque et al 2013). In addition, the most recent studies have developed a method whereby the dietary analysis of consumers can be made with universal primers using predator-specific endonuclease restriction enzymes or blocking primers, including the analysis of small planktonic consumers such as bivalve larvae (Maloy et al 2013).…”

Sympatric elasmobranchs and fecal samples provide insight into the trophic ecology of the smalltooth sawfish

“…Hussey et al 2012a, O'Rorke et al 2012, Thorburn et al 2014. In species with small populations, nonlethal techniques may be the only legal sampling methods, and these minimally invasive techniques include muscle biopsies, drawing blood, and fin clips (Hussey et al 2012a).…”

“…shorelines vs. open water). In addition, DNA sequencing (including highthroughput techniques) of consumer fecal samples can provide insights into specific prey types on a variety of taxonomic scales (Brown et al 2012, O'Rorke et al 2012. For example, 18S rRNA gene techniques have been used to study the diet of species for which there are research gaps or which would otherwise be difficult to study (O'Rorke et al 2012).…”