Universal primers and PCR of gut contents to study marine invertebrate diets

Abstract

Determining the diets of marine invertebrates by gut content analysis is problematic. Many consumed organisms become unrecognizable once partly digested, while those with hard remains (e.g. diatom skeletons) may bias the analysis. Here, we adapt DNA-based methods similar to those used for microbial diversity surveys as a novel approach to study the diets of macrophagous (the deep-sea amphipods Scopelocheirus schellenbergi and Eurythenes gryllus) and microphagous (the bivalve Lucinoma aequizonata) feeders in the deep sea. Polymerase chain reaction (PCR) in conjunction with 'universal' primers amplified portions of the mitochondrial cytochrome c oxidase I (COI) gene for animals ingested by S. schellenbergi and E. gryllus and the 18S rRNA gene for lesser eukaryotes ingested by L. aequizonata. Amplified sequences were combined with sequences from GenBank to construct phylogenetic trees of ingested organisms. Our analyses indicate that S. schellenbergi, E. gryllus and L. aequizonata diets are considerably more diverse than previously thought, casting new light on the foraging strategies of these species. Finally, we discuss the strengths and weaknesses of this technique and its potential applicability to diet analyses of other invertebrates.

Development of a Denaturing High-Performance Liquid Chromatography (DHPLC) Assay to Detect Parasite Infection in Grass Shrimp Palaemonetes pugio

“…certain species with high sensitivity. To overcome these drawbacks, universal primers have been developed to target SSU rDNA of diverse phylum (Bower et al, 2004;Blankenship and Yayanos, 2005). When coupled with universal primers, DH-PLC can be a competent method due to its high sensitivity for separation, which can detect as small as a 2-bp difference in sequence composition (Troedsson et al, 2008b).…”

The feeding ecology of penaeid shrimp in tropical lagoon-estuarine systems

“…The concept of this methodology is founded on the assumption that DNA from consumed organisms is not completely degraded during digestion and therefore could be amplifi ed via PCR and analyzed (Zaidi et al 1999). This PCR-based method is a potentially powerful course for expanding the range and diversity of dietary items detected in stomach contents, especially by generalist feeders (Blankenship & Yayanos 2005). With a fair amount of precaution, careful implementation of controls, and the selection of an optimal molecular marker, this technique has the potential to reveal previously unknown dietary items for many shrimp.…”

“…With a fair amount of precaution, careful implementation of controls, and the selection of an optimal molecular marker, this technique has the potential to reveal previously unknown dietary items for many shrimp. With the continued expansion of DNA databases, we conjecture that PCR-based approaches with universal primers will become increasingly useful in studying shrimp dietary habits (Blankenship & Yayanos 2005).…”

“…However, little is known about the diets of most aquatic invertebrates. Classical analysis methods include the direct observation of diet food and the examination of intestinal contents or feces of recognizable remains of ingested organisms (Blankenship & Yayanos 2005). Therefore, studies of gut contents other methods are used to identify and quantify the food resources of shrimp, providing information on the preferred food available in this environment (Tararam et al 1993).…”

Development of Clamping Probe for Rare DNA Detection using Universal Primers

“…In 1989, Bottger (1989 first demonstrated that a portion of the 16S rRNA gene from Legjonella pneumophila, Escherichia coli or Mycobacterium tuberculosis can be amplified by using one set of universal PCR primers and then sequenced to identify these bacteria. Several targets are available for each taxonomy now using 16S rDNA for bacteria (Baker et al, 2003;Tian et al, 2005), 18S rDNA for eukaryote (Blankenship and Yayanos, 2005), 23S rDNA for marine algae and cyanobacteria (Sherwood and Presting, 2007), and 28S rDNA and ITS for fungi (Mohlenhoff et al, 2001;Iwen et al, 2002). In spite of convenience of the universal primer with ease of application to most species, separation of the amplicons from unknown sample mostly relied on a special treatment such as restriction enzyme (Lin et al, 2004) and denaturing high performance liquid chromatograph (DHPLC) (Troedsson et al, 2008a;Troedsson et al, 2008b).…”