Molecular Detection of Marine Invertebrate Larvae

Abstract

The ecological patterns of many invertebrate larvae remain an ongoing mystery, in large part owing to the difficult task of detecting them in the water column. The development of nucleic-acid-based technology has the potential to resolve this issue by direct identification and monitoring of embryonic and larval forms in situ. We report herein on the successful development and application of nucleic-acid-based sandwich hybridization assays that detect barnacles using rRNA-targeted probes with both group-(order Thoracica) and species-(Balanus glandula) specificity. Primary results include the determination of target 18S rRNA sequences and the construction of "capture" probes for detection of larvae using hybridization techniques. In addition, we modified existing protocols for whole cell hybridization of invertebrate larvae as confirmation of the sandwich hybridization results. We used both hybridization techniques successfully in the laboratory on a plankton time series collected over 3 months, as well as a week-long in situ deployment of the technique in Monterey Bay, CA. The adaptability of this technology promises to be further applicable to various organisms and could be used to enhance our understanding of larval presence in the world's oceans.

Application of a sandwich hybridisation assay for rapid detection of the Northern Pacific seastar, Asterias amurensis

“…Probe design DNA amplification and sequencing of A. amurensis and related seastars (extracted DNA provided by CSIRO) was carried out as described previously, using the universal 18S rDNA primers, 18e and 18P (Goffredi et al 2006). However, there is low sequence diversity between the Asterias species even in this variable region, and initially two general Asteriidae capture probes were designed for testing: Ast2F and Ast351 (Table 1).…”

“…Harvey et al (2009) designed taxon-and group-specific primers, targeting the intergenic spacer regions of the ribosomal DNA, for the development into molecular probes to detect invasive invertebrate larvae in Puget Sound, USA. Sandwich hybridisation assays have been developed for the barnacle, Balanus glandula (Goffredi et al 2006), and for the invasive European green crab, Carcinus maenas (adult tissue and larvae; Jones et al 2008). The SHA chemistry has been coupled with an in situ instrument, the Environmental Sample Processor (ESP), for autonomous environmental monitoring for a range of larval species simultaneously ).…”

“…However, these regions do not always distinguish between closely related species (e.g. Goffredi et al 2006). As an alternative, the ITS region offers a more variable albeit less abundant target for the design of SHA probes.…”

Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton

“…The ESP is an automated fluid handling system that collects and processes biological samples from seawater (Scholin et al, 2009). Current real-time capabilities include array-based detection of target organisms including harmful algal species (Greenfield et al, 2006(Greenfield et al, , 2008Haywood et al, 2007), invertebrate larvae (Goffredi et al, 2006;Jones et al, 2008) and major bacterial and archaeal clades (Preston et al, 2009). Quantitative PCR capabilities are also currently in development (Scholin et al, 2009).…”

“…Quantitative PCR capabilities are also currently in development (Scholin et al, 2009). The ESP can return samples preserved in a saline-ethanol solution, but sample analysis has been primarily limited to in situ hybridization techniques (Goffredi et al, 2006;Greenfield et al, 2006Greenfield et al, , 2008Jones et al, 2008). To extend the scope of laboratory analysis of archived samples, new protocols for sample preservation were developed for gene expression analysis and transcriptomics.…”