As a starting point, we chose to focus on development of ribosomal RNA (rRNA) targeted probes as tools for identifying and estimating the abundance of a variety of marine organisms, and to devise methods for applying those probes in a fashion consistent with their use outside of a conventional laboratory. This effort is rooted in the field of harmful algal bloom (HAB) research and the need to quantify harmful and toxic organisms collected from natural samples. We have explored use of rRNA probes in both whole cell (fluorescent in situ hybridization) and cell-free formats (sandwich hybridization; Scholin et al. 1996, 1997), targeting harmful organisms that span several classes of algae: diatoms, dinoflagellates and raphidophytes. The species studied are found in many coastal regions of the world where they pose substantial public health concerns and economic impacts (Hallegraeff et al. 2003). Blooms of these organisms can also have deleterious impacts and on wildlife (e.g., Scholin et al. 2000).
Utilizing relatively simple off-the-shelf and semi-custom sample processing apparatus we prototype and evaluate the performance of various assays, particularly with respect to their suitability for automation (e.g., Anderson et al. in review, Tyrrell et al. 2001, Scholin et al. 1999, Miller and Scholin 1998). The same techniques developed for use with HAB species have been applied to aid the detection of marine microbes and invertebrate larvae in collaboration with the DeLong and Vrijenhoek labs. We have also worked to establish protocols for sample archival that are also suitable for automation. Our challenge here is to preserve samples in a way that obviates the need for refrigeration or freezing, preserves gross cell morphology when microscopy is necessary, and does not interfere with standard molecular biological techniques like DNA extraction and sequencing (e.g., Miller and Scholin, 2000; Preston and Scholin, unpublished data). Techniques that have proved most useful for near real-time detection of target species and sample archival have served to define functional requirements of a new class of instrumentation – the ESP.
We have conducted extensive field surveys and time series studies of HAB species using traditional microscopy, whole cell probing and cell-free assays, and are now applying the same techniques for studies of picoplankton and invertebrates. For most applications, cell-free analytical methods clearly offer greater analytical throughput and the potential for detecting many more molecular signatures in a single sample simultaneously than techniques based on intact cells. Nevertheless, the whole cell analyses have proved invaluable for evaluating the performance of cell-free assays, and thus remain a centerpiece of our research (e.g., Anderson et al. in review, Lundholm et al. in revision, Miller and Scholin 2000). We have demonstrated how detection of species-specific (HAB) rRNA sequences in the context of recognizable, intact cells does not always equate with detection of the same signature sequences found in sample homogenates (e.g., Anderson et al. and O’Halleron et al. submitted, Tyrrell et al 2002, Scholin et al. 1999). These techniques reveal how organisms’ genetic signatures, as well substances they produce (like toxins), may be transferred through the food web in the absence of recognizable cells indicative of the target species.
At present the majority of our work is focused on developing DNA probe arrays to detect multiple targets in a single sample simultaneously. Results leading up to the present demonstrate that species ranging from marine bacteria to phytoplankton to invertebrate larvae can be detected and in some cases enumerated in near real-time using a common sample collection, preparation and processing protocol that can run on relatively little electrical power. The reagents employed in these assays appear to be stable for extended periods (none used in the ESP require refrigeration), and the chemical reactions themselves are amenable to microfluidic scaling. Different arrays are tailored to detection of specific groups of organisms such as ‘planktonic microbes’, ‘harmful algae,’ or ‘invertebrate larvae,’ etc. (Figure 1).
Working with a company called Orca Research (Seattle, WA), complete, prepackaged tests for harmful algae as well as bulk reagents for the sandwich hybridization assay made to our specification can be purchased for research purposes, making it possible for other groups to use and evaluate assays we have published and patented. Our protocols are widely distributed and utilized by workers outside of MBARI, especially those at University of California at Santa Cruz, Woods Hole Oceanographic Institution, NOAA NOS/NMFS (Seattle, WA and Charleston, SC), Florida Marine Research Institute (St. Petersburg, FL), University of Miami, the Cawthron Institute in New Zealand, and several other labs in Europe. Beyond basic lab research, we have also successfully employed the tests shipboard to achieve near real-time mapping of HAB species in California waters, as well as in the Gulf of Maine and Gulf of Mexico (~10 different species in total). More recently we used this approach for detecting groups of marine bacteria in the Juan de Fuca study area (Preston et al., unpublished data).