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A 3-week diversion of the Orange County Sanitation District effluent discharge into nearshore waters off Newport Beach, CA constituted a considerable injection of secondarily-treated effluent into the coastal ecosystem. The location ≈1.6 km from shore, shallow water depth (≈16 m), volume and nutrient content of the discharge (≈5.3 × 10 8 L day −1 of effluent with inorganic nitrogen concentration >2 mM) during the diversion raised concerns regarding the potential for stimulating phytoplankton blooms and, in particular, blooms of toxic species. Remarkably, phytoplankton standing stocks during the event and shortly thereafter did not reach values associated even with minor blooms historically observed in the region (generally −1 ), although shifts in community composition were observed. Diatom abundances increased early during the diversion, dinoflagellates, phototrophic picoplanktonic eukaryotes and other algae increased mid-diversion, and cyanobacteria ( Synechococcus , Prochlorococcus ) increased near the end of the diversion. Concentrations of domoic acid (a phycotoxin commonly present in the area) remained near or below detection throughout the diversion, and abundances of potentially-harmful algal species were unresponsive. Bacterial biomass increased during the diversion, and equaled or exceeded total phytoplankton biomass in most samples. Abundances of microbial grazers were also elevated during the diversion. We speculate that nutrient uptake by the bacterial biomass, acting in concert with or a response to a negative effect of disinfection byproducts associated with chlorination on phytoplankton physiology, played a significant role in muting the response of the phytoplankton to nutrients released in the effluent.

In this paper, a molecular analytical approach for detecting hydrocarbonoclastic bacteria in water is suggested as a proxy measurement for tracking petroleum discharges in industrialized or pristine aquatic environments. This approach is tested for general application in cold marine regions (freezing to 5 °C). We used amplicon sequencing and qPCR to quantify 16S rRNA and GyrB genes from oleophilic bacteria in seawater samples from two different crude oil enrichments. The first experiment was conducted in a controlled environment using laboratory conditions and natural North Sea fjord seawater (NSC) at a constant temperature of 5 °C. The second was performed in the field with natural Arctic seawater (ARC) and outdoor temperature conditions from −7 °C to around 4 °C. Although the experimental conditions for NSC and ARC differed, the temporal changes in bacterial communities were comparable and reflected oil biotransformation processes. The common bacterial OTUs for NSC and ARC had the highest identity to Colwellia rossensis and Oleispira antarctica rRNA sequences and were enriched within a few days in both conditions. Other typical oil degrading bacteria such as Alcanivorax (n-alkane degrader) and Cycloclasticus (polycyclic aromatic hydrocarbons degrader) were rapidly enriched only in NSC conditions. Both the strong correlation between Oleispira SSU gene copies and oil concentration, and the specificity of the Oleispira assay suggest that this organism is a robust bioindicator for seawater contaminated by petroleum in cold water environments. Further optimization for automation of the Oleispira assay for in situ analysis with a genosensing device is underway. The assay for Colwellia quantification requires more specificity to fewer Colwellia OTUs and a well-established dose-response relationship before those taxa are used for oil tracking purposes.

An observational study was performed in the central Southern California Bight in Spring 2010 to understand the relationship between seasonal spring phytoplankton blooms and coastal processes that included nutrient input from upwelling, wastewater effluent plumes, and other processes. Multi-month Webb Slocum glider deployments combined with MBARI environmental sample processors (ESPs), weekly pier sampling, and ocean color data provided a multidimensional characterization of the development and evolution of harmful algal blooms (HABs). Results from the glider and ESP observations demonstrated that blooms of toxic Pseudo-nitzschia sp. can develop offshore and subsurface prior to their manifestation in the surface layer and/or near the coast. A significant outbreak and surface manifestation of the blooms coincided with periods of upwelling, or other processes that caused shallowing of the pycnocline and subsurface chlorophyll maximum. Our results indicate that subsurface populations can be an important source for “seeding” surface Pseudo-nitzschia HAB events in southern California.

Recent advances in robotic technologies provide new opportunities to conduct high-resolution sampling of patchily distributed zooplankton and associated environmental variables. We used two robots and molecular probes to assess the temporal and spatial variability of zooplankton in water samples obtained from Monterey Bay, California. The Autonomous Underwater Vehicle (AUV) Dorado is a mobile platform that carries ten, 1.8-L bottles ("Gulpers") capable of rapidly acquiring discrete seawater samples, and an extensive sensor suite for gathering contextual environmental data during day-long expeditions. Molecular assays were conducted ex situ at a shore-based laboratory. In contrast, the Environmental Sample Processor (ESP) was deployed as a stationary (moored) device capable of repeatedly “sipping” water to conduct in situ molecular assays and record environmental data during month-long deployments. Molecular analyses were conducted with the sandwich hybridization assay (SHA), which employed 18S ribosomal RNA oligonucleotide probes designed to detect calanoid and podoplean copepods, and the larvae of barnacles, mussels, polychaete worms, brachyuran crabs, and invasive green crabs (Carcinus maenas). Both the stationary and mobile sampling protocols revealed the greatest zooplankton diversity and abundance in relatively warm waters, higher in chlorophyll and lower in salinity and nitrate. Diversity and abundance were least in recently upwelled waters with the inverse conditions. High-resolution sampling revealed that while calanoid copepods were generally associated with elevated chlorophyll, they were most abundant in upwelling fronts, in some cases. These narrow features appear to provide favorable conditions for the growth and aggregation of certain zooplankton.

Summary: ? Marine microbes drive chemical reactions that contribute to Earth's habitability. ? Although molecular techniques are used to monitor oceanic microbial communities, collecting intermittent samples for laboratory analysis limits the usefulness of this approach. ? Instruments that collect and analyze microbes in situ can provide unprecedented resolution of biogeochemical cycles over large spatial and temporal scales. ? We should combine knowledge of key genes with new instruments to obtain time-series information on key marine microbial biogeochemical transformations. The oceans maintain critical ecosystems that, ultimately, determine the habitability of the planet. Moreover, unicellular eukaryotes, bacteria, and archaea are the most abundant organisms within those ecosystems and thus the dominant biomass within the oceans, situated at the base of the food chain that feeds macroscopic invertebrates, fish, and mammals. We are only beginning to uncover the extent of marine microbial genetic diversity and the complex roles that these microbes play in driving the biogeochemistry of global ocean ecosystems. Molecular biology, genomics, microscopy, mass spectrometry, and high-throughput cultivation are already providing useful information on the diversity and activities of marine microflora, including new biochemical pathways. However, in the face of global environmental change, we need to gain a deeper understanding of the distributions of marine microbes and their activities in maintaining ecosystem functions.

We assess the application of the second-generation Environmental Sample Processor (ESP) for the detection of harmful algal bloom (HAB) species in field and laboratory settings using two molecular probe techniques: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). During spring 2006, the first time this new instrument was deployed, the ESP successfully automated application of DNA probe arrays for various HAB species and other planktonic taxa, but non-specific background binding on the SHA probe array support made results interpretation problematic. Following 2006, the DNA array support membrane that we were using was replaced with a different membrane, and the SHA chemistry was adjusted. The sensitivity and dynamic range of these modifications were assessed using 96-well plate and ESP array SHA formats for several HAB species found commonly in Monterey Bay over a range of concentrations; responses were significantly correlated (p < 0.01). Modified arrays were deployed in 2007. Compared to 2006, probe arrays showed improved signal:noise, and remote detection of various HAB species was demonstrated. We confirmed that the ESP and affiliated assays can detect HAB populations at levels below those posing human health concerns, and results can be related to prevailing environmental conditions in near real-time.

The Environmental Sample Processor (ESP) is an autonomous robotic instrument developed at the Monterey Bay Research Aquarium Institute (MBARI) that operates below the ocean's surface, sampling raw seawater and executing a variety of sample manipulation and analytical protocols, in situ. It uses DNA and antibody probes to identify marine planktonic organisms and substances they produce. Initial prototypes of the ESP were hosted on an Intel i486 CPU running a commercial real-time operating system (OS). The application, coded in C++, included a custom ‘macro’ language interpreter to direct biochemical analyses. To achieve greater flexibility and minimize the development effort for the 2nd generation of the ESP (2G ESP), MBARI replaced its ‘macro’ language with a general purpose, open-source scripting language, selecting Ruby for its unique combination of a succinct, English-like syntax with a seamless underlying object-oriented paradigm. The 2G ESP application, aside from custom servo control firmware, is coded entirely in Ruby, hosted on a low-power ARM9 CPU running Linux. Servo control was distributed onto a network of dedicated microcontrollers to cope with the nondeterministic delays inherent in the Linux operating system and Ruby interpreter.

A study of 25 cultures tentatively identified as Pseudo-nitzschia delicatissima (Cleve) Heiden, and originating from geographically widely distributed locations, showed both morphological and genetic variation among strains. Use of rRNA-targeted DNA probes on 17 different strains showed large variation in the hybridization patterns. Detailed morphological studies placed the isolates into three groups. The sample on which the neotype of P. delicatissima is based was also examined, and used to establish the morphological identity of P. delicatissima. Phylogenetic analyses of 16 strains, based on sequences of internal transcriber spacer 1 (ITS1), 5.8S and ITS2 of the nuclear-encoded rDNA, supported the morphological observations and the hybridization studies, and revealed large genetic variation among strains. A combination of the morphological and molecular findings resulted in the description of two new species, P. decipiens sp. nov. and P. dolorosa sp. nov. P. dolorosa has a mixture of one or two rows of poroids in the striae whereas P. delicatissima always has two rows. In addition, P. dolorosa has wider valves and a lower density of poroids. P. decipiens differs from P. delicatissima by a higher density of striae on the valve face as well as a higher density of poroids on the girdle bands. Among the strains referred to P. delicatissima, an epitype was selected. Large genetic variation was found among the P. delicatissima strains and a subdivision into two major clades represent cryptic species.

The sandwich hybridisation assay (SHA) is a DNA probe-based method for rapid identifica- tion and enumeration of toxic micro-algae which uses species specific oligonucleotide probes targeted at ribosomal RNA. It is suited to fragile micro-algal cells which commonly collapse during the fixation stage of sample collection, compromising identifi- cation by traditional microscopy. The assay has been available for research for several years, but was validated and accepted for international accreditation for commercial laboratory use in New Zealand in May 2004 (International Accreditation New Zea- land: ISO 17025). During the validation of the raphidophyte assay, some discrepancies were noted between SHA cell concentration estimates and tra- ditional light microscope cell counts. Higher SHA estimates were recorded when blooms had collapsed but rRNA was still present in sea water. Conversely, higher traditional cell counts occurred when sample delivery was delayed more than 48 h, presumably owing to degradation of rRNA in the live cultures used for the SHA. SHA cell concentration estimates of the toxic diatom bloom-former Pseudo-nitzschia australis were also compared with whole cell for- mat DNA probe counts and traditional microscope counts; SHA counts were comparable for the three methods tested.

Field testing of a ribosomal RNA (rRNA)-targeted sandwich hybridization assay (SHA) for Heterosigma akashiwo (Raphidophyceae) in Puget Sound, WA, USA, has showed that the lower limit of detection is well below the level at which cells pose a danger to fish. Moreover, the assay has proven to be both rapid and easy-to-use. Isolates of H. akashiwo from Australia, Japan, New Zealand, South Korea, Spain and USA were correctly identified using the SHA, indicating that this diagnostic tool could be deployed globally. Samples containing H. akashiwo can be preserved for subsequent SHA analysis using several methods: fixation with acidic Lugol’s iodine followed by room temperature storage, collection onto Durapore filters followed by storage at −70 °C or, alternatively, the filters are mixed with a lysis solution buffer and the sample lysate stored at −70 °C. Additionally, we sought to determine whether the SHA could successfully detect H. akashiwo in the presence of clay that might some day be used to mitigate the impacts of natural H. akashiwo blooms. Results from preliminary laboratory trials indicate that clay at the maximum proposed dosage rate does not interfere with the assay. Thus, it may be possible to use the SHA as a simple means of following the fate of H. akashiwo cells during larger-scale clay mitigation trials.

Pseudo‐nitzschia multistriata (Takano) Takano has been observed in seawater samples from the North Island and from the northern waters of the South Island, New Zealand, with blooms of >1.0 × 106 cells litre‐1 being recorded in December 1997 (austral summer), and minor blooms occurring in both summer and winter 1998 and 1999. The species is notable for its sigmoid form, and for its cross‐reactivity with oligonucleotide probes targeted at P. australis Frenguelli ribosomal RNA. The species is non‐toxic, and for biotoxin monitoring purposes needs to be discriminated from the highly toxic P. australis. It falls in the “delicatissima” complex of Pseudo‐nitzschia H. Peragallo species, and electron microscopy shows a close resemblance to P. delicatissima (Cleve) Heidin, except for the lack of a central larger interspace.

Domoic acid (DA) was first detected in shellfish in New Zealand after the implementation of a comprehensive biotoxin monitoring programme for amnesic, paralytic, diarrhetic and neurotoxic shellfish toxins, following a suspected neurotoxic shellfish poisoning (NSP) event in early 1993. Both phytoplankton monitoring and shellfish flesh testing programmes have led to an extensive database which has helped link species of Pseudo-nitzschia to specific DA outbreaks. In 1994, P. pungens and P. turgidula were associated with DA contamination of shellfish, and cultured isolates of these species proved to be toxin producers. During 1996 the use of species-specific ribosomal RNA (rRNA)-targeted oligonucleotide probes and DA immunoassays led to the discovery of toxin production by P. fraudulenta, and showed the nontoxic P. heimii to be a major bloom former. Pseudo-nitzschia delicatissima, P. pseudodelicatissima and P. multiseries, also identified using rRNA-targeted probes, have been linked to DA contamination of New Zealand shellfish; P. australis is the main cause of DA in scallops. The relative amnesic shellfish poisoning (ASP) risk associated with different species, largely determined by DA immunoassays of cultured isolates, is now used by some regulators to refine risk assessments. Species identification is therefore vital so that shellfish growers, and health and industry officials, can make safe and economically sound harvesting decisions. The development and field trialling of DNA probes is proving invaluable in this context. Copyright © 1999 John Wiley & Sons, Ltd.

We are developing an instrument to conduct molecular biological analyses below the ocean surface, autonomously. The device is known as the Environmental Sample Processor, or ESP. The system is based on a modular design consisting of a core sample processor (the ESP), analytical modules and sampling modules. The core ESP provides the primary interface between the environment and a set of DNA and antibody-based tests that are carried out onboard the instrument in real-time. In addition, the ESP can be used to archive samples for a variety of analyses after the instrument is returned to a laboratory. Sampling modules are devices external to the core ESP that can be added to meet specialized needs, such as operating in the deep-sea (etc). Analytical modules are conceived of as stand-alone devices that can be added to the core ESP to impart different suites of analytical functions downstream of common sample processing operations. At the time of this writing we have worked most extensively on the core ESP and detection chemistries that involve DNA probe and protein arrays. The ESP has been deployed successfully in coastal ocean surface waters. We are also developing a sample collection module and pressure housing suitable for deploying the ESP at depths to 1000m. This version of the instrument is known as the deep-sea ESP, or D-ESP. The long-term objective of the D-ESP program is to provide a molecular analytical capability at deep-sea hot vents and cold seeps. The D-ESP will be deployed using an ROV and later transitioned to benthic moorings and a cabled observatory. Finally, we are just starting work to incorporate a microfluidic analytical module to support assays that require DNA purification and amplification