Chris Scholin

INTER- AND INTRASPECIFIC VARIATION OF THE PSEUDO-NITZSCHIA DELICATISSIMA COMPLEX (BACILLARIOPHYCEAE) ILLUSTRATED BY RRNA PROBES, MORPHOLOGICAL DATA AND PHYLOGENETIC ANALYSES1

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.

Pseudo-nitzschia in New Zealand and the role of DNA probes and immunoassays in refining marine biotoxin monitoring programmes

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.

Monitoring for Heterosigma akashiwo using a sandwich hybridization assay

Abstract 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 −70xa0°C or, alternatively, the filters are mixed with a lysis solution buffer and the sample lysate stored at −70xa0°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.

International accreditation of sandwich hybridisation assay format DNA probes for micro‐algae

Abstract The sandwich hybridisation assay (SHA) is a DNA probe‐based method for rapid identification 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 identification 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 Zealand: ISO 17025). During the validation of the raphidophyte assay, some discrepancies were noted between SHA cell concentration estimates and traditional 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 format DNA probe counts and traditional microscope counts; SHA counts were comparable for the three methods tested.

Pseudo‐nitzschia multistriata (Bacillariophyceae) in New Zealand

Abstract 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.

The Environmental Sample Processor (ESP) - An Autonomous Robotic Device for Detecting Microorganisms Remotely using Molecular Probe Technology

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

Controlling a Robotic Marine Environmental Sampler with the Ruby Scripting Language

The Environmental Sample Processor (ESP) is an autonomous robotic instrument developed at the Monterey Bay Research Aquarium Institute (MBARI) that operates below the oceans 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.

The founding charter of the Genomic Observatories Network

The co-authors of this paper hereby state their intention to work together to launch the Genomic Observatories Network (GOs Network) for which this document will serve as its Founding Charter. We define a Genomic Observatory as an ecosystem and/or site subject to long-term scientific research, including (but not limited to) the sustained study of genomic biodiversity from single-celled microbes to multicellular organisms.An international group of 64 scientists first published the call for a global network of Genomic Observatories in January 2012. The vision for such a network was expanded in a subsequent paper and developed over a series of meetings in Bremen (Germany), Shenzhen (China), Moorea (French Polynesia), Oxford (UK), Pacific Grove (California, USA), Washington (DC, USA), and London (UK). While this community-building process continues, here we express our mutual intent to establish the GOs Network formally, and to describe our shared vision for its future. The views expressed here are ours alone as individual scientists, and do not necessarily represent those of the institutions with which we are affiliated.