Pseudo-nitzschia multiseries (Hasle) Hasle
Pseudo-nitzschia multiseries is a domoic-acid-producing, pennate diatom responsible for Amnesic Shellfish Poisoning in Atlantic Canada.
The distinctive morphological character of P. multiseries is the "multiseriate" striae of the valve face (Hasle et al. 1996).
Pseudo-nitzschia multiseries is a major domoic acid producer on the west coast of North America and has caused shellfish contamination in Atlantic Canada. Thus, P. multiseries is of particular interest to coastal resource managers. Since not all Pseudo-nitzschia produce domoic acid, monitoring of this genus presents the dual challenge of identifying the species and detecting the toxin, using microscopy and various bioassays or chromatography methods. Neither can give real-time data from the field. Faster molecular methods for species identification have been developed and are continually improved. Antibodies have been developed and used for identification of P. multiseries. Whole cell fluorescent LSU targeted probes have been developed for identifying P. multiseries cultures from the west coast of the United States (Miller & Scholin 1996). These probes were used to develop whole cell and sandwich hybridization methods for detection of P. multiseries in natural samples at near real time (Scholin et al. 1997, Miller & Scholin 1998). Detecting domoic acid in the field is more of a challenge. The relatively recent development of ELISA methods for toxin detection decrease limits of detection and increase speed, but are costly and do not report real-time. Some managers use indicator species which concentrate the toxin in their tissues, thus allowing for a less expensive analysis with a higher limit of detection. Mussel bags are most commonly used for this purpose. The possibility of using sand crabs (Emerita analoga) as an indicator species in Monterey Bay has also been explored (Ferdin et al. 2002).
Pseudo-nitzschia multiseries cells have been found to fluoresce blue when excited by UV light, a response typical of some proteins.
Whole genome sequencing of P. multiseries is currently under way.
The SSU of P. pungens is very similar to P. multiseries, but the ITS sequences are much more variable (Manhart et al. 1995). The 5.8S sequences are identical. Restriction fragment length patterns for 13 isolates of P. multiseries and 16 isolates of P. pungens revealed differences between but not within the two species (Manhart et al. 1995).
Microsatellite markers have been developed for P. multiseries and have been used to genotype field isolates and their descendants (Evans et al. 2004). In this study, the number of alleles per locus ranged from 3 to 7 and the heterozygosities ranged from 0.39 to 0.70. Twenty three different genotypes were detected among 25 field isolates, suggesting high genetic variation in natural populations. Mating experiments confirmed that alleles were inherited according to Mendelian ratios.
One of the most well-studied aspects of P. multiseries physiology is the production of domoic acid. P. multiseries can produce domoic acid under constant light (Villac et al. 1993a). Axenic cultures of P. multiseries can produce domoic acid but at much lower levels than non-axenic cultures. Bacteria play an important role in the domoic acid content of P. multiseries cultures and could explain differences in toxicity between laboratory strains (Kaczmarska et al. 2005). Growth rates of laboratory cultures decrease at ammonium concentrations above 200 micromolar (Hillebrand and Sommer 1996). P. multiseries can use urea, glutamine and nitrite for growth in culture (Hillebrand and Sommer 1996). The optimal salinity range for growth of Canadian estuarine isolates was 30-45 (Jackson et al. 1992). These cultures also produced more of the amino acid taurine at higher salinities (Jackson et al. 1992). The upper pH limit for growth of a Canadian isolate was 8.8-8.9, which also had a maximum specific growth rate of 1.01 ± 0.02 per day in the laboratory (100 micromolar photons m-2s-1 and 15 deg C; Lundholm et al. 2004). A Japanese isolate had a maximum specific growth rate of 1.01 ± 0.05 per day and an upper pH limit for growth of 9.0 in the laboratory (100 micromolar photons m-2s-2 and 15 deg C; Lundholm et al. 2004). A Canadian isolate had a specific growth rate of 0.25 per day at 5 deg C, 0.65 per day at 20 deg C and 0.60 per day at 25 deg C (Lewis et al. 1993). The highest stationary phase cell concentrations occurred at 5 - 15 deg C. Domoic acid production was 0.01 pg domoic acid per cell at 5 deg C and 0.51 pg domoic acid per cell at 25 deg C. Changing irradiance from 80 to 180 microEinsteins m-2s-1 did not change growth in this isolate. Growth of Monterey Bay, CA isolates was inhibited by Fe limitation and Cu stress(Maldonado et al. 2002). Domoic acid was released under metal stress conditions. Addition of dissolved domoic acid increased Fe uptake threefold and partially relieved Cu toxic conditions.
Pseudo-nitzschia multiseries is part of the microplankton. Individual cells can be seen with a standard light microscope and can be up to 100 microns in length. Pseudo-nitzschia cells typically form chains of up to 20 cells long that can be seen with the naked eye. In the laboratory, the cell size of a clonal strain will gradually decreases over time due to the nature of diatom cell division. In the field, cell size is restored through sexual reproduction. The average cell volume of a Japanese P. multiseries laboratory isolate was 1122 ± 60 cubic microns (Lundholm et al. 2004).
Pseudo-nitzschia multiseries is found in coastal temperate regions world wide. It is commonly abundant in the autumn and winter.
Many Pseudo-nitzschia blooms occur in the spring and fall, when irradiance is relatively low (Parsons et al. 1998, Mercado et al. 2005). In culture, P. multiseries can out compete other phytoplankton species at low irradiance with a short photoperiod (Sommer 1994). Parasitic oomycetes and chytrids have infected P. multiseries in eastern Prince Edward Island, Canada. Toxic P. multiseries is resistant to the effects of UV light while non-toxic (Hargraves et al. 1993). Experiments using a Japanese and a Canadian strain of P. multiseries found that domoic acid did not have an allelopathic effect on 9 species of phytoplankton (Lundholm et al. 2005).
P. multiseries has been recorded primarily from meso- to polyhaline coastal waters in the temperate zone during the spring and autumn.
Pseudo-nitzschia multiseries reproduces asexually through cell division and sexually (Bates & Davidovich 2002). Since cell length decreases with every division, cells become sexualized when cell length enters a specific range. In P. multiseries the minimum length is 30 microns. Axenic clones of P. multiseries would not undergo sexual reproduction until bacteria were reintroduced (Thompson 2000). Offspring of P. multiseries clones that lose their ability to produce DA in the laboratory can be toxic (Bates et al. 1999). A positive correlation was found between hours of light exposure per 24 hours at approximately 100 micromolar photons per square meter per second and sexual cell production (Hiltz et al. 2000). The greatest production of initial cells per female gamete (26%) occurred in 10 hours of light.
Pseudo-nitzschia was not recognized as a toxic diatom until the first documented incident of ASP occurred in Prince Edward Island, Canada in 1987 when residents ate DA contaminated mussels (Mytilus edulis) from Cardigan Bay estuaries (Bates et al. 1989, Wright et al. 1989). Out of 250 reported illnesses, 107 met the case definition for ASP (Perl et al. 1990). Common symptoms were vomiting, abdominal cramps, diarrhea, incapacitating headache and loss of short-term memory (Perl et al. 1990). Nineteen people were hospitalized, twelve requiring intensive care because of seizures, coma, severe lung congestion, and unstable blood pressure (Perl et al. 1990). Some of the twelve intensive care patients showed additional serious neurological problems including inability to speak, irritability and uncontrollable facial movements (Perl et al. 1990). Four people died, three in the hospital and one three months after apparent recovery (Perl et al. 1990, Teitelbaum et al. 1990). Brain tissue from three of the four dead patients revealed severe cell damage, especially in the hippocampus and amygdala (Perl et al. 1990, Teitelbaum et al. 1990). Of those patients that lived, the more severely affected experienced memory deficits as much as five years after DA consumption (Todd 1993). One patient who suffered short term memory loss also developed epilepsy one year after exposure (Cendes et al. 1995). The causative organism was found to be P. multiseries, which was blooming in Cardigan Bay at the time of the outbreak (November to December) and declined shortly thereafter (Subba Rao et al. 1988, Bates et al. 1989). The identity of the toxin as DA was confirmed by proton nuclear magnetic resonance spectra in mussel tissue, cultured P. multiseries and plankton samples from Cardigan Bay (Bates et al. 1989, Wright et al. 1989). A positive correlation was found between the concentrations of P. multiseries and DA in plankton samples (Bates et al. 1989). No DA was found in cultures of other diatom species, 10 isolated from Cardigan Bay and 12 obtained from a culture collection. Small amounts of DA were found in a local macroalga, Chondria baileyana (Bates et al. 1989). Prior to the 1987 ASP event, DA had not been detected in shellfish (Wright et al. 1989). Mussels from Cardigan Bay and patients’ uneaten mussels were initially tested for PSP toxins using the mouse bioassay; however, the test mice exhibited involuntary scratching of their shoulders with their hind legs, a symptom atypical of PSP (Perl et al. 1990). Mussels were also tested for dangerous bacteria, viruses and chemical residues; none were found. Metabolites, including DA, were extracted from whole mussels and DA was identified using HPLC, high-voltage paper electrophoresis, ion-exchange chromatography and ultraviolet, infrared and mass spectroscopy (Wright et al. 1989). Dissected mussels contained the most toxin in the digestive gland (Wright et al. 1989). The Pacific oyster (Crassostrea gigas) had an immune response to toxic P. multiseries and experienced respiratory acidosis from shell closure (Jones et al. 1995a,b). A toxic culture of P. multiseries had no allelopathic effects on Chrysochromulina ericina, Heterocapsa triquetra, Eutreptiella gymnastica and Rhodomonas marina (Lundholm et al. 2005).