Due to its variable toxicity and cosmopolitan distribution, Pseudo-nitzschia poses a unique management challenge worldwide. An effective monitoring program must include Pseudo-nitzschia identification and enumeration, domoic acid quantification and testing of potential seafood vectors. Since the presence of Pseudo-nitzschia does not guarantee the presence of domoic acid, abundance data alone are rarely a sufficient basis for management decisions. Instructions on how to monitor for Pseudo-nitzschia spp. and domoic acid can be found in the Manual on Harmful Marine Microalgae, edited by G. M. Hallegraeff, D. M. Anderson and A. D. Cembella. Traditional plankton sampling techniques are typically useful for Pseudo-nitzschia monitoring; however, there are situations when this sampling method may be inadequate. Physical and biological processes are known to concentrate Pseudo-nitzschia into subsurface layers from several meters (Ryan et al. 2005) to less than a meter thick (Rines et al. 2002). These cells can be transported long distances and inoculate unexpected blooms at the surface (Bates & Trainer 2006). Pseudo-nitzschia can also be missed when intermingled in colonies of Chaetoceros socialis (Rines et al. 2002) instead of free in the water column. A laser-scattering sensor can detect a patch of P. australis in Monterey Bay, CA, but the ability of the sensor to distinguish between pennate diatom species is limited (Rienecker et al. 2008). Oceanographic characteristics of a region should be taken into account when developing a sampling program so that any heterogenous distributions of Pseudo-nitzschia in the water column can be defined. Several days may pass from the time a water sample is collected to identification of Pseudo-nitzschia species via electron microscopy (generally not available to most routine monitoring programs). Molecular probes have come into use to increase speed of identification and resolution between genetically distinct strains. Molecular probes are designed to adhere to a specific set of molecules associated with a particular algal group and can be used as a basis for detecting these groups in natural samples. There are three types of molecular probes: lectin-binding, antibodies or nucleic acid. Lectin-binding probes have been used to discriminate between species of Pseudo-nitzschia with limited success. Trials with New Zealand derived cultures were able to discriminate between 6 of 7 Pseudo-nitzschia species tested; P. delicatissima and P. pseudodelicatissima could not be distinguished (Rhodes 1998a). All 7 species tested from Spain and all 3 species tested from Korea were successfully identified using lectin binding patterns, but there were differences among strains of the same species (Fraga et al. 1998, Cho et al. 1999). Lectin binding patterns can differ between strains of the same species depending on origin and possibly physiological condition and therefore are not broadly useful as a monitoring tool. Antibodies can be designed to bind to molecules (antigens) like glycoproteins, peptides, nitrogen-containing carbohydrates or toxins. Antibodies have been developed and used for identification of P. pungens and P. multiseries, but immunoassays are much more commonly used to detect DA in shellfish and water samples (Bates et al. 1993b, Vrieling et al. 1996, Rhodes et al. 1998a). DNA probes for detecting species have been targeted at small subunit (SSU), large subunit (LSU) and internal transcribed spacer (ITS) regions of rRNA. There are several types of rRNA targeted probes: whole-cell hybridization (labeling of intact cells), fluorescent in-situ hybridization (FISH), sandwich hybridization (measuring DNA in cell homogenate) and PCR methods (PCR replication of targeted genome). New Zealand is the first country to use molecular probes for routine harmful algal bloom (HAB) monitoring and risk assessment (Rhodes et al. 1998a). Design of these probes depends on obtaining rRNA sequences with desired specificity. This can be a challenge for Pseudo-nitzschia, a group with high intra- and interspecific variability. Whole cell fluorescent LSU targeted probes have been developed for identifying P. australis, P. pungens, P. multiseries, P. heimii, P. fraudulenta, P. delicatissima, P. pseudodelicatissima and P. americana in cultures originating from the west coast of the United States (Miller & Scholin 1996). These probes were then used to develop whole cell and sandwich hybridization methods for detection of species in natural samples at near real-time (Scholin et al. 1997, Miller & Scholin 1998). Probes for P. multiseries, P. pungens, P. fraudulenta and P. delicatissima have been used successfully in the Gulf of Mexico, US west coast, the Wadden Sea and Korean waters (Miller & Scholin 1996, Vrieling et al. 1996, Miller & Scholin 1998, Parsons et al. 1999, Cho et al. 2002). However, not all rRNA probes work in all regions. The probe designed to work on P. pseudodelicatissima from the west coast of the United States did not work on P. pseudodelicatissima in samples from the Gulf of Mexico (Parsons et al. 1999). This could be because of high genetic variability within the P. pseudodelicatissima group, which was later divided into additional species based on morphological and molecular analysis (Lundholm et al. 2003). Designing Pseudo-nitzschia species-specific probes in some regions, like the Chesapeake Bay, are particularly challenging because of high variability within morphologically defined species (H. Bowers pers. comm). In some regions, like the Gulf of Naples, genus-specific probes based on clone libraries have been used to successfully separate species, with multiple probes used to identify P. delicatissima and P. pseudodelicatissima, two morphologically defined species that probably include cryptic species (McDonald et al. 2007). RNA-based detection methods are particularly useful when strain differences and cryptic species are important. After determining the abundance and species of Pseudo-nitzschia present, further management decisions can be made. Some countries use “trigger levels” of abundance to initiate shellfish sampling. In New Zealand, when Pseudo-nitzschia is > 50% of total phytoplankton, a concentration of 5 X 104 cells L-1 will trigger shellfish sampling. When Pseudo-nitzschia is < 50% of total phytoplankton, a concentration of 105 cell L-1 will trigger shellfish sampling. DA is quantified by HPLC and UV detection. If shellfish contain more than 20 mg kg-1 DA, harvesting is stopped until three consecutive samples spaced out over at least 2 weeks have < 20 mg DA kg-1 (Todd 2003). Countries with limits for DA in shellfish are Canada, USA, New Zealand, Chile, Peru and EU member states. The limit is 20 mg kg-1 edible meat. A limit of 30 mg DA kg-1 in cooked viscera of Dungeness crab has been set by the FDA in the United States. In order to minimize economic impact of DA contamination, the EU allows harvest of scallops with whole body burdens of DA between 20 and 250 mg kg-1 if they are sold after total removal of the hepatopancreas (Fernandez et al. 2003).

Author(s): Thessen, Anne

• Comment (0)

There are many methods of detection of DA, which are described in detail in Quilliam (2003). The current global standard for detection of many algal toxins in shellfish is the mouse bioassay (Fernandez et al. 2003, Todd 2003). This involves preparing an extract of the tissue to be tested and injecting it into a test mouse. If the mouse exhibits symptoms, a toxin is considered present. However, the limit of detection of the mouse bioassay for ASP toxin is 40 mg DA kg-1 shellfish, above the regulatory limit of 20 mg DA kg-1 shellfish. High Performance Liquid Chromatography (HPLC) with UV detection is the most commonly used chemical analytical method for DA detection due to its simplicity, speed and reproducibility. However, while the limit of detection is low enough to be used for routine monitoring of shellfish, it is not low enough for work with seawater and plankton. Thus, HPLC methods based on derivatization of fluorescence reagents were developed (Pocklington et al. 1990). Other chromatographic-based assays for domoic acid include thin-layer chromatography (TLC), capillary electrophoresis (CE) and liquid chromatography with detection by mass spectroscopy (LC-MS). When establishing the presence of DA in a new area or in a new species, comparing chromatographic peaks to a DA standard is not conclusive. Spectroscopic data such as ultra-violet or mass spectra are necessary to confirm identification. Receptor based assays and immunoassays are relatively new. Receptor based assays utilize the specificity of a toxin for a particular action site and measure activity of the toxin rather than discrete structural components. The receptor binding assay for domoic acid involves measuring the competition between a known amount of labeled kainic acid and domoic acid in the sample to be tested for glutamate receptors in brain tissue (VanDolah et al. 1997). The newest detection method is an immunoassay or ELISA, which is based on competition of domoic acid in the sample with a DA-conjugated protein for anti-DA antibodies (Garthwaite et al. 1998). The Jellet Rapid Test is a rapid immunodiagnostic test that has been successfully used to detect domoic acid in Scottish waters (Turrell et al. 2008). Receptor-based assays and immunoassays have the advantage of giving low limits of detection, but can be costly. In many early studies on domoic acid and Pseudo-nitzschia, samples were taken quickly and stored frozen until analysis within two weeks because of potential DA degradation (e.g., Bates et al. 1991). Light, temperature and pH were all factors believed to affect domoic acid stability. Over time, studies have shown that domoic acid is not as unstable as first thought. Ambient temperatures, artificial light and repeated chilling and warming do not degrade domoic acid in saline solution (Johannessen 2000); however, domoic acid does degrade under acidic conditions (Quilliam et al. 1989).

Author(s): Thessen, Anne

• Comment (0)

Timing and frequency of sexual reproduction in Pseudo-nitzschia has important implications for the genetic structure of populations. Sexual reproduction should be an important source of genetic variability. Direct evidence of sexual reproduction has been documented in natural populations on the west coast of the United States (Holtermann et al. 2010) and cell size has been found to abruptly increase seasonally (D'Alelio et al. 2006). Molecular methods of examining diversity should be applied to a larger investigation of Pseudo-nitzschia biogeography in order to describe global population dynamics and genetic relatedness between populations in different regions.

Author(s): Thessen, Anne

• Comment (0)

A heavily studied aspect of Pseudo-nitzschia physiology is domoic acid (DA) production. It is commonly known that growth phase, nutrients, temperature, irradiance and bacteria play a role in DA production. A good review of this is Bates (1998). Many studies show Pseudo-nitzschia cultures produce little DA until cell division has stopped. In batch cultures, DA production often starts at the onset of stationary phase and DA content of the cells peaks about one week later. Some cultures produce DA during late exponential phase, possibly because this is a period of transition when some cells have stopped growing and are producing DA while other cells are still dividing. However, one culture of P. cf. pseudodelicatissima from the Northern Gulf of Mexico produced the highest levels of DA during early exponential growth (Pan et al. 2001). In continuous culture, toxin content increases when growth is slowed by decreasing the dilution rate (Pan et al. 1996). This growth effect means that many factors that slow growth would also indirectly increase toxin production. Studies showing that an increased pH will increase toxin production also show that growth rate decreases under these circumstances, making it difficult to know the effect of pH alone (Lundholm et al. 2004). Cultures grown on urea have higher DA production, but also have a slower growth rate (Armstrong Howard et al. 2006). The increase in toxin production when growth slows must be taken into account when investigating factors that affect DA production. Nutrient limitation is widely used to induce DA production in culture, with Si and P limitation commonly used. Initially, it was hypothesized that DA production was specifically linked to Si limitation in P. multiseries, but Pseudo-nitzschia cultures will produce toxin when growth is limited by Si or other factors in the presence of replete nitrogen and light. Cells do not produce DA under nitrogen limitation (Bates et al. 1991). Toxic levels of NH4+ and Cu also limit growth and subsequently increase DA production (Bates et al. 1993a). Low temperature has been found to have a negative affect on cell division and DA production in some strains (Lewis et al. 1993); however, toxic mussels have been harvested from ice-covered water, implying that Pseudo-nitzschia can produce DA at near 0°C (Smith et al. 1993). A culture of P. seriata produced more DA at 4°C than 15°C which could reflect cold water adaptation (Lundholm et al. 1994). Metabolism typically decreases with lower temperatures, but many enzymes involved in the production of DA have differing temperature optima. For example, RUBISCO, the enzyme that fixes atmospheric carbon, operates optimally at a higher temperature (Li et al. 1984, Smith & Platt 1985, Descolas-Gros & de Billy 1987) than nitrate reductase, which transforms NO3‾ into NO2‾ (Packard et al. 1971, Kristiansen 1983, Dohler 1991, Lomas & Glibert 2000). Both fixed carbon and reduced nitrogen are required for DA synthesis. Temperature has an obvious effect on DA production by regulating the speed of multiple enzyme reactions within the cell. No studies have examined the effects of rapid changes in temperature on DA production, although rapid temperature changes can uncouple the light and dark reactions of photosynthesis. Irradiance is, of course, a very important control on DA production since it provides the energy necessary for biosynthesis. Irradiances below 100 μmol photons m-2sec-1 can lead to decreased DA production, a trait that has consequences for mass culture of toxic Pseudo-nitzschia (Whyte et al. 1995). Self-shading in large culture vessels can reduce DA production compared to small cultures in which self-shading is less important (Whyte et al. 1995). P. multiseries can produce DA under constant light (Villac et al. 1993a), but no experiments have been performed to examine the effect of extremely short photoperiods on DA production (Bates 1998). In P. seriata, cultures exposed to a long photoperiod (18:8 L:D) had higher total toxin production than those exposed to a short photoperiod (9:15 L:D) (Fehling et al. 2005). The effects of rapid changes in irradiance on DA production have not been studied, although rapid shifts may temporarily uncouple the light and dark reactions of photosynthesis (Lomas & Glibert 1999). Bacteria play a role in DA production, with the exact mechanisms unknown. Thus far there is no evidence that bacteria can produce DA autonomously (Bates et al. 2004). Axenic cultures of P. multiseries can produce DA but at much lower levels than non-axenic cultures. The variability between DA production in different cultures of the same strain may be partially explained by differences in bacterial flora (Kaczmarska et al. 2005). Bacteria have been observed on the frustule and free-living in culture media. It has been suggested that the bacteria are using organic matter released from the diatom (because their location on the frustule, the raphe and cingulum, are locations where organic matter could be released) while providing the diatom with a co-factor, such as a precursor that enables DA production under physiologically stressful conditions. Addition of amino acids, like proline, stimulated growth in axenic Pseudo-nitzschia cultures (Stewart et al. 1997), which supported suggestions that bacteria may produce specific amino acids that stimulate growth of the diatom, i.e., Pseudo-nitzschia and its attached bacteria live symbiotically (Stewart et al. 1997). However, another hypothesis states that DA is produced to chelate trace metals in competition with bacteria that produce their own chelator, gluconic acid, as the addition of gluconic acid to a Pseudo-nitzschia

Author(s): Thessen, Anne

• Comment (0)

Pseudo-nitzschia is a cosmopolitan genus; however, some tropical and polar species exist as well as coastal and oceanic species (Hasle 1965, Skov et al. 1999, Hasle 2002). Many species of Pseudo-nitzschia are found over a wide range of salinity and temperature (P. pungens) while other species are restricted to a narrow environmental regime (P. prolongatoides and P. turgiduloides). Pseudo-nitzschia pungens, P. heimii, P. inflatula, P. pseudodelicatissima and P. fraudulenta can be found in coastal and oceanic, tropical and temperate waters while P. brasiliana, P. caciantha, P. decipiens, P. micropora and P. sinica have only thus far been found in tropical waters. Pseudo-nitzschia obtusa can be found primarily in arctic coastal regions and P. turgiduloides and P. prolongatoides are restricted to the Antarctic region alone. Pseudo-nitzschia turgidula and P. granii are limited to cold waters. There has been no report of P. seriata from the southern hemisphere while P. subcurvata has only been reported in the southern hemisphere (Skov et al. 1999). Pseudo-nitzschia americana, P. calliantha, P. cuspidata, P. delicatissima and P. linea are found in tropical and temperate coastal waters. Pseudo-nitzschia australis, P. galaxiae, and P. multiseries are found in coastal temperate regions. Pseudo-nitzschia subfraudulenta is a coastal warm water species while P. subpacifica is an oceanic warm water species. Pseudo-nitzschia dolorosa has been found only in upwelling regions. Pseudo-nitzschia lineola has been reported in the open ocean and coastal regions in temperate and polar areas. Pseudo-nitzschia multistriata has been reported mostly in the tropical and temperate Pacific. Many species have a seasonal distribution. In the South China Sea, P. pungens peaks in April, May and June, P. multistriata is present only in spring and P. sinica and P. subpacifica are found in late fall and early winter (Qi et al. 1994). In addition, P. pungens in the colder (1-10°C) waters of the North China Sea is present only in winter and spring while P. pungens in the warmer (21-29°C) East and South China Seas is present year round revealing two ecotypes (Zou et al. 1993). On the west coast of the United States, P. pungens is abundant in the summer and autumn as well as P. fraudulenta, P. subpacifica and P. heimii. Pseudo-nitzschia multiseries is abundant in the autumn and winter while P. delicatissima is abundant in the spring and summer. Pseudo-nitzschia pseudodelicatissima, P. seriata and P. australis are common in the autumn (Fryxell et al. 1997).

Author(s): Thessen, Anne

• Comment (0)

Pseudo-nitzschia blooms can be stimulated by nutrients from two sources: upwelling or mixing events and riverine inputs. Both sources stimulate Pseudo-nitzschia blooms at concentrations of 8 – 22 μM NO3‾, 2.4 – 35 μM Si, 0.2 – 2 μM PO4‾ (Dortch et al. 1997, Scholin et al. 2000, Trainer et al. 2000, Loureiro et al. 2005), but in different temperature and salinity regimes. Pseudo-nitzschia abundances and domoic acid concentrations on the west coast of the United States are associated with low temperature, high salinity and high nutrient conditions typical of upwelling (Villac 1996, Trainer et al. 2000, Trainer et al. 2002). Similarly, upwelling regions off the coast of Portugal contain high concentrations of Pseudo-nitzschia, which are used as upwelling indicators during spring and summer (Moita 2001, Loureiro et al. 2005). Riverine inputs have stimulated toxic Pseudo-nitzschia blooms in many regions and are characterized by lower salinities and higher temperatures than upwelling zones (Bird & Wright 1989, Smith et al. 1990, Horner & Postel 1993, Dortch et al. 1997, Trainer et al. 1998, Scholin et al. 2000, Spatharis et al. 2007). A distinction between nutrients in upwelling and river plumes is that riverine inputs are likely the result of anthropogenic nutrient loading. Sedimentological data show an increase in Pseudo-nitzschia abundance in the Mississippi River plume since 1950, suggesting a response to eutrophication (Parsons et al. 2002). However, in addition to an increase in nitrogen and phosphorus, nutrient ratios in Mississippi River water have also changed, i.e., a decreasing Si:N ratio which is favorable for Pseudo-nitzschia in culture (Turner & Rabalais 1991, Sommer 1994). Other river systems have also affected Pseudo-nitzschia abundances. When the mouth of the Yellow River in China was artificially redirected in 1976, the location of the Pseudo-nitzschia bloom abruptly changed location to follow the river plume (Zou et al. 1993). Pseudo-nitzschia abundance in the plume of the Yangtze River is positively correlated to NO3‾ and PO4‾ concentrations (Zou et al. 1993). In the South China Sea, Pseudo-nitzschia abundances respond to increased land runoff after rainfall (Qi et al. 1994). An analysis of P. delicatissima and P. pseudodelicatissima dynamics and environmental parameters in the Bay of Fundy, Canada show the importance of NO3‾ and NO2‾ concentrations to abundance of these species (Kaczmarska et al. 2007). These coastal studies show a response to riverine nutrients, changing nutrient ratios and eutrophication. Much of the seasonal variability in Pseudo-nitzschia abundance can be explained by regular shifts in wind, light, temperature and river flow. In the northern Gulf of Mexico, Pseudo-nitzschia abundance peaks in spring, corresponding to the average maximum in river flow with another small peak in fall during wind events that mix the stratified water column (Dortch et al. 1997). 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). However, low light may contribute to the demise of autumn blooms (Bates et al. 1998). Day length can affect growth rates, cell yield, toxin production and influence which species of Pseudo-nitzschia becomes dominant (Fehling et al. 2005). Local meteorological phenomenon, such as winds and heavy rainfall events, can stimulate Pseudo-nitzschia blooms. Wind events can be especially important for transporting toxic blooms inland from upwelling sites offshore (Trainer et al. 2000, Trainer et al. 2002) or providing mixing necessary to bring nutrients into the photic zone (Lund-Hansen & Vang 2004). Heavy rainfall after a drought can cause a dramatic increase in Pseudo-nitzschia abundances in the river outflow, such as in Eastern Canada in 1987 (Bates et al. 1998). Larger scale changes in weather such as the El Niño Southern Oscillation can affect Pseudo-nitzschia abundances by controlling upwelling near the west coast of the United States. During weak ENSO years, upwelling is high and therefore so are Pseudo-nitzschia abundances (Fryxell et al. 1997). However, Pseudo-nitzschia can still take advantage of other favorable events, such as increased runoff after rainfall, during strong ENSO years and bloom. Both 1991 and 1998, years with large toxic events on the west coast of the United States, were strong ENSO years. The decline of Pseudo-nitzschia blooms is less studied than initiation. Parasitic fungi may play an important role in the demise of Pseudo-nitzschia blooms (Bates et al. 1998). Parasitic oomycetes and chytrids have infected P. multiseries and P. pungens in eastern Prince Edward Island, Canada. Additionally, fungal parasites have been observed in cells during bloom decline in coastal Washington, USA (Horner et al. 1996) and an unexpected decrease in P. multiseries abundance in the Skagerrak between 1991 and 1993 was suspected to be caused by parasitic fungi (Hasle et al. 1996). Viruses are known to infect marine diatoms (Nagasaki et al. 2004, Nagasaki et al. 2005), but no studies exist on viral infections in Pseudo-nitzschia and the genus may be immune (Caron pers. comm.; Coats pers. comm.). High pH resulting from dense blooms could also lead to bloom decline. Laboratory cultures of several Pseudo-nitzschia species could not continue exponential growth at pH from 8.7 to 9.3 (Lundholm et al. 2004). The exact mechanisms of Pseudo-nitzschia bloom decline are uncertain and could be caused by multiple factors.

Author(s): Thessen, Anne

• Comment (0)

Blooms of Pseudo-nitzschia happen relatively frequently, in some regions seasonally, and in a wide variety of locations. In culture, Pseudo-nitzschia spp. can grow in salinities as low as 6 and as high as 48 and at temperatures as low as 5°C and as high as 30°C with a broad range for optimum growth (Miller & Kamykowski 1986, Jackson et al. 1992, Lundholm et al. 1997, Cho et al. 2001, Thessen et al. 2005).

Author(s): Thessen, Anne

• Comment (0)

Different species in natural populations can demonstrate distinct correlations with environmental characteristics, which suggests seasonal succession of species or regional specificity (Fryxell et al. 1997). Many species may coexist, but different growth and loss rates can lead to complex bloom dynamics and seasonal succession. Several studies looking at molecular data have found potential cryptic species or ecotypes within morphological species (Amato et al. 2007, Amato et al. 2008).

Author(s): Thessen, Anne

• Comment (0)

Pseudo-nitzschia, like many pennate diatoms, can reproduce sexually (Geitler 1935). Clonal cultures of Pseudo-nitzschia will gradually decrease in cell size over time and eventually die if they do not undergo sexual reproduction. This is due to vegetative cell division and splitting of the frustule between two daughter cells. The halves of the frustule fit together like a glass Petri dish, with one side slightly smaller than the other. The daughter cell that receives the smaller of the two frustules will grow a new second frustule inside the first. This cell will be smaller than the initial parent cell. In this way, the average dimensions of the cell gradually decrease until they become so small the culture can no longer survive. However, if cells undergo sexual reproduction, cell size is restored. A Pseudo-nitzschia cell will become sexualized when cell length has decreased below a threshold size, known as the first cardinal point, which in P. multiseries, is approximately 63% of the length of largest cells (Bates & Davidovich 2002). Sexual reproduction must occur before the cells reach a minimum length, which in P. multiseries, is approximately 30 μm (Bates & Davidovich 2002). In P. delicatissima, this size range is from 19-80 μm (Amato et al. 2005). During this size window, cultures of Pseudo-nitzschia can be mixed together to stimulate sexual reproduction. Pseudo-nitzschia is dioecious, meaning that male and female gametes are produced by separate clones and intraclonal mating is rare or absent. These “sexes” are referred to as “+” and “-“ in Pseudo-nitzschia. While no monoecious clones of Pseudo-nitzschia have been reported, mating between two clones of the same sex has been observed, suggesting that a single culture could switch sex under some conditions which have not yet been investigated, or more than one mating type exists (Davidovich & Bates 1998). The sexual cycle differs between pennate and centric diatoms (Drebes 1977, Round et al. 1990). Centrics are characterized by oogamous reproduction involving the formation of flagellated male gametes and non-motile female gametes. The first paper reporting sexual reproduction in P. multiseries caused considerable debate and criticism for claiming that Pseudo-nitzschia, a pennate diatom, had oogamous reproduction (Subba Rao et al. 1991). Observation of flagellated gametes in cultures of P. multiseries was consistent with fungal contamination and was considered anomalous (Rosowski et al. 1992). Mating in Pseudo-nitzschia can be achieved simply by mixing clones of the same species, but opposite sex. Clones must be in good physiological condition to undergo sexual reproduction. This means that clones must be mixed during exponential growth phase, which can be anywhere from 3-6 days after inoculation of a batch culture. Clones must receive a sufficient amount of light during a 24 h period. A photoperiod length up to 16:8 L:D, the maximum studied, will increase gamete and auxospore production (Davidovich & Bates 1998, Hiltz et al. 2000). These results suggest that parent cells must be healthy and photosynthesizing to produce energy for sexual reproduction. Sexual reproduction has been described in P. multiseries, P. pseudodelicatissima, P. calliantha (Davidovich & Bates 1998), P. subcurvata (Fryxell et al. 1991) and P. delicatissima (Amato et al. 2005). Despite some differences in the amount of time necessary to complete sexual reproduction, the mating process is similar in all Pseudo-nitzschia species tested. The first step in sexual reproduction is parental pairing between cells of the opposite sex. Two cells will pair valve to valve, lying parallel with close alignment of the cells. The next stage is gametogenesis. The paired cells divide meiotically and the cell contents divide along the apical plane to form spherical gametes, two per cell. These gametes are identical in appearance and non-flagellated, but the behavior of the gametes differs between sexes. One cell produces two active gametes (- male) and the other two passive gametes (+ female). The frustules of both cells open, permitting the active gametes to enter and fuse with the passive gametes. This is not always successful in both pairs of gametes or in all pairing of parent cells. When it is successful, this fusion takes only 1-2 min. After gamete fusion, the resulting zygote expands to form larger auxospores inside which the initial cell is formed. The entire process, from gamete production to formation of initial cells takes 2-4 days. This process has not been documented in nature for several reasons. Calculations suggest that three years may pass before a cell becomes sexualized (Davidovich & Bates 1998). The entire mating process itself only takes 2-4 days and when mating occurs in the laboratory there are few auxospores compared to vegetative cells. Thus, paired cells and auxospores would be rare in natural populations. Furthermore, it is likely that the coupling between parent cells is weak enough that sampling and preservation techniques can disrupt them: placing mixtures of clonal cultures on an orbital shaker at 170 rpm prevented or reduced sexual reproduction (Gordon 2001, Bates & Davidovich 2002). It is unknown how well gametes and auxospores survive sampling and preservation, if at all. Preliminary work has shown an interesting relationship between epibiont bacteria and Pseudo-nitzschia sexual reproduction. Some axenic clones of P. multiseries would not undergo sexual reproduction until bacteria were reintroduced (Thompson 2000). Other mixtures of axenic clones did undergo sexual reproduction; however, it is possible that there was bacterial contamination. Further work must be done to determine the role of bacteria in sexual reproduction in this species. There is also some evidence that a type of “pheromone” or other chemical is being produced by sexually active Pseudo-nitzschia. Filtrates of sexually reproducing clones induce higher gamete production in other clones (Haché 2000). These results suggest that a chemical is produced that improves gamete production and thus would synchronize gamete production in already sexualized cells, but not necessarily aid in location of other sexualized cells. Sexual reproduction in Pseudo-nitzschia is important for domoic acid (DA) production. Clonal cultures of Pseudo-nitzschia decrease in size over time, as described previously, and also lose their ability to produce DA (Bates 1998). Offspring of P. multiseries clones that lose their ability to produce DA can be toxic, sometimes even more toxic than their parents were initially (Bates et al. 1999). Sibling clones can have significant variability in DA production, which could be accounted for by genetics or by the presence of different types and numbers of epibiont bacteria.

Author(s): Thessen, Anne

• Comment (0)

As a diatom, Pseudo-nitzschia is an important primary producer at the base of the food web. It is consumed directly by a wide variety of organisms from heterotrophic dinoflagellates to planktivorous fish. It can form dense blooms and be an important source of food for these primary consumers, thereby introducing domoic acid into higher trophic levels. As a hydrophilic molecule, domoic acid does not bioaccumulate. Instead, DA is concentrated in the digestive system with little transfer to surrounding tissues and can be quickly eliminated from the body. The toxin is moved through the food chain during blooms when primary consumers with guts full of Pseudo-nitzschia are eaten by secondary consumers. Eventually DA is depurated, but depuration rates can vary, from hours in the blue mussel (M. edulis), Mediterranean cockle (Acanthocardia tuberculatum), and greenshell mussel (Perna canaliculus), to several days in the mussel M. galloprovincialis (Novaczek et al. 1992, Wohlgeschaffen et al. 1992, Mackenzie et al. 1993, Vale & Sampayo 2002). Three bivalves that are very slow to depurate are the razor clam Siliqua patula (> 86 days), the scallop Placopecten magellanicus (> 14 days) and the scallop Pecten maximus (~ 416 days; Wohlgeschaffen et al. 1992, Horner et al. 1993, Douglas et al. 1997, Blanco et al. 2002). Differential DA accumulation is an important factor in commercial species which are often not eaten whole. Most scallops, for example, are dissected and only the adductor muscle is eaten, which is the tissue containing the least amount of DA (Douglas et al. 1997, Blanco et al. 2002). Many commercially harvested animals, such as crabs, fish and cephalopods, retain most of the DA in their viscera, not typically consumed by humans (Horner & Postel 1993, Costa et al. 2003, Costa & Garrido 2004, Costa et al. 2004, Costa et al. 2005). However, studies do show trace amounts of DA in consumable tissues. Animal feeds that consist of whole fish captured during Pseudo-nitzschia blooms can be contaminated and sicken animals far from coasts or toxic blooms (Naar et al. 2002). However, DA in rainbow trout (Oncorhynchus mykiss) feed containing fish meal made from contaminated anchovies did not affect fish health nor lead to contaminated trout (Hardy et al. 1995). Hence, differential distribution of DA in vector tissues can be important for processing of commercially harvested species. Packaging and handling procedures have an effect on which tissues are toxic. During storage, for example, DA can transfer from the digestive system into surrounding tissues (Smith et al. 2006). Freezing and thawing can affect distribution of DA within crab tissues (Hatfield et al. 1995, Costa et al. 2003). Storage in pickling brine or frozen storage can cause DA to leach into the surrounding medium (Leira et al. 1998). Removal or flushing of the digestive tract or hepatopancreas can decrease DA in bivalves (Leira et al. 1998, Campbell et al. 2003). Boiling of toxic animals before ingestion can also reduce DA body burdens by causing the toxin to leach into the boiling media (Costa et al. 2003). Many different types of organisms can have DA in their tissues; however, not all of these organisms are filter feeders or their predators. Scavengers and deposit feeders have also been found to contain DA (Goldberg 2003). Scavengers could become contaminated by eating DA contaminated remains. Deposit feeders could become contaminated by consuming flocs of Pseudo-nitzschia that sink to the benthos at the end of a bloom (Goldberg 2003). Domoic acid has been detected in ocean sediments and sinking particles at high levels, indicating that the toxin can persist after the surface bloom is over and enter benthic food webs (Sekula-Wood et al. 2009) Some carnivores, like the swimming crab Polybius henslowii, can contain high levels of DA but there have been no recorded incidents of poisoning in their predator, the yellow-legged gull (Larus cachinnans) that feeds on them almost exclusively (Alvarez 1968, Munilla 1997, Costa et al. 2003). There may be a limit in the number of trophic transfers over which DA can still be present at high enough concentration to cause a toxic event: to date all recorded DAP and ASP events involved only three trophic levels, Pseudo-nitzschia, a bivalve or a planktivorous fish and a bivalve or fish predator.

Author(s): Thessen, Anne

• Comment (0)

Pseudo-nitzschia was not recognized as a toxic diatom until the first documented incident of Amnesic Shellfish Poisoning (ASP) occurred in Prince Edward Island, Canada in 1987 when residents ate domoic acid (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). No new cases of ASP have been reported since 1987. However, DA has been detected in shellfish (Villac et al. 1993b, Wekell et al. 1994, Rhodes et al. 1998a, Vale & Sampayo 2001) and a small outbreak of “food poisoning” in Oregon in 1992 possibly could have been the result of DA in razor clams consumed by locals (Todd 1993, Wright 1998). Effects of single or multiple exposures to levels of DA too low to cause outward symptoms are unknown; low levels of DA could have negative effects without causing an obvious toxic event. Preliminary work shows a possible risk to mental development of infants whose breastfeeding mothers eat contaminated shellfish and children who eat contaminated shellfish themselves (Grattan et al. 2002). Only DA levels above 40 μg g-1 wet weight of mussel meat will show symptoms in test mice (Todd 1993). During the 1987 event, seemingly unaffected individuals consumed 0.2-0.3 mg kg-1, persons with mild symptoms had consumed 0.9-2.0 mg kg-1 and the most serious cases had consumed 1.9-4.2 mg kg-1. After the 1987 ASP event, the Canadian government enacted a 20 μg DA g-1 mussel flesh action limit, which when exceeded, would authorize closure of the shellfish bed (Waldichuk 1989). This action limit has also been adopted by the European Union, New Zealand, United States and Australia. The first documented case of domoic acid poisoning (DAP) involved brown pelicans Pelicanus occidentalis and Brandt’s cormorants Phalacrocorax penicillatus in 1991 in Monterey Bay (Fritz et al. 1992, Work et al. 1993a,b). The birds exhibited strange behaviors indicative of central nervous system disorder, such as scratching, head weaving, wryneck, clenched toes and loss of righting reflex. Histological examination of brain tissue from dead birds revealed lesions similar to those found in the human brains during the 1987 Canadian ASP event (Scholin et al. 2000). Plankton samples collected in Monterey Bay at the time of the incident showed phytoplankton assemblages to be almost unialgal P. australis at 4 X 104 cells L-1 maximum concentration. Pseudo-nitzschia australis frustules were found in the birds’ stomachs at 1.5 X 106 recognizable P. australis fragments g-1 wet w and in the stomachs of the birds’ prey, the northern anchovy Engraulis mordax (Fritz et al. 1992). This incident showed that a second species of Pseudo-nitzschia could produce DA at sufficient levels to cause a toxic event and that planktivorous fin fish can be vectors of DA to higher trophic levels (Garrison et al. 1992). In 1996, 150 brown pelicans Pelecanus occidentalis died at the tip of the Baja Peninsula, Mexico from feeding on mackerel Scomber japonicus contaminated with DA from an unknown Pseudo-nitzschia sp. (Sierra-Beltran et al. 1997). The birds’ stomachs were empty, indicating recent vomiting, but smears from the digestive tract revealed Pseudo-nitzschia frustules. Viscera from pelicans and mackerel tested positive for DA by HPLC. Another incident in the Gulf of California in 1997 killed 766 common loons Gavia immer and 182 sea mammals belonging to 4 different species including the common dolphin (Sierra-Beltran et al. 1998). Microscopic analysis found P. australis frustules in the stomachs of the common dolphin Delphinus capensis and the sardine Sardinops sagax found inside the dolphin stomachs. Histological examination of dolphin brain tissue showed distinct lesions. Another DAP event occurred in Monterey Bay in 1998, this time affecting over 400 California sea lions Zalophus californianus in addition to marine birds (Scholin et al. 2000, Gulland et al. 2002). Again E. mordax was acting as a vector, delivering DA from a local P. australis bloom (maximum concentration ~1.3 X 105 cells L-1) that had responded to high Si levels, indicative of terrestrial run-off (Scholin et al. 2000). DA was found in E. mordax, sea lion body fluids and plankton samples, but not in mussels, Mytilus edulis. Of the 400 sea lions affected, only 81 were found alive and transported to The Marine Mammal Center where 48 died despite treatment (Scholin et al. 2000, Gulland et al. 2002). All affected animals displayed neurological symptoms: seizures, head weaving, ataxia, unresponsiveness and abnormal scratching. Five females had fetuses detectable by ultrasound, but no detectable fetal heartbeats (Gulland et al. 2002). Histological examination of brain tissue from the 48 dead sea lions revealed lesions in the hippocampus. This event recurred in 2000, with 184 sea lion strandings (Gulland et al. 2002). Between the two events, 129 animals recovered and were released. Eleven re-stranded within four months giving a re-stranding rate of 9%, 0.5% higher than re-stranding rates for sea lions rehabilitated for other reasons (Gulland et al. 2002). Two of these animals had an atrophied hippocampus and were euthanized. Eight animals appeared normal after a week of treatment and re-released (Gulland et al. 2002). Symptoms of ASP and DAP have many similarities consistent with the pharmacology of DA and the effects of its biochemical analog kainic acid and glutamic acid in animal models (Perl et al. 1990, Hampson & Manalo 1998, Schrader & Langlois 2001). The distinctive characteristic of DA poisoning appears to be permanent damage to the hippocampus, which can be detected even after DA has been eliminated from the body (Cendes et al. 1995, Gulland et al. 2002). Long term memory effects and/or chronic siezures have been documented in affected humans (Cendes et al. 1995), but only seizures have been documented in animals (Gulland et al. 2002).

Author(s): Thessen, Anne

• Comment (0)

Domoic acid (MW 311) is a water-soluble, heat stable analogue of the amino acid glutamate (Hatfield et al. 1995, Leira et al. 1998). It was first isolated from Chondria armata and named after the Japanese word for seaweed – domoi (Mos 2001). DA is toxic to vertebrates because it binds to neurons twenty times more powerfully than ordinary neurotransmitters (Teitelbaum et al. 1990), resulting in a massive depolarization of the neuron. The subsequent increase of intracellular Ca2+ causes swelling and cell death. This happens in the hippocampus, the part of the brain associated with memory. Tests in animal models have repeatedly documented DA effects in adults and developing animals. Intraperitoneal injections up to 1.25 mg DA kg-1d-1 cause no signs of toxicity in adult female rats, but doses of 1.75 mg DA kg-1d-1 induced abortions (Khera et al. 1994). Doses of 2 mg DA kg-1d-1 can cause death in pregnant rats. Lactating females given doses of 1 mg DA kg-1d-1 transferred the toxin to their young up to 24 h after exposure, but not enough to cause acute symptoms (Maucher & Ramsdell 2005). Research shows that DA lingers in breast milk longer than in blood plasma and that neonatal rats are much more susceptible to DA than adults (Xi et al. 1997, Doucette et al. 2000, Maucher & Ramsdell 2005). This is probably because in adults, DA poorly penetrates the blood-brain barrier, but in fetuses and newborns, this barrier is incomplete (Mayer 2000). A study comparing the effects of DA on young adult versus old adult rat brains shows equal sensitivity to initial exposure and reduced sensitivity to a second exposure in the younger brains (Kerr et al. 2002). Oral doses have less effect than injection of DA in animals and almost all DA ingested is excreted in feces. This suggests that DA is poorly absorbed from the gut (Iverson et al. 1989). Other studies with rats and cynomolgus monkeys show that DA is well distributed in body water and rapidly excreted, as expected for a hydrophilic compound, and implies that renal function is important for DA removal (Truelove & Iverson 1994). Numerous studies show death, brain damage, reduced learning and memory ability in rats and primates exposed to DA in the laboratory (Jeffery et al. 2004). Fish have long been considered immune to the effects of DA. However, injections of DA can produce neurological symptoms suggesting that fish are susceptible to DA at doses similar to rats and monkeys (Lefebvre et al. 2001). Fish exposed to DA in the field showed high levels of DA in their gut, but DA levels were 1000 X lower in the brain tissue, suggesting that DA uptake from the gut is low. The complete absence of neurological symptoms in fish given oral doses of DA provides more evidence that little DA is taken up through the gut and even less passes the blood-brain barrier. Despite proven susceptibility to injections of DA, fish exposed to DA in the field may not display symptoms. A study of toxicity in fish eggs injected with DA showed effects even at small doses (Tiedeken et al. 2005). Injections of 0.4 mg DA kg-1 reduced hatching success by 40% and injections of 1.2 mg DA kg-1 reduced hatching success by 50%. In the eggs that lived, embryos convulsed at 2 days post fertilization. All surviving eggs injected with 4 mg DA kg-1 had no response to touch at 4 days post fertilization and at 5 days post fertilization had constant rapid pectoral fin movements possibly analogous to the scratching observed in laboratory rodents exposed to DA. Not only are fish susceptible to DA at similar doses as mammals, they are also susceptible to developmental toxicity.

Author(s): Thessen, Anne

• Comment (0)