Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: morphology,toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species

The genus Pseudo-nitzschia includes several species capable of producing domoic acid, the causative agent of Amnesic Shellfish Poisoning. Some of these species have been recorded frequently in the Gulf of Naples. For one of the species, P. multistriata, which has been recurrently found in our sampling area since 1995, this is the first report for European waters. Here we provide further details on the fine structure of this species. Pseudo-nitzschia multistriata was the only one found to produce domoic acid among all the Pseudo-nitzschia species from the Gulf of Naples, and this finding raises the number of potentially toxic species in this genus to nine. Phylogenetic relationships among several Pseudo-nitzschia species were assessed using the hypervariable domains (D1–D3) of the large subunit (LSU) rDNA. The match between the phylogeny obtained and important taxonomic characters used in this genus are discussed. Results show that P. multistriata clusters with wider species lacking a central larger interspace in the raphe. Close genetic relationships were determined between P. fraudulenta and P. subfraudulenta, and between P. pungens and P. multiseries. Genetic differences among these pairs of species are comparable to those among isolates of P. pseudodelicatissima from the Gulf of Naples, indicating high intraspecific genetic diversity of Pseudo-nitzschia species in the relatively conserved LSU region. This could explain the problematic results obtained when testing a match between species-specific Pseudo-nitzschia LSU probes and our sequences.     

Intra- and interspecies differences in growth and toxicity of Pseudo-nitzschia while using different nitrogen sources

Clonal cultures of plankton are widely used in laboratory experiments and have contributed greatly to knowledge of microbial systems. However, many physiological characteristics vary drastically between strains of the same species, calling into question our ability to make ecologically relevant inferences about populations based on studying one or a few strains. This study included 19 non-axenic strains of three species of the diatom Pseudo-nitzschia isolated primarily from the mid-Atlantic coastal region of the United States. Toxin (domoic acid) production and growth rates were measured in cultures using different nitrogen sources (NH₄⁺, NO₃⁻ and urea) and growth irradiances. The strains exhibited broad differences in growth rate and toxin content even between strains isolated from the same water sample. The influence of bacteria on toxin production was not investigated. Both P. multiseries clones produced toxin, yet preferentially used different nitrogen sources. Only two of nine P. calliantha and two of five P. fraudulenta isolates were toxic and domoic acid content varied by orders of magnitude. All three species had variable intraspecies growth rates on each nitrogen source, but P. fraudulenta strains had the broadest range. Light-limited growth rate and maximum growth rate in P. fraudulenta and P. multiseries varied with species. These findings show the importance of defining intra- and interspecies variability in ecophysiology and toxicity. Ecologically relevant functional diversity in the form of ecotypes or cryptic species appears to be present in the genus Pseudo-nitzschia.     

Taxonomical diversity of Pseudo‐nitzschia from the Central Adriatic Sea

The aim of our research was to study the composition of Pseudo‐nitzschia species during a period when neurotoxin domoic acid (DA) was present in shellfish. Sampling was conducted in Ka?tela Bay (Central Adriatic Sea), between November 2015 and January 2016. Concentrations of DA analyzed in various shellfish species were low, below the regulatory limit, while the highest abundance of Pseudo‐nitzschia spp. reached 1.85 × 10⁵ cells L^?1 in the surface layer, at the beginning of November. Within the temperature and salinity range recorded during the investigated period, a positive correlation of Pseudo‐nitzschia spp. abundance was recorded with temperature. Morphological analyses by scanning electron microscopy revealed the presence of five Pseudo‐nitzschia species that had already been reported in the Adriatic Sea – P. calliantha, P. delicatissima, P. fraudulenta, P. pseudodelicatissima /P. cuspidata and P. subfraudulenta – as well as an unknown Pseudo‐nitzschia sp. The composition of the Pseudo‐nitzschia assemblage changed over the investigated period. The species P. pseudodelicatissima/P.cuspidata was found throughout the entire period and the highest diversity was noticed in January, when all six observed species were recorded. These results represent the first taxonomical investigation of the genus Pseudo‐nitzschia in Ka?tela Bay, as well as the first report of DA in shellfish from this area.     

Feeding,egg production,and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudo-nitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female^–1 d^–1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.     

The genus Pseudo-nitzschia (Bacillariophyceae) in continental shelf waters of Argentina (Southwestern Atlantic Ocean, 38–55°S)

The distribution pattern of Pseudo-nitzschia species, associated phytoplankton flora and its relationships with main environmental factors were studied for the first time in continental shelf surface waters of the Argentine Sea (Southwestern Atlantic Ocean, 38–55°S). Both qualitative and quantitative samples, collected during summer and fall 2003, were examined using light and scanning electron microscopy. Results indicated that the genus Pseudo-nitzschia has a wide distribution along the studied area. It was present at low densities, with infrequent peak abundances and appeared most frequently as a minor component of the diatom populations that typically develop on the continental shelf of the Argentine Sea. Moreover, phytoplankton communities were numerically dominated by unidentified phytoflagellates (≤5 μm) throughout almost all samples analyzed. Eight Pseudo-nitzschia species were identified in our study: P. australis, P. fraudulenta, P. heimii, P. lineola, P. pungens, P. cf. subcurvata, P. turgidula and P. turgiduloides. Of these, P. heimii, P. lineola and P. turgiduloides are new records for the Argentine Sea. Their presence in the area is attributable to the influence of southerly cold water masses. Spatial and temporal variations of the environmental parameters recorded in the study area generally determined the distribution of Pseudo-nitzschia species. P. pungens and P. australis were widely distributed and reached high densities, especially in waters with elevated temperatures and salinities (around 15 °C, 33.8 psu) and low nutrients concentrations. On the other hand, P. heimii, P. lineola, P. turgidula and P. turgiduloides showed a more restricted distribution, with lower densities in relatively cold, less saline (8 °C, 32.45 psu) and nutrient-rich waters. From the Pseudo-nitzschia species found throughout this survey, P. australis, P. fraudulenta, P. pungens and P. turgidula are known as domoic acid (DA) producers around the world, but there is little information on the potential toxicity of these species in Argentina.     

Note on the occurrence of Pseudo-nitzschia australis and domoic acid in squid from Monterey Bay, CA (USA)

Over the past decade diatom blooms of domoic acid (DA)-producing Pseudo-nitzschia spp. have been responsible for numerous marine mammal and bird mortalities in Monterey Bay, CA. One possible toxin vector is the market squid, Loligo opalescens, a small pelagic mollusk that plays an important role in the near-shore food web of the California Current ecosystem as a favored vertebrate prey species. This study examined the trophic link between toxic Pseudo-nitzschia and L. opalescens using toxin and stomach content analyses of animals collected from Monterey Bay, CA in 2000. Receptor binding assay data (confirmed by tandem mass spectrometry), demonstrated the presence of DA in squid during a toxic Pseudo-nitzschia event, with P. australis frustules observed in stomach samples. Though DA levels were low (<0.5 μg DA g⁻¹ tissue) in L. opalescens during the study period, it is now clear that this potent neurotoxin can occur in squid and is likely delivered through its krill prey species, which are known to retain DA after feeding on toxic Pseudo-nitzschia. Our findings suggest that further study of the relationship between Pseudo-nitzschia blooms and DA contamination of squid is warranted to better evaluate the potential health risk to humans and wildlife associated with this major commercial seafood species and important prey item.     

Pseudo-nitzschia simulans sp. nov. (Bacillariophyceae), the first domoic acid producer from Chinese waters

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05–1.54 fg cell⁻¹. This is the first toxigenic diatom species reported from Chinese waters.     

Are most of the domoic acid-producing species of the diatom genus Pseudo-nitzschia cosmopolites?

Records from all oceans, most of them published during 1990–2000, and personal unpublished observations of nine Pseudo-nitzschia taxa known as potential domoic acid (DA) producers have been used to outline their geographical distribution. Pseudo-nitzschia seriata f. seriata as the only taxon was found in the North Atlantic Ocean exclusively. The records of P. multistriata were too few and the identification of P. turgidula from the North Atlantic too unreliable to provide an idea about their distribution. Pseudo-nitzschia pungens and the less frequently recorded P. fraudulenta, P. multiseries and P. australis appeared to be cosmopolites. The wide distribution of the apparently most efficient DA producers, P. australis and P. multiseries, is especially noteworthy. The records of P. delicatissima and P. pseudodelicatissima also indicate a cosmopolitan distribution although with the qualification of certain taxonomic and identification ambiguities. Hence, the question of whether most DA-producing Pseudo-nitzschia species are cosmopolites may be answered with a tentative “yes”.     

Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France)

The population dynamics of different Pseudo-nitzschia species, along with particulate domoic acid (pDA) concentrations, were studied from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly. In 2012, three different species were identified during the spring bloom (P. australis, P. pungens and P. fraudulenta) with high pDA concentrations (∼1400 ng l⁻¹) resulting in shellfish harvesting closures. In contrast, the 2013 spring was characterised by a P. delicatissima bloom without any toxic event. Above all, the results show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N < 1), while nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development of either P. delicatissima or P. australis. This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by nutrients, especially nutrient ratios.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Growth characteristics of the diatoms Pseudonitzschia pungens and P. fraudulenta exposed to ultraviolet radiation

Interest in the biology of planktonic, chain-forming Pseudonitzschia species has grown recently after the discovery of toxin production in Pseudonitzschia pungens and related taxa, following the outbreak of shellfish toxicity in Canada in 1987. As part of a broader study on the effects of enhanced ultraviolet light on the growth of bloom-forming phytoplankton, we have examined the growth rates and production of the toxin domoic acid and two additional chemicals [bacillariolides I and II] by Pseudonitzschia pungens varieties and Pseudonitzschia fraudulenta from Narragansett Bay, Rhode Island. Growth of P. fraudulenta is significantly inhibited by enhanced UV, P. pungens var. pungens shows slight inhibition, and P. pungens var. multiseries is unaffected. Production of bacillariolides I and II by P. pungens var. multiseries is similar in enhanced and deleted UV light. Tolerance of UV light by P. pungens var. multiseries appears to be acquired, and persistent. If ambient UV light continues to increase as a result of global ozone depletion, one may expect UV-resistant taxa such as P. pungens var. multiseries to become more prominent in coastal phytoplankton communities.     

A review ofPseudo-nitzschia,with special reference to the Skagerrak,North Atlantic,and adjacent waters

ThePseudo-nitzschia flora of the Skagerrak, North Atlantic, and adjacent waters, comprisingP. pungens, P. multiseries, P. seriata, P. fraudulenta, P. heimii, P. delicatissima, andP. pseudodelicatissima, has been examined. Except forP. australis, allPseudo-nitzschia species shown to produce the toxin domoic acid are present in the area although an outbreak of amnesic shellfish poisoning has never been reported. For comparison of morphological and taxonomic characters,Pseudo-nitzschia seriata f.obtusa, P. australis, P. subfraudulenta, P. subpacifica, P. lineola, P. inflatula, andP. cuspidata have been included in this investigation. Fine details of band structure and poroid occlusions, previously ignored or unresolved, have proven to add to the morphological distinction betweenP. pungens andP. multiseries, P. seriata andP. fraudulenta, P. seriata andP. australis, andP. delicatissima andP. pseudodelicatissima. Additional information on the structure of the proximal mantle compared to that of the valve face has revealed similarities in most of the species but differences betweenP. pungens andP. multiseries. The species seasonal and long-term distributional patterns during the sampling period (October 1978 through September 1993) in the Skagerrak area are outlined. The greatest abundances ofP. seriata, a cold-water species most likely restricted to the northern hemisphere, occurred in the spring, and those of the presumably cosmopolitan diatomsP. pungens, P. multiseries andP. pseudodelicatissima, in the autumn. WhereasP. multiseries seems to have decreased in abundance in the 1990s,P. pseudodelicatissima has apparently increased.     

The copepod Calanus finmarchicus: A potential vector for trophic transfer of the marine algal biotoxin, domoic acid

The marine algal biotoxin, domoic acid (DA), is produced by certain members of the diatom genus Pseudo-nitzschia. This neurotoxin has been responsible for several mass mortality events involving marine birds and mammals. In all cases, the toxin was transferred from its algal producers through marine food webs by one or more intermediate vectors. The ability of some copepod taxa to serve as vectors for DA has been demonstrated; however, the role played in DA trophic transfer by Calanus finmarchicus, which often dominates N. Atlantic zooplankton assemblages and is a primary dietary component of the highly endangered N. Atlantic right whale (Eubalaena glacialis), has been uncertain. In the present study, we examined the ability of C. finmarchicus to consume DA-producing algae and retain the toxin. Results of grazing and toxin accumulation/depuration experiments showed that C. finmarchicus consumed DA-producing Pseudo-nitzschia multiseries regardless of the presence or absence of morphologically similar, but non-toxic, P. pungens, across initial cell concentrations ranging from 1000-4000 cells mL^− 1. Furthermore, C. finmarchicus did not appear to preferentially consume or avoid either Pseudo-nitzschia species tested. After ingestion of P. multiseries, copepods accumulated DA and retained it for up to 48 h post-removal of the toxin source. These findings provide evidence for the potential of C. finmarchicus to facilitate DA trophic transfer in marine food webs where toxic Pseudo-nitzschia is present.     

IDENTIFICATION OF CULTURED PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES-SPECIFIC LSU rRNA-TARGETED FLUORESCENT PROBES1

Some, but not all, marine pennate diatoms of the genus Pseudo-nitzschia H. Peragallo are associated with the production of domoic acid, a naturally occurring amino acid responsible for amnesic shellfish poisoning. Distinguishing between potentially toxic and nontoxic representatives of this genus is time-consuming and difficult because it demands scanning electron microscopy of cleaned frustules. The objective of this work is to speed and ease identification of these organisms by using whole-cell (in situ) hybridization and species-specific large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotide probes. Toward that end, cultures of P. australis Frenguelli, P. pungens (Grunow) Hasle, P. multiseries (Hasle) Hasle, P. fraudulenta (P. T. Cleve) Heiden, P. heimii Manguin, P. delicatissima (P. T. Cleve) Heiden, P. pseudo-delicatissima (Hasle) Hasle, and P. americana (Hasle) Fryxell were screened with a suite of 15 putative species-specific probes. Of those, a subset of eight probes was found that distinguished each species tested. In addition, Pseudo-nitzschia chloroplasts were labeled with a probe directed against a eubacterial-conserved sequence. Identification of new cultures based on their reactivity toward a set of probes agreed with species designations as defined by morphological criteria. Whole-cell hybridization is a rapid, simple, and cost-effective technique for discriminating among cultured Pseudo-nitzschia species.     

Correlation between the presence of Gonyaulax fragilis (Dinophyceae) and the mucilage phenomena of the Emilia-Romagna coast (northern Adriatic Sea)

The formation of massive amounts of suspended mucilaginous organic matter which periodically affects the Adriatic Sea, has been regarded as a complex physico-chemical phenomenon resulting from the production of extracellular material by phytoplankton. Although the exact cause has remained obscure, the mechanism of its formation has usually been considered to be a long-term process, starting after the late winter-early spring blooms, and involving the participation of various algal species, mainly within the diatom group. In this paper we report on the results of a phytoplankton monitoring programme in northern Adriatic seawaters off the Emilia-Romagna coast of Italy which revealed the constant concomitant presence of the dinoflagellate Gonyaulax fragilis (Schütt) Kofoid and mucilaginous formations. In the early stages of the phenomenon the dinoflagellate was clearly observable by microscopic examination in the mucilage, but as the mucilage aged this alga almost completely decomposed and diatom cells increased in number and became predominant. Although characterized by a slow growth rate in culture, in natural seawater G. fragilis was observed to reach cell densities of up to 7.0×10⁶ cells l⁻¹. The results of this study lead us to propose the hypothesis that the appearance of mucilage in the water column of the Adriatic Sea is the consequence of a seasonal growth of this dinoflagellate favoured by specific environmental circumstances.     

The influence of the Pseudo-nitzschia toxin,domoic acid,on microzooplankton grazing and growth: A field and laboratory assessment

We conducted field and laboratory experiments to determine whether the Pseudo-nitzschia-derived metabolite, domoic acid (DA), functions as a microzooplankton grazing suppressant. Using the seawater dilution technique in natural plankton communities along the Pacific Northwest coast, we found no significant relationship between dissolved DA and microzooplankton grazing rate on Pseudo-nitzschia spp. Dilution experiments amended with either 50 or 80 nM dissolved DA also showed no evidence that microzooplankton community grazing was affected by DA. The relationship between Pseudo-nitzschia spp. intracellular DA and microzooplankton grazing was less clear. On a subset of data where small Pseudo-nitzschia spp. cells dominated community composition, an apparent negative relationship between intracellular DA and microzooplankton grazing was observed. However, we provide evidence that this relationship is a microzooplankton response to Pseudo-nitzschia spp. growth rate, rather than cellular DA. In laboratory experiments, two diatom-consuming dinoflagellates, Protoperidinium excentricum and P. pellucidum, were fed single and mixed diets of a toxic and non-toxic Pseudo-nitzschia species and an optimal prey, Ditylum brightwellii. P. excentricum did not grow or ingest either the toxic or non-toxic Pseudo-nitzschia. However, P. pellucidum grew as well on the toxic Pseudo-nitzschia multiseries as it did on D. brightwellii, but did not grow on the non-toxic Pseudo-nitzschia pungens. Both dinoflagellates were capable of growing if Pseudo-nitzschia spp. diets were mixed with D. brightwellii. Addition of dissolved DA also had no negative effect on dinoflagellate growth when fed the optimal diatom diet. We conclude that domoic acid has no functional role in deterring microzooplankton grazing or growth rates. Further, our findings highlight the difficulty of defining the complex mechanisms that regulate predator and prey interactions within microplankton food webs.