An assessment of Pseudo-nitzschia population dynamics and domoic acid production in coastal Louisiana

Over 1200 samples were collected from Louisiana estuarine and coastal shelf waters between 1989 and 2002, and analyzed to examine the population dynamics of Pseudo-nitzschia and to assess the potential threat posed by domoic acid (DA), a potent neurotoxin produced by some members within this toxigenic diatom genus. Results demonstrated that three species in this region (Pseudo-nitzschia multiseries, P. pseudodelicatissima complex, P. delicatissima) produce DA, and that particulate toxin levels were highest (up to 3.05 μg L⁻¹) during the spring bloom, while cellular concentrations were highest in the winter/early spring when P. multiseries was most abundant (up to 30 pg cell⁻¹). These particulate toxin levels are comparable to those seen in other regions (e.g., United States west coast) where DA poisoning events have occurred in the past. Pseudo-nitzschia were most abundant under dissolved inorganic nitrogen-replete conditions coupled with lower silicate and/or phosphate concentrations, and in the early spring months when temperatures were cooler. Pseudo-nitzschia were occasionally well-represented in the phytoplankton assemblage (≥10⁶ cells L⁻¹ in 14% of samples, over 50% of total phytoplankton in 5% of samples), indicating that planktivores (e.g., Gulf menhaden, Brevoortia patronus) may have little choice but to consume Pseudo-nitzschia cells, thereby providing potential vectors for DA transfer to higher trophic levels. By comparison, eastern oysters (Crassostrea virginica) present in estuarine waters may be more exposed to this toxin when Pseudo-nitzschia cells are part of a mixed assemblage, reducing selective grazing by these bivalves. C. virginica may thus represent the most effective vector for DA exposure in humans.     

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Records from all oceans, most of them published during 1990–2000, and personal unpublished observations of nine Pseudo-nitzschia taxa known as potential domoic acid (DA) producers have been used to outline their geographical distribution.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”.     

Host-specific adaptation governs the interaction of the marine diatom,Pseudo-nitzschia and their microbiota

The association of phytoplankton with bacteria is ubiquitous in nature and the bacteria that associate with different phytoplankton species are very diverse. The influence of these bacteria in the physiology and ecology of the host and the evolutionary forces that shape the relationship are still not understood. In this study, we used the Pseudo-nitzschia–microbiota association to determine (1) if algal species with distinct domoic acid (DA) production are selection factors that structures the bacterial community, (2) if host-specificity and co-adaptation govern the association, (3) the functional roles of isolated member of microbiota on diatom–hosts fitness and (4) the influence of microbiota in changing the phenotype of the diatom hosts with regards to toxin production. Analysis of the pyrosequencing-derived 16S rDNA data suggests that the three tested species of Pseudo-nitzschia, which vary in toxin production, have phylogenetically distinct bacterial communities, and toxic Pseudo-nitzschia have lower microbial diversity than non-toxic Pseudo-nitzschia. Transplant experiments showed that isolated members of the microbiota are mutualistic to their native hosts but some are commensal or parasitic to foreign hosts, hinting at co-evolution between partners. Moreover, Pseudo-nitzschia host can gain protection from algalytic bacteria by maintaining association with its microbiota. Pseudo-nitzschia also exhibit different phenotypic expression with regards to DA production, and this depends on the bacterial species with which the host associates. Hence, the influences of the microbiota on diatom host physiology should be considered when studying the biology and ecology of marine diatoms.     

Domoic acid exposure in pygmy and dwarf sperm whales (Kogia spp.) from southeastern and mid-Atlantic U.S. waters

The neurotoxin domoic acid (DA) was detected in urine and fecal samples recovered from pygmy sperm whales (Kogia breviceps) and dwarf sperm whales (Kogia sima) stranding along the U.S. Atlantic coast from 1997 to 2008. Of the 41 animals analyzed from Virginia, North Carolina, South Carolina and Florida, 24 (59%) tested positive for DA at concentrations of 0.4–1.8 ng/mL in urine and 12–13,566 ng/g in feces as determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Feces appeared to be the best indicator of DA exposure in Kogia spp., with 87% of all fecal samples analyzed testing positive for this toxin. Additional stranded animals (n = 40) representing 11 other cetacean species were recovered from the same region between 2006 and 2008 and analyzed by LC–MS/MS, however DA was not detected in any of these individuals. DA is produced naturally by diatoms in the genus Pseudo-nitzschia. Although blooms of DA-producing Pseudo-nitzschia have been associated with repeated large-scale marine mammal mortalities on the west coast of the U.S., there is no documented history of similar blooms on the southeast U.S. coast, and there were no observed Pseudo-nitzschia blooms in the region associated with any of these strandings. The feeding habits of Kogia spp. are poorly documented; thus, the vector(s) for DA exposure to these deep-diving species remains to be identified. Toxin accumulation in these pelagic whale species may be an indication of cryptic harmful algal bloom activity in offshore areas not currently being monitored. This study highlights the need for a better understanding of the role of toxigenic algae in marine mammal morbidity and mortality globally.     

Release and degradation of amnesic shellfish poison from decaying Pseudo-nitzschia multiseries in presence of bacteria and organic matter

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at least 20 days with 1× or 4× bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a potential future mitigation technique.     

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.     

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.     

Interaction between bacteria and the domoic-acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle; can bacteria produce domoic acid autonomously?

The interaction between bacteria and phytoplankton is increasingly becoming recognised as an important factor in the physiology of toxin production and the dynamics of harmful algal blooms (HABs). Bacteria can play a direct or indirect role in the production of biotoxins once solely attributed to microalgae. Evidence implicating bacteria as an autonomous source paralytic shellfish poisoning biotoxins raises the question of autonomous bacterial toxigenesis of the neurotoxin domoic acid (DA), the cause of amnesic shellfish poisoning. Here, we examine whether the previously observed bacterial enhancement of DA production by Pseudo-nitzschia multiseries (Hasle) Hasle may be attributable to independent biotoxin production by the extra-cellular bacteria associated with this diatom. The growth and toxicity of six cultures of xenic P. multiseries clone CLN-1 were followed for 24 days. Up to day 14 (mid-stationary phase), DA production was not statistically different among culture flasks. On day 14, P. multiseries cells were removed by gentle filtration from a set of triplicate flasks, leaving the bacteria in the filtrate. Following the removal of the algal cells, DA in the filtrate ceased to increase. Instead, DA levels continuously declined. A follow-up experiment determined that this was likely caused by photodegradation rather than by bacterial degradation. We conclude that after removing P. multiseries cells, the extra-cellular bacteria remaining in the filtrate were incapable of autonomous DA toxigenesis, even in the presence of P. multiseries exudates. However, scanning electron microscopy revealed that P. multiseries cells harboured epiphytic bacteria, the importance of which can still not be ruled out in DA production.     

Are pelagic seabirds exposed to amnesic shellfish poisoning toxins?

Marine birds have been hypothesized to be underreported victims of harmful algal blooms (HABs). Toxic blooms of Pseudo-nitzschia spp., the primary amnesic toxin producer microalgae, domoic acid (DA) are known to cause massive mortalities of coastal seabirds and marine mammals around the world. However, these fatalities are only detected when birds die nearby the coastline and little is known about possible outbreaks of pelagic seabirds in oceanic areas. Here we aim to understand whether pelagic seabirds are exposed to amnesic shellfish poisoning (ASP) toxins. For this purpose, we tracked pelagic seabirds feeding on small epipelagic fish and squid, reported to be vectors of DA, which are obtained in high productivity zones where intense Pseudo-nitzschia blooms regularly occur. In particular, we tracked Cory’s (Calonectris borealis) and Scopoli’s (C. diomedea) shearwaters breeding in Gran Canaria (Canary Is.) and in Menorca (Balearic Is.) and feeding on the Canary Current region and the Catalonian coast, respectively. We sampled birds for blood at the recovery of the GPS (Global Positioning System) and analyzed it for DA determination by Liquid Chromatography coupled with Tandem Mass Spectrometry (LC–MS/MS). Among the 61 samples analyzed from Gran Canaria, and 87 from Menorca, 31 (50.8%) and 28 (32.2%) from each location presented detectable levels of DA ranging 1.0–10.6 ng mL⁻¹. This work reveals that DA can be detected at variable levels in the blood of ASP-asymptomatic shearwaters and suggests a chronic exposure of shearwaters to DA, highlighting the need for further studies on DA effects. These results are of high relevance due to the vulnerability of these marine birds, which populations are in continuous decline. Since global warming is expected to alter and increase the occurrence of HABs, marine toxins might become an additional stressor for seabirds and exacerbate the already precarious conservation status of many species.     

Pseudo-nitzschia physiological ecology,phylogeny, toxicity,monitoring and impacts on ecosystem health

Over the last decade, our understanding of the environmental controls on Pseudo-nitzschia blooms and domoic acid (DA) production has matured. Pseudo-nitzschia have been found along most of the world's coastlines, while the impacts of its toxin, DA, are most persistent and detrimental in upwelling systems. However, Pseudo-nitzschia and DA have recently been detected in the open ocean's high-nitrate, low-chlorophyll regions, in addition to fjords, gulfs and bays, showing their presence in diverse environments. The toxin has been measured in zooplankton, shellfish, crustaceans, echinoderms, worms, marine mammals and birds, as well as in sediments, demonstrating its stable transfer through the marine food web and abiotically to the benthos. The linkage of DA production to nitrogenous nutrient physiology, trace metal acquisition, and even salinity, suggests that the control of toxin production is complex and likely influenced by a suite of environmental factors that may be unique to a particular region. Advances in our knowledge of Pseudo-nitzschia sexual reproduction, also in field populations, illustrate its importance in bloom dynamics and toxicity. The combination of careful taxonomy and powerful new molecular methods now allow for the complete characterization of Pseudo-nitzschia populations and how they respond to environmental changes. Here we summarize research that represents our increased knowledge over the last decade of Pseudo-nitzschia and its production of DA, including changes in worldwide range, phylogeny, physiology, ecology, monitoring and public health impacts.     

A shift in the dominant toxin-producing algal species in central California alters phycotoxins in food webs

In California, the toxic algal species of primary concern are the dinoflagellate Alexandrium catenella and members of the pennate diatom genus Pseudo-nitzschia, both producers of potent neurotoxins that are capable of sickening and killing marine life and humans. During the summer of 2004 in Monterey Bay, we observed a change in the taxonomic structure of the phytoplankton community—the typically diatom-dominated community shifted to a red tide, dinoflagellate-dominated community. Here we use a 6-year time series (2000–2006) to show how the abundance of the dominant harmful algal bloom (HAB) species in the Bay up to that point, Pseudo-nitzschia, significantly declined during the dinoflagellate-dominated interval, while two genera of toxic dinoflagellates, Alexandrium and Dinophysis, became the predominant toxin producers. This change represents a shift from a genus of toxin producers that typically dominates the community during a toxic bloom, to HAB taxa that are generally only minor components of the community in a toxic event. This change in the local HAB species was also reflected in the toxins present in higher trophic levels. Despite the small contribution of A. catenella to the overall phytoplankton community, the increase in the presence of this species in Monterey Bay was associated with an increase in the presence of paralytic shellfish poisoning (PSP) toxins in sentinel shellfish and clupeoid fish. This report provides the first evidence that PSP toxins are present in California's pelagic food web, as PSP toxins were detected in both northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax). Another interesting observation from our data is the co-occurrence of DA and PSP toxins in both planktivorous fish and sentinel shellfish. We also provide evidence, based on the statewide biotoxin monitoring program, that this increase in the frequency and abundance of PSP events related to A. catenella occurred not just in Monterey Bay, but also in other coastal regions of California. Our results demonstrate that changes in the taxonomic structure of the phytoplankton community influences the nature of the algal toxins that move through local food webs and also emphasizes the importance of monitoring for the full suite of toxic algae, rather than just one genus or species.     

Hypoxia and seasonal temperature: Short-term effects and long-term implications for Acartia tonsa dana

Far more attention has been given to the short-term lethal impacts of reduced dissolved oxygen on commercially important fish and crabs than to the long-term sublethal impacts on these same species, or on lower trophic levels. This study demonstrates that chronic, sublethal effects of hypoxia on the copepod Acartia tonsa, a critical component of many pelagic coastal food webs, can lead to significant decreases in population growth. The results of laboratory experiments conducted at 15 °C (winter) and 25 °C (summer), under conditions of normoxia (Controls), sublethal hypoxia (1.5 ml l^− 1) and lethal hypoxia (0.7 ml l^− 1) show that egg production female^− 1 day^− 1 was significantly lower at 0.7 ml l^− 1 compared to Controls at both temperatures, while egg production female^− 1 day^− 1 was significantly lower at 1.5 ml l^− 1 compared to controls in both summer experiments and in one of the two winter experiments. Survival was significantly decreased in the 0.7 ml l^− 1 treatment compared to Controls and the 1.5 ml l^− 1 treatment. Copepods developed more slowly and matured at smaller adult body sizes at both temperatures under both lethal and sublethal hypoxia compared to normoxia. Under summer temperatures, egg production was reduced by hypoxia exposure on two counts: (1) exposure to hypoxia during development resulted in smaller adults, which translated into lower egg production, and (2) egg production was still significantly lower in hypoxia treatments compared to Controls even when differences in body size were taken into account. While copepods collected in winter and exposed to winter temperatures and hypoxia also matured at smaller body sizes than copepods exposed to normoxia, egg production in winter was almost entirely attributable to this reduction in body size. These results suggest that coastal hypoxia may have a significantly greater impact in the summer months, when copepod populations are most abundant and growing at their most rapid rate of the year. With the anticipated increases in global temperatures, hypoxia may have even greater impacts on pelagic food webs.     

Convergent evolution of morphological and ecological traits in the open-habitat chat complex (Aves, Muscicapidae: Saxicolinae)

Snails in the closely related trochid genera Phorcus Risso, 1826 and Osilinus Philippi, 1847 are ecologically important algal grazers in the intertidal zone of the northeastern Atlantic Ocean and Mediterranean Sea. Here we present the first complete molecular phylogeny for these genera, based on the nuclear 28S rRNA gene and the mitochondrial 16S rRNA and COI genes, and show that the current classification is erroneous. We recognize nine species in a single genus, Phorcus: estimated by BEAST analysis, this arose 30 (±10) Ma; it consists of two subgenera, Phorcus and Osilinus, which we estimate diverged 14 (±4.5) Ma. Osilinus kotschyi, from the Arabian and Red Seas, is not closely related and is tentatively referred to Priotrochus Fischer, 1879. Our phylogeny allows us to address biogeographical questions concerning the origins of the Mediterranean and Macaronesian species of this group. The former appear to have evolved from Atlantic ancestors that invaded the Mediterranean on several occasions after the Zanclean Flood, which ended the Messinian Salinity Crisis 5.3 Ma; whereas the latter arose from several colonizations of mainland Atlantic ancestors within the last 3 (±1.5) Ma.     

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods.     

First record of the dynamics of domoic acid producing Pseudo-nitzschia spp. in Indonesian waters as a function of environmental variability

Within the past few decades, harmful algal blooms (HABs) have occurred frequently in Indonesian waters, resulting in environmental degradation, economic loss and human health problems. So far, HAB related studies mainly addressed ecological traits and species distribution, yet toxin measurements were virtually absent for Indonesian waters. The aim of the present study was to explore variability of the potentially toxic marine diatom genus Pseudo-nitzschia, as well as its neurotoxin domoic acid as a function of environmental conditions in Ambon Bay, eastern Indonesia. Weekly phytoplankton samples, oceanographic (CTD, nutrients) and meteorological (precipitation, wind) parameters were analyzed at 5 stations in the bay during the dry and wet seasons of 2018. Liquid chromatography – tandem mass spectrometry (LC–MS/MS) was used to detect particulate DA (pDA). Vegetative cells of Pseudo-nitzschia spp. and pDA were found in 98.6% and 51.4% of the samples, respectively. pDA levels were low, yet detected throughout the campaign, implying that Ambon Bay might potentially be subject to amnesic shellfish poisoning. The highest levels of both Pseudo-nitzschia spp. cell abundance and pDA were found in the wet season, showing a strong positive correlation between both parameters, compared to the dry season, (r = 0.87 and r = 0.66 (p < 0.01), respectively). Statistical analyses revealed that temperature and mixed layer depth positively correlated with Pseudo-nitzschia spp. and pDA during the dry season, while ammonium showed positive correlations in both seasons. This study represents the first successful investigation of the presence and variability of Pseudo-nitzschia spp. and its neurotoxin DA in Indonesian waters.     

Characterization of a toxic Pseudo-nitzschia spp. bloom in the Northern Gulf of Mexico associated with domoic acid accumulation in fish

A toxic bloom of Pseudo-nitzschia spp. was observed in the Alabama coastal waters of the northern Gulf of Mexico (NGOM) in June 2009 that resulted in the accumulation of domoic acid (DA) in fish. The bloom initiated following a large storm event that likely caused increased groundwater discharge 16–20 days prior to peak densities. Eleven sites, located in littoral shoreline waters and inshore embayments spanning the entire Alabama NGOM coastline, were sampled during peak densities to assess Pseudo-nitzschia species composition and toxicity, and associated water-quality parameters. Small fish (0.27–11.9 g body weight) were collected at six of these sites for analysis of DA content. High Pseudo-nitzschia spp. densities (8.27 × 10⁴–5.05 × 10⁶ cell l⁻¹) were detected at eight sites located in the littoral shoreline and particulate DA was detected at six of these littoral sites (48.0–540 pg ml⁻¹). The bloom consisted primarily (>90%) of Pseudo-nitzschia subfraudulenta, a species previously characterized as forming only a minor component of Pseudo-nitzschia assemblages and not known to produce DA. Pseudo-nitzschia spp. were at low densities or not detected at the inshore sites and DA was detected at these sites. Pseudo-nitzschia spp. density varied along an estuarine gradient, with greater densities occurring in the most saline, clear, and nutrient-poor waters. Cell density was strongly and negatively correlated with silicate (Si) concentrations and the ratios of silicate to dissolved inorganic nitrogen and phosphate (Si:DIN and Si:PO₄). Cell toxin quota was negatively correlated with phosphate, and strongly and positively correlated with the ratio of total nitrogen to total phosphorus (TN:TP). These relationships are consistent with previous observations that indicate Pseudo-nitzschia spp. density and toxicity are likely to be greater in high salinity, high irradiance, and nutrient-poor waters. DA was detected in 128 of 131 (98%) of the fish collected, which included seven primary and secondary consumer species. This is the first demonstration of trophic transfer of DA in this region of the NGOM, indicating that toxic blooms of Pseudo-nitzschia spp. in Alabama coastal waters have the potential to transfer DA to recreationally and commercially important fish species.     

Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9 kDa) and receptor binding BinB (51.4 kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197 M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl β-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC₅₀ dose of 82.3 and 66.9 ng ml⁻¹, respectively, at 48 h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly β strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.     

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

The effect of food limitation on gonad development and egg production of the planktonic copepod Calanus finmarchicus

Calanus finmarchicus, one of the dominant copepods species of the North Atlantic, often encounters low food concentration or quality during the reproductive period; however, our knowledge on the effect of these conditions on reproduction processes is scarce. The present study combines experiments with histological observations to describe the response of C. finmarchicus to limited food focussing on (1) oocyte maturation processes, (2) gonad morphology, (3) egg production rates as a function of spawning frequency and clutch size, and (4) the fuelling of egg production by measuring carbon and nitrogen content of the females. In the laboratory, C. finmarchicus females were exposed to 0, 10, 50, 150 or >300 μg C l⁻¹ for several days. To account for food quality and season, reproductive activity was compared in April and July 1999 between females feeding on diatoms or dinoflagellates. The effect of feeding history was studied in February with females fed and starving prior to the experiment. Feeding conditions had severe effects on oocyte maturation process. Hence, egg production varied significantly with food concentration and quality, season and feeding history due to variation in both clutch size and spawning frequency. Clutch size differed by a factor of 2-4 between food limited and well fed females, and is thus an important parameter for modelling egg production. Changes in clutch size were related to changes of the number of maturing oocytes in the females gonads indicating that the latter can be used to precise the prediction of egg production from preserved samples. The proportion of females carrying at least some mature oocytes was relatively high at low food availability. Apparently, these females used internal body reserves as the carbon and nitrogen content decreased significantly under these conditions. These results indicate that C. finmarchicus embarks on the strategy to enable reproduction in all or many females of a population at low rates when feeding conditions are unfavourable.