Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and Dinophysis acuminata from New Zealand

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).     

LC–MS/MS analysis of okadaic acid analogues and other lipophilic toxins in single-cell isolates of several Dinophysis species collected in Hokkaido,Japan

Quantification of diarrhetic shellfish poisoning (DSP) toxins (okadaic acid analogues), and other lipophilic toxins in single-cell isolates of the dinoflagellates Dinophysis fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus and D. rotundata, collected in coastal waters Hokkaido, Japan in 2005, was carried out by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoyldinophysistoxin-1 (DTX3), pectenotoxin-1 (PTX1), pectenotoxin-11 (PTX11), pectenotoxin-2 (PTX2), pectenotoxin-6 (PTX6), pectenotoxin-2 seco-acid (PTX2sa), yessotoxin (YTX) and 45-hydroxyyessotoxin (45-OHYTX) were quantified by LC–MS/MS. PTX2 was the dominant toxin in D. acuminata, D. norvegica and D. infundibulus whereas both DTX1 and PTX2 were the principal toxins in D. fortii. None of the toxins were detected in D. mitra, D. rotundata and D. tripos. These results suggest that D. fortii is the most important species responsible for DSP contamination of bivalves in Hokkaido. This is the first finding of PTX2 in D. infundibulus, and confirms the presence of PTX2 in Japanese D. acuminata and D. norvegica collected from natural seawater.     

DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: Differences between picked cells and plankton concentrates

This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell⁻¹) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell⁻¹ in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.     

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on OA and DTX1

It has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads^® SP850, Sepabeads^® SP825L, Amberlite^® XAD4, Dowex^® Optipore^® L-493 and Diaion^® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72 h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media.     

DSP toxin contents inDinophysis fortii and scallops collected at Mutsu Bay,Japan

Cell densities of toxic phytoplankton species responsible for diarrhetic shellfish poisoning (DSP) were monitored at a sampling site in Mutsu Bay, Japan, in 1995.Dinophysis fortii almost completely dominated the toxic phytoplankton community. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) contents in bothD. fortii cells and midgut glands of scallops collected at the same sampling site were determined by HPLC — fluorometry. DTX1 was detected fromD. fortii and scallops. The contents of DTX1 inD. fortii changed markedly during the experimental periods (5–252 pg cell^–1). The highest concentration of DTX1 in the midgut glands of scallops coincided with the period of relatively high cell densities ofD. fortii with the highest content of DTX1 (252 pg cell^–1). The results demonstrate that toxin content in the cells is an important factor affecting the toxicity of shellfish.     

Oxidative metabolism by Thalassiosira weissflogii (Bacillariophyceae) of a diol-ester of okadaic acid, the diarrhetic shellfish poisoning

Previous investigations into the comparative toxicity of the diarrhetic shellfish poisoning (DSP) toxins to Thalassiosira weissflogii (Grun.) Fryxell et Hasle found that this diatom oxidatively metabolized okadaic acid diol‐ester (OA diol‐ester) to a more water‐soluble product. This oxidative transformation of OA diol‐ester by the diatom is significant for two reasons. First, it is known that dinophysistoxin‐4 (DTX‐4), the primary DSP toxin produced by the dinoflagellate Exuviaella lima (Ehr.) Butschli, will be hydrolyzed to the diol‐ester following cell rupture (e.g. ingestion by a predator). Second, it implies that the ester, an uncharged, lipophilic intermediate, can easily enter cells and therefore may play an important role in the uptake and transfer of DSP toxins through the food web. It has been suggested that the water soluble DTX‐4 may also be the form in which DSP toxins are excreted from the producing cell. Therefore, the stability of DTX‐4 was examined when incubated either in fresh seawater medium into which washed cells of E. lima were introduced or in seawater medium conditioned by E. lima cells. Rapid hydrolysis of DTX‐4 to the diol‐ester took place in both cases. Thus, regardless of the route by which DTX‐4 is liberated from the cell, either by cell disruption or excretion, the diol‐ester will be the dominant form of the toxin to challenge associated organisms. To examine the metabolism of OA diol‐ester by T. weissflogii in more detail, serial cultures of the diatom were challenged with OA diol‐ester at a concentration of 2.0 μg·mL^?1. The metabolism and fate of the diol‐ester in both cellular and medium fractions were monitored over 3 days using liquid chromatography with either ultraviolet (LC‐UV) or mass spectrometric (LC‐MS) detection. During the course of the experiment, all of the diol‐ester was metabolized. LC‐MS analysis revealed the presence of multiple oxidative products of OA diol‐ester in the medium fraction, including a carboxylic acid derivative. The major metabolites were isolated in sufficient quantity to permit structural elucidation by NMR and MS. All the metabolites identified resulted from oxidation of the diol‐ester side chain with the primary sites of attack at the terminal, subterminal, and unsaturated carbons. OA was found in both cellular and medium fractions, and its production was directly correlated with the metabolism of the diol‐ester. The relative partitioning of both OA diol‐ester and its oxidation products between cells and medium supports the contention that OA diol‐ester can readily enter cells, be metabolized, and then excreted in more water‐soluble forms.     

DIFFERENCES IN THE PRODUCTION AND EXCRETION KINETICS OF OKADAIC ACID,DINOPHYSISTOXIN‐1, AND PECTENOTOXIN‐2 BETWEEN CULTURES OF DINOPHYSIS ACUMINATA AND DINOPHYSIS FORTII ISOLATED FROM WESTERN JAPAN1

We established clonal cultures of Dinophysis acuminata Clap. et Lachm. and D. fortii Pavill. isolated from western Japan and examined toxin production in them, focusing on intracellular production and extracellular excretion. At the end of incubations, the total amounts of pectenotoxin‐2 (PTX‐2), dinophysistoxin‐1 (DTX‐1), and okadaic acid (OA) in the D. acuminata cultures reached up to 672.7 ± 14.7 (mean ± SD), 88.1 ± 2.8, and 539.3 ± 39.7 ng · mL^?1, respectively, and the excreted extracellular amounts were equivalent to 5.1, 79.5, and 79.5% of the total amounts, respectively. Similarly, at the end of incubations, the total amounts of PTX‐2, DTX‐1, and OA in the D. fortii cultures reached up to 526.6 ± 52.6 (mean ±SD), 4.4 ± 0.4, and 135.9 ± 3.9 ng · mL^?1, respectively, and the excreted extracellular amounts were equivalent to 1.8, 80.1, and 86.6% of the total amounts, respectively. Further, we tested the availability of cell debris and dissolved organic substances that originated from the ciliate prey Myrionecta rubra for growth and toxin production in D. acuminata. Although no significant growth was observed in D. acuminata in the medium containing the cell debris and organic substances originated from M. rubra, the toxicity was significantly greater than that in the control (P < 0.05–0.001); this finding suggested the availability of organic substances for toxin production. However, toxin productivity was remarkably lower than that of Dinophysis species feeding on living M. rubra.     

Production of dinophysistoxin-1 and pectenotoxin-2 by a culture of Dinophysis acuminata (Dinophyceae)

The production of diarrhetic shellfish poisoning toxins (okadaic acid analogues and other lipophilic toxins) by a culture of Dinophysis acuminata, fed with the autotrophic ciliate Myrionecta rubra, was confirmed by LC–MS analysis, and the toxin profile compared with that in the field assemblage of the same species. The growth response of D. acuminata to the density of the food organism was also examined in laboratory experiments. In semi-continuous culture experiments, the growth rates of D. acuminata increased with increasing density of M. rubra and a maximum growth rate of 0.67 per day was calculated. In batch culture experiments; the cellular content of PTX2 and DTX1 were 14.7–14.8 and 2.5–4.8 pg cell^?1, respectively. Okadaic acid, dinophysistoxin-3, pectenotoxin-1, pectenotoxin-6, yessotoxin (YTX) and 45-OHYTX were not detected. PTX2 was detected (cellular toxin content: 22 pg cell^?1), but DTX1 was not detected, in an extract of D. acuminata collected from natural seawater at the same location where the cultured D. acuminata specimens were isolated. These results strongly suggest that D. acuminata produces these toxins during cell growth and that environmental factors influence variations in the toxin composition and specific cellular toxicity.     

The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta

Three out of 4 isolates of the Deuteromycete Metarhizium anisopliae were pathogenic for larvae of the tobacco hornworm, Manduca sexta. The most virulent isolate (ME1) grew sparsely in the insect prior to death and caused paralysis of its host. The other 2 pathogenic isolates killed Manduca larvae more slowly, grew profusely in the haemolymph and did not induce symptoms of toxicosis. Toxicosis is apparently due to the production by the fungus of several cyclodepsipeptide toxins, destruxins (DTX). ME1 produced large quantities of DTX in vitro, while other isolates produced less. Destruxin A (DTX A) was recovered from the haemolymph of paralysed, diseased insects infected with ME1, but not with other isolates. It is suggested that DTX may have a pathogenic role, when the toxins are active in causing disease, or an aggressive role, when they facilitate the establishment of the pathogen.     

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth,photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

The phytotoxicity of selected mycotoxins on mature,germinatingZea mays embryos

Mature maize (Zea mays) embryos were exposed to 5, 10 and 25 µg ml^–1 of deoxynivalenol (DON), zearalenone (ZEA), ochratoxin A (OA) and a mixture of zearalenone and deoxynivalenol (ZEA/DON) for 9 days. DON and the ZEA/DON combination were consistently more inhibitory of the measured parameters than either ZEA or OA. Based on the predicted additive values, it would appear that, in combination, ZEA and DON act synergistically to inhibit root and shoot growth. For ZEA alone, a concentration of 5 µg ml^–1 ZEA was generally inhibitory of root and shoot elongation and fresh mass accumulation, while at 10 and 25 µg ml^–1, this toxin had a stimulatory effect on these parameters. For OA, the measured effects on root and shoot growth at 5 and 25 µg ml^–1 were stimulatory, while at 10 µg ml^–1 OA, an inhibitory effect was observed. For all toxins, inhibitory/stimulatory effects were generally more marked for root parameters than for shoot elongation or mass.Abbreviations ADON acetyldeoxynivalenol - AFB₁ aflatoxin B₁ - DAS diacetyoxyscirpenol - DON deoxynivalenol - FB₁ fumonisin B₁ - FHB Fusaium head blight - MON moniliformin - NIV nivalenol - OA ochratoxin A - ZEA zearalenone     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.     

Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK

Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 10² to 10³ cells g⁻¹ fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

First report of a new rapid assay for diarrhetic shellfish poisoning toxins

Jellett Rapid Testing Ltd. has developed a rapid field test kit to screen for diarrhetic shellfish poisoning (DSP) toxins. The new test provides a qualitative (positive/negative) indication of the presence of okadaic acid (OA) and some of its analogues in about 30 min. It is designed as a screening method for regulatory labs to eliminate negative samples, thereby leaving a smaller number of positive samples to be tested with more sophisticated and time-consuming quantitative methods. Due to its simplicity and speed, the Rapid Test for DSP may eventually be used in other applications such as shellfish harvest management and toxin research. The test is based on easy-to-use lateral flow immunochromatographic (LFI) test strips, which operate the same way as Jellett Rapid Testing's Rapid Tests for paralytic shellfish poisoning (PSP) toxins and amnesic shellfish poisoning (ASP) toxins. The sensitivity of the antibodies to some of the analogues of the DSP family of toxins was investigated using pure compounds from the National Research Council of Canada. In the Rapid Test format, okadaic acid, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2) were detected similarly with 50% reduction in test line color intensity at 5 nM for the solutions applied to test strips. One of the DTX-3 esters eliminated the test line at 500 nM, indicating low cross-reactivity, whereas no effect was observed with one of the brevetoxins (PbTx-3), yessotoxin, gymnodimine, spirolide and pectenotoxins PTX2, PTX11, at concentrations up to 1000 nM. In the ELISA format, the distinction between analogues was more apparent than on test strips. Mid-points were at 8 nM for okadaic acid, and 40 nM and 25 nM for DTX1 and DTX2, respectively.     

Leucine Transport Is Affected by Bacillus thuringiensis Cry1 Toxins in Brush Border Membrane Vesicles from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) Midgut

The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K⁺/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K⁺ gradient. Experiments with the voltage-sensitive fluorescent dye 3,3′-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K⁺ gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.     

Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: Differences between picked cells and plankton concentrates

This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell⁻¹) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell⁻¹ in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.