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.     

The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters

The dynamics of Dinophysis acuminata and its associated diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX1) as well as pectenotoxins (PTXs), were investigated within plankton and shellfish in Northport Bay, NY, USA, over a four year period (2008–2011). Over the course of the study, Dinophysis bloom densities ranged from ~10⁴ to 10⁶ cells L⁻¹ and exceeded 10⁶ L⁻¹ in 2011 when levels of total OA, total DTX1, and PTX in the water column were 188, 86, and 2900 pg mL⁻¹, respectively, with the majority of the DSP toxins present as esters. These cell densities exceed – by two orders of magnitude – those previously reported within thousands of samples collected from NY waters from 1971 to 1986. The bloom species was positively identified as D. acuminata via scanning electron microscopy and genetic sequencing (cox1 gene). The cox1 gene sequence from the D. acuminata populations in Northport Bay was 100% identical to D. acuminata from Narragansett Bay, RI, USA and formed a strongly supported phylogenetic cluster (posterior probability = 1) that included D. acuminata and Dinophysis ovum from systems along the North Atlantic Ocean. Shellfish collected from Northport Bay during the 2011 bloom had DSP toxin levels (1245 ng g⁻¹ total OA congeners) far exceeding the USFDA action level (160 ng g⁻¹ total OA of shellfish tissue) representing the first such occurrence on the East Coast of the U.S. D. acuminata blooms co-occurred with paralytic shellfish poisoning (PSP) causing blooms of Alexandrium fundyense during late spring each year of the study. D. acuminata cell abundances were significantly correlated with levels of total phytoplankton biomass and Mesodinium spp., suggesting food web interactions may influence the dynamics of these blooms. Given that little is known regarding the combined effects of DSP and PSP toxins on human health and the concurrent accumulation and depuration of these toxins in shellfish, these blooms represent a novel managerial challenge.     

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.     

Co-occurrence of Dinophysis tripos and pectenotoxins in Argentinean shelf waters

The species Dinophysis tripos is a widely distributed marine dinoflagellate associated with diarrheic shellfish poisoning (DSP) events, which has been recently identified as a pectenotoxin (PTX) producer. In two sampling expeditions carried out during austral autumns 2012 and 2013 along the Argentine Sea (≈38–56° S), lipophilic phycotoxins were measured by tandem mass spectrometry coupled to liquid chromatography (LC–MS/MS) in size-fractionated plankton samples together with microscopic analyses of potentially toxic phytoplankton. PTX-2, PTX-11 and PTX-2sa were recurrently detected in the 50–200 μm fractions, in association to D. tripos. PTX-2 was also widely distributed among the 20–50 μm fractions, mostly related to Dinophysis acuminata. Okadaic acid or its analogs were not detected in any sample. This is the first report of D. tripos related to PTX in the Argentine Sea and the first record of PTX-11 and PTX-2sa for this area. The morphological variability of D. tripos, including the presence of intermediate, small and dimorphic cells, is described. Also, the micro- and mesoplanktonic potential grazers of Dinophysis spp. were explored.     

Toxin production,retention, and extracellular release by Dinophysis acuminata during extended stationary phase and culture decline

Dinophysis spp. produce diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins. The extent to which the dinoflagellate cells retain their toxicity in stationary phase, a period when cells are most toxic, and their transition into cell death is not known. Here we present results on the production, recycling, retention, and release of toxins from a monoculture of Dinophysis acuminata during these two important stages. Once stationary phase was reached, cultures were divided between light and dark treatments to identify if light influenced toxin dynamics. Light was required for long-term cell maintenance (>2 months) of D. acuminata in the absence of prey, however, in the dark, cells in stationary phase survived on reserves alone for four weeks before beginning to decline. Cells maintained relatively constant levels of intracellular OA (0.39 ± 0.03 pg/cell, 0.44 ± 0.05 pg/cell), DTX1 (0.45 ± 0.09 pg/cell, 0.64 ± 0.10 pg/cell) and PTX2 (10.4 ± 1.4 pg/cell, 11.0 ± 1.9 pg/cell) in the dark and light treatments, respectively, throughout stationary phase and into culture decline. Toxin production was only apparent during late exponential and early stationary growth when cells were actively dividing. In general, the concentration of dissolved (extracellular) toxin in the medium significantly increased upon culture aging and decline; cells did not appear to be actively or passively releasing toxin during stationary phase, but rather extracellular release was likely a result of cell death. Light availability did not have an apparent effect on toxin production, quotas, or intracellular vs. extracellular distribution. Together these results suggest that a bloom of D. acuminata would retain its cellular toxicity or potency as long as the population is viable, and that cells under conditions of low light (e.g., at the boundary or below euphotic zone) and/or minimal prey could maintain toxicity for extended periods.     

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin. author for correspondence     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

Harmful effects of Dinophysis to the ciliate Mesodinium rubrum: Implications for prey capture

Toxigenic Dinophysis spp. are obligate mixotrophic dinoflagellates that require a constant supply of prey—Mesodinium rubrum—to achieve long-term growth by means of kleptoplasty. Mesodinium rubrum is, however, a fast moving, jumping ciliate exhibiting an effective escape response from suspensivorous predators. In the present study, a series of laboratory experiments evaluating the motility and survival of M. rubrum in the presence of Dinophysis cells and/or substances contained in their culture medium was designed, in order to assess the mechanisms involved in prey capture by Dinophysis spp. Cell abundance of M. rubrum decreased in the presence of Dinophysis cf. ovum cells producing okadaic acid (OA; up to 7.94 ± 2.67 pg cell⁻¹) and smaller amounts of dinophysistoxin-1 (DTX-1) and pectenotoxin-2 (PTX-2). Prey capture was often observed after the ciliate had been attached to adhesive “mucus traps”, which only appeared in the presence of Dinophysis cells. Before being attached to the mucus traps, M. rubrum cells reduced significantly their swimming frequency (from ∼41 to 19 ± 3 jumps min⁻¹) after only 4 h of initial contact with D. cf. ovum cells. M. rubrum survival was not affected in contact with purified OA, DTX-1 and PTX-2 solutions, but decreased significantly when the ciliate was exposed to cell-free or filtered culture medium from both D. cf. ovum and D. caudata, the latter containing moderate concentrations of free eicosapentaenoic acid and docosahexaenoic acid. The results thus indicate that Dinophysis combines the release of toxic compounds other than shellfish toxins, possibly free PUFAs, and a “mucus trap” to enhance its prey capture success by immobilizing and subsequently arresting M. rubrum cells.     

Effects of different levels of N- and P-deficiency on cell yield,okadaic acid,DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate Prorocentrum lima

Prorocentrum lima (Ehrenberg) Dodge is a cosmopolitan epiphytic dinoflagellate that produces biotoxins which are causative of diarrhetic shellfish poisoning (DPS). Here we report on effects of several nitrogen (N) and phosphorous (P) limited conditions on cell yield, okadaic acid (OA) and dinophysistoxin-1 (DTX-1) contents synoptically with cell carbohydrate, exopolysaccharide (EPS) and cell protein concentrations in a P. lima strain isolated from the Sacca di Goro lagoon (Northern Adriatic Sea). Batch culture experiments were set to assess changes induced by four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P, and 1/50-P) with respect to control nutrient conditions (f/2 medium; NO₃⁻ and PO₄³⁻ concentrations: 883 and 36.3 μM, respectively; N/P ratio: 24). Low nutrients availability determined lower cell yields starting from 1/10-N and 1/3-P levels and the pattern observed was dependent on nutrient dynamics, as shown by N and P analyses performed in culture media during growth. Final cell yield decreased significantly up to 4.7- and 5.6-fold under 1/50-N and 1/50-P-limited levels with respect to control values, while cell volume increased with respect to control (up to 30% and 35% for N- and P-experiment, respectively). On overall, OA concentration ranged from 6.69 to 15.80 pg cell⁻¹, while DTX-1 ranged from 0.12 and 0.39 pg cell⁻¹ resulting in unusual high OA/DTX-1 ratios. The study indicates that protein, carbohydrate, EPS, and toxin concentrations displayed remarkable different patterns under the two kinds of nutrient deficiencies. The main differences can be summarised as: (i) significant decrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no significant changes in protein concentration under P-limitation; (ii) significant increase of cell carbohydrate (up to 2.8-fold and 3.4-fold for N- and P-limitation, respectively) and cell OA amount (up to 1.9-fold and 2.3-fold, N- and P-limitation, respectively) under both N- and P-limitations, however different level-deficiency dependent patterns were displayed under the two nutrient conditions; (iii) significant increase of EPS concentration (up to 6.50-fold) under P-limitation, conversely no significant changes in EPS concentration under N-limitation. Data presented here indicate that P. lima adopts different eco-physiological strategies to face N-limitation or P-limitation. This study provides the first evidence for an increase in EPS production by benthic dinoflagellates under P-limited conditions; the ecological significance of this increase is discussed.     

Toxigenic phytoplankton and concomitant toxicity in the mussel Choromytilus meridionalis off the west coast of South Africa

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5 km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella; (2) several species of Dinophysis, including Dinophysis acuminata, Dinophysis fortii, Dinophysis hastata and Dinophysis rotundata; and (3) members of the diatom genus Pseudo-nitzschia. Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis, but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12 d⁻¹. Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis. Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.     

Binding of diarrheic shellfish poisoning toxins to okadaic acid binding proteins purified from the sponge Halichondria okadai

Okadaic acid (OA) and dinophysistoxin-1 (DTX1) cause diarrheic shellfish poisoning. This article examines the biochemical interactions of the two toxins with novel okadaic acid binding proteins (OABPs) 2.1 and 2.3, originally isolated from the marine sponge Halichondria okadai. First, recombinant OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-³H]OA and the recombinant OABP 2.1 or 2.3 demonstrated the dissociation constant K_d of 1.30 ± 0.56 nM and 1.54 ± 0.35 nM, respectively. Binding of [24-³H]okadaic acid to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity in mouse leukemia P388 cells was inhibited in the presence of the recombinant OABPs 2.1 and 2.3 with an EC₅₀ of 92 ± 8.4 nM and 87 ± 13 nM, respectively. These results suggest that the blockage of OA-induced cytotoxicity by OABPs 2.1 and 2.3 may be involved in regulating symbiotic relationships present in the sponge H. okadai.     

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.     

The relationship between Pseudo-nitzschia (Peragallo) and domoic acid in Scottish shellfish

The diatom genus Pseudo-nitzschia (Peragallo) associated with the production of domoic acid (DA), the toxin reposnsible for amnesic shellfish poisoning, is abundant in Scottish waters. A two year study examined the relationship between Pseudo-nitzschia cells in the water column and DA concentration in blue mussels (Mytilus edulis) at two sites, and king scallops (Pecten maximus) at one site. The rate of DA uptake and depuration differed greatly between the two species with M. edulis whole tissue accumulating and depurating 7 μg g⁻¹ (now expressed as mg kg⁻¹) per week. In contrast, it took 12 weeks for DA to depurate from P. maximus gonad tissue from a concentration of 68 μg g⁻¹ (now mg kg⁻¹) to <20 μg g⁻¹ (now mg kg^‐1). The DA depuration rate from P. maximus whole tissue was <5% per week during both years of the study. Correlations between the Pseudo-nitzschia cell densities and toxin concentrations were weak to moderate for M. edulis and weak for P. maximus. Seasonal diversity on a species level was observed within the Pseudo-nitzschia genus at both sites with more DA toxicity associated with summer/autumn Pseudo-nitzschia blooms when P. australis was observed in phytoplankton samples. This study reveals the marked difference in DA uptake and depuration in two shellfish species of commercial importance in Scotland. The use of these shellfish species to act as a proxy for DA in the environment still requires investigation.     

First evidence of cell deformation occurrence during a Dinophysis bloom along the shores of the Gulf of Tunis (SW Mediterranean Sea)

Never before observed or cited in Dinophysis studies, deformations in Dinophysis acuminata and Dinophysis sacculus are reported throughout their cellular division phases (cytokinesis, and sulcal list regeneration) in 5 in situ cell cycle studies in the Punic harbors of Carthage (northern Tunisia). Two types of deformation were observed: invaginations in the ventral and dorsal margin and protuberances at the base of the left sulcal list. No virus or bacteria were detected with Syber green stain. In situ division rates (μ) varied among seasons and stations for the same species. D. acuminata exhibited moderate (0.22 day⁻¹) to high (0.68 day⁻¹) μ rates which were however very low (0.02–0.17 day⁻¹) for D. sacculus in autumn and moderate (0.21–0.35 day⁻¹) in late spring. In 2009 the seasonal distribution of Dinophysis indicates maximum Dinophysis cf. ovum abundance in March and a high number of D. acuminata in early June, while in 2010 maximum abundance of the same species was found in mid-June.Molecular and genetic studies and staining with specific fluorescent strains should be addressed to hopefully explain these Dinophysis cell deformations during their in situ division.     

Influence of inoculation with plant growth promoting rhizobacteria (PGPR) on tomato plant growth and nematode reproduction under greenhouse conditions

Numerous species of soil bacteria which flourish in the rhizosphere of plants or around plant tissues stimulate plant growth and reduce nematode population by antagonistic behavior. These bacteria are collectively known as PGPR (plant growth promoting rhizobacteria). The effects of six isolates of PGPR Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus cereus, were studied on tomato plant growth and root knot nematode reproduction after 45 days from nematode infection. The highest number of shoot dry weight/g (43.00 g) was detected in the plant treated with S. marcescens; then P. putida (34.33 g), B. amyloliquefaciens (31.66 g), P. fluorescens (30.0 g), B. subtilis (29.0 g), B. cereus (27.0 g) and nematode alone (untreated) 20 g/plant. While the highest number of plant height was observed when plant was treated with S. marcescens, P. fluorescens, P. putida, B. amyloliquefaciens and P. putida 52.66, 50.66, 48 and 48 cm respectively. No significant differences were seen between previous treatments but only had significant differences compared with untreated plant. The highest number of fruit/plant was observed when plants were treated with S. marcescens (10.66), then B. amyloliquefaciens (8.66), P. putida (8), P. fluorescens (8) and B. cereus (7.66). No significant differences between the last 4 treatments, but all had significant differences compared with untreated plants. The highest weight of plant yield (g) was observed with S. marcescens (319.6 g/plant) and the lowest weight of plant yield was observed in plants treated with nematode alone (untreated). On the other hand, the lowest numbers of J₂/10 g of soil (78), galls/root, (24.33) galls/root, egg masses/root (12.66) and egg/egg masses were observed in the plants treated with S. marcescens.     

Scales characterising a high density thin layer of Dinophysis acuta Ehrenberg and its transport within a coastal jet

An investigation into the distribution of Dinophysis spp. in coastal waters off the south coast of Ireland was carried out in July 2007. Dinophysis acuta was present as a sub surface layer containing up to 55,000 cells L⁻¹. The population had a high percentage of viable cells (mean: 89%; median: 94%; n = 24) with a high specific division rate (∼0.55 d⁻¹). The layer, of approximately 5 m thickness, did not coincide with the fluorescence maximum and was present as a patch of horizontal dimension less than 10 km × 7 km. Both conventional and towed undulating CTD used in conjunction with high vertical resolution sampling methods showed the patch of Dinophysis to move with a similar speed and direction as the coastal flow, which ran parallel to the coast in the form of a coastal jet with speed of the order of 6.5–7 km day⁻¹. The implications of the alongshore transport of populations of harmful species in coastal jets for monitoring programmes and predictive models are discussed.     

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.     

Whole body protection against lethal ionizing radiation in mice by REC-2001: A semi-purified fraction of Podophyllum hexandrum

The current study has concentrated on assessment of the radioprotective potential of REC-2001, a semi-purified fraction of rhizomes of Podophyllum hexandrum, in Swiss albino Strain ‘A’ mice exposed to 10 Gy whole-body gamma radiation. Animals were treated with 10 and 15 mg/kg b wt (i.p.) of REC-2001 1 h prior to exposure to a lethal dose of γ-radiation (10 Gy) and observed upto 30 days. For analysis of maximum tolerable dose (MTD), LD₅₀ and acute toxic dose, different concentrations of the extract were administered to animals and their mortality and morbidity status was observed upto 72 h and one week, respectively. Dose reduction factor (DRF) was determined by exposing REC-2001 pre-treated mice to supra-lethal doses of γ-radiation. Endogenous spleen colony forming units (CFU), DNA strand breaks in thymocytes (alkaline halo assay) and lipid degradation was studied to understand the mechanism of radioprotection . A single dose of REC-2001 (10 and 15 mg/kg b wt i.p.) exhibited >90% survival in the pre-treated irradiated group versus no survival in radiation control group. Single doses of upto 75 mg/kg b wt (i.p.) did not cause any mortality (MTD) in mice. REC-2001, a dose of 90 mg/kg b wt, resulted in 50% mortality (LD₅₀), while the LD₁₀₀ was 115 mg/kg b wt REC-2001 exhibited a DRF of 1.62. CFU counts in the REC-2001 treated group were found significantly high (5.33/spleen) as compared to controls. Exposure of thymocytes to 10 Gy radiation resulted in increased halo diameter (45±3 μm) in comparison to untreated controls (8±1 μm). REC-2001 administration (500 μg/ml) decreased the halo diameter to 15±2 μm. Radiation-induced lipid degradation was also inhibited by REC-2001. The present study has revealed that REC-2001 is a promising radioprotective fraction that can be effectively used against lethal doses of γ-radiation after further investigations in higher animal models.     

Molt cycle related changes and effect of short term starvation on the biochemical constituents of the blue swimmer crab Portunus pelagicus

Synthesis and hardening of a new exoskeleton are essential to the arthropod molting process. The present study emphasizes the variations in the levels of hemolymph total free sugars, hepatopancreas glycogen and cuticular proteins during the molting stages of Portunus pelagicus. It also reports the effect of short-term starvation conditions on the biochemical constituents of the hemolymph. Intermolt crabs were subjected to 6 days of starvation and hemolymph samples were taken. Standard biochemical procedures were followed toward the quantification of total proteins, total free sugars and total lipids. The total free sugar level in the hemolymph of P. pelagicus was observed to increase during early premolt D₀ (3.108 ± 0.032 g/ml) and a gradual decrease till late postmolt B stage (0.552 ± 0.124 g/ml), suggesting the need for total free sugars to provide energy for the apolysis process. Increase in the levels of hepatopancreas glycogen was observed from 1225 ± 0.04 μg/mg in early premolt D₀ to 1700 ± 0.3 μg/mg in late premolt D_2–3. This is in correlation with the decreased levels of free sugars during premolt stages, suggesting an increase in the storage of glycogen reserves in the hepatopancreas. Cuticular proteins increased during stage B (2.702 ± 0.093 g/ml) and stage C (3.065 ± 0.012 g/ml), indicating exoskeleton hardening and mineralization. Results of the starvation studies clearly showed a steady decline in the level of total free sugars till day 6 (0.099 ± 0.00 g/ml) when compared to the control (8.646 ± 0.08 g/ml). Gradual decrease of total lipids was also observed from the first day of the experiment (6.088 ± 2.44 g/ml) to the last day of the study (0.401 ± 0.20 g/ml) which was 85% lesser than the control (8.450 ± 0.49 g/ml)suggesting the efficient usage of total sugars to consolidate the loss of energy reserves during starvation. The knowledge of Molt-cycle events can be used as a tool for the evaluation of the developmental state providing a morphological reference system for physiological and biochemical studies related to crab aquaculture. Starvation studies enlightens that increasing carbohydrate levels in crab feed together with good protein content could alleviate the natural effects of starvation, improve farm productivity and reduce the deleterious impact of nitrogen pollution generated by rich-protein feeds used in crab farming.