Paralytic Shellfish Poisoning Toxin-Producing Cyanobacterium Aphanizomenon gracile in Northeast Germany

Neurotoxic paralytic shellfish poisoning (PSP) toxins, anatoxin-a (ATX), and hepatotoxic cylindrospermopsin (CYN) have been detected in several lakes in northeast Germany during the last 2 decades. They are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, and Aphanizomenon. Although no additional sources of PSP toxins and ATX have been identified in German water bodies to date, the observed CYN concentrations cannot be produced solely by Aphanizomenon flos-aquae, the only known CYN producer in Germany. Therefore, we attempted to identify PSP toxin, ATX, and CYN producers by isolating and characterizing 92 Anabaena, Aphanizomenon, and Anabaenopsis strains from five lakes in northeast Germany. In a polyphasic approach, all strains were morphologically and phylogenetically classified and then tested for PSP toxins, ATX, and CYN by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) and screened for the presence of PSP toxin- and CYN-encoding gene fragments. As demonstrated by ELISA and LC-MS, 14 Aphanizomenon gracile strains from Lakes Melang and Scharmützel produced four PSP toxin variants (gonyautoxin 5 [GTX5], decarbamoylsaxitoxin [dcSTX], saxitoxin [STX], and neosaxitoxin [NEO]). GTX5 was the most prevalent PSP toxin variant among the seven strains from Lake Scharmützel, and NEO was the most prevalent among the seven strains from Lake Melang. The sxtA gene, which is part of the saxitoxin gene cluster, was found in the 14 PSP toxin-producing A. gracile strains and in 11 non-PSP toxin-producing Aphanizomenon issatschenkoi, A. flos-aquae, Anabaena planktonica, and Anabaenopsis elenkinii strains. ATX and CYN were not detected in any of the isolated strains. This study is the first confirming the role of A. gracile as a PSP toxin producer in German water bodies.Neurotoxic saxitoxins, also known as paralytic shellfish poisoning (PSP) toxins, as well as neurotoxic anatoxin-a (ATX) and hepatotoxic cylindrospermopsin (CYN), have been detected in several northeast German lakes in the last 2 decades (3, 35). In a survey conducted in 1995 and 1996, ATX was present in 26% of 78 German lakes and PSP toxins were present in 34% of 29 lakes (3). In 2004, a qualitative survey showed that CYN was present in 50% of 127 German lakes investigated (8). Aphanizomenon flos-aquae Ralfs ex Born. et Flah. has been identified as producer of CYN in these lakes (33), but sources of PSP toxins and ATX have yet to be identified in German water bodies.PSP toxins are potent neurotoxic alkaloids produced by marine dinoflagellates and filamentous freshwater cyanobacteria (1, 2, 42). The 21 currently known PSP toxin variants belong to four groups: carbamoyl toxins, decarbamoyl toxins, N-sulfocarbamoyl toxins, and deoxydecarbamoyl toxins (15). Carbamoyl toxins are the most potent PSP toxins, including saxitoxin (STX) and neosaxitoxin (NEO), while deoxydecarbamoyl toxins comprise the least potent PSP toxins (38). PSP toxins block neural sodium ion channels, leading to death through respiratory failure (1).Cyanobacteria belonging to the orders Oscillatoriales and Nostocales, including members of the genera Cylindrospermopsis, Anabaena, and Aphanizomenon, have been identified as PSP toxin producers in freshwater habitats (4). Aphanizomenon gracile Lemmermann and Aphanizomenon flos-aquae strains from China, Portugal, and the United States have been described as PSP toxin producers (9, 23, 31). Both species are abundant members of the Nostocales and are widely distributed in phytoplankton communities in oligotrophic, mesotrophic, and eutrophic water bodies throughout northeast Germany (35).Regarding saxitoxins, Cylindrospermopsis raciborskii (Woloszyńska) Seenayya et Subba Raju strain T3 was recently found to contain a new candidate saxitoxin gene cluster containing around 35 kb of DNA and comprising more than 26 genes (16). This saxitoxin gene cluster was also found in Anabaena circinalis Rabenhorst ex Bornet & Flahault strains from Australia, in Aphanizomenon sp. strain NH5, and in Lyngbya wollei (Farlow ex Gomont) comb. nov. (16).Anatoxin-a, a neurotoxic bicyclic alkaloid, has been detected in freshwater bodies worldwide (4). Anatoxin-a production has been found in Anabaena, Aphanizomenon, Cylindrospermum, Oscillatoria sp., and Phormidium strains (4). Anatoxin-a is a potent agonist for the nicotinic acetylcholine receptor. Its toxic effects include muscle fasciculation, gasping, convulsions, and death by respiratory arrest in vertebrates (2).Cylindrospermopsin is a potent alkaloid hepatotoxin produced by planktonic cyanobacteria of the order Nostocales. It was first detected in Australian Cylindrospermopsis raciborskii strains (12) and is additionally produced by Anabaena bergii Ostenfeld (36), Umezakia natans M. Watanabe (11), Aphanizomenon ovalisporum (Forti) (37), and A. flos-aquae (33). CYN results in liver, kidney, intestinal, and lung damage (13) and inhibits protein synthesis (40).Overall knowledge of the cyanobacterial sources of PSP toxins, ATX, and CYN is scarce. To identify the producers of such toxins, we isolated and investigated 92 Aphanizomenon, Anabaena, and Anabaenopsis strains from five northeast German water bodies dominated by cyanobacteria of the order Nostocales. All strains were morphologically and phylogenetically classified and screened for the presence of toxin-encoding genes and for the ability to produce cyanobacterial toxins using a polyphasic approach including enzyme-linked immunosorbent assay (ELISA) and liquid chromatography with tandem mass spectrometry (LC-MS/MS).     

微小亚历山大藻麻痹性贝类毒素小鼠生物检测

应用小鼠生物检测法对培养的微小亚历山大藻（LJX02株系）麻痹性贝类毒素进行了检测。首先建立微小．[X02亚历山大藻培养技术,利用对数期藻细胞接种,可使藻细胞处于快速生长,并在7d内藻细胞密度最高达到30000ml^-1。从培养株系中提取的藻毒素,经液相质谱分析主要为GTX1／4和GTX2／3,其含量分别为12．23μg／ml和9．742μg／ml,12L藻体培养液中可获得18．35μgGTX1／4和14．61μgGTX2／3毒素量。采用小鼠生物检测法建立了藻毒素剂量-小鼠死亡时间曲线,其曲线方程为Y=74．017X^-1.5896,将死亡时间t变为时间倒数1／t,GTX毒性MU取对数为logMU,得直线回归方程为Y=3．7009X-0．0858（R^2=0．9824）,求得了昆明小鼠一个鼠单位所代表的GTX1／4和GTX2／3毒素剂量分别为0．026μg和0．021μg。     

Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.     

In Silico Analysis of Putative Paralytic Shellfish Poisoning Toxins Export Proteins in Cyanobacteria

Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na⁺ channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H⁺) or sodium (Na⁺) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na⁺ and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na⁺ recognition domain was conserved in both SxtF/M, indicating that Na⁺ can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na⁺ incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na⁺ in toxin export, as well as the motifs L³⁹⁸XGLQD⁴⁰³ (SxtM) and L³⁹⁰VGLRD³⁹⁵ (SxtF) in toxin recognition.     

Contrasting Physiological Responses of Two Populations of the Razor Clam Tagelus dombeii with Different Histories of Exposure to Paralytic Shellfish Poisoning (PSP)

This study describes the physiological performance of two populations of the razor clam Tagelus dombeii from two geographic areas with different histories of exposure to paralytic shellfish poisoning (PSP) linked to the toxic dinoflagellate Alexandrium catenella. Clams from Melinka-Aysén, which are frequently exposed to PSP, were not affected by the presence of toxins in the diet. However, clams from Corral-Valdivia, which have never been exposed to PSP, exhibited significantly reduced filtration activity and absorption, affecting the energy allocated to scope for growth (SFG). Ammonia excretion and oxygen uptake were not affected significantly by the presence of A. catenella in the diet. Measurements of energy acquisition and expenditure were performed during a 12-day intoxication period. According to three-way repeated measure ANOVAs, the origin of the clams had a highly significant effect on all physiological variables, and the interaction between diet and origin was significant for the clearance and absorption rates and for the scope for growth. The scope for growth index showed similar positive values for both the toxic and non-toxic individuals from the Melinka-Aysén population. However, it was significantly reduced in individuals from Corral-Valdivia when exposed to the diet containing A. catenella. The absence of differences between the physiological response of the toxic and non-toxic clams from Melinka-Aysén may be related to the frequent presence of A. catenella in the environment, indicating that this bivalve does not suffer negative consequences from PSP. By contrast, A. catenella has a negative effect on the physiological performance, primarily on the energy gained from the environment, on T. dombeii from Corral-Valdivia. This study supports the hypothesis that the history of PSP exposure plays an important role in the physiological performance and fitness of filter feeding bivalves.     

Photosynthetic Efficiency in Lawson's Bay on the East Coast of India

Seasonal variations in the efficiency of utilization of radiant energy by phytoplankton photosynthesis in a tropical embayment followed the same pattern as that of primary production. showing that incident radiation did not limit phytoplankton production. Peak values in primary production and photosynthetic efficiency coincided with periods of nutrient enrichment caused either by upwelling during the southwest monsoon or by run-off during the northeast, monsoon. A world-wide comparison of published data on photosynthetic efficiencies in different latitudes showed that higher efficiency is achieved in the tropics, where the values are similar to those observed on phytoplankton cultures. Based on the maximum efficiency of photosynthesis attained, three regimes can be recognized in tropical waters: 1) regions of upwelling with a maximum of 6%,. 2) coastal bays with 0.7%, 3) oceanic regions with 0.24%.     

Seasonal Grouping in Female Moufflons in Relation to Food Availability

The social behaviour of ungulates is very often explained by feeding resources. This appears more striking for females since reproduction is very costly in energy terms. We expect female group size to be directly dependent on food availability. Our results on moufflons show that ewe gathering supports this hypothesis during spring, summer and autumn. However, our winter results are contradictory since we observed a stronger tendency of ewes to gather during this season. In the light of recent papers, other ecological or social parameters are discussed to explain this phenomenon.     

On some commensal Turbellaria of the Canadian East Coast

The brachyurans Chionoecetes opilio and Hyas araneus collected from the east coast of Canada harbour two species of commensal turbellarians. Ectocotyla hirudo (Levinsen) and E. multitesticulata Fleming & Burt are found on the gills and branchial chambers of male and female crabs. The molluscs Crassostrea virginica and Mytilus edulis collected from several locations where they are cultured along the Northumberland strait carry two commensal eulecithophoran turbellarians, both on the gills, viz., Urasloma cyprinae (Graff) and Paravortex gemellipara (Linton), the latter being a new host record. Aspects of the biology and life-history of these turbellarians are discussed, especially in relationship to the biology of their hosts.Abbreviations in the figures A Atrium - An Antrum - C Capsule - CO Common Oviduct - E Embryo - Ep Egg-laying pore - GP Genital Pore - I Intestine - O Ovary - Od Oviduct - Og Ora-genital Pore - Ph Pharynx - Sp Sperm - SV Seminal Vesicle - T Testes - Va Vagina - Y Yolk     

Upper age limit of the East Coast Gondwanas, India

The controversial upper age limit of the East Coast Gondwanas is discussed. A reassessment of the evidence furnished by animal and plant fossils is made in view of recent findings. Whereas the flora points towards a Jurassic age, the ammonites and other invertebrate megafossils indicate a Neocomian age. The microfossils support the concept of an Early Cretaceous age. The conflicting evidence of flora and fauna and the probable reliability of each is discussed. Although there is some doubt about the correctness of the floral testimony, the reliability of faunal evidence seems established, and an Aptian or Albian age is favoured as the upper age limit of the East Coast Gondwanas.     

Seasonal variation in the biochemical composition of Mytilus viridis at Ratnagiri on the West Coast of India

1. The seasonal variation in the water, protein, fat and glycogen contents of the mussel, Mytilus viridis has been studied for the year March, 1974 to March, 1975.
2. The water level increased during the monsoon season and decreased in summer.
3. The level of protein, fat and glycogen showed correlation with the reproductive cycle of the mussel.
4. The protein level was high when the mussels were mature and dropped during the breeding period.
5. During sex change from male to female in May the protein level remained high whereas during sex change from female to male in October and November it was low.
6. The fat level was high in mature mussels and declined on spawning.
7. The glycogen level was at its peak in immature mussels and low in mature.

     

Determining the Advantages,Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST)

The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.