Characterization of saxitoxin production and release and phylogeny of sxt genes in paralytic shellfish poisoning toxin-producing Aphanizomenon gracile

The freshwater cyanobacterium Aphanizomenon gracile is one of the most widely distributed producers of the potent neurotoxins saxitoxin (STX) and its derivatives (paralytic shellfish poisoning toxins, PSP toxins). However, the phylogeny of STX biosynthesis genes and the regulation of STX production and release remain poorly studied in the genus Aphanizomenon. In this study, two A. gracile strains from Spanish freshwaters were grown in semi-continuous cultures under three temperatures (15, 20 and 28 °C) and their STX production and release were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). STX production was stable along the temperature range, with 1.4–2.3-fold shifts in biomass-standardized STX contents, and maxima of 0.22 μg equivalent STX mg⁻¹ dry weight 15.3 fg equiv STX cell⁻¹ and 15.1 μg equiv STX mg⁻¹ Chl a. The extracellular fraction was remarkably high (13.6–35.3%), not clearly affected by temperature but with nitrate-depleted medium (BG11₀) inducing a 2-fold increase in extracellular content. STX production and release were not directly related to growth rates. The 16S rRNA phylogenetic analyses in sixteen A. gracile strains from Spanish and German freshwaters showed that PSP-producing A. gracile grouped within a monospecific and highly supported cluster, together with PSP-producing Aphanizomenon sp. NH-5 and clearly separated from a monospecific Aphanizomenon flos-aquae cluster. The sixteen A. gracile strains formed also monospecific and highly supported clusters for PSP-biosynthesis genes (sxtG, sxtI, sxtH and sxtX) together with Aphanizomenon sp. NH-5. This study evidences an elevated extracellular proportion of STX in A. gracile with importance for risk assessment, and supports the identification of Aphanizomenon sp. NH-5 as A. gracile.     

UNRAVELING MOLECULAR DIVERSITY AND PHYLOGENY OF APHANIZOMENON (NOSTOCALES,CYANOBACTERIA) STRAINS ISOLATED FROM CHINA1

Fifty‐three strains of the genus Aphanizomenon isolated from Chinese waters were employed to conduct morphological examination and sequencing of the 16S rRNA gene, rbcLX (RUBISCO), and cpcBA‐IGS gene regions. Based on morphological characteristics, the examined strains were divided into three morphotypes [Aph. flos‐aquae Bréb. ex Bornet et Flahault, Aph. gracile Lemmerm., and Aph. issatchenkoi (Usacer) Proshk.‐Lavr.]. Phylogenetic analysis based on 16S rRNA and rbcLX showed that Aphanizomenon strains could be divided into three main clades (Clade A of Aph. flos‐aquae, Clade B of Aph. gracile, and Clade C of Aph. issatchenkoi), but two additional clades formed by Aph. ovalisporum and Aph. aphanizomenoides were detected in the 16S rDNA‐based topology. All Aph. issatchenkoi strains contained an additional 175 nucleotides from the 779 to 954 nucleotide location in rbcLX region, compared with strains of Aph. flos‐aquae and Aph. gracile. The cpcBA‐IGS‐based phylogenetic tree revealed that Aph. issatchenkoi strains were not discriminated from Aph. flos‐aquae strains; however, a concatenated alignment of 16S rDNA, rbcLX, and cpcBA‐IGS led to the three distinct clades (Aph. flos‐aquae, Aph. gracile, and Aph. issatchenkoi, respectively). It is suggested that the taxonomic revision of Aphanizomenon and Anabaena genera is required to be performed by employing multilocus sequence analysis and polyphasic studies.     

Phylogeography of Cylindrospermopsin and Paralytic Shellfish Toxin-Producing Nostocales Cyanobacteria from Mediterranean Europe (Spain)

Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg⁻¹ [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.     

FIRST REPORT OF THE CYANOTOXIN ANATOXIN‐A FROM APHANIZOMENON ISSATSCHENKOI (CYANOBACTERIA)1

The taxonomy and toxicity of a single‐filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography–mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAWBG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) Proshkina‐Lavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxin‐a but did not produce homoanatoxin‐a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin‐a‐producing species in the Southern Hemisphere and the first report of anatoxin‐a production by A. issatschenkoi.     

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)1

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.     

Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6 °C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27 °C and 30 °C), as well as projected mean (33 °C) and maximum temperatures (36 °C) based on tropical climate change estimates of +6 °C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33 °C and M. flos-aquae at 30 °C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33 °C were found to boost total MC cell quota for three Microcystis strains although further increase to 36 °C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.     

Cholinium carboxylate ionic liquids for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification

The present study is the first report demonstrating that ionic liquids consisting of cholinium cations and linear carboxylate anions ([Ch][CA] ILs) can be used for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification. Six variants of [Ch][CA] ILs were systematically prepared by combining cholinium cations with linear monocarboxylate anions ([C_nH_2n+1–COO]⁻, n = 0–2) or dicarboxylate anions ([HOOC–C_nH_2n+1–COO]⁻, n = 0–2). These [Ch][CA] ILs were analyzed for their toxicity to yeast cell growth and their ability to pretreat kenaf powder for subsequent enzymatic saccharification. When assayed against yeast growth, the EC₅₀ for choline acetate ([Ch][OAc]) was 510 mM, almost one order of magnitude higher than that for 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). The cellulose saccharification ratio after pretreatment at 110 °C for 16 h with [Ch][OAc] (100.6%) was almost comparable with that after pretreatment with [Emim][OAc]. Therefore, [Ch][OAc] is a biocompatible alternative to [Emim][OAc] for lignocellulosic material pretreatment.     

An extremely alkaline novel chitinase from Streptomyces sp. CS495

An extremely alkaline chitinase from Streptomyces sp. CS495 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase was purified 7.0 fold with a 33.9% yield by Sepharose Cl-6B column. The molecular mass of the enzyme (Ch495) was approximately 41 kDa. Ch495 was found to be stable over a broad pH range (5–12.5) and to 50 °C and have an optimum temperature of 60 °C. Ch495 had K_m and V_max values of 1.34 ± 2.9 mg/mL and 889 ± 3.6 mmol/min, respectively using different concentrations of colloidal chitin. N-terminal sequence of Ch495 was APREKINLLYFLGYF. HPLC and TLC analysis of Ch495 shows the production of produced N-acetyl d-glucosamine (GlcNAc) as minor and diacetylchitobiose (GlcNAc)₂ as major products. Ch495 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis which can be used for the biological control of fungus. As being simple in purification, extreme alkalophilic, stable in broad range of pH, ability to produce oligosaccharides, and antifungal activity shows that Ch495 has potential applications in industries as for chitooligosaccharides production used as medical prebiotics or/and for the biological control of plant pathogens in agriculture.     

MORPHOLOGICAL AND 16S rRNA GENE EVIDENCE FOR RECLASSIFICATION OF THE PARALYTIC SHELLFISH TOXIN PRODUCING APHANIZOMENON FLOS‐AQUAE LMECYA 31 AS APHANIZOMENON ISSATSCHENKOI (CYANOPHYCEAE)1

A taxonomic reevaluation of the paralytic shellfish toxin (saxitoxins) producing cyanobacterium Aphanizomenon flos‐aquae Ralfs ex Born. & Flah. LMECYA31 was done using morphology and 16S rRNA gene sequences. We found that strain LMECYA31 was incorrectly identified as Aph. flos‐aquae based on (a) lack of bundle formation in trichomes, (b) shape of terminal cells in the trichomes, (c) lower similarity (<97.5%) in the 16S rRNA gene sequences relative to those of Aph. flos‐aquae, and (d) comparison within a phylogenetic tree of 16S rRNA gene sequences. The shape of the terminal trichome cells and the shape and size of the vegetative cell, heterocyst, and akinete in strain LMECYA31 match characters of Aph. issatschenkoi (Ussachew) Proschkina‐Larvernko. 16S rRNA gene sequences and phylogenetic clusters constructed from 16S rRNA gene sequences support our conclusion that strain LMECYA31 should be Aph. issatschenkoi.     

Interspecific allelopathy in cyanobacteria: Cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa

The biological role of cyanobacteria secondary metabolites is relatively unknown although several possible hypotheses have been discussed. In the following study the effect of cylindrospermopsin (CYN) and metabolites of non-CYN producing Cylindrospermopsis raciborskii strain on growth, alkaline phosphatase (ALP) activity and microcystin-LR (MC-LR) production in Microcystis aeruginosa was evaluated. Higher concentrations of CYN (10 and 50 μg L⁻¹) induced toxicity effects demonstrated by significant growth inhibition and M. aeruginosa cell necrosis. Lower concentrations of CYN (1 and 5 μg L⁻¹) slightly decreased growth rates but significantly up-regulated ALP activity. Moreover, under all studied CYN concentrations MC-LR production strongly decreased. Spent C. raciborskii medium mimicked the CYN action by inducing strong inhibition of M. aeruginosa growth and MC-LR production and through up-regulation of ALP activity. On the other hand, spent M. aeruginosa medium did not affect C. raciborskii growth and no alterations in ALP activity were observed. Co-culturing of these two species resulted in an increase of C. raciborskii contribution at the expense of M. aeruginosa. From the results we conclude that CYN can be involved in interspecific competition in cyanobacteria and that non-CYN producing C. raciborskii strains may produce a hitherto unknown bioactive compound(s) which can mimic CYN action.     

A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9 min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6 ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2–9.6% and 1.3–12.0% respectively. The method was applied to the analysis of aquatic samples (n = 206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n = 22), cylindrospermopsin (n = 25), microcystin-RR (n = 17), microcystin-LR (n = 12), microcystin-LY (n = 1), microcystin-LF (n = 1) and nodularin (n = 5). For microcystins, the levels detected ranged from 0.001 to 1.51 μg/L, with two samples showing combined levels above the guideline set by the WHO of 1 μg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.     

Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences

The taxonomy of Aphanizomenon flos-aquae strain NH-5, a producer of cyanotoxins, was re-evaluated by comparison with six other Aphanizomenon strains using morphological characteristics and 16S rRNA gene sequences. Strain NH-5 was concluded to be improperly identified as Aph. flos-aquae based upon (1) lack of bundle formation in the trichomes, (2) location of akinetes next to heterocytes, (3) lower similarities (less than 97.5%) in the 16S rRNA gene sequences relative to Aph. flos-aquae strains, and (4) comparison within a phylogenetic tree constructed from 16S rRNA gene sequences. The Aphanizomenon strains investigated in this study are classified to four morphological groups as described by the classical taxonomy of Komárek & Kovácik (1989). This classification was supported from the phylogenetic results of 16S rRNA gene sequences. This study also discusses the generic boundaries between Aphanizomenon and Anabaena.     

The effect of diet on longevity,fecundity, and the sex ratio of Clitostethus arcuatus (Rossi) (Coleoptera: Coccinellidae)

Clitostethus arcuatus is a major, cosmopolitan predator of some Aleyrodidae. Field collected adult beetles were reared in the laboratory on different diets: Siphoninus phillyreae eggs, Trialeurodes vaporariorum eggs, Sitotroga cerealella eggs, or an artificial diet consisting of honey, yeast, and pollen. All experiments were conducted at 25 ± 2 °C, 65 ± 5% RH and a photoperiod of 16:8 (L:D) h. Female and male C. arcuatus consumed a mean (± SE) of 61 ± 0.6 and 27 ± 0.9 T. vaporariorum eggs d^? 1, respectively, and a mean of 56 ± 2.2 and 29 ± 1.1 S. phillyreae eggs d^? 1, respectively. Significant differences were noted between sexes and between hosts consumed by female C. arcuatus. No feeding occurred on S. cerealella eggs. Although there was a significant difference between rates of oviposition due to diet, fertility rates on different diets did not show significant differences. The sex ratio of C. arcuatus (female:male) was 51.4:48.6, 55.2:44.8, and 54.6:45:4 when adults fed on T. vaporariorum, S. phillyreae, and artificial diet, respectively. These differences were not significantly different. Average longevity (± SE) was 66.4 ± 2.6, 54.9 ± 2.5; 77.3 ± 6.9, 67.5 ± 7.2; and 86.4 ± 4.5 70.3 ± 3.6 days for female and male C. arctuatus, respectively, on T. vaporariorum, S. phillyreae and artificial diet, respectively, with significant differences between sexes and diets. Although developmental duration on T. vaporariorum was longer than ash whitefly, this difference was not significant (mean 27.68 ± 0.31 and 25.09 ± 0.21 days for predators reared on T. vaporariorum and S. phillyreae, respectively). Given its longevity and fecundity on T. vaporariorum, C. arcuatus may be a good choice for mass release on glasshouse crops infected by greenhouse whitefly.     

Effect of temperature and host species on parasitism,development time and sex ratio of the egg parasitoid Trichogrammatoidea lutea Girault (Hymenoptera: Trichogrammatidae)

The developmental biology of Trichogrammatoidea lutea Girault (Hymenoptera: Trichogrammatidae) was studied at six constant temperatures (18, 21, 24, 27, 30 and 35 °C) on eggs of three lepidopteran host species: Helicoverpa armigera (Hübner) (Noctuidae), Chilo partellus (Swinhoe) (Crambidae) and Cadra cautella (Walker) (Pyralidae). T. lutea did not complete development at 35 °C on any of the three host species. Parasitism levels were highest on H. armigera at 27 °C (58%), C. cautella at 27 and 30 °C (31% and 28%) and C. partellus between 24 and 30 °C (13–17%). Realized progeny of T. lutea per parasitized host egg was influenced by host size. The number of progeny of T. lutea per parasitized host egg was highest on H. armigera, followed by C. partellus and lowest on C. cautella. The sex ratio was female biased on C. partellus, female biased on C. cautella with the exception of 21 °C and close to 1:1 on H. armigera. The rate of development from egg to pupa and egg to adult was fastest on H. armigera and slowest on C. partellus. Lower thresholds for development and degree days (DD) of T. lutea from egg to adult were 12.8 °C and 105.4 DD on H. armigera, 11.3 °C and 141.6 DD on C. partellus and 12.9 °C and 118.2 DD on C. cautella, respectively. Based on these results, H. armigera is the most suitable host for mass rearing of T. lutea for biological control of Lepidoptera pests because of the relatively high parasitism levels, short development time, greater clutch size and balanced sex ratio. C. cautella may also be used although longer exposure times might be required due to lower parasitism levels.     

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).     

Differentiated influence of off-road and on-road cycling practice on balance control and the related-neurosensory organization

This study aimed to determine the sensorimotor strategies privileged by mountain bikers (MTB) and road cyclists (RC) for balance control. Twenty-four MTB and 24 RC (off-road Olympics, world, continental and national champions, Tour-de-France participants, on-road world cup race winner) volunteered to answer a questionnaire about the characteristics of cycling practice and perform a sensory organization test, aiming to evaluate balance control in 6 different sensory situations based upon visual and support surface perturbations (C1^ES to C6^ES). RC balance performances were better than those of MTB both during quiet stance eyes opened (C1^ES, p = 0.011) and when only somatosensory information is disrupted (C4^ES, p = 0.039), highlighting a higher use of vision to control balance in RC. Moreover, a positive correlation was shown in the whole population (MTB + RC) between the visual ratio (R^VIS = C4^ES/C1^ES) and the proportion of riding distance of on-road cycling (ρ = 0.28, p = 0.054). In MTB, the use of proprioception (somatosensory ratio: R^SOM = C2^{ES(eyes closed)}/C1^ES) was increased by a higher intensity of off-road cycling (ρ = 0.49, p = 0.018) and that of vision (R^VIS) by a higher intensity of on-road cycling (ρ = 0.41, p = 0.048). The difference in sensory organization between MTB and RC could be explained by adaptive processes elaborated from environmental stimulations and technical specificities of these disciplines.     

Altered cellular redox status,sirtuin abundance and clock gene expression in a mouse model of developmentally primed NASH

BackgroundWe have previously shown that high fat (HF) feeding during pregnancy primes the development of non-alcoholic steatohepatits (NASH) in the adult offspring. However, the underlying mechanisms are unclear.AimsSince the endogenous molecular clock can regulate hepatic lipid metabolism, we investigated whether exposure to a HF diet during development could alter hepatic clock gene expression and contribute to NASH onset in later life.MethodsFemale mice were fed either a control (C, 7% kcal fat) or HF (45% kcal fat) diet. Offspring were fed either a C or HF diet resulting in four offspring groups: C/C, C/HF, HF/C and HF/HF. NAFLD progression, cellular redox status, sirtuin expression (Sirt1, Sirt3), and the expression of core clock genes (Clock, Bmal1, Per2, Cry2) and clock-controlled genes involved in lipid metabolism (Rev-Erbα, Rev-Erbβ, RORα, and Srebp1c) were measured in offspring livers.ResultsOffspring fed a HF diet developed NAFLD. However HF fed offspring of mothers fed a HF diet developed NASH, coupled with significantly reduced NAD⁺/NADH (p < 0.05, HF/HF vs C/C), Sirt1 (p < 0.001, HF/HF vs C/C), Sirt3 (p < 0.01, HF/HF vs C/C), perturbed clock gene expression, and elevated expression of genes involved lipid metabolism, such as Srebp1c (p < 0.05, C/HF and HF/HF vs C/C).ConclusionOur results suggest that exposure to excess dietary fat during early and post-natal life increases the susceptibility to develop NASH in adulthood, involving altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06 × 10¹⁰ to 1.36 × 10¹⁵ CFU/ml for stomach fluid and 1.92 × 10¹⁰ to 6.10 × 10¹⁵ CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.