Occurrence of tetrodotoxin in the gastropodsRapana rapiformis andR. venosa venosa

Paralytic toxicity was detected in four of 17 specimens and six of 28 specimens of the gastropodsRapana rapiformis andR. venosa venosa, respectively, collected from Chiching (Kaohsiung City) and Nanfangao (Ilan County), Taiwan, in 1988 and 1989. The highest toxicities, calculated as tetrodotoxin (TTX) content, were 140 and 13 mouse units (MU) g^–1 digestive gland inR. rapiformis andR. venosa venosa, respectively. The toxins obtained from each species were purified by ultrafiltration and Bio-Gel P-2 column chromatography. The toxin's specific toxicities (as TTX) were 56 MU mg^–1 (R. rapiformis) and 52 MU mg^–1 (R. venosa venosa). Results of analyses by thin-layer chromatography, cellulose acetate membrane electrophoresis and high-performance liquid chromatography showed that TTX and anhydrotetrodotoxin were responsible for the toxicity. The alkali hydrolysate of each toxin showed maximum absorption at ca. 274 nm, which is the unique absorption for the C₉-base of TTX and its related substances (such as anhydrotetrodotoxin, 4-epitetrodotoxin, etc.). Here, we report for the first time the occurrence of TTX in the gastropodsR. rapiformis andR. venosa venosa.     

Bacterial diversity in mining and non-mining regions with emphasis on plant growth-promoting traits

A total of 20 soil samples were collected from mining and non-mining areas of coal field regions of Dhanbad, India. These samples were grown on different types of selective media and a total of 141 isolates, each having plant growth-promoting (PGP) characteristics were obtained. The isolates were further tested for PGP traits (phosphate solubilisation, production of indole acetic acid, hydrogen cyanide, ammonia and siderophores) and a biochemical analysis was done to determine the prevalent species of the region. All the strains selected showed at least two PGP characteristics, which indicated that they can be used as plant growth promoters. Bacillus and Pseudomonas species were prevalent in both mining and non-mining areas, whereas Rhizobium, Azospirillum and Azotobacter were found mostly in the non-mining area. A total of 48 Bacillus, 29 Pseudomonas, 4 Rhizobium, 11 Azospirillum and 10 Azotobacter species were obtained from the two selected regions. The soil samples from the mining region were poor in nutrient content and thus relatively lower numbers of PGP isolates were attained.     

Selection of poly(R)-3-hydroxybutyric acid utilising bacteria by enrichment,optimisation and compatibility testing for consortia development

Bacterial strains with poly-3-hydroxybutyrate (PHB) degradation potential were isolated from waste yard soil samples of selected industrial sites in Uttarakhand, India, and two microbial consortia were developed, i.e. Consortium I comprises Pseudomonas sp. strain Rb10, Pseudomonas sp. strain Rb11 and Bacillus sp. strain Rb18, and Consortium II is composed of Lysinibacillus sp. strain Rb1, Pseudomonas sp. strain Rb13 and Pseudomonas sp. strain Rb19. The current study involved enrichment selection via liquid and semi-solid media, followed by isolation and screening of bacterial strains using PHB pellets and films. Furthermore, the identification and characterisation was done by triphasic approach. The utilisation of PHB by the characterised strains was confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy. Moreover, the minimum inhibitory concentration of solubilised PHB was found to be 2.5?mg/mL, which was detected through ‘clear zone assay’. Further, the selection and biocompatibility testing of potential isolates were performed for the formation of bacterial consortia. Thus, the present work would provide direct and standardised protocol for screening and selection of potential microbiomes for biodegradation of polymers by overcoming the negative effect of organic solvents. Moreover, indigenously developed consortia would be evaluated for their in situ biodegradation potential against various bioplastic films.     

Occurrence of tetrodotoxin in the starfishes Astropecten polyacanthus and A. scoparius in the Seto Inland Sea

Two starfishes, togemomijigai Astropecten polyacanthus and momijigai A. scoparius were collected from the Seto Inland Sea in October 1983 through November 1984, and assayed for toxicity by the standard method for tetrodotoxin (TTX). Most of the 54 A. polyacanthus specimens assayed were toxic, with the highest toxicity score being 520 mouse units (MU) g^-1. All seven A. scoparius were toxic, with the highest score being 46 MU g^-1. The toxin from A. polyacanthus was purified by a method which consisted mainly of activated charcoal treatment, and chromatography on CM-Sephadex C-25 and Bio-Rex 70 columns. The purified starfish toxin showed a specific toxicity of 4 700 MU mg^-1, a value which was almost comparable to that of authentic TTX. From the thin-layer chromatographic and electrophoretic behavior, along with ¹H-NMR spectrum, A. polyacanthus toxin was identified as TTX.     

Allelopathic effect of Cylindrospermopsis raciborskii extracts on the germination and growth of several plant species

Cylindrospermopsis raciborskii is an invasive cyanobacterium and a potential producer of the alkaloid toxin cylindrospermopsin (CYN). Extracts of two strains of C. raciborskii were tested for their effects on the germination and growth of Lactuca sativa, Phaseolus vulgaris, Pisum sativum and Solanum lycopersicum. Germination was not significantly inhibited for any of the plant species tested, but growth was affected, depending on the species. Root and stem growth in L. sativa was generally stimulated by both strains. Ph. vulgaris root growth was stimulated by both strains but no effect was visible in stem growth. S. lycopersicum root growth was inhibited by both strains and stem growth was inhibited only by the CYN strain. P. sativum root growth was also inhibited by both strains but stem growth was stimulated. CYN accumulation was also differential with toxin transfer to the stem. Ph. vulgaris accumulated the highest CYN concentration. This study suggests that plants behave differently in their response to this toxin and that roots and stems also show different abilities to react and accumulate the toxin. Knowledge of the impact of CYN- and non-CYN-producing cyanobacteria in different plant species and translocation of the toxin to different plant parts is essential for the avoidance of human as well as environmental health hazards.     

As(V)-reduction to As(III) by arsenic-resistant Bacillus spp. bacterial strains isolated from low-contaminated sediments of the Oliveri-Tindari Lagoon,Italy

Five arsenic-resistant bacterial strains designated MT1, MT2, MT3, V1 and V2 were isolated from sediments of the Oliveri-Tindari Lagoon (Italy), which comprises six small lakes whose sediments contain low arsenic concentrations. Phylogenetic analysis of the 16S rRNA gene sequences assigned them to the genus Bacillus. Bacillus sp. strain MT3 showed higher tolerance to As(III) and As(V), as indicated by minimum inhibitory concentrations of 14 and 135 mmol^?1, respectively. Bacillus sp. strain V1 showed growth inhibition at 14 mmol^?1 in the presence of As(III) and at 68 mmol^?1 in the presence of As(V), whereas the arsenic resistance of Bacillus sp. strain MT1 was 10 and 27 mmol^?1 for As(III) and As(V), respectively. The strains Bacillus spp. MT2 and V2 showed low levels of As(III) and As(V) resistance, as it was unable to grow at concentrations>7 and 14 mmol^?1, respectively. The isolated arsenic-resistant Bacillus spp. strains were able to reduce As(V) to As(III), especially Bacillus spp. strain MT3. This study suggests that the isolated bacterial strains play a role in the arsenic biogeochemical cycle of arsenic-poor sediments in the Oliveri-Tindari Lagoon.     

Biodegradation kinetics of cymoxanil in aquatic system

A potential method to detoxify pesticides in aquatic system is using bioremediation. In this study, four microorganisms (Pseudomonas sp (EB11), Streptomyces sp. (EB12), Aspergillus niger (EB13) and Trichoderma viride (EB14) were isolated from cucumber leaves previously treated with cymoxanil using enrichment technique. These strains were evaluated for their potential to detoxify cymoxanil in aquatic system at the concentration level of 5×10^?4M. The effect of pH and temperature on the growth ability of the tested strains was also investigated by measuring the intracellular protein and mycelia dry weight for bacterial and fungal strains, respectively. Moreover, the remaining toxicity of cymoxanil after 28 days of incubation with tested strains was evaluated to confirm the complete removal of any toxic materials (cymoxanil and its metabolites). The results showed that the optimum pH for the growth of cymoxanil degrading strains (bacteria and fungi) was 7. A temperature of 30°C appears to be the optimum for the growth of either fungal or bacterial strains. Pseudomonas sp. (EB11) was the most effective strain in cymoxanil degradation followed Streptomyces sp (EB12), Trichoderma viride (EB14) and Aspergillus niger (EB13), with half-lives of 4.33, 9.5, 17.3 and 24.7 days, respectively. The degradation of cymoxanil by bacterial strains was much faster than fungal one. There is no remaining toxicity of cymoxanil detected in aqueous media previously treated with Pseudomonas sp. (EB11) for 28 days. The results suggest that bioremediation by Pseudomonas sp. (EB11) are promising for the detoxification of cymoxanil in aqueous media.     

The praying mantis (Mantodea) as predator of the poisonous red-spotted newt <Emphasis Type="Italic">Notophthalmus viridescens</Emphasis> (Amphibia: Urodela: Salamandridae)

A Chinese praying mantis, Tenodera sinensis, was observed feeding on a living red-spotted newt, Notophthalmus viridescens. Specimens of that newt’s population are known to contain high concentrations of tetrodotoxin (TTX), a specific blocker of voltage-gated sodium channels. After experimental oral administration of a TTX-solution (1 mg/ml) to adult specimens of four mantis species, all survived high TTX concentrations (up to 30.8 μg/g body mass) as revealed by analysis of their body extracts, but they are rapidly killed by intra-abdominal injection of 1 μg TTX. The toxin was found to be gradually excreted with faeces. As demonstrated by monoclonal antibody-based immunohistochemical technique, TTX does not penetrate the mid-gut membrane, since it was localized only in the gut lumen, but not in the epithelial cells. This prevents the toxin to reach its target, the sodium channels of the insect’s nervous system, and enables the mantids to feed on toxic prey without risking poisoning.     

External secretion of tetrodotoxin from puffer fishes stimulated by electric shock

Four toxic species of puffers (Takifugu pardalis, T. poecilonotus, T. vermicularis and T. niphobles) secreted large amounts of tetrodotoxin into the surrounding water immediately after being stimulated by electric shock. The total amount of toxin secreted ranged from 2 000 to 50 000 MU depending upon the specimen. The amount of toxin secreted seemed to be related to the toxicity of individuals. Non-toxic specimens of T. rubripes did not secrete any toxin. The stimulation always caused inflation of puffers, and this is generally accepted as a repelling behavior of these species. The secreted tetrodotoxin may act as a repellent to predators.     

Tissue distribution of the amnesic shellfish toxin,domoic acid,in<Emphasis Type="Italic"> Octopus vulgaris</Emphasis> from the Portuguese coast

Domoic acid (DA), the amnesic shellfish toxin, is a food-web-transferred algal toxin that has been detected in many marine organisms from copepods to whales. However, cephalopods, which are important members of the food chain, have never been implicated in DA transfer or accumulation. Here, we present data showing relevant values of DA detected in the common octopus (Octopus vulgaris) from the Portuguese continental coast. Even though DA is hydrophilic and is not expected to be accumulated in the tissues, DA was always detected in our octopus tissue samples. Tissue distribution of DA revealed that the digestive gland and the branchial hearts are the main organs of DA accumulation. Highly variable DA concentrations, ranging from 1.1 to 166.2 g DA g^–1, were observed in the digestive glands. Low levels of DA were detected in the digestive tract (stomach and intestine) and could be a consequence of high digestion rates or a result of non-exposure to toxic vectors during the sampling period. In fact, octopus prey, such as bivalves, crustaceans and fishes, are known to occasionally work as DA vectors. Consequently, DA uptake into octopus tissues is likely sporadic. Similar low levels were detected in the kidney, gills, systemic heart, posterior salivary glands and mantle, and no DA was found in either the gonads or the ink sac. These data are the necessary first step towards achieving an understanding of the accumulation of phycotoxins in O. vulgaris.Communicated by S.A. Poulet, Roscoff     

Comparisons of genetic population structures in four intertidal brachyuran species of contrasting habitat characteristics

Genetic population structures along the Japanese coast, analyzed by sequence data from the mitochondrial DNA COI region, were determined for four intertidal brachyuran species in the superfamily Thoracotremata (Ocypode ceratophthalma, Gaetice depressus, Chiromantes dehaani and Deiratonotus japonicus), which were characterized by different habitat requirements. O. ceratophthalma (seashore; supratidal sand) and C. dehaani (estuarine; supratidal marsh) showed no significant genetic differentiation among Japanese populations. The Japanese populations of O. ceratophthalma, however, were found to genetically differentiated from the Philippine population. G. depressus (seashore; intertidal cobbles) exhibited significant genetic differentiation between the Amami-Ohshima population and other local populations. D. japonicus (estuarine; intertidal cobbles) showed significant genetic differentiation among many local populations separated by about 30–1,200 km. The different patterns of genetic population structure recorded for the four species, thus, do not simply correspond to habitat type.     

Comparisons of genetic population structures in four intertidal brachyuran species of contrasting habitat characteristics

Genetic population structures along the Japanese coast, analyzed by sequence data from the mitochondrial DNA COI region, were determined for four intertidal brachyuran species in the superfamily Thoracotremata (Ocypode ceratophthalma, Gaetice depressus, Chiromantes dehaani and Deiratonotus japonicus), which were characterized by different habitat requirements. O. ceratophthalma (seashore; supratidal sand) and C. dehaani (estuarine; supratidal marsh) showed no significant genetic differentiation among Japanese populations. The Japanese populations of O. ceratophthalma, however, were found to genetically differentiated from the Philippine population. G. depressus (seashore; intertidal cobbles) exhibited significant genetic differentiation between the Amami-Ohshima population and other local populations. D. japonicus (estuarine; intertidal cobbles) showed significant genetic differentiation among many local populations separated by about 30–1,200 km. The different patterns of genetic population structure recorded for the four species, thus, do not simply correspond to habitat type. An erratum to this article can be found at     

Cellular damage of plant pathogenic fungi by antifungal compounds produced by Bacillus spp. isolates

The use of microorganisms as biological control agents (BCAs) has become an effective alternative to chemical means of controlling plant pathogens. The antagonistic and inhibitory activity of 71 Bacillus spp. strains, which were isolated from different Mexican sites, were tested against several phytopathogen fungi, Fusarium oxysporum, Fusarium equiseti, Fusarium avenaceum, Bipolaris spp. and Alternaria spp. From the antagonism study, the strain ELI149 showed a marked inhibition of growth against all tested fungi; therefore crude metabolites from this strain were extracted using ethyl acetate and amberlite resin and probed against the same fungi as well as strains of Mucor sp., Penicillium spp. and Paecilomyces spp. The results indicated that amberlite was more suitable for extraction of secondary metabolites with antifungal activity. Finally, observation of cell damage in the tested pathogenic fungi showed marked morphological changes on reproductive structures in all tested fungi indicating that antibiosis was the mechanism of the antagonistic effect. These results suggest that metabolites from the Bacillus strains have a wide spectrum of antibiotic activities, which can be used as biocontrol agents for controlling fungal plant diseases of agricultural importance.     

Selective predation on mature male Byblis japonicus (amphipoda: Gammaridea) by the barface cardinalfish,Apogon semilineatus

Ontogenetic change of habitat depths of Nautilus pompilius in the Philippines (Tañon Strait) and Fiji is considered by comparing ¹⁸O/¹⁶O ratios in septa and cameral fluid of live-caught specimens and ambient sea-water. ¹⁸O values of cameral liquid become heavier with decreasing volume within a chamber, which may be due to isotopic fractionation during discharge across the siphuncular wall. All of the seven Philippine and Fiji specimens analyzed show a distinct change in ¹⁸O from light values in the first seven septa to heavier values in the succeeding septa. Two different isotopic temperature scales are obtained for the Fiji and Philippine populations, suggesting a differential vital effect of metabolism between them. Sightly light ¹⁸O values in Septa 1 to 7 and hatching at relatively high temperatures in aquaria both suggest that N. pompilius hatch at the shallowest depths within their inhabitable vertical range. Depths of postembryonic animals in the Philippines and Fiji waters estimated from the isotopic temperature-depth diagrams range from 120 to 160 m and from 440 to 520 m, respectively, both of which correlate well with capture records. More than several tens of small-scale ¹⁸O cycles are detected in the sequence of nacreous layers within the single septum of a submature Philippine specimen. This can be interpreted as reflecting daily vertical migration.     

Tetrodotoxin affects survival probability of rough-skinned newts (Taricha granulosa) faced with TTX-resistant garter snake predators (Thamnophis sirtalis)

Lethal chemical defenses in prey species can have profound effects on interactions with predators. The presence of lethal defenses in prey can correct the selective imbalance suggested by the life-dinner principle in which the fitness consequences of an encounter between predator and prey should be much greater for the prey species than the predator. Despite the apparent adaptive advantages of lethality the evolution of deadly prey presents a fundamental dilemma. How might lethal defenses confer an individual fitness advantage if both predators and prey die during interactions? We examined the interaction between the rough-skinned newt (Taricha granulosa), which contains a powerful neurotoxin called tetrodotoxin (TTX), and the common garter snake (Thamnophis sirtalis). In some sympatric populations, Th. sirtalis have evolved physiological resistance to TTX. Whether the newts’ toxin confers protection from snake predators or has been disarmed by the snakes’ physiological resistance has not yet been directly tested. In predator–prey trials, newts that were rejected by snakes had greater concentrations of TTX in their skin (4.52 ± 3.49 mg TTX/g skin) than those that were eaten (1.72 ± 1.53 mg TTX/g skin). Despite the plethora of taxa that appear to use TTX defensively, this is the first direct and quantitative demonstration of the antipredator efficacy of TTX. Because the survival probability of a newt (and thus fitness) is affected by individual TTX concentration, selection can drive the escalation of toxin levels in newts. The variable fitness consequences associated with both TTX levels of newts and resistance to TTX in snakes that may promote a strong and symmetrical coevolutionary relationship have now been demonstrated.     

Effects of UV-light on biochemical composition of silk gland of silkworm,Bombyx mori L. fed an artificial diet

The effects of UV-light on biochemical parameters of posterior silk gland of mulberry silkworm, Bombyx mori L. were investigated in two popular bivoltine hybrids, Jufang (Chinese) × Chenxin (Japanese) and Chenxin × Jufang fed an artificial diet. Larvae of the first day of fifth instar were used for the present investigation. The larvae were irradiated with 20 W of UV-light at the intensity of 40 cm above the larvae for one of three exposure times (30, 60 or 120 min) just before feeding. UV treatment produced no significant effects at 30 min. However, at 60 or 120 min there was a significant decrease in silk gland weight and the levels of total protein, free amino acids, RNA and carbohydrates. UV-light thus affects the biosynthetic activities of silk glands.     

Predators usurp prey defenses? Toxicokinetics of tetrodotoxin in common garter snakes after consumption of rough-skinned newts

Snakes are common predators of organisms, such as amphibians, with toxic defenses that can be lethal to other predators. Because snakes do not have the option of dissecting prey into edible versus inedible components, they face a full dose of any chemical defenses encountered during attempted predation. This limitation has likely resulted in intense selection favoring the evolution of alternative mechanisms for dealing with prey toxins. These mechanisms can be physiological (e.g., resistance to prey toxins) or behavioral (e.g., toxin sampling and rejection). When physiological resistance arises, the possibility of bioaccumulation of a toxin results. We examined the coevolutionary interaction between the common garter snake (Thamnophis sirtalis) and the rough-skinned newt (Taricha granulosa), which contains a powerful neurotoxin called tetrodotoxin (TTX). In some populations syntopic with newts, individuals of T. sirtalis have evolved resistance to TTX. We examined the persistence of TTX in T. sirtalis after administration of an oral dose of TTX to investigate the possibility that snakes are sequestering TTX. The half-life of TTX in snake liver was estimated at 8.1?days. Accordingly, clearance of 99% of a single dose of TTX averages 61?days. Negative fitness consequences of intoxication during and after newt consumption may be balanced by co-opting the newts?? chemical defense for protection from the snakes?? own predators. Accounting of the coevolutionary dynamic between snakes and newts must incorporate post-consumption affects of lingering TTX.     

Stylochus mediterraneus (Turbellaria: Polycladida), predator on the mussel Mytilus galloprovincialis

The polyclad Stylochus mediterraneus Galleni has been found associated with the mussel Mytilus galloprovincialis Lmk., on which it feeds. Polyclads allowed to feed freely on groups of mussels of different sizes preyed mainly on small mussels <25 mm in length. The predation rate (number of mussels eaten per no. of polyclads per no. of days) ranged between 0.07 and 0.33. The average amount of food ingested by 1 polyclad feeding on mussels 16 to 25 mm or 26 to 35 mm in length was 7.1 and 11.2 (dry weight) mg per day, respectively. Before penetrating the mussels, the worm first straddles the valves at the posterior edge of the shell and then, after having digested the posterior adductor muscle, removes and swallows the soft parts of the prey.     

Chemical defense in pelagic octopus paralarvae: Tetrodotoxin alone does not protect individual paralarvae of the greater blue-ringed octopus (Hapalochlaena lunulata) from common reef predators

Some pelagic marine larvae possess anti-predator chemical defenses. Occasionally, toxic adults imbue their young with their own defensive cocktails. We examined paralarvae of the greater blue-ringed octopus (Hapalochlaena lunulata) for the deadly neurotoxin tetrodotoxin (TTX), and if present, whether TTX conferred protection to individual paralarvae. Paralarvae of H. lunulata possessed 150 ± 17 ng TTX each. These paralarvae appeared distasteful to a variety of fish and stomatopod predators, yet food items spiked with 200 ng TTX were readily consumed by predators. We conclude that TTX alone does not confer individual protection to paralarvae of H. lunulata, and that they possess an alternative defense. In larger doses, tetrodotoxin is a deterrent to the predatory stomatopod Haptosquilla trispinosa (mean dose = 3.97 μg/g). This corresponds to 12–13 paralarvae per predator based on the TTX levels of the clutch we examined. Thus, the basic assumption that individual paralarvae of H. lunulata are defended by TTX alone was disproved. Instead, functionality of TTX levels in paralarvae may arise through alternative selective pathways, such as deterrence to parasites, through kin selection, or against predator species not tested here.     

Combined use of earthworm (Alma millsoni) and bacterium (Bacillus sp.) improved the bioremediation of spent engine oil contaminated soil

This study was carried out to assess the effectiveness of the combined use of earthworm (Alma millsoni) and bacterium (Bacillus sp.) in the bioremediation of spent engine oil (SEO) contaminated soils. A. millisoni were collected from the Botanical Garden of University of Ibadan, Nigeria. The stock culture of hydrocarbonoclastic Bacillus sp. was used for the bioremediation study. A set-up of eight pots containing 1000?g soil sample and 20?g of cow dung were mixed with 100, 75, 50 and 0?mL SEO respectively. Each of the set up was subjected to bioremediation agents; A. millisoni alone, Bacillus sp alone, A. millisoni and Bacillus sp, no treatment (control) in duplicate. Treatment of 100?ml SEO contaminated soil with combined A. millisoni and Bacillus sp resulted in significantly (P? bacterium > vermi-remediating agents. Earthworms exposed to 100?mLSEO-contaminated soil had higher CAT, SOD, and GPx activities compared to the control. Findings indicated that A. millisoni with Bacillus sp. can synergistically improve bioremediation of SEO contaminated soils.