Evolution and detection of cyanobacterial hepatotoxin synthetase genes

Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are Microcystis, Anabaena, and Planktothrix. Nodularins are produced by strains of Nodularia spumigena in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (mcyE) and the orthologous nodularin synthetase gene F (ndaF) sequences. General primers were designed to amplify the mcyE/ndaF gene region from microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that mcy genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that nda genes evolved from mcy genes. The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general mcyE primers, and in 91% of the lakes with the three genus-specific mcyE primers. Potential microcystin-producing Microcystis were detected in 70%, Planktothrix in 63%, and Anabaena in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of mcyE genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. Genus-specific probe pairs for microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the mcyE/ndaF genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. Analysis of the mcy and nda gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of Microcystis, Planktothrix, and Anabaena, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.

Development of cat's claw creeper leaf-tying moth Hypocosmia pyrochroma (Lepidoptera: Pyralidae) at different temperatures: Implications for establishment as a biological control agent in Australia and South Africa

The leaf-tying moth Hypocosmia pyrochroma Jones (Lepidoptera: Pyralidae), a native of sub tropical South America, has been introduced as a biological control agent for cat’s claw creeper, Dolichandra unguis-cati (L.) Lohman (Bignoniaceae), in Australia and South Africa. So far there has been no evidence of its field establishment in either country. A narrow temperature tolerance is a potential limiting factor for the establishment of weed biological control insects in novel habitats. In this study, we evaluated the effect of seven constant temperatures (12–40 °C) on the survival and development of H. pyrochroma in temperature-controlled cabinets. Temperatures between 20 and 30 °C were the most favorable for adult survival, oviposition, egg hatching, and larval and pupal development. Adult survival (12–40 °C) and egg development (15–35 °C) showed tolerance for wider temperature ranges than oviposition, and larval and pupal development, which were all negatively affected by both high (>30 °C) and low (<20 °C) temperatures. The degree-day (DD) requirement to complete a generation was estimated as 877 above a threshold temperature of 12 °C. Based on DD requirements and an obligatory winter diapause of pupae from mid-autumn to mid-spring, the potential number of generations (egg to adult) the leaf-tying moth can complete in a year in Australia or South Africa range from one to three. A climate-matching model predicted that the inland regions of both Australia and South Africa are less favorable for H. pyrochroma than the coastal areas. The study suggested that H. pyrochroma is more likely to establish in the coastal areas of Australia where most of the cat’s claw creeper infestations occur, than in South Africa where most of the cat’s claw creeper infestations are inland.

Biological control of Mikania micrantha in Papua New Guinea and Fiji using the rust fungus Puccinia spegazzinii

Mikania micrantha Kunth (Asteraceae), commonly known as ‘mile-a-minute’, is a neotropical plant species now found in 17 Pacific island countries and territories, invading small cropping areas and plantations, thereby reducing productivity and food security. In 2006, a biocontrol project on M. micrantha commenced in Fiji and Papua New Guinea (PNG). The distribution of M. micrantha as well as baseline data such as plant growth rates and socio-economic impacts were determined before the importation of any biocontrol agents. Mikania micrantha was recorded in all 15 lowland provinces in PNG and on all major islands in Fiji. Plants grow about 3.2cm/day in PNG and about 1.9cm/day in Fiji. A socio-economic survey, involving over 370 respondents in over 220 villages from 15 provinces in PNG, found that 78% of respondents considered M. micrantha a serious weed and about 44% had M. micrantha, which they needed to weed at least fortnightly, in over a third of their land. Over 80% of respondents used slashing and/or handpulling as the preferred method of weed control. About 40% of respondents considered that M. micrantha reduced crop yield by more than 30%. In Fiji, 52 respondents from four islands participated in the survey. Over 60% of respondents in Fiji considered M. micrantha a serious weed and 23% had about 30% of their farm lands infested with the weed. Only 15% of respondents needed to weed at least fortnightly, with 56% using slashing and/or hand-pulling as the preferred means of control. Over 65% of respondents estimated that they lost at least 30% of potential crop yield to M. micrantha. Nearly 90% of respondents used M. micrantha as a medicinal plant to treat cuts and wounds. The life history of the rust Puccinia spegazzinii de Toni (Pucciniales: Pucciniaceae), originating from Ecuador, and imported into PNG and Fiji in 2008, was studied. P. spegazzinii is a microcyclic and autoecious rust and has a life cycle of 18-22 days. An efficient culturing and field release method was developed. Since 2008, the rust has been released at over 450 sites in 15 provinces in PNG, establishing at nearly 70 sites in four provinces. From some sites, the rust has spread over 7 km in 12 months. In Fiji, the rust has been released at over 80 sites, on four of the main islands, namely Viti Levu, Vanua Levu, Taveuni and Ovalau, and has established at 20 sites on Viti Levu and Vanua Levu. Plant growth studies and field monitoring in PNG showed that P. spegazzinii can significantly reduce the growth and density of M. micrantha and offers great potential for the control of this weed.

Assessing the potential of the rust fungus Puccinia spegazzinii as a classical biological control agent for the invasive weed Mikania micrantha in Papua New Guinea

The rust fungus Puccinia spegazzinii was introduced into Papua New Guinea (PNG) in 2008 as a classical biological control agent of the invasive weed Mikania micrantha (Asteraceae), following its earlier release in India, mainland China and Taiwan. Prior to implementing field releases in PNG, assessments were conducted to determine the most suitable rust pathotype for the country, potential for damage to non-target species, most efficient culturing method and potential impact to M. micrantha. The pathotype from eastern Ecuador was selected from the seven pathotypes tested, since all the plant populations evaluated from PNG were highly susceptible to it. None of the 11 plant species (representing eight families) tested to confirm host specificity showed symptoms of infection, supporting previous host range determination. A method of mass-producing inoculum of the rust fungus, using a simple technology which can be readily replicated in other countries, was developed. Comparative growth trials over one rust generation showed that M. micrantha plants infected with the rust generally had both lower growth rates and lower final dry weights, and produced fewer nodes than uninfected plants. There were significant correlations between the number of pustules and (a) the growth rate, (b) number of new nodes and (c) final total dry weight of single-stemmed plants placed in open sunlight and between the number of pustules and number of new nodes of multi-stemmed plants placed under cocoa trees. The trials suggest that field densities of M. micrantha could be reduced if the rust populations are sufficiently high. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.

Mitochondrial DNA diversity of Cleruchoides noackae (Hymenoptera: Mymaridae): a potential biological control agent for Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae)

Carpintero and Dellap, (Hemiptera: Thaumastocoridae) is a native Australian sap-feeding insect that has become invasive and seriously damaging to commercially grown in the Southern Hemisphere. Lin and Huber (Hymenoptera: Mymaridae) was recently discovered as an egg parasitoid of the Thaumastocoridae in Australia. Mitochondrial DNA (mtDNA; cytochrome oxidase subunit I, COI) sequence diversity amongst 104 individuals from these native populations revealed 24 sequence haplotypes. The COI haplotypes of individuals collected from the Sydney and Southeast Queensland clustered in distinct groups, indicating limited spread of the insect between the regions. Individuals collected from Perth in Western Australia were represented by four COI haplotypes. Although this population is geographically more isolated from other populations, two COI haplotypes were identical to haplotypes found in the Sydney region. The results suggest that has recently been introduced into Perth, possibly from the Sydney area. The high mtDNA diversity and limited spread that is suggested for is in contrast to the lack of geographic associated mtDNA diversity and extensive spread of . If implemented as a biological control agent, this factor will need to be considered in collecting and releasing .

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Biological and economic management strategy evaluations of the eastern king prawn fishery

Stock assessment of the eastern king prawn (EKP) fishery, and the subsequent advice to management and industry, could be improved by addressing a number of issues. The recruitment dynamics of EKP in the northern (i.e., North Reef to the Swain Reefs) parts of the fishery need to be clarified. Fishers report that the size of the prawns from these areas when they recruit to the fishing grounds is resulting in suboptimal sizes/ages at first capture, and therefore localised growth overfishing. There is a need to assess alternative harvest strategies of the EKP fishery, via computer simulations, particularly seasonal and monthly or lunar-based closures to identify scenarios that improve the value of the catch, decrease costs and reduce the risk of overfishing, prior to implementing new management measures.

Issues of Digital Ecosystems Approach to Biological Ecosystem Simulation Model Fit Optimization

This study examines the application of digital ecosystems concepts to a biological ecosystem simulation problem. The problem involves the use of a digital ecosystem agent to optimize the accuracy of a second digital ecosystem agent, the biological ecosystem simulation. The study also incorporates social ecosystems, with a technological solution design subsystem communicating with a science subsystem and simulation software developer subsystem to determine key characteristics of the biological ecosystem simulation. The findings show similarities between the issues involved in digital ecosystem collaboration and those occurring when digital ecosystems interact with biological ecosystems. The results also suggest that even precise semantic descriptions and comprehensive ontologies may be insufficient to describe agents in enough detail for use within digital ecosystems, and a number of solutions to this problem are proposed.

Prospects for the classical biological control of Calotropis procera (Apocynaceae) using co-evolved insects

Calotropis procera (Apocynaceae), a native of tropical Africa, the Middle East and the Indian subcontinent, is a serious environmental and rangeland weed of Australia and Brazil. It is also a weed in Hawaii in USA, the Caribbean Islands, the Seychelles, Mexico, Thailand, Vietnam and many Pacific Islands. In the native range C. procera has many natural enemies, thus classical biological control could be the most cost-effective option for its long-term management. Based on field surveys in India and a literature search, some 65 species of insects and five species of mites have been documented on C. procera and another congeneric-invador C. gigantea in the native range. All the leaf-feeding and stem-boring agents recorded on Calotropis spp. have wide host range. Three pre-dispersal seed predators,the Aak weevil Paramecops farinosus and the Aak fruit fly Dacuspersicus in the Indian subcontinent, and the Sodom apple fruit fly Dacus longistylus in the Middle East have been identified as prospective biological control agents based on their field host range. In Australia and Brazil, where C. procera has the potential to spread across vast areas, pre-dispersal seed predators would help to limit the spread of the weed. While the fruits of C. procera vary in size and shape across its range, those from India are similar to the ones in Australia and Brazil. Hence, seed-feeding insects from India are more likely to be suitable due to adaptation to fruit size and morphology. Future survey efforts for potential biological control agents should focus on North Africa.