Gabriel Visnovsky

A rapid method for the isolation of eicosapentaenoic acid-producing marine bacteria

Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements. A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species. By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (-) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography-mass spectrometry and the content of EPA was confirmed by gas chromatography. From a pool of 2.0 x 10(8)CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria.

Inclusion of chloroplast genes that have undergone expansion misleads phylogenetic reconstruction in the Chlorophyta

PREMISE OF THE STUDY Chlorophytes comprise a substantial proportion of green plant diversity. However, sister-group relationships and circumscription of the classes Chlorophyceae, Trebouxiophyceae, and Ulvophyceae have been problematic to resolve. Some analyses support a sister relationship between the trebouxiophycean Leptosira and chlorophyceans, potentially altering the circumscription of two classes, also supported by a shared fragmentation in the chloroplast gene rpoB. We sought to determine whether the latter is a synapomorphy or whether the supporting analyses are vulnerable to systematic bias. METHODS We sequenced a portion of rpoB spanning the fragmented region in strains for which it had not previously been sampled: four Chlorophyceae, six counterclockwise (CCW) group (ulvophyceans and trebouxiophyceans) and one streptophyte. We then explored the effect of subsampling proteins and taxa on phylogenetic reconstruction from a data set of 41 chloroplast proteins. KEY RESULTS None of the CCW or streptophyte strains possessed the split in rpoB, including inferred near relatives of Leptosira, but it was found in all chlorophycean strains. We reconstructed alternative phylogenies (Leptosira + Chlorophyceae and Leptosira + Chlorellales) using two different protein groups (Rpo and Rps), both subject to coding-region expansion. A conserved region of RpoB remained suitable for analysis of more recent divergences. CONCLUSIONS The Rps sequences can explain earlier findings linking Leptosira with the Chlorophyceae and should be excluded from phylogenetic analyses attempting to resolve deep nodes because their expansion violates the assumptions of substitution models. We reaffirm that Leptosira is a trebouxiophycean and that fragmentation of rpoB has occurred at least twice in chlorophyte evolution.

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

The potential of baculoviruses to be employed as insecticides is known since more than 75 years ago (Benz, 1986). To date, over 30 different baculoviruses are used to control several insect plagues in agriculture, horticulture and forestry (Moscardi, 1999). The use of baculovirus as insecticides is based on a set of useful properties, such as pathogenicity, specificity, narrow host range, environmental persistence, ability to act synergistically with other natural enemies of the pest and ability to induce artificial epizootics. Despite these advantages, very few baculoviruses have become widely used as insecticides, standing out as some successful examples the use of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) to control the velvetbean caterpillar in soybean crops in Latin America, Cidia pommonella granulovirus (CpGV) to fight the codling moth attacks in fruit orchards, and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) to control the armyworm in vegetable crops under cover in Europe (Moscardi, 1999). The causes of the limited acceptance of baculoviruses as insecticides are diverse, including slow speed of action, problems to register and market these biological insecticides and difficulties to produce them at an appropriate scale. The technologies currently used to produce insecticidal baculoviruses are based on the infection of susceptible insect larvae (Black et al., 1997). However, the implementation of processes to produce baculovirus in insect larvae is hampered by several limitations: high labour requirements, lack of expertise in standardization and validation of such processes, difficulties in scaling production to levels consistent with the profitability of the process and difficulties to properly control both the process production and product quality. While several improvements in production systems in insect larvae have been described in the last years which could help overcome some of the problems described above (van Beek & Davis, 2007), it has been also proposed that the adoption of an alternative technology based on the viral propagation in insect cell cultures could enable the development of well standardized, controlled and scalable production processes for insecticidal baculoviruses (Szewczyk et al., 2006).

Production of the Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line in stirred reactor and airlift reactor

The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), is one of the main plagues for soybean crops. Velvetbean caterpillar larvae are susceptible to be infected by occlusion bodies of the baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a biological insecticide. The insect cell line saUFL-AG-286 produces very high yields of occlusion bodies of AgMNPV in suspension cultures done in the low-cost serum-free medium UNL-10 in shake-flasks. However, its ability to adapt to conditions of industrial production in bioreactors was unknown. The aim of this study was to characterize the growth of saUFL-AG-286 cell cultures in UNL-10 medium, as well as its capability to replicate AgMNPV in two different bio-reactors at laboratory scale. The cell line was able to adapt to conditions that can be used at industrial scale, both in an airlift reactor and a stirred reactor, although the former was better than the last to support the cell growth. The infection with AgMNPV in the airlift reactor produced a high yield of occlusion bodies, with very low production of budded virus, the progeny used as inoculums. On the other hand, infection in the stirred reactor yielded high titers of budded virus. These results suggest that a feasible strategy for scaling-up the production of AgMNPV might involve the use of airlift reactors for the scaling-up of cell suspension cultures and the final production of occlusion bodies, while the scaling-up of the viral inoculums being carried out under conditions as those existing in stirred reactors.

Alpine Scenedesmaceae from New Zealand: new taxonomy

Cultures from samples collected from the alpine zone in Canyon Creek, Canterbury, New Zealand, revealed three taxa belonging to Scenedesmaceae. Identifications were made based on morphological data (light microscopy and transmission and scanning electron microscopy) and sequences of 18S ribosomal DNA. Desmodesmus abundans is a commonly recorded species worldwide and in New Zealand, but Desmodesmus granulatus is a new record for this country. Coelastrella multistriata var. grandicosta is described as new to science. Cryptodesmus ellipsoideus, previously isolated from the alpine zone at Arthurs Pass, is transferred to Coelastrella ellipsoidea on the basis of new ultrastructural and molecular information. Analysis of 18S ribosomal DNA in Coelastrella indicates likely intragenomic recombination and possible horizontal transfer of introns between taxa in this genus. http://zoobank.org/urn:lsid:zoobank.org:pub:2D9CA9F7-72DE-462B-8201-2725F4F9C91F

Novel alpine algae for New Zealand: Klebsormidiales

Abstract The alpine zone of Mt Philistine, Arthurs Pass National Park, is home to at least three species of algae in the Klebsormidiales. Klebsormidium dissectum was described from the site previously. A distinctive filamentous species, Klebsormidium crenulatum, has now been found that is larger than other New Zealand strains and is a new record for New Zealand. A unicellular species, Interfilum terricola, is also described as another new record for New Zealand. Phylogenetic analyses of the rbcL gene and the ITS-1-5.8S-ITS2 region show that New Zealand Klebsormidium is not monophyletic. The very close relationship between New Zealand and Belgian I. terricola illustrates the readiness with which transoceanic dispersal must occur in this genus. Our analyses of individual and concatenated genes suggest the paraphyly of the genus Klebsormidium, that the ancestral condition of the genus may have been the larger form, and that the incorporation of Klebsormidium into Interfilum is likely in the future, when more knowledge of diversity and relationships has accumulated.

Genome Sequences of Two Single-Stranded DNA Viruses Identified in Varroa destructor

ABSTRACT Varroa destructor is a ubiquitous and parasitic mite of honey bees, infecting them with pathogenic viruses having a major impact on apiculture. We identified two novel circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses from V. destructor sampled from a honey bee hive near Christchurch in New Zealand.

Cell culture for production of insecticidal viruses

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.

Identification, library synthesis and anti-vibriosis activity of 2-benzyl-4-chlorophenol from cultures of the marine bacterium Shewanella halifaxensis

Summer Gut Syndrome (SGS) is caused by various Vibrio bacterial species and can have negative effects on aquaculture farms worldwide. In New Zealand, SGS is caused by Vibrio harveyii infecting King Salmon (Oncorhynchus tshawytscha). To find leads for the prevention of SGS, we screened the inhibitory effects of 16 strains of Shewanella upon V. harveyii growth in competitive solid phase cultures. The detailed investigation of Shewanella halifaxensis IRL548 revealed 2-benzyl-4-chlorophenol (1), a known, commercially available antibacterial agent, as the major bioactive component. Synthesis of a small library of congeners to confirm the natural product identity and to provide a structure-activity relationship for the observed activity was also completed. Compound 1 exhibits moderate activity against two pathogenic microorganisms.

Effect of nutrients on the growth of a new alpine strain of Haematococcus (Chlorophyceae) from New Zealand: Effect of nutrients on Haematococcus

The microalga Haematococcus lacustris is a source of astaxanthin used widely in aquaculture, pharmaceutical, and cosmetic industries. A new strain of Haematococcus (LCR‐26C‐1f) isolated from the New Zealand alpine zone was evaluated in this study. The influence of vitamins, micronutrients, various carbon and nitrogen sources were investigated to maximize biomass production in batch cultures using shake flasks. Supplementation of vitamins consisting of thiamine, biotin, and cyanocobalamin improved the cell density by 40% over the vitamin‐free medium. Out of the individual vitamins tested, thiamine was shown to be necessary to maintain high cell densities. The best nitrogen source tested was nitrate in the form of sodium nitrate, at a 40 mM concentration. Heterotrophic growth yielded much lower cell densities compared to autotrophic growth. The micronutrients iron and manganese were essential for growth. However, the best growth was obtained using a micronutrient mix that included iron, copper, cobalt, zinc, manganese and molybdenum.