Saprolegnia parasitica, an oomycete pathogen with a fishy appetite: new challenges for an old problem

Water moulds (oomycetes) of the order Saprolegniales, such as Saprolegnia and Aphanomyces species, are responsible for devastating infections on fish in aquaculture, fish farms and hobby fish tanks. Members of the genus Saprolegnia cause Saprolegniosis, a disease that is characterised by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. Up till 2002, Saprolegnia infections in aquaculture were kept under control with malachite green, an organic dye that is very efficient at killing the pathogen. However, the use of malachite green has been banned worldwide due to its carcinogenic and toxicological effects and this has resulted in a dramatic re-emergence of Saprolegnia infections in aquaculture. As a consequence Saprolegnia parasitica is now, economically, a very important fish pathogen, especially on catfish, salmon and trout species, and warrants further investigation to develop new alternative control strategies.     

Host-parasite relationship of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica in the Argentinean Patagonian coast

The association of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica is described. The identity of the green alga was confirmed by molecular studies; the alga was found within the hemocytes that infiltrate the connective tissue of the geoduck siphons. Cytological characteristics of hemocytes were not altered by algal infection; very often the algae were seen enveloped by a digestive vacuole within the hemocyte cytoplasm, evidencing diverse degrees of resorption. Connective cells of siphons were rarely infected by C. parasitica. The mean prevalence of C. parasitica was higher (82%) in San Matías Gulf (42°00′S, 65°05′W) than in San José Gulf (45%) (40°32′S, 64°02′W); except for spring, when the two locations showed no differences in prevalences (80%). Independently of location, season and host size, infected geoducks showed lower condition index values than uninfected ones. Regarding other bivalve species, only one specimen of the razor clam Ensis macha was found infected, and none of the oysters Ostrea puelchana and Pododesmus rudis and scallop Aequipecten tehuelchus was parasitized by the green alga.     

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.     

Repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale, against the maize weevil, Sitophilus zeamais

The repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale (Zingiberaceae), against the maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae), was investigated in four-way olfactometer bioassays. Results showed that vacuum distilled A. melegueta and Z. officinale extracts were repellent towards adult S. zeamais both in the absence and the presence of maize, Zea mays, grains. Bioassay-guided liquid chromatographic fractionation of the distillates showed that fractions containing oxygenated compounds accounted for the repellent activity. Coupled gas chromatography-mass spectrometry (GC-MS), followed by GC peak enhancement and enantioselective GC using authentic compounds, identified 3 major compounds in the behaviourally active fractions of A. melegueta and Z. officinale to be (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool in a ratio of 1:6:3, and 1,8-cineole, neral and geranial in a ratio of 5.48:1:2.13, respectively. The identification of these behaviourally active compounds provides the scientific basis for the observed repellent properties of A. melegueta and Z. officinale, and demonstrates the potential for their use in stored-product protection at the small-scale farmer level in Africa.     

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1

We characterized a novel Bacillus thuringiensis isolate native to China (HS18-1) that shows a spherical crystal harboring two major proteins of about 70 and 130 kDa, and contains three novel cry genes (cry4Cb1, cry30Ga1, cry54-type). Furthermore, the cry4Cb1 and cry30Ga1 genes were expressed in Escherichia coli BL21 (DE3): pLysS. Insecticidal activity tests showed that the cry4Cb1 protein exhibited larvicidal activity against Aedes aegypti (Diptera) and the cry30Ga1 protein was toxic to both A. aegypti and P. xylostella (Lepidoptera).     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Giardia lamblia: Immunogenicity and intracellular distribution of GHSP-115, a member of the Giardia head-stalk family of proteins

Giardia lamblia, a protozoan causing diarrheal outbreaks, is one of the main pathogens monitored in developed countries. Immunoscreening of G. lamblia expression library using the monoclonal antibodies (mAb) against G. lamblia, identified a subset of antigenic proteins in this protozoan, which are proteins belonging to GHSP (Giardia head-stalk protein), GHSP115, GHSP138, and GHSP180. In order to map the epitope region of GHSP115, the corresponding open reading frame was dissected into three parts and expressed as recombinant proteins with histidine tags. Western blot analysis of these recombinant proteins with mAbs reacting with GHSP115 indicated that one-third of the C-terminus of GHSP115 showed immunoreactivity with the mAb. Intracellular location of GHSP115 was examined both in trophozoites and encysting cells of G. lamblia by an immunofluorescence assay, indicating that location of GHSP115 varies during encystation. These results suggest that GHSP115 is an abundant and antigenic protein, which is differentially localized during life cycle of G. lamblia.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

Detection of ‘long-haired’ Saprolegnia (S. parasitica) isolates using monoclonal antibodies

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs (S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptor’s C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.     

Expression of recombinant HAO3 from an Iranian isolate of Hyalomma anatolicum anatolicum in Pichia pastoris and evaluation of its antigenicity

Hyalomma anatolicum anatolicum tick is considered as one of the main problem of ruminants’ productivity in endemic countries such as parts of Africa, the Middle East and India. The disease is economically important and hence, its control and eradication is a priority. This problem reinforces the need for alternative approach like vaccine to control tick infestations instead of continuous application of acaricide which led to the natural selection of the acaricide-resistant ticks. Therefore, the present study provided evidence for the construction of transformant containing the chromosomally integrated multi-copy expression cassettes of HAO3, its successful and efficient expression in Pichia pastoris yeast and purification of the secreted protein by ultrafiltration (UF) system in a high level yield and purity.The result of antigenicity assay for the rHAO3 protein pointed well toward its capability for the elicitation of antibody response in immunized rabbits. Interestingly, the results indicated that the expressed HAO3 protein reacted well with mid gut antigen (MGAg) and rBm86 (Gavac) antisera in ELISA and western blot assays making it evident that the epitopes present in expressed protein are well recognized by the antibodies against MGAg and rBm86 proteins. Moreover, the presence of cross-reactive epitopes between rHAO3 protein with its native antigen from mid gut cells was also determined.     

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the ¹⁵N-AATS-labeled protein, the transformed B. choshinensis was cultured in ¹⁵N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a ¹⁵N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The ¹⁵N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.