Expression of Thioredoxin/Thioredoxin Reductase System Genes in Aphid-Challenged Maize Seedlings.

Thioredoxins (Trxs) and thioredoxin reductases (TrxRs) encompass a highly complex network involved in sustaining thiol-based redox homeostasis in plant tissues. The purpose of the study was to gain a new insight into transcriptional reprogramming of the several genes involved in functioning of Trx/TrxR system in maize (Zea mays L.) seedlings, exposed to the bird cherry-oat aphid (Rhopalosiphum padi L.) or the rose-grass aphid (Metopolophium dirhodum Walk.) infestation. The biotests were performed on two maize genotypes (susceptible Zlota Karlowa and relatively resistant Waza). The application of real-time qRT-PCR technique allowed to identify a molecular mechanism triggered in more resistant maize plants, linked to upregulation of thioredoxins-encoding genes (Trx-f, Trx-h, Trx-m, Trx-x) and thioredoxin reductase genes (Ftr1, Trxr2). Significant enhancement of TrxR activity in aphid-infested Waza seedlings was also demonstrated. Furthermore, we used an electrical penetration graph (EPG) recordings of M. dirhodum stylet activities in seedlings of the two studied maize varieties. Duration of phloem phase (E1 and E2 models) of rose-grass aphids was about three times longer while feeding in Waza plants, compared to Zlota Karlowa cv. The role of activation of Trx/TrxR system in maintaining redox balance and counteracting oxidative-induced damages of macromolecules in aphid-stressed maize plants is discussed.

Effect of manuka honey on biofilm-associated genes expression during methicillin-resistant Staphylococcus aureus biofilm formation.

Methicillin-resistant Staphylococcus aureus (MRSA) are among the most important biofilm-forming pathogens responsible for hard-to-treat infections. Looking for alternatives to antibiotics that prevent biofilm formation, we investigated the effects of manuka honey on the transcriptional profile of genes essential for staphylococcal biofilm formation using qRT-PCR. mRNA from two hospital MRSA strains (strong and weak biofilm producer) were isolated after 4, 8, 12 and 24 h from cells grown in biofilm. Manuka honey at 1/2 minimum biofilm inhibition concentration (MBIC) significantly reduced MRSA cell viability in biofilm. Manuka honey downregulated the genes encoding laminin- (eno), elastin- (ebps) and fibrinogen binding protein (fib), and icaA and icaD involved in biosynthesis of polysaccharide intercellular adhesin in both weakly and strongly adhering strain compared to the control (untreated biofilm). Expression levels of cna (collagen binding protein) and map/eap (extracellular adherence protein-Eap) were reduced in weakly adhering strain. The lowest expression of investigated genes was observed after 12 h of manuka honey treatment at 1/2 MBIC. This study showed that the previously unknown mechanism of manuka honey action involved inhibition of S. aureus adhesion due to reduction in expression of crucial genes associated with staphylococcal biofilm.

Effect of trans-Cinnamaldehyde on Methicillin-Resistant Staphylococcus aureus Biofilm Formation: Metabolic Activity Assessment and Analysis of the Biofilm-Associated Genes Expression.

The effects of trans-cinnamaldehyde (TC) on transcriptional profiles of biofilm-associated genes and the metabolic activity of two methicillin-resistant Staphylococcus aureus (MRSA) strains showing a different degree of adherence to polystyrene, were evaluated. Metabolic activity of S. aureus in biofilm was significantly decreased in the presence of TC at 1/2 minimum biofilm inhibition concentration (MBIC). Expression levels of the genes encoding laminin binding protein (eno), elastin binding protein (ebps) and fibrinogen binding protein (fib) in the presence of TC at 1/2 MBIC were lower than in untreated biofilm in both the weakly and strongly adhering strain. The highest decrease of expression level was observed in case of fib in the strongly adhering strain, in which the amount of fib transcript was 10-fold lower compared to biofilm without TC. In the presence of TC at 1/2 MBIC after 3, 6, 8 and 12 h, the expression level of icaA and icaD, that are involved in the biosynthesis of polysaccharide intercellular adhesin, was above half lower in the weakly adhering strain compared to biofilm without TC. In the strongly adhering strain the highest decrease in expression of these genes was observed after 3 and 6 h. This study showed that TC is a promising anti-biofilm agent for use in MRSA biofilm-related infections.

Changes in the content of thiol compounds and the activity of glutathione s-transferase in maize seedlings in response to a rose-grass aphid infestation.

The rose-grass aphid (Methopolophium dirhodum Walk.) is a major pest of maize (Zea mays L.), but little is known about the biochemical interactions between M. dirhodum and its host plant. Thiol compounds and glutathione S-transferase (GST) play a crucial role in the defense responses of maize to biotic stress factors, including aphids. The purpose of this research was to evaluate the impact of M. dirhodum herbivory on the total thiol (TT), protein bound thiol (PT), reduced glutathione (GSH) and oxidized glutathione (GSSG) contents as well as the activity of GST in three varieties of Z. mays (Zlota Karlowa, Ambrozja and Plomyk), that were classified as aphid-susceptible, aphid-relatively resistant and aphid-resistant, respectively. The earliest and strongest aphid-triggered alterations in the levels of TT, PT and GSH, and the greatest induction of GST activity, were recorded in the resistant Plomyk seedlings in relation to the relatively resistant Ambrozja and the susceptible Zlota Karlowa.

Transcriptional profiling of catalase genes in juglone-treated seeds of maize (Zea mays L.) and wheat (Triticum aestivum L.).

The major aim of the present study was to investigate the influence of juglone (JU; 5-hydroxy-1,4-naphthoquinone) treatments on the expression level of Cat1, Cat2 and Cat3 genes, encoding the respective catalase isozymes in maize (Zea mays L.) and wheat (Triticum aestivum L.) seeds. In parallel, germination efficiency, catalase (CAT) activity and hydrogen peroxide (H2O2) content in juglone-exposed cereal seeds were assessed. Juglone applications significantly stimulated abundance of three target catalase transcripts as well as induced CAT activity and generation of H2O2 in both maize and wheat kernels. Furthermore, germination process of juglone-affected maize seeds was more severe suppressed than in case of wheat kernels. The role of juglone in triggering the oxidative stress as well as antioxidative responses in seeds of the studied model cereal species are discussed.

Expression of the Biofilm-Associated Genes in Methicillin-Resistant Staphylococcus aureus in Biofilm and Planktonic Conditions.

The role of genes that are essential for development of Staphylococcus aureus biofilm during infection is not fully known. mRNA from two methicillin-resistant S. aureus strains that formed weak and strong biofilm on polystyrene plates were isolated at five time points from cells grown in biofilm and planktonic culture. Quantitative real-time PCR analysis showed that the expression levels of investigated genes under biofilm conditions were significantly higher than under planktonic conditions. The expression levels of the gene encoding elastin binding protein (ebps) and laminin binding protein (eno) were significantly increased in biofilm at 3 h, both in strongly and weakly adhering strain. The peak expression of fib gene encoding fibrinogen binding protein was found at 6 and 8 h in the case of strongly and weakly adhering strain, respectively. The expression of icaA and icaD genes in both strains was significantly higher under biofilm conditions when comparing to planktonic cells during 12 h. The expression level of the genes encoding binding proteins and the glucosamine polymer polysaccharide intercellular adhesin (PIA) slowly decreased after 24 h. Finally, we found that the expression levels of genes encoding binding factors in weakly adhering strain were significantly lower than in strongly adhering strain.

Coexistence of Borrelia burgdorferi s.l. genospecies within Ixodes ricinus ticks from central and eastern Poland.

The purpose of the study was to assess the prevalence and coinfection rates of Borrelia burgdorferi sensu lato genotypes in Ixodes ricinus (L.) ticks sampled from diverse localities in central and eastern regions of Poland. In years 2009-2011, questing nymphs and adults of I. ricinus were collected using a flagging method at 18 localities representing distinct ecosystem types: urban green areas, suburban forests and rural woodlands. Molecular detection of B. burgdorferi s.l. genospecies was based on amplification of a fla gene using nested PCR technique, subsequent PCR-RFLP analysis and bidirectional sequencing. It was revealed that 45 samples (2.1%) harboured two different B. burgdorferi s.l. genospecies, whereas triple infections with various spirochetes was found in 11 (0.5%) individuals. Generally, the highest average coinfection rates were evidenced in arachnids gathered at rural woodlands, intermediate at suburban forests, while the lowest were recorded at urban green areas. Overall, single spirochete infections were noted in 16.3% (n = 352/2,153) ticks. Importantly, it is the first report evidencing the occurrence of Borrelia miyamotoi (0.3%, n = 7/2153) in I. ricinus populations within central Poland. Circumstantial variability of B. burgdorferi s.l. genospecies in the common tick individuals sampled at various habitat types in central and eastern Poland was displayed. The coexistence of two or three different spirochete genospecies in single adult ticks, as well as the presence of B. miyamotoi were demonstrated. Therefore, further studies uncovering the co-circulation of the tested bacteria and other human pathogens in I. ricinus ticks are required.

Mechanism of entomotoxicity of the Concanavalin A in Rhopalosiphum padi (Hemiptera: Aphididae).

The toxicity effect of Concanavalin A (Canavalia ensiformis lectin, ConA) to bird cherry-oat aphid, Rhopalosiphum padi L. (Hemiptera: Aphididae), was investigated in the laboratory by using artificial diets containing ConA concentrations. Bird cherry-oat aphid performance was affected by the presence of Con A in artificial diets. The lectin added into the liquid diet increased the prereproductive period, mortality, and the average time of generation development (T) and decreased fecundity and the intrinsic rate of natural increase (rm). In attempt to unravel the mode of action of ConA, the interaction of the lectin with insect gut and the effect of ConA on feeding behavior were investigated. Extract of gut of treated grain aphid demonstrated DNA fragmentation, and this was accompanied with an increase in caspase 3 activity. Moreover, addition of ConA to the sucrose-agarose gels reduced salivation and passive ingestion of fluids from the gel. The results indicate that the insecticidal activity of ConA on R. padi may involve effects on death of the gut epithelial cells and effects on feeding behavior. This can be employed to create plants that are resistant to aphids.

Expression profiling of selected glutathione transferase genes in Zea mays (L.) seedlings infested with cereal aphids.

The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2•-) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2•- was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2•- generation in comparison with the Tasty Sweet genotype.

Are naringenin and quercetin useful chemicals in pest-management strategies?

The effects of two polyphenolic flavonoids (flavanone naringenin and flavonol quercetin) on development, fecundity, and mortality of the pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), were determined in vitro, on an artificial diets. Also determined in vitro (DC EPG method), on sucrose-agarose gels, were the effects of flavonoids on the probing and feeding behavior of adult apterae. When added to a liquid diet, higher concentrations of studied flavonoids increased the developmental time, the pre-reproductive period, and mortality and decreased fecundity and the intrinsic rate of natural increase of A. pisum. In most events associated with stylet activity (as indicated by EPG waveform g-C), differences in probing behavior did not statistically differ between the control gel and those with flavonoids; quercetin at 10, 100, and 1,000 µg cm-3 prolonged the number of gel penetrations; and quercetin only at 10,000 µg cm-3 prolonged the time the first g-C waveform was observed. Addition of flavonoids to the gels generally reduced passive ingestion from fluids of the gels (EPG waveform g-E2). At higher concentrations (>1,000 µg cm-3) the flavonoids completely stopped salivation (EPG waveform g-E1) and passive ingestion from fluids of the gels (EPG waveform g-E2). In events associated with active ingestion (EPG waveform g-G), however, differences in feeding behavior did not statistically differ between the control gel and those with flavonoids. The present findings demonstrate detrimental effects of the flavanone naringenin and flavonol on the behavior of the pea aphid. This can be employed in a biotechnological projects for plant breeding resistant to herbivores, including aphids.

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

Despite senescence-induced chlorophyll depletion in plants has been widely studied, the enzymatic background of this physiologically regulated process still remains highly unclear. The purpose of this study was to determine selected biochemical properties of partially purified fractions of chlorophyllase (Chlase, chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from leaves of three Prunus species: bird cherry (Prunus padus L.), European plum (Prunus domestica L.), and sour cherry (Prunus cerasus L.). Secondarily, this report was aimed at comparing seasonal dynamics of Chlase activity and chlorophyll a (Chl a) content within investigated plant systems. Molecular weight of native Chlase F1 has been estimated at 90 kDa (bird cherry) and approximately 100 kDa (European plum and sour cherry), whereas molecular mass of Chlase F2 varied from 35 kDa (European plum) to 60 kDa (sour cherry). Furthermore, enzyme fractions possessed similar optimal pH values ranging from 7.6 to 8.0. It was found that among a broad panel of tested metal ions, Hg(+2), Fe(+2), and Cu(+2) cations showed the most pronounced inhibitory effect on the activity of Chlase. In contrast, the presence of Mg(+2) ions influenced a subtle stimulation of the enzymatic activity. Importantly, although Chlase activity was negatively correlated with the amount of Chl a in leaves of examined Prunus species, detailed comparative analyses revealed an incidental decrement of enzymatic activity in early or moderately senescing leaves. It provides evidence that foliar Chlase is not the only enzyme involved in autumnal chlorophyll breakdown and further in-depth studies elucidating this catabolic process are required.

Molecular screening for Bartonella henselae and Borrelia burgdorferi sensu lato co-existence within Ixodes ricinus populations in central and eastern parts of Poland.

The presented study aimed at establishing the prevalence and co-infection rates of Bartonella henselae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from the central and eastern parts of Poland. The common tick individuals were gathered in the years 2008-2009. Questing ticks were sampled by dragging a white woollen flag over lower vegetation at 17 localities within diverse types of habitats: urban recreational green areas (city parks and squares), suburban forests and rural woodlands throughout the investigated regions of Poland. Detection of B. henselae in tested tick specimens was based on PCR amplification of the citrate synthase (gltA) gene, while screening for the presence of B. burgdorferi s.l. DNA was carried out by analyzing fragments of two genes: the flagellin (fla) and outer surface protein A (ospA). A total number of 1,571 I. ricinus ticks were sampled: 865 (55.1%) nymphs, 377 females (24.0%) and 329 males (20.9%). The application of PCR assays revealed that 76 (4.8%) tick samples were B. henselae-positive, B. burgdorferi s.l. DNA was detected in 194 specimens (12.3%), whereas the co-existence of these pathogens was evidenced in 22 tested ticks (1.4%). Furthermore, the occurrence of bartonellae and co-circulation of analysed microorganisms in I. ricinus was affirmed only within adult individuals, while presence of the screened spirochetes was ascertained in both nymphal and adult ticks. It should be stressed that the suburban woods of Warsaw and rural forests in Warsaw County characterized the highest prevalence levels of dual infection with investigated tick-borne pathogens, whereas the lowest co-infection rates were recorded in tick populations inhabiting rural forests in Plock County and forested areas in Korczew-Mogielnica (within the Nadbuzanski Landscape Park).

Antimetabolic effect of phytohemagglutinin to the grain aphid Sitobion avenae fabricius.

The insecticidal activity of plant lectins against a wide range of insect species have been intensively studied. Understanding the mechanism of the toxicity of lectins is one of the studied aspects. In the present research, the first step was determine the effect of phytohemagglutinin (PHA) on the development, fecundity and mortality of grain aphid. Next, the effect of PHA lectin on the activity of such enzymes as: α- and ß-glucosidases, alkaline (AkP) and acid (AcP) phosphatases, aminopeptidase N and cathepsin L involved in the metabolism of sugar, phosphorus and proteins of an adult apterae aphids was investigated. The PHA lectin added into the liquid diet increased the pre-reproductive period, mortality of Sitobion avenae, the time of generation development and decreased its fecundity and the intrinsic rate of natural increase. In addition, activity of α-glucosidase, alkaline phosphatase and aminopeptidase N of adult apterae exposed to PHA were reduced. The results indicate that the insecticidal activity of PHA on S. avenae may involve changes in activity of the enzymes in the midgut and it may be part of its toxicity.

Molecular evidence of Anaplasma phagocytophilum and Babesia microti co-infections in Ixodes ricinus ticks in central-eastern region of Poland.

The aim of the study was to elucidate the distribution of Anaplasma phagocytophilum and Babesia microti co-infection in Ixodes ricinus populations within the central-eastern region of Poland. The prevalence of analysed tick-borne human pathogens in single and polymicrobial infections in I. ricinus ticks were analysed using the conventional and nested PCR techniques. A total number of 1,123 questing tick individuals (291 females, 267 males and 565 nymphs) were collected at different ecosystems (municipal parks, suburban forests, and woodlands). In the presented study, 95 samples of ticks (8.5%) were infected with A.phagocytophilum, 3.1% (n=35) with B. microti, whereas the co-existence status of these human pathogens was detected in 1.8% (n=20) of all tested samples. It has been demonstrated that the prevalence of co-infection status was the highest among females of I. ricinus (11 samples, 3.8%), whereas the lowest within tested nymphs (5 samples, 0.9%). Ticks collected at city parks in Warsaw and suburban areas of this town characterized the highest prevalence of co-infections (3.3 and 4.8%, respectively). Furthermore, it was established that co-infection rates of ticks inhabiting woodlands within Kampinos National Park and Nadbuzanski Landscape Park were similar and reached the levels of 1.4% (n=5) and 1.1% (n=4), respectively.

Induced proteolysis within the bird cherry leaves evoked by Rhopalosiphum padi L. (Hemiptera, Aphidoidea).

The objectives of this study were to elucidate the impact of bird cherry-oat aphid (Rhopalosiphum padi L.) feeding on functioning of the proteolytic machinery in bird cherry leaves. Biochemical analyses proved that R. padi feeding in tissues of primary host stimulated activity of the two major fractions of proteinases (extracted at the optimal pH values: 5.0 and 7.0). Additionally, it has been demonstrated that aphids' feeding on bird cherry led to a decline in levels of albumins and globulins (main protein fractions in P. padus leaves). The opposite tendency, regarding the amounts of these protein fractions was ascertained at the phase of disappearance of R. padi population on tested shoots. Furthermore, it is reported that an increase in activity of the analysed enzymes and a decline in the content of tested protein fractions, were proportional to density of aphid individuals developing on P. padus side shoots. It is hypothesized that long-term R. padi feeding may lead to intensifying the catabolic processing of proteins by the activated proteolytic machinery in bird cherry leaves. The multi-level biological functions of endogenous plant proteinases and their significance in triggering the defense reactions in aphid-infested plant tissues are discussed.