Wioletta Wieloch

Evolution of NIN-Like Proteins in Arabidopsis, Rice, and Lotus japonicus

Genetic studies in Lotusjaponicus and pea have identified Nin as a core symbiotic gene required for establishing symbiosis between legumes and nitrogen fixing bacteria collectively called Rhizobium. Sequencing of additional Lotus cDNAs combined with analysis of genome sequences from Arabidopsis and rice reveals that Nin homologues in all three species constitute small gene families. In total, the Arabidopsis and rice genomes encode nine and three NIN-like proteins (NLPs), respectively. We present here a bioinformatics analysis and prediction of NLP evolution. On a genome scale we show that in Arabidopsis, this family has evolved through segmental duplication rather than through tandem amplification. Alignment of all predicted NLP protein sequences shows a composition with six conserved modules. In addition, Lotus and pea NLPs contain segments that might characterize NIN proteins of legumes and be of importance for their function in symbiosis. The most conserved region in NLPs, the RWP-RK domain, has secondary structure predictions consistent with DNA binding properties. This motif is shared by several other small proteins in both Arabidopsis and rice. In rice, the RWP-RK domain sequences have diversified significantly more than in Arabidopsis. Database searches reveal that, apart from its presence in Arabidopsis and rice, the motif is also found in the algae Chlamydomonas and in the slime mold Dictyostelium discoideum. Thus, the origin of this putative DNA binding region seems to predate the fungus–plant divide.

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C(16:0), C(16:1), C(18:0), C(18:1), C(18:2), C(18:3), C(20:0) and C(20:1). At high concentrations spore germination was inhibited by C(7:0), C(8:0), C(9:0), C(10:0), C(12:0), C(18:2) and C(18:3) and hyphal growth was merely retarded by C(5:0), C(6:0), C(6:2), C(14:0), C(16:0), C(16:1), C(18:0,) C(18:1), C(20:0) and C(20:1). The presence of C(15:0) at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C(16:0) and C(18-20) fatty acids. Low concentrations of C(5:0), C(6:0), C(6:2) and C(7:0) enhanced sporulation. Fatty acids C(5-12) as well as C(18:3), C(20:0) and C(20:1) decreased the ability of fungal colonies to infect G. mellonella while C(16:1) elevated it thus suggesting that C(16:1) may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C(6:0), C(6:2,) C(7:0), C(9:0), C(12:0), C(16:1), C(18:2), C(18:3), C(20:0) and C(20:1); other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.

The Composition of the Cuticular and Internal Free Fatty Acids and Alcohols from Lucilia sericata Males and Females

GC, GC–MS, and HPLC–LLSD analyses were used to identify and quantify cuticular and internal lipids in males and females of the blow-fly (Lucilia sericata). Sixteen free fatty acids, seven alcohols and cholesterol were identified and quantitatively determined in the cuticular lipids of L. sericata. Cuticular fatty acids ranged from C6 to C20 and included unsaturated entities such as 16:1n-9, 18:1n-9, 20:4n-3 and 20:5n-3. Cuticular alcohols (only saturated and even-numbered) ranged from C12 to C20 in males and C10 to C22 in females. Only one sterol was found in the cuticular lipids of both males and females. 23 free fatty acids, five alcohols and cholesterol were identified in the internal lipids. Internal fatty acids were present in large amounts—7.4xa0mg/g (female) and 10.1xa0mg/g (male). Only traces of internal alcohols (from C14 to C26 in males, from C14 to C22 in females) were found in L. sericata. Large amounts of internal cholesterol were identified in L. sericata males and females (0.49 and 0.97xa0mg/g of the insect body, respectively).

Cuticular and internal n-alkane composition of Lucilia sericata larvae, pupae, male and female imagines: application of HPLC-LLSD and GC/MS-SIM

The composition of cuticular and internal n-alkanes in Lucilia sericata larvae, pupae, and male and female imagines were studied. The cuticular and internal lipid extracts were separated by HPLC-LLSD, after which the hydrocarbon fraction was identified by GC/MS in selected ion monitoring (SIM) and total ion current (TIC) modes. The cuticular lipids of the larvae contained seven n-alkanes from C23 to C31. The major n-alkane in L. sericata larvae was C29 (42.1%). The total cuticular n-alkane content in the cuticular lipids was 31.46 μg g-1 of the insect body. The internal lipids of L. sericata larvae contained five n-alkanes ranged from C25 to C31. The most abundant compound was C27 (61.71 μg g-1 of the insect body). Eighteen n-alkanes from C14 to C31 were identified in the cuticular lipids of the pupae. The most abundant n-alkanes ranged from C25 to C31; those with odd-numbered carbon chains were particularly abundant, the major one being C29:0 (59.5%). Traces of eight cuticular n-alkanes were present. The internal lipids of L. sericata pupae contained five n-alkanes, ranging from C25 to C31. The cuticular lipids of female imagines contained 17 n-alkanes from C12 to C30. Among the cuticular n-alkanes of females, C27 (47.5%) was the most abundant compound. Four n-alkanes, with only odd-numbered carbon chains, were identified in the internal lipids of females. The lipids from both sexes of L. sericata had similar n-alkane profiles. The cuticular lipids of adult males contained 16 n-alkanes ranging from C13 to C31. C27 (47.9%) was the most abundant cuticular n-alkanes in males. The same n-alkanes only with odd-numbered carbon chains and in smaller quantities of C27 (0.1%) were also identified in the internal lipids of males. The highest amounts of total cuticular n-alkanes were detected in males and females of L. sericata (330.4 and 158.93 μg g-1 of the insect body, respectively). The quantities of total cuticular alcohols in larvae and pupae were smaller (31.46 μg g-1 and 42.08 μg g-1, respectively). The internal n-alkane contents of larvae, pupae, and male and female imagines were significantly higher than the cuticular n-alkane contents (153.53, 99.60, 360.06 and 838.76 μg g-1 of the insect body, respectively).

Antimicrobial activity of alcohols from Musca domestica

SUMMARY Information on the stimulatory and inhibitory effects of cuticular alcohols on growth and virulence of insecticidal fungi is unavailable. Therefore, we set out to describe the content of cuticular and internal alcohols in the body of housefly larvae, pupae, males and females. The total cuticular alcohols in larvae, males and females of Musca domestica were detected in comparable amounts (4.59, 3.95 and 4.03 μg g−1 insect body, respectively), but occurred in smaller quantities in pupae (2.16 μg g−1). The major free alcohol in M. domestica larvae was C12:0 (70.4%). Internal alcohols of M. domestica larvae were not found. Among cuticular pupae alcohols, C12:0 (31.0%) was the most abundant. In the internal lipids of pupae, only five alcohols were identified in trace amounts. The most abundant alcohol in males was C24:0 (57.5%). The percentage content of cuticular C24:0 in males and females (57.5 and 36.5%, respectively) was significantly higher than that of cuticular lipids in larvae and pupae (0.9 and 5.6%, respectively). Only two alcohols were present in the internal lipids of males in trace amounts (C18:0 and C20:0). The most abundant cuticular alcohols in females were C24:0 (36.5%) and C12:0 (26.8%); only two alcohols (C18:0 and C20:0) were detected in comparable amounts in internal lipids (3.61±0.32 and 5.01±0.42 μg g−1, respectively). For isolated alcohols, antimicrobial activity against 10 reference strains of bacteria and fungi was determined. Individual alcohols showed approximately equal activity against fungal strains. C14:0 was effective against gram-positive bacteria, whereas gram-negative bacteria were resistant to all tested alcohols. Mixtures of alcohols found in cuticular lipids of larvae, pupae, males and females of M. domestica generally presented higher antimicrobial activity than individual alcohols. In contrast, crude extracts containing both cuticular and internal lipids showed no antifungal activity against the entomopathogenic fungus Conidiobolus coronatus, which efficiently kills adult house flies.

Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane

Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date. C. coronatus isolate 3491 inducing 100% mortality of G. mellonella last instar larvae exposed to sporulating colonies, was cultivated at 20 °C in minimal medium. Post-incubation filtrates were used as a source of fungal metabolites. A two-step HPLC (1 step: Shodex KW-803 column eluted with 50 mM KH(2)PO(4) supplemented with 0.1 M KCl, pH 6.5; 2 step: ProteinPak™ CM 8HR column equilibrated with 5 mM KH(2)PO(4), pH 6.5, proteins eluted with a linear gradient of 0.5 M KCl) allowed the isolation of coronatin-1, an insecticidal 36 kDa protein showing both elastolytic and chitinolytic activities. Addition of coronatin-1 into primary in vitro cultures of G. mellonella hemocytes resulted in rapid disintegration of spherulocytes freely floating in culture medium and shrinkage of plasmatocytes adhering to the bottom of culture well. Coronatin-1 stimulated pseudopodia atrophy and, in consequence, disintegration of nets formed by cultured hemocytes. After incorporation of coronatin-1 into planar lipid membrane (PLM) ion channels selective for K(+) ions in 50/450 mM KCl solutions were observed. Potassium current flows were recorded in nearly 70% of experiments with conductance from 300 pS up to 1 nS. All observed channels were active at both positive and negative membrane potentials. Under experimental conditions incorporated coronatin-1 exhibited a zero current potential (E(rev)) of 47.7 mV, which indicates K(+)-selectivity of this protein. The success of the purification of coronatin-1 will allow further characterization of the mode of action of this molecule, including ability of coronatin-1 to form potassium channels in immunocompetent hemocytes.

Application of Two-Way Hierarchical Cluster Analysis for the Identification of Similarities between the Individual Lipid Fractions of Lucilia sericata

The composition of the cuticular and internal lipids of larvae and pupae of Lucilia sericata was studied using chromatographic techniques. The lipids from both stages of L. sericata had similar free fatty acid (FFA) profiles and also contained alcohols and cholesterol. The range of the number of C‐atoms detected for these classes of compounds was to some extent similar in larvae and pupae, but the relative amounts of each class differed between stages. Saturated as well as unsaturated FFAs with even and odd numbered C‐atom chains were present in both cuticular and internal lipids. The alcohol fractions of L. sericata were represented by free, straight‐chain primary alcohols containing an even number of C‐atoms. The lipid composition of male and female L. sericata adults and the hydrocarbon composition of all stages of L. sericata had previously been analyzed. To have a full overview of the lipid composition and to identify similarities or dissimilarities between the individual lipid fractions in this insect species, two‐way hierarchical cluster analysis (HCA) was performed using also the data from these previous publications. The content of FFA 18u2009:u20091 (n‐9) was noticed to be very high in the cuticular fractions of larvae and pupae as well as in all internal fractions (male, female, larvae, and pupae) and low in the cuticular fractions of male and female imago. The contents of FFAs 16u2009:u20090 and 16u2009:u20091 (n‐9), cholesterol, and the n‐alkanes n‐C31, n‐C29, n‐C27, n‐C25, and n‐C23 varied between particular fractions, whereas the amounts of other compounds were similar in all fractions.

Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes.

Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva-1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.