Responses of microbial components of the rhizosphere to plant management strategies in a semiarid rangeland

Summary Easily soluble heavy-metal fractions from different soils, a garbage-sewage sludge compost and peat were extracted by EUF. Blanks were determined by extracting distilled water. As the rubber seal of the extraction chamber contained Zn, the obtained Zn values were not reliable. The relative standard deviations of extracted micronutrients were 29.1% for Fe and 20.5% for Mn, Fe, Mn, Zn, Cu, Pb, Cd and Cr were not only found in the filters but also in the extracts.The extraction of CrIII and CrVI solutions showed that CrVI mainly migrated into the anode extract. CrIII was found mainly in the cathode filter and cathode extract, a smaller part however was obviously oxidized to CrVI and migrated into the anode extract. Consequently, CrIII and CrVI in soils could not be distinguished unequivocally by EUF.The amounts of Zn, Cu, Pb and Cd extracted by EUF from various substrates were small compared with the quantities extracted by 2N HCl. The heavy metal contents of the leaves were mostly in the order of those of the EUF extract.Several vineyard soils as well as peat were mixed with increasing quantities of Grünsalz (green salt), a fertilizer consisting mainly of iron sulphate. High amounts of Grünsalz (100–200 g/200 g soil) were necessary to raise soluble Fe in calcareous soils. In peat, however, small Grünsalz additions (1 g/50 g peat) were sufficient. Soluble Mn and Cu increased too when Grünsalz was added to soil or peat. These results give valuable information on how grapevine chlorosis can be reduced by the use of Grünsalz or mixtures of peat and Grünsalz.     

Experiments on the reproductibility of results from EUF soil extracts with possible improvements resulting from these experiments

Summary The reproducibility of EUF-Ca, K, Na, P, B, NO₃–N and EUF extractable N for fraction 1 (200 V, 20° C, 30 minutes) was within a CV range of 4.54–12.28%.The summation of results for fraction 1 (200 V, 20°C, 30 minutes) and fraction 2 (400 V, 80°C, 5 minutes) gave an improved CV range of 4.23–8.81%.EUF-Mg obtained from fraction 1 (200 V, 20°C, 30 minutes), fraction 2 (400 V, 80°C, 5 minutes) and the acid fraction gave a CV of 6.81%.Results from the acid fraction gave poor reproducibility for Zn, (CV=45.1%). The CV of 12.5% for Mn is low compared to the CV of 30–40% usually obtained.The recovery of Zn, Mn and Mg from the acid fraction was improved. The reproducibility of Zn and Mg was also improved by (a) reducing the sources of contamination; (b) introducing a second filter at the cathode; (c) steeping the filters for 48 hours.     

A comparison of electro-ultrafiltration and quantity/intensity measurements of soil potassium with its uptake by ryegrass in Scottish soils

Summary Electro-ultrafiltration (EUF) and quantity/intensity (Q/I) parameters of soil K were compared for 14 soils from each of three soil series. The K desorbed by EUF during the first 10 min (K₁₀) was closely correlated with the equilibrium activity ratio (AR₀) for soils of the same series, but differences between series reflected the soil K-buffering capacity, indicating that K₁₀ includes loosely held exchangeable K and is not strictly an intensity measurement. EUF values were compared with conventional soil test methods for predicting K-uptake and dry-matter yield of ryegrass grown in the glasshouse. Correlation coefficients between K uptake at the first cut were 0.80 for K₁₀, 0.88 for K_a (the initially labile K derived from the Q/I curve), 0.92 for K₃₅ (desorbed by EUF in 35 min) and 0.97 for K_ex (1.0M ammonium acetate extraction).     

Evaluation of the electro-ultrafiltration technique (EUF) as a measure of the K supplying power of Quebec soils

The electro-ultrafiltration (EUF) method has been used to evaluate the short-term and long-term supplying power of soils for many essential plant nutrients. The objective of this study was to compare the capacity of EUF with other extraction techniques to predict the plant availability of soil K and K fertilizer responsiveness by 10 cuts of alfalfa (Medicago sativa L.) growing over a 366-day period. Increasingly higher average concentrations of soil K were extracted by EUF at 50 V and 20°C (29 mg kg^-1), EUF at 200 V and 20°C (48 mg kg^-1), 0.002 M SrCl₂ (55 mg kg^-1), EUF at 200 V and 80°C (85 mg kg^-1), 0.1 M HCl (105 mg kg^-1), Mehlich 3 (119 mg kg^-1), 1 M NH₄OAc (120 mg kg^-1) and boiling 1 M HNO₃ (601 mg kg^-1). The large content of vermiculitic minerals in the silt and clay fractions is responsible for EUF desorbing more K in 55 minutes than NH₄OAc in 29 out of 30 soils. The total amount of K desorbed by EUF at 80°C was as effective as Mehlich 3-extractable K in predicting K uptake for the first three cuts and was best among the extracting procedures after boiling 1 M HNO₃ in predicting the long-term K supply, the uptake of K from non-exchangeable sources and the relative yield of alfalfa over 10 cuts. The desorption of soil K with EUF provides a better evaluation of the K-supplying power of Quebec soils than the extractants currently used, especially on a long-term basis.Contribution no. 396.     

Application of the EUF procedure in sugar beet cultivation

Summary The relationship between the EUF-nutrient fractions in the soil on the one hand and the nutrient uptake of sugar beet as well as root yield and quality (polarization, α-amino N etc.) on the other is described on the basis of results obtained over several years in surveys conducted in farmers' fields (5000–6000 fields under sugar beet per year) and in field experiments (25–35 sites per year). Statistically significant close correlations with the respective parameters were found for the following EUF nutrient fractions: EUF-NO₃, EUF-P, EUF-K, EUF-Na, EUF-B and EUF-Mn. Within five years it was possible to determine the EUF-nutrient values which are required for the production of 9 t sugar/ha. These EUF values are the following: Ca: 65–70 mg/100 g at 20°C K: 11–15 mg/100 g at 20°C (depending on the clay content) Mg: 3–5 mg/100 g at 20°C Na: 2–3 mg/100 g at 20°C P: 1.4–1.6 mg/100 g at 20°C For calculation of the N fertilizer requirements of sugar beet it is suggested to use the sum of the EUF-extractable N amounts. It was found in Austria, Yugoslavia and Denmark over a period of 3 years that the EUF-N value of 1 mg/100 g soil determined between June and September was equivalent to 40 kg N/ha. If, for example, the analysed soil contains 3 mg EUF-N/100 g, 3×40=120 kg N/ha will be available to the sugar beet crop in the following year.     

Preliminary investigations on available and potentially available phosphorus using electro-ultrafiltration (EUF)

Summary Preliminary investigations were carried out on calcareous silty loam (clay content 25%, pH 8.0) at the Cotton Research Institute, Multan to determine the solubility and desorption rates of phosphorus at various soil depths throughout the cotton growing season using the EUF technique. The treatments included two applications of single superphosphate, equivalent to 0, 75, 150, 250 and 350 kg P₂O₅/ha. There was no significant difference in seed cotton yield between the five treatments. EUF-extracted phosphorus decreased with depth in all samples. There was an increase in phosphorus concentration during the growing season, but most of the increase was noted in the slowly available phosphorus fraction (10–30 minutes EUF aliquots). This was attributed to continuous transformation of phosphorus into less soluble calcium phosphate forms. The available phosphorus fraction (0–10 minute EUF aliquots) remained small throughout the growing season. It was concluded that this fraction was in equilibrium with the slowly available phosphorus fraction and was continually being replenished as crop uptake continued. A high level of effectively available phosphorus (0–30 minute EUF aliquots) was recovered in all five treatments and was above the sufficiency level of 12 ppm phosphorus reported by Nemeth and Makhdam⁷. There was therefore no difference between treatments in phosporus concentration nor in phosphorus uptake by plants. This work was carried out under UNDP/FAO Project-PAK 73/026     

Influence of soil type and potassium and magnesium fertilization on the release of potassium and magnesium by electro-ultrafiltration and ammonium acetate-lactate extraction

Summary The effect of increasing potassium and magnesium fertilization during 15 years on the amounts of K and Mg extracted by AL-solution and desorbed by Electro-Ultrafiltration (EUF) was determined in four Swedish soils. In all soils, with increasing potassium fertilization the amounts of K extracted by AL and desorbed by EUF increased, and the amounts of Mg-AL and Mg-EUF decreased. Magnesium fertilization had no significant effect on the K values but increased both Mg-AL and Mg-EUF. The EUF-values were lower than the corresponding AL-values, but the ratio of EUF-desorbed to AL-extracted amounts of K and Mg varied depending on soil type as well as on fertilization rate. The ratio of K-EUF to K-AL increased and the ratio of Mg-EUF to Mg-AL decreased with increasing potassium fertilization, whereas magnesium fertilization decreased the ratio of Mg-EUF to Mg-AL.     

Pressurized liquids as an alternative green process to extract antiviral agents from the edible seaweed <Emphasis Type="Italic">Himanthalia elongata</Emphasis>

Pressurized liquid extraction (PLE), an environmentally friendly technique, was used to obtain antiviral compounds from the edible seaweed Himanthalia elongata. The antiviral properties of PLE extracts (hexane, ethanol, and water) were evaluated against herpes simplex virus type 1 (HSV-1) at different stages during viral infection. Pre-treatment of Vero cells with 75 μg mL⁻¹ of ethanol extract inhibited virus infection by approximately 90%, whereas the same concentration of water and hexane extracts reduced the virus infectivity to 78% and 70%, respectively. Moreover, ethanol extract was also more effective against HSV-1 intracellular replication than water and hexane extracts. The antiviral activity of water PLE extract was found to correlate with polysaccharides, since the polysaccharide-rich fraction isolated from this extract showed higher antiviral activity than the original water extract. A GC–MS characterization of the hexane and ethanol extracts showed that the antiviral activity of the hexane extract seemed to be related with the presence of fucosterol; meanwhile, in the case of the ethanol extract, other compounds, besides fucosterol, could be involved in this activity. Results demonstrated that PLE was an appropriate technique to obtain antiviral agents from H. elongata. These antiviral compounds were in addition to polysaccharides, which are the antiviral agents usually proposed when studying seaweeds.     

Variability of the healthy human proteome

The aim of this review is to analyze results of studies on characteristics of protein variability and diversity of posttranslational modifications of proteins in healthy humans. Numerous studies have demonstrated that a proteomic profile is characterized by significant intra- and inter-individual variability, and quite often natural (“normal”) variability of some proteins can be comparable to changes observed in pathological processes. Results obtained by our research group have demonstrated high intra-individual variability of serum low-molecular subproteome of healthy volunteers, certified by a special medial committee (the coefficient of variation (CV) of 42.6%). The proteins characterized by high variability under normal conditions (e.g. haptoglobin — 0–40 mg/ml; lysozyme — 0.01–0.1 mg/ml; C-reactive protein — 0.01–0.3 mg/ml) cannot be considered as potential biomarkers of diseases. On the contrary, proteins and peptides characterized by insignificant dispersion in healthy population (such as albumin ( CV = 9%); transferrin-(CV = 14%); C3c complement (CV = 17%), α-1 acid glycoprotein (CV = 21%), α-2-macroglobulin (CV = 20%); transthyretin fragment (CV = 28.3%) and α2-HS-glycoprotein βchain (CV = 29.7%)) can provide valuable information about the state of health. Thus, studies of plasticity in the proteomic profiles of healthy humans will help to correct reference intervals used in clinical proteomics.     

In vitro morphogenetic responses and comparative analysis of phthalides in the highly valued medicinal plant Ligusticum porteri Coulter & Rose

The morphogenetic response of Ligusticum porteri, a medicinal and ceremonial plant, was investigated as part of the conservation strategy of this wild species and was compared to that of a cultivated species, Petroselinum crispum. Seeds were germinated in half strength Murashige and Skoog medium. Plantlets were excised into root, cotyledon, petiole, stem and leaf explants and cultured in an induction medium supplemented with the range of 0–18.09 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 0–21.48 μM α-naphthaleneacetic acid in combination with 0–13.31 μM 6-benzylaminopurine. Calli derived from leaf, seeds, petiole, stem and roots, mature aerial parts and roots extracts of L. porteri and P. crispum were analyzed by thin layer chromatography and gas chromatography coupled to mass spectrometry. 3-Butylidenephthalide (6.3%) was identified along with other 23 compounds from mature aerial parts extract of L. porteri and also in its roots (20.8%). 3-n-Butylphthalide (0.7%) and 3,6,7,-trimethoxy-isobenzofuran-13(H)-one (4.9%) were identified from the roots of P. crispum. 3-Butylidenephthalide was identified from two petiole (0.9%; 0.26%) and one stem (0.8%) callus extracts of L. porteri. This is the first report on phthalides production from in vitro cultures of L. porteri. The results indicated that in vitro cultures of this plant possess the biosynthetic machinery for the biosynthesis of these highly valuable compounds.     

Characterisation of the magnesium status of soils by means of the EUF-Mg fractions

Summary On the basis of pot experiments carried out on soils with different Mg contents using ryegrass, it could be established that Mg uptake by ryegrass is closely related to Mg in the EUF extracts. The Mg contents in the EUF extracts are therefore suitable indices of actually and potentially available Mg in soils.The ratio between Mg in the EUF extracts and Mg deposited on the electrode + filter varies. The absolute amount of Mg in the EUF extracts increases with increasing Mg content of the soils. The higher, however, the total desorbable Mg the lower are the Mg contents in the EUF extracts in comparison with the Mg contents deposited on electrode + filter. Besides total desorbable Mg the EUF-Mg fractions are influenced as well by the organic and inorganic CEC and by soil pH. The amount of EUF filtrate flowing through filter and electrode also has an influence on the amount of Mg in the EUF filtrate.Consequently for a proper evaluation of available soil Mg total EUF-Mg (Mg in the EUF filtrate + Mg deposited on electrode + filter) should be used.     

Antioxidant properties of <Emphasis Type="Italic">Galium verum</Emphasis> L. (Rubiaceae) extracts

The antioxidant properties of methanol extracts of Lady’s Bedstraw (Galium verum L., Rubiaceae) herb from two different localities in Serbia were evaluated. Antioxidant activity was assessed in four different model systems. Free radical scavenging capacity (RSC) was examined by measuring the scavenging activity of extracts on 2,2-diphenyl-1-pycrylhydrazil (DPPH) and hydroxyl radical (OH), as well as on hydrogen peroxide. In addition, the protective effects of lipid peroxidation (LP) in corn oil were evaluated by the TBA-assay using the Fe²⁺/ascorbate system of induction. The amount of dried extract, the content of total phenolics, flavonoids and chlorophylls was also determined. Extracts from both locations expressed very strong scavenger activity, reducing the DPPH^⊙ (IC₅₀=3.10 μg/mland 8.04 μg/ml) and OH radical formation (IC₅₀=0.05 μg/ml and 0.54 μg/ml) and neutralising H₂O₂ (IC₅₀=4.98 μg/ml and 3.80 μg/ml), in a dose dependant manner. Also, examined extracts showed notable inhibition of LP (IC₅₀=11.69 μg/ml and 19.47 μg/ml). The observed differences in antioxidant activity could be partially explained by the levels of phenolics (2.44–4.65 mg and 4.57–5.16 mg gallic acid equivalents/g dry extract), flavonoids (6.38–10.70 μg and 15.56–17.96 μg quercetin equivalents/g dry extract) and chlorophylls in the investigated Lady’s Bedstraw extracts.     

Polysaccharides from the red seaweed Bostrychia montagnei: chemical characterization

Bostrychia montagnei was submitted to aqueous extraction at 25 and 85 °C. The purified polysaccharide extracts represent ∼ 17% of the dried alga. Galactose is the principal monosaccharide component of these extracts (60.8–70.4 mol%). 3,6-Anhydrogalactose and its 2- O-methyl derivative are also present in smaller amounts (16.2–22.0 mol%), as well as other methylated sugars, such as 6- O- (6.5–7.8 mol%) and 2-O-methylgalactose (0.2–2.1 mol%). Xylose (4.1–8.1 mol%) and glucose (0.7–2.6 mol%) were also detected. The aqueous extracted polysaccharides (25 °C) were separated by anion-exchange chromatography into six sulphated galactan fractions with negative specific rotations and another two with high xylose contents and positive specific rotations. The sulphated galactans all have an agar type backbone modified by partial O-methyl substitution on O-6 or O-2 of the galactosyl units. The latter substitution is also present in varying degrees of 3,6-anhydrogalactose. This revised version was published online in June 2006 with corrections to the Cover Date.     

SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP) for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05) at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively.     

Improved methods of analysis for betaines in <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> and its commercial seaweed extracts

Beneficial effects of seaweeds and their extracts on crop performance have been attributed to a variety of compounds, including the betaines which are quaternary ammonium betaines. Methods of analysis of betaines published thus far suffer from low sensitivity, lack of baseline separation of individual betaines and from interference from other sample constituents. A rapid cleanup protocol and a sensitive LC-MS/MS method of analysis were developed to afford baseline separation of four betaines in the brown alga Ascophyllum nodosum and its commercial seaweed extract. Using this method, the presence of glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine and laminine in A. nodosum, and commercial extracts derived from A. nodosum, were confirmed and quantified. The major betaine present was γ-aminobutyric acid betaine accounting for 0.008–0.014% of the dry weight of the seaweed and 0.014–0.027% of the dry weight of the commercial extracts. Seasonal variation in betaine content was observed. Differences in the total betaine content were observed between A. nodosum of the yellow (0.011–0.017% dry weight) and the olive green (0.017–0.021% dry weight) coloured morphologies.     

Antifungal Activity in Ethanolic Extracts of <Emphasis Type="Italic">Carica papaya</Emphasis> L. cv. Maradol Leaves and Seeds

Bioactive compounds from vegetal sources are a potential source of natural antifungic. An ethanol extraction was used to obtain bioactive compounds from Carica papaya L. cv. Maradol leaves and seeds of discarded ripe and unripe fruit. Both, extraction time and the papaya tissue flour:organic solvent ratio significantly affected yield, with the longest time and highest flour:solvent ratio producing the highest yield. The effect of time on extraction efficiency was confirmed by qualitative identification of the compounds present in the lowest and highest yield extracts. Analysis of the leaf extract with phytochemical tests showed the presence of alkaloids, flavonoids and terpenes. Antifungal effectiveness was determined by challenging the extracts (LE, SRE, SUE) from the best extraction treatment against three phytopathogenic fungi: Rhizopus stolonifer, Fusarium spp. and Colletotrichum gloeosporioides. The leaf extract exhibited the broadest action spectrum. The MIC₅₀ for the leaf extract was 0.625 mg ml⁻¹ for Fusarium spp. and >10 mg ml⁻¹ for C. gloeosporioides, both equal to approximately 20% mycelial growth inhibition. Ethanolic extracts from Carica papaya L. cv. Maradol leaves are a potential source of secondary metabolites with antifungal properties.     

Litre-scale microbial fuel cells operated in a complete loop

Using the anode effluent to compensate the alkalinization in a bio-cathode has recently been proposed as a way to operate a microbial fuel cell (MFC) in a continuous and pH neutral way. In this research, we successfully demonstrated that the operation of a MFC without any pH adjustments is possible by completing the liquid loop over cathode and anode. During the complete loop operation, a stable current production of 23.2 ± 2.5 A m⁻³ MFC was obtained, even in the presence of 3.2–5.2 mg O₂ L⁻¹ in the anode. The use of current collectors and subdivided electrical circuitries for relative large 2.5-L-scale MFCs resulted in ohmic cell resistances in the order of 1.4–1.7 mΩ m³ MFC, which were comparable to values of ten times smaller MFCs. Nevertheless, the bio-cathode activity still needs to be improved significantly with a factor 10–50 in order achieve desirable current densities of 1,000 A m⁻³ MFC. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Antimicrobial activity of the extracts from fruits of <Emphasis Type="Italic">Rumex</Emphasis> L. species

This study was designed primarily to investigate the antibacterial and antifungal activity of the extracts from fruits of six Rumex L. species: R. acetosa L., R. acetosella L., R. confertus Willd., R. crispus L., R. hydrolapathum Huds. and R. obtusifolius L. The 7 Grampositive and 7 Gram-negative bacteria strains and 5 fungal ones were tested by agar and broth dilution method. Determination of minimal inhibitory concentration (MIC) revealed that the extracts from R. confertus, R. crispus, R. hydrolapathum and R. obtusifolius exerted differential inhibitory effect on the growth of Gram-positive bacteria — staphylococci (MIC=62.5–125 μg/mL) and Gramnegative bacteria — Escherichia coli ATCC 3521, Proteus mirabilis, Pseudomonas aeruginosa (MIC=125→500 μg/mL); MIC values determined by agar dilution method were somewhat higher. The same extracts inhibited also the growth of fungi — Candida spp. or Trichophyton mentagrophytes ATCC 9533 (MIC=250–500 μg/mL), as found by agar dilution method. The total content of polyphenols (11.66–78.36 mg/g), anthracene derivatives (0.26–12.93 mg/g) and tannins (4.00–11.16%) was also determined.     

Wearing a raincoat: exocrine secretions contain anti-wetting agents in the oribatid mite, <Emphasis Type="Italic">Liacarus subterraneus</Emphasis> (Acari: Oribatida)

Liacarus subterraneus is a large, soil-dwelling oribatid mite species that possesses a conspicuously shiny, clean and not wettable cuticular surface. The exocrine cuticular chemistry of this species was investigated by means of gas chromatography–mass spectrometry. Besides a fraction of hydrocarbons and a terpene, hexane extracts of whole mite bodies exhibited free carboxylic acids and their glycerides as main components. The compounds were arranged in three distinct extract profiles. Based on data from individual extracts, (1) the majority (more than 3/4) of specimens showed large amounts of 1,2-dioctanoyl-glycerol (and three other related esters) but no (or only traces of) free carboxylic acids. (2) In about 1/8 of extracts, free acids (mainly octanoic (caprylic) acid) and glycerides were detected. This second type of profile highly varied with respect to the relative abundance of acids and esters. (3) The third profile (in about 7% of specimens) exclusively exhibited free acids and no (or only traces of) glycerides. In addition, a few extracts exhibited no components at all. The extract compounds most likely originate from the lipid layer of the cerotegument of L. subterraneus. The cuticle of individuals that possessed extractable cerotegumental compounds (profile I, II, III) exhibited strong water repellent properties, while the cuticle of individuals that possessed no components in their extract did not. After hexane extraction, water repellent properties got lost. The distinct extract profiles detected most likely portray the stepwise generation of an anti-wetting, exocrine surface lipid layer of glycerides: If this layer is lost, fatty acids may be discharged again (profile III) and may subsequently esterify (profile II) to larger and more stable esters (diacyl-glycerols), eventually building up the “raincoat” (mainly profile I) of L. subterraneus.