Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid-state fermentation of deoiled Jatropha curcas seed cake

Large amount of seed cake is generated as by-product during biodiesel production from Jatropha seeds. Presence of toxic phorbol esters restricts its utilization as livestock feed. Safe disposal or meaningful utilization of this major by-product necessitates the degradation of these phorbol esters. The present study describes the complete degradation of phorbol esters by Pseudomonas aeruginosa PseA strain during solid state fermentation (SSF) of deoiled Jatropha curcas seed cake. Phorbol esters were completely degraded in nine days under the optimized SSF conditions viz. deoiled cake 5.0 g; moistened with 5.0 ml distilled water; inoculum 1.5 ml of overnight grown P. aeruginosa; incubation at temperature 30 °C, pH 7.0 and RH 65%.SSF of deoiled cake seems a potentially viable approach towards the complete degradation of the toxic phorbol esters.     

Antifungal activity of the termite alkaloid norharmane against the mycelial growth of Metarhizium anisopliae and Aspergillus nomius

Antifungal activity of norharmane, a β-carboline alkaloid found in termites (Isoptera, Rhinotermitidae) was tested against two entomopathogenic fungi, Metarhizium anisopliae and Aspergillus nomius. It was determined that, at physiological concentration (10 μg ml⁻¹), norharmane had no significant effect on A. nomius mycelial growth rate but reduced M. anisopliae growth rate by 11.9%. Contrary to previous findings, we suggest that norharmane has a limited role in disease resistance against fungal pathogens in individual subterranean termites, and we discuss the potential role of this chemical at a colony level.     

Insecticidal activity and chemical composition of the essential oil of Artemisia vestita from China against Sitophilus zeamais

In our screening program for new agrochemicals from local wild plants, essential oil of Artemisia vestita Wall (Asteraceae) was found to possess strong insecticidal activity against maize weevil, Sitophilus zeamais Motsch. Essential oil of aerial parts of A. vestita was obtained from hydrodistillation and was investigated by GC and GC–MS. The main components of essential oil were grandisol (40.29%), 1,8-cineol (14.88%) and camphor (11.37%). The essential oil of A. vestita possessed strong fumigant toxicity against S. zeamais adults with a LC₅₀ value of 13.42 mg/L air. The essential oil of A. vestita also showed contact toxicity against S. zeamais adults with a LD₅₀ value of 50.62 mg/adult.     

Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity

Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F₂ mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F₂ plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker‐assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.     

Microwave assisted alkali-catalyzed transesterification of Pongamia pinnata seed oil for biodiesel production

In this study, microwave assisted transesterification of Pongamia pinnata seed oil was carried out for the production of biodiesel. The experiments were carried out using methanol and two alkali catalysts i.e., sodium hydroxide (NaOH) and potassium hydroxide (KOH). The experiments were carried out at 6:1 alcohol/oil molar ratio and 60 °C reaction temperature. The effect of catalyst concentration and reaction time on the yield and quality of biodiesel was studied. The result of the study suggested that 0.5% sodium hydroxide and 1.0% potassium hydroxide catalyst concentration were optimum for biodiesel production from P. pinnata oil under microwave heating. There was a significant reduction in reaction time for microwave induced transesterification as compared to conventional heating.     

The use of warmed water treatment to induce protective immunity against the bacterial cold-water disease pathogen Flavobacterium psychrophilum in ayu (Plecoglossus altivelis)

We investigated the induction of protective immunity against bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum by warmed water treatment in ayu (Plecoglossus altivelis). Fish were immersed in a live bacterial suspension (10⁷ CFU mL⁻¹) for 30 min and placed in 700 L concrete tanks. The 28 °C warmed water treatment lasted 3 days and began 1, 6, and 24 h after immersion in the live bacterial suspension. A naïve control fish group was immersed in a sterilized modified Cytophaga (MCY) broth instead of the bacterial suspension. Fourteen days after the immersion, agglutination antibody titers against F. psychrophilum were measured by using micro-titer methods. Fish were then exposed to a bacterial bath to infect them with live F. psychrophilum, and cumulative mortality was monitored. Fish treated with warmed water at 1, 6, and 24 h after immersion in the live bacterial suspension had cumulative mortalities of 36%, 30%, and 18%, respectively, all of which were significantly lower than the cumulative mortality of the naïve control fish (90%). Treated fish also showed high antibody titers against F. psychrophilum in agglutination tests. These results demonstrate that warmed water treatment could not only cure BCWD but also immunize the fish against the causative agent F. psychrophilum.     

Leaf press juice from Bryophyllum pinnatum (Lamarck) Oken induces myometrial relaxation

Aims

The use of preparations from Bryophyllum pinnatum (Lamarck) Oken (Kalanchoe pinnata (Lamarck) Persoon) in tocolysis is supported by clinical evidence. We studied here the effect of B. pinnatum leaf press juice and its chemical fractions on the response of human myometrial strips. No data are available if the influence on myometrial strips of the juice differs from that of its components in the chemical fractions, in order to increase the pharmacological effect.

Methodology

In vitro study to test the effect of repeated addition of B. pinnatum leaf press juice (BPJ) and its chemical components in several dilutions (undiluted, 1-10%) on myometrium strips hang up in a myograph chamber. Chemical analysis is including HPLC, MPLC with Sephadex LH-20 and TLC.

Results

All test solutions are inhibiting contractility by reducing the amplitude and the area under the curve (AUC) of the contractions. Undiluted BPJ and its undiluted chemical fraction 4 are reducing most effective these two parameters: the amplitude was at 78% of the baseline (95% CI (77-89); p < 0.05) at the second addition of the BPJ and at 70% (95% CI (50-90); p < 0.05) of the first addition of fraction 4; the AUC was at 82% (95% CI (69-95); p < 0.05) of the baseline at the first addition of the press juice and at 51% (95% CI (27-74); p < 0.05) at the first addition of fraction 4. The BPJ decreased amplitude and AUC significantly faster and increased frequency significantly faster than the control. Fractions could be tentatively assigned to bufadienolids, flavonoids and cinnamic acids. Fraction 4, accounted for flavonoids, increased the frequency of the contractions most effectively: 557% of the baseline (95% CI (316-797); p < 0.05) at the first addition.

Conclusion

Leaf juice of B. pinnatum and its flavonoid fraction are most effective in relaxing myometrial strips by inducing frequency.     

Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml⁻¹. After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml⁻¹), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.     

Biopesticidal value of selected essential oils against pathogenic fungus, termites, and nematodes

The biopesticidal potential of six plant-derived essential oils (mint [Mentha arvensis], ajwain [Carum capticum], lemongrass [Cymbopogon citrates], clove [Eugenia caryophyllata], cedarwood [Cedrus deodara], and eucalyptus [Eucalyptus globulas]) was evaluated against Odontotermes obesus (termites), Fusarium oxysporum (plant pathogenic fungi), and Meloidogyne incognita (nematodes). In the case of termites, a “no-choice” bioassay revealed that the mint oil gave the best results (100% mortality in 30 min with 10% oil and in 10 h with 0.12% oil) followed by the lemongrass and ajwain oils. The disc diffusion method was adopted to test the anti-fungal activity of the essential oils and it was found that the clove oil gave the maximum inhibition measured in terms of the average inhibition zone diameter (5.3 ± 0.2 cm with 10% oil and 6.6 ± 0.9 cm with 20% oil), followed by the ajwain oil. To check the anti-nematicidal activity of the essential oil, in-vitro growth chamber experiments revealed that eucalyptus oil was the most efficient (100% mortality in 6 h with 1000 ??l l⁻¹ oil and in 30 h with 125 ??l l⁻¹ oil), followed by the ajwain oil. The use of the crude oils at low concentrations provided satisfactory results at the laboratory level against these pathogens, and needs further evaluation in field trials.     

Isolation of anti-Candida albicans compounds from Markhamia obtusifolia (Baker) Sprague (Bignoniaceae)

An increase in clinical cases of Candidiosis globally as well as fungal resistance to drugs prompted the search for novel anti-Candida albicans agents from plant sources. Leaf extracts of Markhamia obtusifolia were screened for activity against C. albicans in vitro. An acetone extract obtained following serial exhaustive extraction contained mainly the active components with at least four active zones on the bioautogram. Bioassay guided fractionation of this extract led to the isolation of three compounds which inhibited the growth of three C. albicans strains. Based on spectroscopy studies (NMR and MS), the compounds were identified as 3β-hydroxyurs-12-en-28-oic acid, ursolic acid (1) 3β, 19α-dihydroxyurs-12-en-28-oic acid, pomolic acid (2) and 2β, 3β, 19α -trihydroxy-urs-12-en-28-oic acid, 2-epi-tormentic acid (3). The most active compound was 3β, 19α-dihydroxy-12-ursen-28-oic acid (2) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL for C. albicans isolated from dog and 25.0 µg/mL for C. albicans from cat and ATCC 90028 at 24 h following incubation. However, at 48 h of incubation MICs were > 400 µg/mL for all the three compounds isolated. This study indicated that M. obtusifolia could be a potential source of active principles against C. albicans.     

The role of multiple partners in a digestive mutualism with a protocarnivorous plant

Background and aims

The protocarnivorous plant Paepalanthus bromelioides (Eriocaulaceae) is similar to bromeliads in that this plant has a rosette-like structure that allows rainwater to accumulate in leaf axils (i.e. phytotelmata). Although the rosettes of P. bromelioides are commonly inhabited by predators (e.g. spiders), their roots are wrapped by a cylindrical termite mound that grows beneath the rosette. In this study it is predicted that these plants can derive nutrients from recycling processes carried out by termites and from predation events that take place inside the rosette. It is also predicted that bacteria living in phytotelmata can accelerate nutrient cycling derived from predators.

Methods

The predictions were tested by surveying plants and animals, and also by performing field experiments in rocky fields from Serra do Cipó, Brazil, using natural abundance and enriched isotopes of ¹⁵N. Laboratory bioassays were also conducted to test proteolytic activities of bacteria from P. bromelioides rosettes.

Key Results

Analyses of ¹⁵N in natural nitrogen abundances showed that the isotopic signature of P. bromelioides is similar to that of carnivorous plants and higher than that of non-carnivorous plants in the study area. Linear mixing models showed that predatory activities on the rosettes (i.e. spider faeces and prey carcass) resulted in overall nitrogen contributions of 26·5 % (a top-down flux). Although nitrogen flux was not detected from termites to plants via decomposition of labelled cardboard, the data on ¹⁵N in natural nitrogen abundance indicated that 67 % of nitrogen from P. bromelioides is derived from termites (a bottom-up flux). Bacteria did not affect nutrient cycling or nitrogen uptake from prey carcasses and spider faeces.

Conclusions

The results suggest that P. bromelioides derive nitrogen from associated predators and termites, despite differences in nitrogen cycling velocities, which seem to have been higher in nitrogen derived from predators (leaves) than from termites (roots). This is the first study that demonstrates partitioning effects from multiple partners in a digestion-based mutualism. Despite most of the nitrogen being absorbed through their roots (via termites), P. bromelioides has all the attributes necessary to be considered as a carnivorous plant in the context of digestive mutualism.     

Fatty Acid Diversity is Not Associated with Neutral Genetic Diversity in Native Populations of the Biodiesel Plant Jatropha curcas L.

Jatropha curcas L. (Euphorbiaceae) is a shrub native to Mexico and Central America, which produces seeds with a high oil content that can be converted to biodiesel. The genetic diversity of this plant has been widely studied, but it is not known whether the diversity of the seed oil chemical composition correlates with neutral genetic diversity. The total seed oil content, the diversity of profiles of fatty acids and phorbol esters were quantified, also, the genetic diversity obtained from simple sequence repeats was analyzed in native populations of J. curcas in Mexico. Using the fatty acids profiles, a discriminant analysis recognized three groups of individuals according to geographical origin. Bayesian assignment analysis revealed two genetic groups, while the genetic structure of the populations could not be explained by isolation‐by‐distance. Genetic and fatty acid profile data were not correlated based on Mantel test. Also, phorbol ester content and genetic diversity were not associated. Multiple linear regression analysis showed that total oil content was associated with altitude and seasonality of temperature. The content of unsaturated fatty acids was associated with altitude. Therefore, the cultivation planning of J. curcas should take into account chemical variation related to environmental factors.     

Determination of antimicrobial and phytochemical compounds of Jatropha curcas plant

The aim of this study was to explore the effectiveness of different parts of J. curcas plant against some selected human pathogens as antimicrobial agent which are known to cause diseases and to check antioxidant and phytochemicals from different plant sections of J. curcas. Plant extracts were analyzed by quantification of antimicrobial and phytochemical compounds. This study reveals that 20% ethanol stem extract of J. curcas showed maximum antibacterial activity (40 ± 0.0 mm) against Klebsiella pneumonia. Water extract of root of J. curcas also inhibited E. coli (35.25 ± 0.35 mm). The growth of K. pneumonia and Agrobacterium tumifaciens were also ceased when ethanol extract of J. curcas root applied to check their potential as antimicrobial agent. The results also revealed that fungal species, Aspergillus niger, and Pencillium notatum noted the maximum antifungal activity in ethanol extract of flower and methanol extract of root (38.5 ± 0.7 mm) and (27.25 ± 0.35 mm) respectively. Phytochemicals and many secondary metabolites were present in J. curcas extracts such as alkaloids, steroids, tannins, glycosides, flavonoids, saponins, courmerin, and phenolic compounds. It also showed the highest density of color in the different parts of plant extract of J. curcas. Similarly, biochemical primary metabolites were observed at maximum amount of biochemical in different parts of J. curcas, and correlated with antimicrobial activity. The study concluded that J. curcas has great potential as antibacterial agent and cure various human pathogens.     

Lipase-mediated Transformation of Vegetable Oils into Biodiesel using Propan-2-ol as Acyl Acceptor

Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 °C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7^th cycle when methanol was used as an acyl acceptor, under standard reaction conditions. Revisions requested 22 December 2005; Revisions received 26 January 2006     

Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantis using TaqMan-MGB assay in contaminated metalworking fluids

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degradation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, was found to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantis in MWF is difficult to study using standard culturing techniques. To overcome this, a study was conducted to design a specific quantitative real-time PCR (qPCR) assay using TaqMan^®MGB (minor groove binding) probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeeping gene sequence was selected for designing a TaqMan^® MGB primer-probe pair using the Allele ID^® 5.0 probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly with P. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primer-probe pair’s sensitivity was 10¹ colony forming units (CFU) ml⁻¹. The assay provided no amplification with other closely related Pseudomonas species found in MWFs indicating its specificity. It was successful in identifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 10⁴ and 10⁶ CFU ml⁻¹. The designed TaqMan^® MGB probe thus can be successfully used for the subspecies-specific identification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.     

Studies on Guizotia abyssinica L. oil: Biodiesel synthesis and process optimization

Guizotia abyssinica seeds, a common bird feedstock, have been explored for the potential of biodiesel synthesis. The oil was extracted from the seeds by solvent extraction and composition of G. abyssinica oil was examined. The reaction parameters for biodiesel synthesis have been optimized. Temperature, oil: methanol ratio, catalyst type and catalyst concentration were found to have significant role on ester conversion. According to this study, the maximum yield of ester (98.7%) can be obtained with optimized sodium methoxide catalyst dosage (0.6%) at an operational temperature of 65 °C. Methyl ester of G. abyssinica oil was also studied for its oxidation stability and low temperature properties. Further, the synthesized product was blended in diesel at 5–20% ratios and evaluated for physico-chemical properties.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C_4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C₈) with K_m and k_cat values of 71 μM and 14,700 s⁻¹, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a K_m of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The K_d for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (K_s) for ThDP and (K_c) for Mg²⁺ were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC₅₀ values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (−7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.     

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.