Geanne K.N. Santos

Chemical composition, oviposition deterrent and larvicidal activities against Aedes aegypti of essential oils from Piper marginatum Jacq. (Piperaceae)

The essential oils of leaves, stems and inflorescences of Piper marginatum, harvested in the Atlantic forest in the State of Pernambuco, Brazil, were obtained by hydrodistillation. GC and GC-MS analyses revealed the presence of 40 components accounting, respectively, for 99.6%, 99.7% and 99.1% of the leaf, stem and inflorescence oil, the most abundant being (Z)- or (E)-asarone and patchouli alcohol. The essential oil of the inflorescences exhibited potent activity against the 4th instar of Aedes aegypti with LC(10) and LC(50) values of 13.8 and 20.0 ppm, respectively. Furthermore, the inflorescence oil did not interfere in the oviposition of A. aegypti females when assayed at 50 ppm. These properties suggest that P. marginatum oil is a potential source of valuable larvicidal compounds for direct use or in conjunction with baits in traps constructed to capture eggs and larvae.

Oviposition-Stimulant and Ovicidal Activities of Moringa oleifera Lectin on Aedes aegypti

Background Natural insecticides against the vector mosquito Aedes aegypti have been the object of research due to their high level of eco-safety. The water-soluble Moringa oleifera lectin (WSMoL) is a larvicidal agent against A. aegypti. This work reports the effects of WSMoL on oviposition and egg hatching of A. aegypti. Methodology/Principal Findings WSMoL crude preparations (seed extract and 0–60 protein fraction), at 0.1 mg/mL protein concentration, did not affect oviposition, while A. aegypti gravid females laid their eggs preferentially (73%) in vessels containing isolated WSMoL (0.1 mg/mL), compared with vessels containing only distilled water (control). Volatile compounds were not detected in WSMoL preparation. The hatchability of fresh eggs deposited in the solutions in the oviposition assay was evaluated. The numbers of hatched larvae in seed extract, 0–60 protein fraction and WSMoL were 45±8.7 %, 20±11 % and 55±7.5 %, respectively, significantly (p<0.05) lower than in controls containing only distilled water (75–95%). Embryos were visualized inside fresh control eggs, but not within eggs that were laid and maintained in WSMoL solution. Ovicidal activity was also assessed using stored A. aegypti eggs. The protein concentrations able to reduce the hatching rate by 50% (EC50) were 0.32, 0.16 and 0.1 mg/mL for seed extract, 0–60 protein fraction and WSMoL, respectively. The absence of hatching of stored eggs treated with WSMoL at 0.3 mg/mL (EC99) after transfer to medium without lectin indicates that embryos within the eggs were killed by WSMoL. The reduction in hatching rate of A. aegypti was not linked to decrease in bacteria population. Conclusions/Significance WSMoL acted both as a chemical stimulant cue for ovipositing females and ovicidal agent at a given concentration. The oviposition-stimulant and ovicidal activities, combined with the previously reported larvicidal activity, make WSMoL a very interesting candidate in integrated A. aegypti control.

Nutritional restriction of omega-3 fatty acids alters topographical fine tuning and leads to a delay in the critical period in the rodent visual system

The development and maturation of sensory systems depends on the correct pattern of connections which occurs during a critical period when axonal elimination and synaptic plasticity are involved in the formation of topographical maps. Among the mechanisms involved in synaptic stabilization, essential fatty acids (EFAs), available only through diet, appear as precursors of signaling molecules involved in modulation of gene expression and neurotransmitter release. Omega-3 fatty acids, such as docosahexaenoic acid (DHA), are considered EFAs and are accumulated in the brain during fetal period and neonatal development. In this study, we demonstrated the effect of omega-3/DHA nutritional restriction in the long-term stabilization of connections in the visual system. Female rats were fed 5 weeks before mating with either a control (soy oil) or a restricted (coconut oil) diet. Litters were fed until postnatal day 13 (PND13), PND28 or PND42 with the same diets when they received an intraocular injection of HRP. Another group received a single retinal lesion at the temporal periphery at PND21. Omega-3 restriction induced an increase in the optical density in the superficial layers of the SC, as a result of axonal sprouting outside the main terminal zones. This effect was observed throughout the SGS, including the ventral and intermediate sub-layers at PND13 and also at PND28 and PND42. The quantification of optical densities strongly suggests a delay in axonal elimination in the omega3(-) groups. The supplementation with fish oil (DHA) was able to completely reverse the abnormal expansion of the retinocollicular projection. The same pattern of expanded terminal fields was also observed in the ipsilateral retinogeniculate pathway. The critical period window was studied in lesion experiments in either control or omega-3/DHA restricted groups. DHA restriction induced an increased sprouting of intact, ipsilateral axons at the deafferented region of the superior colliculus compared to the control group, revealing an abnormal extension of the critical period. Finally, in omega-3 restricted group we observed in the collicular visual layers normal levels of GAP-43 with decreased levels of its phosphorylated form, p-GAP-43, consistent with a reduction in synaptic stabilization. The data indicate, therefore, that chronic dietary restriction of omega-3 results in a reduction in DHA levels which delays axonal elimination and critical period closure, interfering with the maintenance of terminal fields in the visual system.

Differential vulnerability of substantia nigra and corpus striatum to oxidative insult induced by reduced dietary levels of essential fatty acids

Oxidative stress (OS) has been implicated in the etiology of certain neurodegenerative disorders. Some of these disorders have been associated with unbalanced levels of essential fatty acids (EFA). The response of certain brain regions to OS, however, is not uniform and a selective vulnerability or resilience can occur. In our previous study on rat brains, we observed that a two-generation EFA dietary restriction reduced the number and size of dopaminergic neurons in the substantia nigra (SN) rostro-dorso-medial. To understand whether OS contributes to this effect, we assessed the status of lipid peroxidation (LP) and anti-oxidant markers in both SN and corpus striatum (CS) of rats submitted to this dietary treatment for one (F1) or two (F2) generations. Wistar rats were raised from conception on control or experimental diets containing adequate or reduced levels of linoleic and α-linolenic fatty acids, respectively. LP was measured using the thiobarbituric acid reaction method (TBARS) and the total superoxide dismutase (t-SOD) and catalase (CAT) enzymatic activities were assessed. The experimental diet significantly reduced the docosahexaenoic acid (DHA) levels of SN phospholipids in the F1 (~28%) and F2 (~50%) groups. In F1 adult animals of the experimental group there was no LP in both SN and CS. Consistently, there was a significant increase in the t-SOD activity (p < 0.01) in both regions. In EF2 young animals, degeneration in dopaminergic and non-dopaminergic neurons and a significant increase in LP (p < 0.01) and decrease in the CAT activity (p < 0.001) were detected in the SN, while no inter-group difference was found for these parameters in the CS. Conversely, a significant increase in t-SOD activity (p < 0.05) was detected in the CS of the experimental group compared to the control. The results show that unbalanced EFA dietary levels reduce the redox balance in the SN and reveal mechanisms of resilience in the CS under this stressful condition.

Effects of Croton rhamnifolioides Essential Oil on Aedes aegypti Oviposition, Larval Toxicity and Trypsin Activity

Although numerous reports are available concerning the larvicidal potential of essential oils, very few investigations have focused on their mechanisms of action. In the present study, we have investigated the chemical composition of the leaf oil of Croton rhamnifolioides during storage and its effects on oviposition and survival of larvae of the dengue fever mosquito Aedes aegypti. In addition, we have established a possible mechanism of action for the larvicidal activity of the essential oil. GC-MS analyses revealed marked differences in the composition of oil that had been freshly isolated and that of a sample that had been stored in a sealed amber-glass vial under refrigeration for three years. However, both fresh and stored oil exhibited substantial larvicidal activities with LC50 values of 122.35 and 89.03 ppm, respectively, and oviposition deterrent effects against gravid females at concentrations of 50 and 100 µg·mL−1. These results demonstrate that the larvicidal effect of the essential oil was unchanged during three years of storage even though its chemical composition altered. Hence, the essential oil could be used in the preparation of commercial products. In addition, we observed that the trypsin-like activity of mosquito larvae was inhibited in vitro by the essential oil of C. rhamnifolioides, suggesting that the larvicidal effect may be associated with inhibition of this enzyme.

Larvicidal isoxazoles: Synthesis and their effective susceptibility towards Aedes aegypti larvae

Twenty 3,5-disubstituted isoxazoles have been synthesized and tested against fourth instar Aedes aegypti larvae. In the synthesis of title compounds, modifications have been made in the C-5 side-chain with a view to test their larvicidal activity. These isoxazoles have been obtained by 1,3-dipolar cycloaddition of arylnitrile oxides to terminal alkynes which furnished the desired products in 20% to 79% yields. A comparative study of the larvicidal activity between 3-(3-aryl-isoxazol-5-yl)-propan-1-ols and 3-(3-aryl-isoxazol-5-yl)-propionic acids clearly demonstrated that the latter compounds possess much better larvicidal activity than the former. We also tested two esters, viz., methyl 3-[3-(phenyl)-isoxazole-5-yl] propionate and methyl 3-[3-(4-chlorophenyl)-isoxazole-5-yl] propionate, where the latter presented an excellent larvicidal profile.

Dopaminergic cell populations of the rat substantia nigra are differentially affected by essential fatty acid dietary restriction over two generations.

Essential fatty acids play a crucial role in the activity of several neurotransmission systems, especially in the monoaminergic systems involved in cognitive and motor aspects of behavior. The present study investigated whether essential fatty acid dietary restriction over two generations could differentially affect dopaminergic cell populations located in the substantia nigra rostro-dorso-medial (SNrm) or caudo-ventro-lateral (SNcv) regions which display distinct neurochemical profile and vulnerability to lesions under selected pathological conditions. Wistar rats were raised from conception on control or experimental diets containing adequate or reduced levels of linoleic and α-linolenic fatty acids, respectively. Stereological methods were used to estimate both the number and soma size of tyrosine hydroxylase (TH)-immunoreactive neurons in the SNrm and SNcv. TH protein levels were assessed with Western blots. Long-term treatment with the experimental diet modified the fatty acid profile of midbrain phospholipids and significantly decreased TH protein levels in the ventral midbrain (3 fold), the number of TH-positive cells in the SNrm (∼20%) and the soma size of these neurons in both SNrm (∼20%) and SNcv (∼10%). The results demonstrate for the first time a differential sensitivity of two substantia nigra dopaminergic cell populations to unbalanced levels of essential fatty acids, indicating a higher vulnerability of SNrm to the harmful effects induced by docosahexaenoic acid brain deficiency.

Evaluation of the Activity of the Essential Oil from an Ornamental Flower against Aedes aegypti: Electrophysiology, Molecular Dynamics and Behavioral Assays.

Dengue fever has spread worldwide and affects millions of people every year in tropical and subtropical regions of Africa, Asia, Europe and America. Since there is no effective vaccine against the dengue virus, prevention of disease transmission depends entirely on regulating the vector (Aedes aegypti) or interrupting human-vector contact. The aim of this study was to assess the oviposition deterrent activity of essential oils of three cultivars of torch ginger (Etlingera elatior, Zingiberaceae) against the dengue mosquito. Analysis of the oils by gas chromatography (GC)—mass spectrometry revealed the presence of 43 constituents, of which α-pinene, dodecanal and n-dodecanol were the major components in all cultivars. Solutions containing 100 ppm of the oils exhibited oviposition deterrent activities against gravid Ae. aegypti females. GC analysis with electroantennographic detection indicated that the oil constituents n-decanol, 2-undecanone, undecanal, dodecanal, trans-caryophyllene, (E)-β-farnesene, α-humulene, n-dodecanol, isodaucene and dodecanoic acid were able to trigger antennal depolarization in Ae. aegypti females. Bioassays confirmed that solutions containing 50 ppm of n-dodecanol or dodecanal exhibited oviposition deterrent activities, while a solution containing the alcohol and aldehyde in admixture at concentrations representative of the oil presented an activity similar to that of the 100 ppm oil solution. Docking and molecular dynamics simulations verified that the interaction energies of the long-chain oil components and Ae. aegypti odorant binding protein 1 were quite favorable, indicating that the protein is a possible oviposition deterrent receptor in the antenna of Ae. aegypti.

Extract of Bowdichia virgilioides and maackiain as larvicidal agent against Aedes aegypti mosquito

The larvicidal activities of extracts of three hardwood species (Hymenaea stigonorcapa, Anadenanthera colubrina and Bowdichia virgilioides) against 4th instar larvae of Aedes aegypti were evaluated using WHO guidelines. Extracts of H. stignocarpa and A. colubrina showed weak activity. The highest larvicidal effect was obtained with the cyclohexane extract of the heartwood of B. virgilioides, which caused 100% mortality at concentrations at 50 and 100 µg/mL. Fraction toluene/EtOAc (8:2) from this extract showed larvicidal activity (LC₅₀ = 34.90 ± 1.27 µg/mL). A mixture of two compounds identified as medicarpin and maackiain exhibited a very good larvicidal activity (sub-fraction 2, LC₅₀ = 17.5 ± 1.87 µg/mL) and maackiain showed to be a strong larvicidal compound (LC₅₀ = 21.95 ± 1.34 µg/mL). This result can be of value in the search for new natural larvicidal compounds from other hardwood plant extracts and presents the first report of B. virgilioides being used to control a mosquito vector.

The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the dengue vector Aedes aegypti

The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC50 measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid.