Toxicological effects of chemical constituents from Piper against the environmental burden Aedes aegypti Liston and their impact on non-target toxicity evaluation against biomonitoring aquatic insects.

Dengue is the most rapidly spreading mosquito-borne viral disease in the world. The mosquito, Aedes aegypti, also spreads Yellow fever, Chikungunya, and Zika virus. As the primary vector for dengue, Ae. aegypti now occurs in over 20 countries and is a serious concern with reports of increasing insecticide resistance. Developing new treatments to manage mosquitoes are needed. Formulation of crude volatile oil from Piper betle leaves (Pb-CVO) was evaluated as a potential treatment which showed larvicidal, ovipositional, and repellency effects. Gut-histology and enzyme profiles were analyzed post treatment under in-vitro conditions. The Pb-CVO from leaves of field collected plants was obtained by steam distillation and separated through rotary evaporation. The Pb-CVO were evaluated for chemical constituents through GC-MS analyses revealed 20 vital compounds. The peak area was establish to be superior in Eudesm-7(11)-en-4-ol (14.95%). Pb-CVO were determined and tested as four different concentrations (0.25, 0.5, 1.0, and 1.5 mg/L) of Pb-CVO towards Ae. aegypti. The larvicidal effects exhibited dose dependent mortality being greatest at 1.5 mg Pb-CVO/10 g leaves. The LC50 occurred at 0.63 mg Pb-CVO/L. Larva of Ae. aegypti exposed to Pb-CVO showed significantly reduced digestive enzyme actions of α- and ß-carboxylesterases. In contrast, GST and CYP450 enzyme levels increased significantly as concentration increased. Correspondingly, oviposition deterrence index and egg hatch of Ae. aegypti exposed to sub-lethal doses of Pb-CVO demonstrated a strong effect suitable for population suppression. Repellency at 0.6 mg Pb-CVO applied as oil had a protection time of 15-210 min. Mid-gut histological of Ae. aegypti larvae showed severe damage when treated with 0.6 mg of Pb-CVO treatment compared to the control. Non-toxic effects against aquatic beneficial insects, such as Anisops bouvieri and Toxorhynchites splendens, were observed at the highest concentrations, exposed for 3 h. These results suggest that the Pb-CVO may contain effective constituents suitable for development of new vector control agents against Ae. aegypti.

Development of an eco-friendly mosquitocidal agent from Alangium salvifolium against the dengue vector Aedes aegypti and its biosafety on the aquatic predator.

Plant extracts with their enriched chemical constituents have established potential alternative mosquito control agents. In this research, we developed an eco-friendly mosquitocidal agent from Alangium salvifolium leaves against the dengue and Zika virus vector Aedes aegypti and we investigated its biosafety on the mosquito aquatic predator Toxorhynchites splendens. Results showed that the methanolic extract of A. salvifolium leaves was composed by eight main compounds, with major peak area for hexadecenoic acid (21.74%). LC50 and LC90 values calculated on Ae. aegypti fourth instar larvae were 104.80 and 269.15 ppm respectively. The methanolic extract tested at 100 ppm decreased the α-ß carboxylesterase and SOD ratio significantly and upregulated the GST and CYP450 level. The A. salvifolium methanolic extract displayed significant repellent and adulticidal activity at 100 and 400 ppm respectively. The treatment with 100 ppm of the methanolic extract led to 210 min of protection from Ae. aegypti bites. Four hundred parts per million of the extract showed 98% adult mortality within 30 min from the treatment. Lastly, biosafety assays on the mosquito aquatic predator Tx. splendens showed that the toxicity of the A. salvifolium extract was significantly lower if compared to the cypermethrin-based treatments. The methanolic extract of A. salvifolium showed a maximum of 47.3% mortality rate at the concentration of 1000 ppm, while 0.7 ppm of cypermethrin achieved 91.3% mortality rate on Tx. splendens. Overall, our study enhances basic knowledge on how to improve natural larvicidal agents against dengue and Zika virus mosquito vector with harmless responses on non-target aquatic predators.

Toxicological effects of Sphaeranthus indicus Linn. (Asteraceae) leaf essential oil against human disease vectors, Culex quinquefasciatus Say and Aedes aegypti Linn., and impacts on a beneficial mosquito predator.

Use of environmentally friendly, decomposable natural products for effective vector control has gained considerable momentum in modern society. In this study, essential oil of Sphaeranthus indicus (Si-EO) was extracted and further phytochemical screening revealed fourteen compounds with prominent peak area percentage of 24.9 and 22.54% in 3,5-di-tert-butyl-4-hydroxybenzaldehyde and benzene,2-(1,1-dimethylethyl)-1,4-dimethoxy, respectively. The Si-EO was further evaluated for their larvicidal response against Culex quinquefasciatus and Aedes aegypti at different dosages (62.5, 125, 250 and 500 ppm). The Si-EO displayed prominent larvicidal activity at higher concentration (500 ppm) against both species of mosquitoes. The LC50 and LC90 values of oils were observed at 130 and 350 ppm against C. quinquefasciatus larvae and at 140 and 350 ppm against A. aegypti larvae, respectively. Repellent bioassay established higher protection rate at 200 ppm up to 120 min against both the mosquitoes. However, adulticidal response displayed higher mortality rate only at 700 and 800 ppm against C. quinquefasciatus and A. aegypti, respectively. Toxicological screening against mosquito predator Toxorhynchites splendens revealed that the Si-EO was harmless even at the concentration of 1500 ppm. Overall, these results suggest that the Si-EO plays a significant role as a new bio-rational product against ecological burden mosquito vectors which provides an eco-friendly alternative to synthetic pesticides.

Acute toxicity of chemical pesticides and plant-derived essential oil on the behavior and development of earthworms, Eudrilus eugeniae (Kinberg) and Eisenia fetida (Savigny).

Comparative toxicity of two chemical pesticides (temephos and monocrotophos) versus a plant-derived betel leaf oil Piper betle (L.) to earthworm Eudrilus eugeniae (Kinberg) and redworm Eisenia fetida Savigny, historically: Eisenia foetida (Savigny 1826), was evaluated. Mortality rate was more prominent in temephos at 100 µg concentration to both the earthworms in filter paper test (FPT) as well as 10 mg concentration in artificial soil test (AST). In contrast, P. betle does not display much mortality rate to both the earthworms even at 1000 mg of treatment concentrations. The lethal concentration (LC50) value was observed at 3.89 and 5.26 mg/kg for temephos and monocrotophos against E. eugeniae and 3.81 and 5.25 mg/kg to E. fetida, respectively. Whereas, LC50 value of betel leaf oil was only observed at 3149 and 4081 mg/kg to E. eugeniae and E. fetida, respectively. Correspondingly, the avoidance or attraction assay also displayed that earthworms were more sensitive to the soil containing chemical pesticides. Whereas, the avoidance percentage was decreased in the P. betle oil. Similarly, sublethal concentration of chemical pesticides (5 and 6.5 mg) significantly reduced the earthworm weight and growth rate. However, P. betle oil did not change the developmental rate in the duration of the assay (2, 7 and 14 days) even at 4000 mg treatment concentration. The enzyme ratio of CAT and SOD was also affected significantly after exposure to the chemical pesticides (6.5 mg/kg). Hence, our study implied the risk assessment associated with the chemical pesticides and also recommends plant-derived harmless P. betle oil against beneficial species as an alternative pest control agent.

Chemicals isolated from Justicia adhatoda Linn reduce fitness of the mosquito, Aedes aegypti L.

Extracts from Justicia adhatoda L. (Acanthaceae) strongly reduced the fitness of the mosquito, Aedes aegypti Linn. The methanolic extracts inhibited several enzymes responsible for protecting insects from oxidative and other damage, including glutathione-S-transferase, superoxide dismutase, cytochrome P450, and α- and ß-esterases. They increased repellency (maximum repellency at 100 ppm) in host-seeking adult females using the "arm-in cage assay." Histopathological examination showed the extracts led to serious midgut cell damage. Justicia adhatoda extracts led to reduced fecundity and oviposition of gravid females compared to controls. The extracts led to substantially reduced A. aegypti survival. We infer that the extracts have potential to reduce pathogen transmission by suppressing population growth of A. aegypti, and possibly other mosquito species.

Comparative analysis of mosquito (Diptera: Culicidae: Aedes aegypti Liston) responses to the insecticide Temephos and plant derived essential oil derived from Piper betle L.

Resistance to treatments with Temephos or plant derived oil, Pb-CVO, between a field collected Wild Strain (WS) and a susceptible Laboratory Strain (LS) of Ae. aegypti were measured. The Temephos (0.1mg/L) showed the greatest percentage of mosquito mortality compared to Pb-CVO (1.5mg/L) in LS Ae. aegypti. However, WS Ae. aegypti was not significantly affected by Temephos (0.1mg/L) treatment compare to the Pb-CVO (1.5mg/L). However, both strains (LS and WS) when treated with Pb-CVO (1.5mg/L) displayed steady larval mortality rate across all instars. The LC50 of Temephos was 0.027mg in LS, but increased in WS to 0.081mg/L. The LC50 of Pb-CVO treatment was observed at concentrations of 0.72 and 0.64mg/L for LS and WS strains respectively. The enzyme level of α- and ß-carboxylesterase was reduced significantly in both mosquito strains treated with Pb-CVO. Whereas, there was a prominent deviation in the enzyme ratio observed between LS and WS treated with Temephos. The GST and CYP450 levels were upregulated in the LS, but decreased in WS, after treatment with Temephos. However, treatment with Pb-CVO caused both enzyme levels to increase significantly in both the strains. Visual observations of the midgut revealed cytotoxicity from sub-lethal concentrations of Temephos (0.04mg/L) and Pb-CVO (1.0mg/L) in both strains of Ae. aegypti compared to the control. The damage caused by Temephos was slightly less in WS compared to LS mosquito strains.

Impact of Terminalia chebula Retz. against Aedes aegypti L. and non-target aquatic predatory insects.

Aedes aegypti Linn is one of the most important mosquito species. The vectors are responsible for causing deadly diseases like dengue and dengue hemorrhagic fever. Several chemical pesticides used to control these dengue vectors caused severe toxic significances on human health and other non-target beneficial insects. Therefore the current investigation has been made to access the bio-efficacy of the crude seed extracts of T. chebula against the dengue vector Ae. aegypti. The GC-MS analysis of crude seed extracts of T. chebula identified nine chemical compounds with major peak area in the 1,2,3-Benzenetriol (61.96%), followed by Tridecanoic acid (09.55%). Ae. aegypti larvae showed dose dependent mortality rate was observed between the treatments. Prominent protection rate at greater concentrations of 100ppm and moderate protection at 75 and 50ppm was observed in the repellent assay. Lethal concentration (LC50 and LC90) of fourth instar larvae of Ae. aegypti was observed in 138 and 220ppm concentration respectively. Similarly, the seed extracts showed 100% adulticidal activity at the concentration of 400ppm at 30min of exposure time. Phytochemicals present in the seed extracts of T. chebula significantly affects the major portions of the midgut tissues of Ae. aegypti at the concentration of 100ppm. The toxicological evaluation of seed extracts also proved non-toxic towards the A. bouvieri and Tx. splendens aquatic predatory insects. Hence, the present result suggest that bio-rational plant derived T. chebula could be incorporated in the dengue vector control and have no adverse effects on non-target beneficial insects.

Potential mode of action of a novel plumbagin as a mosquito repellent against the malarial vector Anopheles stephensi, (Culicidae: Diptera).

Plumbagin was isolated and characterized from the roots of Plumbago zeylanica using chromatography: TLC, Column chromatogram, HPLC, FTIR and 1H NMR. The isolated pure compounds were assayed for potency as inhibitors of: acetylcholine esterase (AchE), glutathione S-transferases (GST), superoxide dismutase (SOD), cytochrome P450 and α, ß-esterase, and for repellency with Anopheles stephensi at four different concentrations (25, 50, 75 and 100ppm). The enzyme assay against the pure compound reveals that the level of esterase and SOD was decreased significantly in contrast the level of GST and cytochrome P450 was increased significantly. Our results suggests that novel Plumbagin has significantly alters the level of enzyme comparable to the control. Evaluations resulted in Plumbagin producing maximum repellency scores against An. stephensi mosquitoes in dose dependent manner with highest repellence was observed in the 100ppm. Histological examination showed that the midgut, hindgut and muscles are the most affected tissues. These tissues affected with major changes including separation and collapse of epithelial layer and cellular vacuolization. The results support the utility of plant compound Plumbagin for vector control as an alternative to synthetic insecticides, however, more vigorous field trials are needed to determine viability under natural conditions.

Toxicity and physiological effect of quercetin on generalist herbivore, Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny.

A novel flavonoid, quercetin, was isolated from the medicinal plant Euphorbia hirta L. through chromatography techniques including: TLC, Column chromatography, NMR and then screened for toxicity to larvae of Spodoptera litura Fab. Bioassays were used to analyze pupal weight, survival rate, fecundity, egg hatchability, population growth index, Nutritional index and histopathology of treated larvae at a range of E. hirta extract concentrations. Results of toxicity assays demonstrated that, 6 ppm of quercetin caused 94.6% mortality of second, 91.8% of third, 88% of fourth, and 85.2% of fifth instars respectively. The lethal concentrations (LC50 and LC90) was calculated as 10.88 and 69.91 ppm for fourth instar larvae. The changes in consumption ratio and approximate digestibility produced a reduction in growth rates. Histopathology examinations revealed that the cell organelles were severely infected. Analyses of earthworm toxicity effects resulted in significantly lower rates compared to synthetic insecticides (chloropyrifos and cypermethrin). These results suggests that the botanical compound (quercetin), could have a part as a new biorational product which provides an ecofriendly alternative. Validation of the potential of quercetin, still needs to be demonstrated under field conditions, where formulation will be important in maintaining the activity.

Target and non-target toxicity of botanical insecticide derived from Couroupita guianensis L. flower against generalist herbivore, Spodoptera litura Fab. and an earthworm, Eisenia foetida Savigny.

Botanical insecticides may provide alternatives to synthetic insecticides for controlling Spodoptera litura (F.) and they are target specific, biodegradable, and harmless to mammals. Eight natural chemical compounds with larvicidal activity were identified from fraction F6 of C. guianensis flower extract. Probit analysis of 95% confidence level exposed an LC50 of 223ppm against S. litura third instar larvae. The growth and development of S. litura was affected in sub-lethal concentrations of fraction F6 (50, 100, 150 and 200ppm) compared to controls. Similarly nutritional indices values decreased significantly compared to controls. Fraction F6 also damaged the gut epithelial layer and brush border membrane (BBM). This study also resolved the effects of toxicity to non-target earthworm treated with fraction F6 and chemical pesticides (monotrophos and cypermethrin) and the results showed that fraction F6 had no harmful effect on E. fetida. Further, fraction F6 was eluted and sub fractions F6c (50ppm) showed high mortality against S. litura third instar larvae. Octacosane from fraction F6c was established and confirmed using IR spectrum and HPLC. The time of retention of fraction F6c was confirmed with the octacosane standard. Fraction F6 of C. guianensis extract caused dose-dependent mortality towards S. litura. Octacosane in fraction F6c was establish to be the prominent chemical compound associated with causing mortality but other compounds present in the fraction F6 were shown to be associated with changes in development of S. litura at low dosages. S. litura at low dosage. Therefore, these findings suggest that octacosane may be one of the major insecticidal compounds affecting S. litura survival.

Anti-dengue efficacy of bioactive andrographolide from Andrographis paniculata (Lamiales: Acanthaceae) against the primary dengue vector Aedes aegypti (Diptera: Culicidae).

The current study investigated the toxic effect of the leaf extract compound andrographolide from Andrographis paniculata (Burm.f) against the dengue vector Ae. aegypti. GC-MS analysis revealed that andrographolide was recognized as the major chemical constituent with the prominent peak area compared with other compounds. All isolated toxic compounds were purified and confirmed through RP-HPLC against chemical standards. The larvicidal assays established at 25ppm of bioactive compound against the treated instars of Ae. Aegypti showed prominent mortality compared to other treated concentrations. The percent mortality of larvae was directly proportional to concentration. The lethal concentration (LC50) was observed at 12ppm treatment concentration. The bioactive andrographolide considerably reduced the detoxifying enzyme regulations of α- and ß- carboxylesterases. In contrast, the levels of GST and CYP450 significantly increase in a dose dependent manner. The andrographolide also showed strong oviposition deterrence effects at the sub-lethal dose of 12ppm. Similarly, the mean number of eggs were also significantly reduced in a dose dependent manner. At the concentration of 12ppm the effective percentage of repellency was greater than 90% with a protection time of 15-210min, compared with control. The histopathology study displayed that larvae treated with bioactive andrographolide had cytopathic effects in the midgut epithelium compared with the control. The present study established that bioactive andrographolide served as a potential useful for dengue vector management.

Developmental response of Spodoptera litura Fab. to treatments of crude volatile oil from Piper betle L. and evaluation of toxicity to earthworm, Eudrilus eugeniae Kinb.

Evaluations of biological effects of (Pb-CVO) the crude volatile oil of Piper betle leaves on the tobacco cutworm Spodoptera litura were conducted. Pb-CVO was subjected to GC-MS analysis and twenty vital compounds were isolated from the betel leaf oil. Pb-CVO was tested at four different concentrations (0.25, 0.5, 1.0 and 1.5%) against S. litura. The treated insects exhibited dose depended mortality. The mortality rate was significantly higher at the 1.0 and 1.5% Pb-CVO. The LC50 (Lethal concentration) were observed at 0.48% Pb-CVO. Larval and pupal durations increased in all treatment concentrations (0.25, 0.3, 0.4 and 0.5%) whereas, pupal weight decreased compared to control. Adult longevity of S. litura was reduced in all treatments but predominantly in the 0.4 and 0.5% Pb-CVO. Correspondingly, mean fecundity rate was reduced at all concentrations compared to control. Histological studies of larvae mid-gut profiles of S. litura were severely damaged in 1.0 and 1.5% and showed abnormalities in mid-gut cells with 0.25 and 0.5% Pb-CVO treatments. Earthworm toxicity illustrated that 0.1% of chemical insecticides (monocrotophos and cypermethrin) varied widely in their contact toxicities compared to 0.5 and 1.0% Pb-CVO and control in both contact filter paper and artificial soil test. These findings suggest that twenty essential compounds of betel leaf oil were significant inhibitors of the development and caused behavioral changes of S. litura. Treatment with betel leaf oil at these concentrations had no adverse effect on earthworm populations.

Toxicity of Alangium salvifolium Wang chemical constituents against the tobacco cutworm Spodoptera litura Fab.

Widespread use of synthetic pesticides has resulted in the development of insecticide-resistant populations of pests and harmful effects on human health and the environment. There is a need to identify alternative pest management strategies to reduce our reliance on conventional chemical pesticides. In recent years the use of botanical pesticides for protecting crops from insect pests has assumed greater importance. Methanol extract of Alangium salvifolium (L.f.) Wang has potential insecticidal activity against Spodoptera litura Fab. The active fractions were identified through chromatographic techniques as F-IV (Rf value=0.45) and F-VI (Rf value=0.63) and were subjected to GC-MS (GCMATE II). Fifty, 100 and 200ppm of active fractions were applied to fourth instar larvae and the mortality increased with higher concentrations. Relative consumption rate, relative growth rate, efficiency of conversion of ingested food and efficiency of conversion of digested food values all decreased in treated larvae, but approximate digestibility rate increased after treatment. The hydrolytic enzymes, such as acid phosphatase, alkaline phosphatase and the glycolytic enzyme lactate dehydrogenase were inhibited in treated larvae compared with controls. The histopathology study revealed that the epithelial columnar cells were enlarged, completely atrophied; intercellular spaces were swollen, and also noted a cytoplasmic ooze of cell material that mixed with food column. The present study clearly showed the active fractions from A. salvifolium as potential botanicals to control the larvae of S. litura. This is the first report for nutritional indices, enzymatic activities and histological effects of A. salvifolium chemical constituents against S. litura. Thus probably, this will be used as an alternative for synthetic pesticides against the polyphagous pest like S. litura.

Physiological and biochemical effects of botanical extract from Piper nigrum Linn (Piperaceae) against the dengue vector Aedes aegypti Liston (Diptera: Culicidae).

The leaves of Piper nigrum L. (Piperaceae) were evaluated for chemical constituents and mosquito larvicidal activity against the larvae of Aedes aegypti. GC and GC-MS analyses revealed that the crude extracts contain 16 compounds. Thymol (20.77%) and ç-elemene (10.42%) were identified as the major constituents followed by cyclohexene, 4-ethenyl-4-methyl-3-(1-methylethenyl)-1-(1 methylethyl)-, (3R-trans) (7.58%), 4,6-octadienoic acid, 2-acetyl-2-methyl-, ethyl ester (6.98), 2(3H)-furanone, 3,4-bis(1,3-benzodioxol-5-ylmethyl) dihydro-, (3R-trans) (6.95%), 1-naphthalenol, 1,2,3,4,4a,7,8,8a-octahydro-1,6-dimethyl-4-(1-methylethyl)-, [1R-(1à,4á,4aá,8aá)]-(Cedreanol) (5.30%), trans-2-undecen-1-ol (4.48%), phytol (4.22%), 1,6-cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-,[s-(E,E)] (3.78%) and 2,6-dimethyl-3,5,7-octatriene-2-ol, Z,Z (2.39%). Larval mortality was observed after 3 h of exposure period. The crude extract showed remarkable larvicidal activity against Ae. aegypti (LC50 = 34.97). The larvae of Ae. aegypti exposed to the P. nigrum, significantly reduced the activities of α- and ß-carboxylesterases and superdioxide. Further, P. nigrum extract was severely affecting the mosquito gut cellular organelles. Based on the results, the chemical constituents of crude extracts of P. nigrum can be considered as a new source of larvicide for the control of Ae. aegypti.