Target and non-target toxicity of fern extracts against mosquito vectors and beneficial aquatic organisms.

Dengue and malaria are significant mosquito-borne diseases that are rapidly spread worldwide, mainly in temperate countries. Pteridophytes were identified to be a significant source of novel mosquitocidal agents. The present research was to explore the eco-friendly larvicides from methanol extracts of ferns, viz., Actiniopteris radiata, Adiantum caudatum, Cheilanthes swartzii, Hemionitis arifolia and Lycopodium clavatum. The larvicidal potential of the extracts screened using larvae of dengue vector Aedes aegypti (III and IV instar) and malarial vector Anopheles stephensi (III and IV instar), showed 10-100% mortality rates. Biosafety assessment was made on embryos of Danio rerio and Artemia nauplii. The phyto-constituents of the methanol extract of A. radiata leaves were identified through gas chromatography-mass spectrometry (GC-MS). Methanolic leaf extracts of A. radiata, A. caudatum and C. swartzii exhibited larvicidal activity against III and IV instar larvae of Ae. aegypti (LC50: 37.47, 74.51 and 152.38 and 67.58, 95.89 and 271.46 ppm) and An. stephensi (LC50: 70.35, 112.12 and 301.05 and 113.83, 175.30 and 315.19 ppm), respectively. The GC-MS of the methanol extract of A. radiata leaves revealed the presence of 7 phyto-components among which, Carbamic acid, phenyl-, (2-Nitrophenyl) methyl ester (1), Benzoic acid, 3- methylbenzoate (2) and 4-(benzylimino)- 1,4-dihydro-1-(p-toluoylmethyl) pyridine (3) were dominant. Biosafety assessment of methanol extract of A. radiata leaves on embryos of Danio rerio (Zebra fish) and Artemia nauplii (micro crustacean) revealed that there were no destructive or teratogenic effects. To conclude, the larvicidal activity and insignificant toxicity to non-target aquatic organisms of A. radiata leaves makes it a potential and environment safe biocontrol agent against dengue and malarial vectors.

Structural characterization and evaluation of mosquito-larvicidal property of silver nanoparticles synthesized from the seaweed, Turbinaria ornata (Turner) J. Agardh 1848.

The silver nanoparticles synthesized from Turbinaria ornata (To-AgNPs) showed spherical with crystalline nature (20-32 nm) was evaluated against fourth instar larvae of three mosquitoes. The maximum activity of To-AgNPs was recorded on Aedes aegypti followed by Anopheles stephensi and Culex quinquefasciatus with the following lethal concentration values (µg/ml): LC50 of 0.738, 1.134, and 1.494; and LC90 of 3.342, 17.982, and 22.475, respectively. The obtained respective values (µg/ml) vis-a-vis aqueous extract (To-AE) were: 2.767 and 40.577; 4.347 and 158.399, and 7.351 and 278.994. The findings revealed that To-AgNPs could form a base for the development of an eco-friendly, low-cost pesticide.

Response of three cyprinid fish species to the Scavenger Deterrent Factor produced by the mutualistic bacteria associated with entomopathogenic nematodes.

The symbiotic bacteria, Photorhabdus and Xenorhabdus associated with entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema, respectively, produce a compound(s) called the Scavenging Deterrent Factor (SDF). SDF deters a number of terrestrial insect scavengers and predators and one bird species from feeding on host insects killed by the nematode-bacterium complex but has not been tested against aquatic vertebrates. Moreover, the Heterorhabditis-Photorhabdus association is believed to have evolved in an aquatic environment. Accordingly, we hypothesized that SDF will deter fish from feeding on nematode-killed insects and tested the responses of three omnivorous fresh water fish species, Devario aequipinnatus, Alburnoides bipunctatus, and Squalius pursakensis, to SDF in the laboratory. When the fish were exposed to Galleria mellonella larvae killed by the Heterorhabditis- or Steinernema-bacterium complex at 2 or 4days post-infection, all three fish species made several attempts to consume the cadavers but subsequently rejected them. However, all fish species consumed freeze-killed control larvae. In a choice test, when D. aequipinnatus or A. bipunctatus were offered a pair of nematode-killed larvae, both fish species rejected these cadavers; when offered a nematode-killed larva and a freeze-killed larva, both fish species consumed the freeze-killed larva but not the nematode-killed one. In further tests with D. aequipinnatus, there was no significant difference in the number of 2-day-old Bacillus thuringiensis subsp. kurstaki-killed (Btk) larvae consumed compared to freeze-killed larvae, but significantly fewer 4-day-old Btk-killed larvae were consumed compared to freeze-killed larvae. When D. aequipinnatus was fed G. mellonella larvae killed by the symbiotic bacteria, the fish rejected the cadavers. When given freeze-killed or nematode-killed mosquito (Aedes aegypti) larvae, the fish consumed significantly more of the former larvae (99%) compared to the latter (55%). When D. aequipinnatus was placed in a symbiotic cell-free supernatant for 18h, a significant reduction in consumption of freeze-killed larvae compared to cell-free Btk or control broth supernatant was observed. We showed that SDF protects the nematode-killed insects from being consumed by omnivorous fishes and suggests that they will have minimal effects on recycling of EPNs in the aquatic environment.

Idaein chloride induced p53 dependent apoptosis in cervical cancer cells through inhibition of viral oncoproteins.

Host dependent expression of early HPV oncoproteins, E6 and E7 play central role in the formation of cervical carcinoma. Presently, we have shown that the cyanidin analog, idaein chloride treatment induced dose dependent apoptosis (IC50 = 2.579 µg/ml) in HPV positive - HeLa cells. Flow cytometric analysis showed arrest of cell cycle at G1 phase with an increased sub G1 cell population on 12th h of exposure. The recorded reduced expression levels of cell cycle proteins - cyclin D, cdk 4 and cdk 6 confirmed the occurrence of cell cycle arrest at G1 phase. In addition, the idaein chloride significantly inhibited the expression of E6 and E7 proteins, resulting in p53 re-expression and hence triggering of p53 dependent apoptosis. This has been further supported by the recorded variations in the expression patterns of p21/WAF, pRb and E2F regulatory proteins. In case of mitochondrial apoptotic markers, the expression of Bax was restored and Bcl-2 level got decreased at 12th h. Cleaved caspases 3 & 9 and PARP were also observed after 3 h of treatment. Interestingly, the epigenetic regulatory enzymes (DNMTs) were inhibited by idaein chloride. Thus, idaein chloride could be a potent source for developing a drug against cervical carcinoma.