Layout 1 [Journal of Entomological and Acarological Research 2014; 46:1920] [page 59] Abstract Silver nanoparticles (AgNPs) were synthesized from the latex of the medicinally important plants Euphorbia milii, Euphorbia hirta, Ficus racemosa and Jatropha curcas. Synthesized AgNPs were characterized by UV-Vis spectrophotometry, scanning electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, Fourier transformed infrared spectroscopy, particle size, and zeta potential analysis. Potency of latex and latex-synthesized AgNPs was evaluated against the 2nd and 4th instar larvae of Aedes aegypti and Anopheles stephensi. The lowest lethal concentration 50 (LC50) value among the different types of plant latex studied was observed for latex of E. milii (281.28±23.30 and 178.97±37.82 ppm, respectively) against 2nd instar larvae of Ae. aegypti and An. stephensi. E. milii latex-synthesised AgNPs showed a high reduction in LC50 compared with its latex; i.e., 8.76±0.46 and 8.67±0.47 ppm, respectively, for 2nd instars of Ae. aegyp- ti and An. stephensi. LC50 values of AgNPs synthesized using the latex of E. hirta, F. racemosa and J. curcas were lower than those of the latex of the respective plants; i.e., 10.77±0.53, 9.81±0.52, 12.06±0.60 and 8.79±0.51, 9.83±0.52, 9.60±0.51 ppm, respectively, for 2nd instars of An. stephensi and Ae. aegypti. Similarly, as compared with the plant latex, lower LC50 values were reported for latex-synthesized AgNPs against 4th instars of Ae. aegypt and An. stephensi. Results showed that all the types of plant latex investigated have the potential to convert silver nitrate into AgNPs showing a spectrum of potent mosquito larvicidal effects, indicating the possibility of further exploration of the bioeffi- cacy of latex and latex-synthesized AgNPs against vectors of public health concerns. Introduction About 3.3 and 2.5 billion people, respectively, are at risk of malaria and dengue worldwide, with a higher frequency in the population of sub-Saharan Africa (SSA) (WHO, 2009, 2011). In India, 1.49 million cases of malaria, 28,292 cases of dengue, 767 and 108 deaths were reported from malaria and dengue in 2010 (NVBDCP, 2011). The above figures indicate the global impact of mosquito-transmitted diseases with respect to loss of national productivity due to mortality and mor- bidity. Mosquito species such as Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus are widely distributed in the tropical and sub- tropical zones, acting as vectors of diseases like malaria, dengue, filar- iasis, Japanese encephalitis, yellow fever, and chikungunya (WHO, 2009). To control the outbreak of mosquito-borne diseases, attention should be given to targeting the larval stage of mosquitoes, which are unable to fly and are present in the breeding habitat. Devising a con- trol methodology should therefore be relatively easy for the larval stage. During the past several decades, organophosphates such as temephos and fenthion, and insect growth regulators such as difluben- zuron and methoprene, have been used to control mosquito larvae (Yang et al., 2002). Insecticides of microbial origin, such as Bacillus thuringiensis, have also been employed for larval control (Raghvendra et al., 2011). However, continued and indiscriminate use of these insecticides creates problems such as insecticide resistance, environ- mental pollution and toxicity to human and non-target organisms (Raghvendra et al., 2011). To combat these shortcomings of chemical insecticides, research has shifted toward products of biological origin (Patil et al., 2012a; Karunamoorthi, 2013). Use of products of plant origin to control mosquito larvae has been shown to be an exciting alternative to traditional methods of larval management, as they are not associated with the problems noted above (Shaalan et al., 2005, Borase et al., 2013). For example, root and leaf extracts of Plumbego zeylanica and Cestrum nocturnum (Patil et al., 2011b), leaf extracts of Ocimum sanctum, Phyllanthus emblica (Murugan et al., 2012), and hydrodistillate extracts of Mentha piperita, Ocimum basilicum, Zingiber officinale, and curcuma longa (Kalaivani et al., 2012) have been used against mosquito larvae of An. stephensi, Ae. aegypti and Cu. quinquefasciatus. The use of phytosynthesized silver nanoparticles as a larvicidal Correspondence: Satish V. Patil, School of Life Sciences, North Maharashtra University, Post Box 80, Jalgaon 425001, Maharashtra, India. Tel.: +91.257.2257421 - Fax: +91.257.2258403. E-mail: satish.patil7@gmail.com Key words: plant latex, mosquito biolarvicidal, silver nanoparticles, Anopheles stephensi, Aedes aegypti. Acknowledgements: Hemant P. Borase is DST-INSPIRE fellow (Grant File No. DST/INSPIRE Fellowship/2011[149].), Chandrashekhar D. Patil is thankful to CSIR (Ref: 09/728 (0028)/2012- EMR-I) for the award of senior research fellowship. Received for publication: 12 September 2013. Revision received: 26 December 2013. Accepted for publication: 17 January 2014. ©Copyright H.P. Borase et al., 2014 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2014; 46:1920 doi:10.4081/jear.2014.1920 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. Journal of Entomological and Acarological Research 2012; volume 44:eJournal of Entomological and Acarological Research 2014; volume 46:1920 Mosquito larvicidal and silver nanoparticles synthesis potential of plant latex H.P. Borase,1 C.D. Patil,1 R.B. Salunkhe,1 C.P. Narkhede,1 R.K. Suryawanshi,1 B.K. Salunke,1 S.V. Patil1,2 1School of Life Sciences, North Maharashtra University; 2North Maharashtra Microbial Culture Collection Centre (NMCC), North Maharashtra University, India No n- co mm er cia l u se on ly [page 60] [Journal of Entomological and Acarological Research 2014; 46:1920] agent instead of chemical insecticides is gaining importance because of their safety to users as well as nontarget species, and the novelty of their mechanism of action (Marimuthu et al., 2011; Patil et al., 2012b). Several plants have been screened successfully for silver nanoparticle synthesis, such as Plumeria rubra. (Patil et al., 2011a), Pergularia daemia (Patil et al., 2012a), Acacia arabica (Thakur et al., 2013), Cadaba indica lam leaf extract (Kalimuthu et al., 2013), Euphorbia tiru- calli, and Alstonia macrophylla (Borase et al., 2013), as described in a review by Gan & Li (2012). Chemical and physical methods of nanosyn- thesis have shortcomings such as the use of toxic chemicals and high temperatures. To address these, the use of living organisms such as plants and microorganisms (bacteria and fungi) for nanoparticle syn- thesis is gaining momentum. Latex is a milky to transparent sap produced in some plants and stud- ied mostly with respect to rubber production, interactions with insects as a plant defense mechanism, and in explorations of different pharma- cological activities (Kekwick, 2007). The latex-producing plants E. milii, E. hirta, F. racemosa and J. curcas used in the present study are available in large quantities locally in India and have been reported in the literature for their medicinal applications as well as for their active biochemical constituents (Table 1). For these reasons and because of the potent mosquito larvicidal activity showed by plant Plumeria rubra and Pergularia daemia and synthesized AgNPs in our earlier study (Patil et al., 2011a; 2012a), we wanted to investigate the potential of other types of plant latex as eco-friendly mosquito larvicidal agents, and as precursors for environmentally benign silver nanoparticle synthesis. Materials and methods Plant material E. milii, E. hirta, F. racemosa and J. curcas growing in the vicinity of Jalgaon, India, were used as sources of fresh latex. Latex was collected in the early morning during March, 2013, by making a small incision near the youngest leaves and at the ends of branches. Extruded latex was collected in sterile tubes (10 mL). Tubes were kept at 4°C to stop coagulation until the time of the experiments. Phytochemical characterisation of latex Latex samples were subjected to qualitative tests for the presence of different metabolites as reported by Kokate (1999) and Patil et al. (2012b). Synthesis of silver nanoparticles One mL of fresh latex was added to 100 mL of an aqueous solution of silver nitrate (100 ppm). The flask was incubated on a rotary shaker (28°C at 120 rpm). Simultaneously, controls containing latex with Milli-Q deionized water and silver nitrate solution alone were main- tained under the same conditions. Solutions were observed periodical- ly for any colour change. Test organisms For the laboratory trials, locally collected early 2nd and 4th instar larvae of Ae. aegypti and An. stephensi were used as experimental specimens. The larvae were kept in plastic enamel trays containing dechlorinated tap water, and were maintained as reported by Kalimuthu et al. (2013). Mosquito larvicidal bioassay Different concentrations of latex and AgNPs were prepared in dechlorinated tap water. Larvicidal activity was assessed using the pro- cedure of WHO (1996) with some modifications and as per the meth- ods of Patil et al. (2011b, 2012b). Twenty five 2nd and 4th instar larvae were taken in four batches in 249 mL of water, and 1.0 mL of the desired concentration of latex plus AgNPs were added. The control was set up with dechlorinated tap water. The numbers of dead larvae were counted after 24 h of exposure, and the percent mortality was recorded for the average of four replicates. The experimental media, in which 100% mortality of larvae occurred, was selected for the dose-response bioassay (data not shown). Dose response bioassay Based on the preliminary screening results, crude latex extract of the experimental plants plus synthesized AgNPs were subjected to a dose-response bioassay for larvicidal activity against the larvae of Ae. aegypti and An. stephensi. Different concentrations ranging from 62.25 Article Table 1. Medicinal properties and chemical constituents of latex producing plants used for analysing larvicidal and silver nanoparticle synthesis potential. Botanical name Common name Family Medicinal Chemical References (vernacular name) property constituents Euphorbia milii Milli Crown of throrn Euphorbiaceae Molluscicidal Miliin, serine proteas, Yadav et al. (2006); (Christ Plant) flavons, triterpenoids, steroids, steroidal glycoside, alkaloids Euphorbia hirta Tawa-tawa Euphorbiaceae Antihelminthic, Sterols, alkaloids, tannins, Iwu (1993); (Dudhi) repellent, antifeedant glycosides, triterpenoids, Wei et al. (2005); and controlling Plutella alkenes, phenolic acids, Kumar et al. (2002); xylostella and nematicidal choline and shikimic acid Parekh & Chanda (2007); and against roundworm Rajeh et al. (2012) like guinea worm Ficus racemosa Cluster Fig Tree Moraceae Anti-inflammatory, Racemosic acid, Khan & Sultana (2005); (Udumbara) antidiarrheal, clears horsevoice triterpenes Li et al. (2004); and chemomodulatory, larvicides Rahuman et al. (2008) Jatropha curcus Bagbherenda Euphorbiaceae Nematicidal, fungicidal, Triglycerols, sterols, Sharma & Trivedi (2002); (Jungli erand) mosquito (Ochlerototatus oils, phorbal esters, Gübitz et al. (1999) triseriatus) larvicidal, glucanase protein insecticidal activities No n- co mm er cia l u se on ly to 2000 ppm (for the latex) and 0.625 to 20 ppm (for the synthesized AgNPs) were prepared, and numbers of dead larvae were counted after 24 h of exposure; percent mortality was reported from the average of four replicates. Statistical analysis Mortality was calculated using Abbott’s formula (Abbott, 1925). The dose-response data were subjected to probit regression analysis (Finney, 1971). The lethal concentrations in parts per million (LC50, LC90) and the 95% confidence intervals of LC50 (upper confidence limit) and (lower confidence limit) were calculated. Characterisation of silver nanoparticles AgNPs solutions were centrifuged at 10,000 rpm for 10 min (REMI, Cooling centrifuge, C-24 BL, India); the pellet obtained was resuspend- ed in water and used to analyse surface plasmon resonance of the sil- ver nanoparticles using a UV-Vis spectrophotometer (Shimadzu 1601, Tokyo, Japan) at the resolution of 1 nm from 200 to 800 nm. Other tech- niques used for AgNPs characterization included Fourier-transformed infrared spectroscopy (FT-IR) (Shimadzu, Prestige 21, Tokyo, Japan), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) (HITACHI- S4800, Tokyo, Japan), X-ray diffraction (XRD) (Brucker D8 Advance, Karlsruhe, Germany), particle size, and zeta potential analy- sis (Zetasizer, Malvern Instrument Ltd, Westborough, MA, USA). Results Synthesis and characterisation of AgNPs Transformations of AgNO3 to AgNPs were clearly indicated by a colour change of AgNO3 from colourless to yellowish brown, depleting all the plant latex within 5 to 20 min of latex addition, without agglomeration and indicating synthesis of stable AgNPs (Figure 1A). Latex of E. milii showed the fastest colour change among all types of latex tested (within 5 min). Synthesized nanoparticles were characterized by UV-Visible spectroscopy showing a surface plasmon resonance band at 410 to 450 nm, which arises due to the conduction of free electrons on the surface of AgNPs (Figure 1B) (Smitha et al., 2008). Absorption maxima at 440, 433,419, and 444 nm were observed for AgNPs synthesized from E. milii, E. hirta, F. racemosa and J. curcas, respectively. Similar results have been shown by Borase et al. (2013) and Thakur et al. (2013). Absorbance of AgNPs synthesized from the latex of E. milii was found to be higher than the other types of plant latex under study. E. milii latex-fabricated AgNPs show a smaller size of 208 nm with a zeta potential of –9.19 mV (Figure [Journal of Entomological and Acarological Research 2014; 46:1920] [page 61] Article Figure 1. A) Tubes showing colour change of AgNPs: a. silver nitrate solution, b. E. milii, c. E. hirta, d. F. Racemosa, and e. J. curcas. B) UV spectra of AgNPs. C and D) Particle size and zeta potential analysis of AgNPs produced from E. milii latex. Figure 2. A and B) Scanning electron microscopy and energy dispersive X-ray image of AgNPs synthesized from E. milii. No n- co mm er cia l u se on ly [page 62] [Journal of Entomological and Acarological Research 2014; 46:1920] 1C and D). AgNPs from E. hirta, F. racemosa and J. curcas showed larger size particles having low stability, as compared with E. milii-synthesized AgNPs (data not shown). SEM imaging showed high density of spherical size, monodispersed AgNPs (Figure 2A). EDX spectra confirmed the presence of elemental silver in the samples, as there was a strong signal for the silver atom (Figure 2B). Fourier transformed infrared spectroscopy analysis showed the pres- ence of different functional groups corresponding to proteins, alka- loids, tannins, saponins and other plant metabolites (Figure 3A). A peak at 670.03 cm–1 was assigned to N-H wag of amines of proteins, 701.91 cm–1 as a C-H deformation in carbohydrates, 3439.96 cm–1 for Ar- OH, O-H and N-H for phenols, alcohols and amides, and 2997.48 cm–1 for the C=O bond found in terpenoids and flavonoids. The remaining peaks also indicate the presence of proteins, flavonoids, saponins and other plant metabolites, as evidenced by qualitative phytochemical analysis (Table 2). XRD analysis revealed the crystalline nature of AgNPs. Other peaks in the XRD may arise due to biomolecules capped on the AgNPs surface (Figure 3B). Mosquito larvicidal bioassay The plant latex under study and AgNPs fabricated from the latex were used to analyse their potency against the 2nd and 4th instar larvae of Ae. aegypti and An. stephensi. The results of larvicidal bioassays of the plant latex are presented in Tables 3 and 4, and that of the plant latex-synthe- sized AgNPs are presented in Tables 5 and 6. All tested plant latex and synthesized AgNPs showed larvicidal efficacy within 24 h of exposure. Mortality rate (Y) was positively related to the dose (X), indicating that mortality is dose-dependent. Latex materials from all the plants tested were less toxic than the synthesized AgNPs to both mosquito species. Among the AgNPs tested, the AgNPs synthesized from the latex of E. milii were highly effective against An. stephensi (LC50=8.76 ppm, LC90= 17.11 ppm), and the AgNPs from J. curcas was highly effective against Ae. Aegypti (LC50=9.43ppm, LC90=18.20 ppm). All the plants used in the present study showed LC50 values less than 13 ppm, which could be an important factor in determining a practical larvicidal dose. Discussion Latex producing plants secrete milky fluid from a network of laticifer cells, in which subcellular organelles intensively synthesize proteins and secondary metabolites (Lopes et al., 2009). The biological importance of latex fluids is still unclear and knowledge of their physiological role is still limited (Ramos et al., 2007). Ramos et al. (2009) presented first evi- dence for the use of Calotropis procerra (Ait.) R.Br.-secreted proteolytic enzymes as chemical agents against Ae. aegypti larvae. Plant latex has been reported to have a negative effect on several insect functions such as egg hatch, larval growth and survival (Giridhar et al., 1984; Morsy et al., 2001; Ramos et al., 2006). The chemico-physical method of nanopar- ticle synthesis involves the use of toxic substances (sodium borohydrate, polyvinylpyrrolidone) that are harmful to the environment. Our method of AgNPs synthesis using latex, which has an abundance of proteins, enzymes and secondary metabolites, is novel, eco-friendly, and does not require toxic chemicals. Previous studies have demonstrated the involve- ment of proteins, polyphenols and carbohydrates in the synthesis of Article Table 2. Phytochemical analysis of plant latex. Sr. No. Metabolites E. milii E. hirta F. racemosa J. curcas 1 Protein + + + + 2 Carbohydrates + – + – 3 Terpenoids + + + + 4 Alkaloids – + + + 5 Phenolics + + + + 6 Flavonoids + + + + 7 Tannin + + – – 8 Saponins + + + + 9 Glycosides + – + – +=present; –= absent. Sr., serial number. Figure 3. A and B) Fourier transformed infrared spectroscopy and X-ray diffraction spectrum of AgNPs synthesized from E. milii.No n- co mm er cia l u se on ly metal nanoparticles (Gan & Li, 2012). Nanoparticles produced using chemical methods are of a defined size and shape due to the use of a sin- gle reducing and capping agent. In biological synthesis, diverse particle size and shape is observed because of multiple reducing and capping agents. Consequently, isolation, purification and scale-up of compounds responsible for nanoconversion of silver represent potentially valuable alternatives to chemical synthesis. Duran et al. (2011) discussed involvement of the enzyme NADPH- dependent nitrate reductase in production of AgNPs, while Vigneshwaran et al. (2006) showed the role of reducing sugars in AgNPs production, AgNPs synthesis were also reported from combina- tion of reducing agents and terpenoids (Shankar et al., 2004), polyols, eugenol, quinines and Phyllanthin (Jha et al., 2009; Kasthuri et al., 2009; Singh et al., 2010). The plant latex used in the present study also showed the presence of proteins and secondary metabolites (ter- penoids, tannins, alkaloids and others), so we may preliminarily con- clude there is an interaction of enzymatic and non-enzymatic com- pounds in AgNPs formation. Corbel et al. (2007) showed that increased insecticide resistance in mosquitoes is due to increased activity of enzymes involved in insecti- cide metabolism (e.g., esterases, oxidases, glutathione-S-transferase) and mutation in the target sites of insecticide action. This can be cor- roborated with how AgNPs exhibit their larvicidal action. AgNPs have a high surface area-to-volume ratio, which imparts to them many types of biocidal and catalytic activities. Also, in latex-mediated nanosynthe- sis, capping of latex metabolites on the surface of the AgNPs, in addi- tion to imparting stability, also increases their larvicidal action. The higher mortality at lower doses is consistent with earlier reports of AgNPs produced from leaf extracts of Nelumbo nucifera (LC50=0.69 ppm, LC90=2.15 ppm) against An. subpictus and Cu. quinquefasciatus (LC50=1.10 ppm, LC90=3.59 ppm) Thirunavukkarasu et al. (2010). Marimuthu et al. (2011) reported bioactivity of Mimosa pudica-synthe- sized AgNPs against the larvae of An. subpictus, Cu. quinquefasciatus, and R. microplus (LC50=13.90, 11.73 and 8.98 ppm), respectively. AgNPs synthesized using Tinospora cordifolia extract were tested against the larvae of An. subpictus (LC50=6.43 mg/L) and Cu. quinquefasciatus (LC50=6.96 mg/L) (Jayaseelan et al., 2011). Shaalan et al. (2005) reported that varying results obtained in lethal concentration values can be due to differences in the levels of toxicity among the insecticidal components of different plants, and the effect of plant extracts can vary significantly depending on plant species, plant part, age of the plant part, extraction solvent, seasonal variation, and mosquito species In prokaryotic systems, AgNPs have multiple targets for biocidal effects by causing structural damage (Kim et al., 2007), generation of reactive oxygen species, interfering with DNA replication, and reacting with the thiol enzyme group (Liau et al., 1997; Feng et al., 2000). Patil et al. (2012b) also pointed out the antagonistic effect of AgNPs on enzymes and proteins regardless of the Gram characteristics in bacte- ria. The mechanism of larvicidal action of silver nanoparticles requires more detailed study. Conclusions Studies were conducted to evaluate the potential mosquito larvicidal activity of plant latex and latex-synthesized AgNPs. Our results suggest the possibility of addressing the problem of emerging mosquito resist- ance to chemical insecticides by using latex, latex-synthesized AgNPs, or combinations of chemical insecticides with latex and AgNPs, which could be considered an alternative larval eradication tactic that could help reduce the burden of toxic chemical insecticides on the environ- ment and non-target organisms. [Journal of Entomological and Acarological Research 2014; 46:1920] [page 63] Article Table 3. Larvicidal activity of latex against 2nd instars larvae of Aedes aegypti and Anopheles stephensi. Mosquito species Plant latex LC50±SE 95% fiducial limits LC90±SE 95% fiducial limits Regression (mg L–1) (LCL-UCL) (mg L–1) (LCL-UCL) equation Aedes aegypti E. milii 281.28±23.30 234.87-327.91 752.27±51.56 665.59-874.59 Y=9.58+0.00941X E. hirta 675.26±39.73 601.94-760.27 1422.69±88.19 1272.29-1626.91 Y=33.63+0.0112X F. racemosa 726.69±42.33 647.66-815.91 1555.16±90.48 1399.16-1761.71 Y=3.49+0.0111X J. carcus 746.98±48.52 655.56-848.55 1768.99±109.92 1580.74-2022.16 Y=4.49+0.00993X Anopheles stephensi E. milii 178.97±37.82 95.93-248.31 909.88±73.06 788.93-1087.58 Y=11.9+0.00772X F. racemosa 549.52±54.24 441.85-658.39 1809.71±134.66 1584.20-2130.08 Y=7.62+0.00820X E. hirta 568.74±46.84 477.61-664.21 1621.64±111.01 1433.66-1881.56 Y=6.70+0.00916X J. carcus 755.70±49.04 294.70-391.19 1772.58±112.93 1579.869-2033.902 Y=4.37+0.00985X LC50, 50% lethal concentration; SE, standard error; LCL, lower confidence limit; UCL, upper confidence limit; LC90, 90% lethal concentration. Table 4. Larvicidal activity of latex against 4th instars larvae of Aedes aegypti and Anopheles stephensi. Mosquito species Plant latex LC50±SE 95% fiducial limits LC90±SE 95% fiducial limits Regression (mg L–1) (LCL-UCL) (mg L–1) (LCL-UCL) equation Aedes aegypti E. milii 638.11±36.53 571.00-716.69 1299.02±80.07 1162.58-1484.72 Y=3.45+0.0116X E. hirta 683.69±39.32 611.31-768.07 1408.23±86.27 1260.96-1607.78 Y=3.33+0.0114X F. racemosa 777.43±43.49 697.64-870.85 1563.74±93.01 1404.19-1777.41 Y=2.55+0.0113X J. carcus 798.89±46.00 713.78-896.79 1678.54±99.45 1507.56-1906.32 Y=3.00+0.0108X Anopheles stephensi E. milii 761.11±43.43 680.80-853.64 1580.75±93.51 1420.08-1795.11 Y=3.02+0.0111X E. hirta 783.42±42.89 704.43-875.06 1560.04±89.73 1405.42-1764.95 Y=2.38+0.0115X F. racemosa 884.69±45.65 800.79-982.26 1681.22±92.00 1521.86-1889.82 Y=1.43+0.0115X J. carcus 919.31±52.52 822.32-1031.26 1930.60±113.96 1734.54-2191.33 Y=2.68+0.010X LC50, 50% lethal concentration; SE, standard error; LCL, lower confidence limit; UCL, upper confidence limit; LC90, 90% lethal concentration. 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Mosquito species Plant AgNPs LC50±SE 95% fiducial limits LC90±SE 95% fiducial limits Regression (mg L–1) (LCL-UCL) (mg L–1) (LCL-UCL) equation Aedes aegypti J. carcus 9.43±0.48 8.53-10.46 18.20±0.97 16.50-20.41 Y=9.08+0.009X E. milii 9.49±0.48 8.61-10.53 17.60±0.96 15.93-19.79 Y=0.884+1.14X E. hirta 10.67±0.54 9.57-11.85 20.00±1.10 18.08-22.51 Y=0.808+1.06X F. racemosa 11.44±0.65 10.26-12.86 23.07±1.43 20.63-26.38 Y=1.89+0.925X Anopheles stephensi E. milii 9.95±0.49 9.05-11.02 18.13±0.97 16.45-20.33 Y=0.515+1.14X J. carcus 10.01±0.51 9.07-1.10 19.01±1.02 17.24-21.33 Y=1.13+1.10X F. racemosa 11.76±0.60 10.66-13.7 21.98±1.22 19.86-24.77 Y=0.723+1.00X E. hirta 12.63±0.66 11.44-14.07 23.39±1.35 21.07-26.49 Y=0.525+0.95X LC50, 50% lethal concentration; SE, standard error; LCL, lower confidence limit; UCL, upper confidence limit; LC90, 90% lethal concentration. Table 5. Larvicidal activity of latex synthesized AgNPs against 2nd instars of Aedes aegypti and Anopheles stephensi. Mosquito species Plant AgNPs LC50±SE 95% fiducial limits LC90±SE 95% fiducial limits Regression (mg L–1) (LCL-UCL) (mg L–1) (LCL-UCL) equation Anopheles stephensi E. milii 8.76±0.46 7.91-9.74 17.11±0.94 15.48-19.24 Y=1.82+1.13X E. hirta 10.77±0.53 9.78-11.91 20.11±1.06 18.27-22.5 Y=0.84+1.07X F. racemosa 9.81±0.52 8.85-10.93 19.34±1.07 17.47-21.78 Y=1.69+1.06X J. carcus 12.06±0.60 10.97-13.36 22.00±1.19 19.94-24.71 Y=0.36+1.01X Aedes aegypti E. milii 8.67±0.47 7.81-9.68 17.62±1.01 15.89-19.92 Y=2.04+1.11X E. hirta 8.79±0.51 7.82-9.87 19.51±1.17 17.50-22.18 Y=3.21+1.01X F. racemosa 9.83±0.52 8.88-10.93 19.14±1.06 17.31-21.54 Y=1.53+1.07X J. carcus 9.60±0.51 8.67-10.69 18.96±1.05 17.14-21.35 Y=1.81+1.07X LC50, 50% lethal concentration; SE, standard error; LCL, lower confidence limit; UCL, upper confidence limit; LC90, 90% lethal concentration. No n- co mm er cia l u se on ly D.H., CHO M.H., 2007 - Antimicrobial effects of silver nanoparti- cles. - Nanomed. Nanotechnol. Biol. 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[Journal of Entomological and Acarological Research 2014; 46:1920] [page 65] Article No n- co mm er cia l u se on ly