Diseases caused by mosquito are of a serious threat to modern world in many aspects such as mortality . Mosquitoes are the vectors responsible for many diseases that include most commonly malaria and lymphatic filariasis. Culex quinquefasciatus species bite man persistently and transmit a neglected disease known as filariasis in the tropical regions. The Anopheles species are of more interest because they are responsible for transmitting malaria . Larviciding is the act of sinking mosquito densities in their propagation places before they grow into adults . Culex quinquefasciatus is domestic mosquito specie found in the vicinity of human habitat. Biological control can be achieved by the application of nanoparticles obtained through plant-mediated synthesis which is less toxic and eco-friendly .
The phyto-mediated synthesized nanoparticles can be a rapid, simple, cost effective and environmentally safer biopesticide for controlling the malarial vector . Green chemistry is generally the use of methods and techniques to eliminate or reduce the generation or use of feedstock, byproduct, product, reagents, and solvents, etc. that are detrimental to human health or to the environment . Much literature has reported the green plant-mediated synthesis of nanoparticles which is more favored by researchers due to its eco-friendliness over photochemical reduction, heat evaporation, electrochemical reduction, and chemical reduction. Some of these reducing agents reported for larvicidal activity include actinobacterium, Streptomyces sp. , Streptomyces sp. , Aganosma cymosa leaf extract , bud extract of Polianthus tuberose , bark extract of Terminalia arjuna [ , petal extracts of Tagetes sp. and Rosa sp. .
Palmyra palm with the scientific name Borassus aethiopum is one of the trees usually referred to as the Palms. It is a member of the family Arecaceae and economically and medicinally useful. For example it is used as vegetable  and commonly in West Africa . This study reports the green synthesis of Ag-Co hybrid bimetallic nanoparticles using the aqueous root extract of palmyra palm, their partial characterization using UV-Visible and FT-IR spectrophotometers as well as their larvicidal effect on first, second and third/fourth instars of Culex quinquefasciatus larva.
2. Materials and Methods
2.1. Palmyra Root Sample Collection and Preparation
Palmyra root samples were dug from Kalorgu in Kaltungo Local Government Area of Gombe State. They were transported to Chemistry Laboratory of Gombe State University in polythene bag. The root samples were washed several times with water and distilled water to removed impurities. About 100 g was crushed using pestle and mortar and transferred to a 250 ml beaker. It was placed on a magnetic stirrer and 100 ml of distilled water was added. The mixture was warmed for 1 h with continuous stirring at 60˚C to extract the phytochemicals. It was then filtered and kept for the synthesis of silver—cobalt nanocomposite.
2.2. Test Larvae Collection
The test larvae (Culex quinquefasciatus) were obtained from stagnant open water bodies in Gombe town.
2.3. Green Synthesis of Silver-Cobalt Bimetallic Nanoparticles
One hundred milliliters of the prepared palmyra palm root extract was mixed with a solution of 500 ml containing 250 ml each of 0.01mol/dm3 AgNO3 and CoCl2 (1:5 v/v) gradually on a hot plate at 80˚C while stirring for 30 minutes in a 600 ml beaker . Change in color of the reaction mixture from light pink to light brown was visually noticed. The solution was stored for 24 hours after which the nanoparticles obtained were evaporated and dried in an oven at 105˚C.
2.4. Ultraviolet-Visible Spectroscopic Investigation
Optical measurement was carried out using UV-Visible Spectrophotometer model 6705 for the wavelength between 250 to 800 nm by placing 1 mL sample of the supernatant used for the synthesis in 1 × 1 cm cuvettes operated at a resolution of 1 nm and de-ionized water as the blank solvent.
2.5. Fourier Transform Infrared Spectrophotometry Analysis
The dried synthesized Silver-Cobalt bimetallic nanoparticles and root extract of Palmyra palm were characterized using Fourier Transform Infrared Spectroscopy. This was done to determine which functional groups were involved in the bio-reduction process. PerkinElmer Spectrum Version 10.03.09 was used.
2.6. Larvicidal Bioassay
Twenty larvae (first, second and third/fourth instar) each were placed in a beaker to which 5 ppm of the synthesized Ag/Co bimetallic nanoparticles diluted with de-ionized water was added to make the solution 100 ml. Test of this concentration against each instar was replicated twice. In each case, a control comprising of 20 larvae in 100 ml de-ionized water was used as reference. The test was carried out for further concentration of 10, 20, 25 and 50 ppm. The mortality data was collected after 24 hours  and the percentage mortality was calculated as follow:
2.7. Statistical Analysis
All data were analyzed using SPSS 16.0. Probit analyses for LC50, LC90, chi square as well as correlation analysis were evaluated.
3. Results and Discussions
3.1. Optical Measurement Using UV-Visible Spectrophotometer
The formation of the Ag-Co bimetallic nanoparticles was first noticed by change of color of the mixture of Ag-Co salt and the extract from milky, Figure 1(D) to light brown, Figure 1(E) within 15 minutes as a result of the surface Plasmon resonance which is due to the collective oscillation of the free conduction band electrons which is excited by the incident electromagnetic radiation .
Figure 1. 0.01 M CoCl2 (A), 0.01 M Ag(NO3) (B), Aqueous root extract Palmyra palm (C), Mixture of Ag-Co immediately after addition (D), and Ag-Co BMNPs formation (E).
The supernatant liquid was used for UV-Vis spectroscopic analysis which is frequently used to characterize synthesized metal nanoparticles. The maximum absorption peak was found at 420 nm (Figure 2).
3.2. FTIR Analysis
FT-IR spectroscopy was used to investigate the functional groups involved in the reducing and capping process. The FT-IR spectra of the root extract and that of the biosynthesized Ag-Co BNPs are shown in Figure 3 and Figure 4 respectively. The FT-IR spectra of the Ag-Co displayed peaks due to O-H stretching frequency at 3389.93 cm−1, medium sharp peak for C-H absorption at 2924.28 cm−1, C=C stretching at 1633.48 cm−1, C=N stretching, C-C stretching and the C-O at 1541.53 cm−1, 1384.59 cm-1 and 1046.88 cm−1 bands respectively. Similar result was reported by . These have replaced those observed in the spectrum of the root extract which were peaks at 3443.26 cm−1, 2929.48 cm−1, 1651.28 cm−1, 1384.15 and 1080.12 cm−1 respectively. Most notably is the appearance of a prominent peak at 1541.53 cm−1 due to C=N stretching which was absent in the root extra spectra and the disappearance of the peaks at 1162.66 cm−1, 986.29 cm−1, 861.19 cm−1 and 525.69 cm−1. This variation is due to various metabolites such as tannins and saponins that may be present because no existing literature reported the photochemical. The active metabolites are responsible for the bioreduction .
3.3. Larvicidal Results
The larvicidal activity was evaluated by recording the mortality of Culex quinquefasciatus larvae when exposed to different concentrations of the Ag-Co bimetallic nanoparticles represents after 24 hours. It was found be concentration dependent. Moreover, development stage is also a factor because the Ag-Co BMNPs showed better activity against 1st instars, followed by 2nd instars and 3rd or 4th instars. Table 1 and Figure 5 represent the % mortality rates at concentrations of 5, 10, 20, 25 and 50 ppm. The outcome of the result showed effective larvicidal effect for all the 3 instars with LC50 and LC90 values obtained as: 1st instar (LC50 = 5.237 ppm, LC = 49.240 ppm), 2nd instar (LC50 = 9.310 ppm, LC90 = 94.969 ppm) and 3rd or 4th instar (LC50 = 13.626 ppm, LC90 = 105.542 ppm). These results are comparable with that obtained by Elijah et al. (2016) for the larvicidal activity of Ag NPs where the LC50 and LC90 values after 24 h exposure were 4.43 ppm and
Figure 2. UV-visible spectrum for Ag-Co BMNPs.
Figure 3. FT-IR spectrum for root extract of Borassus aethiopum.
Figure 4. FT-IR spectrum for Ag-Co BMNPs.
Figure 5. Ag-Co BMNPs larvicidal activity on Culex quinquefasciatus larvae.
Table 1. Effect of different concentrations of Ag-Co BMNPs on Culex quinquefasciatus larvae.
LC50 and LC90 are the lethal concentrations that would kill 50 and 90% of the exposed larvae respectively; r is the regression co-efficient and χ2 Chi square.
8.37 ppm against 3rd and 4th instars. The LC50 and LC90 values also have some correlation with the result obtained by Kanayairam and Ravichandran (2016) whose LC50 = 10.59, 11.10, 11.90, 12.71 ppm; LC90 = 32.11, 35.12, 37.48, 42.17 ppm, for 1st, 2nd, 3rd and 4th instars respectively. A similar result was also observed by Shanmugasundaram and Balagurunathan  with LC50 = 48.98 mg/L, r = 0.956 and χ2 value of 14.307).
Secondary metabolites of the root extract of Palmyra palm were used to synthesize Ag-Co bimetallic nanoparticles using AgNO3 and CoCl2 metal salts. Their formation was visually noticed by a color change and further characterized using UV-Visible and FT-IR spectrophotometers. These plant-mediated nanoparticles were active against Culex quinquefasciatus mosquito larvae.
We acknowledged Gombe State University for opportunity to work in various laboratories for this research.
 Roopan, S.M., Rohit Madhumita, G., Rahuman, A.A., Kamaraj, C., Bharathi Shanmugasundaram, T. and Balagurunathan, R. (2015) Mosquito Larvicidal Activity of Silver Nanoparticles Synthesized Using Actinobacterium, Streptomyces sp. M25 against Anopheles Subpictus, Culex quinquefasciatus and Aedes aegypti. Journal of Parasitic Distribution, 39, 677-684.
 Ramanibai, R. and Velayutham, K. (2015) Bioactive Compound Synthesis of Ag Nanoparticles from Leaves of Melia azedarach and Its Control for Mosquito Larvae. Research Journal of Vet Science, 98, 82-88.
 Anjali, R. (2017) Mosquito Larvicidal Activity of Green Silver Nanoparticle Synthesized from the Bud Extract of Polianthus tuberosa L. International Journal of Nanotechnology and Applications,11, 17-28.
 Gopinath, K., Chandran, S. and Ayyakannu, A. (2013) Green Synthesis, Characterization of Silver, Gold and Bimetallic Nanoparticles Using Bark Extract of Terminalia Arjuna and Their Larvicidal Activity Against Malaria Vector, Anopheles Stephensi. International Journal of Recent Scientific Research, 4, 904-910.
 Ismaila, M., Khana, M.I., Sher, B.K., Khan, M.A., Khana, K.A. and Abdullah, M.A. (2018) Green Synthesis of Plant Supported Cu-Ag and Cu-Ni bimetallic Nanoparticles in the Reduction of Nitrophenols and Organic Dyes for Water Treatment. Journal of Molecular Liquids, 260, 78-90.
 Akinsiku, A.A., Dare, E.O., Ajani, O.O., Joan, A., Ademosun, O.T. and Samuel, O.A. (2018) Room Temperature Phytosynthesis of Ag/Co Bimetallic Bimetallic Nanoparticles Using Aqueous Leaf Extract of Canna indica. Proceedings of 2nd International Conference on Science and Sustainable Development, 173, 1-14.
 Rajesh, K., Padmavathi, K.C., Ranjani, A., Gopinath, P.M., Dhanasekaran, D. and Archunan, G. (2013) Green Synthesis, Characterization and Larvicidal Activity of AgNPs against Culex quinquefasciatus and Aedes aegypti Larvae. American Journal of Drug Discovery and Development, 3, 245-253.
 Giovanni, B. and Marimuthu, G. (2017) Green-Synthesized Mosquito Ovipositor Attractants and Ovicides: Towards a Nanoparticle-Based ‘‘Lure and Kill’’ Approach. Journal of Cluster Science, 28, 287-308.
 Amita, H., Snehali, D. and Naba, K.M. (2016) Mosquito Larvicidal Activity of Cadmium Nanoparticles Synthesized from Petal Extracts of Marigold (Tagetes sp.) and Rose (Rosa sp.) Flower. Journal of Parasitic Distribution, 40, 1519-1527.
 Barot, S. and Gignoux, J. (1999) Population Structure and Life Cycle of Borassus aethiopum mart: Evidence of Early Senescence in a Palm Tree. Biotropica, 31, 439-448.
 Igwe, O.U. and Ekebo, E.S. (2018) Biofabrication of Cobalt Nanoparticles Using Leaf Extract of Chromolaena odorata and Their Potential Antibacterial Applications. Research Journal of Chemical Sciences, 8, 11-17.
 Surendra, A. (2013) TV Low-Cost and Eco-Friendly Phytosynthesis of Silver Nanoparticles Using Cocos nucifera coir Extract and Its Larvicidal Activity. Industrial Crops Production, 43, 631-635.
 Kumar, K.M., Mandal, B.K., Sinha, M. and Krishnakumar, V. (2012) Terminalia chebula Mediated Green and Rapid Synthesis of Gold Nanoparticles. Spectrochimica Acta, 86, 490-494.