Larvicidal activities of the leaves of Niger Delta mangrove plants against Anopheles gambiae

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1 Sky Journal of Microbiology Research Vol. 2(7), pp , October, 2014 Available online ISSN X 2014 Sky Journals Full Length Research Paper Larvicidal activities of the leaves of Niger Delta mangrove plants against Anopheles gambiae Tariwari C. N. Angaye 1, Elijah I. Ohimain 1 *, Ebimiere P. Siasia 1, Philips I. Asaigbe 2 Oladuni A. Finomo 3 and 1 Toxicology Research Group, Department of Biological Sciences, Niger Delta University, Bayelsa State, Nigeria. 2 Department of Genetics and Biotechnology, University of Calabar, Cross-River State, Nigeria. 3 Department of Medical Laboratory Service, Federal Medical Center, Bayelsa State, Nigeria Accepted 24 September, 2014 The application of plant-derived pesticide as larvicides has emerged as a desirable eco-friendly and sustainable approach in malaria control programmes. This emergence is indicative of the fact that the synthetic pesticides induce environmental toxicity, and even the application of drugs merely abates the morbidity burden due to possible re-infection. The morbidity burden of malaria in the Niger Delta is becoming hyper-endemic due to the water-lodged nature of the terrain, which provides favorable breeding ground for most vectors. The efficacy of n-hexane, Chloroform and methanolic extracts of the leaves of 4 Niger Delta Mangrove plants (Rhizophora mangle, R. racemosa, Avicennia germinans and Languncularia racemosa), were investigated against 4 th instar larvae of Anopheles gambiae, in a 24 h two-phased (Rapid and Final Screening), static non-renewal test. Results of the rapid screening test shows that; Chloroform extracts of A. germinans and L. racemosa induced total mortality above 500 ppm, compared to R. mangle (LC 50 = ppm) and R. racemosa (LC 50 = ppm). Results of the final screening shows significant toxic activities amongst all mangrove species, with the highest activities in the methanolic extracts (R. mangle LC 50 = ppm; R. racemosa LC 50 = ppm; A. germinans LC 50 = ppm; L. racemosa LC 50 = ppm). While hexane extracts had LC 50 values of , , and ppm for R. mangle, R. racemosa, A. germinans and L. racemosa respectively. The results confirm that mangrove plants in the Niger Delta can be used as a larvicide in the control of malaria. Key words: Niger Delta, mangrove plant, Anopheles gambiae, larvicide, rapid screening, final screening. INTRODUCTION The Niger Delta is home to about 20 million people belonging to more than 40 different ethnic groups. Its floodplain is made up of 7.5% of Nigeria s total land mass, which is classified into four ecological zones. These zones are made up of coastal barrier island, mangrove swamp forest, freshwater swamp and lowland rainforest. Core States in the Niger Delta includes; Delta, Bayelsa, Rivers and Akwa Ibom. A Delta is that part of a country s land mass through which its rivers systems go into the ocean. The Niger Delta have three basic tributaries (River-Forcados to the west, River-Nun central, River-Orashi to the east), linked to a major parent tributary originating from a northernmost offshoot at Ebueto. *Corresponding author. eohimain@yahoo.com. Generally, the description of these basic tributaries forming the Niger Delta, assumes a triangular shape, hence a triangular Delta. The term mangrove is indicative of an outstanding ecosystem, dominated by highly fecund halophytic trees and shrubs. Mangrove plants are endemic in tropical and sub-tropical swamps of tidalcoastal areas and deltaic swamps, which have adapted to harsh environment that border between inland and coastal areas (Miles et al., 1999; Spalding et al., 2010), covering a total global area of approximately 15,429,000 ha (Miles et al., 1999). Although, the origin of the context mangrove seems enigmatic, nevertheless it is believed to have been coined from several words (mangue in Portuguese; manglier in French or mangi- mangi in Malaysia), in conjunction with the English word grove (Siasia 2014; Berjak et al., 1977). The most dominant mangrove plants in Nigeria are found in the Niger

2 46 Sky. J. Microbiol. Res. Delta. Endemic species includes, red mangrove (Rhizophoraspp), white mangrove (A. germinans) as well as the black mangrove (L. racemosa). The Niger Delta ecosystem host varieties of keystone species, which has become a resource to mankind. On the other hand, it provides a favorable breeding ground for some vectors, especially mosquitoes. Consequently, vector-borne diseases are becoming a threat in the Niger Delta. Globally, statistic abounds that about 3.5 billion persons are affected by vector-borne diseases. In addition, statistical report of the World Health Organization rates malaria as the most devastating vector-borne disease, in terms of its overall mortality and morbidity burden (Angaye, 2013). In developing continent like Africa, the primary species responsible for malaria is A. gambiae (Okomu et al., 2007). Amongst the insects, mosquitoes ranks major transmitter of vector-borne diseases compared to any other arthropods group (Ghosh et al., 2012). Furthermore, there are about 3,500 species of mosquitoes belonging to about 41 genera (Bassey et al., 2013), of which only about transmit malaria in nature (NCID, 2004). These vectors are endemic in over 100 countries globally, with an annual global morbidity rate of over 700 million people (Ghosh et al., 2012). Consequently, the Niger Delta terrain provides brusquely breeding environment for mosquitoes, which are becoming hyper-endemic, especially amongst indigent people who lack the capacity and resources to combat this dreaded disease. Several commendable malaria control programmes in the Niger Delta have been adopted by government. Notwithstanding, these programmes have only abate the morbidity burden, while the source of re-infection (i.e. breeding sites), thrives. It will be most desirable to incorporate larviciding in such programmes in order to achieve more sustainable measures. The larvae are easier to control in their breeding phase because they are stationary (Ublom et al., 2012), and besides they are curbed before they attain maturity (Ohimain et al., 2014). In addition, plant-derived pesticides are eco-friendly, and have faster degradability compared to synthetic pesticides (Angaye et al., 2014; Ohimain et al., 2014). Previous studies have shown efficacy of mangrove plant against most pathogens and vectors; these includes, insecticidal, fish poison, mosquito repellency, antiviral, as well as anti-microbial activities (Radfar et al., 2011; Miles et al., 1999; Kokpal et al., 1992, 1996). Notwithstanding, the effectiveness of various extracts (chloroform, hexane and methanolic extracts), against A. gambiae has not been investigated, as such it was necessary to investigate the larvicidal activities of selected Niger Delta Mangrove plants against A. gambiae. MATERIALS AND METHODS Collection of plant materials/culture of larvae The leaves of mangrove plants were collected from Ogonokom, Fimie Ama (N ʹ E ʹ) and Amadi-Ama (N ʹ E ʹ), communities in Rivers State, Nigeria. The plant were identified in the University of Port-Harcourt Herbarium, and transported to the laboratory for the bioassay. The larvae (A. gambiae), used for this investigation were cultured from the wild using baits as described by Ohimain et al. (2014). The development of the larvae was closely examined for the conspicuous emergence of swimming wrigglers. The larvae were transported to the laboratory. Prior to the bioassay, the larvae were transferred to a plastic enamel tray for acclimatization and identification. Plant extraction process The leaves of the plants (R. mangle, R. racemosa, A. germinans and L. racemosa), were shade-dried at 28 ± 2 C. Furthermore, the shade-dried leaves were mechanically pulverized with mortar and pestle. 200 g of the pulverized plants were distinctly macerated in 500 ml chloroform, hexane and methanol for 72 h. The respective filtrates were concentrated to dryness in a rotary evaporator and preserved at -4 C until it was ready for use. Phytochemical screening Phytochemical screenings of the plants were investigated following standard protocols with slight modification (AOAC, 1995). All the prepared plant extracts (Chloroform, hexane and methanolic extracts), were subjected to preliminary phytochemical screening in order to unravel the secondary metabolites such as alkaloids, flavonoids, tannins, saponin and glycosides. The degree of abundance were rated as absent (-), present (+) and present in abundance (++) (Onyenekwe et al., 2013). Rapid and final screening test The rapid screening test was carried out in order to determine the range of activity (ROA). Concentrations of 1000 and 500 ppm were used to screen the larva for total (100%) mortality within 24 h, only extract(s) which showed 100% mortality at 500 ppm (i.e. 500 ppm) during the rapid screening was used for the final screening to determine the minimal lethal dose. Experimental setup Triplicate samples of 20 healthy 4 th instar larvae were carefully placed in the solution of the respective extracts

3 Angaye et al. 47 Table 1. Phytochemical screening of various extracts of the different species of Mangrove plant. Species Solvent Used Phytochemicals present Saponin Alkaloid Glycosides Flavonoids Tannins R. mangle Methanol Hexane Chloroform R. racemosa Methanol Hexane Chloroform A.germinans Methanol Hexane Chloroform L. racemosa Methanol Hexane Chloroform Keys: ++= Present in abundance; +: Present;- = Absent. Table 2. Result of rapid screening for various extracts of the different species of the plant. Plant Extraction method Mortality Rates (%) 1000ppm 500ppm R. mangle Methanol Extract 100±0.00% 100±0.00% Chloroform Extract 100±0.00% 100±0.00% R. racemosa Methanol Extract 100±0.00% 100±0.00% Chloroform Extract 100±0.00% 100±0.00% A. germinans Methanol Extract 100±0.00% 100±0.00% Chloroform Extract 100±0.00% 85.79±0.91% L. racemosa Methanol Extract 100±0.00% 100±0.00% Chloroform Extract 100±0.00% 93.34±1.06% following standard protocol (WHO, 2013). A 24 h exposure static non-renewal test was employed for the investigation, which was performed in accordance with standard protocol (WHO, 2013) with slight modification. At varying concentrations, the mortality rates of the larvae were observed and the mean mortality and standard error was recorded. Temephos (1 ppm) was used as the positive control, while 500 ml of distilled water attuned with 2.5 ml of 10% dimethyl sulfoxide was used as the negative control (Dibua et al., 2013). With slight modification, the screening was carried out in two phases (rapid and final screening), following the protocols of Bassey et al. (2013) and Agboola et al. (2011). Statistical analysis The mean mortality and standard deviation of data from the experiment were collated, after which they were further subjected to statistical analysis (with 5% error), to estimate the median lethal concentration, based on statistical package (Microsoft Excel, 2013 version). RESULTS The results, demonstrates the larvicidal efficacy of the Niger Delta Mangrove plants (R. mangle, R. racemosa, A. germinans and L. racemosa), extracted with n-hexane, chloroform and methanol screened against 4 th instar larvae of A. gambiae larvae are presented in Tables 1 and 2 and Figure 1. Results of the preliminary phytochemical screening (Table 1), generally indicate a diversity of phytochemicals amongst all extracts of the mangrove species. These phytochemicals (Saponin, Alkaloids, Glycosides, Flavonoids and Tannins), were found present in the plant samples tested. However, the most similar in phytochemicals where R. mangle and R. racemosa, which indicated almost similar phytochemicals in all its extracts as opposed to A. germinans and L. racemosa. Generally, saponin which was most conspicuously indicated was absent in the hexane extract of R. racemosa, Alkaloid was also absent in the methanolic extracts of both R. mangle and R. racemosa species, as well as, Glycosides and Tannins in

4 48 Sky. J. Microbiol. Res. Figure 1a: Concentration-Mortality rate curve for R. mangle Figure 1b: Concentration-Mortality rate curve for R. racemosa Figure 1c: Concentration-Mortality rate curve for A. germinans Figure 1d: Concentration-Mortality rate curve for L. racemosa Note: N/A means not applicable due to higher LC100 value exceeding 500ppm Figure 1. Result of final screening for various extracts of the different species of the plant. the Hexane extract. Furthermore, Flavonoids was found in methanol extract of R. mangle but absent in R. racemosa. On the other hand, the indication of phytochemicals in A. germinans and L. racemosa, followed a different trend as shown in Table 1. Results of the rapid screening phase (Table 2), demonstrate the larvicidal activities for all three extracts of the of the four Niger Delta mangrove plants screened against the larvae of An. gambiae. Concentrations ranging from ppm were used, in order to detect the range of activity (ROA). Besides the chloroform extracts of A. germinans and L. racemosa, whose minimal lethal concentrations exceeds 500 ppm, every other extracts entered the final screening phase (Table 2). The final screening of the bioassay (Figure 1), shows significant larvicidal activities amongst all extracts of the mangrove species, with the highest mortality induced by the methanolic extracts of R. mangle LC 50 = ppm; R. racemosa LC 50 =150.00ppm; A. germinas LC 50 = ppm and L. racemosa LC 50 = ppm. Hexane extracts induced LC 50 values of , , and ppm for R. mangle, R. racemosa, A. germinas and L. racemosa respectively. The chloroform extracts of R. mangle (LC 50 = ppm) and R. racemosa (LC 50 = ppm) induced moderate toxicity as opposed to their respective co-extracts (i.e. A. germinas and L. racemosa whose minimal lethal concentrations values exceeds 500 ppm). In addition, the negative control induced no mortality throughout the bioassay, while the positive control induced total mortality at 1 ppm. Discussions Although the chloroform extracts of A. germinans and L. racemosa indicated toxic metabolites as well as toxicological activities, the administration of high dosage of these extracts (i.e.lc 100 > 500 ppm), may not favour their application as larvicides, since the breeding sites of the larvae (i.e. water bodies), co-habits other nontargeted species that plays vital role in the food-chain (Angaye, 2013), and besides serve as water source for human settlements aligning the coastal regions of the Niger Delta.

5 Angaye et al. 49 Furthermore, compared to A. germinans and L. racemosa, the results of the LC 50 values (Figure 1), indicates a close relationship between the toxicities of R. mangle and R. racemosa as well as amongst the various extracts (Methanol>chloroform>Hexane). This relative toxicological convergence between R. mangle and R. racemosa and their relative divergence amongst other species (A. germinans and L. racemosa), as well as amongst different solvent extracts was earlier indicated in the phytochemical screening (Table 1). In the same vein, an earlier study, a 50-characted dendogram show close relationship between R. mangle and R. racemosa (Siasia, 2014). From the foregoing, mangrove plant (especially Rhizophora species), is have induced various degrees of toxicities due to its phytochemicals and high salinity. For instance, previous studies, shows that Rhizophora species contain salt, tannins which can be used for the treatment of systemic ailment such as; pile, diarrhea, dysentery and even bark has found application as mosquito repellant (Miles et al., 1999). Some of the phytochemicals indicated by the plants used in our study (Table 1), were similarly identified by previous authors as therapeutics, due to certain metabolites they possess. For instance, Kandil et al. (2004) quantified the metabolites in the leaves of R. mangle as carbon-based condensed tannins and other polyphenolics (about 23% of the total leaf dry weight), such as flavonol glycosides. The polyphenolics play vital role, which serves as chemical defense mechanism against pathogens and other environmental stress. Furthermore, it could be observed over the years that scientist now prefer plant derived larvicides over commercial synthetics due to problems of environmental toxicity and the recalcitrance of certain active ingredients. Therefore, there is a rigorous research across the diversity of plants, in which significant results have been obtain plants such as Aristolochia saccata roots - effective against A. Albopictus larvae (Das et al., 2007), Leaves, Bark, Stem and Root of Jatropha curcas against A. gambiae (Ohimain et al., 2014), bark and root of Azadirachta indica against A. Gambiae (Angaye et al., 2014), Datura stramonium, Lantana camara and Tridaxprocumbens on Aedes aegyptil. (Rajasekaran and Duraikannan, 2012), Elaeagnus indica and Maesa indica on A. aegypti (Shivakumaret al., 2013). Furthermore, the larvicidal activities of plants varies among different plant organs. That is to say, while the leaves of a given plant are effective on the larvae, for other plants it could be either root or stem. This is in line with Patil et al. (2010) where the roots extracts of P. zeylanica and B. aegyptica showed high mortality against A. aegypti and A. stephensi. Conclusion Up until now, the control of malaria in the Niger Delta still remains a major challenge due to the nature of the terrain, which provides favorable breeding sites for most vectors, especially mosquitoes. The use of synthetic pesticides has induced environmental toxicity to nontargeted species, especially the co-habitants of the vector s breeding site. Since most extracts of the four mangrove plants under study shows significant toxic activities, mangrove plants could thus, be recommended as larvicides for the control of mosquitoes in the Niger Delta. Further study is still required to unravel if these larvicidal components are also found in other organs of the plant. 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