Entomocidal properties of Monodora myristica (Dunal, 1831) and Conyza sumatrensis (Retzius, 1742-1821) extracts: Studies on two dipterous insect pests Anopheles gambiae (Giles, 1902) and Culex quinquefasciatus (Say, 1823)

Anopheles gambiae (Giles, 1902) and Culex quinquefasciatus (Say, 1832) mosquitoes are the main vectors of human malaria and lymphatic filariasis, respectively. This study aims to analyze the larvicidal, pupicidal and adulticidal properties of Monodora myristica (Dunal, 1831) and Conyza sumatrensis (Retzius, 1742-1821) extracts against An. gambiae and Cx. quinquefasciatus. The experiment was conducted in the laboratory at ambient temperature of 28 °C ± 2 °C and 75% ± 5% relative humidity. The results showed that M. myristica and C. sumatrensis extracts significantly affect all stages of An. gambiae and Cx. quinquefasciatus tested. The mosquitocidal toxicity of the two plant extracts is dosage dependent. Antilarval activity of M. myristica at rate 500 mg/L and 1,000 mg/L caused 100% mortality of An. gambiae larvae while it evoked 80% and 100% mortality of Cx. quinquefasciatus larvae. The same trend of results were also obtained on the anti-pupal and adulticidal toxicity of M. myristica and C. sumatrensis extracts. As larvicides, pupicides and adulticides, the LC50s and LC90s, after 24 h varied across plant extracts and mosquito species. C. sumatrensis attained LC50 and LC90 at higher concentration than M. myristica. On An. gambiae larvae, the LC50s after 24 h, varied from 86.95 mg/L (M. myristica) to 131.73 mg/L (C. sumatrensis). Similarly, the LC90s after 24 h on An. gambiae larvae, varied from 278.39 mg/L (M. myristica) to 131.73 mg/L (C. sumatrensis). For Cx. quinquefasciatus larvae, the LC50s after 24 h , varied from 391.41 mg/L (M. myristica) to 898.20 mg/L (C. sumatrensis). The seed extract of M. myristica exerted the best pupicidal activity among the two tested extracts with LC50 and LC90 values of 140.61 mg/L and 520.35 mg/L on An. gambiae, respectively, followed by leaf of C. sumatrensis with LC50 and LC90 values of 157.59 mg/L and 781.86 mg/L on An. gambiae, respectively. More concentrations were require to achieve 50% and 90% death of Cx. quinquefasciatus pupae. On adulticidal activity, seed of M. myristica exerted LC50 and LC90 values of 122.79 mg/L and 502.99 mg/L on An. gambiae, respectively, followed by leaf of C. sumatrensis with LC50 and Received August 11, 2018 Accepted August 30, 2018 Released August 31, 2018


Introduction
The dipterans are the most important orders of hexapods with veterinary and medical importance, which can transmit many pathogenic parasites causing diseases such as malaria and filariasis among the rural dwellers in the world (Sanei-Dehkordi et al., 2018).Anopheles and Culex mosquitoes are most dangerous vectors commonly find in tropical region (Okorie et al., 2014).About 40% of the world population live in this region (Pal et al., 2014) and this shows how preventive measurement is essential in this area.Despite the efforts made by World Health Organization and researchers over the past decades to decline the mortality rate of malaria and lymphatic filariasis all over the world, malaria disease are still household illness in the tropical regions of the world (Ileke et al., 2017;Vatandoost et al., 2018).Human malaria is the most important vector-borne disease caused by Plasmodium species transmitted by anopheline mosquitoes.About 212 million peoples were suffering from malaria with 429,000 deaths recorded worldwide (WHO, 2016).Culex species is the most medically important vectors of human pathogens causing etiologic agents of different forms of encephalitis, Rift valley fever and lymphatic filariasis that are still predominant in the tropical region (Vatandoost et al., 2018).Immediate intervention in the control of these vectors in the rural area where the breeding of mosquitoes appeared permanent or semi-permanent as a result of ignorance of the vectors on the part of rural dwellers.
Vectors control programme have been intensified by medical entomologists and parasitologists throughout the world with the use of botanicals in lieu of synthetic chemical insecticides which are toxic to untargeted organisms and natural enemies as well as high cost of purchase in the management of malaria and lymphatic filariasis vectors (WHO, 2013;Vatandoost et al., 2018).
Monodora myristica is a tropical tree of the Family Annonaceae.The plant is about 35 m high and 2 m in diameter.It has a clear trunk and branches horizontally (Fournier et al., 1999).The leaves are alternately arranged and drooping with the leaf blade being elliptical, oblong or broadest towards the apex and tapering to the stalk (Weiss, 2002).It is a flowering plant with the seeds containing 5%-9% of a colourless essential oil (Weiss, 2002).The plant is used as stimulants, stomachic, for headaches, sores and also as insect repellent.The seeds are also made into necklaces (Weiss, 2002).Previous studies have shown that its polar extracts possess insecticidal properties against cowpea beetle, Callosobruchus maculatus (Okosun and Adedire, 2010;2017).It also possess ant-larval against Aedes albopictus (Tankeu et al., 2016) Conyza sumatrensis is an annual, biennial or perennial herbaceous plant that belong to Family Asteraceae.The genus Conyza consists of about 80-100 described species (Beentje, 2002;Chai et al., 2008).The oil of C. sumatrensis have antimicrobial and antifungal effects (Deans et al., 1992).Liu et al. (2012) reported the antifugi activity of C. sumatrensis against Phoma macrostoma.
In view of the current trend of developing plant based insecticides as a substitute to synthetic chemical insecticides, this study was undertaken to evaluate the larvicidal, pupicidal and adulticidal properties of M. myristica (Dunal, 1831)

Collection and rearing of larva, pupa and adult mosquitoes
Mosquito baits, consisting of shallow containers with a large surface area was established under a partial shade in an open field by filling the white bucket with rain water.10 g of yeast (Bakers' yeast) were sprinkled on the surface of the water to serves as source of foods for the nourishment of larvae.Wild mosquitoes were allowed to freely visit the baits and to lay eggs.This was monitored for 4-6 days for the development of the egg and first larva instar.These larvae were taken into the laboratory for identification into species levels (Gillies and De Meillon, 1968).The An. gambiae and Cx.quinquefasciatus larvae were separated from the mixed culture and transferred into another plastic container containing rain water to get a pure culture of each of the two dipterous insects.Some of the larvae were used for the larvicidal tests.The An. gambiae and Cx.quinquefasciatus larvae were further nurtured to pupae for 4-6 days for pupicidal tests.
Adult mosquitoes that emerged were fed with 10% sucrose solution and periodically fed with blood of 5-7 weeks restrained chick (Afolabi et al., 2018).The reared mosquitoes were maintained at 28 °C ± 2 °C and 75% ± 5% relative humidity, 12 h light followed by 12 h dark photoperiod.

Collection of plant materials and extraction
The pieces and air dried for 14 days in the laboratory.The seeds of M. myristica were also air dried for 21 days before pulverized into fine powders using an industrial electric pulverizing machine at the Department of Animal Production and Health Laboratory of the Federal University of Technology Akure.The powders were further sieved to pass through 1 mm² perforations and kept in an air-tight plastic containers for storage before use at ambient temperature 28 °C ± 2 °C.About 300 g of M. myristica and C. sumatrensis powders were soaked separately in an extraction bottle containing 600 mL of absolute methanol for 72 h.The mixture was stirred occasionally with a glass rod and extraction was terminated after 72 h.Filtration was carried out using a double layer of Whatman No. 1 filter papers and solvent evaporated using a rotary evaporator at 30 °C to 40 °C with rotary speed of 3 to 6 rpm for 8 h (Udo, 2011).The resulting extracts was air dried in order to remove traces of solvent.The extracts were kept in labeled plastic bottles till when needed.Ileke Braz.J. Biol.Sci., 2018, v. 5, No. 10, p. 347-358.

Preparation of standard stock solution
Standard stock solutions were prepared by dissolving 2 g of the crude extracts in 1 L of water.From these stock solutions, different concentrations of 62.5 mg/L, 125 mg/L, 250 mg/L, 500 mg/L and 1,000 mg/L were prepared and these aqueous solutions were used for the various experiment.

Larvicidal, pupicidal and and adulticidal bioassay
100 mm of aqueous solutions of the various plant extracts at various concentrations of 62.5 mg/L, 125 mg/L, 250 mg/L, 500 mg/L and 1,000 mg/L were each put in a labelled transparent bowl.25 days old larvae of Anopheles and Culex mosquitoes were introduced separately into the various plant extracts.They were replicated four times and water was used as control.The number of dead larvae were counted and recorded accordingly after 24 h of treatment.Dead larvae were those incapable of rising to the surface or without the characteristic diving reaction when the water was disturbed (WHO, 2006;2009).

% Larval Mortality = Number of dead larvae Number of larvae introduced
x 100 1 Similar experiment as described above was carried out for An.gambiae and Cx.quinquefasciatus pupae.22 days old pupae of An. gambiae and Cx.quinquefasciatus were introduced separately into the various M. myristica and C. sumatrensis concentrations.They were replicated four times and water was used as control.The number of dead pupae were counted and recorded accordingly after 24 h of treatment.
20 An. gambiae and Cx.quinquefasciatus adults were introduced into separate round bottom conical flask that contain suspended filter papers soaked with 62.5 mg/L, 125 mg/L, 250 mg/L, 500 mg/L and 1,000 mg/L M. myristica and C. sumatrensis extracts separately for adult bioassay.They were replicated four times and water was used as control.Mortality of adult insect was accessed after 24 h of exposure.

Statistical analysis of data
Percentage larvae, pupae and adults mortalities were estimated and corrected according to Abbott's Formula (Abott, 1925).The log-Probit model analysis (Finney, 1971) was done to the data recorded in the larvicidal, pupicidal and adulticidal bioassay to assess the 50% and 90% lethal concentrations.All values are means of four replicate followed by ± Standard error of the mean.Mean followed by the same letters, superscript at the end of each value, down the column are not significantly different (p > 0.05) from one another using Tukey's Test.
At concentration 1,000 mg/L, both plant extracts had 100% mortality on An. gambiae an effect that was not significantly different (p > 0.05) from mortality of Cx. quinquefasciatus larvae.Based on the results obtained, M. myristica caused more mortality of An. gambiae than Cx.quinquefasciatus larvae.
Both M. myristica and C. sumatrensis extracts had 100% mortality on An. gambiae pupae an effect that was not significantly different (p > 0.05) from mortality of Cx. quinquefasciatus pupae.M. myristica caused more mortality of An. gambiae than Cx.quinquefasciatus pupae.

Fumigant toxicity of plant extracts on An. gambiae and Cx. quinquefasciatus adults
Figure 2 showed the fumigant toxicity of M. myristica and C. sumatrensis extracts on An. gambiae and Cx.quinquefasciatus adults after 24 h of exposure.

Discussion
The utilization of botanicals in vectors management is gaining interest as results of the hazard associated with synthetic chemical insecticides such as toxic wastes hazard and toxicity on nontargeted organism's couple with high cost of purchase.Exploitation of low cost materials, such as agricultural wastes (cocoa pod, orange peel, cowpea pod), used in rural settlement against vectors of malaria and lymphatic filariasis may lead to promising control strategies in in developing countries (Sukumar et al., 1991;Tankeu et al., 2016).Many tropical plants have been reported to contain bioactive compound against stored product pests and vector of malaria (Adedire and Ajayi, 1996;Adedire and Lajide, 1999;Shaalan et al., 200;Adedire, 2003;Adedire et al., 2011, Akinkurolere et al., 2011, Ileke and Ogungbite, 2015;Ileke et al., 2015;2016;Awosolu et al., 2018).Presently, larvicidal, pupiidal and adulticidal properties of M. myristica seeds and C. sumatrensis leaves extracts on two dipterous insect pests.An. gambiae and Cx.quinquefasciatus has been investigated.
According to the results of this study, the M. myristica and C. sumatrensis extracts showed insecticidal effect on all stages of An. gambiae and Cx.quinquefasciatus tested.The mosquitocidal toxicity is dosage dependent; the higher the concentration, the higher the mortality rate of the vectors developmental stages.Anti-larval activity of M. myristica at rate 500 mg/L and 1,000mg/L caused 100% mortality of An. gambiae larvae while it evoked 80% and 100% mortality of Cx. quinquefasciatus larvae.The same trend of results were also obtained on the anti-pupal and aduticidal toxicity of M. myristica and C. sumatrensis extracts the two dipterous insects.The insecticidal potential of M. myristica extracted with five different solvents against cowpea bruchid, Callosobruchus maculatus have been reported by Okosun and Adedire (2010;2017).Adedire (2003) made similar observation on the toxicity of Nutmeg in the control of Callosobruchus maculatus.The insecticidal activity of the M. myristica extract against two dipterous insects in this study may be due to the presence of various bioactive compounds such as alkaloids, terpenoids, phenolics, tannins and flavonoids (Vindhya et al., 2014;Tankeu et al., 2016).Emeasor et al. (2005) ascribed the effectiveness of M. fragrans to give a long term protection to cowpea seeds as result of active components such as phellandrene, pcymene and limonene, which possess pesticidal properties.
Conyza sumatrensis is an annual, biennial or perennial herbaceous plant that belong to family Asteraceae.Exposure of An. gambiae and Cx.quinquefasciatus larvae, pupae and adults to a range of concentrations (62.5 mg/L-1,000 mg/L) caused mortalities ranged between 20%-100%.The toxicity of C sumatrensis to all the developmental stages of Cx. quinquefasciatus tested may be due to presence of limonene content in the essential extract as suggested by Sfara et al. (2009) and Kassir et al. (1989)

Table 1 .
Toxicity of plant extracts on larvae An. gambiae and Cx.quinquefasciatus after 24 h of exposure.

LC 50 and LC 90 values calculated for the tested plant extracts LC 50
Cx. quinquefasciatus larvae, pupae and adults is presented in Table2 and 3.The LC 50 of M. myristica extract was 86.95 mg/L while C. sumatrensis extract was 131.73 mg/L for An.gambiae larvae.The concentration of M. myristica and C. sumatrensis extracts to cause 50% death of Cx. quinquefasciatus larvae were 103.83 mg/L and 189.48 mg/L, respectively.The LC 90 of M. myristica extract was 278.39 mg/L while C. sumatrensis extract was 648.98 mg/L The concentration of M. myristica and C. sumatrensis extracts required to evoke 90% death of An. gambiae pupae were 520.35 mg/L and 781.86 mg/L, respectively.The LC 90 of M. myristica and 138.46 mg/L and 270.52 mg/L for Cx.quinquefasciatus adults, respectively.The concentration of M. myristica and C. sumatrensis extracts required to evoke 90% death of An. gambiae adults were 502.99 mg/L and 981.25 mg/L, respectively.The LC 90 of M. myristica and and LC 90 of the M. myristica and C. sumatrensis extracts on An. gambiae and C. sumatrensis extracts to cause 90% death of Cx. quinquefasciatus larvae were 391.41 mg/L and 898.20 mg/L, respectively.The concentration of M. myristica and C. sumatrensis extracts required to evoke 50% death of An. gambiae pupae were 115.22 mg/L and 157.59 mg/L, respectively.The LC 50 of M. myristica extract was 140.61 mg/L while C. sumatrensis extract was 197.49mg/L for Cx.quinquefasciatus pupae.myristicaand C. sumatrensis extracts to cause 50% death.The LC 50 of M. myristica and C. sumatrensis extracts Ileke Braz.J. Biol.Sci., 2018, v. 5, No. 10, p. 347-358.were

Table 2 .
LC 50 of M. myristica and C. sumatrensis extracts on An. gambiae and Cx.quinquefasciatus larvae, pupae and adults.

Table 3 .
LC 90 of M. myristica and C. sumatrensis extracts on An. gambiae and Cx.quinquefasciatus larvae, pupae and adults; Syzygium guineense, M. myristica and Zanthoxylum heitzii against Aedes albopictus.The seed extract of M. myristica exerted the best pupicidal activity among the two tested extracts with LC 50 and LC 90 values of 140.61 mg/L and 520.35 mg/L on An. gambiae, respectively, followed by leaf of C. sumatrensis with LC 50 and LC 90 values of 157.59 mg/L and 781.86 mg/L on Cx. quinquefasciatus pupae.This ascribed to the report of Tankeu et al. (2016) who worked on larvicidal activities of three extracts against Aedes albopictus.On Adulticidal activity, seed of M. myristica exerted the best among the two tested plants with LC 50 and LC 90 values of 122.79 mg/L and 502.99 mg/L on An. gambiae, respectively, followed by leaf of C. sumatrensis with LC 50 and LC 90 values of 215.05 mg/L and 981.25 mg/L on Conclusion African nutmeg, M. myristica seeds and C. sumatrensis leaves extracts have shown significant larvicidal, pupicidal and adulticidal properties on An. gambiae and Cx.quinquefasciatus that causes human malaria and lymphatic filariasis.The two tested plants can be integrated into pest management programmes to combat human malaria and lymphatic filariasis vectors breeding site in Nigeria.The two plants are readily available in the trophic, ecofriendly, biodegradable and medicinal.I recommend formulation of M. myristica seeds which have the lowest LC 50 and LC 90 after 24 h for field evaluation in other to solve the problem of wild migration of vectors to human's settlement.