CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

Mosquitoes are insects which belong to the phylum Arthropoda, class insecta, order dipteraand

familycullicidae (Service, 2008). About 41 genera of mosquitoes and approximately 3500

Species have been described, many of which are known vectors of human and zoonotic diseases.

The family cullicidae has three medically important genera; Anopheles (malarial vector in man),

Culex (vector of West Nile virus in man), and Aedes (vector of dengue, yellow fever and filarial

diseases) (Nasir et al., 2015). Mosquitoes spread more diseases than any other group of

Arthropod, mosquito-borne diseases is the major health problem in both man and veterinary

Sectors (Mekhlafi et al., 2015).

Malaria, dengue fever.chikungunya, filariasis and Japanese encephalitis are transmitted by

mosquitoes affecting humans and animals worldwide, causing millions of death every year

(Johnson and Singh, 2017). Mosquitoes spread diseases to humans causing millions of death

every year. In 2017, malaria

alone caused 435000 deaths (WHO, 2018). Mosquitoes are distributed worldwide, occurring

throughout tropical and temperate regions and northwards into the arctic circle but are absent in

Antarctica and few islands (Service, 2008). Aedes mosquitoes are found in tropical, sub-

tropical, and in some temperate climates worldwide (Service. 2008).

Mosquitoes are vectors of disease causing agents found within almost all tropical and subtropical

countries. They are responsible for the transmission of pathogens causing some of the life
threatening and debilitating diseases of man, such as; malaria, yellow fever, dengue fever,

chikungunya, filariasis, encephalitis, etc (Chandra et al., 2008; ICMR, 2003). Lymphatic

filariasis is a significant public health and economic problem in many tropical and subtropical

regions of the world, including. Not only the man is affected by mosquitoes but also they

transmit diseases among animals like viral diseases “fowl pox” of chicken, meyxomatosis of

rabbits, rift valley of sheep and encephalitis of horses and birds. Moreover, their vicious biting

and hovering sound cause a lot of annoyance especially in urban areas; this cause great

discomfort; psychological one.

The control of mosquito at the larval stage is necessary and efficient in integrated mosquitos’

management. During the immature stage, mosquitoes are relatively immobile; remaining more

concentrated than they are in the adult stage. Since the discovery of DDT, mosquito control

approach has been almost completely based on synthetic organic insecticides. But the extensive

use of synthetic organic insecticides during the last five decades have resulted in environmental

pollution and also in the development of physiological resistance in major vector species in

addition to the increased costs of insecticides. Hence forth, there appeared a continuous search

for selective pesticides that could be safe to non-target organisms as well as to the environment,

but at the same time, have the knock out ability only to the target organisms. Such pesticides can

be used ad jointly with biological control agents without any detrimental effects.

Plant constituents have been suggested as alternative sources for larval mosquito control

products, in part, because they constitute a potential source of bioactive chemicals that often

produce only minor adverse effects on non-target organisms and the environment. Essential oils

often act at multiple and novel target sites, thereby reducing the potential for resistance

(Sukumaret al. 1991, Wink 1993, Kostyukovskyet al. 2002, Priestley et al. 2003, Isman 2006).
These constituents are widely available with some being relatively inexpensive compared with

plant extracts such as pyrethrum and neem (Isman 2001). Much effort has been focused on plants

and their essential oils as potential sources of commercial larvicides because they meet the

criteria of minimum risk pesticides (USEPA 1996, 2004).

With the development of resistance to conventionally used synthetic insecticides, vector

management has become acutely problematic. Hence more attention has been focused on

botanicals. Therefore this study is carried out to determine the Larvicidal activity of aqueous

extract of AnarcardiumOccidentale on aedesaegypti.

1.2 Statement of the Problem

More than 2 billion people living in the tropics have been infected by mosquito-borne diseases,

such as malaria, chikungunya, dengue fever, lymphaflariasis, yellow fever and Japanese

encephalitis (Mahardika et al., 2014). Malaria alone caused 435,000 deaths in 2017

(WHO,2018). Chemical insecticides is commonly considered to be the most effective control

strategies against mosquitoes but they have negative impacts on man and the environment (Igbai

et al., 2018). The recently released WHO Global report in the insecticide resistance in Malaria

vectors 2010-2016 shows that resistance to the four commonly used insecticide classes –

Pyrethroids, organochlorines, carbamates and organophosphates is widespread in the WHO

regions of Africa, America, South-East Asia and many more(WHO, 2018).

1.3 Aim and Objective of the Study

The aim of this research study is to determine the larvicidal potential of aqueous extract of

cashew leaves (AnacardiumOccidentale) on aedesaegypti mosquito larvae.

1.3.2 Objectives
i. To determine the effects of the aqueous extract on the developmental duration of mosquitoes.

ii. to determine the larvicidal efficacy of the leaf extracts on aedesaegyptilarvae.

iii. to determine the median lethal concentration (LC50) and the 90% lethal concentration (LC90)

of the extract on aedesaegypti.

1.4 Significance of the Study

Repeated use of synthetic insecticides for mosquito control has disrupted natural biological

control systems and led to resurgence in mosquito populations. It has also led to the development

of resistance (WHO, 1992). Synthetic insecticides are toxic and adversely affect the environment

by contaminating soil, water and air. There is need to find alternatives to these synthetic

pesticides. Controlling the vectors using various methods can interrupt disease transmission.

Plants are considered rich sources of bioactive chemicals and may be an alternative source of

mosquito control agents.

The emergent consciousness of the hazards of extreme use of pesticides worldwide has led

researchers to search for safer and more environment friendly another methods for insect pest

control. Therefore, widespread studies are accepted out to screen plants as insect increase control

agents. Over the last two to three decades, better concentration has been alert on the bioactivity

of Phytochemicals for their potential as pesticides against phytophagous insects. Plant derived

pest control agents are biodegradable, non-toxic to non-target organisms, non-constant in nature,

as well they do not promote drug resistance. Secondary metabolites are very much toxic to many

insect species and more than 2000 plant species are known to possess some larvicidal properties.

Biopesticides supply an alternative to synthetic pesticides since of their commonly low

environmental contamination, low toxicity to humans and other advantages. Botanical properties
of plant source with larvicidal properties have been tried as an indigenous technique for

organized range of insect pests in the modern past. The use of plant extracts for insect control has

numerous attractive features as these are usually more recyclable, less dangerous and a rich

storehouse of chemicals of various biological actions. In addition, herbal source give a lead for

discovering new insecticides.

1.5 Research Methodology

Materials were sourced from journals, articles, textbooks, archives and Internet.

1.6 Organization of the Study

The work is divided into five chapters. Chapter one is an introduction. Chapter two is literature

review. Chapter three focuses on materials and methods. Chapter Four is on result. Chapter five

comprises discussion, conclusion and recommendation.

 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Plant Description of AnarcardiumOccidentale(Cashew)

The cashew (AnacardiumOccidentale) is a member of the Anarcardium genus of the

Anarcardiaceae family. It is a small tree, with leaves that are alternate, simple, entire, obtuse, and

borne on short leaves stalks. The flowers are abundant, small and fragrant, and are produced in

terminal, loose panicles. The tree can grow as tall as 14 metres(46 feet), but the dwarf cultivars,

growing up to 6m( 20ft), prove more profitable, with earlier maturity and greater yields.

Cashew leaves are leaves of the cashew tree. They are oval in shape with fine pronounced

veins and midribs. Each leaf is 10 to 20 centimeters long, by 7 to 12 centimeters in width.

Cashew leaves are best eaten when they are young and are a purplish green at the tips, and a

brighter green on the bottom. When young, the leaves have a texture like hard spinach. They

have a tangy, astringent taste. Cashew leaves are available all year round. The leaves and young

shoots are used in Salads and as a medicinal herb.

Research has shown that cashew leaves are rich in antioxidants and have antifungal, anti-

parasitic, anti-bacterial, antiseptic and anti- inflammatory properties. In Peru and India, they are

chewed and used as toothpaste, and are further used to treat toothaches and gum problems. They

may be pulped to make mouthwashes. In Africa, they are used to treat diabetes and Malaria.

They contain vitamin B and Vitamin C. They are a fair source of iron and calcium, and also

contain zinc, magnesium, phosphorus, manganese, sodium and potassium. Young cashew leaves

may be eaten fresh out of hand, like lettuce leaves. They are commonly included among the

leaves in “Ulam” salads in Malaysia. They may be dipped in a spicy sauce and eaten as a snack,
or may be used as a garnish for fish and sambal dishes. Their astringent taste helps to bring

coolness to spicy dishes. To store Cashew leaves, place them in a bag in the refrigerator, where

they will last for several days

Fig 1.Anacardiumoccidentale leaves ( Cashew Leaves)

Source: https://www.wikipedia.org/cashewleaves.com
2.2 Taxonomical Classification of AnacardiumOccidentaleleaves( Cashew leaves)

Kingdom: Plantae

Phylum: Angiosperms

Class: Eudicots

Order: Sapindales

Family: Anacardiaceae

Genus: Anacardium

Species: AnarcardiumOccidentale

2.3 Phytochemical Composition of Anarcardiumoccidentale

2.3.1 Saponin

Saponins are glycosides with distinctive foaming characteristics. They are natural detergents

found in certain plants. They are found in many plants especially certain desert plants. Saponins

are freely soluble in both organic solvents and water (Hostettman and Marston, 1995). Saponins

have detergent or surfactant properties because they contain both water soluble and fat soluble

components. They are amphipathic compounds, possessing both hydrophilic and lipophilic

portions. They are therefore surface active agents and can be used as emulsifiers. At

concentrations between 200-500ppm, saponins exits as monomers, above 500ppm they aggregate

as micelles with a molecular weight of approximately 100,000Daltons.

2.3.2 Tannin

They are general descriptive names for a group of polymeric phenolic substances capable of

tanning leather or precipitating gelatin from solution, a property known as astringency. Their

molecular weight ranges from 500 to 3000KiloDaltons. The tannin compounds are widely

distributed in many species of plants, where they play a role in protection from predation, and

perhaps also as pesticides, and in plant growth regulation (Katie et al., 2006). The astringency

from the tannins is what causes the dry and puckery feeling in the mouth following the

consumption of unripened fruit or red wine (McGee, 2004). Likewise, the destruction or

modification of tannins with time plays an important role in the ripening of fruit and the aging of

wine. Tannins are divided into two groups, hydrolysable and condensed tannins. Hydrolysable

tannins are based on Gallic acid, usually as multiple esters with D-glucose, while the more

numerous condensed tannins (often called proanthocyanidins) are derived from flavonoid

monomers. Tannins may be formed by condensation of flavan derivatives which have been

transported to woody tissues of plants. Alternatively, tannins may be formed by polymerization

of quinine units.

2.3.3 Terpenoids

The terpenoids include several sesquiterpene lactones such as: vernolide, vernodalol, vernolepin,

vernodalin, vernomygdin, hydroxyvernolide, vernodalinol, vernomenin, vernolic, 11, 13-

dihydrovernodalin, 11, 13- dihydrovernorodeline, 4, 15-dihydrovernodalin, 1, 2, 3, 15, 11, 13, 2’,

3’-octahydrovernodalin and epivemodalol(Njanet al., 2008). Some of these compounds may be

associated with the ability of Vernoniaamygdalina to regulate blood sugar (Njanet al., 2008;

Nwaoguikpe, 2010; Amaechiet al., 2018).

2.3.4 Flavonoids

The flavonoids found in this plant are quercetin, kaempferol, and catechins. In higher plants,

flavonoids are involved in the filtration of ultraviolet light, nitrogen fixation, cell cycle

inhibition; as chemical messengers and for the protection of plants against certain diseases.

Flavonoids are known to demonstrate two major mechanisms of action (either through direct

scavenging of free radicals ;or interfering some enzyme activities such as with nitric oxide

synthase activity or xanthine oxidase activity) (Nijveldtet al., 2001).

2.4 Larvicidal Activities of Bio-active Components of Different Plants on Mosquito Species

The secondary compounds of plants make up a vast repository of compounds with a wide range

of biological activities. Phytochemicals have a major role in mosquito control programs. The

bioactive plant ingredients can be obtained from the whole plant or from specific parts by

extraction with different types of polar and nonpolar solvents. Most studies report active

compounds as steroidal saponins. Saponins are freely soluble in both organic solvents and water.

Saponins are naturally occurring glycosides with the ability of forming soapy lather when shaken

with water and classified as triterpenesaponin or steroidal saponins on the basis of the structural

features of the aglycone moieties (Sakagamiet al., 2012). Bioassay-guided fractionation of

Achyranthesaspera led to the separation and identification of a saponin as a potential mosquito

larvicidal compound (Bayavan, 2008).

Flavonoids are hydroxylated phenolic substances that occur as a C6-C3 unit linked to an

aromatic ring. They are synthesized by plants in response to microbial infection (Fessender,

1982, Dixon et al., 1983). Their activity is probably due to their ability to complex with

extracellular and soluble proteins of bacterial cell walls. More lipophilic flavonoids may also
disrupt microbial membranes (Tsuchiya et al., 1996). They have been found to be effective

against a wide array of microorganisms (Casley, 1997). Several members of Asteraceae family

are reported to have mosquito larvicidal activity due to the presence of several flavonoids and

thiophenes (Srivastavaet al., 2008). Flavonoid 7-methoxyaromadendrin which is present in

Trixisvauthieri extracts accounts for its mosquito larvicidal activity (Macedoet al., 1997).

Flavonoids are important secondary metabolites in higher plants and classified into flavones,

isoflavones, flavonols, flavanones, catechins and anthocyanidins, based on the skeletons of

aglycone moieties (Sakagamiet al., 2012). The methanolic extract of Tagetesminuta was found to

contain Aglycan flavonoids which may be the effective components that have larvicidal effect

(Hadjiakhoondiet al., 2005).

Tannin is a general descriptive name for a group of polymeric phenolic substances capable of

tanning leather or precipitating gelatin from solution. Their molecular weights range from 500 to

300 (Haslam, 1996). They are found in every part of the plant such as leaves, wood, fruits, bark

and roots. They are divided into two groups, hydrolysable and condensed tannins. Tannins may

be formed by condensation of flavan derivatives that have been transported to woody tissues of

plants. Many human physiological activities such as stimulation of phagocytic cells, host

mediated tumour activity and a wide variety of anti-infection actions have been assigned to

tannins (Haslam, 1996). The growth of many fungi, bacteria, yeast and viruses are inhibited by

tannins. Tannins in fruits thus serve as a natural defense mechanism against microbial infections

(Samy and Gopalakrishnakone, 2008). The antimicrobial properties of hydrolysable tannins

could be associated with the hydrolysis of ester linkage between gallic acid, usually as multiple

esters with D-glucose which eventually affects the biosynthetic steps in the synthesis of cell wall
and cell membrane (Lim et al., 2006). A cardiac glycoside was reported to have a caricidal effect

against larva and adult stages of the camel tick (Al-Rajhlet al., 2003).

The fragrance of plants is carried in the essential oil fraction. These oils are secondary

metabolites that are highly enriched in compounds based on isoprene units. They are called

terpenes. Terpenes are a class of molecules that typically contain either ten or fifteen carbon

atoms built from a five-carbon building block called isoprene. If three isoprenes are present, as in

farnesol, the category is called sesquiterpenes which is a common group of plant poisons.

Broadly, terpenoids are grouped on the basis of isoprene units present (Shrivastava and Patel,

2007).

A new himachalenesesquiterpenoidhugonianene A (135), was identified as the major compound

responsible for larvicidal effect of Hugoniabusseana extract. It exhibited a high activity against

Anopheles gambiae (Magadula and Erasto, 2009). Terpenoids are usually present in essential oils

as hemiterpenes, monoterpenes or sesquiterpenes and as their derivatives. High concentrations of

monoterpenes in plants may repel many potential predators but may also attract other animals.

According to research, many plants of Asteraceae family have recorded larvicidal activity. The

ethanol extract of aerial parts of T. minuta against Aedesfluviatilis exhibited high activity with an

LC50 value of 1.0 ppm (Macedoet al., 1997) while the methanol extract of the same plant

showed an LC50 value of 2.5 ppm against Anopheles stephensi (Hadjiakhoondiet al., 2005).

Ethanol extract of Ecliptapaniculata also in the family recorded an LC50 value of 3.3 ppm

against Aedesfluviatilis (Macedoet al., 1997). However petroleum ether extracts of

Centratherumanthelminticumfruits and leaves were comparatively less active with an LC50

value of 162.60 and 522.94 respectively (Srivastavaet al., 2008).

In Kenya, research work has been carried out to determine the mosquito larvicidal activity of

various plant extracts. According to (Matasyohet al., 2008) extracts of Aloe turkanensis, Aloe

ngongensis and Aloe fibrosa exhibit activity against the third instar larvae of Anopheles gambiae.

Roots of Vernoniaadoensis were found to have toxicity against Culexquinguefasciatus with a

mortality of 80 % at a concentration of 2000 mg/l (Swamyet al., 2014). Essential oils and non-

volatile compounds from Faragopsisangolensis (Rutaceae) leaves have also demonstrated

larvicidal activity against Anopheles gambiae (Mudalunguet al., 2013). Research work that was

done on cultures of Sterum species (J05289) has shown that they have larvicidal compounds that

can be used to control mosquitoes and spread of diseases by the vectors (Chirchiret al., 2013).

2.5 Antimalaria

Extracts of leaves and root bark showed antimalarial activity against Plasmodium berghei which

causes malaria when tested in vivo in mice and against Plasmodium falciparumin vitro (Kambizi

and Afolayan, 2001). These extracts suppress the growth of malaria parasites. The leaf extract

has also been observed to restore the efficiency of drugs like chloroquine after the malaria

parasites had developed resistance towards them (Iwalokun, 2008; Njanet al., 2008; Egharevbaet

al., 2014).

2.7 Aedes mosquitoes

Aedesaegypti, the yellow fever mosquito, is the major global vector of dengue fever,

chikungunya, Zika fever, Mayaro and yellow fever viruses, and other disease agents which are

poorly controlled in Africa and often go unrecognized (Weetmanet al., 2018). The mosquito can

be recognized by black and white markings on its legs and a marking in the form of a lyre on the

upper surface of its thorax. Its distribution and abundance has been favored by poor water supply
and sanitation infrastructure, including poorly designed tanks and jars and unreliable piped water

supply. (Koenraadtet al., 2006; Nagao et al., 2006; Strickmanet al., 2003).

According to the World Health Organization it has been reported that about 129 countries are

suffering from dengue and there is increase in the number of people who get infected with

dengue (WHO, 2019).

The larvae feed on bacteria, grow over a period of weeks until they reach the pupa stage and

have a comb-scale like silk pattern (Fig 1). The lifespan of an adult Ae.aegypti is two to four

weeks depending on conditions, but the eggs can be viable for over a year in a dry state, which

allows the mosquito to re-emerge after a cold winter or dry spell (McBride et al., 2014;

Weetmanet al., 2018). Both Aedes species like to deposit their eggs in natural reservoirs.

Favorites are forks in trees, or manmade reservoirs like old (car) tyres, empty bottles, vases,

etc.Aedes has a shorted and darker siphon compared to Culex. At the early instars of Aedes larva,

the siphon remains soft which gets darker and harder in the later instars. Moreover, it cannot

transmit any disease at this (larva) stage due to the lack of proboscis which they use to bite

humans (MdShakhawat et al., 2022; Arista-Jalife et al., 2020)

Figure 1: Larvae of AedesMosquitoes (Russell, 2015)

2.8 Larvicidal Phytochemicals

Phytochemicals literally mean “plant chemicals”. They are biologically active, naturally

occurring chemical compounds found in plants, which provide health benefits for humans further

than those attributed to macronutrients and micronutrients. They protect plants from disease and

damage and contribute to the plant’s colour, aroma and flavor. Phytochemicals such as rotenone,

nicotine and pyrethrum have been used as pesticides by man before the advent of synthetic

insecticides. Various members of the families Annonaceae, Asteraceae, Meliaceae, Myrtaceae

and Piperaceae produce chemical compounds which act as antifeedants, repellents, biocides or

growth inhibitors detrimental to many insect species (Singh, 2016).The insecticides of plant

origin are commonly used in the form of aqueous/solvent extracts, powders, slurries, volatiles

and oils, or as shredded segments (Mohammed et al., 2018).

Toxicity of plant extracts towards larvae of insects has been reported by several authors.

Myristicin extracted from P. mullesua effectively killed fourth instar larvae of Spilarctiaobliqua
(Walker) (Lepidoptera: Arctiidae) after 24 hours following topical application (Srivastavaet al.

2001). In an experiment to assess the efficacy of crude seed extract of sugar-apple

(Annonasquamosa L.) against larvae of the diamondback moth, Plutellaxylostella L.

(Lepidoptera: Plutellidae), 84 % larval mortality was achieved at 0.5 % concentration. Activity

of the sugar-apple aqueous extract compared favourably with 1 % rotenone and pyrethrum, two

well-known commercial insecticides of botanical origin (Leatemia and Isman 2004). Larvicidal

and repellent potential of Moringaoleiferaagainst malarial vector, Anopheles stephensiListon

(Insecta: Diptera: Culicidae) has been studied and it was revealed that phytochemicals derived

from M. oleiferaseeds extracts are effective mosquito vector control agents and the plant extracts

could be used for further integrated pest management programs (Prabhuet al., 2011)

2.9 Mosquito Repellants

Mosquito repellents appear to act by masking odor cues given off by a person or by direct

repellency to female mosquitoes seeking a blood meal. Plant products have been used

traditionally by human communities in many parts of the world against the vectors and the

different species of insects(Saniet al., 2019). Many scientists have reported plants and natural

derived chemicals which are non toxic to man and domestic animals as useful basis for

development of safer and more effective mosquito insecticides. The search for natural herbal

preparations that do not produce any adverse effects in the non-target organisms and are easily

biodegradable remains a top research issue for scientists associated with alternative vector

control strategies (Govindrajan, 2010). Phytochemicals explored from plant sources can act as

larvicides and can be responsible for the interruption of the transmission of mosquito-borne

diseases at the individual as well as at the community level (Govindrajan et al., 2008).
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Study Area

The plants were collected from AnyigbaDekina LGA KogiState.The town is situated on latitude

7.493366 and longitude 7.173639 and it is located 120KM east of Lokoja, the state Capital.

Anyigba shares common boundaries in the North with Ajiolo, Abocho and Biraidu, and South

with Abejukolo-egume, and Ofakaga. Anyigba fall within the southern Guinea Savannah with no

portable water supply, and characterized by dry and wet seasons.

3.2 Collection of Anacardium occidentaleLeaves

The Plants were collected from the study area (Anyigba, Kogi State) and brought to the

Department of Plant Science and Biotechnology, Prince AbubakarAudu University (PAAU),

Anyigba for identification and then air dried in the department of Animal and Environmental

Biology PAAU, Anyigba.

3.3 Preparation of Leaf Extract

The collected leaves were rinsed with distilled water and dried at room temperature for 4 weeks

and then taken to the university Biochemistry laboratory, where the dried leaves were pulverized

into powder by stainless electrical blender and stored in an air tight container. The extraction was

carried out for 48 hours and the powdered material was used for further phytochemical analysis.

The extraction was done in Biochemistry laboratory of Prince AbubakarAudu University. 500

grams of the pulverized plant was measured into a conical flask and was diluted with 5000mls of
eva water. It was tightly covered and left for 48 hours for proper mixture. After 48 hours, the

mixture was filtered through vacuum filtration and then concentrated through a water bath.

3.4 Identification and Collection of Mosquito Larvae.

The larva were identified to be aedesaegypti by an entomologist using an identification key for

arthropod vector. The larval stage of mosquito for this study was sourced from the root of a

pawpaw tree that has been cut down.

3.5 Materials and Reagents.

Pipette, Dropper, Disposable tubes, Strainer, beakers, measuring cylinder, ice bath (for grinding),

plastic tubes, water, scoop, mosquito larvae (biological materials).

3.6 Extraction of phytochemicals

The leaves of Anacardiumoccidentale were collected and washed thoroughly with borehole

water so as to remove dirt, air-dried in a shaded area and grinded. Water was then added then left

for twenty four hours. The mixture was allowed to stay for a certain period with occasional

shaking to allow the solvent to extract the phytochemicals. The extract was then filtered and the

quantity of the phytochemical present in the extract using appropriate analytical techniques was

determined.

3.7Larvicidal Assay

Bioassay for the larvicidal activity was carried out according to World Health Organization

standard, with minor modifications (WHO, 2005). A stock solution was prepared and stored in a

refrigerator at 15°C. 20 healthy third larvae instar were collected and placed in the 500mls

plastic beaker containing 1000mls of water from their natural growth medium with various
concentration of the aqueous extract ranging 500ppm, 250ppm, 125ppm, 62.50ppm, and

31.25ppm in triplicates. Three controls were also setup. The beakers were covered with muslin

cloth to prevent the entry of strange materials. Mortality count of larvae was monitored at regular

intervals i.e. 3, 6, 12, 24 and 48 hours after treatment. Larvae was considered dead if they settle

and remain motionless in the bottom of the bowl with no response to light or mechanical

stimulus or not recovering life functions even after being transferred to their growth medium.

3.8 Statistical Analysis

Using Abbot's formula, percentage mortality was calculated and with the aid of Statistical

Package for the Social Sciences (SPSS), regression analysis was done and the LC 50 and LC95 were

estimated at 95% Confidence Limit. Upper and Lower Boundaries for the Extract were also

determine.
 CHAPTER FOUR
RESULTS

4.1 Larvicidal Efficacy of Aqueous Extract of Anarcadium occidentalis on Aedes aegyptii

Table 4.1 shows the result of the Larvicidal effect of different concentrations (500, 250, 125, 62.5, and

31.5 ppm) of Aqueous Extract of Anarcadium occidentalis on Aedes aegyptii within a range of 5 minutes

to 48 hours. As observed from the results, no significant difference is observed from 5 mins to 1 hr period

of exposure to extract. which denotes that within the first period of 3 hours of exposure, no effect of the

extract was observed on the larva Aeds aegyptii. But a significant difference was observed from 3 hrs to

48 hrs. The highest level of significance is observed within the period of 48 hours. This reveals that at 24

hrs, the there is high effect of Anarcadium occidentale on the Larva of Aedes aegyptii.

Across the concentration, no significant difference is observed in the control, and 500 ppm. A significant

difference was observed in 48 hrs across the 500 ppm conc.

Across the 250 ppm the highest level of significance was observed at 3hrs and 48 hrs. no significant

difference was observed in the remaining periods of time. Across the 125 ppm no significant difference

was observed in 5 minutes to 1 hr time but a significance was observed the first 3 hour period. Across the

62.5ppm, no significant difference was observed in 5 minutes to 48 hr time, and finally across the 31.5

ppm no significant difference was observed in 5 min 50 24 hours but a significant difference was

observed in 48 hour of time.

Parame 5 mins 10 mins 15 mins 30 mins 1 hr 3hrs 6hrs 12hr 24hr 48hr
ters

Control 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.00±0.00 a 0.33±0.58a 0.33±0.58a 0.33±0.58a 2.67±0.58a
a

500 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 1.67±1.15ab 2.33±1.52ab 2.33±1.52ab 2.33±1.52ab 7.33±2.30b

250 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.00±0.00a 1.67±0.58ab 1.67±0.58ab 1.67±0.58ab 12.00±3.00c

125 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 1.00±0.00ab 3.67±1.52b 3.67±1.52b 3.67±1.52b 7.33±3.21b

62.5 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.69±1.15ab 1.67±1.15ab 1.67±1.15ab 1.67±1.15ab 5.33±1.15ab

31.5 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 2.33±1.52b 3.00±2.00b 3.00±2.00b 3.00±2.00b 8.00±2.64bc

Table 4.1 Larvicidal Efficacy of Aqueous Extract of Anarcadium occidentalis on Aedes aegyptii
4.2 Lethal Concentration of Anarcadium occidentale
The lethal concentration observed in this indicates that at 24 hours an LC 50 of 47.24 and at 48 hrs a lethal

concentration of 9.83 was observed. This indicates that within 24 hours, more lethality was observed and

almost half the population of the mosquito larva was reduced and weakened.
Table 4.2 Lethal Concentration of Anarcadium occidentalis on Aedes aegyptii

LC50 Period

47.24 24hr

9.83 48hrs
 CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion
It seems that the concentration of aqueous leaf extract had no significant effect on the mosquito

larvae during the initial period (from 5 minutes to 1 hour), but a significant difference emerged

after 3 hours and became more pronounced at 48 hours, particularly among the concentrations of

500 ppm, 250 ppm, and 125 ppm. This pattern suggests that the effects of the extract take some

time to manifest and become noticeable. It would be beneficial to conduct further research to

understand the mechanism behind this delayed but significant impact. The result of this study

denotes that within the first period of 3 hours of exposure, no effect of the extract was observed

on the larva Aedes aegyptii. But a significant effect was observed from 3 hrs to 48 hrs. The

highest effect was observed within the period of 48 hours. This reveals that at 48 hrs, there is

high effect of Anarcadium occidentale on the Larva of Aedes aegyptii. This result is contrary to

the work of Mukhopadhyaya (2010) who observed a higher efficacy at 24 hours. In their study,

the highest efficacy was observed in 12 ppm which contradicts the result in this study whose

highest efficacy was observed in 500, 250, and 125 ppm. The results of the lethal concentration

of 47.24 for 24 hrs contradicts the result observed in Ali and El-Rabaa (2010) who observed a

LC50 conc of 41.25 at 48hr. higher efficacy observed in the 500, 250, and 125ppm concentration

might be because of the high level in concentration. The findings from this study are in

agreement with the findings from the work of Yousaf and Zuharah (2015) in which 100 %

mortality was observed by mosquito larva after being administered with a high concentration of

Anarcadium occidental. The findings in this study also contradicts the findings of de Azevedo et

al. (2021). They had the highest efficacy of vegetable leaf on mosquito larva at 120hrs after

exposure to the concentration of 2500 ppm of the extract. The present findings corporate earlier
findings of Macedo et al. (1997) who showed that ethanol extract of Tagetes patula was less

active and only 50% larvae were killed at higher concentration (100 ppm).

5.2 Conclusion

Our present observation reveals that Ae. aegypti larvae are highly susceptible to Anarcadium

occidentalis extract at (500, 250, and 125 ppm). The highest Lethal concentration was observed

in 24 hrs with the LC50 of 47.24. in conclusion, the result in this study proves that there is high

efficacy of Anarcadium occidentalis on the Ae. Aegyptii. However, further studies on the

identification of the active principles involved and their mode of action and field trials need

presently other investigations.

5.3 Recommendation

Further research study needs to be carried out to help understand the effect of the chemical

component of anarcadium extract. This will help in observing the full efficacy of the extract. The

results also emphasized the need of further research and investigation to find out the bioactive

compounds of anarcadium extract and their activities against other vector pests. This may help in

enhancement of the bioactivity of their phytochemicals and replacement of the synthetic

insecticides in future.
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