CHAPTER ONE

1.0 INTRODUCTION

Several diseases are transmitted by arthropod vectors (Leitner et al., 2015) of

which mosquito is one of them. Mosquitoes are responsible for the transmission of

diseases such as malaria, Lymphatic filariasis, and dengue fever among others

(WHO, 2017) most especially in the tropical regions of the world. Out of these

diseases transmitted by mosquitoes, malaria is the most devastating in terms of the

number of incidence, prevalence, morbidity and mortality (WHO, 2017). It is

transmitted by the mosquitoes of the genus Anopheles and it is caused by the

protozoa of the Plasmodium genus (Singh et al., 2014). Examples of Anopheles

species include Anopheles gambiae, Anopheles culicifacies, Anopheles stephensi,

Anopheles fluviatilis, Anopheles minimus, Anopheles dirus and Anopheles

sundaicus among others with Anopheles gambiae being the prominent one among

them all (WHO, 2015). Five species of Plasmodium namely Plasmodium

falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and

Plasmodium knowlesi are responsible for human malaria (Oddoux et al., 2011) but

P. falciparum and P. vivax are most notorious in terms of the number of cases they

are responsible for (WHO, 2018)They were an estimated 219 million cases of

malaria with 435,000 of them resulting in death in 2017 (WHO, 2018). In order to

stop or reduce the transmission of malaria parasites, there is need to control the
vector population. Several methods such as habitat modification (Jacups et al.,

2011), the use of synthetic chemical insecticides (WHO, 2018). and the use of

biological means such as employing natural predators, parasites and parasitoids to

control the populations of mosquitoes (WHO, 2018). The major method that have

been employed in the control of mosquitoes is the use of synthethic insecticides

such as chlorodane, aldrin, Dieldrin, dichlorodiphenyl trichoroethane (DDT)

among others (Jacups et al., 2011).

However, these chemical have been shown to have negative effect in the

dynamics of the earth’s ecosystem resulting in resistance of mosquito species

to the chemicals, environmental pollution and toxicity to human and other non-

targeted organisms (Mahmood et al., 2015). Hence, there have been clamor by

various quarters to develop a safe means to combat the proliferation of insect

vector species (Kwenti, 2017). This has led to the focus on developing

insecticides from botanical sources that are safe to use in the environment due

to the fact that they are easily degradable and less toxic to humans and non-

targeted organisms (Kwenti, 2017). Apart from the hazards caused by the use

of established pesticides and insecticides to man and livestock, they are also

very expensive to purchase; therefore, they are no more within the reach of

many Nigerians. Moreover, chemical control technology is subject to the

predicament of unstable foreign exchange. In the light of these shortcomings, it

 becomes inevitable to research into local sources of insecticides that would be

cheap and readily available to individuals. Moreover, treatment using

medicines of natural origin is gaining momentum nowadays on account of

increasing concern about potentially harmful synthetic additives (Mahmood et

al., 2015).

Cymbopogon citratus, commonly known as lemon grass and other

Cymbopogon species is a tall, coarse grass with a strong lemon taste. Lemon grass

is a perennial herb widely cultivated in the tropics and sub-tropics, and it

designates two different species; East Indian Cymbopogn flexuosus (DC.) and

West Indian, Cymbopogon citratus (Kwenti, 2017). It is a tropical plant, grown

as an ornamental in many temperate areas with maximum a height of about 1.8m

and its leaves 1.9cm wide covered with a whitish bloom (Bashir et al., 2010).

Biologically active compounds derived from selected plants species such as

Cymbopogon citrates, Ocimum gratissiumum, Hyptis sauveolen, Acarcia

Arabica, Azadirachta indica and Eleusive indica have been commonly used in the

past to control insects in many tropical counties (Bashir et al., 2010). The

essential oils obtained from the leaves and stems of this plant are used as remedy

for several health problems like fever, throat inflammations, ears or eyes a typical

example is the use of the leaves in the Eastern Nigeria to treat various heart

disorders (Laura Salvia et al., 2014). Research by (Moser and Joshi 2013),
indicated that globally the most important use of neem was as an insecticide.

Neem contains several aromatic compounds that can be used to repel insects from

biting humans and animals. Neem oil mixed with coconut oil gave up to 98.03%

protection against the mosquito, Anopheles culicifacies, in all-night biting tests

conducted in Gujarat, India (Ojewumi and Owolabi 2012). Neem oil also

provided more than 75% protection against A. fluviatilis, Aedestaeniorhynchoides

and Mansonia uniformis. Burning neem oil in a room is also said to repel

mosquitos and other biting insects11.

1.2 Statements of the problem

Mosquito-transmitted diseases remain a main source of illness and death

(Laura Salvia et al., 2014). Despite decades of malaria control efforts, malaria

continues to be a major worldwide public health issue with 3.3 billion persons at

risk in 106 countries and territories in the tropical and subtropical areas (Laura

Salvia et al., 2014). It is one of the significant reasons for maternal and childhood

morbidity and mortality, including low birth weight, stillbirths, and early infant

death in sub-Saharan Africa (WHO, 2018). Among 500 species of Anopheles

mosquitoes known globally, more than 50 species can transmit malaria from the

bite of the infected female Anopheles spp. (Moser and Joshi 2013). Presently,

there is no effective prophylactic anti-malarial vaccine and no suitable preventive

measure other than vector control is available (Moser and Joshi 2013). Thus,
protection from mosquito bites is one of the best approaches to reduce the disease

incidence.

This situation calls for a re-evaluation of the strategies for effective and

sustainable vector management. In addition to the anthropogenic habitats, there

are natural water bodies scattered around most urban areas that can support

mosquito breeding. This makes potential larval habitats numerous and larval

control seems a difficult. Therefore the data on these mosquito species would be

relevant in predicting their public health implications and planning strategies that

would help control diseases associated with these mosquito vectors in an endemic

environment.

1.3 Justification

Female Anopheles mosquitoes are the vectors of human malaria. The use of

chemical insecticides for vector control has hampered with environmental

pollution and insect. This suggests the need for the development of more potent

and environment-friendly insecticides for effective control of malaria. This

research will show the larvicidal activities of selected Nigerian plants species of

Neem and lemon grass leaf extracts against, Anopheles gambiae. Most plants

contain compounds that they use in preventing attack from phytophagous (plant

eating) insects. These chemicals fall into several categories, including repellents,
feeding deterrents, toxins, and growth regulators. Most can be grouped into five

major chemical categories:

i. Nitrogen compounds (Primarily Alkaloids),

ii. Terpenoids,

iii. Phenolics,

iv. Proteinase inhibitors,

v. Growth regulators

Although the primary functions of these compounds is defence against

phytophagous insects, many are also effective against mosquitoes and other biting

Diptera, especially those volatile components released as a consequence of

herbivory (Moser and Joshi 2013). In spite of innovations and revolutions in

diagnostics, drug therapy, and vector control measures, the global burden of

vector-borne diseases especially mosquito-borne diseases remains unacceptably

high. It is hoped that these findings from this study will promote and improve

effective vector control decision making.

1.4 AIM AND OBJECTIVES

1.4.1 Aim of the study

 To evaluate the insecticidal activity of two Nigerian plant species of Neem

and lemon grass extract against Anopheles gambiae

1.4.2 Objectives of the study

1. To examine insecticidal activity of two selected Nigerian plant species of

Neem and lemon grass extract against Anopheles gambiae

2. To determine larvicidal activities of selected Nigerian plants species of

Neem and lemon grass leaf extracts against, Anopheles gambiae.

3. To determine the properties of Nigerian plant species Lemon grass

(Cymbopogon flexuosus) and Neem tree (Azadirachta indica L.) leaves

extract against Anopheles gambiae.

 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Anopheles gambiae

The Anopheles gambiae complex consists of at least seven morphologically

indistinguishable species of mosquitoes in the genus Anopheles. The complex was

recognised in the 1960s and includes the most important vectors of malaria in sub-

Saharan Africa, particularly of the most dangerous malaria parasite, Plasmodium

falciparum (Yakob, 2021). It is one of the most efficient malaria vectors known.

The An. gambiae mosquito additionally transmits Wuchereria bancrofti which

causes Lymphatic filariasis, a symptom of which is elephantiasis (Yakob, 2021).

Figure 1: Anopheles gambiae

Scientific classification

 Kingdom: Animalia

 Phylum: Arthropoda

 Class: Insecta

 Order: Diptera

 Family: Culicidae

 Subfamily: Anophelinae

 Genus: Anopheles

 Species complex: A. gambiae

Species: A. arabiensis, A. bwambae, A. melas, A. merus, A. quadriannulatus,

Anopheles gambiae sensu stricto, Anopheles coluzzii, Anopheles amharicus

2.2 The Plasmodium parasite

Human malaria cases are usually due to five Plasmodium species,

Plasmodium falciparum, P. vivax, P. malariae, P. ovale (two types) and P.

knowlesi (Duval, 2009). Plasmodium falciparum is the most lethal. Approximately

1.38 billion people are at risk of P. falciparum malaria. High levels of P.

falciparum malaria endemicity are common in Africa (Hay, 2017). In the Americas

and Asia, sub-tropical regions have predominantly P. vivax malaria, while the

tropical regions have a mix of P. falciparum and P. vivax malaria (Lorenz et al.,

2021). Plasmodium vivax malaria often has milder symptoms but it can stay

dormant for months or even years in patients, which makes it difficult to cure

people and identify carriers (Lorenz et al., 2021). Plasmodium malariae and P.

ovale malaria are less common. The recently described human malaria cases of P.

knowlesi are mostly limited to Malaysia, where they can be common (Mondal et

al., 2016).

2.3 Mosquito vectors of the genus Anopheles

All human malaria vectors belong to mosquitoes of the genus Anopheles

(Diptera: Culicidae). Worldwide, 52 Anopheles species are primary malaria

vectors (most of these are indicated in Figure 2) (Hay, 2017). There are seven

dominant Anopheles species (or species complexes) in Africa including Anopheles

merus, An. moucheti and An. nili in addition to the ones mentioned in Figure 2. In

Europe and the Middle East, there are six primary vectors species (Hay, 2017). In

the case of the Americas the nine dominant Anopheles species (or species

complexes) are An. albitarsis, An. marajoara plus the seven mentioned (Hay,

2017). In Pacific Asia, however, 26 Anopheles species (and species complexes) are

the primary vectors of human malaria which, apart from those mentioned include

An. aconitus, An. balabacensis, An. campestris, An. donaldi, An. koliensis, An.

lesteri, An. letifer, An. leucosphyrus, An. ludlowae, and An. nigerrimus (Hay,

2017).

2.3.1 Causes of Malaria in Human

Among the parasites of the genus Plasmodium four species have been

identified which can cause disease in humans:

 Plasmodium falciparum

 Plasmodium vivax

 Plasmodium ovale

 Plasmodium malariae

 Plasmodium knowlesi
Figure 2: Plasmodium falciparum

Source: Wikipedia 2016

 Scientific classification

 Domain: Eukaryota

 Phylum: Apicomplexa

 Class: Aconoidasida

 Order: Haemosporida

 Family: Plasmodiidae

 Genus: Plasmodium

 Species: P. relictum and others of the genus

2.4 Life cycle and ecology of malaria vectors

 Anophelines exhibit complete metamorphosis, which means that after

hatching the larva passes through a pupal stage to develop into an adult (Figure 3).

The adult female mosquito deposits eggs on water at night (Lorens et al., 2021).

Ovipositing females show a preference for certain types of aquatic habitats which

depends on abiotic and biotic factors. Anopheles gambiae prefer small, sunlight

and transient, and often turbid habitats to lay their eggs (Service, 2012). Anopheles

stephensi and An. culicifacies prefer fresh water over sea water (Lorens et al.,

2021). Anopheles albimanus mostly lay eggs in habitats with cyanobacteria

(Lorens et al., 2021). Anopheles gambiae lay more eggs in water with bacteria and

fewer eggs in water that contains conspecific larvae or contained predators, e.g.,

tadpoles and back swimmers (Kosova and Jonida, 2003). Eggs lie individually on

the water surface and are boat-shaped. Anopheline eggs cannot resist drought

(Peach and Gries, 2016). Anopheles gambiae eggs do not hatch after being exposed

to 45 °C for 10 min. In a tropical climate, dry soil temperature can reach 40 - 50

°C, however, temperature of moist soil rarely exceeds 35 °C (Baylis, 2017).

Anopheles gambiae eggs can, therefore, survive and hatch on moist soil (Baylis,

2017). Anopheles larvae are legless. They breath air and feed on particulate matter

(microorganisms, diatoms, bacteria, algae, detritus of decaying plants, etc.) at the

water surface and, therefore, spend more or less all of their time at the air-water

interface (Baylis, 2017). Anopheles stephensi larvae prefer shade and can be found
in man-made breeding sites in urban areas and rice fields, and polluted and saline

water habitats in rural areas (Hay, 2017). Anopheles gambiae sensu stricto and An.

Arabiensis.

Figure 3: Stages of Anopheles life cycle

 Mosquitoes go through four stages in their life cycles: egg, larva,

pupa, and adult or imago. The first three stages—egg, larva, and pupa—are largely

aquatic. Each of the stages typically lasts 5 to 14 days, depending on the species

and the ambient temperature, but there are important exceptions (Kosova and

Jonida, 2003). Mosquitoes living in regions where some seasons are freezing or

waterless spend part of the year in diapause; they delay their development,

typically for months, and carry on with life only when there is enough water or

warmth for their needs. For instance, Wyeomyia larvae typically get frozen into

solid lumps of ice during winter and only complete their development in spring.

The eggs of some species of Aedes remain unharmed in diapause if they dry out,

and hatch later when they are covered by water. Eggs hatch to become larvae,

which grow until they are able to change into pupae. The adult mosquito emerges

from the mature pupa as it floats at the water surface. Bloodsucking mosquitoes,

depending on species, sex, and weather conditions, have potential adult lifespans

ranging from as short as a week to as long as several months. Some species can

overwinter as adults in diapause (Kosova and Jonida, 2003).

2.4.1 Breeding

In most species, adult females lay their eggs in stagnant water: some lay near

the water's edge while others attach their eggs to aquatic plants. Each species

selects the situation of the water into which it lays its eggs and does so according
to its own ecological adaptations. Some breed in lakes, some in temporary puddles.

Some breed in marshes, some in salt-marshes. Among those that breed in salt water

(such as Opifex fuscus), some are equally at home in fresh and salt water up to

about one-third the concentration of seawater, whereas others must acclimatize

themselves to the salinity (Wigglesworth, 2007). Such differences are important

because certain ecological preferences keep mosquitoes away from most humans,

whereas other preferences bring them right into houses at night. Some species of

mosquitoes prefer to breed in phytotelmata (natural reservoirs on plants), such as

rainwater accumulated in holes in tree trunks, or in the leaf-axils of bromeliads.

Some specialize in the liquid in pitchers of particular species of pitcher plants, their

larvae feeding on decaying insects that had drowned there or on the associated

bacteria; the genus Wyeomyia provides such examples — the harmless Wyeomyia

smithii breeds only in the pitchers of Sarracenia purpurea (Crans et al., 2013).

However, some of the species of mosquitoes that are adapted to breeding in

phytotelmata are dangerous disease vectors. In nature, they might occupy anything

from a hollow tree trunk to a cupped leaf. Such species typically take readily to

breeding in artificial water containers. Such casual puddles are important breeding

places for some of the most serious disease vectors, such as species of Aedes that

transmit dengue and yellow fever. Some with such breeding habits are

disproportionately important vectors because they are well-placed to pick up

pathogens from humans and pass them on. In contrast, no matter how voracious,

mosquitoes that breed and feed mainly in remote wetlands and salt marshes may

well remain uninfected, and if they do happen to become infected with a relevant

pathogen, might seldom encounter humans to infect, in turn (Crans et al., 2013).

2.4.2 Eggs and Oviposition

Figure 4: Electron micrograph of a mosquito egg

Mosquito habits of oviposition, the ways in which they lay their eggs, vary

considerably between species, and the morphologies of the eggs vary accordingly.

The simplest procedure is that followed by many species of Anopheles; like many
other gracile species of aquatic insects, females just fly over the water, bobbing up

and down to the water surface and dropping eggs more or less singly. The bobbing

behavior occurs among some other aquatic insects as well, for example mayflies

and dragonflies; it is sometimes called "dapping" (Crans et al., 2013). The eggs of

Anopheles species are roughly cigar-shaped and have floats down their sides.

Females of many common species can lay 100–200 eggs during the course of the

adult phase of their life cycles. Even with high egg and intergenerational mortality,

over a period of several weeks, a single successful breeding pair can create a

population of thousands (Crans et al., 2013).

Figure 5: An egg raft of a Culex species.

Some other species, for example members of the genus Mansonia, lay their eggs in

arrays, attached usually to the under-surfaces of waterlily pads. Their close

relatives, the genus Coquillettidia, lay their eggs similarly, but not attached to

plants. Instead, the eggs form layers called "rafts" that float on the water. This is a

common mode of oviposition, and most species of Culex are known for the habit,

which also occurs in some other genera, such as Culiseta and Uranotaenia.

Anopheles eggs may on occasion cluster together on the water, too, but the clusters

do not generally look much like compactly glued rafts of eggs (Marten and Reid

2007).

In species that lay their eggs in rafts, rafts do not form adventitiously; the

female Culex settles carefully on still water with its hind legs crossed, and as it

lays the eggs one by one, it twitches to arrange them into a head-down array that

sticks together to form the raft (Marten and Reid 2007).

Aedes females generally drop their eggs singly, much as Anopheles do, but

not as a rule into water. Instead, they lay their eggs on damp mud or other surfaces

near the water's edge. Such an oviposition site commonly is the wall of a cavity

such as a hollow stump or a container such as a bucket or a discarded vehicle tire.

The eggs generally do not hatch until they are flooded, and they may have to

withstand considerable desiccation before that happens (Marten and Reid 2007).

They are not resistant to desiccation straight after oviposition, but must develop to
a suitable degree first. Once they have achieved that, however, they can enter

diapause for several months if they dry out (Marten and Reid 2007). Clutches of

eggs of the majority of mosquito species hatch as soon as possible, and all the eggs

in the clutch hatch at much the same time. In contrast, a batch of Aedes eggs in

diapause tends to hatch irregularly over an extended period of time. This makes it

much more difficult to control such species than those mosquitoes whose larvae

can be killed all together as they hatch. Some Anopheles species do also behave in

such a manner, though not to the same degree of sophistication (Huang et al.,

2006).

2.5 Malaria transmission

Plasmodium is transmitted from one infected person to another by

mosquitoes. An Anopheles mosquito injects Plasmodium intradermally into the

human body, in the form of sporozoites during a blood meal. Only a few

sporozoites (10-30) are injected at a time, and they migrate through the skin cells.

Some of the injected sporozoites reach the blood circulation and are rapidly

transferred to the liver. Plasmodium sporozoites develop and multiply into

thousands of newly-formed merozoites in the liver cells, which are then released

into the bloodstream. The merozoites attach to and enter red blood cells and

replicate asexually within them. The red blood cells subsequently rupture and

release the merozoites, which attach to adjacent red blood cells and the asexual
replication cycle is initiated again (Sawada et al., 2017). The sexual stages, male

and female gametocytes, are also produced in the red blood cells. The male and

female gametocytes are taken up by a mosquito whilst feeding. Inside the mosquito

mid-gut, the gametocytes fuse to form a zygote that develops into a motile

ookinete. The ookinete enters the mid-gut epithelium and migrates to the outer

layer of the mid-gut, called the basal lamina. Here, the ookinete transforms into an

oocyst and after several mitotic divisions forms sporozoite buddings which contain

sporozoites. The sporozoites are released into the haemolymph and are transferred

to the salivary glands. These sporozoites are injected into the human skin by an

Anopheles female while blood feeding (Sawada et al., 2017). The development

period of Plasmodium spp. in the mosquito is sensitive to temperature. As a result,

malaria is limited to areas where the temperature is suitable for the Plasmodium

spp. to complete their development within the life span of the adult mosquito

(Peach and Gries, 2016). At 15 - 17 °C the complete development of P. vivax takes

38 days, which is longer than the adult mosquito life time. However, at 22 - 23 °C

complete development occurs in 14 days, so at this temperature P. vivax malaria

can be effectively transmitted. In the case of P. falciparum, the development period

is 9 - 10 days at 28 °C and 22 days at 20 °C (Peach and Gries, 2016). The intensity

of malaria transmission can be expressed by the stability index, which is the

average number of bites by an average mosquito during a normal mosquito

lifetime. If the index is >2.5 it indicates stable malaria transsmission, a value

between 0.5 and 2.5 indicates intermediate malaria transmission and <0.5 indicates

unstable malaria transmission. Stable malaria is transmitted by an Anopheles with

a frequent biting habit, moderate to high longevity and living at a temperature

favourable for the rapid development of Plasmodium. Stable malaria is difficult to

control as even a low density of Anopheles can maintain transmission (Peach and

Gries, 2016)). Vectorial capacity is the daily rate at which future inoculations

arise from a currently infective person. It directly depends on the man-biting rate,

feeding habits (anthropophilic (preference for human blood meals) vs zoophilic

(preference for animal blood meals)) and life expectancy of anophelines. Each

species has a different behaviour and, therefore, the vectorial capacity in a certain

area depends on the local Anopheles species. Anopheles gambiae s.s. and An.

funestus are highly anthropophilic and bite indoors. Anopheles arabiensis mostly

bites outdoors and is more opportunistic as it equally feeds on humans and animals

(Hay, 2017). Anopheles stephensi is mainly an urban mosquito but also found in

rural areas where, unlike in urban areas, it has a secondary role in malaria

transmission (Peach and Gries, 2016). One of the reasons for this secondary role is

that An. stephensi is zoophilic. In the urban areas An. stephensi feeds on humans

due to unavailability or low number of animal hosts and, therefore, plays a primary

role in malaria transmission (Hay, 2017). A study at a refugee camp in Pakistan

showed that sponging cattle with insecticide reduced malaria transmission as much

as indoor spraying of houses because it targeted the zoophilic malaria vectors

(Peach and Gries, 2016). It is, therefore, important to understand the distinct

behaviour of each Anopheles species even within a species complex in order to

control the transmission of malaria.

2.5.1 Malaria control

Presently, there are two strategies to control malaria, vector control and drug

treatment (Djouaka et al., 2011). Currently, no vaccine is available for the

prevention of malaria (Djouaka et al., 2011). Vector control aims to reduce vector-

human contact by targeting the larval or adult stage of Anopheles. The use of

larviciding is limited and considered suitable mostly for application in urban and

peri-urban areas where breeding sites are well-defined and approachable

(President’s Malaria Initiative, 2011; WHO, 2010). Adult mosquito control is the

main vector control approach that is currently used. This is based on

epidemiological models which predict that targeting adult female mosquitoes is the

most effective way of reducing malaria transmission (WHO, 2010). Adult control

involves indoor residual spraying (IRS), space spraying and insecticide treated bed

nets (ITNs) (Djouaka et al., 2011). The latter include long-lasting treated bed nets

(LLINs) and prevent mosquitoes from biting people. Only chemicals that are

approved by WHOPES (WHO Pesticide Evaluation Scheme) can be utilised for

IRS and treating bed nets. Approved chemicals belong to four insecticide classes:

organochlorines, carbamates, organophosphates and pyrethroids (Olayemi et al.,

2011). Indoor residual spraying targets female mosquitoes that enter the house and

rests, on the walls and iling, before and/or after a blood meal. The Global Malaria

Eradication Programme (1955 - 1969) was based on the use of IRS against the

vector mosquitoes. The programme caused significant reductions in the global

malaria burden, particularly in Asia, Latin America, North America, Southern

Africa and Europe (WHO, 2006). The global eradication programme was halted in

1969 for technical and logistical reasons, leaving much of malaria in Africa

untouched. In 1997 a regional partnership initiative known as the African Initiative

on Malaria Control was launched by representatives of the World Bank, WHO,

UNICEF and others. This initiative provided the foundation for the launch of the

Roll Back Malaria programme in 1998. With renewed vigour, malaria control was

initiated in most malaria-endemic countries. Presently, IRS is recommended for the

control of malaria by 71 countries, 32 of which are in Africa and eight in Southeast

Asia. In some countries such as Botswana, Mozambique, Namibia, South Africa,

Swaziland and Zimbabwe, it is the primary vector control intervention (WHO,

2010). Indoor residual spraying is known to be effective in areas with unstable

malaria transmission; however, its effectiveness in areas with stable malaria is not

well established (Oduola et al., 2012). In contrast to IRS, an insecticide-treated bed

net (ITN) targets the female mosquito which is attracted by a human host.

Insecticide-treated bed nets are more cost effective than IRS and have a high

community acceptance (Olayemi et al., 2011). There are indications that

insecticide-treated bed nets provide better protection than IRS (Oduola et al.,

2012). Nearly 254 million ITNs were distributed in Africa between 2008 and 2010

and a very high percentage (80%), of these available ITNs, is being used (WHO,

2010). Space spraying is reserved for application during emergencies to control

epidemics and is, therefore, used only in areas with unstable malaria transmission

((Oduola et al., 2012). Drugs such as chloroquine, amodiaquine, sulfadoxine-

pyrimethamine, primaquine and artesunate target different stages of Plasmodium in

the human body ((Oduola et al., 2012). Chloroquine is used for the treatment of P.

vivax malaria while P. falciparum malaria is treated by artemisinin-based drugs

(WHO, 2010).

 Vector Control

Malaria vector control aims to protect people against infective malaria mosquito

(Anopheles spp.) bites by reducing vector longevity, vector density and/or human-

vector contact. It also includes the reduction of malaria transmission at human

community level and hence the incidence and prevalence of human infection and

disease. The two broadly used interventions for vector control are long lasting

insecticide treated bed nets (LLINs) and indoor residual spraying (IRS). Both

LLINs and IRS are directed against adult mosquitoes and involve the use of

chemical insecticides (WHO, 2010). Currently twelve insecticides, which belong

to four chemical classes (organophosphates, carbamates, pyrethroids and

organochlorines), are recommended by the WHO Pesticide Evaluation Scheme

(WHOPES) for IRS and among them only pyrethroids are recommended for use on

LLINs ((Oduola et al., 2012). WHO, 2010). These four chemical classes have only

two different modes of action, i.e., the inhibition of acetylcholinesterase and

modulation of voltage-gated sodium channels (Oduola et al., 2012). The

development of resistance in mosquitoes against these two modes of action

compromises the vector control strategies currently being used in malaria- endemic

countries (Hay, 2017). Although insecticide resistance can be managed, it requires

early detection and reporting of resistance followed by determining the type of

resistance mechanisms, the Anopheles spp. most resistant, the area over which

resistance has spread and why resistance emerged. Once all these data have been

collected and analysed, an informed decision on which insecticide or combination

of insecticides to use in local malaria vector control programme can be made

(Djouaka, 2011). However, managing resistance will face difficulties because:

  Recommended chemicals have only two modes of action

 Scaling up of IRS and LLINs (required for effective vectors control) puts a

large pressure on the selection for resistance

 It is likely to be many years before new chemicals become available as

alternative options for malaria control programmes (Oduola et al., 2012).

In addition, even when both LLINs and IRS are effective they are limited in

preventing malaria transmission due to early feeding and/or outdoor dwelling

Anopheles spp. (Oduola et al., 2012). It is, therefore, important to incorporate

interventions that provide additional protection to humans against malaria by

targeting the resistant and/or outdoor. Anopheles population into malaria control

programmes. Larval control can provide this additional protection.

 Larval control

Larval control targets the aquatic stages of Anopheles mosquitoes which results in

reduced adult emergence from breeding sites and, therefore, reduced numbers of

adult mosquitoes in surrounding areas. Larval control is a cost-effective approach

and is especially suitable for localities where breeding sites are few and/or fixed

and easy to identify, map and treat ((Oduola et al., 2012). Utzinger, 2001; WHO,

2010). Such areas include include highlands, desert-fringes, urban settings and

areas prone to epidemics. Larval control is also effective in rural areas when
carried out systematically (Oduola et al., 2012). Larval control can complement

current malaria control strategies by reducing the adult population including

resistant and/or exophagic Anopheles populations (Oduola et al., 2012). Over the

last decade the potential of larval control has been increasingly acknowledged on

the basis of both historical successes and contemporary research. Historically,

larval control was carried out with petroleum oils, chemicals and/or environmental

management. Considering the issues of environmental safety and resistance

development, petroleum products and chemicals are not suitable for larval control.

Many non-chemical tools for larval control are available but they have rarely been

utilised to the full extent for the control of Anopheles larvae.

2.6 Azadirachta Indica

Neem or Margosa is a botanical cousin of mahogany. It belongs to the

family Meliaceae. The latinized name of Neem - Azadirachta indica - is derived

from the Persian:

 Azad = Free,

 dirakht = Tree,

 i - Hind = of Indian Origin

which literally means: 'The Free Tree of India'.

 Azadirachta indica, commonly known as neem, nimtree or Indian lilac,

(Oduola et al., 2012). and in Nigeria called dogoyaro or dogonyaro, (Barstow and

Deepu, 2018) is a tree in the mahogany family Meliaceae. It is one of two species

in the genus Azadirachta, and is native to the Indian subcontinent and most of the

countries in Africa. It is typically grown in tropical and semi-tropical regions.

Neem trees also grow on islands in southern Iran. Its fruits and seeds are the source

of neem oil. Neem is an attractive broad-leaved, evergreen tree which can grow up

to 30m tall and 2.5m in girth. Its trunk usually straight is 30-80 cm in diameter. Its

spreading branches form a rounded crown of deep-green leaves and honey-scented

flowers as much as 20m across.

Figure 6: Neem
  Scientific Classification

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Eudicots

Clade: Rosids

Order: Sapindales

Family: Meliaceae

Genus: Azadirachta

Species: A. indica

 Description

Neem is a fast-growing tree that can reach a height of 15–20 metres (49–66 ft), and

rarely 35– 40 m (115–131 ft). It is deciduous, shedding many of its leaves during

the dry winter months. The branches are wide and spreading. The fairly dense

crown is roundish and may reach a diameter of 20–25 m (66–82 ft). The neem tree

is similar in appearance to its relative, the chinaberry (Melia azedarach). (Oduola

et al., 2012). The opposite, pinnate leaves are 20–40 cm (8–16 in) long, with 20 to

30 medium to dark green leaflets about 3–8 cm (11⁄4–31⁄4 in) long. The terminal

leaflet often is missing. The petioles are short.

 White and fragrant flowers are arranged in more-or-less drooping axillary

panicles which are up to 25 cm (10 in) long. The inflorescences, which branch up

to the third degree, bear from 250 to 300 flowers. An individual flower is 5–6 mm

(3⁄16–1⁄4 in) long and 8–11 mm (5⁄16–7⁄16 in) wide. Protandrous, bisexual flowers

and male flowers exist on the same individual tree. The fruit is a smooth

(glabrous), olive-like drupe which varies in shape from elongate oval to nearly

roundish, and when ripe is 14–28 mm (1⁄2–11⁄8 in) by 10–15 mm (3⁄8–5⁄8 in). The

fruit skin (exocarp) is thin and the bitter-sweet pulp (mesocarp) is yellowish-white

and very fibrous. The mesocarp is 3–5 mm (1⁄8–1⁄4 in) thick. The white, hard inner

shell (endocarp) of the fruit encloses one, rarely two, or three, elongated seeds

(kernels) having a brown seed coat (Oduola et al., 2012).

2.6.1 Therapeutic Role of Azadirachta indica

Products made from neem trees have been used in the traditional medicine

of India for centuries, (Oduola et al., 2012). but there is insufficient clinical

evidence to indicate any benefits of using neem for medicinal purposes. In adults,

no specific doses have been established, and short-term use of neem appears to be

safe, while long-term use may harm the kidneys or liver; in small children, neem

oil is toxic and can lead to death (YashRoy and Gupta 2000). Neem may also cause

miscarriages, infertility, and low blood sugar (Oduola et al., 2012).

  Other Uses

 Tree: the neem tree is of great importance for its anti-desertification

properties and possibly as a good carbon dioxide sink. It is also used for

maintaining soil fertility.

 Fertilizer: neem extract is added to fertilizers (urea) as a nitrification

inhibitor. Animal feed: neem leaves can be occasionally used as forage for

ruminants and rabbits

 Teeth cleaning: neem has traditionally been used as a type of teeth-cleaning

twig.

2.7 Cymbopogon flexuosus

Cymbopogon flexuosus, also called Cochin grass, East-Indian lemon grass or

Malabar grass, is a perennial grass native to India, Sri Lanka, Burma, and

Thailand. It is placed in the genus Cymbopogon (lemongrasses).

Figure 7: Cymbopogon flexuosus

Scientific classification

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Monocots
 Clade: Commelinids

Order: Poales

Family: Poaceae

Subfamily: Panicoideae

Genus: Cymbopogon

The genus Cymbopogon constitutes of approximately 140 species that show

widespread growth across the semi-temperate and tropical regions of Asian,

American and African continents. Australia and Europe are home to only a few

species of lemon grass. Also known as ‘Squinant’ in English, lemon grass is

known by various other colloquial names throughout the world. The members of

the Cymbopogon genus produce volatile oils and thus are also known as aromatic

grasses (Adhikari et al., 2013).

A strong lemon fragrance, a predominant feature of this grass, is due to the

high citral content of its oil. The redolence of the oil enables its use in soaps,

detergents, etc. As a good source of citral, it finds an application in the perfumery

as well as food industries. It is also the starting material for the manufacture of

ionone’s, which produce Vitamin A. (Viabhav et al., 2013).

Lemon grass contains several bioactive compounds that impart medicinal value to

it. Considerable evidence is available for its ethno pharmacological applications.4

According to the WHO, herbal medicine is considered as an important part of the

healthcare industry by more than two-thirds of the population in developing

countries.5 Apart from an overall description of lemon grass, this review article

also highlights its medicinal properties that make it a potent herb for

pharmacognostic applications.

2.7.1 Taxonomical and Botanical Description

Lemon grass is a perennial monocotyledonous grass which can grow upto 6

feet in height and 4feet in width. It grows in clusters. It has long, slender, drooping

brightgreen leaves that measures from 1.3-2.5cm in width and 3feet in length.

Leaves are simple with entire margins. Flowers grow on spikes. It has a lengthy

inflorescence ranging from 30-60cm (Adhikari et al., 2013). The floral

arrangement of this scented grass gives it the name ‘Cymbopogon’. Cymbo

Essential oils are extracted from flowers, herbs, trees and various other plant

materials. These oils contain a mixture of chemical compounds. Terpenes

associated with aldehydes, alcohols and ketones form the major chemical

component of such essential oils. AH (Zaibunnisa et al., 2013). Apart from being

used to manufacture of perfumes, soaps, cosmetics and detergent, citronella oil

also finds an application in the pharmaceutical industry. The extraction of this

essential oil is classified as clean technology (Zaibunnisa et al., 2013). Lemon

grass contains 1-2% of essential oil on a dry weight basis.10 Lemon grass oil is
also known as citronella oil. Steam and hydro distillation are the conventional

methods of its extraction. These procedures are however time consuming

(Zaibunnisa et al., 2013). An innovative Microwave-Assisted Hydro distillation

(MAHD) not only reduces the extraction time but also retains the quality of oil.7

The benefits of microwave radiation aided oil extraction technique over

pogoncitratus is a common inhabitant of Southeast Asia (Zaibunnisa et al., 2013).

Taxonomic details of this herb are as follows: (Adhikari et al., 2013).

2.7.2 Extraction of Lemon Grass Oil

Essential oils are extracted from flowers, herbs, trees and various other plant

materials. These oils contain a mixture of chemical compounds. Terpenes

associated with aldehydes, alcohols and ketones form the major chemical

component of such essential oils (Adhikari et al., 2013). Apart from being used to

manufacture of perfumes, soaps, cosmetics and detergent, citronella oil also finds

an application in the pharmaceutical industry. The extraction of this essential oil is

classified as clean technology.8,9 Lemon grass contains 1-2% of essential oil on a

dry weight basis (Desai and Parikh, 2015). Lemon grass oil is also known as

citronella oil. Steam and hydro distillation are the conventional methods of its

extraction. These procedures are however time consuming (Desai and Parikh,

2015). An innovative Microwave-Assisted Hydrodistillation (MAHD) not only

reduces the extraction time but also retains the quality of oil.7 The benefits of
microwave radiation aided oil extraction technique over hydro distillation have

also been reported (Desai and Parikh, 2015). Pressurized liquid extraction using

nitrogen gas, is a novel technique and was found to yield better quality of oil in

comparison to Soxhlet extraction and hydrodistillation methods.10 Supercritical

extraction of citronella oil with CO2 under high pressure has also been investigated

(Desai and Parikh, 2015).

2.7.3 Medicinal Properties of Lemon Grass and its Oil

Lemon grass has been traditionally used to remediate a plethora of medical

conditions. This is due to the broad spectrum of secondary metabolites that it

produces. It has been used to treat fever, cough, elephantiasis flu, leprosy, malaria

and digestive problems among many other illnesses. The use of lemon grass in

Ayurveda is still relevant today due to its therapeutic value. Conventional medicine

has a lot of adverse effects. Therefore plant-based medicine has become a popular

alternative for synthetic medicine. Thus, this herbaceous plant may find many

applications in the pharmaceutical industry (Garg et al., 2012).

Indonesian Scientists have investigated and confirmed the ability of β-

citronellol-the major component of “Sereh Wangi” (colloquial name for Lemon

grass in Indonesia) oil, to bring about a reduction in weight of rats fed with a high

fat diet. Inhalation of vapors of β-citronellol enhances the sympathetic nerve

activity of the rats that leads to the increased activity in the adipose tissue resulting

in weight loss. The findings of this study were significant due to the fact that β-

citronellol caused a reduction in body mass without affecting the concentration and

activity of the liver enzymes (Garg et al., 2012). Weight loss is only one of the few

pros of citronella grass.

Maintenance of oral health is an important aspect of daily routine. Gingivitis

and periodontitis are oral health conditions caused due to dental plaque. Many

reports confirm that these dental caries are risk factors for ischemic stroke and

cardiovascular disease. The antagonistic activity of lemon grass against the

planktonic and biofilm forms of Candida dubliniensis, a common oral pathogen

has been reported. Citronella grass may be used in formulating herbal drugs for

oral healthcare (Garg et al., 2012).

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Area

The study was carried out in the insectary laboratory, University of Abuja,

main campus along Nnamdi Azikiwe Airport Road, FCT, Abuja. The University of

Abuja is situated at lat 9° 32n and long 50°10e with a land mass of 11,824
hectares, Abuja has rich soil for cultivation and enjoys equable climate that is

neither too hot (35° c) none too cold (22°c) all year Round. The neem samples was

collected at Giri, Gwagwalada and the lemon grass was collected at Iddo,

Gwagwalada, Abuja while the mosquito larval of Anopheles gambiae was

collected at Gausau, Abuja.

Plate 1: University of Abuja permanent site

Source: portal.uniabuja.com. 2020

3.1 Collection of Mosquito Larva

The mosquito larvae of Anopheles gambiae were collected from stagnant

water in an exposed bowl at Gausau, F.C.T. Abuja. The larvae were brought to the

insectary laboratory department of biological sciences, University of Abuja,

Gwagwalada, Abuja. with enclosed mosquito net and then transferred into a beaker

with clear tap water covered with mosquito net for safety precautions in case of

fast emergence of the larvae into adult mosquito (Kamsuk et al., 2007).

3.2 Extraction of plant materials

Cymbopogon flexuosus (Lemon grass) and Azadirachta indica (Neem tree)

leaves were washed separately with distilled water, shade dried, cut into small

pieces and grinded them then kept in insectary laboratory. The method of Olubiyi

et al., (2013) was employed and modified for preparation. 100 g of the

Cymbopogon flexuosus and Azadirachta indica powdered leaves were weighed and

dissolved in 1000 ml of the solvent and allowed to soak for 72 hours at room

temperature but stirred regularly at every 12 hours. The solvent was mixed with

distilled water. The resultant suspension was filtered into a 1000 ml beaker using

muslin cloth reinforced with Whatsman's No. 1 filter paper (Ohia et al., 2013).
Plate 1: A pictorial view of Neem tree Azadirachta indica
Plate 2: A pictorial view of Lemon grass Cymbopogon flexuosus
3.2 Anti-larval activity of Cymbopogon flexuosus and Azadirachta indica

extracts

Twenty (20) larval of An. gambiae were introduced into Cymbopogon

flexuosus and Azadirachta indica extracts separately at concentrations 20 mg/L, 40

mg/L, 80 mg/L, 120 mg/L and 160 mg/L with a set of control containing tap water.

All tested concentrations were replicated four times. The number of dead larvae

were counted and recorded accordingly after 24 hours of exposure. Dead larvae

were those unable of rising to the surface or without the characteristic diving

reaction when the water was disturbed (WHO, 2006).

% Larval Mortality = Number of dead larva X100

Number of larvae introduce

3.4 Statistical analysis

Data were subjected to analysis of variance and means were separated using

the new Duncan’s multiple range test. The log-Probit model analysis was carried

out on larvicidal bioassay results to assess the 50% lethal concentration (LC50)

and 90% lethal concentration (LC90).

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