LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

INTRODUCTION:
Several mosquito species belonging to Aedes , Culex , Anopheles are vectors for the pathogens of
disease like communicable disease dengue, malaria, chikungunya etc. (Redwane et al., 2002). These
diseases are also liable for huge economic losses, both in terms of health-care costs and lost
productivity, mostly incountries that may least afford them. Malaria, with 300-500 million new cases
annually and approximately 2.5 million annual deaths, is one in every offoremost devastating diseases
affecting humans.

Presently organophosphate, organochlorine and artificial pyrethroid insecticides ends up in the

multiple insecticides resistance. Malaria vectors in india are resistance to DDT Deltamethrin ,
Malathion ( Ragavendra and Subbarao 2002).

The extensive uses of synthetic organic insecticides during the last five decades have resulted in
environmental hazards and also within the development of physiological resistance in major vector
species. This have necessitated the necessity for search and development of environmentally
safe,biodegradable and low cost indigenous methods for vector control.

Phytochemical obtain from plant sources can act as larvicides, insects growth regulators, repellents
and might play important role within the interruption of the transmission of mosquito-borne disease at
the individualin addition as at the community level. (choochote et al., 2004 mullai, 2007., Mathew et
al., 2009) Thus, one in every of the approaches for control of those mosquito- borne diseases is that
the interruption of disease transmission by killing or preventing mosquitoes from bitting people in
general.

Number of plant families are known to provide alkaloids, phenolics and oils which areused for insect
control since a protracted time. Theywere called as insect killers and were embloyed by Romans and
Chineses. Most of the issues of mosquito vector borne diseases occur in low-incometropical
communities but these communities havethe advantage of access to thousands of species ofplants
which can be contain useful phytochemicalsfor control of both agriculturally and medically important
insects. Ethiopia is endowed with uniquehabitats that harbor many endemic species ofplants. Of the
6500-7000 species of vascular plantsin Ethiopia, 12% are endemic.

Plants are been used since past times to repel/kill blood-sucking insects within the human history and
even now, in many parts of the globe people are practicing plants substances to repel/kill the
mosquitoes and other blood-sucking insects. The phytochemicals obtained from plant sources can act
as larvicides, insect growth regulators, repellents and ovipositional attractants. Mosquitoes within the
larval stage are attractive targets for pesticides because mosquitoes breed in water, and thus, it’s easy
to cater to them during this habitat. The employment of conventional pesticides within the water
sources, however, introduces many risks to people and/or the environment. Natural pesticides,
especially those derived from plants, are splendid during this aspect.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

The medicinal plants are Cymbopogon (C.) citrates, Cymbopogon nardus, Vitex negundo, Lavandula
had insects/mosquitoes repellent properties among the agricultural residents. The aim of this present
investigation was to guage the larvicidal activity against Aedes, anopheles, culex.

REVIEW OF LITERATURE:

PLANT:
Lavender is a genus of 47 known species of flowering plants in the mint family, Lamiaceae. It
is native to the Old World and is found from Cape Verde and the Canary Islands, Europe across to
northern and eastern Africa, the Mediterranean, southwest Asia to southeast India.

Many members of the genus are cultivated extensively in temperate climates as ornamental plants for
garden and landscape use, for use as culinary herbs, and also commercially for the extraction
of essential oils. The most widely cultivated species, Lavandula angustifolia, is often referred to as
lavender, and there is a colour named for the shade of the flowers of this species. Despite its use over
centuries in traditional medicine and cosmetics, there is no high-quality clinical evidence that lavender
has any effects on diseases or improves health.

Scientific Classification:

Kingdom: Plantae

Order: Lamiales

Family: Lamiaceae

Subfamily: Nepetoideae

Tribe: Ocimeae

Genus: Lavandula

DESCRPTION:
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

The genus includes annual or short-lived herbaceous perennial plants, and shrub-like

perennials, subshrubs or small shrubs.

Leaf shape is diverse across the genus. They are simple in some commonly cultivated species; in other
species they are pinnately toothed, or pinnate, sometimes multiple pinnate and dissected. In most
species the leaves are covered in fine hairs or indumentum, which normally contain the essential oils.

Flowers are borne in whorls, held on spikes rising above the foliage, the spikes being branched in
some species. Some species produce coloured bracts at the apices. The flowers may be blue, violet or
lilac in the wild species, occasionally blackish purple or yellowish. The calyx is tubular. The corolla is
also tubular, usually with five lobes (the upper lip often cleft, and the lower lip has two clefts)

PHYTOCHEMICALS:
Some 100 individual phytochemicals have been extracted from lavender oil, including major contents
of linalyl acetate (30-55%), linalool (20-35%), tannins (5-10%), and caryophyllene (8%), with lesser
amounts of sesquiterpenoids, perillyl alcohols, esters, oxides, ketones, cineole, camphor, beta-
ocimene, limonene, caproic acid, and caryophyllene oxide. The relative amounts of these compounds
vary considerably among lavender species.

MEDICINAL USES:
The stress relief benefits of lavender are safe, gentle and dependable. Lavender has been used by
herbalists for hundreds of years. It has a mild pleasant gentle aroma. It is easy to grow and is therefore
relatively inexpensive.Lavender flower is calming and relaxing both physically and emotionally and is
particularly useful for easing tension headaches, and other physical aches and pains.It is very useful
for easing the digestive upsets and gas which often accompany stress. Lavender has been used to ease
depression as it is said to be very helpful for people who are “stuck” in repetitive patterns of thinking
about a particularly stressful or traumatic event or circumstance in their lives.It is also often used for
encouraging deep restful sleep and stress relief. Lavender soothes the respiratory system, making
breathing for stress relief easier.The physical constituents of lavender thought to be the source of the
healing benefits include: flavonoids, coumarins, triterpenes and of course volatile oils for which
lavender is famous.

LAVENDER OIL:
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Commercially, the plant is grown mainly for the production of lavender essential oil of lavender.
English lavender (Lavandula angustifolia) yields an oil with sweet overtones, and can be used in
balms, salves, perfumes, cosmetics, and topical applications. Lavandula × intermedia, also known
as lavandin or Dutch lavender, yields a similar essential oil, but with higher levels
of terpenes including camphor, which add a sharper overtone to the fragrance.

The lavandins Lavandula  × intermedia are a class of hybrids of L. angustifolia and L. latifolia. The

lavandins are widely cultivated for commercial use, since their flowers tend to be bigger than those of
English lavender and the plants tend to be easier to harvest, but lavandin oil is regarded by some to be
of a lower quality than that of English lavender, with a perfume less sweet.

MOSQUITO:
Anopheles is a genus of mosquito first described and named by J. W. Meigen in 1818. About 460
species are recognised; while over 100 can transmit human malaria, only 30–40 commonly transmit
parasites of the genus Plasmodium, which cause malaria in humans in endemic areas. Anopheles
gambiae is one of the best known, because of its predominant role in the transmission of the most
dangerous malaria parasite species (to humans) – Plasmodium falciparum.

Mosquitoes in other genera (Aedes, Culex, Culiseta, Haemagogus, and Ochlerotatus) can also serve as

vectors of disease agents, but not human malaria.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Scientific classification

Kingdom: Animalia

Phylum: Arthropoda

Class: Insecta

Order: Diptera

Family: Culicidae

Subfamily: Anophelinae
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Genus: Anopheles

EVOLUTION:
The ancestors of Drosophila and the mosquitoes diverged 260 million years
ago.The culicine and Anopheles clades of mosquitoes diverged between 120 million years
ago and 150 million years ago. The Old and New World Anopheles species subsequently diverged
between 80 million years ago and 95 million years ago. Anopheles darlingi diverged from the African
and Asian malaria vectors ∼100 million years ago. The Anopheles gambiae and Anopheles
funestus clades diverged between 80 million years ago and 36 million years ago. A molecular study of
several genes in seven species has provided additional support for an expansion of this genus during
the Cretaceous period

The Anopheles genome, at 230–284 million base pairs (Mbp), is comparable in size to that

of Drosophila, but considerably smaller than those found in other culicine genomes (528 Mbp–1.9
Gbp). Like most culicine species, the genome is diploid with six chromosomes.

The only known fossils of this genus are those of Anopheles (Nyssorhynchus) dominicanus Zavortink
& Poinar contained in Dominican amber from the Late Eocene (40.4 million years ago to 33.9 million
years ago) and Anopheles rottensis Statzcontained in German amber from the
Late Oligocene (28.4 million years ago to 23 million years ago).

SYSTEMATICS:
The genus Anopheles Meigen (nearly worldwide distribution) belongs to the subfamily Anophelinae
together with another two genera: Bironella Theobald (Australia only) and Chagasia Cruz
(Neotropics). The taxonomy remains incompletely settled. Classification into species is based on
morphological characteristics – wing spots, head anatomy, larval and pupal anatomy, chromosome
structure, and more recently, on DNA sequences. In the taxonomy published by Harbach et al in 2016,
it was shown that three species of Bironella: confusa, gracilis, and hollandi are phylogenetically
similar Anopheles kyondawensis than other Bironella species. The same phylogeny also argues that,
based on genetic similarity, Anopheles implexusis actually divergent from the common ancestor to
the Anopheles genus, raising new questions regarding taxonomy and classification.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

The genus has been subdivided into seven subgenera based primarily on the number and positions of
specialized setae on the gonocoxites of the male genitalia. The system of subgenera originated with the
work of Christophers, who in 1915 described three subgenera: Anopheles (widely
distributed), Myzomyia (later renamed Cellia) (Old World)
and Nyssorhynchus (Neotropical). Nyssorhynchus was first described as Lavernia by Frederick
Vincent Theobald. Frederick Wallace Edwards in 1932 added the subgenus Stethomyia (Neotropical
distribution). Kerteszia was also described by Edwards in 1932, but then recognised as a subgrouping
of Nyssorhynchus. It was elevated to subgenus status by Komp in 1937, and it is also found in the
Neotropics. Two additional subgenera have since been recognised: Baimaia (Southeast Asia only) by
Harbach et al. in 2005 and Lophopodomyia (Neotropical) by Antunes in 1937.

Two main groupings within the genus Anopheles are used: one formed by

the Celia and Anopheles subgenera and a second by Kerteszia, Lophopodomyia and Nyssorhynchus.
Subgenus Stethomyia is an outlier with respect to these two taxa. Within the second
group, Kerteszia and Nyssorhynchus appear to be sister taxa.

The number of species currently recognised within the subgenera is given here in
parentheses: Anopheles (206species), Baimaia (1), Cellia (216), Kerteszia (12), Lophopodomyia (6), 
Nyssorhynchus (34) and Stethomyia (5).

Taxonomic units between subgenus and species are not currently recognised as official zoological
names. In practice, a number of taxonomic levels have been introduced. The larger subgenera
(Anopheles, Cellia and Nyssorhynchus) have been subdivided into sections and series which in turn
have been divided into groups and subgroups. Below subgroup but above species level is the species
complex. Taxonomic levels above species complex can be distinguished on morphological grounds.
Species within a species complex are either morphologically identical or extremely similar and can
only be reliably separated by microscopic examination of the chromosomes or DNA sequencing. The
classification continues to be revised.

Subgenus Nyssorhynchus has been divided in three sections: Albimanus (19 species), Argyritarsis (11

species) and Myzorhynchella (4 species). The Argyritarsissection has been subdivided
into Albitarsis and Argyritarsis groups.

The Anopheles group was divided by Edwards into four

series: Anopheles (worldwide), Myzorhynchus (Palearctic, Oriental, Australasian and
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Afrotropical), Cycloleppteron (Neotropical) and Lophoscelomyia (Oriental); and two

groups, Arribalzagia (Neotropical) and Christya (Afrotropical). Reid and Knight (1961) modified this
classification and consequently subdivided the subgenus Anopheles into two
sections, Angusticorn and Laticorn and six series. The Arribalzagia and Christya Groups were
considered to be series. The Laticorn Section includes the Arribalzagia (24
species), Christya and Myzorhynchus series. The Angusticorn section includes members of
the Anopheles, Cycloleppteron and Lophoscelomyia series.

All species known to carry human malaria lie within either the Myzorhynchus or the Anopheles series.

LIFE STAGES:
Like all mosquitoes, anophelines go through four stages in their life cycles: egg, larva, pupa,
and imago. The first three stages are aquatic and together last 5–14 days, depending on the species and
the ambient temperature. The adult stage is when the female Anopheles mosquito acts
as malaria vector. The adult females can live up to a month (or more in captivity), but most probably
do not live more than two weeks in nature.[9]

Eggs

Anopheles egg

Adult females lay 50–200 eggs per oviposition. The eggs are quite small (about 0.5 × 0.2 mm). Eggs
are laid singly and directly on water. They are unique in that they have floats on either side. Eggs are
not resistant to drying and hatch within 2–3 days, although hatching may take up to 2–3 weeks in
colder climates.[9]
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Larvae

Anopheles larva, about 8 mm long

Feeding position of an Anopheleslarva (A), compared to that of a nonanopheline mosquito (B)

The mosquito larva has a well-developed head with mouth brushes used for feeding, a large thorax and
a nine-segment abdomen. It has no legs. In contrast to other mosquitoes, the Anopheles larva lacks a
respiratory siphon, so it positions itself so that its body is parallel to the surface of the water.[9] In
contrast, the feeding larva of a nonanopheline mosquito species attaches itself to the water surface
with its posterior siphon, with its body pointing downwards.

Larvae breathe through spiracles located on the eighth abdominal segment, so must come to the
surface frequently. The larvae spend most of their time feeding on algae, bacteria, and other
microorganisms in the surface microlayer. They dive below the surface only when disturbed. Larvae
swim either by jerky movements of the entire body or through propulsion with the mouth brushes.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Larvae develop through four stages, or instars, after which they metamorphose into pupae. At the end
of each instar, the larvae molt, shedding their exoskeletons, or skin, to allow for further growth. First-
stage larvae are about 1 mm in length; fourth-stage larvae are normally 5–8 mm in length.

The process from egg-laying to emergence of the adult is temperature dependent, with a minimum
time of seven days.

The larvae occur in a wide range of habitats, but most species prefer clean, unpolluted water. Larvae
of Anopheles mosquitoes have been found in freshwater or saltwater marshes, mangrove swamps, rice
fields, grassy ditches, the edges of streams and rivers, and small, temporary rain pools. Many species
prefer habitats with vegetation. Others prefer habitats with none. Some breed in open, sun-lit pools,
while others are found only in shaded breeding sites in forests. A few species breed in tree holes or the
leaf axils of some plants.[9]

Pupae
Pupa is also known as tumbler.The pupa is comma-shaped when viewed from the side. The head
and thorax are merged into a cephalothorax with the abdomen curving around underneath. As with the
larvae, pupae must come to the surface frequently to breathe, which they do through a pair of
respiratory trumpets on their cephalothoraces. After a few days as a pupa, the dorsal surface of the
cephalothorax splits and the adult mosquito emerges.[9] The pupal stage lasts around 2–3 days in
temperate areas.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Adults

Resting positions of adult Anopheles (A, B), compared to a nonanopheline mosquito (C)

The duration from egg to adult varies considerably among species, and is strongly influenced by
ambient temperature. Mosquitoes can develop from egg to adult in as little as five days, but it can take
10–14 days in tropical conditions.

Like all mosquitoes, adult Anopheles species have slender bodies with three sections: head, thorax and
abdomen

The head is specialized for acquiring sensory information and for feeding. It contains the eyes and a
pair of long, many-segmented antennae. The antennae are important for detecting host odors, as well
as odors of breeding sites where females lay eggs. The head also has an elongated, forward-
projecting proboscis used for feeding, and two maxillary palps. These palps also carry the receptors
for carbon dioxide, a major attractant for the location of the mosquito's host.

The thorax is specialized for locomotion. Three pairs of legs and a pair of wings are attached to the
thorax
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

The abdomen is specialized for food digestion and egg development. This segmented body part
expands considerably when a female takes a blood meal. The blood is digested over time, serving as a
source of protein for the production of eggs, which gradually fill the abdomen.

Anopheles mosquitoes can be distinguished from other mosquitoes by the palps, which are as long as
the proboscis, and by the presence of discrete blocks of black and white scales on the wings. Adults
can also be identified by their typical resting position: males and females rest with their abdomens
sticking up in the air rather than parallel to the surface on which they are resting.

Adult mosquitoes usually mate within a few days after emerging from the pupal stage. In most species,
the males form large swarms, usually around dusk, and the females fly into the swarms to mate.

Males live for about a week, feeding on nectar and other sources of sugar. Males cannot feed on blood,
as it appears to produce toxic effects and kills them within a few days, around the same lifespan as a
water-only diet. Females will also feed on sugar sources for energy, but usually require a blood meal
for the development of eggs. After obtaining a full blood meal, the female will rest for a few days
while the blood is digested and eggs are developed. This process depends on the temperature, but
usually takes 2–3 days in tropical conditions. Once the eggs are fully developed, the female lays them
and resumes host-seeking.

The cycle repeats itself until the female dies. While females can live longer than a month in captivity,
most do not live longer than one to two weeks in nature. Their lifespans depend on temperature,
humidity, and their ability to successfully obtain a blood meal while avoiding host defenses.

In a study by the London School of Hygiene & Tropical Medicine researchers found that female
mosquitoes carrying malaria parasites are significantly more attracted to human breath and odours than
uninfected mosquitoes. The research team infected laboratory-raised Anopheles gambiae mosquitoes
with Plasmodium parasites, leaving a control group uninfected. Then tests were run on the two groups
to record their attraction to human smells. Female mosquitoes are particularly drawn to foot odours,
and one of the tests showed infected mosquitoes landing and biting a prospective host repeatedly. The
team speculates that the parasite improves the mosquitoes' sense of smell. It may also reduce its risk
aversion.

INSECTISIDE RESISTANCE:
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Insecticide-based control measures (e.g. indoor spraying with insecticides, bed nets) are the principal
ways to kill mosquitoes that bite indoors. However, after prolonged exposure to an insecticide over
several generations, mosquito populations, like those of other insects, may evolve resistance, a
capacity to survive contact with an insecticide. Since mosquitoes can have many generations per year,
high levels of resistance can evolve very quickly. Resistance of mosquitoes to some insecticides has
been documented with just within a few years after the insecticides were introduced. Over 125
mosquito species have documented resistance to one or more insecticides. The evolution of resistance
to insecticides used for indoor residual spraying was a major impediment during the Global Malaria
Eradication Campaign. Judicious use of insecticides for mosquito control can limit the evolution and
spread of resistance. However, use of insecticides in agriculture has often been implicated as
contributing to resistance in mosquito populations. Detection of evolving resistance in mosquito
populations is possible, so control programs are well advised to conduct surveillance for this potential
problem.In Malawi and other places, a shrub known as mpungabwi (Ocimum americanum) is used to
repel mosquitoes.

METHODOLOGY
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

PLANT COLLECTION
Maturated fresh leaves of Lavandula angustifolia were collected from Coimbatore District, Tamil
Nadu.

MOSQUITO COLLECTION
The larvae and adults of the mosquito Culex quinquefasciatus were collected from National Centre
For Disease Control, Mettupalayam.

PREPARATION OF PLANT EXTRACT

The plant material was washed and dried at room temperature.10g of fresh leaves were weighed and
taken. The leaves are dissolved in 100ml of distilled water and 100ml of methanol. The extract were
kept at refriegator for 72 hours. After that extract was filtered using Whatt mann filter paper. The
filterate were collected and shade dried at room temperature. The filterate was dissolved in petroleum
ether solvent.

PREPARATION OF OIL EXTRACT

Lavandula angustifolia oil was brought commercially from shops. From that 1ml of the oil was taken
and dissolved in 9ml of the alcoholic water.

LARVICIDAL BIOASSAY
In the larvicidal bioassay early fourth instar larvae of Culex quinquefasciatus were exposed to
different concentration of methanolic, aqueous and oil extracts Lavandula angustifolia leaves .
Standard WHO protocol( Annexure 1) with slight modification was adapted for the study. The study
was conducted in glass beakers.larvicidal activity at test concentration of 25, 50, 75 and 100 ppm were
assessed. Twenty five healthy larvae were released into each 250ml glass beakers containing the test
concentration. Larval mortality was observed 24 hours post treatment. A total of five trials with three
replicates per trial for each concentration were carried out.distilled water as control was run
simultaneously. The larval per cent mortality was calculated and when control mortality ranges from
5-20% it was corrected by using Abbott’s formula. The determination of LC50 and LC 90 values
were calculated using log probit paper.

REPELLENT BIOASSAY
In this bioassay adult Culex quinquenfasciatus mosquito were exposed to different concentration of
metanolic ,aqueous and oil extracts of Vitex negundo leaves. Standard ICMR protocol (Annexure2)
with slight modifications was followed. This study will be carried out in the laboratory maintained at
27 degree Celsius. The study was carried out in mosquito cages (2 cubic feet) containing plastic
bowls. DEET (N,N-dimethyl -3-methylbenzamide) is used as positive control,while distilled water is
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

used as negative control. Study will be carried out against each individual species in replicates of
three.the test concentrations 25, 50, 75, and 100 ppm were assessed. About one hundred 3to 5 days
old mosquitoes are released into a each cage containing different concentrations of extract .Five
landing counts will be made at 0, 1, 2, 4 and 6 hours. Percentage repellency will be calculated.

DATA ANALYSIS
Data from all the replicates should be pooled for analysis.LC50 and LC90 values are calculated from a
log dosage probit mortality regression line using log probit paper

ANNEXURE 1
Larvicidal assay

Initially the mosquito larvae are exposed to a wide range of test concentrations and a control to find
out the activity range of the materials under test. After determining the mortality of larvae in this
wide range of concentrations a narrower range (of 4-5 concentrations, yielding between 10% and
95% mortality in 24 h or 48h) is used to determine LC50 and LC90 values.

Batches of 25 third or fourth instar larvae are transferred by means of strainers, screen loops or
droppers to small disposable test cups or vessels, each containing 100-200ml of water. Small,
unhealthy or damaged larvae should be removed and replaced. The depth of the water in the cups or
vessels should remain between 5cm and 10cm; deeper levels may cause undue mortality.

The appropriate volume of dilution is added to 100ml or 200ml water in the cups to obtain the desired
target dosage, starting with the lowest concentration. Four or more replicates are set up simultaneously
with tap water, to which 1ml alcohol is added. Each test should be run three times on different days.
For long exposures larval food should be added to each test cup, particularly if high mortality is noted
in control. The test containers are held at 25-28 degree Celsius and preferably a photoperiod of 12h
light followed by 12h dark.

After 24 h exposure larval mortality is recorded. For slow acting insectides, 48h reading may be
required. Moribund larvae are counted and added to dead larvae for calculating percentage mortality.
Dead larvae are those that cannot be induced to move when they are probed with a needle in the
siphon or the cervical region. Moribund larvae are those incapable of rising to the surface or not
showing the characteristic diving reaction when the water is disturbed. The results are recorded on the
form provided, where the LC50 and LC90 values and slope and heterogeneity analysis are also
noted. The form will accommodate three separate tests for six concentrations, each of four replicates.

Larvae that have pupated during the test period will negate the test. The more than 10% of the control
larvae pupate in the course of the experiment; test should be discarded and repeated. If the control
mortality is between 5% and 20% , the mortalities of treated groups should be corrected according to
Abbott’s formula

Mortality % = X-Y÷X×100
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Where X= percentage survival in the untreated control and Y= percentage survival in the treated
sample. ( WHO protocol)

ANNEXURE 2
These studies will be carried out in the laboratory maintained at 27±2oC and 80% ± 10% relative
humidity using laboratory-reared strains of vectors in cloth cages (2 cubic feet). Studies will be
carried out against each individual species in replicates of three. In each of the replicate cage, one
hundred 3 to 5-days old sugar-fed female mosquitoes will be held. Mosquitoes that are pre-starved for
12 h or more prior to testing will be used for the experiment. These conditions and format of
experiment given below need standardisation for different species. Use of positive control is essential
in these tests. DEET (N,N-diethyl-3- methylbenzamide), the active ingredient of most commercially
available repellents can be used as a positive control against which the effectiveness of the candidate
repellent is tested. In the cage, five plastic bowls with sugar-soaked cotton (10% in water) should be
placed at four opposing corners and one in the middle. These bowls should be treated separately with
three different specified concentrations of the formulation, one specified concentration of DEET
(known synthetic repellent compound as positive control) and with only sugar-soaked cotton
(negative control). The four treated bowls will be placed in four opposing corners while the untreated
negative control in the middle on the floor of the cage. Five minute landing counts will be made at 0,
1, 2, 4 and 6 h. The cups will be removed between the exposure intervals. Mean percent repellency for
each percent formulation and species will be calculated based on the data of the three replicates at the
given times of observation. Percent repellency will be calculated using the formula given below.

(a) No. landing on negative control – No. landing on treated with repellent

------------------------------------------------------------------------------------------- X 100

No. landing on negative control

(b) No. landing on negative control – No. landing on treated with DEET

------------------------------------------------------------------------------------------- X 100

No. landing on negative control

Where, (a) provides % repellency of the candidate repellent and (b) % repellency

of DEET. Efficacy of the candidate repellent can be assessed relative to DEET ( ICMR protocol).

RESULT AND DISCUSSION

The larvicidal activity of Lavandula angustifolia leaf crude extract at different concentrations is
represented in table 1.The data shows that 100% mortality rate of mosquito larvae was observed
at 25, 50, 75,100 and 200 ppm of concentrations.And at the concentration of 200 ppm 100%
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

mortality rate was recorded within 12 hours of the treatment for 50 ppm after 36 hours and for 25
ppm it was recorded after 48 hours of the treatment.the efficacy of crude extract on the mosquito
larvae showed lesser activity when the concentrations of the same was decreased to 10 and 5 ppm
which showed 75 and 45% mortality rate respectively within 48 hours of the treatment. The
control did not show any mortality the mean and standard error also showed in Table 1.( DR
Nayak J.B and Rajani B
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Concentration Time n=20

(hours)

12 24 36 48 Total % Mean SE(+/-)

5 3 5 7 9 9 45 2.25 1.291

10 7 10 12 15 15 75 3.75 1.683

25 10 14 18 20 20 100 5.00 2.217

50 14 17 20 - 20 100 6.66 1.436

100 18 20 - - 20 100 10.00 0.500

200 20 - - - 20 100 20.00 0.000

Control 0 0 0 0 0 0 0.000 0.000

TABLE 1

Krishnan et.al (2007) reported 50% mortality rate for the metanolic extract of Lavandula
angustifolia leaves at the concentration of 41 ppm. As earlier results are in support of the present
study of Lavandula angustifolia leaf extract may be considered as the potential control against
mosquito larvae which is eco friendly in nature. I also except this result for my study.

ANNEXURE 1

The mosquito larvae are exposed to a wide range of test concentrations and a control to find out
the activity range of the materials under test. After determining the mortality of larvae in this wide
range of concentrations, a narrower range (of 4–5 concentrations, yielding between 10% and 95%
mortality in 24 h or 48 h) is used to determine LC50 and LC90 values.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

Batches of 25 third or fourth instar larvae are transferred by means of strainers, screen loops or
droppers to small disposable test cups or vessels, each containing 100-200ml of water. Small,
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST ANOPHELES

unhealthy or damaged larvae should be removed and replaced. The depth of the water in the cups
or vessels should remain between 5cm and 10cm; deeper levels may cause undue mortality.

The appropriate volume of dilution is added to 100ml or 200ml water in the cups to obtain the
desired target dosage, starting with the lowest concentration. Four or more replicates are set up
simultaneously with tap water, to which 1ml alcohol is added. Each test should be run three times
on different days. For long exposures larval food should be added to each test cup, particularly if
high mortality is noted in control. The test containers are held at 25-28 degree Celsius and
preferably a photoperiod of 12h light followed by 12h dark.

After 24 h exposure larval mortality is recorded. For slow acting insectides, 48h reading may be
required. Moribund larvae are counted and added to dead larvae for calculating percentage
mortality. Dead larvae are those that cannot be induced to move when they are probed with a
needle in the siphon or the cervical region. Moribund larvae are those incapable of rising to the
surface or not showing the characteristic diving reaction when the water is disturbed. The results
are recorded on the form provided, where the LC50 and LC90 values and slope and heterogeneity
analysis are also noted. The form will accommodate three separate tests for six concentrations,
each of four replicates.

Larvae that have pupated during the test period will negate the test. The more than 10% of the
control larvae pupate in the course of the experiment; test should be discarded and repeated. If
the control mortality is between 5% and 20% , the mortalities of treated groups should be
corrected according to Abbott’s formula

Mortality % = X-Y÷X×100

Where X= percentage survival in the untreated control and Y= percentage survival in the treated
sample. ( WHO protocol)

ANNEXURE 2
These studies will be carried out in the laboratory maintained at 27±2oC and 80% ± 10%
relative humidity using laboratory-reared strains of vectors in cloth cages (2 cubic feet).
Studies will be carried out against each individual species in replicates of three. In each of the
replicate cage, one hundred 3 to 5-days old sugar-fed female mosquitoes will be held.
 LARVICIDIAL ACTIVITY OF LAVANDULA ANGUSTIFOLIA AGAINST
ANOPHELES

Mosquitoes that are pre-starved for 12 h or more prior to testing will be used for the
experiment. These conditions and format of experiment given below need
standardisation for different species. Use of positive control is essential in these tests.
DEET (N,N-diethyl-3- methylbenzamide), the active ingredient of most
commercially available repellents can be used as a positive control against which the
effectiveness of the candidate repellent is tested.
In the cage, five plastic bowls with sugar-soaked cotton (10% in water) should be
placed at four opposing corners and one in the middle. These bowls should be treated
separately with three different specified concentrations of the formulation, one
specified concentration of DEET (known synthetic repellent compound as positive
control) and with only sugar-soaked cotton (negative control). The four treated bowls
will be placed in four opposing corners while the untreated negative control in the
middle on the floor of the cage. Five minute landing counts will be made at 0, 1, 2, 4
and 6 h. The cups will be removed between the exposure intervals. Mean percent
repellency for each percent formulation and species will be calculated based on the
data of the three replicates at the given times of observation. Percent repellency will
be calculated using the formula given below.
(a) No. landing on negative control – No. landing on treated with repellent
X 100
No. landing on negative control
(b) No. landing on negative control – No. landing on treated with DEET
X 100
No. landing on negative control
Where, (a) provides % repellency of the candidate repellent and (b) % repellency
of DEET. Efficacy of the candidate repellent can be assessed relative to DEET ( ICMR protocol

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