Research Proposal

IDENTIFICATION OF MOSQUITOCIDAL COMPOUNDS FROM n-

HEXANE EXTRACT OF CALENDULA ARVENSIS AGAINST CULEX
QUINQUEFASCIATUS

Author: Ijaz Ullah

Doctor of Philosophy
DEPARTMENT OF ZOOLOGY/ANIMAL
SCIENCES
Chinese Scholarship Council
 INTRODUCTON

The big issue to the man’s health, especially in regions of poor resources are insect-borne
diseases (Karunamoorthi and Ilango., 2010). Nearly world’s half population is at risk and insect-
borne diseases are about over 17% of total diseases that lead to over 1 million deaths per anum
(WHO 2014). It negatively affects human lives in terms of poverty, socio- economic
development and social debility (Jang et al., 2002). According to a report of WHO (2014),
mostly poor people of the society are affected because of less knowledge about insect-borne
diseases and unhygienic condition.

Mosquito specie is thought to be as deadly harmful insect on the planet (Earth) because of their
potency to carry thousands of devastating diseases worldwide (Karunamoorthi, 2012).
Anthropophagic behavior (prefer to feed on human) are highly showed by varieties of mosquito
while in order to obtain the blood meals. Prevalence of Culex quinquefasciatus is widespread and
occur across urban and semi-urban areas; the Anopheles mostly in rural areas, and Aedes mainly
in and around urban areas (WHO, 2015).

Mosquitoes transmit parasites and pathogens of various diseases. It is estimated that every year
at least 500 million people in the world suffer from one or the other tropical diseases that include
malaria, lymphatic filariasis, schistosomiasis, dengue, leishmaniosis, strypanosomiasis, yellow
fever, Japanese encephalitis and other arbo-viral diseases. One to two million deaths due to
malaria is reported annually worldwide. At least 120 million people in 73 countries in Africa,
India, Southeast Asia, and Pacific Islands affected by lymphatic filariasis. These diseases not
only result in high levels of morbidity and mortality, but also cause great economic loss and
social disruption on developing countries such as Pakistan, India, China, etc. About 40% of
global filariasis burden is contributed by India alone and it count about 720 crores economic loss
annualy (Hotez et al., 2004).

The genus Culex consists of many species, which mostly act as a vector for lymphatic filariasis,
WN virus etc. Culex quinquefasciatus (Cx. quinquefasciatus) is a vector of filariasis. In many of
the tropical countries, human filariasis is a major public health hazard and remains a challenging
socioeconomic issue (Udonsi, 1986). Wuchereria bancrofti (W. bancrofti) is a parasite
transmitted by mosquito Cx. quinquefasciatus causing lymphatic filariasis is found to be more
endemic in the Indian subcontinent. According to Rajasekariah (1991) reported Culex
quinquefasciatus infects approximately more than hundred millions of persons per year globally.
In tropical world, lymphatic filariasis remains endemic, and its control is a major goal to
improved public health worldwide.

Mosquito Control
For the prevention, control and eradication of insect-borne diseases such substances, application
and means should be used that will have no harmfully affects the ecosystem. So, one have to use
methods of eco-friendly (which include proper sanitation and water management), plants base
chemicals and biological control measures (like utilization of biological toxins and natural
enemies) may prove fruitful in controlling insect-borne diseases. Consciousness & Wakefulness
of people is needed related to worse effect of chemical insecticides and efficacy and eco-friendly
nature of plant based insecticides. E.g. when following the recommended application rates and
best mosquito control practices, using of Bacillus thuringiensis subsp. israelensis (Bti) against
mosquito, chironomids (non-target organism) which is among the organism of high sensitivity
was not effected by phytochemicals (Lagadic et al., 2016).

Over decades, vector control was thought to be best strategy to overcome the insect borne
disease associated illness and deaths worldwide (Karunamoorthi., 2011). Various alkaloids,
phenolic, oils and several secondary metabolites are synthesized by plants, which possess strong
insecticidal potential and were used by Chinese and Romans in the ancient days (Heintz and
Downum, 1987).

The previous research in M.Phil was investigated to explore the larvicidal, pupicidal, repellent
and mosquito adulticidal potential of n-hexane extracts of Calendula arvensis and Elsholtzia
fruticosa against lymphatic filariasis vector Culex quinquefasciatus in a search for effective and
affordable natural bio products to be used in the control of vectors, in which Calendula arvensis
showed best result.

In the Doctoral study I want to extend the previous research to identify those phytochemicals of
Calendula arvensis which are responsible for mosquito larvicidal, pupicidal and adulticidal
activity of both plants’ extracts. Also to observe the ovicidal, growth regulatory and adult
emergence inhibition activity of mentioned plants’ extracts against other harmful mosquito
species.

OBJECTIVES
Keeping in view the importance of botanical insecticides, the present research will be conducted
with the following objerctives:

• To screen the larvicidal, pupicidal and adulticidal activities of native medicinal plants i.e.
Calendula arvesis.

• To identify the phytochemicals of Calendula arvensis those are responsible for insecticidal
activity.
 MATERIALS AND METHODS

Plant material

The aerial part of the Calendula arvensis will be collected from the selected area of research.
The plant will be identified by texanomist in the subject of Botany.

Extraction

The whole plant of Calendula Arvensis and will be cleaned and shade dried. The plant will be
finely ground and the powder form will be soaked in n-hexane for three days. The soaked plant
materials will be filtered through Whatman filter paper no.42. The filtrate will be evaporated on
a rotary evaporator under reduced pressure at 45º C. The n-hexane extract will be stored in
refrigerator for use.

Larvicidal bioassay

For conduction of larvicidal activity, larvae will be collected from the same site in a large plastic
jar and brought to the laboratory. The immature stages will be reared in laboratory by using
mosquito cages till adulthood. The adult will be blood fed after initial sucrose feeding. The
mosquito will breed and lay eggs in water containing jar provided in the cage. The eggs will
hatch into larvae and I will be able to pick up and use 3rd and 4th instar larvae for larvicidal
bioassay. The larvicidal activity will be assessed by the WHO (1996) guidelines for larvicidal
activity.

Adulticidal bioassay

After the conduction of larvicidal activity, samples of 3rd and 4th instars larvae of mosquito will
be collected with the help of a plastic dipper and dropper from stagnant sewage water. The
immature stages will be reared in a plastic jars in laboratory in mosquito cage. The larvae will
be reared to adulthood in the laboratory. The adulticidal activity will be conducted by using
WHO standard procedure (WHO, 1981).
Column Chromatoghraphy

Column chromatography of the sample solution will be done in order to isolate the
fractions and identify the phytochemicals.

Analysis of Data

The larvicidal activity of extracts will be presented as percent mortality of larvae by using the
following formula; percent mortality = Number of dead larvae ⁄ Total exposed larvae X 100.
The results will be presented as mean ± standard deviation of three replicates. The dose-
response data will be subjected to linear Regression analysis to point out the relationship
between the increase in extract concentration and larval mortality. For the calculation of LC50
values the data will be subjected to Probit Regression Analysis. For all these analysis computer
software SPSS 16.0 will be used.
 REFFERENCES

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