CHAPTER TWO

MATERIALS AND METHOD

Chemicals

A known insecticide (e.g Snipper,DDT), Ethyl alcohol. All other reagents were of analytical grade,
and solvents were redistilled before use.

Equipment

What are the equipment involve in this studies?

Plants collection and identification

Location and time (months and year) you collected your plant material. How did you identify
and authenticate the plant (Herbarium used and who authenticated the plant)?

Plant processing

How did you prepare the sample from collection point to utilization for the extract point

Extraction of essential oils

What type of extraction method did you used and what and how did you achieved. To include
formular for %yield calculation

Thin Layer Chromatography (TLC) Analysis

Write on TLC methods Commented [OT1]: Use these paper to write the
phytochemical and TLC methods:
https://seer.ufrgs.br/index.php/dar/article/view/125038/85
625

https://etflin.com/article/160
Essential oil combinations

Individual essential oils and their combinations tested against Anopheles mosquito

EOs and their combinations Plant Code Formulation

EOs C. citratus CCEO 10% C. citratus EO+90% ethyl alcohol

 C. citratus CCEO 20% C. citratus EO+80% ethyl alcohol

E. t ETEO 10% E. t EO+90% ethyl alcohol

E. t ETEO 20% E. t EO+90% ethyl alcohol

S. aromaticum SAEO 10% S.aroma EO+90% ethyl alcohol

S. aromaticum SAEO 20% C. citratus EO+90% ethyl alcohol

Combinations FM1 7.5% C. citratus EO + 7.5% E. t EO +

+ 7.5% S. aromaticum EO + 77.5%
ethyl alcohol

FM2 10% C. citratus EO + 10% E. t EO +

10% S. aromaticum EO + 70% ethyl

alcohol

EO-Essential oil, CCEO- C. citratus essential oil, ETEO- Eucalyptus torifolia essential oil, SAEO-
Syzygium aromaticum essential oil, FM1- Formula 1, FM2- Formula 2

Mosquito Rearing (This is done for you, I want you to rephrase all in your own style but the
content must remain same)

The eggs of Anopheles Mosquito were collected within the Okada communities. The mosquito
colony was kept under a standard laboratory conditions and photoperiod of 12-h light and 12-h
dark. The eggs of the mosquito were brought to hatch in a plastic tray containing 1000 ml of clean
water. The larvae were fed with fish food pellets for 12–15 days until they pupated. The pupae
were not fed with any food. One hundred new pupae were collected in a 300-ml plastic cup
containing 200 ml of clean water, transferred into an insect cage (the size of 30 × 30 × 30 cm3),
and left lying until developed into adults. Mosquito adults were provided with 5% glucose solution
as food, soaked in cotton sheets. Two-day-old female adults (not yet fed with blood meal) were
collected as subjects for a World Health Organization (WHO 2018) susceptibility test.

World Health Organization Susceptibility Test (This is done for you, I want you to rephrase
all in your own style but the content must remain same)
Knockdown and mortality tests against Anopheles mosquito were performed using the World
Health Organization (WHO 2018) Susceptibility Test. Twenty-five 2-day-old female mosquitoes
(not yet fed with blood meal) and ten 3-day-old female houseflies were exposed to 2 ml of each
formulation, which was dropped onto a filter paper (Whatman® No.1) the size of 12 × 15 cm2 for
1 h in a treatment tube (44 mm in diameter and 125 mm in length) then transferred to a non-
treatment tube. The knockdown rate was recorded at 1 h, and the mortality rate was recorded at 24
h after exposure. Each treatment was performed in five replicates. Ten percent (w/v) cypermethrin
and 70% v/v ethyl alcohol were used as positive control and negative control, respectively. The
criterion for knockdown and mortality was no movement of any of the mosquito body parts (Fig.
1). The distinction between knockdown and mortality was that knockdown was an occurrence
recorded at 60 min after exposure while mortality was an occurrence recorded at 24 h after
exposure. Knockdown (KR%) and mortality rates (MR%) were calculated by the following
formula:

NK
Knockdown Rate (KR%) = ----------------- X 100
NT

ND
Mortality Rate (MR%) = ----------------- X 100
NT
where NK is the total number of knocked-down adults; ND is the total number of dead adults; and
NT is the total number of treated adults.
The means of these rates were analyzed and compared by analysis of variance (ANOVA) and
Duncan’s Multiple Range Test (DMRT). Susceptibility levels were classified according to WHO
criteria: Susceptible (S) means 98–100% of mosquito mortality, Possible Resistant (PR) means
80–97% of mosquito/housefly mortality, and Resistant (R) means less than 80%of
mosquito/housefly mortality.

Adulticidal Bioassay (This is done for you, I want you to rephrase all in your own style but
the content must remain same)

Adulticidal activities were performed using the CDC bottle bioassays (CDC, 2010; Brogdon &
McAllister, 1998). In this bioassay, essential oils and combinations of the three plants were
screened at varying concentrations. Two hundred fifty ml Wheaton bottles with screw lids were
properly cleaned and dried, and then they were coated with 1ml solution of the desired essential
oil and the combinations of the tested plants by swirling assuring complete coating of the bottle
and its cap. Similarly, 1ml of ethyl alcohol was added to the control bottle handled as before. Adult
mosquitoes (20, aged 2–5 days) fed on 10% sucrose solution were released to each bottle, including
the control bottle with the help of mouth aspirator. The opening of each bottle was closed with
their lids after introduction of mosquito adults. The exposure time was set to 2 h. After that, the
mosquitoes were transferred into 250 ml capacity plastic cups using aspirator, where they were
provided with 10% sucrose solution and the mortalities were checked after 24 h. The experiment
was done a standard laboratory conditions, 12:12 light and dark photoperiod in the insectary.

Ref
WorldHealthOrganization (WHO) (2018) Test procedures for insecticide resistance monitoring in
malaria vector mosquitoes-2nd ed. IOP Publishing PhysicsWeb.
https://apps.who.int/iris/handle/10665/250677. Accessed 11 June 2019.

Brogdon WG, McAllister JC. Simplification of adult mosquito bioassays through use of time-
mortality determinations in glass bottles. J Am Mosq Control Assoc. 1998;14:159–64.
38. CDC.Centers for Disease Control and Prevention. Guideline for evaluating insecticide
resistance in vectors using the CDC bottle bioassay. 2010.