Effects of Lemon grass (Cymbopogon citratus), San Francisco

(Codiaeum variegatum) and Tawa- tawa (Euphorbia hirta) on the

Growth and Development of the Mosquito, Aedes aegypti,
a Vector of Dengue Virus
Jerouen Paul D. Lumabao
Daniel R. Aguinaldo National High School, Matina, Davao City, Philippines
(Presented in the Intel International Science and Engineering Fair (ISEF) 2016 at
Phoenix, Arizona, USA on May 7-13, 2016)
Chito R. Napitan, Ed.D.-Research Mentor

ABSTRACT

The mosquito, Aedes aegypti, the principal vector of dengue virus has caused an
alarming health issue worldwide. Commercial mosquito insecticides are readily available, but
the vectors develop resistance to these and may cause harm and pose negative effects on
living organisms and to the environment. This study evaluated the crude extracts of Lemon
grass (Cymbopogon citratus), San Francisco (Codiaeum variegatum) and Tawa-tawa
(Euphorbia hirta) leaves on the hatchability of mosquito eggs, and the mortality, abdominal
morphology and behavior of 4th instar A. aegypti larvae. Mosquito eggs and larvae were
exposed to 500 µL of each crude extract and then observed after 24 hours. Mean level of
hatchability and mean level of mortality were recorded and the larvae were observed under a
compound light microscope. Results showed significant differences on the hatchability and
mortality manifested in the treatment using San Francisco crude extract. This means no eggs
hatched out of the ten eggs introduced in the San Francisco crude extract, manifesting its
ovicidal potential. Also, all ten larvae introduced in San Francisco crude extract did not survive
indicating its larvicidal potential. Qualitative analyses showed that the extracts from Tawa-tawa
and Lemon grass caused abnormalities on the abdominal morphology and behavior of the 4th
instar A. aegypti larvae when compared with the control. The results showed a great potential
for the production of organic insecticides from the plant extracts to control A. aegypti, vector of
the dengue virus.

INTRODUCTION

The prevalence of dengue has become one of the main health concerns in
tropical and sub-tropical countries worldwide, wherein over 2.5 billion people with 40%
of the world’s population is severely affected, composed of 50-100 million infections
every year and about 2.5% of affected people die (World Health Organization, 2014).

In the Philippines, 19,946 alleged dengue cases were recorded by the

Department of Health (DOH) in the first three months of 2015, greater than by 6.49%
recorded in the same period of 2014 (Geronimo, 2015). On the other hand, Davao City

1
has the highest number of cases recorded in Region XI, a total of 1,633 cases in the
first semester of 2015 was reported (Colina IV, 2015).

Aedes aegypti, which is the principal vector of the dengue virus, increases the
threat of disease to humans. When a gravid female A. aegypti bites a human for food,
she injects saliva with the dengue virus into the host. Since the virus can be spread
from adult to egg, it is certain that the virus will survive until the next summer and when
heavy rains come (Dengue Virus Net, 2015).

In early times, the use of synthetic chemical larvicides and insecticides are said
to be effective and has an immediate action towards mosquito control. But the
excessive, continual and unsystematic use of synthetic chemicals has led to more
alarming problems like resistance to insecticides, toxicity to non-target organisms, lethal
impacts on the individuals and residual effects on the environment and its
destabilization (Jirakanjanakit, Rongnoparut, Saengtharatip, Chareonviriyaphap,
Duchon, Bellec, & Yoksan, 2007; Sarwar, Ahmad & Toufiq, 2009).

With this, researches are now focused towards the use of plants and organic
substances as potential mosquito control agents to prevent the expected toxicity and
resistance problems which are by product of using chemical insecticides (Warikoo,
Wahab & Kumar, 2011).

Lemon grass (Cymbopogon citratus), San Francisco (Codiaeum variegatum) and

Tawa-tawa (Euphorbia hirta) leaves were used in this study. Reports showed that
several groups of phytochemicals such as alkaloids and steroids have insecticidal
activities (Shaalan, Canyonb, Younesc, Abdel-Wahaba, & Mansoura, 2005) and
saponins and tanins have larvicidal effect (El Hag, Nadi, & Zaitoon, 1999) which are
present in these plants. The study aims to explore and investigate the effects of different
plant leaves crude extracts on the hatchability of mosquito eggs, mortality, behavior and
morphology of 4th instar A. aegypti larvae aside from the recent phytochemicals and
larvicidal studies conducted.

This study can contribute towards unravelling new knowledge and researches in
understanding the life, behavior and nutrition of mosquito that will eventually lead to
dengue control through different plant substances that are readily available in the
environment.

MATERIALS AND METHODS

 
 
 This study is composed of three phases: Phase I - Plant Extract Preparation,
Phase II - Mosquito Experimentation, and Phase III – Data Collection and Analysis. All
experimental procedures were done in the Biology Laboratory of Daniel R. Aguinaldo
National High School (DRANHS) except the phytochemical screening which was done
at the University of Immaculate Conception, Science Resource Center, Davao City. The
unit of analysis in this study are the eggs of mosquito and 4th instar larvae of A. aegypti.
There were ten eggs introduced in the petri plates per treatment.

Phase I - Plant Extract Preparation

Collection of Plant Material

The leaves of Lemon grass (Cymbopogon citratus), San Francisco (Codiaeum

variegatum) and Tawa-tawa (Euphorbia hirta) were collected from the neighborhood of
GSIS Heights, Matina, Davao City. The authentication of the plants was done by Arceli
G. Yebes, Agriculturist, Department of Agriculture, Bureau of Plant Industry XI.

Crude Extract Preparation

The freshly cut leaves were cleansed thoroughly with tap water, cut into smaller
pieces and air dried for two days. The totally desiccated leaves were soaked each in 1 L
of 95% ethanol for 48 hours. The ethanol extraction followed the method of Vital and
Rivera (2009). The ethanol extracts were then underwent to modified extraction using
oil bath technique in order to get the crude extracts.

Phytochemical Screening

Phytochemical screening was performed at the University of the Immaculate

Conception, Science Resource Center, Davao City by Russell Caesar P. Enoc, Junior
Analyst I and certified by Ludivina M. Porticos, MBA, MS, PRC License No. 6237,
Director.

Phase II - Mosquito Experimentation

Collection of Mosquito Samples

Collection of mosquito samples were done using the Ovicidal- Larvicidal (OL)
trapping method by the University of the Philippines and DOST. Nine clean black cans
were used and lawanit paddles were placed in each can. The can was filled with ¾ tap
water and the ovitraps were then installed in the dark corners of nine different rooms in
DRANHS, wherein gravid female A. aegypti mosquitoes likely to lay their eggs. After six
days, the ovitraps were collected and the lawanit paddles were air dried for two days.

 
 
 The completely dried lawanit paddles were examined under the magnifying glass
and the eggs present in the paddle were collected manually. The eggs were placed in a
sealed white bowl and left to stay until further use.

Hatchability and Mortality Set-up

Five petri plates were used in the set-up, one for each treatment (Treatment 1 –
lemon grass crude extract, Treatment 2 - San Francisco crude extract, Treatment 3 –
tawa-tawa crude extract, and Treatment 4 – normal saline solution (negative control)
and Treatment 5- pulverized Piper nigrum pellet solution (positive control). Ten eggs
were introduced in each plate and 500µL or .5 mL of each extract and solution were
poured using a micro pipette. After every 24 hours for three days, the number of eggs
that hatched in each treatment were counted for the hatchability and number of dead
larvae were counted for mortality.

Morphological Observation and Evaluation

Five petri plates were prepared for the morphological observation and
experimentation representing each treatment. Ten eggs were introduced in each plate.
After planting the eggs, they were left to grow until they turned into 4th instars larvae.
After acquiring the 4th instars, 500µL or .5 mL of each extract and solution were poured
using a micro pipette. After 24 hours, the larvae were observed using a compound light
microscope. Behavioral changes, morphological deformities, and internal abnormalities
were observed and recorded.

Identification of A. aegypti larvae

To verify that the samples used in the study were A. aegypti, the comb scales
were examined under a compound light microscope. Comb scales of A. aegypti were
single row and pitched fork in shape (Rueda, 2004). The certification for identification of
A. aegypti species was done by Ryan H. Bonsato, R.N., Research Assistant in the
School-Based Mosquito Abundance Model (SMAM) Project funded by UP-Manila,
DOST, PCHRD.

All throughout the data experimentation, the temperature in degree Celsius and
relative humidity (RH) of the laboratory were gathered using a thermo hygrometer in
order to establish and monitor ecological factors on the hatchability of mosquito eggs
and development of mosquito (Lopes, Holcman, Barbosa, Domingos, & Barreiros, 2014;
Beserra, Castro, Jr., Santos J., Santos T., & Fernandes, 2006)

Disposal of Mosquito Samples

 
 
 The A. aegypti eggs and larvae were subjected to hot water after the
experimentation that is a traditional way of killing mosquito larvae (Buckner, 1934).
Furthermore, liquids with mosquito eggs and larvae were thrown in cemented dry
ground to ensure total death of the organisms.

Phase III - Data Collection and Analysis

After administering the crude extracts and solution in the treatment groups, the
data for the number of hatched eggs and dead larvae were taken every 24 hours within
three days and for the morphological observation, larvae were observed under a
compound light microscope in order to evaluate and record the changes, abnormalities
and deformities in the structure particularly in the abdomen of the larvae, as well as its
behavior. Images of the larvae were captured using an ASUS Zenfone 5.

Statistical Analysis

One-way ANOVA: One-way Analysis of Variance was used to discover if the hypothesis
will support the study specifically in testing the significant differences in the hatchability
of mosquito eggs and mortality of larvae under different treatments.

Mean: Determine the average hatchability of mosquito eggs and mortality of larvae.

In testing the difference, the study used .05 level of significance. All data
gathered from the study was encoded using IBM SPSS software.

Research Design

This study used a Mixed Method Design wherein a quantitative analysis was
utilized in the hatchability of eggs and mortality of larvae under different treatment
groups. While qualitative analysis was made in the morphological study of 4th instar A.
aegypti larvae.

RESULTS AND DISSCUSSIONS

Identification of Aedes aegypti larvae

In order to confirm that the larvae were A. aegypti, comb scales were observed
and identified under a compound light microscope (Figure 1). Comb scales of A.aegypti
were described as single row with a pitch fork shape (Rueda, 2004).

 
 
 Figure 1. Comb scales of A.aegypti larvae.100x magnification

Phytochemical Screening Results

Table 1 shows the phytochemical screening performed at the University of the

Immaculate Conception, Science Resource Center, Davao City. Alkaloids were found to
be only present in Lemon grass. While saponins and tannins, except steroids, were
present in all plant extracts.

Table 1. Phythochemical Analysis

Phythochemicals Plant Extracts

Tawa- Lemon San
tawa Grass Francisco
Alkaloids - + -
Saponins + + +
Tannins + + +
Steroids - - -
+, presence; -, abscence

Effect of crude extracts on Hatchability of A. aegypti eggs

Table 2. Mean hatchability of A. aegypti eggs after three days of treatments

Average No.of Mean Hatchability* of

Treatment
Hatched Eggs Aedes aegypti eggs
a
Lemon Grass Crude Extract 1 0.100
a
San Francisco Crude Extract 0 0.000
b
Tawa-tawa Crude Extract 6 0.600
b
Normal Saline Solution (Negative control) 2 0.200
b
Pulverized Piper nigrum Pellet Solution 7 0.700
Treatment mean having different letters are significantly different at α= 0.05 level of significance
*Mean hatchability (Total no. of eggs/No. of replicates per treatment)

 
 
The San Francisco crude extract compared to that of Tawa-tawa and Lemon Grass
shows the lowest mean level, manifesting the ovicidal potential of the plant. The
phytochemical screening confirmed that San Francisco leaves contain saponins and
tannins which according to Akinyemi, et al. (2005) have larvicidal and insecticidal
potential.

Comparison on Mortality Count of A. aegypti larvae among Treatments

Presented in Table 3 is the result of the ANOVA to compare the mortality count
of larvae among treatments. Result shows that that there is a significant difference (F =
15.113, p < 0.05) on the mean mortality count of larvae among the treatments.

Table 3. ANOVA on Mortality Count of Aedes aegypti Larvae among Treatments

Sum of df Mean F Sig.

Squares Square

Between 7.120 4 1.780 15.113 .000

Groups
Within 5.300 45 .118
Groups
Total 12.420 49

Table 4 shows the post hoc test among treatments. San Francisco has the highest
mortality mean which shows that it has a larvicidal potential against A. aegypti
mosquito, same as to the pulverized Piper nigrum L. pellet solution (positive control).
Although, there was no larvicidal activity reported on San Francisco, Garcia (1990)
showed molluscicidal properties against Oncomelania hupensis quadrasi or Freshwater
Grastropods, suggesting that the presence of an inherent pesticidal component in San
Francisco leaves. On the other hand, Briones, Garbo, Casa, and Bion (2012) concluded
that the aqueous and pelletized extracts of P. nigrum exhibit larvicidal activity.

Table 4. Mean Mortality of 4th instar A. aegypti Larvae among Treatments

Treatment Average No. of Dead Mean Mortality of
th th
4 instar Larvae A.aegypti 4 instar
Larvae
b
Lemon Grass Crude Extract 3 0.300
a
San Francisco Crude Extract 10 1.000
b
Tawa-tawa Crude Extract 2 0.200
Normal Saline Solution (Negative b
0 0.000
Control)
a
Pulverized Piper nigrum Pellet Solution 8 0.800
Treatment mean having different letters are significantly different at α= 0.05 level of significance.

 
 
Abdominal Morphology and Behavioral Observations

When the 4th instar larvae of A. aegypti were subjected with the different
treatments, the following were observed: (1) aggressive and restless behavior of the
organism, (2) constricted or twisted alimentary canal, and (3) darkening or blackening of
abdomen.

(A) (B)
Figure 2. Aggressive anal gill biting of Aedes aegypti larvae treated with Piper nigrum
solution (A) and Tawa-tawa crude extract (B) under 40x magnification

(A) (B) Normal

Figure 3. Constricted abdomen subjected to crude extract of Tawa-tawa (A) and
Darkening or Blackening of abdomen subjected to Piper nigrum solution (B) under 10x
magnification

(A) (B) Normal

Figure 4. Exposure of larvae treated with Lemon grass crude extract (A), Tawa-tawa
crude extract (B) (from left to right) under 10x magnification

 
 
 Figure 5. Dead 4th instar larvae subjected to San Francisco crude extract under 4x
magnification

The results showed that the crude extracts of lemon grass, Tawa-tawa and San
Francisco and the P.nigrum solution have effects towards the abdominal morphology
and behavior of the larvae after every 24 hours for three days. The microscopic
observations of the morphological features of the larvae in the control groups and
treatment groups did not show any absence of the larval organs such as eyes,
antennae, mouth, head, thorax, siphon, and ventral brush. It was observed that the
larvae became aggressive, restless and excited after subjected to P. nigrum and Tawa-
tawa (Figure 2). These symptoms are similar to what Sharma, Kumar, and Tripathi,
(2015) have observed. They suggested presence of nerve poisons similar to the
extracts of Achyrantes aspera that they studied which could have acted as cytolysin
producing neurotoxic impact on A. aegypti larvae. Moreover, these findings are in
accordance with the study conducted by Warikoo and Kumar (2013) in the larvae of A.
aegypti when assayed on the extracts of Agave mexicana, indicating that the extracts
act as nerve poisons; though, the aggressive and uncoordinated movements along with
other toxic symptoms were observed at relatively different time intervals. The larvae
exposed to crude extract of tawa-tawa and P. nigrum solution exhibited darkening or
blackening of abdomen and twisted or constricted abdomen (Figure 3). Comparable
abnormalities were also reported by Khater and Shalaby (2008) when the larvae of
Culex pipiens showed aberrations such as pigmented and twisted larvae after exposure
to some commercially available plant oils. Fluids and dark spots were also observed in
the larvae (Figure 4) which may indicate damaged abdomen. In recent studies, the
larvae of A. aegypti exposed to A. aspera extracts also showed distorted midgut with
loss of pigmentation and partial or total cell destruction. Related findings in the midgut of
A. aegypti were reported by Chaithong et al. (2006) after exposure of larvae to pepper
extracts.

 
 
CONCLUSION

Based on the results of the study, it was found out that San Francisco crude
extract prevents the hatching of mosquito eggs thus preventing it to become larvae
which also showed high mortality count of the organism. In the qualitative observations,
results showed that crude extracts of lemon grass and tawa-tawa and the pulverized P.
nigrum pellet solution exhibited aberrations on the abdominal morphology and behavior
of the larvae. These occurrences observed showed the ability of these plant substances
to inhibit growth and development of the disease-causing insect. With these findings,
plant extracts have the potential as organic insecticides for vector control in the combat
against dengue disease.

RECOMMENDATIONS

For further research:

1. utilize various concentration of the plant extracts to establish significant

differences in the treatment groups
2. compare the effectivity of the extracts to a commercially available insecticide

ACKNOWLEDGMENT

The researcher would like to express his sincerest thanks and gratitude to Mrs
Suzette A. Bermejo, Mr. Jim Boy P. Pasia and Chito R. Napitan Ed.D for the guidance
and assistance that lead to the success and fulfilment of this study. To GOD be all the
the glory.

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