The Influence of the Ethanol Extract of Bitter Vine (Mikania

micrantha Kunth.) on the Mortality, the Hatchability of the Eggs and

the Larval Growth of Aedes aegypti Linn.

Nursal

and A. Hardiansyah

Department of Biology, Universitas Sumatera Utara, Jl. Dr. T. Mansyur No.9, Medan, Indonesia

Keywords: Mikania micrantha, Mortality, Growth, Aedes Aegypti.

Abstract: The research on the impact of the ethanol extract of M. micrantha leaf on the mortality, egg hatchability and

larval growth of A. aegypti had been conducted using a Complete Randomized Design (CDR) with five

treatments and replications. The mortality tests on 3rd instar larva with concentration treatments of the ethanol

extract of M. micrantha leaves at 0.2%,0.4%,0.6%,0.8%, and 0.1%generated a LC

value of 0.58%.The

ethanol extract of M. micrantha leaves at a sub-lethal concentration of 0,1%,0,2%,0,3%,0,4%,0,5%indicated a

significant impact on the mortality, egg hatchability and larval growth (p≤0,05).A sublethal concentration at

0,4% of the plant was effective in suppressing the egg hatchability at a percentage of 41,6%, larval

development into pupa at a percentage of 19.5% and pupae transformation into imago a percentage of

63,3%.

1 INTRODUCTION

The control of the vector-borne disease can be

conducted using chemical compounds such as

synthetic insecticides, but this may cause losses such

as resistance, death of untargeted, or human

poisoning. World Health Organization has

advocated finding alternatives to control these issues

through biological or environmental control

methods, by using natural chemicals derived from

the plants (Indonesian Department of Health, 2010).

Floras in Indonesia have mass potentials to be

utilized as an alternative for plant-based insecticides

using the secondary metabolites they produce

(Boesri et al., 2015). Organic insecticides are

generally pesticides whose active ingredients come

from plant parts that are toxic to insects and have

secondary metabolites containing various bioactive

compounds (Thamrin, M. et al., 2007).

Various plants, including weeds, have secondary

metabolite compounds that can be used for self-

defense against pests and diseases (Tampubolon,

2018). Bitter Vine (Mikania micrantha) is one of the

potential weeds and has been proven as an effective

plant-based insecticide because it contains secondary

metabolites that can kill insects (Salam et al., 2014).

Based on the phytochemical analysis results, the

leaf extract of M. micrantha contains active

substances in the form of secondary metabolites

such as alkaloids, saponins, flavonoids, steroids,

tannins, and terpenoids (Polakitan et al., 2017). M.

micrantha also has other specific active substances

called mikanolide and dihydromichiolide. These

substances belong to the sesquiterpene group

commonly found in the plants of the Asteraceae

family (Tripathi et al., 2012). Noshirma & Willa

(2016), in their research, mentioned that these

phenolic metabolites might cause stomach poisoning

which can interfere the digestive system of A.

aegypti larvae, so the larvae fail to develop and

eventually die. “interfere with the digestive system”

Plant-based insecticides also work specifically

by damaging the growth of eggs, larvae, and pupae;

inhibiting skin turnover; disrupting insect

communication; inhibiting the female reproduction;

reducing appetite; blocking the insect ability to eat;

and repelling the insects (Sudarmo, 2005). Only a

few studies on the leaves of M. micrantha as an

organic insecticide have been conducted, so a test is

required to see the effect on the egg hatchability,

mortality and development of A. aegypti larvae as

one of the efforts in controlling the number of A.

aegypti mosquitoes through monitoring in the larval

450

Nursal, . and Hardiansyah, A.

The Inﬂuence of the Ethanol Extract of Bitter Vine (Mikania micrantha Kunth.) on the Mortality, the Hatchability of the Eggs and the Larval Growth of Aedes aegypti Linn..

DOI: 10.5220/0010204500002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 450-455

ISBN: 978-989-758-556-2

phase. Thus, the object of this study was how the

ethanol extract of M. micrantha leaves affect the egg

hatchability, mortality, and development of A.

aegypti larvae. The study was conducted to

determine the effect of ethanol extract of M.

micrantha leaves on larval mortality, egg

hatchability, and development of A. aegypti larvae.

2 METHODS

2.1 Animal Subject Rearing

Rearing was carried out to keep and breed the

animal subjects to provide the eggs and larvae of the

A. aegypti. It was conducted at the Institute of

Environmental Health and Infection Disease Control

(BTKL-PPM) Class 1 Medan.

2.2 General Architecture

This research is an experiment using a completely

randomized design (CRD) with five extract

concentrations (with one control) and five iterations

of 25 larvae and A. aegypti eggs.

2.3 Ethanol Extract of M. micrantha

Leaves

Five kilograms of M. micrantha leaf samples were

washed and dried for five days, then crushed using a

blender to form a powder. The powder was weighed

as much as 1 kg then macerated with ethanol for 144

hours and stored in Erlenmeyer. During the

maceration process, the stirring was carried out

every day until obtained macerate. The obtained

macerate was then filtered and evaporated with a

Vacuum Rotary Evaporator until all the ethanol

evaporated into a thick extract. This extract would

be stored in a silica gel desiccator (Hamidah et al.,

2015).

2.4 Observation of Test Parameter

2.4.1 The Mortality Test on the Third Instar

A. aegypti Larva

The toxicity test of the ethanol extract of M.

micrantha leaves on the mortality of the third instar

A. aegypti larvae was conducted using six

concentration variants with five replications, namely

K1 = 0.2%, K2 = 0.4%, K3 = 0.6%, K4 = 0,8%, K5

= 1%, and K0 = 0% (control). A total of 25 third

instar A. aegypti larvae were put into separate

exposure medium containing 100 ml of each extract.

The temperature and humidity in the treatment room

as and the exposure medium were set as the standard

measurement. The observation was performed 24

hours after the exposure. The larval mortality rate

can be calculated using the formula below.

𝐿𝑎𝑟𝑣𝑎𝑙 𝑀𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦







𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐷𝑒𝑎𝑑 𝐿𝑎𝑟𝑣𝑎𝑒

𝑇𝑜𝑡𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐿𝑎𝑟𝑣𝑎𝑙 𝑆𝑢𝑏𝑗𝑒𝑐𝑡𝑠

𝑥 100

(1)

Furthermore, the da

ta analyzed for regression to

obtain the LC50 value using Microsoft Excel 2013.

2.4.2 Egg Hatchability and Growth of A.

aegypti Larvae at Sublethal

Concentration

The sublethal test was conducted to determine the

egg hatchability and larval development of A.

aegypti until the imago phase. Twenty-five eggs of

A. aegypti were put into a test cup containing 100 ml

of ethanol extract of M.micrantha leaf with sublethal

concentrations, based on the results of the mortality

test, at P0 = 0% (control), P1 = 0.1%, P = 2, 0.2%,

P3 = 0.3%, P4 = 0.4% P5 = 0.5% with five

replications. The observation of the egg hatchability

was monitored every 24 hours for 72 hours (3 days)

(WHO, 2005).

𝐸𝑔𝑔 𝐻𝑎𝑡𝑐ℎ𝑎𝑏𝑖𝑙𝑖𝑡𝑦 %



𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 ℎ𝑎𝑡𝑐ℎ𝑒𝑑 𝑒𝑔𝑔𝑠

𝑇𝑜𝑡𝑎𝑙 𝑜

𝑓

𝑒

𝑔𝑔

𝑠

𝑥 100

(2)

The testing of the development of A. aegypti was

observed from the hatched eggs into larval stages in

24 hours for ten days. It aims to determine the

number of successful larvae

transformed into pupae

and pupae into the imago. This can be calculated

using the formulas below.

𝐿𝑎𝑟𝑣𝑒  𝑃𝑢𝑝𝑎𝑒







𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐷𝑒𝑣𝑒𝑙𝑜𝑝𝑒𝑑 𝑃𝑢𝑝𝑎𝑒

𝑇𝑜𝑡𝑎𝑙 𝑜

𝑓

𝐿𝑎𝑟𝑣𝑎𝑒

𝑥 100

(3)

𝑃𝑢𝑝𝑎𝑒  𝐼𝑚𝑎𝑔𝑜







𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐷𝑒𝑣𝑒𝑙𝑜𝑝𝑒𝑑 𝐼𝑚𝑎𝑔𝑜

𝑇𝑜𝑡𝑎𝑙 𝑜

𝑓

𝑃𝑢𝑝𝑎𝑒

𝑥 100

(4)

2.5 Statistical Analysis

The obtained data from each observation variables

were recorded and arranged in tabular form. The

The Inﬂuence of the Ethanol Extract of Bitter Vine (Mikania micrantha Kunth.) on the Mortality, the Hatchability of the Eggs and the Larval

Growth of Aedes aegypti Linn.

451

generated quantitative data (dependent variables)

were tested for their significance on the impact of

the treatment groups (independent variables) with

the help of a statistical computer program, namely

the SPSS (release 22). The test sequence began with

a normality test, homogeneity test, one-way

ANOVA test for data with repeated observations

(more than two times).

If in the ANOVA test, there is a significant

difference (p <0.05) in the treatment group, then the

test will be continued using the Post Hoc-Duncan

analysis at a level of 5%. At the end of the study, it

can be determined which concentration of ethanol

extract of M. micrantha leaves has the most

significant and practical effect on the egg

hatchability and larval growth of A. aegypti.

3 RESULTS AND DISCUSSION

3.1 Larval Mortality

Based on the test results, the mortality rate of the

third instar A aegypti larvae was obtained after

being treated with ethanol extract of M. micrantha

leaves. It can be seen in Figure 1.

Figure 1: Influence of ethanol extract of M. micrantha

leaves on the mortality rate of the third instar A. aegypti

larvae (24-hour observation). Note: K0: 0% (control), K1:

0.2%, K2: 0.4%, K3: 0.6%, K4: 0.8% and K5: 1%. The

number followed by the same letter in the figure was not

significantly different in the Duncan test at a rate of 5%.

Figure 1 shows that the greater the concentration

of the treatment given, the higher the percentage of

larval mortality rate, compared to the K0 (control)

treatment. In the K0 treatment, no larvae mortality

was found. Larval mortality began to occur in the

K1 treatment with the lowest percentage of mortality

rate at 16%, while the highest mortality was in the

K5 treatment at a percentage of 88%. Based on the

results shown in Figure 1, the lethal concentration

value of 50% (LC50) can be determined by using

probit analysis.

The result of the probit analysis showed that the

LC50 value is at 0.58% after 24 hours. The

regression calculations illustrated the relationship

between the extract concentration of M. micrantha

leaf and larval mortality. This can be obtained using

the equation of y = 0.44 + 21.0x with a regression

coefficient of r2 = 0.98, which is shown in Figure 2.

Figure 2: Graph of regression analysis on the effect of the

ethanol extract concentration of M. micrantha leaves with

the percentage of A. aegypti larval mortality.

Fitmaya (2006) stated that the higher the

concentration of the insecticide given, the higher the

content of the active substance so it can increase

metabolic obstruction of the larvae subject which led

to the increasing percentage of its death. Haisya, N.

et al., (2013) mentioned that the leaves of M.

micrantha contain several active compounds in the

form of secondary metabolites such as alkaloids,

flavonoids, tannins that are insecticides.

Each active substance has different work

principles in impacting larval mortality. According

to Cania (2013), alkaloids serve as a stomach

poison. Alkaloids, in the form of salts, can degrade

the cell membranes to damage the cells and also

disrupt the larval nerve system by inhibiting the

action of the acetylcholinesterase enzyme and

causing larvae to undergo paralysis and die.

Flavonoids act as a respiratory poison, causing

larvae death. Tannins play a role in reducing the

ability to digest the food by suppressing the activity

of digestive enzymes (Haditomo, 2010).

The lower the LC50 value of a substance means

that the substance has higher activity in killing

experimental animals since it requires a lower

concentration to kill the animals simultaneously

(Chang, 2004).

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

452

3.2 Egg Hatchability and Development

of A. aegypti Larvae in Sublethal

Concentration

The tests on egg hatchability and larval development

were performed using LC

at sublethal

concentrations (0.58%) of 0.5%, 0.4%, 0.3%, 0.2%

and 0.1%.

Table 1: The influence of ethanol extract of M. micrantha

leaf on the percentage of the egg hatchability, and the

larvae-pupae and pupae-imago growth of A. aegypti in

the 14-days observation.

Treatm

ent

Numb

Egg

Hatchabil

ity (%)

Larvae-

Pupae

Developm

ent

(%)

Pupae –

imago

Developm

ent

(%)

P0 25

85.3

92.0

P1 25

80.8

68.4

87.0

P2 25

73.6

66.4

82.3

P3 25

65.6

44.3

78.3

P4 25

41.6

19.5

63.3

Note that P0= 0 % (control), P1= 0.1%, P2=

0.2%, P3= 0.3% , P4= 0.4%, and P5= 0.5%. The

number followed by the same letter in the

same

column

was not significantly different in the

Duncan test at a rate of 5%.

3.2.1 The Egg Hatchability

Table 1. shows that the ethanol extract of M.

micrantha leaf at treatments of P1, P2, P3, P4, and

P5 can decrease the rate of the egg hatchability, and

development of larvae-pupae and pupae-imago

compared to P0 (control). The number of hatched

eggs decreases with increasing concentration of the

given treatment. The lowest number of hatched eggs

was at P5 treatment at a percentage of 26.4%, while

the highest hatchability rate at P1 treatment at

80.8%.

The difference in egg hatchability is thought to

be caused by the secondary metabolite content of the

extract which disrupts the metabolism in the egg, so

the egg fails to hatch. Salam et al., (2014) stated that

M. micrantha leaves contain active substances such

as flavonoids and tannins. The decline in the

percentage of the egg hatchability is due to the

flavonoids that enter the egg through the diffusion

process on the surface of the eggshell.

3.2.2 Development of Larvae - Pupae

Table 1 showed that the ethanol extract of M.

micrantha leaf in all treatments was able to suppress

the development of the larvae-pupa stage. The

highest percentage of the successful growth of

larvae-pupa was in P1 treatment at 68.4% while the

lowest occurred in P5 treatment at 11.5%. This

shows that during exposure, the ethanol extract of

M. micrantha leaves affected the development of

larvae into pupae. The higher concentration of

ethanol extract of M. micrantha leaves led to the

failure of larvae to become pupae, thus reducing the

percentage of successful development

During the observation, the larval phase showed

a tendency to require a longer time to develop into

pupae, which was around 10-12 days, compared to

the control treatment, which only took 8-10 days.

The larvae also tend to experience changes in body

size which is larger than the larvae in the control

treatment group. This may be caused by the

extracted content of M. micrantha leaf, in the form

of Mikanolide, and belongs to the sesquiterpene

group, which has a structural similarity to the

juvenile hormone.

Bowers (1971) stated that the metamorphosis

stage from larvae to pupae is sensitive and complex.

The distribution of juvenile hormones in a long time

can have effects such as the formation of large

larvae. Elimam et al., (2009) also revealed that the

levels of the juvenile hormone could directly

determine the larval stage to become a pupa that will

last a long time.

3.2.3 Development of Pupae - Imago

The living pupae were observed their development

into the imago. The percentage of pupa-imago was

obtained from the comparison of the number of

imagoes formed with the total number of pupae.

Table 1 shows that the highest percentage of

successful pupa-imago development was in the P1

treatment at 87.0%, while the lowest was in the P5

treatment at 50.0%. All ethanol extracts of M.

micrantha leaves tended not to affect the percentage

of developmental failure of the pupae-imago stage

compared to the control treatments.

This can be seen from the high percentage of

pupae-imago. This may be related to the pupae that

no longer needs food, which means that the pupae

do not consume the extract solution anymore so that

the pupa can avoid the toxic effects of the ethanol

extract of M. micrantha leaves.

During the observation, it could be seen that the

growth of pupae to imago needed a longer time,

The Inﬂuence of the Ethanol Extract of Bitter Vine (Mikania micrantha Kunth.) on the Mortality, the Hatchability of the Eggs and the Larval

Growth of Aedes aegypti Linn.

453

which was around 5-6 days. (Yulidar & Wilya,

2015) stated that the normal time for pupae to

develop into imago is 3-4 days.

The pupae stage is a fasting phase where the

body is wrapped in a layer called the puparium. The

increase in the time it takes for a pupa to become an

imago may be due to the pupa trying to survive by

extending its maturation period into an imago, so the

pupa remains protected by a protective layer that

wraps its body from exposure to toxic extracts.

Typically, at the fourth instar larvae, there is a

decrease in the secretion of juvenile hormone by

corpora allata, but with the exposure to the ethanol

extract of M. micrantha leaves, which was thought

to have an effect like juvenile hormone, induced the

pupae to prolong its development into an imago

(Habibi, 2011).

Based on the results of the study on the effect of

the ethanol extract of M. micrantha leaves on the

egg hatchability and the development of A. aegypti,

it can be determined that the most significant

concentration which can reduce the rate of the egg

hatchability and the larval development is the P4

treatment with a concentration of 0.4 %.

The same thing occurred in the research of

Nursal & Hardiansyah (2018). The dichloromethane

extract of the leaves of the bitter melon (Momordica

charantia L.), basil (Ocimum basilicum L.), and

lemongrass (Cymbopogon winterianus) can reduce

the percentage of the egg hatchability and the larval

development (larvae- pupae and pupae – adult) of

Aedes aegypti. Likewise, with Nursal & Yeanny

(2019), the ethanol extract of the leaves of bitter

melon (Momordica charantia L.) and basil (Ocimum

basilicum L.) can also reduce the hatchability of the

eggs and the growth (larvae-pupae and pupae-adult)

of Aedes aegypti mosquitoes.

4 CONCLUSIONS

Based on the test results, it can be concluded that the

LC50 concentration of the ethanol extract of M.

micrantha leaves on the mortality of third instar

larvae was at 0.58%. The sublethal concentration of

the ethanol extract of M. micrantha leaves has a

significant influence on the eggs hatchability and

larval development. The ethanol extract

concentration of M. micrantha at 0.4% was effective

in reducing the rate of the eggs hatchability and the

larval development into pupae, and the pupae

development into imago by 41.6%, 19.5%, and

63.3% respectively.

REFERENCES

Boesri, H., Heriyanto, B., Wahyuni, S., Handayani, &

Suwaryono, T. (2015). Toxicity Test of Numerous

Plant Extracts against Aedes aegypti Larvae Dengue

Hemorrhagic Fever Vector. Vektora, 7, 29–38.

Bowers, W. S. (1971). Insect Hormones and Their

Derivatives as Insecticides. Bull. Org. Mond. Sante;

Bull. Wld Hlth Org, 44, 381–389.

Cania, E. (2013). The Effectiveness of Larvacides Legundi

Leaf Extract (Vitex trifolia) Against Aedes aegypti

Larvae. Medical of Journal Lampung University,

22(4), 52–60.

Chang, P. S. (2004). Cinnamon Oil May Be an

Environmentally Friendly Practice, With the Ability to

Kill Mosquito Larvae.

Elimam, A. M., Elmalik, K. H., & Ali, F. (2009).

Larvicidal Adult Emergence Inhibition and

Oviposition Deterrent Effects of Foliage Extract from

Ricinus communis L. against Anopheles arabiensis

and Culex quinquefasciatus in Sudan. Tropical

Biomedicine, 26(2), 130–139.

Fitmaya, A. (2006). The Activity Test of the Ethanol

Extract Larvalide at 96% of Sweet Starfruit (Averrhoa

carambola L.) Leaves against the Third Instar

Anopheles aconitus Larvae and its Thin Layer

Chromatography. Universitas Muhammadiyah

Surakarta.

Habibi, S. (2011). Juvenile Hormone (JH) As a Supporter

And Controller Of Insect Life. Proceedings of

National Seminar of Universitas Terbuka Banten.

Haditomo, I. (2010). The Effect of Larvaside Extract of

Clove (Syzygium aromaticum L.) Leaves Against

Aedes aegypti L. Universitas Sebelas Maret.

Haisya, N., Asfi, R. L., & Riris, P. S. (2013). Bitter Vine

(Mikania micrantha H.B.K.) as natural alternative

antibacterial and its study against bacterial common

as a causative agent in cattle mastitis in Indonesia.

Hamidah, H. S., Mukarlina, L., & Linda, R. (2015). The

ability of the extract of the convex vine (Mikania

micrantha H.B.K) leaves as a bioherbicide of Weed

Melastoma affine D.Don. Protobiont, 4(1), 89–93.

Indonesian Department of Health. (2010). Epidemiology

Window Bulletin Topics of Dengue Hemorrhagic

Fever (2nd ed.). Surveillance and Epidemiology Data

Center.

Noshirma, M., & Willa, R. W. (2016). Biological

larvicides used in the Control of Dengue Fever Vector

in Indonesia. SEL, 3(1), 31–40.

Nursal, & Hardiansyah, A. (2018). The effectiveness of

dichloromethane extract of various plants on eggs

hatchability, and life cycle of Aedes aegypti L.

mosquitoes. Journal of Physics: Conference Series.

Nursal, & Yeanny, S. M. (2019). The Egg Hatchability

and the Development of Aedes aegypti Mosquitoes in

Ethanol Extracts of the Leaves of Bitter Melon

(Momordica charantia L.) and Basil (Ocimum

basilicum L.). IOP Conference Series: Earth and

Environmental Science.

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

454

Polakitan, I. R., Fatimawali, & Leman, M. A. (2017).

Inhibitory Test of the Extract of Bitter Vine (Mikania

micrantha) against Streptococcus mutans Growth.

Pharmacon, 6(1), 1–8.

Salam, D. M., Mukarlina, L., & Diba, F. (2014).

Coptotermes curvignathus Holmgren Soil Termite

Control Using The Extracts of Bitter Vine Leaf

(Mikania micrantha Kunth.). Protobiont, 3(2), 87–92.

Sudarmo, S. (2005). Plant-Based Pesticides. Kanisius

Publishing.

Tampubolon, K. (2018). The Potential of Secondary

Metabolites of Weeds as Organic Pesticides in

Indonesia. Journal of Cultivation, 17(3), 683–693.

Thamrin, M., Asikin, S., Mukhlis, & Budiman, A. (2007).

Potential of Swamp Land Flora Extract as Plant-Based

Pesticides. Swamp Field Research Center, 35–54.

Tripathi, R. S., Khan, M. L., & Yadav, A. S. (2012).

Biology of Mikania micrantha H.B.K. A Review.

WHO. (2005). Guidelines for laboratory and field testing

of mosquito larvicide.

Yulidar, & Wilya, W. (2015). Life Cycle of Aedes

Aegypti on Laboratory Scale. SEL, 2(1), 22–28.

The Inﬂuence of the Ethanol Extract of Bitter Vine (Mikania micrantha Kunth.) on the Mortality, the Hatchability of the Eggs and the Larval

Growth of Aedes aegypti Linn.

455