EVALUATION OF THE LARVICIDAL ACTIVITY

OF ARDISIA TOMENTOSA CRUDE WATER EXTRACT AGAINST MOSQUITO

LARVAE OF GENUS AEDES

_______________________________________________________________________

A Research Paper

Presented to the faculty of the

Department of Science

In partial fulfilment of the requirements of the subject

Research I

______________________________________________________
By

Macaraeg, Bea

Moreno, Katherine Leigh M.

January 2021
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CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the survey of related literature and studies taken from

books, journals, and electronic sources to strengthen the study's conceptualization.

Related Literature

Mosquitoes are known to be carriers of many causing pathogens. It may cause

more ailments than in any other class of arthropods worldwide. Aedes Aegypti is known

to bear Dengue and yellow fever (Dhanasekaran et al, 2014). Anopheles stephensi carries

malaria and filarial disease by Culex. The rise in mosquitoes with Zika is not attributable

to many breeding sites in the metropolitan agglomeration. The emerging insecticide

resistance issues involving various insecticides, the reason for new outbreaks, are

attributed to insects' improved vulnerability to current chemical insecticides. Vector-

borne diseases contribute to a significant disease burden in Eastern Visayas (Region 8),

Philippines. The spread of the disease can be disrupted by vector control using different

methods. However, the systematic and unbalanced use of chemical insecticides has

created problems like enhancing mosquito population resistance to synthetic insecticides

(Dhanasekaran, 2014).

Disrupting disease spread by killing or preventing mosquitoes from infecting

humans is one of the measure to counter these mosquito-borne diseases. Working on

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alternate vector management, finding herbal preparations that have no negative effects on

non-target species and are easily biodegradable remains a key research priority.

Due to debilitating morbidities and high death rates that come with mosquito-

borne diseases, some reports have prioritized the mosquito population's regulation by

concentrating on its life cycle. Larvicide mosquito management requires the spraying of

industrial insecticides. However, these synthetic insecticides cause environmental

degradation, residual effects, and resistance through their indiscriminate use. Different

synthetic insecticides include organochlorine, organophosphorus, carbamates, pyrethrins,

and pyrethroids (Al Fadhli et al. 2020). Thus, the need arises for an environmentally

friendly alternative to synthetic insecticides. Here, the researchers presented the

evaluation of larvicidal efficacies of Ardisia water extract prepared from different

soaking times against 5-day old Aedes larvae. Aedes aegypti undergoes maturation,

begins with the embryo and progresses toward adult form. This insect spends its life

cycle strongly dependent on the environment.

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Aedes larvae. Aedes aegypti is the main global host of dengue viruses that belong

to the Culicidae tribe and the Diptera family. An Aedes Aegypti lives the life cycle from

the aquatic pupal stage to the adult aerial stage. Environmental conditions such as light

and temperature significantly influence larval growth. Mosquito management is mostly a

concern of discovering the target organisms, biological and ecological requirements

whenever possible. (Baltazar, Clare, and Salazar, Nelia; "Philippine Insects an

Introduction," Diliman, Quezon City, University of the Philippines Press, 1979) The

female Aedes aegypti has mouthparts modified for sucking blood from its hosts. The

female mosquitoes need it to mature their eggs.

Female mosquitoes will lay eggs on the water container's inner side; the eggs will

hatch and release mosquito larvae when they enter the water. Female mosquito larvae can

go through four stages: egg, larvae, pupae, and adult, male and female. The female

mosquito can only have one mating since Aedes females are only fertilized once in her

existence. Following that, the female mosquito will lay eggs any time she draws blood.

The reproductive span of a mosquito ranges from 8 days to 12 days, depending on the

weather situation. Higher temperatures result in shorter lifespans for mosquitoes. More

mosquitoes will emerge as its eggs hatch out and rise to maturity.
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If a female mosquito sucks blood on someone sick during a second blood intake,

the mosquito has acquired the infection; it consumes the blood that she has ingested. As a

result, precise detection of the virus and a specific protein will get into the stomach

lining. The virus can infiltrate and replicate inside the stomach cell. It will infect all

organs, especially the ovaries, establishing direct transmission from the female mosquito

to its eggs. Viruses are also multiplying in the salivary glands. The virus can infect

individuals; this is known as horizontal transmission. Since the female mosquito's

salivary glands are affected, as it bites, the female mosquito will emit saliva when

consuming blood ration, and this saliva will carry the infection.

Assume a female mosquito fed on a person and became contaminated with the

diseases. It will then consume the individual's blood and pass it on to the next female

mosquito. If the virus is permitted to continue in the mosquito's intestine, a specific

protein spreads centrifugally across the stomach lining. The disease it brings will move

through the stomach cell, where it will replicate. Within mosquitoes, the virus can cause

organs to die except the ovaries, and then there will be a clear transfer of the virus from

the female eggs. As well, viruses like hepatitis A, B, and C can become a concern. When

a female mosquito bites, it will emit infectious saliva. This saliva acted as a carrier to

bring the illness, including the virus.

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Due to debilitating morbidities and high fatality rates of mosquito-borne diseases,

some researchers prioritize control of the mosquito population by targeting its different

life stages (egg, larvae, pupa, and adult). Many published studies on the role of plant-

derived compounds target the larval stage of mosquitoes (Vikram, et al. 2012).

Insecticides cause air degradation, their residue is unavoidable, and their improper use

renders resistance inevitable (Ramaswamy, et al. 2007). Different synthetic insecticides

include organochlorine, organophosphorus, carbamates, pyrethrins, and pyrethroids

(Ahmad, September 2019). Thus, the need arises for an alternative to synthetic

insecticides. Searching for new secondary metabolites in plants for their potential to

serve as new control agents in countries with a profound herbal heritage, has gained

popularity among scholars. A variety of plants have been found to be insecticidal.

Mosquito-borne infections - Mosquitoes bring many illnesses that are

transmitted mainly through mosquito bites on humans. The persistence and propagation

of viruses by mosquitoes also depend on various factors such as vector compatibility,

temperature, and mosquito life cycle. A disease needs a host vulnerable to a capable

vector and the involvement of a pathogen. Mosquito-borne conditions may impact

anybody, from an infant to someone over sixty years old. All four of these living

elements must be present. A pathogen's continued existence inside a geographic area

involves two things; first, the pathogen that triggers the disease must be present in that

particular location. Second, the pathogen’s source in that location must be the region that
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provides the food source for mosquito larvae (and, in certain instances, humans).

Susceptible hosts may be contaminated by a pathogen (4) Species of mosquitoes which

can spread the pathogen from reservoirs to susceptible hosts, either mechanically or

biologically.

Vaccination and a combination of nonsteroidal anti-inflammatory medications

(NSAIDs) are the cornerstones of preventing dengue infections. Environmental

influences such as the type of housing, environmental conditions, artificial water-holding

tanks, the population density of the area, and impacted flood-prone areas all have a

significant impact on pathogenic microorganisms. Ardisia species is related to

Myrsinaceae and Theophrastaceae. A syndrome seen in angiosperms that do not occur in

gymnosperms is inside the Caryophyllales and is "free-central" placentation. (Anderberg,

Stahl, 1995) call Ardisia plants like herbs, climbers, shrubs, and tiny trees that often have

swollen twig bases. The genus Ardisia contains more than 200 species, typically

cultivated in warm and tropical areas (Bailey, 1928). In the Philippines, 68 species of

Ardisia occur, of which 60 are endemic.

The Genus Ardisia. Ardisia tomentosa is a species in the genus of Ardisia.

Almost There are 500 varieties of Ardisia in the world's tropical and subtropical regions

(Jianhong, 2001). Ardisia plants are common in China and utilized as medicinal herbs for

over 900 years. The species are in India, Indonesia, Malaysia, Thailand, Vietnam, and

Taiwan (Jamal et al., 2016). More than 60 species of Ardisia plants can be found in the

Philippines, and most of these are native to the region. According to Ong and Kim

(2014), the Philippine tribe used Ardisia Elliptica to cure diarrhea.

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Biolarvicides. Natural products originating from plants have a tremendous

potential to manage vectors, providing they are reasonably harmless to advantageous

non-target organisms and the environment. [ CITATION Pit07 \l 1033 ]. Locally available

plant-derived biolarvicide can be used to manage the mosquito population, decrease

reliance on costly mosquito control devices, and encourage local attempts to strengthen

the public health system. For example, “the impact of Bacillus sp on the larval gut of

Culex quinquefasciatus” confirms the fast-acting potentials of toxins produced from

Bacillus thurigensis israelensis and Bacillus sphaericus, active constituents. The mortality

observed in Culex quinquefasciatus larvae at all serial dilutions attest to the efficacy of

Vectoax CG biolarvicide at low concentrations. Studies have also shown that the eco-

friendliness of biolarvicides is beneficial to non-target organisms and, at the same time,

has the potential to deter the proliferation of adult mosquitoes.

In recent years, several experiments have been placed out to establish plants'

insecticidal properties in attempts to combat mosquitoes. A recent and significant

problem in parasitology is reducing the repetitive exposure to pesticides that foster

mosquitos' tolerance and pose substantial risks to human health and the ecosystem.

(Roman et al. 2019)

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Related Studies

Various studies have been conducted on evaluating plant extracts against various

essential species of mosquito larvae, including Culex quinquefasciatus, Anopheles

stephensi and Aedes aegypti. Various solvents of different polarities have been used to

separate bioactive components from candidate plants.

A study by Chapagain (2008) offers an overview of the quest for an alternative

route to the development of larvicide from Balanites Aegyptiaca plants. Root explant

cultures and their larval action against mosquito larvae of Aedes aegypti have been

evaluated. A concentration of 500 ppm or more kills 100 percent of the population of

research larvae. Compared to the monitor, 50 parts per million showed no difference in

larval mortality (0 ppm). These findings indicate saponins from root explant B cultures in

vitro. Aegyptiaca can be used as a larvicidal agent.

Another research by Véronique Masotti et al. (2012) states that the larvicidal

behavior of Molinieri extracts was higher (from 50 ppm (K = 9.488, DDL = 4, P < 0.001)

than that of A.Glutinosa. After 48h of exposure, measured lethal concentrations (LC50)

were low, with 9091 and 9898 ppm for A. and Molinieri. Nevertheless, A. Molinieri is an
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environmentally sustainable biolarvicide. Both pesticide activity and protection purposes

may be of interest in cultivating its community.

The larvicidal behavior of extracts of Clitoria Ternatea flowers influenced the

early 4th instar larvae of Aedes mosquito vectors, according to Ravindran et al. (2020).

The research reveals the immense efficacy of flower extracts against the primary vector

of dengue fever. Also, from rudimentary extracts, bioactive molecules are successful and

can be produced as biolarvicides for vector control programs for Aedes mosquitoes.

B.L. Aliero published an article on the Larvaecidal impact of Azadirachta indica

(neem) aqueous extracts on Anopheles mosquito larvae. The research studied the

susceptibility of the larvae to pure seed oil and leaf/bark extracts for 12 hours, resulting in

mortality of 100, 98 and 48 percent, respectively. Dilution of these extracts has also

culminated in larva mortality. The study indicates that neem seed oil and leaf extract

possess properties that could produce and used to control tropical mosquitoes.

The research performed on C. Occidentalis (Linn.) Petroleum ether and N-butanol

extracts screened for larvicidal bioassay. The median potency of petroleum ether is

around 1.6 times less efficient and is slightly higher relative to its use of N- butanol

extract. These extracts are cheap, simple to manage, and safer for mosquito larva control

items, say Deepak Kumar et al., respectively. The findings may help the quest for newer

molecules to combat the mosquito larvae-causing properties of new chemicals.

Conceptual Framework

This study is anchored on the concept of natural products research. Natural

products research aims to develop innovative and effective natural derived active
compounds with novel applications of interest to human resources. Many metabolites,
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also known as secondary metabolites, are organic compounds that aren't specifically
involved for an organism's growth, development and reproduction of an organism.
Secondary metabolites have a variety of functions that vary from species to species. As
the Philippines boasts a high degree of biodiversity, new species with innovative
behaviors will likely be discovered. Plant-derived larvicides must be grown in due time to
control and reduce mosquito-borne diseases and increase the ecosystem and the public's
safety.

The risk of insect vector-borne infections and related illnesses must be controlled
and accomplished by producing highly-efficient and low-cost larvicides. Studies on crude
water extracts as potential larvicides are low-cost and can be quickly applied to society.
This reason is one of the advantages of the method used in this work. Ardisia plant
belongs to the family Primulaceae and is endemic in the Philippines. To date, this plant
has been under-studied. This research on the characterization of Ardisia's behavior
against mosquito larvae is, therefore, a pioneering work.
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Definition of Terms

Aedes Mosquito - Ae. Aegypti has a thin and usually black colored body. First,

the abdomen and thorax variations are peculiar, and second, their light and dark patterns

are unordinary. Their contrasting dark and light bands on the legs often give them a

distinctive appearance. Females are further distinguished by the belly's outline, usually at

the top of the abdomen, and the maxillary palps (sensory structures associated with the

mouth section), shorter than the proboscis. Aedes mosquitoes characteristically keep their

bodies low and parallel to the surface, with the proboscis pointing backwards when

landed.

Bioassay - Refers to the test method employed in calculating the reaction of

Aedes aegypti to the extract Ardisia Tomentosa.

Concentration - The same amount of one element found in another substance.

Crude ethanoic extract - As used in the analysis, this applies to the impure

material derived from Ardisia Tomentosa leaves after extracting ethanol from the

extraction process. Like proboscis, though female A. aegypti has reasonably smooth and

not bushy or thick.

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Endemic plant - A type of plant is limited to a single geographic area, such as an

island or river basin [ CITATION Enc09 \l 1033 ]. In this research, this applies to plants that

are present only in a specific place and nowhere else in the world

LC50 - The threshold level of a toxin that can kill 50% of organisms exposed to

it.

Larvae - An embryonic category of species that transitions to the adult shape.

Larvicide - An insecticide specifically designed to shorten the larval period of the

insect.

Mortality Pace - The number or frequency of deaths in a defined timeframe

compared to population [ CITATION Web02 \l 1033 ]. This analysis applies to the number of

dead larvae over some time while the extract was added.

Plant Extract. A condensed solution produced from various plant sections, such

as bulbs, barks, stems, leaves and fruits. This research refers to a solution extracted from

tagpo fruit peels using ethanol as a solvent obtained by maceration.

References:
Al Fadhli, A. H., Jamal, W. Y., & Rotimi, V. O. (2020). Prevalence of carbapenem-

resistant Enterobacteriaceae and emergence of high rectal colonization rates of blaOXA-

181-positive isolates in patients admitted to two major hospital intensive care units in

Kuwait. PLOS ONE, 15(11), e0241971. https://doi.org/10.1371/journal.pone.0241971

 13

Anderberg, A. A., & Ståhl, B. (1995). Phylogenetic interrelationships in the order

Primulales, with special emphasis on the family circumscriptions. Canadian Journal of

Botany, 73(11), 1699–1730. https://doi.org/10.1139/b95-184

Cyclopedia of American Horticulture. L. H. Bailey. (1902). Botanical Gazette, 33(6),

467–468. https://doi.org/10.1086/328251

Deepalakshmi, S., & Jeyabalan, D. (2017). Studies on Mosquitocidal and biological

activity of endemic plants of Nilgiris Hills against filarial vector, Culex quinquefasciatus

(Say) (Insecta: Diptera: Culicidae). International Journal of Advanced Research in

Biological Sciences (IJARBS), 4(3), 137–151.

https://doi.org/10.22192/ijarbs.2017.04.03.016

Garg, V. (2020, December 16). Osteoarthritis: Lifestyle Changes and Alternative

Therapies. EMediHealth. https://www.emedihealth.com/osteoarthritis-care.html

Jeffries, C. L., White, M., Wilson, L., Yakob, L., & Walker, T. (2020). Detection of Cell-

Fusing Agent virus across ecologically diverse populations of Aedes aegypti on the

Caribbean island of Saint Lucia. Wellcome Open Research, 5, 149.

https://doi.org/10.12688/wellcomeopenres.16030.2

Kay, B., & Nam, V. S. (2005). New strategy against Aedes aegypti in Vietnam. The

Lancet, 365(9459), 613–617. https://doi.org/10.1016/s0140-6736(05)17913-6

Khan, M. A., & Ahmad, W. (2019). Synthetic Chemical Insecticides: Environmental and

Agro Contaminants. Sustainability in Plant and Crop Protection, 1–22.

https://doi.org/10.1007/978-3-030-23045-6_1
 14

Li, S., Wang, P., Yuan, W., Su, Z., & Bullard, S. H. (2016). Endocidal Regulation of

Secondary Metabolites in the Producing Organisms. Scientific Reports, 6(1).

https://doi.org/10.1038/srep29315

Mathew, N., Anitha, M. G., Bala, T. S. L., Sivakumar, S. M., Narmadha, R., &

Kalyanasundaram, M. (2008). Larvicidal activity of Saraca indica, Nyctanthes arbor-

tristis, and Clitoria ternatea extracts against three mosquito vector species. Parasitology

Research, 104(5), 1017–1025. https://doi.org/10.1007/s00436-008-1284-x

Mohan, D. & Ramaswamy, M.. (2007). Evaluation of larvicidal activity of the leaf

extract of a weed plant, Ageratina adenophora , against two important species of

mosquitoes, Aedes aegypti and Culex quinquefasciatus. African Journal of

Biotechnology (ISSN: 1684-5315) Vol 6 Num 5. 6.

Ong, H. G., & Kim, Y.-D. (2014). Quantitative ethnobotanical study of the medicinal

plants used by the Ati Negrito indigenous group in Guimaras island, Philippines. Journal

of Ethnopharmacology, 157, 228–242. https://doi.org/10.1016/j.jep.2014.09.015

Pavela, R., Maggi, F., Iannarelli, R., & Benelli, G. (2019). Plant extracts for developing

mosquito larvicides: From laboratory to the field, with insights on the modes of action.

Acta Tropica, 193, 236–271. https://doi.org/10.1016/j.actatropica.2019.01.019

Pharmacognostical Standardization of Pterygota Alata (Roxb.) Bark. (2018).

International Journal of Green and Herbal Chemistry, 7(1).

https://doi.org/10.24214/ijghc/gc/7/1/16772

Pitasawat, B., Champakaew, D., Choochote, W., Jitpakdi, A., Chaithong, U.,

Kanjanapothi, D., Rattanachanpichai, E., Tippawangkosol, P., Riyong, D., Tuetun, B., &
 15

Chaiyasit, D. (2007). Aromatic plant-derived essential oil: An alternative larvicide for

mosquito control. Fitoterapia, 78(3), 205–210.

https://doi.org/10.1016/j.fitote.2007.01.003

Tanawat, C., Sedthapong, L., & Wallapa, W. (2020). Screening seven commercial

essential herb oils for larvicidal activity against the mosquito Aedes aegypti (Linnaeus), a

vector of the dengue virus. Journal of Applied Pharmaceutical Science.

https://doi.org/10.7324/japs.2020.10706

1. You stated in page 5 that “mosquitoes bring many illnesses that are transmitted

mainly through a mosquito bite on humans”. This implies that there are

alternative methods for infection. What are the other ways that mosquitoes can

transmit diseases to humans other than "bites”?

2.