Efficacy of Male Coconut Beetle (Rhynchophorus Ferrugineus) Extract

Against Escherichia coli

A Thesis Presented to
the Undergraduate School Faculty
San Pedro College, Davao City

In Partial Fulfilment
Of the Requirements for the Degree
BACHELOR OF SCIENCE IN BIOLOGY

By

CATIGARA, Pauline
GENITA, Elane
ENGKONG, Lenzi Jael.

First Semester 2014

Chapter 1

Introduction

The red palm weevil, Rhynchophorus ferrugineus, is a species of snout beetle also known
as the Asian palm weevil or sago palm weevil. Originally red palm weevil is from Asia which
was spread throughout Africa and Europe then reaching to the Mediterranean. Red palm weevil
was well known as a pest that damage palm trees (S. T. Murphy1 and B. R. Briscoe2 ). They are
usually attracted to unhealthy palm trees, but often attack healthy palm trees too.
Natural antibiotics are those remedies that are capable of inhibiting the growth of
microorganisms or eliminate them without significant toxicity to the human or animal host. They
are remedies that are able to prevent or cure many diseases. There are advantages in natural
antibiotics. They do not present so many side effects for animals or humans. And they are cheap
and easily available. Some antibiotics can be extracted from insects that can be used to naturally
kill or inhibit the growth of bacteria.
Chemical studies are needed to discover in order to find active compounds that are
actually present within insect bodies. There is a therapeutic potential of insects that has an
immense contribution to biodiversity conservation, as well as opening a perspective for the
economic and cultural valorization of animals which are traditionally regarded as useless.
Different human cultures all over the world have been using some insects as a source of
medicine. Chemical studies show that there are active compounds found inside insect bodies.
Bioactive molecules have been identified in pheromone of insects. Some insects that are being
used as a source of medicine are pests especially in plants. One of those pests is found in young

palm trees- the Red Palm Weevil (Rhynchophorus ferrugineus). Usually palms less than 20 years
of age are being attacked. It is considered as the most damaging insect pest of palms in the
world. Red palm weevils are native to Southeast Asia and are very active during day and night
time. (Hancock and Chappel, 1999).
Knowing that insects could be a possible sources of antibiotics, we, the researchers were
motivated to conduct a research study which would utilize the Red Palm Weevil Rhynchophorus
ferrugineus which are commonly regarded to as pests in the coconut farms. We hope to
determine the efficacy of male coconut beetle extract against Escherichia coli. We would like
also to determine what specific concentration of the extract is most effective in inhibiting the
growth of Escherichia coli.

Review of Related Literature

Red Palm Weevil

Red palm weevil has its own defensive mechanism against predators and microorganism
despite of being a pest of palm trees. Its outer layer of the skin covering its body has an
antimicrobial activity of those adults, larvae and as well as of eggs. The antimicrobial activity
was tested against gram positive bacterium: Bacillus subtilis (Ehrenberg) Cohn and Bacillus
thuringiensis Berliner, the Gram-negative bacterium Escherichia coli Escherich, and the
entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae
(Metchnikoff) Sorokin. Another examination was being conducted using the hemolymph of red
palm weevils larvae infected by Pseudomonas aeruginosa (Schroter) Migula, E. coli and
staphlococcus aureus Rosenbach. The extraction of the polar surface from the adult and large
larvae of red palm weevil inhibits gram positive bacteria and the B. bassianas growth, but not
the growth of E. coli and M. anisopliae, the same way to the larvaes hemolymph and the surface
extracts of both small larvae and eggs seemed not to show any inhibition.
Red palm weevil has become domesticated in isolated areas in every country of south eastern,
southern and western Asian according to OEPP/EPPO (2005:2008). Today, red palm weevil was
dispersed in Europe, Africa, Oceania and Asia (Zhang et al., 2003; Al-Ayedh, 2008; Yuezhong et
al., 2009) continuing its spreading to Mediterranean basin. The rapid spread was cause by
abundant shipment of plants from one country to another and by the practicing of the intensive
modern date palm-farming was also favoured by this pest (Faleiro, 2006). The damaged trunk of
palms secreted a semiochemical called kairomone which the red palm weevil is attracted and
here the development of larvae occurs. Eventually this palm will die and collapsed. Female red
palm weevil lays 58-531 eggs that hatch in 1-6 days (faleiro, 2006). The complex in discovering

of its attack at an early stage of infestation is due to the red palm weevils concealed tissue borer.
There have been several attempts to isolate pathogens of the red palm weevil ( Gindin et al.
2006,: El sufty et al. 2007,: Guerri-Agullo et al. 2008,: Salamana et al. 2004) which
cytoplasmatic polyhedrosis virus was discovered (Gopinadhan et al. ) Pseudomonas aeruginosa
(Schroter) Migula and yeast isolated from the RPWs hemolymph but none of these has the
possible potential biocontrol agent because their application in natural condition is limited
( Salama et al. 2004). Humoral and cuticular resistances in insects include inductive
antimicrobial peptides (Meisteet a., 2000;Schmid-Hempel, 2005) where these will be found
mainly in the circulating hemocytes, in the cells of the fat body and to a lesser extent cuticular
cells, midgut, salivary glands and reproductive organs (Schmid-Hempel, 2005). A variety of
antimicrobial peptides have been representing to date from insects (Saido-Sakanaka et al., 2005;
Turillazzi et al., 2004; 2006). Based on the observation of S. Longo from his unpublished data
the larvae have the capability to inhibit microbial growth on the rearing substratum. His aim to
his research was to analyze the outdoor and indoor defense systems of red palm weevil not only
analyzing alone but also they trying to understand whether there are different stages of red palm
weevil by using microbiological techniques that inhibits the different growth of microorganisms
including entomopathigens. S. Longo used two approaches: first target is to analyze the external
surface of the red palm weevil, whereas the second he investigated the activity of humoral part of
larval hemolymph. S. Longo regulated a preliminary chemical analysis of the fraction with
antimicrobial activity by MALDI-TOF mass spectrometry.
There is a study that entitled Pharmacological action of integument extracts of red palm
weevil, Rhynchophorus ferrugineus on albino mice. These extracts were prepared from the
tergum and sternum of both sex (male and female) red palm weevil, or called Ryhnchophorus

ferrugineus, they were using acetone, chloroform and diethyl ether. Pharmacological actions
were examined in mice; male Swiss albino mice were used in this study. All of these extracts
showed certain behavioral actions that included central nervous system inhibition and
stimulation with no effects on pain perception or temperature regulation.
Males and females insects Ryhnchophorus ferrugineus were dissected to remove the tergum and
sternum parts. Known weights were extracted separately with one of the following solvents:
acetone, chloroform or diethyl ether extracts were centrifuged at 4000 rpm (revolution per
minute) for 20 minutes. The supernatant were collected and then evaporated to obtain the
residues. These residues were then suspended in 0.25% aqueous solution of sodium carboxyl
methyl cellulose. (ScienceHu,2012)

A study that entitled Coleopteran Antimicrobial Peptides: Prospects for Clinical

Applications. Antimicrobial peptides (AMPs) are activated in response to infected area and have
important roles in vertebrate and invertebrate immune systems. AMPs act directly against
pathogens and have both wound healing and antitumor activities. Although coleopterans
(commonly called beetles) comprise the largest and most diverse order of eukaryotes and occupy
an earlier branch than Drosophila (fruit flies) in the holometabolous (complete metabolism)
lineage of insects, their immune system has not been studied extensively. Initial research reports,
however, indicate that coleopterans possess unique immune response mechanisms, and studies of
these novel mechanisms may help to further elucidate innate immunity. Recently, the complete
genome sequence of Tribolium (flour beetle) was published, boosting research on coleopteran
immunity and leading to the identification of Tribolium AMPs that are shared by Drosophila and
mammals, as well as other AMPs that are unique. AMPs have potential applicability in the

development of vaccines. They review coleopteran AMPs, their potential impact on clinical
medicine, and the molecular basis of immune defense.
Cationic peptides have emerged as important targets for the development of therapeutics against
bacteria, fungi, viruses, and parasites. They are key effector molecules in host defense through
direct and indirect antimicrobial activity. Furthermore, in vertebrates, these peptides mediate a
variety of cellular processes such as immunomodulation, wound healing, and tumorigenesis
(formation of tumors). These roles provide opportunities for the development of therapeutic
products and vaccines. Antimicrobial peptides are attractive molecules for the development of
clinical and veterinary therapeutics because they are fast acting and effective against susceptible
pathogens, are less likely to cause the emergence of resistance compared to traditional
antibiotics, have low toxicity to mammalian cells, and their mode of action tends to be more
physical rather than targeted at metabolic pathways. ( Int J Microbiol,2012)

Conceptual Framework

Efficacy of male coconut beetle(Rhynchophorus Ferrugineus) extract against E.coli.

Independent Variable

Dependent Variable

Male Coconut beetle extract

Effectiveness against E.
coli

(different concentrations)

Figure 1: Conceptual Paradigm of the Study

Statement of the Problem

This study aims to know the Efficacy of male coconut beetle(Rhynchophorus
Ferrugineus) extract against E.coli. Specifically, it aimed to answer the following questions:
1. What is the effect of male coconut beetle(Rhynchophorus Ferrugineus) extract
against E.coli?
2. What is the relationship between the different concentrations of male coconut beetle
extract against E.coli?
3. What concentration of male coconut beetle extract is most effective in controlling the
growth of E.coli?

Chapter 2

Methodology

Research Design
This experiment will use experimental research design. It is concerned with determining
the antibacterial property of the beetle extract against E. coli. We will use random sampling
technique in collecting our sample.
Research Locale
The experiment will be conducted at San Pedro College Laboratory. However the beetles
will be collected at Marilog District, Davao city.
Subjects of the Study
In conducting this study, male coconut beetle (Rhynchophorus Ferrugineus) will be used
as test subject for extraction to be used as an antibiotic against E.coli.
Procedures
A. Culturing E.coli
The E. coli will be obtained from San Pedro Laboratory and will be cultured. The
cultured E.coli will be placed in agar plates and will be stored until the beetle extract will be
ready for testing.
B. Crude extraction of beetle
The beetle will be collected from Marilog District, Davao City. Approximately 10 beetles
will be collected for the test. The beetle will be crushed using mortar and pestle and will
be centrifuged at 5000 RPM for 10 mins. The suspension will be removed and will be
filtered using a filter paper. The liquefied beetle extract will be placed in a 500 ml beaker.
C. Preparation of beetle extract
Using three test tubes, the extract will be differentiated into different concentrations by
adding distilled water in different quantity.

Concentrations
1st Concentration
2nd Concentration
3rd Concentration

Beetle Extract
20 ml
40 ml
80 ml

Distilled Water
80 ml
60 ml
20 ml

D. Paper Disc Method

Strains of bacteria will be spread over to 3 sterile agar plates using sterile cotton buds.
Sterile paper discs of 5mm will be placed at the center of each agar plates containing the
bacterium. The paper discs then will be loaded with 5 drops of the beetle extract.
Bacterial cultures will then be incubated for 18-20 hours before observing the zone of
inhibition. Cultures will be examined of antibacterial activity as zone of growth inhibition
and the results will be tabulated.
Limitations of the study
In this study, only three concentrations of beetle extract will be used, the 20ml, 40ml,
and 80ml. we will only use the E.coli bacteria as our test organisms. In each concentration, three
replicates will be used. The study will be conducted in San Pedro College Laboratory.
E. Positive Control and Negative Control
We will use for our positive control and distilled water for our negative control.