EFFECT OF ALLELOCHEMICALS FLAVONE ON DIGESTIVE ENZYMES ACTIVITY IN

BANANA STEM WEEVIL (ODOIPOROUSLONGICOLLIS)

A PROJECT REPORT
SUBMITTED BY

STEPHY JOSE TFARSCH029

GREESHMA R TFARSCH015
MUHAMMED MARFIN VK TFARSCH003
ATHUL M SURESH TFARSCH013
ASRITH K TFARSCH012

In partial fulfilment for the award for the degree

Of
BACHELOR OF SCIENCE – CHEMISTRY
In
FATHIMA ARTS AND SCIENCE COLLEGE, MOOTHEDAM
Malappuram, Kerala
Affiliated to University of Calicut
March 2019
 CERTIFICATE

This is to certify that this dissertation entitled ‘’EFFECTS OF ALLOLOCHEMICALS

FLAVONE ON DIGESTIVE ENZYMES ACTIVITY IN BANANA STEM WEEVIL
(ODOIPOROUSLONGICOLLI)’’ is a authentic record of the project work carried out by
STEPHY JOSE (TFARSCH029), GREESHMA R (TFARSCH015), MUHAMED
MARFIN V.K (TFARCH003), ATHUL MSURESH (TFARSCH013), ASRITH K
(TFARSCH012), student of BSC chemistry in FATHIMA ARTS AND SCIENCE
COLLEGE, MOOTHEDAM under my supervision in partial fulfilment of the
requirements for the Bachelor Degree in Chemistry of the University of Calicut.

Moothedam
28-02-2020 AMRITHA MINNU K
 DECLARATION

We hereby declare that the project entitled “EFFECT OF ALLELOCHEMICALS FLAVONE ON

DIGESTIVE ENZYMES ACTIVITY IN BANANA STEM WEEVIL (ODOIPOROUSLONGICOLLI)” is a
record of original and independent research work carried out in the KINFRA TECHNO
INDUSTRIAL PARK MALAPPURAM, during the period of October 1-6th 2019 by us under
the guidance of external guide Dr.Vijesh Varghese submitted to the Calicut university in
partial fulfilment for requirement of the degree of Bachelor of science 2017 -2020 now part of
this has previously formed the basis for the award of any degree/diploma/fellowship or other
similar titles of this or any other university or society

MOOTHEDAM STEPHY JOSE

March 2020 GREESHMA R

MUHAMMED MARFIN V K
ATHUL M SURESH
ASRITH K
 ACKNOWLEDGMENT

I thank god, the almighty for helping me to successfully complete this project work. I
express my sincere gratitude to Dr Vijesh Varghese for his guidance and encouragement
this project work.
I wish to extend my heartfelt thanks to Ms Amritha minnu k, deputy of chemistry of my
college for her support and necessary help.
I wish to express my thanks to the principal of the college, Dr Ravindran pillai for
providing facilities.
I thankful to the teaching and non teaching staffs of the department and my beloved
friends for their valuable suggestion and co-operation during the project work

STEPHY JOSE

GREESHMA R
MUHAMMED MARFIN V K
ATHUL M SURESH
ASRITH K
 CONTENTS

CHAPTERS PAGES

INTRODUCTION 1-8

REVIEW OF LITERATURE 9-18

AIM AND OBJECTIVES 19

MATERIAL AND METHOD 20-24

RESULT AND DISCUSSION 25-35

SUMMARY AND CONCLUSION 36

REFERENCE 37-45

1. Introduction
 Bananas and plantains are one of the world’s important food commodity and
rank fourth in terms of gross value, exceeded only by rice, wheat and milk/milk
products. These are important staple food crops in the humid and sub-humid
tropical regions of the world. Banana provides nourishment and a well-balanced
diet to millions of people around the globe and contributes to livelihood through
crop production, processing and marketing (Padmanaban and Sundararaju,
1999). India is the largest producer of banana with an annual production of 11.7
million tonnes on 404,000 ha, contributing to 27% of the world production and
about 37% of the total fruit crop production in the country. India has a rich
genetic diversity of banana with more than 90 distinct clones. Depending on the
contribution of Musa acuminata and Musa balbisiana,the cultivars have been
classified into genomic groups. Banana is grown under diverse conditions and
production systems and hence selection of varieties is based on needs and
situations. Around 20 cultivars viz. Dwarf Cavendish, Robusta, Monthan,
Poovan, Nendran, Red banana, Safed Velchi, Basrai, Ardhapuri, Rasthali,
Karpurvalli, Karthali and Grande Naine etc. are commercially cultivated
(Sidhaet al., 2007).

1.1. Banana stem weevil

The banana stem weevil (BSW) or banana pseudostemborer,

Odoiporuslongicollis Oliver (Coleoptera: Curculionidae) is one of the most
important pests of bananas and plantains. The adult weevils are black-coloured
and measure 23-39 mm. Red-coloured morphs of the BSW are also encountered
in certain banana-growing areas of India. Based on mating studies, it has been
concluded that the colour difference is not due to sexual dimorphism but is a
phenomenon of non-sex limited variation and of sympatric (Nahifet al., 2000).
The weevils are predominantly nocturnal in habit, although during cloudy days
and cooler months they may fly during the daytime. They often confine
themselves within the pseudostem and in the decomposing tissues of harvested
pseudostems. All life stages of the weevil are present throughout the year in the
infested plant. Adults are strong fliers and in this way, move from plant to plant.
The BSW has a long life span and many adults live for a year. The sex ratio
of adults encountered in banana gardens is 1:1.17 (male:female) (Nahifet al.,
2000). The sensory structures present on the rostrum of the weevils provide a
key for sex differentiation (Charles et al., 1996). The pre-oviposition period is
15-30 days and the adult weevils mate throughout the day and night. The mean
number of eggs laid by a female following a single mating is nine eggs at the
rate of one egg per day. Gravid females lay yellowish white, elliptical eggs by
inserting the ovipositors through ovipositional slits cut by the rostrum on the
outer epidermal layer of the leaf sheath of the pseudostem down to the air
chambers. Oviposition takes place only in the leaf sheaths. The number of eggs
deposited is considerably reduced as the number of weevils increases, indicating
the existence of a spacing pheromone, epideictic compounds which act as a
deterrent to conspecific females (Dutt and Maiti, 1972)

Kingdom Animalia
Phylum Arthropoda
Class Insecta
Order Coleoptera
Family Curculionidae
Genus Odoiporus
 Species Odoiporuslongicollis .

Eggs are cream in colour and cylindrical in shape with rounded ends.
Typically, eggs are 3.14 mm in length and 1.1 mm in diameter. The incubation
period ranges from 3 to 8 days. The emerging larvae are fleshy, yellowish white
and apodous. The larvae feed on tissues of the succulent sheath by tunnelling
extensively and may reach as far as the true stem. If larvae emerge during the
advanced pre-flowering stage of the plant, the ascending flower bud and the
peduncle inside the pseudostem can be eaten and damaged, resulting in non-
emergence of the flower bud which decays inside the pseudostem (Ranjith and
Lalitha, 2001). In severely infested plantations, more than 20% plants do not
flower due to this reason. The depth of the tunnel made by the larvae
rangesbetween 8to10 cm. The tunnels are widespread and may go as high as the
fruit peduncle or to the lowermost collar region near the rhizome. The larvae
pass through five instars. The fifth instar larvae enters a non-feeding pre-pupal
stage and constructs a cocoon by winding short pieces of fibrous materials of
the sheath around its body. The pupa is exarate and present inside the cocoon.
The developmental rates are highly dependent on climatic factors with the
duration of the life stages longer in the winter season than in the summer. Under
laboratory conditions the duration from egg to adult stage is 44 days.
Adult BSW are attracted by the volatiles released by the banana plants.
Infestation of the weevil normally starts in 5-month-old plants. Early symptoms
of the infestation are the presence of small pinhead-sized holes on the stem,
fibrous extrusions from bases of leaf petioles, adult weevils and exudation of a
gummy substance from the holes on the pseudostem.
 Fig 2. Life cycle of Odoiporuslongicollis.(Padmanabanet al., 2001)

During the advanced stages of infestation, the stem, when split open, exhibits
extensive tunnelling both in the leaf sheath and in the true stem. Rotting occurs
due to secondary infection of pathogens and a foul odour is emitted. When the
true stem and peduncle are tunnelled after flowering, the fruits do not Adult
stem weevil and grub feeding on leaf sheath. develop properly, presenting a
dehydrated condition with premature ripening of the bunch itself. Stem weevil
infestation interferes with the translocation of nutrients and water, retards
growth and development and increases susceptibility to wind lodging, which is
more commonly associated with nematode infestation. Weakening of the stem
by larval tunnelling may result in breakage by wind or inability to bear the
weight of the maturing bunch. It is estimated that the stem weevil causes 10-
90% yield loss depending on the growth stage of the crop and management
efficiency. The severity of the loss is greater when infestation occurs at the early
vegetative stage (5 months old) (Padmanabanet al., 2001).
The BSW is believed to have originated in South and South East Asia, which
is also the centre of origin of the present day bananas and plantains. This insect
is found in India, China, Malaysia, Indonesia and Thailand and is a key pest of
bananas and plantains, posing a great threat to banana production systems in
these countries (Padmanaban and Sathiamoorthy, 2001). Pest density may vary
from field to field. The weevil prefers plantains and highland bananas,
particularly ’Pome’ types. Total crop failure will result in farms where the
weevils are not managed efficiently. Such crop failures are not uncommon in
banana production systems in India.
1.2. Control of Banana stem weevil

Measures to curb BSW damage vary widely depending upon the type of
banana production systems practised. Large plantations resort to regular
application of chemical insecticides to control the weevil. Resource-limited
marginal farmers cultivating banana as a subsistence crop are unable to
undertake chemical pesticide interventions on a regular basis. In this situation,
cultural control strategies assume greater significance due to their ease of
application and their compatibility with other methods of control. Natural
enemies including arthropods, entomopathogenic nematodes and
entomopathogens have great potential to reduce the population of the weevils
in severely infested gardens. Screening Musa germplasm for resistance to BSW
has the potential to identify the source of resistance genes that could be used
in plant breeding programmes (Sidhaet al., 2007).
Chemical control

Control of BSW is an elusive and complex problem as the life cycle of the
pest may be completed within the pseudostem. Application of organochlorine
insecticides is no longer carried out due to the possible development of
insecticide resistant weevil strains and environmental concerns. Currently stem
injection of a systemic organophosphorus compound (e.g. monocrotophos) is
extensively used in controlling the pest. As well as stem injection, other
insecticide application methods may be used, such as swabbing along with
surfactants, swabbing with mud slurry containing the candidate insecticide,
spraying and fumigation of the spaces between the leaf sheaths in the
pseudostem. Fumigation of banana plants using Celphos (aluminium
phosphide tablets), especially during the vegetative phase is phytotoxic and
should be discouraged (Valmayoret al., 1994).

Cultural control

Field sanitation is imperative in the control of this pest. Dried old leaves
must be removed to allow the detection of early symptoms of weevil
infestation and to increase the efficacy of chemical application. Suckers should
be pruned periodically and infested pseudostems must be removed from the
field and destroyed. Banana stumps kept in the field after harvest must be
removed and destroyed as they serve as weevil refuges and breeding sites.
Investigations made at the National Research Centre for Banana (NRCB) in
India have indicated that traps could be efficiently used to monitor and reduce
the adult weevil population. Among the disc-on-stump and longitudinal split
pseudostem traps, the disc-on-stump traps with higher exudations of plant
fluids have been found to be more effective. However, in general, banana corm
weevil outnumbered the BSW in the traps (Padmanabanet al., 2001). Use of
pheromones to trap and destroy weevil populations is under investigation at
NRCB.

Biological control

Research on biological control of BSW is inadequate and research reports

are scanty. Two species of earwigs feedingon larvae and pupae are reported
from China. There is a report of an acarid mite parasitizing larvae and adults.
Release of an ectoparasitic mite, Uropodia sp. on adult BSW had been tried for
its control with limited success. Metarhiziumanisopliae, an entomopathogenic
fungus, effected more than 90% mortality under laboratory conditions. Fungal
pathogens such as Fusariumsolani, Mucorheimalis,
AspergillusnigerandScopulariopsisbrevicaulis have also been isolated from field
populations of the BSW. Although these entomopathogenic fungi caused more
than 90% mortality in the laboratory, there is a long way to go before they can
be used in the field, as the safety of these fungi to non-target organisms is still
to be tested and efficient mass production systems and application methods
need to be devised. Entomopathogenic nematodes are yet to be isolated from
BSW endemic areas.

Host plant resistance

Host plant resistance may offer a long-term solution to the problem.

Screening trials and surveys to determine the resistance to BSW need further
work although it appears that the pest, with the aid of a host of sensory
structureslocated on the antennal tip and mouthparts, exhibits a high degree
of host plant preference. Resistance to BSW seems to depend on the
morphological and anatomical characteristics of the banana leaf sheath along
with the interaction of the chemicals present in plant sap, thus suggesting a
combination of antixenosis and antibiosis mechanisms. Through field screening
of 212 banana accessions of various genomes, Charles et al.(1996) identified 27
accessions that exhibited tolerance to the pest (Sidhaet al., 2007). A t NRCB,
laboratory screening of 119 accessions led to the identification of a high
degree of resistance in Musa balbisianaclones, such as Bhimkol, Athiakol,
Elavazhai and Sawai. In general, plantains are the most preferred host.

The BSW is believed to have originated in South and South East Asia, which
is also the centre of origin of the present day bananas and plantains. This insect
is found in India, China, Malaysia, Indonesia and Thailand and is a key pest of
bananas and plantains, posing a great threat to banana production systems in
these countries (Charles et al.,1996). Pest density may vary from field to field.
The weevil prefers plantains and highland bananas, particularly ’Pome’ types.
Total crop failure will result in farms where the weevils are not managed
efficiently. Such crop failures are not uncommon in banana production systems
in India.
1.3. Allelochemicals

Allelochemicals representing numerous chemical groups have been isolated

from over 30 families of terrestrial and aquatic plants. Some of the compounds
also have been isolated from soil in quantities sufficient to reduce plant
growth. Although selected allelochemicals are believed to influence plant
densities and distributions, none isolated from higher plants have been
considered active enough for development as commercial herbicidal products.
Almost all herbicidal allelochemicals exist in plants in nontoxic, conjugated
forms. The toxic moiety may be released upon exposure to stress or upon
death of the tissue. The most successful use of allelochemicals in weed control
has been management of selectively toxic plant residues. For example, rye
residues have controlled weeds effectively in a variety of cropping systems.
Several weed species may interfere with crop growth through chemicals
released from their residues. A number of noxious perennial species appear to
exploit allelochemicals in their interference processes.

Fig 3. Molecular structure of the flavone backbone with numbers.

Flavones, are a class of flavonoids based on the backbone of 2-

phenylchromen-4-one. Flavones are common in the food supply, mainly from
spices, and red–purple fruits and vegetables. Common flavones include
apigenin, luteolin, tangeritin, chrysin, and 6-hydroxyflavone.Flavones have no
proven physiological effects in the human body and no antioxidant food value.
Following ingestion and metabolism, flavones, other polyphenols, and their
metabolites are absorbed poorly in body organs and are rapidly excreted in the
urine, indicating mechanisms influencing their presumed absence of metabolic
roles in the body. However, some of the flavones are used against Arthropods
in several literatures.
2. Review of literature

Pests of horticulture importance are evolving rapidly with changing climatic

conditions, intensive farming practices, and constant selection pressures
exerted through insecticides. With worldwide interest in environmental
protection, chemical insecticides have become objects of scientific and popular
protest. Critics charge chemical insecticides of their danger in provoking the
development of resistant strains of pests, sabotaging ecological systems, and
poisoning the environment. These liabilities of chemical insecticides have
paved way to nonchemical methods, which use natural processes and
mechanisms against insect pests. Of several natural processes available for
exploitation of management of insect pests, semiochemicals are less exploited
inspite of their ability in making integrated pest management (IPM) programs
sustainable in the long run.

In India, banana is the second most important fruit crop next to mango.
Itranks first in production and third in area among fruit crops grown in India.
Itsavailability, affordability, varietal range, taste, nutritive and medicinal value
enablesit as the favourite fruit among all classes of people. Banana accounts
for 13%of the total area and 33% of the production of fruits. Production is
highest inMaharashtra (3924.1 thousand tones) followed by Tamil Nadu
(3543.8 thousandtonnes). Among banana growing states of India, Maharashtra
has the highestproductivity of 65.70 metric tonnes /ha as against national
average of 30.5 tonnes/ha. The other major banana producing states are
Karnataka, Gujarat, AndhraPradesh and Assam.

Status in India
 Banana has occupied a top position in India’s booming fruit industry
withan annual production of 13.5 MT from an area of 4.0 lakh ha
(Sathiamoorthyet al., 2000). Banana is widely grown in India on about 2,70,000
hectares andcovers about 16 per cent of total fruit grown area. Banana is
attacked by severalinsect pests during different growth stages of the plants.
More than 470 speciesof insects and mites recorded have been recorded
attacking banana. Of these,250 are foliage feeders, 10 are pseudostem borers,
70 feed on roots and rhizomes,130 feed on fruits and flowers, and more than
10 are disease vectors. In addition,58 species of ants have been collected from
Central American banana plantations.Earlier Roy and Sharma (1952) have also
documented several insect pestsattacking banana in India. The pests feeding
on banana seedling are bananastem borer, banana aphid and banana scale
moth. Amongst them, banana stemborer is the most destructive insect pest
and causing considerable damage tothe commercial production of banana in
India.

Economic Importance

Banana is a very popular fruit due to its low price and high nutritive
value.It is consumed in fresh or cooked form both as ripe and raw fruit and is a
richsource of carbohydrate and vitamins B. It is also a good source of
potassium,phosphorus, calcium and magnesium. The fruit is easy to digest,
free from fatand cholesterol. Banana powder is used as the first baby food. It
helps in reducingrisk of heart diseases when used regularly and is
recommended for patients suffering from high blood pressure, arthritis, ulcer,
gastroenteritis and kidneydisorders. Processed products, such as chips, banana
puree, jam, jelly, juice,wine and halwa can be made from the fruit. The tender
stem, which bears theinflorescence is extracted by removing the leaf sheaths
of the harvestedpseudostem and used as vegetable. Plantains or cooking
bananas are rich instarch and have a chemical composition similar to that of
potato. Banana fibre isused to make items like bags, pots and wall hangers.
Rope and good qualitypaper can be prepared from banana waste.
(http://nhb.gov.in/report_files/banana/BANANA.htm).

Insect Pests of National Significance

Banana and plantain are susceptible to a large magnitude of insect pestsand

diseases. Some insect pests and diseases are quite serious and contagiousand
can easily spread with the planting materials. After establishment, they
arepersistent and very hard to or practically impossible to manage. There are
severalinsect pests causing damage to the banana plants at different
developmentalstages and thus reducing the yield potential of successful
banana cultivation.Insect pests of banana can cause significant damage to
fruits (e.g., BananaRust thrips, CheatanophothripssignipennisBagnall, flower
thrips, ThripsflorumSchumtz; Fruit fly; moths/caterpillars, scales), leaves (e.g.,
cyclamenmite, Phytonemuspallidus; Banana lacewing bug,
StephanitistypicusDistant;leaf thrips, HelionothripskadaliphilusRamak; Banana
leaf eating caterpillar,SpodopteralituraFabricius; Bag worm), corms and
pseudostems (e.g., Bananarhizome weevil, CosmopolitussordidusGermar;
Banana stem weevil,OdoiporuslongicollisOlivier), and can transmit important
plant pathogens(e.g., Banana aphid, PentalonianigronervosaCoquerel transmit
banana bunchytop virus). Damage due to insects can greatly reduce the
marketability of bananafruits.Apart from insect pests, some of the non-insect
pests are also worthmentioning for causing damage to the banana plants. They
include- bananasnails, ants, mites, rodents, and nematodes. A thorough
knowledge of agro-ecosystemanalysis of the banana and plantain is essential
to Integrated PestManagement of insect pests affecting banana. Wild birds,
bats, and rodents notonly feed on banana fruits but often construct nests
within the bunch, poised tofeed their young ones when the bananas ripen.
Rats are also a serious problemfor islanders dependent on bananas, therefore
bananas should be harvested justbefore or at the first sign of rat damage, then
hung upside-down in a rat-freeenvironment until ripe.

Insects are the only animals giving man a real battle for supremacy. They
have been upon this earth for about 300 million years and have developed
special adaptations to live under various environmental conditions. Insects
become "pests" of man when their existence conflicts with his profit,
convenience and welfare. Arthropod pests are responsible for global pre- and
post-harvest crop losses of approximately 20-50 % of potential production and
for transmitting a number of the world's most important plant and animal
diseases. There are many weapons for insect control found in today's arsenal,
but no method is without drawbacks. Insects can be controlled by legislative
methods, physical methods (direct killing of insects using heat, light, X-rays and
so on, reduce reproductive capacity or to attract them to something that will
lull them), genetic method (sterile male method), ecological methods (involve
removal, destruction, modification or isolation of materials that might favour
the survival of an insect pest), biological methods (regulation of pests using
predators, parasitoids, nematodes and microbial agents) and chemical
methods (using natural or synthetic chemicals that act as insecticides,
repellents, attractants, antifeedants and chemosterilants). Genetically
modified crops have been developed that have resistance against insect attack.

Chemical tools for insect control have evolved rapidly in the last 100
years, starting with inorganic agents such as lime, sulk and arsenicals, and
natural products such as pyrethrum, rotenone and nicotine. These chemicals
are considered as first generation pesticides. A series of dramatic discoveries
during the late 1930s provided new synthetic insecticides of unprecedented
power and range of activity that further shifted the emphasis to the chemical
approach of insect control. The wonder insecticide DDT was the first of the
second generation pesticides (organochlorine, organophosphate and
carbamate), which revolutionized insect control.

The conventional pesticides, many of which are basically neuro-

toxicants, which are widely used for the control of insect pests, are highly
toxic, non- selective and persistent in nature (Carson, 1962). The reckless use
of these broad- spectrum second-generation pesticides on a large scale could
constitute an ecological disaster of the first rank. Their continued use has led
to the development of insecticide resistance (emergence of multi-resistant
strains of pests), resurgence of pest populations, change in the pest status,
destruction of beneficial insects, bio-magnification of residues and hazards to
the health of higher animals including human beings. Pesticides have thus
been responsible in causing a considerable deterioration of environmental
quality.

This complicated situation has compelled us to change our attitude and

to search for other safer alternatives and eco-friendly technologies, ultimately
leading to the birth of bio-pesticides.

Botanicals in Pest Management

The use of plant extracts as insecticides can be dated back to at least

4000 years. Rig Veda (2000 BC) makes reference on to the use of poisonous
plant for pest control. There are more than 3,08,000 plant species on our
earth. Among these, approximately 2400 plant species have been recorded as
being useful for pest control. Among the plants evaluated, neem has emerged
as number one and most effective source of pesticide. In India alone, neem has
been evaluated against 105 species of insects, 12 species of nematodes and 9
species of fungi (Singh and Kataria, 1991). Schmutterer and Singh (1995) listed
417 species of insects susceptible to neem the world over. These include
almost all the key pests of agriculture. Though a number of plants were
screened in the first half of 20th century and various reviews published, only
three insecticides, viz. nicotine from Nicotianatabacum, rotenone from Derris
sp. and pyrethrum from Chrysanthemum cinerariaefolium were
commercialized and only pyrethrum survived in the second half of the 2oth
century. The other two were gradually pushed out from the market by
synthetic insecticides. Pyrethrum is still being used and is considered as an
ideal insecticide because it is much safer, non- persistent and at the same time
exhibits high toxicity against insect pests. The pesticidal plants remained
neglected ever since the powerful synthetic pesticides entered the market.
These plants were not systematically screened for activity and many plants,
which may have biological activities, could not come to light.

Bio-pesticides of plant origin being indigenous resources with

insecticidal, repellent, antifeedant and insect growth regulatory action are in
use for over a century to minimise losses due to pests in storage commodities.
Generally these pesticides do not possess a quick knockdown effect unlike
synthetic insecticides, which are currently being used in IPM. However, these
pesticides have many advantages over synthetic insecticides.

1.Pesticidal plants possess least or no mammalian toxicity and thus

cause no health hazards

2. No environmental pollution and minimum risk of development of

insect resistance to these pesticides

3. Surface persistence for a longer period of time; no adverse effect on

grain viability, cooking quality and millingrecovery
 4. Unlike synthetic pesticides, which have only one active compound and
ehbit only one type of biological effect, plant derived compounds may have
more than one biological effect

5. Because of renewability, they fit in well in sustainable agriculture and

6. Less expensive and easily available.

Role of plant secondary metabolites in pest control

Insects and plants originated almost simultaneously about 300 million

years ago, and since then insects and plants have been fighting for their
survival. During this long evolutionary history of attacker (insects) and attacked
(plants), plants have developed ways and means to combat the attacker. Of
the variousdefensive mechanisms developed, chemicals elaborated by the
plants are the most important one. Plants are nature's 'chemical factories'
providing the richest source of chemicals on earth.

These chemicals that plants produce to protect themselves against

insect attack are known as secondary plant substances. These are produced as
by- products of major biochemical pathways. Chemically, they include
alkaloids, terpenoids, phenolics as well as many other compounds. Many of
these chemicals have been successfully exploited by humanity for the control
of arthropod pests (Swain, 1977). These chemicals deter feeding, disrupt
development, provide barrier to attack, assist with wound healing, disrupt

digestion and many areneurotoxic to herbivorous pests.

The ultimate practical objective of insect control is to lessen the extent

ofinsect damage to man's possessions or health by the suppression or
preventionof insect outbreaks. From the records of efforts and successes
during a century of the practice of economic entomology, it seems highly
unlikely that it will be possible to eradicate or exterminate any species of
insects. Though many major pests have been brought down to minor status by
the use of chemicals, cultural practices and sterilization by radiation and
chemicals, constant and massive use of synthetic pesticides in certain crops
has led to the development of resistant strains of insects. There have been
several reports of insecticides inducing the production of detoxifying enzymes
that contribute to the development of resistance in insects (Nigam and Misra,
1998).

Like pest management in field crops, insect pest management in stored

grains also involve the use of synthetic insecticides, bio-control agents, sex
pheromones etc. For stored grains, there is also the possibility of using
fumigants and manipulating storage atmosphere with relatively more ease.
Practically none of these approaches have individually been proved
satisfactory to overcome the problem of insects in stored grains. The necessity
of employing various compatible approaches in an integrated manner is now
being increasingly felt (Prakash et al., 1987 a).

Grains treated with emulsifiable concentrate of synthetic insecticides

protect them from insect attack for varying periods of time. The effective dose
of different insecticides tested/ recommended varies greatly with type of
grains, type of storage structures, type of insect pests and storage period.
Effective concentration of the insecticide like malathion which is considered as
a safe grain protectant has to be constantly increased because of the problem
of resistance. Presently, the concentration that proves effective in controlling
insect pests in stored commodities are too high and may lead to health
hazards. It is therefore, desirable to use synthetic insecticides as direct grain
protectants exclusively on grains that are used only as seeds (Prakash et al.,
1987 b).

Phyto-products are promising alternatives to synthetic insecticides for

protection of seed grains during storage. Mixing of dried neem leaves with
stored grains and keeping them between folds of clothes to ward off insects
are rather well known practices still followed in many developing countries. In
Sri Lanka, farmers burn neem leaves to generate smoke for fumigation of
stored paddy to kill insects. Plant products being liberally available, these
indigenous sources of insecticides and insect repellents have been in use for
more than a century in India. The insecticidal property of plants is not very
quick as compared to that of synthetic insecticides and fumigants. A number of
plant products have been in use against insect pests in stored grains including
rice to minimize storage losses due to insects. However, only a few products
are generally adopted at the farmers level (Prakash et al., 1986). Majority of
farmers are not fully aware of the utility of plant products for the purpose of
grain protection.

Worldwide, rice is an important staple food and it is regularly attacked

by several insect-pests particularly at storage. India is one of the leading
producers of rice in the world accounting 20 percent of the global rice
production. It contributes 52 percent of total food grain production and 55
percent cereal production in India. West Bengal is the highest producer of rice
among all the states of India and therefore, it is popularly known as “Rice Bowl
of India”. Storage pests are the major threat of rice and cause significant losses
worldwide. It is estimated that 5-10% of the world’s grain production is lost
due to ravage of insect-pests (Adam, 1998). According to the estimates of
International Grain Council (IGC), the mean quantity losses of grains by the
store grain pests in India are approximately 10% of the annual production,
which is about 12 million tons per year (Kuzmanov and Dimitrov, 2009).

Rice weevil, Sitophilus oryzaeL. (Curculionidae; Coleoptera) is one of the

most destructive pests of stored cereal grains worldwide and consequently it
has been the subject of concern to all the grain handlers. It is a common pest
of warm climate and attacks paddy, wheat, maize and almost all cereals and
their products. Especially such type of insects is very active in warm and humid
climate. India is considered to be the native land of this weevil. It is also one of
the serious pests of commercial grain in transport train wagons, bullock carts
and ships. Its’ young grub bore into the grains and feed inside the contents
rendering them unfit for consumption. They are mainly controlled with
synthetic chemicals like phosphine, organophosphates, and synthetic
pyrethroids. However, the continuous and indiscriminate use of these
chemicals has led to the emergence of insect populations with high levels of
resistance, health hazard due to residual toxicity and environmental pollution.
An alternative is the use of herbal insecticides which could solve these
problems (Khare, 1994).

There are two main types of chemicals used in the control of insect pests
of stored products. They are contact insecticides and respiratory poisons or
fumigants. A contact insecticide is a poison, which is able to penetrate the
insect cuticle and thereby enter the body tissues. A fumigant is a gas or vapour,
which is taken into the body of the insect through its respiratory system.
Contact insecticides can confer long-term protection (usually referred to as the
residual effect), but often tend to be somewhat specific in their effect upon
insect species and to produce more resistance than the respiratory poisons.
Fumigants provide no residual effect, but unlike contact insecticides, have the
power to penetratethroughout stacks or bulks and to become absorbed into
individual grains or kernels, killing all stages of insect life within (Page and
Lubatti, 1963).

Among the contact insecticides, pyrethrum is the best-known natural

insecticide, the important constituents of which are known as pyrethrins. Their
toxicity to insect can be greatly increased through addition of a synergist, such
as piperonylbutoxide. Pyrethrum is virtually non-toxic to man and is therefore
very safe to be used on food. The efficacy of seed treatment with five synthetic
pyrethroids along with malathion was studied against C. maculatus (Patilet al.,
1994) on pigeon pea seed at 4, 8 and 12 weeks after treatment. Decamethrin,
fenvalerate and cyfloxylate were the most effective insecticides, recording
higher adult mortality, lower egg laying and adult emergence. Permeth,
cypermethrin or fenvalerate resulted in high mortality against C. chinensis
(Gupta et al., 1995 a) and deltamethrin is found to be most toxic to C.
maculatus and C. chinensis than cypermethrin, permethrin and fenvalerate
(Ramzan, 1995). Rahman and Yadav (1987) suggested that toxicity due to dust
was higher than that with solutions. Most of the insect species were initially
susceptible to all the insecticides, but mortality declined with time
(Chakanyaka and Giga, 1993).

Pathak and Jha (1999) tested four insecticides namely deltamethrin,

chloropyrifos-methyl, etrimfos and malathion against Sitophilus sp. and
Sitotrogacerealella and found that deltamethrin was the most toxic insecticide.
Bioactivity and residual toxicity of deltamethrin, mixed with rice bran and rice
husk when used were reported by Usharani (1996). Food grains mixed with
these chemicals were offered to the pest insects like T. castaneum, S. oryzae,
R. dominica, C. maculatusand C. cephalonica. Hundred per cent mortality was
reported. It was also noted that a single application of deltamethrin prevented
insect infestation for a period up to 20 months.
 Combined action of methyl quinone, aggregation pheromone and
pirimiphos methyl on T. castaneum larvae was studied by Mondal(1993).
Results showed that methyl quinone acted as a synergist, increasing larval
mortality. When food grains were treated with sodium tetraborate and boric
acid, a significant reduction in oviposition and adult emergence of C. analis was
noticed (Khan et al., 1998). The relative toxicity of seven fumigants on the four
life cycle stages of C. chinensis were studied by Adu and Muthu (1985). The
results revealed that phosphine followed by methyl iodide were the most toxic
to the insect stages, while methyl formate and ethyl formate were the least
potent. The general trend of susceptibility of life cycle stages of the insect to
the fumigant was egg > adults > larva > pupa.

The organochlorines, DDT and BHC were used prior to 1954 as grain
protectant but gradually malathion was adopted as prophylactic spray at 50
mg/m2. The continuous use of this resulted in a gradual rise in the dose up to
150mg /m2 to reach the level of effective kill (Yadav, 1987). Larvae of T
granarium, C. maculatus, Lasiodermaserricorneand S. oryzae among beetles
and E. cautella and C. cephalonica from moths were found least susceptible to
most of the insecticides. Persistent toxicity of some insecticides namely
malathion, DDVP (dichlorovos), methoxychlor, thanite, pyrethrin and pyrethrin
with PBO (piperonylbutoxide) were studied against C. chinensis by Dhariet al.
(1977)

Role of botanicals inpest management

During the development of organochlorine, organophosphorus and

carbamate insecticides, the toxicological studies conducted were restricted to
the immediate mammalian toxicity, which was found to be within safe limits.
However, the indiscriminate long-term use resulted in bio-accumulation
(because of their non- degradability) and have caused serious health hazards
to human beings and have upset the ecosystem. Moreover, it has been
observed that insects have developed resistance to a number of these
chemicals (Ayyangar and Nagasampagi, 1990). This has prompted the scientists
to turn towards nature and a search for safer insect control agents from
botanicals, i.e., plant products all over the world.

In recent years, many researchers have focused on the search for natural
products derived from terrestrial plants as natural insecticides. Terrestrial
plants are known to contain a rich source of bioactive metabolites which show
antifeedant, repellent and toxic effect in a wide range of insects (Ukehet al.,
2009). The diatomaceous earth (DE) is also considered to be as an alternative
to the traditional insecticides. The DEs are natural in origin and composed of
the fossils of diatoms that in general act as desiccants when applied on the
cuticle of insects (Athanassiouet al., 2004 ).

During the co-evolution of plants and insects, plants have biosynthesized

a number of secondary metabolites to serve as defence chemicals against
insect attack. These defence chemicals may serve as insecticides, antifeedants,
oviposition deterrents, growth inhibitors, juvenile hormone mimics, anti-
juvenile hormones, moulting hormones, anti-moulting hormones, attractants
and repellents. This understanding of co-evolution by chemists and biologists
has been responsible for evolving a new strategy that envisages the use of
plant defence chemicals against insects.

In India there are several plants known for their insecticidal property and
are popularly used as pesticides. The most important and popular among these
is neem, which has international recognition. Probably no plant is known to
possess such a diverse biological activity as neem. Neem has been evaluated
against 105 species of insects, 12 species of nematodes, and at least 9 species
of fungi (Singh, 1990). Neem, unlike synthetic insecticides, has diverse
behavioural and physiological effects on insects and these effects are governed
by about 30 compounds. A majority of phyotochernical work has been
conducted on plants of the genera Azadirachta and Melia. From these plants
more than one hundred limonoid type triterpenes have been isolated many of
which have insect growth regulatory or antifeedant activities. Though, the
insect managing quality of neem was known to Indians since time immemorial,
a breakthrough in its use was made at Indian Agricultural Research Institute,
New Delhi, in 1960 and was subsequently confirmed under field conditions
(Jotwani and Sircar, 1967; Butterworth and Morgan, 1971; Yadava and
Bhatnagar, 1987). Several reports have appeared on the insecticidal,
antifeedant, growth regulatory, oviposition deterring, anti-hormonal and anti-
fertility activities of neem against a broad spectrum of insects. The above
mentioned activities for the oil expelled from seeds and leaves, leaf extracts,
seed extracts, neem cake, bit extracts and various isolated compounds (viz.,
azardirachtin, nimbin, nimbidin etc.) have been demonstrated. Feeding
deterrency and repellency of neem oil (Saxenaet al., 1981);
ovipositiondeterrency of neem seed kernel (Islam, 1984 and Ayyangar and Rao,
1989) and antifeedant property of neem leaf extract (Sosamma and Shiela,
1994) were reported on various agricultural pest.

3. AIM and OBJECTIVES

 Chemicals involved in conveying information in intra- and inter-
specificinteractions between organisms are termed ‘infochemicals’ and
constitute asubcategory of semiochemicals (Dicke and Sabelis, 1988).
Infochemicals playan important role in the biology of many insect species.
These are the chemicalcompounds act as signal by transferring information
between individuals. It is anew term given to semiochemicals recently (Dicke
and Sabelis, 1988). Anunderstanding of their role in plant-herbivore-carnivore
interactions can be usedin the development of tools for the enhancement of
environmentally benignalternatives to synthetic pesticides (Tinzaaret al.,
2002). Chemicals producedby plants and insects play a major role in the
behavioural responses that determinethe performance, survival and
development of the insects (Vet and Dicke, 1992). The main aim of the present
study is to evaluate the effect of allelochemical flavone on digestive enzymes
activity in banana stem weevil Odoiporouslongicollis.

The objectives of the present study are

1. Collection of banana stem weevil Odoiporouslongicollis.

2. Treatment with 10 mg flavone on banana stem weevil

Odoiporouslongicollisfor 24 and 48 hours.

3. Assessment of total protein, total carbohydrate and total lipid levels in

both control and treated insects.

4. Estimation of digestive enzymes like alpha-amylase, Lipase and

protease activityin both control and treated insects.

4. Materials and methods

4.1 Experimental animals:

Odoiporuslongicollisadult insects were collected from Malappuram

district, Kerala, India and insects were maintained in insect rearing cages at
room temperature with banana stem as diet.

4.2 Chemicals:

Flavone, Lead acetate, Gallic acid, Tannic acid, Linalool, Picric acid,
Pyrogallol, Bovin serum albumin, Catechol, glutathione reduced, DTNB,
NADPH, oxidized glutathione, acetylthiocholine iodide and sodium azide were
purchased from Himedia , India. And all other chemicals and reagents used are
analytical grade purchased from SRL, India.

4.3 Glassware and plasticware

Glassware from Borosil and Schott Duran; plasticware from Tarsons and
Laxbrowereused after washing with the detergent and rinsing in distilled
water. They were then driedin hot air oven at 70oC before use.

4.4 Treatment of insects

When plant substanceswere tested as inducers, stock concentration of

the allelochemical flavone was prepared by dissolving 100mg flavone in 1 ml
acetone. Treatment was performed on adult Odoiporuslongicollisinsect using
standard topical application procedure followed by ( Kranthi, 2005). Single
concentration100 µl of flavone from stock and 100 µl acetoneitself used as
control and is applied on the dorsal surface of the prothoracic region of the
insect by using a Hamilton repeating dispenser.20 numbers of insects were
used for the study. After treatment insects were released in 9 mM diameter
petri dishes. The treatment was given for 24 and 48 hours. Atthe end of the
experiments, insects were removedfrom petri dishes and usedfor enzyme
assays. No mortality was observeddue to the treatments (Yu, 1984).

4.5 Preparation of tissue extracts

Total 20 numbers of insects were weighed and rinsed with acetone (2×5
ml) to remove surface residues. Whole insect of both control and treated
group were dissected and homogenate using ice cold normal saline with the
help of a mortar and pestle. The homogenates were centrifuged at 10000 RPM
for 20 minute at 40C to obtain supernatant which was then used for the
enzyme analysis.

4.6 ESTIMATE THE AMOUNT OF PROTEIN

Estimation of protein by Lowry’s method(Lowry et al., 1951)

Aim:

Estimate the amount of total protein by Lowry‘s method.

Principle:

Under alkaline condition the divalent copper ions forms a complex with
peptide bonds in which it is reduced to a monovalent ion. Monovalent copper
ion and the radical group of tyrosine, tryptophan and cysteine react with Folin
reagent to produce an unstable product that becomes reduced to
molybdenum/tungsten blue

Reagents

A. 2% Na2CO3 in 0.1 N NaOH

B. 1% Na-K Tartrate in H2O

C. 0.5% CuSO4.5 H2O in H2O

D. Reagent I: 48 ml of A, 1 ml of B, 1 ml of C

E. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water

F. BSA Standard - 10 mg BSA dissolved in 100 ml of distilled water

Procedure:

• 0.2 ml to 1 ml of BSA working standard in 5 test tubes and make up to 1ml

using distilled water.

• The test tube with 1 ml distilled water serves as blank.

• Add 4.5 ml of Reagent I (Lowry concentrate) and incubate for 10 minutes.

• After incubation add 0.5 ml of reagent II (Folin‘s reagent) and incubate for 30
minutes

• Measure the absorbance at 650 nm and plot the standard graph.

• Estimate the amount of protein present in the given sample from the
standard graph.

4.7 ESTIMATE THE AMOUNT OF CARBOHYDRATE

Determination of Total Carbohydrate by Anthrone Method (Hedge and

Hofreiter., 1962)

Carbohydrates are the important components of storage and structural

materials in the plants. They exist as free sugars and polysaccharides. The basic
units of carbohydrates are the monosaccharides which cannot be split by
hydrolysis into simpler sugars. The carbohydrate content can be measured by
hydrolysing the polysaccharides into simple sugars by acid hydrolysis and
estimating the resultant monosaccharides.
Principle

Carbohydrates are first hydrolysed into simple sugars using dilute

hydrochloric acid. In hot acidic medium glucose is dehydrated to
hydroxymethyl furfural. This compound forms with anthrone a green coloured
product with an absorption maximum at 630 nm.

Materials

1. 2.5 N HCl

2. Anthrone reagent: Dissolve 200 mg anthrone in 100 mL of ice-cold 95%

H2SO4. Prepare fresh before use.

3. Standard glucose: Stock—Dissolve 100 mg in 100 mL water.

4. Working standard—10 mL of stock diluted to 100 mL with distilled water.

Store refrigerated after adding a few drops of toluene.

Procedure

1. Weigh 100 mg of the sample into a boiling tube.

2. Hydrolyse by keeping it in a boiling water bath for three hours with 5 mL of

2.5 N HCl and cool to room temperature.

3. Neutralise it with solid sodium carbonate until the effervescence ceases.

4. Make up the volume to 100 mL and centrifuge.

5. Collect the supernatant and take 0.5 and 1 mL aliquots for analysis.

6. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the working
standard. 0‘serves as blank.

7. Make up the volume to 1 mL in all the tubes including the sample tubes by
adding distilled water.
8. Then add 4 mL of anthrone reagent.

9. Heat for eight minutes in a boiling water bath.

10. Cool rapidly and read the green to dark green colour at 630 nm.

11. Draw a standard graph by plotting concentration of the standard on the

Xaxisversus absorbance on the Y-axis.

12. From the graph calculate the amount of carbohydrate present in the
sample tube.

4.8 ESTIMATE THE AMOUNT OF LIPID

Estimation of lipid by Zak’s method (Zak., 1954)

Aim:

To estimate the amount of cholesterol in an unknown food sample.

Principle:

Cholesterol in glacial acetic acid gives a red colour with ferric chloride
and polar sulphuric acid. This reaction has been employed by ZAK‘S to estimate
the cholesterol in an unknown food sample.

Reagents required:

1. Stock Standard Solution: About 100 mg of cholesterol was dissolved and

made up to 100 ml with glacial acetic acid (concentration 1 mg / ml).

2. Working Standard: About 10 ml of stock solution was made up to 100 ml

with ferric chloride acetic acid reagent (concentration in 0.1 mg / ml).

3. Ferric chloride of 0.05% in acetic acid.

4. Sulphuric acid.

5. Glacial acetic acid.

Procedure:

0.5 ml to 2.5 ml of working standard were Pipetted out into a clean test
tubes. The volume was made up to 5.0 ml with ferric chloride and 3.0 ml of
concentrated sulphuric acid were added. The test tubes were kept at room
temperature for 15 minutes. The pinkish red colour formed was measured at
540 nm. Standard graph was drawn for the values obtained. From the standard
graph the amount of cholesterol present in the food sample can be calculated.

4.9 DIGESTIVE ENZYME ASSAY

4.9.1 α- Amylase enzyme assay

α-Amylase assay was performed according to the method as described by

Miller et al, (1959). The activity of enzyme was determined based on the
reduction in blue colour intensity results from enzyme hydrolysis of starch
using 3, 5-dinitrosalicylic acid (DNSA) reagent. Approximately 200 µl (2.5mg
protein) of enzyme solution was dissolved in 2 ml of 1% soluble starch solution
containing 1.0 ml of 0.2 M phosphate buffer pH 8.0 and incubated at 40°C for
30 minutes. The reaction was stopped by adding 2 ml of DNSA reagent and the
reaction tubes were kept in boiling water bath for 10 minutes and cooled. After
cooling 5 ml distilled water was added to the test tubes and the absorbance
was read at 540 nm. The optical density of the blue-coloured solution was
measured at 540 nm in a spectrophotometer against maltose as standard. One
unit is defined as the amount of enzyme required to liberate one µmoles of
reducing sugar per minute.

4.9.2 Assay of lipase activity

 The activity of lipase was determined by the method of Tsujitaet al. (1989).
100μl of different tissue extracts was mixed with 180μlρ-nitrophenylAcetate (50
mM) as a substrate, and 1.72 ml of sodium phosphate buffer solution (1 MpH =
7). This was then incubated at 37°C. The absorbance was read at 405 nm. One
unit of enzyme releases 1.0 µmoles of p-nitrophenyl per minute at pH 7.0, at
37°C using p-nitrophenylacetate as the substrate.

4.9.3 Assay of protease activity

General protease activity of different tissues was determined using
azocasein as the substrate (Garcia-Carrenoet al., 1994). The reaction mixture
was 80 μl of 2% azocasein solution in 40 mM sodium phosphate buffer of pH
7.2 and 30 μl enzymes. The reaction mixture was incubated at 37°C for 60 min.
Proteolysis was stopped by the addition of 300 μl of 10% trichloro-acetic acid
(TCA). Appropriate blank in which TCA was added first to the substrate, were
prepared for each assay. Precipitation was achieved by cooling at 4°C for 120
min, and the reaction mixture was centrifuged at 10,000 rpm for 10 min. An
equal volume of 1 N NaOH was added to the supernatant. The absorbance was
recorded at 440 nm.

5. Results and Discussion

 Losses of agricultural production due to pests and diseases have been
estimated at 37 % in Europe and worldwide. Most damage is caused by
arthropods and the methods available today for protecting plant crops against
insect predation are heavily dependent on environmentally-aggressive
chemicals and that have been estimated to reduce losses by only about 7 %
(Oerkeet al., 1994). This fact justifies the necessity for the research and
development of alternative approaches to this problem (Carlini and Fatima,
2002).

Pesticides of biological origin have been intensively investigated for the

past 30 years. An effort has been made to find an alternative to conventional
insecticides. The alternative should be able to reduce health and envi-
ronmental impacts (Copping and Menn, 2001). There has been a worldwide
interest in the development of alternative strategies, including the re-
examination of using plant derivatives against agriculturally important insect
pests. Plant-derived materials are more readily biodegradable. Some have low
toxicity to humans as well as natural enemies, and are more selective in action
(Rahman and Siddiqui, 2004).

Numerous methods for weed management in different systems have

been developed over thedecades. Of these, herbicide use and hand and
mechanical weeding are the most reliable weed control methods. Herbicides
use is increasingly being adopted for weed control and for increasing
cropproduction . However, herbicide-resistant weeds are the major constraint
to weed control in modernagriculture. Additionally, hand weeding and
mechanical weed control methods are expensive and labor intensive.
Furthermore, weed control via chemical herbicide use reportedly
negativelyimpacts the environment and human health. Extensive exposure to
chemical herbicides may causenumerous health issues such as reproductive
problems, neurologic impairment, pancreatic cancer, immunemalfunction, and
silicosis. Furthermore, herbicides are unaffordable to small-scale farmers
andunsuitable for organic farming. These challenges associated with the use of
herbicides and mechanicalweed control methods in land cultivation make it
imperative to develop novel, environmentallyfriendly methods. The global
interest in alternative, sustainable, and innovative technologies forweed
management that not only reduce chemical herbicide use but are also
environmentally andhealth-friendly and decrease agricultural production cost
is increasing (Ghimireet al., 2019).

Plant defenses against insect herbivores are mediated, in part, by

enzymes that impair digestive processes in the insect gut. Little is known about
the evolutionary origins of these enzymes, their distribution in the plant
kingdom, or the mechanisms by which they act in the protease-rich
environment of the animal digestive tract (Chen et al., 2007).The transgenic
expression of insecticidal proteins such as α-amylase and protease inhibitors is
also beingevaluated as a potential protective strategy against insects (Schuler
et al., 1998).

5. 1. Estimation of Total protein, Carbohydrate and Lipid content

Carbohydrates along with protein and lipids form the principal classes of
organic compounds. In insects and other organisms, carbohydrates contribute
to the structures and functions and can be found in the nuclei, and membrane
of cells and also in the extracellular haemolymph and in the supporting
tissue.The carbohydrates are involved at all level of cellular
organization.Metabolism of carbohydrates insects is more or less similar as
that occur in other animals. Insects possess exoskeleton rich in
aminopolysaccharide and they also possess disaccharide, trehalose which acts
as storage of glucose.The carbohydrates are involved in regulating and
maintaining life processes. The origin, nature and fate of carbohydrate during
growth, metamorphosis, flight, reproduction, and embryonic development are
varied in different insect species. In most of the insects carbohydrates are
present as glycogen and trehalose which can be readily converted into glucose.

Proteins are the most abundant biological macromolecules, present in all cell
and all parts of calls. Proteins occur in great variety of different kind of forms
relatively small peptide with molecular weight in millions. Proteins are
responsible for diversity of functions of different organism and in different
organism and in different cell of same organism. This dynamic molecule
invariably essential to life. They have an important role in the cellular turnover.
As constituent of cell membrane protein have a major role in process of
interaction between intra cellular media.

Lipids are used in so many insects as energy source, hormone precursors and
structural members. It is stored in different regions in the insect body. Also
lipids located in the egg playan important role in meeting the energy needs for
developing embryo

Table 1: Estimation of Total protein, Carbohydrate and Lipid content

Samples Protein Carbohydrate Lipid

Concentration Concentration Concentration
(mg/ml) (mg/ml) (mg/ml)
Control 2.67 4.62 2.45
T1 2.14 2.06 1.07
T2 1.03 1.79 0.56
 T1- Treatment with Flavone for 24 hours, T2- Treatment with Flavone for
48 hours

Table 1 showed the result of total protein, carbohydrate and lipid content of
control and both treated group (24 and 48 hours). When treated with
allelochemical flavone it is showed that there is reduction in protein,
carbohydrate and lipid content of both 24 and 48 hour treated group when
compared with control group. The protein concentration in control group has
2.67 mg/ml but in 24 and 48 hour treated group protein concentration is 2.14
mg/ml and 1.03 mg/ml respectively. In the case of carbohydrate it is showed
that there is reduction in carbohydrate content of both 24 and 48 hour treated
group when compared with control group. The carbohydrate concentration in
control group has 4.62 mg/ml but in 24 and 48 hour treated group protein
concentration is 2.06 mg/ml and 1.79 mg/ml respectively. In the case of lipid
also showed that there is reduction in lipid content of both 24 and 48 hour
treated group when compared with control group. The lipid concentration in
control group has 2.45 mg/ml but in 24 and 48 hour treated group protein
concentration is 1.07 mg/ml and 0.56 mg/ml respectively.

Figure 1: Estimation of Total Protein

The present results are in agreement with the work of Susheelaet al

(1994) who conducted the toxic effect of pesticides on the survival and
proximate composition of Tubifextubifex. The decrease in protein content in
both the nymphal and adult stages is attributed to the acceleration of tissue
proteolysis, which may be due to the increased utilization of protein during
stress conditions (Ibid).The synthesis of protein takes place in fat body cell
under the control of hormones, which also differ in different insects and may
depend on a series of internal and external factors (Chen et al., 1976). If there
is any change in these by any toxicants it may lead to the depletion in the
synthesis of body metabolites.The present results are in agreement with the
data obtained by Surendranath (1996). A marked decrease in total proteins in
the body and silk gland cells was observed in the 5 th instar silk worm larva
exposed to lethal and sublethal doses of Fenithrothion and Ethion, indicating
the breakdown/degradation of proteins.

Figure 2: Estimation of Total Carbohydrate

It has been observed that the total carbohydrate content had been
reduced in all the treatments when compared to the control. This may be due
to the stress condition that occurred in the insects during the treatment. In
most insects, carbohydrates reserves are present as glycogen and trehalose
and both the reserves can be ready converted into glucose for the support of
all life processes. Metamorphic changes in insect are usually accompanied by
substantial depletion of their carbohydrate reserves. During this period,
glycogen and treholase supply glucose which provides an energy source and a
substrate for the synthesis of pupal and adult tissues, especially the cuticle. In
addition to this, the stress condition that occurred in the insects during the
adverse condition causes a drastic reduction in the amount of trehalose. In this
experiment, the changes are probably due to the treatment with
allelochemical. The obtained results show that the activity of carbohydrate was
affected in treatments and was lower than that obtained with untreated
insects. Change in carbohydrate indicates the role of allelochemical in altering
the physiological balance in insects.Kurita et al., (1981) notice depletion of
carbohydrate in Lohitagrandis due to the action of various hormones.
Bouayadet al.,( 2013) noticed enhanced activity of hydrolytic enzymes and
decreased amount of trehalose in Plodiainterpunctella on treatment with
Moroccan plant extracts. Hatem et al., (2012) reported the effect of some bio
insecticides and Igrs on American Bollworm, Helicoverpaarmigera and noticed
a drastic increase in the activity of the enzyme trehalose and reduced amount
of trehalose.

The increase in the glucose level at different hours exposure may be due
to break down of insect blood trehalose into glucose. In insect haemolymph
the synthesis of trehalose takes place using glucose molecule present in the
body fluids. Due to this reason there was a retention of haemolymph
carbohydrates with minimal loss in the excreta. Since trehalose is a
disaccharide, and it cannot easily be diffused through alimentary canal and
hence it prevents the loss through excreta. The trehalose acts as a stored sugar
in insects under starvation or stress, obviously the activity of insect increases
and hence utilization of glucose becomes high. To fulfill the requirement a
trehalose molecule converted into two molecules of glucose with the help of
trehalase (Wyatt; 1967; Bhavaneet al, 1991 and Bhanotet al., 1992). The
increase in glucose content may also due to the conversion of glycogen to
trehalose and then to glucose to full fill the energy requirement in insects
(Bucher and Klingenberg, 1958). The above results are in agreement with the
results obtained by Ramanna (1992), where he observed the increase in
carbohydrate contents of Oryctesrinocerosb

Figure 3: Estimation of Total Lipid

It is well known that stored lipids are most important and convenient
reservoirs of metabolic energy, which fulfill prolonged energy demand in
insects in stress. Physiologically lipids play an important role in insect survival.
Once lipid metabolism is inhibited most of the normal physiological activities of
insect get obstructed that result in to death.

After the treatment with flavone the lipid level is declined as compared to
control in banana stem weevil. Similar trend was noted in A. annua treated
Culex larvae and lipid content showed 25.0% reduction. This reduction in lipid
profile indicates a negative effect of the extract on lipid metabolism and
peroxidation. The decline in lipid quantity may be due to shift in energy
metabolism towards lipid catabolism as the result of insecticidal stress induced
by the extract. This observation is identical to the findings of Loharet al,, (1993)
who found that Tenebrio molitor suffered lipid depletion in haemolymph, fat
bodies and oocytes when exposed to malathion. Saket al reported the decline in
lipid content due to shift in energy metabolism to lipid catabolism due to
insecticidal stress induced by Pimpalaturionellae (2006). Moreover, it has
reported by Senthilkumaret al that total lipid were reduced in An. stephensi
larvae treated with some plant extracts and it is suggested that it might be due to
physiological stress conditions induced by the extracts (2009).

5.2 Estimation of specific activity of digestive enzymes

Allelochemicals are generallysecondary plant metabolites, which are

part of a wide range of chemicals synthesized by plants, not directly involved in
plant growth and development. Nowadays, allelopathic plants are being used
forweed control and natural herbicide development. The environmental- and
biodiversity-conservingnature of allelopathic weed control has made it more
popular than mechanical methods and chemicalherbicides. Some plant species
exude useful biochemicals (also called allelochemicals or phytochemicals)that
inhibit/suppress seed germination and weed growth. These phytochemicals
are generallyphenolics (such as tannins), alkaloids, steroids, terpenes,
saponins, and quinones that affect the growthand development of certain
plant species. The mechanism of action of some allelochemicals issimilar to
that of chemical herbicides, making them ideal for weed management. The
allelochemicalsproduced by donor plants are often detrimental to their own
species—an autotoxic effect .These phytochemicals are totally/partially water
soluble, making them more environmentally friendly than chemical herbicides.
They are characterized by higher O 2- and N2-rich molecules and few halogen
substitutes. Allelopathy has a positive effect on soil, as it improves nutrient
availability tocrops by increasing microbial activity. Allelochemicals are exuded
from different plant parts atdifferent ratios, and their exudation varies within
different cultivarsDigestion is a process in which ingested macromolecules by
insects break down to smallerones to be absorbable via epithelial cells of
midgut. Several enzymes based on foodmaterials have critical roles in this
process. Any disruption in their activity disables insectsto provide their
nutrients for biological requirements. Several studies demonstrated theeffect
of botanical insecticides on feeding parameters of insects by demonstrating
foodconsumption (Sitthinoiet al., 2017)

Samples Α-amylase Protease Lipase

Specific Activity Specific Activity Specific Activity
(µmoles/min/mg (µmoles/min/mg (µmoles/min/mg
protein) protein) protein)
 Control 321.02 14.29 1540.57
T1 244.20 12.33 852.91
T2 177.42 8.77 435.17
 T1- Treatment with Flavone for 24 hours, T2- Treatment with Flavone for
48 hours

Consumption of allelochemicals may effect changes in metabolic

detoxicationsuch that toxicity of the same or other allelochemicals is markedly
alteredin subsequent exposures. One of the primary mechanisms responsible
is thatof enzyme induction, which has been documented to occur in all the
majordetoxication system (Terriere, 1984). A second mechanism is enzyme
inhibition due toexposure to phytosynergists (plant compounds that function
as synergists of simultaneously occurring toxins).Many studies have shown
that the activity of detoxication enzymes increaseswhen insects are fed
particular allelochemicals or host plants. Indeed,induced activity has often
been interpreted as evidence for the involvement ofa specific enzyme system
in the metabolism of a substrate of interest. Suchconclusions, however, are
best considered tentative; not all enzyme substratesare inducers, and not all
inducers are substrates (Gunderson et al., 1986). The number of cases in which
allelochemicals have been shown to induceenzymes that are responsible for
their own metabolism is relatively small, butsufficient to indicate that this
biochemical response is important in insect-plantinteractions.
Figure 4: Effect of flavone on the level of α-amylase activity

In the present investigation it has been observed that the activity of

enzymes decreased in almost all treated insecticides. The reduction in the
activity of enzymes may be due to the insecticidal impact on enzyme synthesis.
As the enzyme involved in various physiolgical processes including protein
synthesis, the concentration of enzyme might be affected in response to
chemical stress (Rekhaet al, 2000 and Karuppaswamyet al, 2001).
Figure 5: Effect of flavone on the level of protease activity

Figure 5: Effect of flavone on the level of lipase activity

In the present investigation, the activity of digestive enzymes was used

as a parameter for studying the effect of flavone. α-amylase (a-1,4-glucan 4-
glucanohydrolase, EC 3.2.1.1) is an enzyme that degrades starch (first to
oligosaccharides and then in turn to maltose and glucose), by hydrolyzing α-
1,4-glucan bonds (Terra and Ferriera, 2005).Amylase, which splits starch and
glycogen to maltose, has been demonstrated in the digestive secretions of
many insects. Two distinct types of amylase have been identified in plants and
animals. Once the saccharogenic or exoamylase, rapidly splits off maltose from
the straight chains of glucose units and only slowly affects the colour reaction
of the polysaccharide with iodine, the other, "amyloclastic" "dextrinogenic", or
endoamylase, attacks the interior of the polysaccharide chain rapidly reducing
the colour reaction with iodine and producing only a slow increase in maltose
concentration(James and Thomas, 1986).

In our study, treatment of banana stem weevil adult with flavone

showed a reduction in the activity level of α-amylase. This reduction increased
with contacting time period of the allelochemical. Shekariet al. (2008) also
showed the α-amylase activity level in elm leaf beetle treated with A.
annuaextract, decreased after 24 h and sharply increased after 48 h. Artemisia
annuaextract caused the reduction of α-amylase activity in PierisrapaeL. as was
shown by Hasheminiaet al. (2011).Saleem&Shakoori (1987) showed that
sublethalconcentrations of pyrethroids decreased the a-amylase activity in the
larval gut of the beetleTriboliumcastaneumHerbst. The reduction of α-amylase
activity by plant compound could be due to the plant defense compounds that
act on insect gut enzymes, α-amylases, and proteinases (Franco et al. 2002).
Also, the reduction of this enzyme activity could be due to a cytotoxic effect of
different allelochemicals on epithelial cells of the midgut, which synthesize α-
amylase (Jbilouet al. 2008).

Proteases hydrolyze proteins to amino acids, classified as endopeptidases

(EC 3.4.21-24) and exopeptidases (EC 3.2.4.11-19), and based on their catalytic
mechanism (Pascual-Ruiz et al. 2009). Our results showed that flavone caused a
reduction of the protease activity in banana stem weevil. This reduction
increased with contact period of flavone, which coincides with other reports on
insects. It has also been reported that digestive protease of C. medinaliswas
suppressed by extracts of VitexnegundoL. and AzadirachtaindicaA. Juss
(Senthil-Nathan et al. 2006c). Compared with the controls, protease activity was
reduced using 0.013 and 0.026 (gram leaf equivalent/ml) gle/ml concentrations
of A. annuamethanol extract 48 h post-treatment in G. pyloalislarvae as was
demonstrated by Khosraviet al. (2011). Johnson et al. (1990) showed that
botanical insecticides may interfere with the production of certain types of
proteases and disable them to digest ingested proteins.
Lipases play a major role in storage and lipid mobilization. These
enzymes are also the basic components in many of physiological process like,
reproduction, growth, and defense against pathogens. Senthil-Nathan et al.
(2006c) showed that treating C.medinalis, the rice leaf folder, with Btk(Bacillus
thuringiensisKurstaki), NSKE (neem seed kernal), and VNLE (Vitexnegundoleaf
extract) (azadirachtin and neem components) sharply decreased the activity
level of lipase in the midgut. Azadirachtin inhibits peristalsis, reduces enzyme
production as food moves through gut, inhibits midgut cell replacement, and
reduces feeding (Anuradha and Annadurai 2008).
6. SUMMARY AND CONCLUSION

The present study was undertaken to evaluate the allelochemical flavone

effect on digestive enzyme activity in Banana stem weevil. The biological
compounds (Protein Carbohydrate, Lipid, Alpha-amylase, Protease and Lipase)
were successfully extracted and estimated.The Odoiporuslongicollis extract
recorded the maximum expression of biomolecules are Protease, lipid, protein
and carbohydrates. The lipid content showed highly positive relationship
between 24 and 48 hrs compared with control group. From the above study it
is concluded that the lipase enzyme and alpha-amylase enzyme activities not
showed much significancetreated Banana stem weevil. In short, flavone is a
potential insecticide and cause toxic effect on Banana stem weevil as an in
vivoand thereby inhibiting the growth and multiplication in the plant. Its also
make biochemical changes in the Banana stem weevil leads to change the
biochemical pathway.
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