CHAPTER 4

Discussion

Discussion

161

Plants contain several chemicals by birth and purpose of the

presence of these chemicals is defending the plant from pest attack.

Each plant species consist of different chemicals and role of these

chemicals in protecting plant from pest and diseases is well established

since a long time. We investigated the chemicals present in brinjal,

Solanum melongena as plant’s static defense and also confirmed their

biological activity. Extraction, isolation, identification and

characterization study of S. melongena revealed the presence of several

chemicals such as, phenolic and alkaloid compounds and our

experimental results indicated their role in acting against certain pests

and diseases . Among the different solvents used for the extraction of

phytochemicals from shade-dried leaf and fruit of S. melongena,

Methanol proved to be efficient solvent, which could extract phenolic

compound, caffeic acid methyl ester (CME) and alkaloid compounds

solamargine and solasonine. The isolated compounds were evaluated for

thie pesticidal potential against certain agricultural and stored product

pests and were shown to be efficient pest control chemicals. This

indicates the static defense role of above chemicals CME, solamargine

and solasonine isolated from S. melongena plant.

The leaf and fruit extracts of S. melongena and its chromatographic

fractions showed conspicuous antifeedant and growth inhibitory effects

against the larvae of L. orbonalis, S. litura and A. janata. The behaviour of

the insects showed that the larvae occasionally sampled the treated food,

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162

which suggests reduced feeding of treated food or its rejection. The

methanol eluted fraction from fruit extract of S. melongena showed

excellent feeding deterrent activity against the test larvae. It is evident

from the results for the first time that pest repellent activity of extracts

from the well-known brinjal plant are ideal candidates for a natural

method of pest-control.

Several investigators have reported the antifeedant activity of

botanicals against S. litura (Ulrichs et al., 2008, Sreelatha et al., 2009)

and A. janata (Devanand and Usha Rani, 2008). Pavunraj et al. (2011)

have reported that leaf extract of Pergularia daemia (Forssk) Choiv. and

its column eluted ethyl acetate fraction exhibit good antifeedant activity

against Helicoverpa armigera (Hub.) and S. litura.

Amongst the chemical defensive strategies developed by the plant,

leaf phenolics generated due to insect feeding play a major role in

controlling the herbivore damage. It was interesting to study the effects

of different phenolic acids on the growth and development of feeding

herbivore. The results of feeding deterrent bioassays showed significant

reduction in feeding by the test larvae in choice experiments with food

containing CME than the test phenolic standards CA and CGA. Ding et

al. (2011) have reported the accumulation of induced phenyl propanoids

especially ferulic and p-coumaric acids, in response to Sitodiplosis

nonagriodies (Gehin) (Diptera: Cecidomyiidae) feeding on wheat tissues.

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According to Harborne (1988) the induction of plant phenolics due

to insect feeding makes unpalatable to herbivorous insects. In the

present study it is confirmed that S. melongena fruits containing

hydroxycinnamic acid derivative, CME was responsible for feeding

inhibition activity against the test larvae. The isolated glycol alkaloids

solasonine and solamargine from the fruits of S. melongena, failed to

show the antifeedant activity in test insects at all concentrations tested

by leaf disc assay. These compounds have not shown the potential for

controlling the lepidopteran larvae by feeding inhibition. Along with

antifeedant activity, the methanol extracts of S. melongena were potent

growth inhibitors to L. orbonalis, S. litura and A. janata tested. These

extracts were quite effective in reducing growth of three lepidopteran

larvae in the oral feeding bioassay.

The growth inhibition activities of the extracts of several Meliaceae

plants such as A. indica (Agrawal and Mall, 1988), Melia azedarach (Al-

Sharook et al., 1991), M. toosendan (Chen et al., 1995) and Aglaia species

(Koul et al., 1997) have been extensively evaluated on several insect

pests. Ethyl acetate extract from Syzygium lineare Wall (Myrtaceae)

(Jeyasankar et al., 2010) and methanol extract of M. dubia (Meliaceae)

(Koul et al., 2000) have also showed growth inhibitory activity against S.

litura. In the present experiments also the larval growth inhibitory

activity of methanol eluted fractions from S. melongena plants was

observed. Predominantly, fruit extract of S. melongena eluted with

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164

methanol produced most potent growth inhibitor against L. orbonalis, S.

litura and A. janata by oral feeding assay as compared to leaf extract. The

findings of the study also revealed that crude extracts as well as solvent

eluted chromatographic fractions were toxic to the test insects when

evaluated by contact method.

Janprasert et al. (1993) have reported that the isolated fractions

and compounds from Aglaia odorata have feeding inhibition and growth

regulating activities against S. littoralis. In addition to feeding deterrent

activity, CME and solasonine also exhibited growth inhibitory activity

against the test larvae, S. litura, A. janata and L. orbonalis. A number of

workers in their respective studies have shown that plant derived

compounds act as an effective feeding deterrents and growth inhibitors

against many insect species. Such as Sreelatha et al. (2010) reported

that a new benzil derivative extracted from Derris scandens Benth

(Leguminosae) exhibited antifeedant and growth inhibitory efficacy

against A. janata larvae when tested by oral feeding method. Likewise the

isolated compounds khayanolide B from the stem bark of Khaya

senegalensis (Desr) A Juss (Meliaceae) (EI-Aswad et al., 2003), and

extracts and drimanes of Drimys winteri J.R. Forster et G. Forster

(Winteraceae) (Zapata et al., 2009) exhibited most potent feeding

deterrent and growth inhibitory activities against the cotton leaf worm, S.

littoralis.

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It is reported that plant phenolic compounds play a crucial role in

plant defense against herbivore attack (Schoonhoven et al., 2005). For

example, when isoquercitrin was added to the diet of the budworm, H.

virescens larval growth was reduced to 90% (Hedin et al., 1983). Rutin is

major trichome component of tomato (Solanaceae), which on adding to

the diets of fruitworms, H. zea inhibits larval growth (Isman and Duffey,

1982). The reduction in larval growth is proposed to result from the

alkylation of amino acids or proteins by o-quinones and subsequent

reduction in the nutritive quality of foliage. The presence of derivative

form of caffeic acid as caffeic acid methyl ester (CME) in the fruits of S.

melongena as well as solasonine showed larval growth inhibition against

test lepidopteran pests. In the present study a significant increase in

growth inhibitory responses has been observed when CME and

solasonine were present in the food of test larvae. However, no such

increase was observed with solamargine.

The results on insect development revealed that the crude extract

from S. melongena fruits, its methanol eluted fraction and the isolated

pure compounds, CME and solasonine disrupted developmental cycle of

larvae by reducing the pupal weight, formation of pupal-adult

intermediates and/or causing pupal mortality. A reduction in pupal

weight and emergence of deformed adults suggested that CME and

solasonine interfere with the mechanisms of the normal development of

the insects, which are hormonally regulated. This type of delayed

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166

development and appearance of malformations have also been reported

from azadirachtin treated S. litura (Rao and Subrahmanyam, 1987),

plumbagin treated H. armigera (Krishnayya and Rao, 1995) and a new

benzil derivative extracted from Scandens Benth (Leguminosae) treated

A. janata larvae (Sreelatha et al., 2010).

During insect development the shedding of the cuticle known as

moulting or ecdysis occurs. Moulting affects the entire body wall as well

as all internal parts that are formed as invaginations of the wall.

Collectively all changes that involve growth, moulting and development

are known as morphogenesis. Similar results were obtained when last-

instar larvae of S. litura, S. mauritia, Ephestia kuehniella Zell. and

Manduca sexta were subjected to azadirachtin (Jagannadh and Nair,

1992).

In the present study, the normal development of larvae of three

major lepidopteran pests, L. orbonalis, S. litura and A. janata treated with

isolated fractions was arrested, which led to malformation of pupae and

adults. The regulation in growth and development was also observed

when CME and solasonine were provided through oral ingestion. It

indicated the larval intolerance to the treatments. Adult abnormality was

significantly increased with the increase in concentration of test products

and days of exposure. The reduction in the overall growth might be due

to the disturbed digestive physiology and other metabolic activities of the

larvae after ingesting treated food. This type of disturbances not only

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167

reduces pupal weight but also results in poor growth, development and

production of pupal deformities and finally poor emergence of adults.

Similar to present findings, Deborah et al. (2001) have also reported that

extracts of Trichilia americana exhibit growth reduction in S. litura.

The proteolytic activity of three lepidopteran larvae and their

sensitivity to crude extracts and their isolated compounds, CME,

solamargine and solasonine have been studied. The chemical

solamargine was found least responsive then others. Houseman et al.

(1989) stated that the extracts from the digestive tracts of insects from

many families, particularly those of lepidoptera contains serine

proteases. These serine proteases are responsible for protein digestion

and consequently for the supply of amino acids needed for the

development. Serine proteases, as a group of digestive enzymes, have

also been detected in guts of other Spodoptera species (Jongsma et al.,

1996). The serine classes of proteinases such as trypsin, chymotrypsin

and elastase which belong to a common protein superfamily are

responsible for initial digestion of proteins in the gut of plant herbivores

(Garcia-Olmedo et al., 1987). These proteinases involved in cleavage of

polypeptide chains into short peptides which are then cleaved by

exopeptidases to generate amnio acids, the end products of protein

digestion (Lawrence and Koundal, 2002).

In the present study, the regulation of proteases in the midgut of L.

orbonalis, S. litura and A. janata was recorded, which could be targeted

Discussion

168

with natural products for active insect control. Midgut trypsin,

chymotrypsin and elastase showed diverse level of susceptibility towards

CME and solamargine extracted from the fruit of S. melongena plant and

other test standard compounds. Trypsin has been found to be the

predominant and most active protease enzyme in lepidopterous larvae.

Similar to present observation lower activities of chymotrypsin and

elastase have also been reported by Broadway and Duffey (1986). In this

study, the interesting results were recorded in midgut extracts of L.

orbonalis, S. litura and A. janata with the presence of CME and

solamargine in their food. Gut proteases like trypsin, chymotrypsin and

elastase activities were significantly inhibited by the isolated compounds

from S. melongena, CME and solamargine. This leads to the growth

retardation in the test larvae due to reduction in protein metabolism.

The larvae fed on food containing phenolic compounds, CGA and

solasonine showed higher proteolytic activity in midguts as compared to

those fed with CME, solamargine and solvent applied control. The poly-

phytophagous insects, adapt easily to exogenous protease inhibitor

chemical in their food. Since these insects generally have complex

digestive biochemistries and compensate for the loss of proteolytic

activity by either increasing affected proteases or by expressing novel

proteases insensitive to the ingested protein inhibitors (Broadway, 1997;

Gatehouse et al., 1997; Brousseau et al., 1999). From the results, it was

noticed that the hyper-production of proteases in response to ingested

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169

compounds leads to an extra load on the insect for essential amino acids,

resulting in retardation of insect growth. It may be stated that the

phenolic compounds, CME and CG and alkaloids, solamargine and

solasonone taken as oral ingestion have the potential to regulate the gut

proteolytic activities of three lepidopteran pests L. orbonalis, S. litura and

A. janata by oral ingestion.

The experiments with S. melongena fruit crude extracts,

chromatographic fractions, and the isolated pure compounds indicated

the toxic potential of the S. melongena fruit chemicals against stored

grain insects. Particularly, the isolated compound, CME exhibited greater

insecticidal activity in a short duration of time to all the test insects and

indicated the potential of this pure compound rather than other alkaloid

compounds, solasonine and solamargine, isolated from the fruit extract

of S. melongena. Among the test insects, R. dominica showed slight

tolerance to the crude methanol extracts of the fruits and also to column

fractions. The increased tolerance of R. dominica toward the plant

extracts treatments were also reported previously (Usha Rani et al.,

2011). Bioactivity of phytochemical compounds against stored product

pests depends upon several factors such as the chemical composition of

the crude extracts and varied susceptibility of target species. For

example, neem (Azadirachta indica A. Juss) seed extracts are reported to

demonstrate a remarkable insecticidal activity against nearly 200 species

of insects (Lowery and Isman, 1995). Some plant derived materials are

Discussion

170

highly effective against insecticide resistant strains of insect pests

(Arnason et al., 1989; Ahn et al., 1997).

The pure compound (CME) isolated from S. melongena fruit

material appeared to be highly active giving significant percentage of

mortality of all the four major stored product pests tested, irrespective of

the species difference. This may be due to the easy penetration of

isolated pure chemical into the insect cuticles, when compared with

crude and bio-guided column fractions. In contact method, the test

concentration and exposure time played an important role in producing

the lethal effects. Insect mortality and the duration of the exposure were

found directly proportional in all the treatments of pure compounds. The

concentrations employed also played a major role in determining the

efficacy of CME. Though, the compound, CME at the lower concentration

failed to exhibit toxic symptoms 24 hr after treatment, however, the

percentage mortality has been enhanced with increased duration of

exposure to the compound (up to 72 hr). The pure compound showed

significant mortality to S. oryzae and C. chinensis with the lowest

concentration after 72 hr of treatment compared to other test insects, R.

domonica and T. castaneum. Thus results demonstrated that lower

concentrations with longer exposure time may cause higher mortality

comparable to higher concentrations. It is concluded that an adequate

exposure time is crucial for the effectiveness of CME because the insect

movement increases the cuticular contact with the compound. The test

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171

isolated compounds, solasonine and solamargine failed to show the

effective toxicity to all the test insects at concentrations tested in both

contact and fumigation mode.

In the fumigant study, the pure compounds extracted were more

effective than crude extracts against all the tested insects in sealed

containers with in 48 hr after treatment. However, crude extracts of S.

melongena showed significant mortality against four major stored pests

after 72 hr of treatment by fumigation method in a delayed mode. In case

of column fractions (methanol) also the considerable fumigant action was

caused by the presence of concentrated levels of fumigant molecules only

after 72 hr of treatment. Owing to the insufficient amounts of volatiles

released from all the test compounds at 24 hr, less mortality was caused

to the test insects. After 48 hr of fumigant action, the containers were

saturated with the chemicals and no increased mortality was found with

the test compounds. Thus contact application appears to be superior

over the other method employed as a lesser quantity of the test material

was required to achieve a 100% kill. Also it appears that the chemical

penetration in to insect body through oral or cuticular route is higher in

this method of application. Form this study it was established that the

test compounds were more effective in contact mode than vapour mode

toxicity.

The author has not come any reference so far reporting the efficacy

of brinjal plant extracts against the stored grain pests. Therefore, it

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172

appears that this is the first report on the insecticidal activity of S.

melongena and its isolated compound CME against stored product insect

pests. Thus the product based on S. melongena fruit extract or its active

compound, CME may have potential to control the destructive stored

grain insects. It has been well recognized that some plant extracted

insecticidal compounds may be developed into products suitable for pest

control, because they are selective to insects and have no or little

harmful effect against non-target organisms or the environment

(Schmutterer, 1992; Isman, 2000). Although the results of the present

study were promising with regard to the use of isolated compounds from

fruit extracts of S. melongena for the protection of stored grain products,

further investigations are required before using them as grain

protectants.

Feeding deterrent effects of S. melongena leaf and fruit crude

extracts and its chromatographic fractions were studied using flour disc

bioassay method against four stored grain pests. An important feeding

deterrent effect was observed for fruit extract from S. melongena at the

highest concentration tested. Whereas, leaf extract was not effective in

controlling the test stored grain pests, S. oryzae, T. castaneum, R.

dominica and C. chinensis through reduction in feeding. Amongst the

eluted chromatographic fractions, methanol fraction significantly

reduced feeding of treated food against all the test insects. Methanol

extraction of fruits of S. melongena showed good feeding deterrent

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173

activity against stored product pests. Many reports are available to

support the above data, for example, Park et al. (1997) screened the

methanol extracts from 77 oriental medicinal plant species belonging to

42 families for their larvicidal and antifeedant activities against different

insect pests. Liu et al. (2007b) also reported control of T. castaneum and

Sitophilus zeamais Motschulsky using Chinese medicinal plants in his

research work. Pascual-Villalobos and Robledo (1999) screened 57 plant

species from 21 different botanical families from southeastern Spain for

anti-insect activity using the stored-grain pest T. castaneum as test

insect. Likewise Deborah et al. (2001) screened crude methanol extracts

of 39 plant samples from 6 species of Trichilia collected in Costa Rica for

growth inhibition activity using S. litura. Not only this, according to

Simmonds (2000) about 6250 species of plants have been screened since

1985 for various insecticidal activities.

A diverse range of allelochemic compounds of low molecular

weights are present in plants and play an important role against insects.

The feeding deterrence activities of isolated compounds, CME, solasonine

and solamargine from S. melongena also tested against four stored grain

pests. The insect feeding was significantly inhibited in all treatments with

CME after 7 days of treatment. Whereas, the normal feeding activity was

recorded in food containing test alkaloids, solasonine and solamargine

and solvent (control). It was found that C. chinensis was more susceptible

to all the test extracts tested than other insects. The reduction in growth

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174

rate of S. oryzae, T. castaneum, R. dominica and C. chinensis possibly

may be due to feeding deterrent and contact toxic effects of test

compounds. Similar mode of actions has been reported by Sackett et al.

(2007) by using furoquinoline alkaloids against S. litura and Trichoplusia

ni (Hubner) as a dietary supplement. On comparison of isolated phenolic

compound, CME with other two isolated glycol alkaloid compounds,

solasonine and solamargine for their effect on insect feeding; it was

observed that the glycol alkaloids treated food was consumed by test

insects without any repellent effect. Hence, it may be stated that these

alkaloid compounds have no adverse effect on S. oryzae, T. castaneum, R.

dominica and C. chinensis up to 7 days of feeding. Since CME treated

food was found less damaged by insect feeding, it may be stated that it is

one such allelochemic compound that showed promising results in

feeding deterrence assays. Further studies on mode of action of CME

interfering nutritional metabolism of the test insects are required to

exploit this molecule in the management of stored product insect pests.

The findings thus suggest that CME being a feeding deterrent may serve

as a starting point for developing an effective insect control agent.

The crude leaf extracts and their eluted fractions of S. melongena

at higher concentrations have shown excellent repellent effects against S.

oryzae, T. castaneum, R. dominica and C. chinensis in the present study.

It is reported that the essential oils and their constituent monoterpenoids

act as neurotoxins against insects (Keane and Ryan 1999; Enan, 2001;

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175

Kim et al., 2003). Obeng-Ofori et al. (1997) found that the monoterpenoid

1.8-cineole was highly repellent and toxic against several stored product

pests such as Sitophilus granarius L., S. zeamais, Tribolium confusum

Jacquelin du Val and Prostephanus truncatus Horn. A number of herbs

and their isolated compounds, when being studied for pharmaceutical

uses, it was found that they might not be responsible for the insecticidal

or feeding-deterrent activity against S. zeamais and T. castaneum (Liu et

al., 2007b). Although the insecticidal mode of action of the plant extracts

used in the present study has not been determined, it may be pointed

out that leaf and fruit extracts of S. melongena are likely to act as

neurotoxins presumably owing to their high phenolic contents.

It is highly advantageous when plant extract possess repellent

activity, as it prevents the pest approach to damage the commodity. The

repellent effects of phytochemicals on stored product pests depend on

several factors amongst which the chemical composition of the crude

extracts and insect susceptibility towards the test compounds are the

main. Since fruit extract from S. melongena exhibited greater repellent

effects against the test insects, it demonstrates a scientific rationale for

the incorporation of the fruit powder of plants in to grain protection

practices of commodities in the South Indian communities. Additionally,

this research provides a scientific basis for extracting and applying

phytochemicals from S. melongena for stored product protection in the

South Indian region. Moreover, provided with a proper formulation and

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176

dosage, crude extracts may be exploited for use against insect infestation

at the small scale farmers’ level, since they may be more economical,

effective and less cumbersome than application of dried foliage and their

isolated compounds. The plant chemicals tested at lower concentrations

have also shown antifeedant activity to some extent against the test

insects. This may be an added advantage that the residues left to sub

lethal doses after the application of test products may cause the feeding

deterrence leading to slow or ill development of the insect. Though, the

development of resistance by pests and vectors against botanicals has

not been reported so far. The studies will be required to ascertain the

possibilities of resistance development to these isolated compounds.

Interestingly, present studies showed varying toxic properties of

chloroform, ethyl acetate, hexane and methanol extracts of S. melongena

plant. Amongst them methanol extract showed high level of toxicity

against adults of stored grain insects S. oryzae, T. castaneum, R.

dominica and C. chinensis. The toxic activity of extracts with other

solvents was comparatively low. This differential type of toxicities may be

due to the nature of the compounds and their solubility in the solvents.

Suppressing progeny production of stored product pests after contact

with treated commodities with toxic substances is of great importance, as

this would de-facilitate invading adults from establishing a sustainable

population.

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The present results indicate that S. oryzae, R. dominica, T. castaneum

and C. chinensis are all susceptible to test chemicals which limit their

progeny under laboratory conditions. This is a frequent finding in studies

assessing the efficacy of plant extracts in mediating the mortality and

suppression of progeny production by stored product pests (Huang et al.,

1997; Paneru et al., 1997; Obeng-Ofori et al., 1998). Belmain et al. (

2001) reported that Cassia sophera L. leaf powder mixed with different

commodities at 1% and 5% concentrations significantly reduced F1

emergence of S. zeamais, C. maculatus and R. dominica in laboratory

experiments, although such treatments did not affect progeny production

of P. truncates. For long term protection of stored grains, the suppression

of progeny production is more important than mortality (Athanassiou et

al., 2004). The present findings suggest that progeny production of test

insect species is suppressed in grains treated with products extracted

from the fruits of S. melongena. Inhibition of adult emergence is an

important phenomenon while evaluating potential of botanicals. It is

found that the crude extracts and chromatographic fractions and also

isolated pure compound, CME may be used successfully as grain

protectants against stored product pests. In contact mode of evaluation

CME was found more effective in inhibiting the adult emergence fed on

treated grains than isolated alkaloids. Previously several compounds

have been shown to be highly effective to suppress the insect emergence,

leading to grain protection with progeny control against most of the

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178

major stored product pests (Athanassiou et al., 2004; Athanassiou et al.,

2005; Vayias et al., 2006; Daglish and Nayak, 2006; Stathers et al.,

2008).

It has been well recognized that some plant-derived insect control

agents could be incorporated into an Integrated Pest Management

strategy, since these agents are selective to pests, have no or little

harmful action against non-target organisms and the environment and

demonstrate a differential mode of action against pests (Arnason et al.,

1989; Schmutterer, 1992; Hedin et al., 1997). The results of the present

study indicated that the brinjal fruit extract has vital components toxic

in nature. The major advantage of the plant materials tested in the

present study is their high volatility, which is a desirable characteristic

for insecticidal preparations acting as fumigants for the control of stored

product pests (Coats et al., 1991; Konstantopoulou et al., 1992;

Regnault- Roger and Hamraoui, 1995; Ahn et al., 1998). Based on the

results of this study, plant extracts of S. melongena and its isolated pure

phenolic compound, CME and two alkaloid compounds, solasonine and

somargine can be considered as potent insecticides, antifeedants and

progeny production inhibitors. Hence they become suitable for the

control of pests in stored commodities.

For the practical use of these natural extracts and their active

ingredients as novel grain protectants, further research is required as far

as safety issues for human health are concerned. Other areas requiring

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179

attention are development of cost effective formulations with improved

and persistent efficacy, anti-feedant potency and stability against the

stored product pests along with organoleptic qualities of treated

commodity.

The Environmental Protection Agency in 1994 set up a separate

branch to evaluate and facilitate the registration of such products under

the Biopesticides & Pollution Division (BPPD) to help and speed up the

registration processes. Active principles from many plants have been

recognized, isolated, purified and formulated as insecticides.

In the study, it was observed that insect damage to brinjal plants

has induced changes in primary or nutritive compounds and subsequent

quantitative changes in the plant biochemical and enzymatic levels. In

most of the insect wounded plants or due to herbivory, there has been

increase in the levels of biochemicals such as phenols, carbohydrates,

proteins and amino acids. An induction in oxidative enzymes peroxidase

(POD), catalase (CAT) and superoxide dismutase (SOD) activities and

increase in other enzymes phenylalanine ammonia lyase (PAL) and

polyphenol oxidase (PPO) level has also been observed. An obvious

difference in several biochemical parameters of L. orbonalis infested

plants was found in comparison to un-infested brinjal plants. The plant

responses mainly depended on the type of insect damage in fruits, as

this is related to the severity of plant damage. Fruit borers that mainly

drill into the fruit cause considerably higher changes in the plants as

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180

compared to other wounded or damaged plant parts. The increased levels

of carbohydrates in S. melongena infested plants suggest their role in

plant defense mechanism by inducing the signaling pathways in plants.

The induced defense may be direct or indirect.

The studies of Watanabe and Kitagawa (2000) showed that plant

hopper, Nilaparvata lugens infestation causes physiological changes that

reduces photosynthesis or alter the translocation of photosynthates.

Translocation may cause reduction in carbohydrate content in L.

orbonalis damaged S. melongena plants. Studies of Flinn et al. (1990)

revealed that potato leafhopper, Empoasca fabae damage increased total

non-structural carbohydrates of alfalfa from 49 to 465% over the un-

damaged plants. N. lugens infestation caused an increase in the free

amino acids and a decrease in the soluble protein (Sogawa, 1971). The

present data on protein and amino acid content confirm the above

observation. This decrease in nutrient content may induces several

changes in plants, which finally makes plants less palatable for insects

and in turn their development held up.

An increase in POD activity is rather a common phenomenon of

induced plant responses in all plant systems. The increase of the POD

activity in herbivore infested plants can be attributed to the fact that

these are the key enzymes that participate in several plant cell wall

building processes (Chittoor et al., 1999). Huckelvohen et al. (1999) and

Bestwick et al. (1998) had described that the activation of POD might

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181

result in production of hydrogen peroxide and might involve in

hypersensitive cell death. The final products of such enzymatic activities

would be considered anti nutritive because they cannot be effectively

digested and assimilated by herbivore insects (Constabel, 1999). The

same results were observed in L. orbonalis infested brinjal plants after 72

hr of infestation. The increased CAT activity in L. orbonalis infested

plants seems to be related to the plant response to the extent of feeding

plant cell damage. Bi and Felton (1995) observed similar results with H.

zea feeding on soybean plant. Chen et al., (1993) reported that the

increased levels of CAT is known to be involved in increasing the cell wall

resistance and acting as local signals involved in induction of defense

genes. Most lepidopteran insects cause extensive damage to plant tissues

while feeding, whereas this was not so common due to the homopteran

feeding. Normally, the homopteran insects feed from the contents of

vascular tissues by inserting a stylet between the overlying cells, thus

limiting cell damage and due to this reason the minimize induction of a

wounding response in plants are observed. The decrease in the activity of

CAT in L. orbonalis damaged brinjal plants was however, similar to that

observed in Russian wheat aphid infestation (Mohase and Van der

Westhuizen, 2002).

There was a diminutive change observed in the SOD activity due to

biotic stress induced by L. orbonalis in leaf and fruit extracts of brinjal in

comparison to control or undamaged plants. L. orbonalis feeding damage

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182

causes an increase in the SOD activity in brinjal plants. Increased SOD

enzyme activity has been shown to interfere with insect feeding, growth

and development, facilitate microbial infection and finally cause death

(Shapiro et al., 1987; Wang et al., 1996). Though L. orbonalis larvae are

internal feeders and feed inside the brinjal fruits, the induction of

increase in oxidative enzyme activity was found in the fruits as well as in

leaves of infested brinjal plants. The induction of the enzyme might also

be involved in the biosynthetic pathway promoting the induced volatile

emission, which has been observed in earlier studies (Usha Rani et al.,

2007). In earlier studies on pathogen infested groundnut and pest

infested castor plants, changes in oxidative enzymes were also observed

(Usha Rani and Jyothsna, 2009; Jyothsna et al., 2009).

There was an enhanced activity of the defensive enzymes, PAL and

PPO in both the leaf and fruit extracts of L. orbonalis damaged plants

compared to undamaged (Control) plants after 24 to 96 hr after

treatment. The first committed enzyme in the phenylpropanoid and

flavonoid pathways involved in biosynthesis of phytoalexins, lignins and

salicylic acid associated with disease resistance expression has been

discussed by Mauch-Mani and Slusarenko (1996). Activation of PAL in A.

densiflorus was reported to directly affect accumulation of secondary

toxic compounds, such as phytoalexins, phenolic compounds that might

be released through root exudates and inhibit fungal spore germination

and growth (Chenyang et. al., 2001). Similar results were recorded in L.

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183

orbonalis infested brinjal plants after 48 to 72 hr of infestation. The

damaged plants showed significant induction in PAL and PPO enzyme

activities in both fruit and leaf extracts. PAL is considered as the key

enzyme in phenols biosynthesis, since it catalyses the first reaction in

the general pathway of phenylpropanoid biosynthesis, which includes the

formation of flavonoids and hydroxycinnamic acids. The induction of PAL

activity in L. orbonalis damaged leaves and fruits of brinjal plants

indicated that PAL enhancement may be due to ROS generation, which

occurs as primary reaction in response to biotic stress caused by L.

orbonalis larvae. Induced activity of PAL, the key enzyme in

phenylpropanoid pathway indicated the biosynthesis of phenolic

compounds in damaged plants. The induction in biosynthesis of phenolic

compounds in plants have an impact on the herbivore feeding behaviour

that leads to the reduction in further damage caused by L. orbonalis in

brinjal plant system.

Phenolics, a group of secondary metabolites, play very important

roles in plants particularly protection against herbivore attack

(Hahlbrock and Scheel, 1989). In regard to insect pests, phenolics act as

digestion inhibitors and antioxidants by producing free radicals (Appel,

1993). According to Ananthakrishnan et al. (1991) increase in total

phenols are considered as elements of induced resistance in hosts

against herbivory. III and IV instar larvae of L. orbonalis voraciously fed

on brinjal fruits and tender shoots and cause extensive damage. By

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184

comparing the increase in level of foliar and fruit phenolics in case of L.

orbonalis damage, it can be stated that the feeding of L. orbonalis induce

higher amount of foliar and fruit phenolics. From the quantitative

analysis of foliar and fruit extracts significant increase in phenolic

compounds was observed with L. orbonalis infested S. melongena plants

after 48 to 72 hr of damage, when compared with undamaged brinjal

plants after same period of time. This increase however, declined after 96

hr indicating a rapid response. It indicated that the brinjal plants’

response to the specific pest, L. orbonalis was more and as a result

higher amount of phenolic acids were accumulated. Phenols are the

metabolic products in phenylpropanoid pathway and induce in response

of plants to herbivore stress, an increase in phenols accumulation

indicated that the secondary metabolisms of leaves and fruits were

enhanced.

Phenolic acids are structurally diverse class of phytochemicals,

which play major roles in plants protection against herbivore attack

(Hahlbrock and Scheel, 1989). Total amount of phenolic acids (presented

as sum of phenolic compounds) was lowest in control plants and

increase was observed after insect’s infestation. A similar result of low

level free phenolics was observed in control soybean plants and increase

in their content after exposure to stress factors (Janas et al., 2002).

The induction or accumulation of higher quantities of phenolics

acids was due to rapid herbivore feeding on brinjal plant. Normally the III

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185

instar L. orbonalis larvae feeds on brinjal fruits up to pupation and at the

time of voracious feeding by larvae plant responds to the herbivore and

accumulates phenolics in leaves and fruits of the plant. This raid

accumulation impacts on the further feeding by insect herbivore. Jain

and Yadav (2003) have reported that the increase in quantity of total

phenols might be attributed to defense mechanism. The increase in

phenolics in relation to resistance has been reported in Brassica by

Singh (2004). As discussed earlier, higher PPO and PAL activity in brinjal

plant fruits infested by the III instar larvae of L. orbonalis was observed.

This increase in the defensive enzyme activity is associated with the

resistance reaction, which could be due to increased phenolic acid

concentration.

From the analysis of total phenolics, it was observed that increase

in phenolic acids was comparatively higher in case of fruit extracts than

leaf extracts of damaged plants. It is known that fruit borer pests too

induce systemic chemicals in the entire plants on which they feed and

cause changes in the uninfested leaves of the fruit borer infested plants.

Earlier reports indicate that mode of feeding plays an important role in

inducing plant responses (Usha Rani, 2006). The observation recorded

revealed that the changes in brinjal plant were a systemic response, that

the leaves of the fruit borer infested plants also bring changes in leaf

phenolics. But this change was quite low, when compared with the fruit

phenolics. An increase in phenolics, considered a common reaction to

Discussion

186

herbivory (Karban and Baldwin, 1997) has been correlated with its

negative effects on the pest larva (Haukioja and Niemela, 1977). It is

reported by Hori (1973) that Lygus bug feeding on sugar beets results in

increased quinines, which inhibits subsequent bug feeding.

The severe damage in the plant system by herbivore was its

duration of feeding on the plants. The duration of pest feeding also has a

profound impact on phenolic production. Though the duration of feeding

or the extent of damage is same in herbivore infestation, the differences

in the period of infestation and availability of phenolic acids in the leaf

and fruit tissues of brinjal were critically observed in the present study.

Phenolic acid levels in L. orbonalis infested leaf and fruit extracts reached

to maximum level after 72 hr of infestation, which remained above the

control levels for 96 hr of post infestation.

Individual phenolic profiles from L. orbonalis infested fruit extracts

quantified by HPLC analysis suggest that the quantities of chlorogenic,

caffeic, vanillic, cinnamic, chlorobenzoic and synergic acids increased

whereas, coumaric acid decreased in comparison to undamaged fruit

extracts. In case of leaf extracts, quantities of few phenolic acids like

chlorogenic, caffeic, vanillic, coumaric and chlorobenzoic acids were

increased, when compared with undamaged leaf extracts. Unlike fruit

extracts, cinnamic and synergic acids were not noticed in leaf extracts.

The chlorogenic, caffeic and chlorobenzoic acids were quantitatively

increased in brinjal plant up to 72 hr after feeding. Hildebrand et al.

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187

(1986), Usha Rani and Jyothsna (2009), Jyothsna et al. (2009) and

Felton et al. (1992) have found that increased concentration in phenolic

compounds is according to the extent of tissue damaged by feeding

insects or due to pathogen infection. Interestingly, in case of L. orbonalis

infested plants the presence of caffeic acid was recorded even after 96 hr

after infestation. Among the seven phenolic acids monitored, chlorogenic,

caffeic and chlorobenzoic acid were maximum followed by cinnamic acid.

From the leaf and fruit analysis for phenolic compounds, the major

phenolic acids of brinjal plant exhibited good potency against the test

insects, through reduction in feeding and growth of insect herbivore

leading to less damage in the plant.

HPLC results suggest that caffeic acid is one of the phenolic acids,

which play a major role in biosynthesis of lignin (Boerjan et al., 2003).

Coumaric, caffeic and ferulic acids are central intermediates of lignin

biosynthesis. Thus the increase in caffeic acid may be correlated with

lignin biosynthesis. Lignin and other phenolics can strengthen cell walls

and therefore can be anti-nutritional (Brodeur-Campbell et al., 2006;

Schroeder et al., 2006). It is interesting to study the effects of different

phenolic acids on the feeding, herbivore growth, mortality and behaviour.

The results of antifeedant and growth inhibitory bioassays

indicated a significant interaction between the plant feeding and

herbivore performance. Herbivory modify the leaf and fruit chemical

structure, which affect the leaf and fruit suitability to pest larvae.

Discussion

188

Ingestion of caffeic acid methyl ester and caffeic acids deterred the

feeding of the test lepidopteran L. orbonalis, S. litura and A. janata. It is

noteworthy that compounds with less toxicity and having antifeedant

properties were increased due to infestation by L. orbonalis larvae.

Summers and Felton (1994) proposed that the induction of oxidative

stress may be an important component of phenolic toxicity in

lepidopteran larvae. Induced accumulation of phenyl propanoids in

response to insects feeding was reported, especially for the ferulic and p-

coumaric acids in wheat tissues as a response to Sitodiplosis mosellana

(Ding et al., 2000). Chlorogenic acid and rutin, major phenolic

constituents inhibit early larval growth of the fruit worm, H. zea when

added to its artificial diet (Isman and Duffey, 1982). When the tomato

fruit worm H. zea or the beet army-worm S. exigua feed on tomato

foliage, a substantial amount of the ingested chlorogenic acid is oxidized

to chlorogenoquinone, a highly reactive electrophile by PPO in the insect

gut.

The reduction in larval growth is proposed to result from the

alkylation of amino acids or proteins by o-quinones and subsequent

reduction in the nutritive quality of foliage. The cinnamic acid derivatives

like chlorogenic acid and rutin represent model phenolics in the study of

plant antiherbivore defense due to their ubiquitous occurrence among

terrestrial plants and well documented toxicity to insect herbivores

(Isman and Duffey, 1982; Harborne, 1991).

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189

The results presented show much higher biosynthesis of phenolic

acids in brinjal plant leaves and fruits after insects feeding. Thus it can

be suggested that feeding of the herbivorous insects induce antibiosis

based on accumulation of the phenolic acids in brinjal plants. The

summing up of results of this study show that the leaf phenolics are

modified quantitatively due to pest feeding and further these enhanced

phenolics have profound effects on feeding herbivore larval performance

and mortality.

Plant synthesized semio-chemicals that act as insect behaviour

modifying chemicals can be used as tools for management of insect pest

populations (Powell and Pickett, 2003). These chemicals are capable of

inducing a variety of responses in plants, including induction of defenses

against pathogens and herbivores (Walters et al., 2002) and modification

of volatile profiles (Dicke, 1999). The modification of the volatile profile of

plants could repel herbivores. In this way the immigration of

lepidopteran insect, L. orbonalis into a brinjal field could be limited.

The results demonstrated that damaged plant leaf and fruit

extracts of S. melongena increase parasitization efficiency of egg

parasitoid T. chilonis. They indicate that the volatile chemicals extracted

from the surface of L. orbonalis infested brinjal leaf (DLC) and fruit (DFC)

cause arrestment of parasitoids in host vicinity and stimulate oviposition

and orientation by T. chilonis. The parasitoid’s stronger response to

damaged leaf and fruit volatile extracts in comparison to undamaged leaf

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190

(UNDLC) and fruit (UNDFC) volatile extracts indicated that this response

was specific to the volatile chemicals present in L. orbonalis infested leaf

and fruit chemicals. The present findings support previous studies that

have shown the influence of hydrocarbons and terpene compounds on

the behaviour of Trichogramma spp (Jones et al., 1973; Ananthakrishnan

et al., 1991). Potting et al. (1999) stated that parasitoid uses mainly

plant-derived olfactory stimuli in its orientation towards infested plants.

Egg parasitoids locate hosts by using a variety of signals.

Parasitoids use chemical stimuli from their herbivorous hosts or

the host’s food plant during their host searching process (Vinson, 1976;

Vet and Dicke, 1992). Volatile stimuli (synomones) from certain plants

were preferred by Trichogramma pretiosum Riley in an olfactometer and

increased its parasitism rate (Nordlund et al., 1985). Although host-

derived stimuli are reliable indicators of host presence, but they are

difficult to detect at a long distance and are usually used as contact

stimuli. In contrast, plant-derived volatiles are generally more easily

detectable at a long distance (Vet et al., 1991). Many parasitoid species

are known to discriminate in-flight between uninfested plants and host-

infested plants. The evidences are now accumulating in support of

natural enemies that use specific herbivore induced chemical volatiles

emitted by herbivore infested plants (Turlings et al., 1990; Dicke, 1994).

It has been shown that the responsiveness of a number of insect

Discussion

191

parasitoids to volatile odour cues can be increased, if the parasitoid is

allowed to oviposit in the presence of those cues (Turlings et al., 1993).

Few terpene and hydrocarbons are important class of compounds that

were found in the pest damaged leaf and fruit extracts. From the GC-

GCMS analysis of infested leaf (DLC) and fruit (DFC) and un-infested leaf

(UNDLC) and fruit (UNDFC) extracts; few terpene chemicals, α-pinene,

limonene, sabinene hydrate and linalool and few hydrocarbon

compounds such as pentadecane, hexadecane, nonadecane, eicosane,

docosane, tricosane, tetracosane, pentacosane, hexacosane and

octacosane were detected. This finding supports previous work

demonstrating that these compounds existed in pest infested cotton

plants (Loughrin et al., 1995). The monoterpene (E)-β-ocimene emitted

from lima bean leaves after damage by S. littoralis, comprised the

majority (64 to 69%) of the total emissions (Arimura et al., 2007).

The results of Lozano et al. (2000) indicated that α-pinene and 2-

decanone were intricately involved in the attraction and location of the

parasitoids, Dendrosoter protuberans and Cheiropachus quadrum towards

their host, Phloeotribus scarabaeoides. The emission of volatile terpenes

after insect feeding and the subsequent attraction of the infesting insect’s

natural enemies have been shown to occur in several plants by Dicke

(1994). Piperitone 6-isopropyl-3-methylcyclohexane-2-one is present in

the floral volatiles of Tanacetum vulgare L. and is responsible for luring

Lobesia botrana (Gabel et al., 1992). Quantitatively these terpene and

Discussion

192

hydrocarbon chemicals significantly induced in higher concentrations in

damaged plants (DLF and DFC) than in un-damaged ones (UNDLC and

UNDFC). Due to the higher concentrations of these chemicals they may

play an important role in the arrestment responses of T. chilonis by

stimulating oviposition on the treated surfaces. It is presumed that the

brinjal plants undergo physiological changes due to the stress of the L.

orbonalis larval feeding that results in the emission of the chemical

signals.

The GC and GC-MS results combined with the observational

outcomes demonstrate clear evidence of the presence of several

supplementary chemicals in the L. orbonalis infested plants. This

suggests that the quantity of monoterpene and long chain hydrocarbons

are crucial for the arrestment and oviposition stimulation of T. chilonis.

Despite the fact that L. orbonalis leads a concealed life style within the

fruits of the brinjal plants, the parasitoids were able to locate and

identify the pest infested plants among several non infested brinjal plants

and surrounding weed plants. This is strong evidence that the brinjal

plants infested with advance staged L. orbonalis larvae emit volatile

chemicals into the surrounding air, which disperses over large distances.

While there is no particular benefit to T. chilonis, which is an

endoparasitoid of the eggs to identify the larval infested plants, they are

able to discriminate and land on plants infested with IV instar stage

Discussion

193

larvae. It is presumed that this may indicate the likely occurrence of

adults or host eggs on nearby plants.

The major compounds identified as attractant and oviposition

stimulants for egg parasitoids are terpenes and hydrocarbons, which

generally serve many functions in insect behaviour. Parasites have

evolved the ability to utilize these volatile terpene and hydrocarbon cues

produced by plants as primary cues in locating their host insects. These

compounds seem to elicit a fixed behavioural pattern exhibited by

antennal searching of a contaminated area, followed by ovipositor

probing in some parasitoids. In the present findings the induction of

quantitatively higher concentrations of α-pinene, limonene and linalool in

L. orbonalis damaged fruit extracts were found. From the results, it was

observed that extracted volatile chemicals from the damaged fruits led to

significantly higher rates of oviposition and orientation responses by egg

parasitoid, T. chilonis than infested leaf extracts. The similar findings

were noted in a number of other studies. The introduction and over

expression of certain terpene synthases genes in to a host as well as a

non host plant under a constitutive promoter can lead to an enhanced

release of specific terpenes that attract parasitoids searching for their

host (Schnee et al., 2006; Chen et al., 2007).

Mainly pentadecane, heptadecane, eicosane, docosane, tricosane,

pentacosane and hexacosane were the major hydrocarbons present

exclusively in both damaged leaf and fruit extracts of S. melongena

Discussion

194

plants. They may be important cues for host location and ovipositional

stimulation in T. chilonis towards host plant. Wackers and Lewis (1994)

studies by using egg parasitoid, T. japonicum in host location behaviour

support the above results. Certain chemicals such as pentadecane,

heptadecane, eicosane, docosane, tetracosane, pentacosane and

hexacosane were isolated and identified from leaf and fruit extracts. They

have shown increase parasitism (oviposition response) by the parasitoid

T. chilonis in both dual and multiple choice tests, when applied to host

eggs. Whereas, the above compounds showed significant orientation

response in both four-choice olfactometer and culture tube bioassays

against T. chilonis. These compounds were assumed to contribute to an

increase oviposition and orientation responses by T. chilonis, because

these hydrocarbon compounds have a high number of carbon atoms.

They might act as contact stimulants, whereas the chemicals with less

than 10 carbon atoms are more volatile and might attract parasitoids to

the near vicinity of the host eggs.

The increased stimulation of oviposition and orientation by the

extracts of L. orbonalis infested plants may be due to the presence of

chemicals in the leaf and fruit extracts that are emitted through leaf and

fruit surfaces. It was observed only in the fruits infested with advance

staged larvae and larval feeding inside the fruit. Because of this, it is

presumed that the quantity of the chemicals released might depend on

the quantity of feeding inside the galleries and possibly the quantity of

Discussion

195

excretion. This prediction is based on the observation that the damaged

fruit and leaf extracts produced greater orientation and host searching

responses followed by high oviposition by T. chilonis in the laboratory

behavioural bioassays (Usha Rani et al., 2007).

On infestation of fruits by L. orbonalis, the induced changes were

observed in both damaged fruits as well as in undamaged leaf surfaces.

This was achieved due to the systemic response of the brinjal plant.

Fatouros et al. (2005) stated that if this systemically induced odour

attracts natural enemies to the vicinity of the plant then local cues

restricted to the oviposition or feeding site can facilitate fine scale

orientation. Hence, it is unlikely that parasitoids use chemical cues

emanating from the both leaf and fruit surfaces as cues in locating the

egg masses of L. orbonalis to oviposit over them.

The results of this study revealed numerous parallels with other

plant species in which the volatile emission physiologically characterized

(Pare and Tumlinson, 1999). T. chilonis can discriminate between odours

emitted from undamaged leaves and fruits and from infested with or

damaged by its host L. orbnalis. Based on these findings, it can be

concluded that brinjal plants under the stress of herbivory, L. orbonalis

emit volatile chemicals through their leaf and fruit surfaces. These

volatile bouquets serve as signals for mated female parasitoids T.

chilonis. Therefore, plant volatiles take on the functional role of

synomones that are sequestered within plant tissue and T. chilonis rely

Discussion

196

on these chemicals to locate long distance cues from the host habitat. A

systematic isolation, identification and the synthesis of these chemical

cues may lead to the development of synomonal compounds that

enhance the bio control potency of the Trichogramma species.