2. REVIEW OF LITERATURE

It is a prerequisite to gather all the necessary and existing information about a

problem, before the initiation of any proposed research work. As the present study deals

with the effect of plant protease inhibitors on Bactrocera cucurbitae, so, the present review

has been documented under three sections i.e. 1)Introduction to plant protease inhibitors,

2) Their effect on insects and 3) A brief introduction to the melon fruit fly, B. cucurbitae,

the experimental insect model selected for the present study.

2.1 INTRODUCTION TO PLANT PROTEASE INHIBITORS:

Biosynthesis of protease inhibitors is a defense mechanism of plants which they have

generated during co-evolution with the herbivores. Protease inhibitors are often found in

high concentrations in seeds and tubers, particularly in those of the Graminae,

Leguminosae and Solanaceae families (Sadasivam and Thyumanavan, 2003). These

inhibitors are diverse in number but demonstrate specificity towards various proteolytic

enzymes.

2.1.1 Classification and Occurrence:

Proteolytic enzymes catalyze the cleavage of peptide bonds in proteins. They have

been classified (recognized by International Union of Biochemistry and Molecular

Biology, IUBMB) according to their mechanism of catalysis and the amino acid present in

their active center as:

1) Serine proteinases

2) Cysteine proteinases

3) Aspartic proteinases

4) Metalloproteinases

� The Serine Protease inhibitors:

Serine protease inhibitors with a serine and histidine residue in their active center are

widespread in the plant kingdom. They fall into a number of structurally distinct

subfamilies based on their amino acid sequences (Bode and Huber, 1992) such as the

Kunitz family, the Bowman-Birk family, Potato I inhibitor family, Potato II inhibitor

family, Squash inhibitor family, Cereal trypsin inhibitor family and Mustard trypsin

inhibitor family.

The Kunitz (Soybean Trypsin inhibitor) inhibitors are ~ 20kDa proteins, with one

polypeptide chain or two disulphide-linked chains, with an arginine residue in their single

active site (Carlini and Grossi-de-Sa, 2002). They have been identified in a wide range of

plant species belonging to different families like Papilionidae: winged bean, Erythrina

latissima; Erythrina crystagall; Glycine maxima; Cesalpinoideae: Peltophorum africanum;

Mimosidae: Adenanthera pavonina; Albizzia julibrissi; Acacia elata; Acacia sieberana;

Gramineae: Hordeum vulgare; Triticum aestivum and Secale cereale (Garcia-Olmedo et

al., 1987).

Bowman-Birk (Soybean Trypsin-Chymotrypsin inhibitor) type inhibitors are double

headed small polypeptides (8kDa), binding simultaneously and independently to two

separate proteinase molecules such as Trypsin and chymotrypsin (Bode and Huber, 1992).

Inhibitors of Bowman-Birk type have been found in the seeds of many members of

Leguminoseae family (Garcia-Olmedo et al., 1987) i.e. soybean, Glycine max; kidney

bean, Phaseolus vulgaris; azukibean, Vigna angularis; mungbean, Vigna radiate L.

Wilczek; cowpea, Vigna unguiculata; limabean, Phaseolus limensis; groundnut, Arachis

hypogaea; black-eyed pea, Vigna unguiculata unguiculata; runner bean, Phaseolus

coccineus; lentil, Lens culinaris; perennial horse gram, Macrotyloma axxillare; apple-Leaf

(Rain Tree), Lonchocarpus capassa; kiaat tree, Pterocarpus angolensis and garden vetch,

Vicia angustifolia.

Potato inhibitor family is also referred to as chymotrypsin inhibitor I because of its

strong specificity towards chymotrypsin, although it inhibits subtilisin, pronase, as well as

some other alkaline microbial proteases. They are non-covalent tetramers of four different

subunits, each having a molecular weight of 10kDa. Potato II inhibitor family has a

molecular weight of 20kDa and is composed of four distinctly different protomers (Birk,

2003).

Squash inhibitor family (Cucurbitaceae) consists of inhibitors of Trypsin that were

isolated from squash, Cucurbita maxima; zucchini, Cucurbita pepo; melon, Momordica

spp and cucumber, Cucumis sativus seeds. The members of this family are very small

molecules having molecular weight of about 3kDa (Wieczorek et al., 1985). They inhibit

trypsin, plasmin, kallikerin and cathepsin G.

Numerous inhibitors have been found in cereal grains such as barley, H. vulgare; rye,

S. cereale; wheat, T. aestivum; maize, Zea mays; rice, Oryza sativa; Sorghum spp and

oats, Avena sativa that belong to barley trypsin inhibitor family. A single polypeptide chain

of molecular weight of about 13kDa with five disulfide bridges, serves as a prototype for

this group (Odani et al., 1983).

� The Cysteine Protease Inhibitors (Cystatins):

The term cystatin refers to proteins which have a cysteine residue in their active

center and specifically inhibit the activity of papain and related cysteine proteinases like

cathepsin B, H etc. Cystatins have been found to be evolutionarily related, forming the

‘cystatin superfamily’ (Oliveira, 2003), members of which are divided into three groups

(or families) of closely related proteins which comprise two animal Cystatins and one

family from plant Cystatins or phytocystatins. Among plant Cystatins, the oryzacystatin

from rice seeds was the first inhibitor of plant origin considered as member of cystatin

superfamily and was related to chicken egg white cystatin (Barrett et al., 1986).

Phytocystatins are divided into two groups, one that possess single domain, and the second

that possess multiple domains, e.g. multicystatin from potato tubers and tomato leaves

(Walsh and Strickland, 1993; Bolter, 1993). The molecular mass of the purified

phytocystatins ranges between 5 to 87kDa and are stable at extreme pH and temperature. A

number of higher plants, both monocot and dicot species have phytocystatins such as

pineapple, Ananas comosus; potato, Solanum tuberosum; corn, Zea mays; rice, O. sativa;

cowpea, Vigna unguiculata; mungbean, V. radiata; tomato, Solanum lycopersicum; wheat,

T. aestivum; barley, H. vulgare; rye, S. cereale; millet, Pennisetum glaucum and apple

fruit, Malus domestica (Abe et al., 1987, 1992; Hines et al., 1991; Gruden et al., 1997;

Ryan et al.,1998).

� Aspartic Inhibitors:

Aspartic protease inhibitors are with an aspartic group and have been recently isolated

from sunflower, barley, and cardoon, Cyanara cardunculus flowers (Park et al., 2000;

Kervinen et al., 1999 and Frazao et al., 1999).

� Metalloproteinase Inhibitors:

This family of Inhibitors is not very well studied and only a few reports are available

about them. They have a metallic ion (Zn2+, Ca

2+ or Mn

2+) in their active center. Plants

have evolved at least two families of metalloproteinase inhibitors, the

metallocarboxypeptidase inhibitor family in potato and tomato plants and Cathepsin D

inhibitor family in potatoes (Rancour and Ryan, 1968; Keilova and Tomasek, 1976).

Table1: Families of Plant protease inhibitors reported in PLANT-PIs Database (De

Leo et al., 2002)

Plant Protease Inhibitor Family PLANT-PIs Code

Serine PIs

Bowman-Birk protease Inhibitors BBI

Kunitz Soybean Trypsin Inhibitor KNI

Cereal Trypsin/α-amylase Inhibitors BRI

Potato Type I Inhibitors PI 1

Potato Type I Inhibitors PI 2

Squash Inhibitors SQI

Mustard Trypsin Inhibitor MSI

Cysteine PIs CYS

Metallocarboxypeptidase Inhibitors MCI

2.1.2 Physiological Role of Protease Inhibitors in Plants:

1) Regulation of Proteolysis

The function of the inhibitors is to control proteolysis within cells, organelles or

fluids, when limited proteolysis is important for the biochemical or physiological process

(Ussuf et al., 2001). Inhibitors of endogenous proteinases of rice seed have been studied in

great detail. Oryzacystatins I and II suppress the activity of seed Cysteine proteinases

(Oryzains), which cleave glutelin, the major storage protein of rice (Abe et al., 1987). The

ability to act on endogenous seed proteinases was also found in corn (Abe et al., 1992) and

wheat (Kurodo et al., 2001). In addition to Cystatins, proteolytic activity in seeds is

regulated by proteinase inhibitors of other families. A Trypsin inhibitor contained in

quiescent seed of lettuce (Lactuca sativa) suppresses the activity of a Trypsin-like

proteinase, expressed in the course of germination (Shain and Mayer, 1965).

2) Apoptosis

Role of Protease inhibitors in modulation of apoptosis or programmed cell death

has been identified. Cysteine proteinase plays an important role in the regulation of

programmed cell death leading to hypersensitive reaction, following pathogen attack. It is

suggested that in plants balance between the Cysteine proteinase and Cysteine proteinase

inhibitor activity regulates the programmed cell death (Ussuf et al., 2001).

3) Storage

In early reports, the idea that proteinase inhibitors may serve as storage proteins in

plants was advanced and evidence for the same was provided by observations of the high

content of inhibitors in seeds and other storage organs of plants (Ryan, 1973; Richardson,

1977). It was also demonstrated that certain protease inhibitors belong to the same protein

families as storage proteins proper, an observation suggesting a common origin (Mosolov

and Valueva, 2005).

4) In Response to Injury

Protease inhibitor gene expression has been detected in leaves of several species

following wounding, suggesting their role in protecting plants from insect attack and

microbial infection. Studies have shown that protease inhibitors of plant origin are capable

of suppressing the activity of enzymes contained in the digestive tract of insects (Ryan,

1990). In addition to insect pests, the potential of inhibitors in developing transgenic plants

with enhanced resistance to other pathogens, e.g. nematodes, fungi, bacteria and viruses,

the survival and invasion of which require proteolytic activities has also been explored

(Fan and Wu, 2005). Plant protease inhibitors confer natural as well as engineered

protection against nematode attack (Atkinson et al., 2003; Urwin et al., 2003).

2.1.3 Mode of Action of Protease Inhibitors:

Extensive research on protease inhibitors has provided a basic understanding of the

mechanism of action that applies to most serine inhibitor families and probably to the

cysteine and aspartyl inhibitor families as well. All serine inhibitor families from plants are

competitive inhibitors of the protease that they inhibit with a similar standard mechanism

as proposed by Laskowski and Kato (1980). According to this mechanism inhibition

occurs as a consequence of binding of the active-site substrate-binding region of a

proteinase to the corresponding substrate-like region (reactive site) on the surface of the

inhibitors. The reactive site peptide bond after hydrolysis (i.e. in the modified inhibitor)

acquires a newly formed carboxyl terminal residue designated as P1 and an amino terminal

residue designated as P1’. The reactive site residue P1 generally corresponds to the

specificity of the cognate enzyme. According to Walker et al. (1998) the inhibitor binds to

the active site on the enzyme to form a complex with a very low dissociation constant (107

to 1014 at neutral pH values), thus effectively blocking the active site. A binding loop on

the inhibitor usually ‘locked’ into conformation by a disulphide bond, projects from the

surface of the molecule and contains a peptide bond (reactive site) cleavable by the

enzyme. The inhibitor thus directly mimics a normal substrate for the enzyme mechanism

of peptide bond cleavage to proceed to completion i.e. dissociation of the product (Walker

et al., 1998).

2.1.4 Uses of Plant Protease Inhibitors:

The knowledge of therapeutic possibilities of protease inhibitors in the treatment of a

wide range of disorders, such as pancreatitis, shock, allergy and inflammation associated

with enhanced proteolytic activities had resulted in several kallikerin trypsin inhibitor

based drugs (Richardson, 1977). An epidemiological study of the decreased occurrence of

breast, colon, and prostatic cancers in vegetarian populations is suggestive of a role of

plant protease inhibitors in preventing these cancers (Birk, 1993). By now, a variety of

studies have demonstrated that protease inhibitors can suppress several stages of

carcinogenesis, including initiation, promotion, and progression. Plant proteases inhibitors

that regulate human physiological processes e.g. cell signaling/migration, digestion,

fertilization, growth, differentiation, immunological defense, wound healing and apoptosis,

have great potential in therapeutic applications. Plant protease inhibitors are also seen as

potential candidates for insect control programme by means of transgenic plants as a good

number of plant PIs have shown anti-insect activity against lepidopteran and coleopteran

pests (Lawrence and Koundal, 2002)

2.2 EFFECT OF PLANT PROTEASE INHIBITORS ON INSECTS:

2.2.1 EFFECT ON GROWTH AND DEVELOPMENT OF INSECTS:

According to Zeng et al. (1993) insect’s susceptibility to PIs could be measured by

different parameters i.e. survival, development time, fecundity of insect pest and growth

patterns. These parameters were appropriate measures of the effects of PIs on the insect’s

population dynamics. In literature a number of reports are available on the effect of plant

PIs on growth and development of various insects belonging to different orders.

2.2.1.1 Effect of Serine PIs on Growth and Development of Insects:

Lepidoptera

In Lepidoptera, most of the work related to protease inhibitors has been done on legume

pod borer, Helicoverpa armigera (Hubner), although some reports on other lepidopterans

like cutworm, Spodoptera litura (Fab.), fall armyworm, Spodoptera frugiperda

(J.E.Smith), European corn borer, Ostrinia nubilalis (Hubner), cabbage looper,

Trichoplusia ni (Hubner), codling moth, Cydia pomonella (L.), sugarcane borer, Diatrea

sachharalis (Fabricius), Mediterranean flour moth, Anagasta kuehniella (Zeller) are also

available.

Effects of Soybean Kunitz trypsin inhibitor (SBTI) and Soybean Bowman-Birk

trypsin-chymotrypsin inhibitor (SBBI) on newly molted third instar larvae of corn

earworm, H. armigera were studied by Johnston et al. (1993). They found 70% and 100%

mortality by 22nd day in SBBI (0.750 mM) and SBTI (0.234mM) treated diet, respectively.

A decrease in total larval biomass and mean larval weight was also observed. Harsulkar et

al. (1999) investigated the efficacy of PIs from three host plants (Chickpea, Cicer

arietinum; pigeon pea, Cajanus cajan and cotton, Gossypium arboretum) and three non-

host plants (groundnut, A. hypogea; winged bean, Psophocarpus tetragolonubus and

potato, S. tuberosum) in retarding the growth of H. armigera larvae. There was a 3 to 4

fold reduction in weight gain in the larvae fed with non-host PIs, also, food intake was

drastically reduced in larvae showing growth retardation. Pupation too was delayed by

more than ten days. In 2002, Gatehouse et al. reported 20 fold lower larval weight in H.

armigera fed on chymostatin incorporated artificial diet, though there was no significant

difference in the survival of larvae on inhibitor containing base diet (without inhibitor).

Similarly, in 2003, Telang and coworkers studied the effect of bitter gourd protease

inhibitors (BGPI) on growth and development of H. armigera by incorporation of BGPI at

four different doses [3, 6, 9 and 12 trypsin inhibitor units (TIU)/g of feed] into artificial

diet. A significant reduction of about 43% in larval weight of H. armigera was observed

with a diet containing BGPIs (12 trypsin inhibitor units/g) although the loss in larval

weight was not consistently dose dependent. Larval mortality was 30% in larvae fed on

diet containing 6TIU/g feed whereas it reduced to 10% at highest dose of 12 TIU/g. Larval

and pupal periods were also reduced. Pupal weight was reduced in a dose dependent

manner by about 26% at the highest dose of BGPIs as compared to control. BGPIs also

affected fertility and fecundity in the adults emerging after larval treatment. Egg laying

capacity of adult females reduced significantly from 357 eggs/female in control to 113

eggs/female in diet with 9TIU/g while hatching of larvae from eggs reduced from 88% in

control to 25% (6TIU/g feed) and 0% (9 and 12 TIU/g).

Gujar et al. (2004) studied fusion effect of crude proteinase inhibitor extracts from

seeds of different crop plants (black gram, chickpea, chuckling vetch, finger millet, French

bean, green gram, horse gram, lentil, pea and soybean) with Bacillus thruingenesis var.

Kurstaki HD-1 against neonate larvae of H. armigera by diet incorporation method. The

larval mortality due to crude PIs alone (5% seed weight equivalent) ranged from 4 to 19%,

the maximum with pea. A mixture of B. thuringenesis (10ppm) and PI (5% seed weight

equivalent) was synergistic in larval mortality with respect to PIs of pea, chuckling vetch,

lentil, soybean, chickpea, French bean and black gram and antagonistic with respect to

those of finger millet, horse gram and kidney bean. The larval growth reduction with crude

PIs alone ranged from 17.9 to 53.1%, the maximum reduction being with soybean and

minimum with lentil. Similar results were reported by Shukla et al. (2005) as they reported

decrease in larval survival of H. armigera from 90% in larvae reared on control diet to

49% in larvae reared on SBTI (1mg/ml diet) impregnated diet. Fresh and dry weights of

the larvae were lower in control diet as compared to the larvae reared on diets containing

SBTI. Percentage pupation (of the total larvae released) ranged from 40% in SBTI treated

diet to 90% in untreated control diet. Percentage adult emergence was also reduced with

SBTI treatment. They also studied the effect of SBTI (1 and 2%) through surface treatment

of artificial diet and reported 16.67 and 20% mortality as compared to 10% in control.

Larval weights and percentage pupation also decreased in the treated diet.

A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) purified by

Srinivasan et al. (2005) was efficient in inhibiting the larval growth and development of H.

armigera as was evident by 64% and 47% weight reduction in third instar and fourth instar

larvae, respectively fed on diet containing inhibitor from chickpea as compared to control.

By the fifth instar larval (18th day) stage, the average inhibitor fed larvae weighed 202mg,

39% lower than the control (330.8mg). In the same year (2005), Tamhane et al. also

reported 12 to 24% weight reduction in the first generation larvae fed on proteinase

inhibitors from Capsicum annum leaves. Also, pupation of 32 to 44% larvae was delayed

by minimum of 3 days after exposure to PI. Egg laying capacities of adults and egg

hatching were reduced by 60 and 40%, respectively. They also reported larval weight

reduction of 53% in the second generation under the effect of C. annum PIs (CAPI) along

with dose-dependent larval mortality of 23, 30 and 40% in 1X, 3X and 6X CAPI,

respectively. In addition to this, significant decrease in pupal weight was also observed in

CAPI fed groups as compared to control.

In 2007, Bhavani and coworkers bioevaluated Subabul (Leucaena leucocephala)

proteinase inhibitors against H. armigera and reported significant reduction in growth

among larvae fed with inhibitor incorporated artificial diet which weighed 11mg/larvae as

compared to 58mg/larvae in the control. Control larvae pupated on day 17 onwards

whereas in treatment larval period was delayed up to day 24. Larval growth period was

extended by 4 and 6 days in larvae fed on high molecular weight Subabul TI (HSTI) and

low molecular weight Subabul TI (LSTI), respectively. They also experimented with

surface treated chickpea leaves and seeds (water-imbibed) and observed that highest

weight was gained in control (404 mg on 16th day) followed by HSTI (192mg on 22

nd day)

and LSTI (160mg on 22nd day). Also, the larval growth period was extended up to ten days

on HSTI while LSTI fed larvae did not complete their life cycle. Mortality rate was 100%

in LSTI whereas it was 80% in HSTI treatments. Pupal weight too was reduced from

235mg in control to 212mg in HSTI while no pupation occurred in LSTI. Kansal et al.

(2008) purified trypsin inhibitor from mung bean (V. radiata ) seeds which showed good

anti-insect potential against H. armigera as a marked decline in survival of larvae was

observed with increase in concentration of mung bean trypsin inhibitor in the diet. The

larval mortality was 33, 53 and 60% at 10,000 (T1), 20,000 (T2) and 30,000 (T3) TIU,

respectively. A reduction of 78% was observed in larval weight at T3 and larval growth

was also extended by 6, 10 and 15 days in T1, T2 and T3, respectively. Heath et al. (1997)

reported a significant reduction in the growth of budworm, Helicoverpa punctigera

(Wallengren) after the ingestion of diet incorporated with 0.26 % w/v of Nicotiana alata

PIs.

Effects of PIs from various sources like Momordica charantia and Archidendron

ellipticum have been studied against cutworm, S. litura. In 1995, Mcmanus and Burgess

studied the effects of SBTI on growth and digestive proteases of larvae of cotton cutworm,

S. litura. They observed that larvae fed on base diet grew the most, followed by those

consuming 0.21% SBTI than those fed 0.5% SBTI. In 2003, Telang et al. reported a

reduction in larval weight of S. litura fed on artificial diet containing bitter gourd PIs,

maximum reduction being 70% at the highest concentration of 12 trypsin inhibitor units/g

after 10 days of feeding. Larval mortality ranged from 10 to 20% whereas reduction in

pupal weight was by 30%. Larval period was not affected but pupal period was delayed by

up to 3 days. Fertility was drastically affected as the egg laying capacity reduced from

300eggs/female in control to 260eggs/female in treated larvae, however fecundity was not

affected. Bhattacharya et al. (2007) isolated a trypsin inhibitor from A. ellipticum (AeTI)

and studied its bioinsecticidal activity against S. litura. They reported reduction in larval

weight of first to fifth instar and retardation in growth rate as it fell over 85% on the

experimental diet as compared to larvae growing on control diet. Also the larval survival

was affected as AeTI resulted in 76% mortality by the fifth instar stage. Contradictory to

above studies Paullilo et al. (2000) observed that chronic ingestion of semi purified

soybean inhibitors did not result in a significant reduction in growth and development of

fall armyworm, S. frugiperda. The results suggested that S. frugiperda was able to adapt

physiologically to dietary PIs.

In 1978, Steffens and coworkers studied the effect of two purified inhibitors on the

development of European corn borer, O. nubilalis. They reported that SBTI, incorporated

at levels of 2-5% in the diet, inhibited the growth of the larvae and delayed pupation, but

did not prevent completion of the life cycle whereas the corn trypsin inhibitor had no effect

on the growth and metamorphosis of the larvae. Larocque and Houseman (1990) had

perceived that in O. nubilalis, Soybean trypsin inhibitor (Kunitz) incorporated at levels of

2-5% in the diet inhibited growth and delayed pupation but did not prevent completion of

the life cycle.

Broadway (1995) reported that dietary amendment with cabbage PIs reduced larval

growth by 35% and adult emergence by 66% in cabbage looper, T. ni at 1% in diet. The

anti-metabolic properties of soybean proteinase inhibitor (SBI) on growth of neonate

larvae of the sugarcane borer, D. sachharalis have been evaluated by Pompermeyer et al.

(2001). It was observed that larvae fed on control diet weighed more than those fed on SBI

diet. Consumption of SBI diet also delayed the developmental time to pupation and adult

emergence, significantly (P>0.05). About 56% of larvae reared on SBI diet presented six

instars as compared to five instars in control fed larvae. Significant negative effect on

reproductive potential of D. sachharalis has been observed under consumption of SBI.

Oviposition commencement was significantly delayed by six days (an 18% increase)

compared to the control fed adults.

Trypsin PI isolated from seeds of Peltophorium dubium fed to Mediterranean flour

moth, A. kuehniella reduced larval weight by 50% (at 1%) and reduced %age survival to

44.3% (at 1.6%) as compared to control (Macedo et al., 2003). Potato protease inhibitors

and soybean trypsin inhibitor when added to artificial diet reduced the growth rate of the

codling moth, C. pomonella (Markwick et al., 1995), however, the results were more

pronounced in the larvae fed with diets containing a combination of potato PI and

carboxypeptidase inhibitor. More recently, Liao et al. (2007) isolated a trypsin inhibitor

from Cassia obtusifolia which was potent against cabbage white butterfly, Pieris rapae

(Linnaeus) as it delayed growth and resulted in 30% decrease in larval weight.

The anti-metabolic studies of soybean Kunitz inhibitor done against cabbage moth,

Mamestra brassicae (L.) by Chougule et al. (2008) showed a decrease in mean larval

weight. The larvae were ~25% smaller than the control diet fed larvae. Sivakumar et al.

(2005) reported that the gut proteinases of rice moth, Corcyra cephalonica (Stainton) and

diamond back moth, Plutella xylostella (Linnaeus) were inhibited by inhibitors from finger

millet, Eluesine coracana Gaertneri and little millet, Panicum sumatrense Roth.

Coleoptera

Birk and Applebaum (1960) found that partially purified PIs from soybeans inhibited

the growth of red flour beetle, Tribolium castaneum (Herbst) larvae. Oliviera et al. (2002)

incorporated papain PIs from seeds of Algaroba tree, Prosopis juliflora into diet of cowpea

weevil, Callosobruchus maculatus (F.). These inhibitors had detrimental effects on

development of the cowpea weevil. They caused a reduction in 50% of the average larval

mass. Studies done by Gomes et al. (2005) described effects of novel PIs from seeds of

chickpea (C. arietinum) against cotton boll weevil, Anthonomus grandis (Boheman). They

observed that at 1.5% (w/w), C. arietinum trypsin inhibitor (CaTI) caused severe

development delay, several deformities and a mortality rate of ~45%. Macedo et al. (2004)

perceived that when Kunitz type inhibitor, isolated from A. pavonina seed was fed into diet

of cowpea weevil, C. maculatus, it caused 50% mortality, reduced fecundity and

oviposition and severe deformations occurred in the adults emerged from treated larvae. In

2005, Araujo et al. isolated a trypsin inhibitor from Tamarind tree (Tamarindus indica)

seeds which was effective against C. maculatus. It caused 50% mortality (LD50) of larvae

at 3.6% and reduced larval mass by 50% (ED50) at 3.2%. The inhibitors from finger and

little millet were effective against gut proteinases of rice weevil, Sitophilus oryzae (L.) as

reported by Sivakumar and co-workers in 2005.

Diptera

Among dipterans most of the studies have been done on fruit fly, Ceratitis capitata

Weidmann and stable fly, Stomoxys calcitrans (L.). Spates (1979) studied the effect of

soybean trypsin inhibitor on the fecundity of female stable flies, S. calcitrans. He found

that when 3mg SBTI/ml was added to plasma, serum or erythrocyte diets, fecundity was

reduced to zero and when SBTI was added to whole blood or defibrinated blood, fecundity

was reduced by 71 and 79%, respectively.

A proteinaceous trypsin inhibitor from Crotolaria pallida seeds was effective

against C. capitata larvae as it caused 27% mortality and 44.4% mass decrease (at 1%

w/w), however, the action was constant at higher doses from 2-4% (w/w) with 15%

mortality and 38% mass decrease (Gomes et al., 2005). In the same year, Araujo et al.

reported that the concentration of trypsin inhibitor purified from T. indica seeds in the

artificial diet necessary to cause 50% mortality (LD50 ) of C. capitata larvae was 3.6% and

that to reduce 50.0% (ED50) larval mass was 3.2%. Further, the mass of fruit fly larvae

were affected at 53.2% and produced ~34% mortality at a level of 4.0% w/w of TTI. In

2006, Silva et al. reported that C. capitata larvae were susceptible to SBTI which affected

larval mass at ED50 3.01% when incorporated into artificial diet. The larvae were more

affected at initial inhibitor concentrations of 0.25% and 0.5%, causing 20% mortality, but

at high SBTI concentrations mortality decreased to 10%.

Orthoptera

Plant PIs have been reported to be effective against two insects of the order

orthoptera i.e. black field cricket, Teleogryllus commodus (Walker) and grasshopper,

Melanoplus sanguinipes (Fab.).

Growth and survival responses were determined for the black field cricket, T.

commodus fed on 6 different protease inhibitors [SBTI, Potato protease inhibitor (POT-1 &

POT-2), Wheat germ trypsin inhibitor (WGI-1), Cowpea trypsin inhibitor (CpTI), Bovine

pancreatic trypsin inhibitor (BPTI)] either singly or in combination, at a range of

concentrations in diets containing 3 different levels of casein (Burgess et al., 1994). Over a

six week period, SBTI, POT-1, POT-2 and BPTI caused dramatic reductions in cricket

survival on the low casein (0.5%) diet. On 1% casein diet, SBTI and POT-2 killed nearly

all crickets while on 3% casein diet; the inhibitors tested had comparatively little effect on

survival. Overall, only 1.5% of crickets fed on protease inhibitors in a 0.5% casein diet

survived the 12-week period compared with 39% of blank controls. This figure increased

to 15% survival for protease inhibitor treatments on 1% casein diet compared to 69% of

blank controls, and 40% on the high casein diet, on which 71% of controls survived. On

the 0.5% casein diet, in addition to causing mortality, all 5 protease inhibitors which were

added at 0.1% (SBTI, WGI-1, POT-1, POT-2 and CpTI) effectively reduced cricket growth

when compared to the blank control (p<0.05) . On 1% casein diet none of the inhibitors

significantly reduced growth at 0.03%, but all 6 were effective at 0.1% (p< 0.05).

Increasing the concentration from 0.1 to 0.3 % caused a slight decrease in growth for most

protease inhibitors.

Hinks and Hupka (1995) reported that when soybean trypsin inhibitor was

incorporated into diet of grasshopper, M. sanguinipes, detrimental effects like high

mortality during development, delayed growth and low reproductive rate were observed.

Hymenoptera

Malone et al. (1995) and Burgess et al. (1996) observed toxic effects of soybean

trypsin inhibitor (SBTI) on adult European honey bees (Apis mellifera L.) as it

significantly reduced the longevity of bees fed at 1, 0.5 or 0.1% SBTI (w/v) but not at

0.01% or 0.001%. In another study conducted by Malone et al. in 1998, POT 1 and POT 2

fed to newly emerged honey bees at different doses in either sugar syrup (0.2 or 0.01%

w/v) or pollen food (1 or 2% w/w) significantly reduced lifespan, with the effect of pollen

treatment being greater than the syrup treatment. Pham-Delegue et al. (2000) studied the

effect of SBTI and BBI on survival and learning abilities of honeybees, A. mellifera and

found that SBTI at 0.1mg/ml sucrose solution and BBI at 1mg/ml sucrose solution

significantly increased mortality and also reduced the level of conditioned response to the

odor of floral volatile (linalool).

Bioassays using potato aphid, Macrosiphum euphorbiae (Thomas) reared on

artificial diets supplemented with SBBI showed impairment of aphid parasite, Aphelinus

abdominalis (Dalman) that developed on intoxicated aphids. SBBI was detected in only

larval parasitoids while no PI could be detected in adult parasitoids that emerged from PI-

intoxicated aphids (Azzouz et al., 2005).

Hemiptera

In 1997, Tran et al. conducted trials to identify protease inhibitors effective against

cereal aphids on wheat. They studied five inhibitors [POT1, POT2, potato

carboxypeptidase inhibitors, SBTI, and Lima bean trypsin inhibitor (LBTI)] against three

insects i.e. Russian wheat aphid, Diuraphis noxia (Mordnilko); green bug, Schizaphis

graminum (Rondani) and bird cherry oat aphid, Rhopalosiphum padi (L.). Two of these

inhibitors, POT1 and POT2, increased mortality among late instar aphids and reduced

production of nymphs in feeding trials. SBTI produced statistically significant increase in

mortality after 24, 48 and 72h as compared to controls. The lima bean inhibitors showed

little effect on any of the 3 species.

2.2.1.2 Effect of Cysteine PIs on Growth and Development of Insects:

Coleoptera

Orr et al (1994) reported that potato multicystatin (PMC) given in the artificial diet

caused a dose-dependent inhibition of growth in neonate and second instar southern corn

rootworm, Diabrotica undecempunctata howardii (Barber) and western corn rootworm,

Diabrotica virgifera virgifera (Leconte). Neonate southern corn rootworm and second

instar western corn rootworm had similar sensitivity to the inhibitor (50% inhibition at 25-

43.8µg/cm2), whereas second instar southern corn rootworm were about 5-fold less

sensitive. In contrast to southern corn rootworm larvae, western corn rootworm growth

was completely halted by PMC. Long term exposure of southern corn rootworm larvae to

PMC suggested that the larvae become less sensitive to the inhibitor during development.

In 2003, Oppert et al. studied the effect of cysteine PIs from potato on red flour

beetle, T. castaneum and reported that larval growth was suppressed although no

significant mortality was observed. In 1995, Bolter and Jongsma studied the effect of

cysteine PIs in potato leaves (4% of total protein) on larvae of Colorado potato beetle,

Leptinotarsa decemlineata (Say) and reported a significant (p<0.05) lower larval weight

of fourth instar on Methyl Jasmonate-treated plants as compared to control plants.

However, larval mortality was not significantly different. Cysteine PI, E-64 had a profound

effect on L. decemlineata larval growth, development and survival as well as on adult

fecundity (Bolter and Latoszek-Green, 1997). However, the number of insects surviving to

the adult stage did not decrease below 26% with increasing E-64 concentration above

1.5µg/cm2 leaf surface. The development time to the pupal stage increased from 13 days in

control larvae to 21 days at a concentration of 1.5 µg/cm2. The most significant

effect of

dietary E-64 was on adult fecundity, with mated females reared on untreated leaves laying

an average 62±5.7 eggs daily in the first 10 days, and those maintained on 0.5 µg/cm2

laying only 16±2.4 eggs/day. Females given 1 µg/cm

2 laid few eggs if any, but started

producing egg masses as large as control insects about 5-days after being switched to

control leaves.

Zhu-Salzman et al. (2003) constructed a recombinant fusion protein, scN-rGSII

that retained both cysteine protease inhibitor soycystatin N (scN) and Griffonia

simplicifolia lectin II (rGSII) and studied its effect on cowpea bruchid beetle, C.

maculatus. They reported a synergistic delay in insect development (i.e. the delay caused

individually by scN and rGSII) with scN-rGSII recombinant protein as compared to the

additive effect exhibited by the 1:1 mixture of scN to rGSII. In the same year Zhu-

Salzman et al. further reported that the soybean cysteine protease inhibitor, soyacystatin N

(scN), had a negative impact on the growth and development of C. maculatus, however,

the developmental delay and feeding inhibition caused by dietary scN occurred only

during the early developmental stages (1st, 2

nd and 3

rd instars) of the bruchid. The 4

th instar

larvae reared on scN diet (adapted) exhibited rates of feeding and development which were

comparable to those feeding on an scN-free diet (unadapted) prior to pupation.

More recently Chandra and Pandey (2008) found that larval leaf feeding, survival,

pupation and adult emergence of Indian alfalfa weevil, Hypera postica Gyll. was

significantly decreased by cysteine PIs like p-hydroxy-mercuribenzoic acid, cystatin and

E-64 at a concentration of 0.1 and 0.5%.

Thysanoptera

Recombinant potato cystatin and equistatin were fed to adult females to test in vivo

biological effect of inhibitors on the oviposition rate of western flower thrips, Frankliniella

occidentalis (Pergande) (Thysanoptera) (Annadana et al., 2002). A gradual reduction in

oviposition rate to about 45% of control was observed when the thrips were reared on these

PIs for a period of 5 days, with no increase in mortality.

Hemiptera

Ashouri et al. (1998) studied the effect of oryzacystatin I (OCI) on two-spotted

stinkbug, Perillus bioculatus (Fab.), predator of Colorado potato beetle, L. decemlineata

by chronic feeding for 18 days on prey loaded with 1-16µg OCI/day. Mortality of treated

females was negligible, but fertility was reduced by up to 50%. Additional dose-dependent

effects in reproducing females included delayed oviposition, reduced fecundity, lower egg

mass size, and reduced egg eclosion incidence. Females fed for 18 days on OCI at ≤4

µg/day returned to normal oviposition when switched to prey without OCI after 18 days of

treatment, but negative effects persisted for at least 10 days at higher doses.

2.2.1.3 Effect of Aspartic PIs on Growth and Development of Insects:

The scanning of literature revealed only two reports regarding the effect of aspartic

PIs on insects. Bolter and Jongsma (1995) reported no significant effect of Methyl

jasmonate induced aspartic proteinase inhibitors in potato leaves on development and

survival of L. decemlineata. In 2008, Chandra and Pandey studied the effects of aspartic

PIs, Antipain and Pepstatin on the alfalfa weevil, H. postica and found that pepstatin

significantly reduced feeding after 72h of treatment. After 72h, it also reduced the

development period from larvae to pupae.

There is no report available on the effect of plant metalloproteinase inhibitor on the

growth or development of any insect.

2.2.2 EFFECT OF PLANT PROTEASE INHIBITORS ON GUT ENZYMES OF INSECTS

2.2.2.1 Effect of Serine PIs on Gut Enzymes

Lepidoptera

In 1993, Broadway evaluated six species of Lepidoptera for their susceptibility to

serine proteinase inhibitors from cabbage. Trypsin and chymotrypsin activity from larval

cabbage white, P. rapae and green-veined white, Peiris napi (Linnaeus) were not

significantly inhibited (0-18%) in vitro by cabbage PIs, while the serine proteinase activity

in the midguts of larval P. xylostella was moderately inhibited (40-50%). The activity of

enzymes in T. ni, Asian gypsy moth, Lymantria dispar (Linnaeus) and H. zea was

substantially inhibited (55-85%) by cabbage PIs. Following ingestion of Cabbage PIs, the

predominant trypsin-like enzyme(s) in the midgut of larval L. dispar and H. zea were

resistant to inhibition by inhibitors (13-18% inhibited). These results were confirmed for

H. zea and T. ni feeding on PIs in tomato foliage.

Feeding larvae of H. armigera on a diet containing 0.234mM SBTI significantly

reduced the trypsin-like enzyme activities found in their gut contents when compared with

the levels found in larvae fed on the control diet (Johnston et al., 1993). Harsulkar et al.

(1999) investigated the efficacy of PIs from three host plants (Chickpea, C. arietinum,

pigeonpea, Cajanus cajan and cotton, Gossypium arboretum) and three non-host plants

(groundnut, A. hypogea, winged bean, Psophocarpus tetragolonubus and potato, S.

tuberosum) in inhibiting the activity of gut proteases of H. armigera. The larvae fed on

chick pea PIs showed 91% casienolytic and 72% azocaseinolytic activity, whereas, in non-

host PI fed larvae, the caseinolytic and azo-caseinolytic activities were in the range of 35-

37% and 25-30%, respectively. BAPNAse (benzoyl arginyl para-nitroanilide) activity,

which measures trypsin like proteases, was found to be lowest (19%) in winged bean PI -

fed larvae, 22% in groundnut PI-fed larvae, 42% in potato PI-II fed larvae and 89% in

chickpea PI-fed larvae. They also analyzed inhibition of HGP activity and found that PIs

from the host group of plants comprising chickpea, pigeon pea, and cotton showed 45%,

55% and 38% inhibition of HGP activity at pH 7.8 and 33%, 48% and 40% inhibition at

pH 10.0, respectively. On the other hand, PIs from the non-host plants of H. armigera

(groundnut and winged bean) along with potato PIs (PI-I, PI-II and PI-III), showed total

inhibition of HGP activity at both pHs except groundnut PIs, which inhibited HGP activity

up to 84% at pH 10.

In another study Patankar and coworkers (2001) had found that the gut proteinases

of H. armigera fed on chickpea were strongly inhibited by serine proteinase inhibitors

antipain (80%), leupeptin (80%), pefblac (85%) and aprotinin (70%). They also reported

that the gut proteinases of the two larval instar stages showed contrasting trends in the

level of inhibition by chickpea and winged bean PIs. Gut proteinases of the second instar

were resistant to inhibition by chickpea PIs and 50% were inhibited by winged bean PIs.

However, Gut proteinases activity of fifth instar larvae was totally inhibited by winged

bean PIs and partially by chickpea PIs (40%). The differential susceptibilities of the

proteinases to plant PIs observed in the two larval instars suggested the dynamic nature of

expression of the gut proteinases possessing different specifities during the course of larval

development. Gatehouse et al. (2002) studied the effect of chymostatin, an inhibitor of

chymotrypsin on H. armigera and reported an increase in Leucine aminopeptidase (LpNA-

hydrolysing) activity but elastase-like (SAAPLpNA-hydrolyzing) and trypsin-like

(BApNA-hydrolyzing) activty were lower as compared to control. Telang et al. (2003)

observed that PIs from bittergourd (M. charantia) (BGPIs) were strong inhibitors of H.

armigera gut proteases (HGP). Biochemical investigations showed that BGPIs inhibited

more than 80% HGP activity. Telang et al. in 2005 purified two serine proteses from the

midgut H. armigera, one protease HGP-1 was capable of hydrolyzing a synthetic substrate

of Elastase and was inhibited by elastatinal. The second protienase, HGP-2, was inhibited

by a trypsin inhibitor. Furthermore, they studied the interaction of HGP-1 and HGP-2 with

PIs from host and non-host plants and reported that HGP-1 was not only sensitive to a PI

from chickpea (host) but it was also able to degrade it. The same PI from chickpea was

able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host)

showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of

chickpea seed proteins from digestion by both the HGPs. Chougule et al. (2003) reported

inhibition of H. armigera gut proteinases (HGP) by inhibitors from 36 pigeon pea, Cajanus

cajan (L.) Millsp. cultivars and stronger inhibition by wild relatives which was up to 87%

by Rhynchosia PIs. Gujar et al. (2004) reported that midgut proteinase inhibition of

neonate larvae of H. armigera with the crude proteinase inhibitor extracts from seeds of

different crop plants (black gram, chickpea, chuckling vetch, finger millet, French bean,

green gram, horse gram, lentil, pea and soybean) ranged from 9.3 to 60.9%, being lowest

with black gram and highest with Soybean. Two proteinase inhibitors, CapA1 and CapA2,

purified from C. annum leaves inhibited 60-80% HGP (Helicoverpa gut proteinase)

activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of

HGP activity was observed in various instar (II to IV) larvae reared on artificial diet

(Tamhane et al., 2005). The partial purification of HGP isoforms, their characterization

with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-

like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85%

chymotrypsin-like activity of HGPs was insensitive to these inhibitors. In 2007, Bhavani et

al. studied the effects of two Subabul (L. leucocephala) proteinase inhibitors [high

molecular weight Subabul TI (HSTI) and low molecular weight Subabul TI (LSTI)]

against H. armigera and reported that HSTI and LSTI inhibited the gut proteinases from

larvae fed on artificial diet significantly (41.40% and 64.36%) compared to the gut

proteinases (27.80 and 38.90%) from larvae fed on chickpea seeds. The results revealed

that LSTI was a stronger inhibitor of insect gut proteinases. Kansal et al. (2008) purified

trypsin inhibitor from different varieties of mung bean (V. radiata) seeds which showed

inhibition against H. armigera gut proteases, the maximum inhibition being with Pusa

Vishal variety. Srinivasan et al. (2005) identified and purified a low expressing PI from

chickpea seeds. They isolated and cloned the coding sequence for the same into a yeast

expression vector and produced a recombinant protein which did not inhibit chymotrypsin

activity but exhibited stoichiometric inhibition of trypsin, comparable to soybean Kunitz

trypsin inhibitor. The recombinant protein exhibited greater inhibition of total Helicoverpa

gut protease activity as compared to SBTI.

Heath et al. (1997) isolated five serine PIs (one chymotrypsin and four trypsin

inhibitors) from ornamental tobacco, Nicotiana alata and studied their effect on the gut

protease activity of budworm, H. punctigera. They reported a significant decrease in the

gut protease activity both in vitro and in vivo. The effects of cowpea trypsin inhibitor

(CpTI), SBTI and SBBI on the hydrolysis of various synthetic substrates in tobacco

budworm, Heliothis virescens (Fabricius) larval gut extracts were determined by Johnston

et al. (1995). All three plant PIs were effective against both the trypsin like and

chymotrypsin-like enzymes. However, the inhibitors differed greatly in the concentrations

required to give 50% inhibition of activity. Both CpTI and SBTI were approximately 50

times more effective against the trypsin-like enzyme than against the chymotrypsin-like

enzyme. In contrast, SBBI was approximately more effective at inhibiting the

chymotrypsin-like activity being approximately twice as inhibitory as SBTI and 40 times

inhibitory than CpTI. SBTI was most effective at inhibiting the trypsin-like enzyme being

approximately 400 times more effective than SBBI and 30 times inhibitorier than CpTI.

Only SBTI had a significant effect against the enzyme activity hydrolyzing the Elastase

substrate, S(Ala)3pNA. This activity was due to a separate enzyme from the main two

types, as it was not inhibited by CpTI or chymostatin and was only slightly inhibited by

SBBI. The effects of plant protease inhibitors on azocasein and azoalbumin hydrolysis

were also determined. Results from measurements of initial rates of proteolysis showed

inhibition by approx. 20% in the presence of the lower concentration of inhibitor (15µg/ml

CpTI, 5µg/ml SBBI or 0.05µg/ml SBTI). The higher concentration of each inhibitor

(500µg/ml CpTI, 50µg/ml SBBI or 30µg/ml SBTI) reduced proteolysis by about 60-70%

in each case. However, the levels of inhibition were gradually reduced as the enzyme

assays were continued over a longer time.

In 1995, Mcmanus and Burgess studied the effects of SBTI on growth and

digestive proteases of larvae of cotton cutworm, S. litura. They observed that larvae fed on

base diet grew the most, followed by those consuming 0.21% SBTI than those fed 0.5%

SBTI. In vitro titration of the SBTI against each enzyme at its optimal pH revealed that the

inhibitor was most effective at retarding the trypsin like activity, only slightly effective

against the elastase-like and chymotrypsin esterase-like activity, but completely ineffective

against leucine amino peptidase. The activities of serine proteases involved in the digestion

mechanism of the S. litura was examined (in vitro and in vivo) following feeding of PIs

from A. ellipticum (AeTI) by Bhattacharya et al. (2007). They found that AeTI inhibited

the trypsin like activities of the midgut proteases of fifth instar larvae of cutworm by 70%

in vitro. Under feeding trials, influence of AeTI on the larval gut physiology indicated a 7-

fold decrease of trypsin like protease activity and a 5-fold increase of chymotrypsin-like

activity. They also suggested that although the early (1st to 3

rd) larval instars of S. litura

were susceptible to the trypsin inhibitory action of AeTI, the later instars may facilitate the

development of new serine proteases, insensitive to the inhibitor. In the same year again

the efficacy of Caesalpinia bonduc trypsin inhibitor (CbTI) was checked against S. litura

gut proteinases by assessing the inhibition of larval gut trypsin like activity by

Bhattacharya et al. They reported that dosage of CbTI required for inhibition of gut

proteolytic activity was ~1.5-fold lower than that of SBTI by X-ray contact print method.

These results were confirmed by trypsin inhibition assays using the esterolytic substrate

TAME. On incubating the crude larval gut trypsin with CbTI, there was ~68% reduction in

the gut tryptic activity. Comparatively however, bovine trypsin was ~25% more

susceptible to CbTI than larval gut trypsin.

In the year 2000, Paulillo et al. reported that S. frugiperda was able to physiologically

adapt to dietary PIs by altering the compliment of proteolytic enzymes in the insect

midguts as they studied the effect of semi purified soybean protienase inhibitor on

digestive enzymes of fall armyworm. Alfonso et al. (1997) found high levels of trypsin-

like activity in gut extracts from S. frugiperda last larval instars. Different genetic variants

of the major barley trypsin inhibitor were active against this gut enzyme. None of the other

larval digestive protease activities (chymotrypsin-like, Elastase-like, Leucine

aminopeptidase-like and carboxypeptidase A and B-like) were inhibited, indicating that the

barley inhibitor is specific towards trypsin-like enzymes. The inhibitory effects of

recombinant Maize PI (rMPI) on the digestive proteolytic activities of cotton leaf worm,

Spodoptera littoralis Boisduval larvae were studied by Tamayo et al. in 2000. Hydrolysis

of the general substrate casein-resurofin was inhibited by rMPI. Thus, the preincubation of

0.5-1.0µg of MPI with midgut extracts resulted in 40% inhibition of proteolytic activity.

Increasing the amount of inhibitor did not result in higher percentages of inhibition. The

effect of MPI on the midgut proteolytic activities responsible for hydrolysis of the

synthetic substrates, SA3pNA, SA2PLpNA and SA2PPpNA was also determined and the I50

value observed for SA3pNA was 0.45µM. A potent inhibitory effect on the chymotrypsin

activities (SA2PLpNA and SA2PPpNA-hydrolyzing activities) was observed.

Chougule et al. (2008) analysed proteolytic activities in soluble protein extracts from

cabbage moth, M. brassicae using specific peptide substrates and PIs and found that serine

proteinases were the major enzymes detected, with chymotrypsin-like and trypsin–like

activities being responsible for approximately 62% and 19% of the total proteolytic

activity towards a non-specific protein substrate. Only small amounts of Elastase-like

activities could be detected. SBTI ingestion by the larvae had induced inhibitor-insensitive

trypsin-like activity. Qualitative and quantitative changes in protienase activity bands after

gel electrophoresis of gut extracts were evident in SBTI-fed larvae when compared with

controls, with increase in levels of most bands, appearance of new bands, and a decrease in

the major protienase band present in extracts from control insects.

Soybean PIs inhibited corn borer, O. nubilalis trypsin, chymotrypsin and total

proteolysis by midgut preparations (in vitro) but when ingested (in vivo) in the diet an

increased tryptic and decreased chymotryptic activity was observed by Larocque and

Houseman (1990). Proteolytic activity of elastase and leucine aminopeptidase (LAP) from

the codling moth larvae, C. pomonella under the influence of various plant PIs [(POT I),

eglin C, Kunitz soybean trypsin inhibitor (SBTI), lima bean trypsin inhibitor (LBTI),

Cowpea trypsin inhibitor (CpTI), winged bean trypsin inhibitor (WBTI) and

carboxypeptidase protease inhibitor (CPI)] was measured by Markwick et al. (1995). The

two elastase inhibitors significantly lowered the elastase like activity compared with that in

water controls. However, it was higher than control in larvae fed with the four trypsin

inhibitors SBTI, LBTI, CpTI and WBTI. Also, WBTI significantly lowered trypsin

activity. CpTI had a suppressive effect on trypsin activity although it was non-significant.

Macedo et al. (2003) purified a trypsin inhibitor from the seeds of P. dubium

(PDTI) and studied its inhibitory potential against trypsin-like proteinases extracted from

different lepidopteran larvae like A. kuehniella, Angoumois grain moth, Sitotroga

cerealella (Oliv.), C. cephalonica, S. frugiperda, D. sachharalis and A. gemmatalis. The

larval digestive enzymes extracted from the six insect species were trypsin-like enzymes

and were clearly inhibited by PDTI, except for A. gemmatalis for which there was only

weak inhibition. The inhibitory activity of PDTI against insect proteases, expressed as a

percentage of that against trypsin, ranged from ca. 60% to 95% for D. sachharalis and A.

kuehniella, respectively. A novel trypsin inhibitor (PPTI) was purified from the seeds of

the native Brazilian tree Poecilanthe parviflora (Benth) by Garcia et al. (2004) which

inhibited trypsin-like activity in midguts of larval D. sachharalis, A. kuehniella, S.

frugiperda and C. cephalonica.

A trypsin inhibitor from Cassia obtusilolia seeds showed significant inhibitory

activity against trypsin-like proteases present in the larval midgut of P. rapae as reported

by Liao et al. in 2007. A novel trypsin-papain inhibitor, named PdKI-2, purified from the

seeds of Pithecelobium dumosum by Oliviera et al. (2007) was effective against digestive

proteases from Indian meal moth, Plodia interpunctella (Hubner) and cotton leafworm,

Alabama argillacea (Hubner), with 48.7 and 13.6% inhibition, respectively.

Coleoptera

The activity of Kunitz trypsin inhibitor from Algaroba seeds (P. juliflora) (PjI) was

studied towards bruchid larvae by Oliviera et al. (2002). Among the different proteinases

tested, PjI showed a high inhibitory activity against cysteine proteolytic activity of bean

bruchid, Acanthoscelides obtectus (Say) (76%) and cowpea weevil, C. maculatus (72%).

The results also showed moderate inhibitory activity towards digestive proteinases from

pod weevil, Mimosestes mimosae (Fabricius), with approximately 42% inhibition, and low

inhibitory activity against digestive proteolytic activity from Mexican bean weevil, Z.

subfasciatus with 19.1% inhibition. Similarly, Gomes et al. (2005) found that C. maculatus

enzymes were strongly susceptible (74.4 ± 15.8) to the trypsin inhibitor from C. pallida

(CpaTI), and LD50 and ED50 being 3.0 and 2.17%, respectively. Araujo et al. (2005) found

remarkable activity of trypsin inhibitor from Tamarind (TTI) against enzymes from cotton

boll weevil, A. grandis (29.6%), Z. subfasciatus (51.6), C. maculatus (86.7%) and lesser

grain borer, Rhyzopertha dominica (Fabricius) (88.2%). In 1996, Bian et al. studied the

effect of a number of serine PIs on the larval midgut proteinase activities from poplar tree

borer, Anoplophora glabripennis (Motchulsky) and found that the proteolytic activity of

midgut extract was inhibited most by the two wheat germ trypsin inhibitors. Many trypsin

inhibitors, particularly those of the Kunitz family of plant origin, had little effect. On the

other hand, inhibitors of subtilisin, Elastase and chymotrypsin classes of serine

endopeptidase were all effective inhibitors of casein hydrolysis. A trypsin-papain inhibitor,

PdKI-2, purified from the P. dumosum seeds by Oliviera et al. (2007) was effective against

digestive proteases from bruchids, Z. subfasciatus and C. maculatus, with 74.5 and 70%

inhibition, respectively.

Diptera

Spates (1979) studied the effect of SBTI on stable fly, S. calcitrans and observed

an increase in the enzyme activity in midgut homogenates. The proteolytic activity of

midgut homogenates prepared from males that fed on blood meal diets with and without

inhibitors indicated that de novo synthesis of digestive enzymes did occur.

In diptera, the effect of trypsin inhibitor from three different plants has been

investigated on Mediterranean fruit fly, C. capitata. In 2005, Gomes et al. studied the

effect of trypsin inhibitor from C. pallida (CpaTI) on C. capitata and found that enzymes

were susceptible (100 ± 7.3 %) to CpaTI. In the same year Araujo et al. recorded 52.9 %

inhibition of enzyme activity of C. capitata with a trypsin inhibitor from Tamarind (TTI).

Later on in 2006, Silva et al. found that enzymes from C. capitata guts were strongly

susceptible to SBTI in the in vitro assays, producing inhibition of 90%.

Orthoptera

The black field cricket, T. commodus and grasshopper, M. sanguinipes are the only

two insects of the order coleoptera which have been studied under the influence of plant

PIs. Burgess et al., 1994 reported that the gut trypsin activity of T. commodus was reduced

with wheat germ trypsin inhibitor (WGI-1), on all three base diets containing 3 different

levels of casein (0.5%, 1% & 3%).

Hinks and Hupka (1995) studied the effects of soybean trypsin inhibitor

incorporated diet on grasshopper, M. sanguinipes (Fab.) and found that the protein

concentration in the regurgitate varied significantly with diet, and was in the following

order: wheat sap+SBTI > wheat sap > oat sap+SBTI = oat sap. Trypsin and chymotrypsin

activities were in the same order as protein concentration. Small increases in trypsin

activity were observed in the regurgitate from grasshoppers fed sap+SBTI from either

plant, compared to the sap alone. A 20-fold increase in chymotrypsin activity was observed

in the regurgitate of grasshoppers fed wheat sap+SBTI compared to the regurgitate in those

fed wheat sap. A 4.5-fold increase in chymotrypsin activity was observed in the regurgitate

of grasshoppers fed oat sap+SBTI compared to the regurgitate of those fed oat sap alone.

Small non-significant increases in trypsin activity were observed in the grasshoppers fed

sap+SBTI from either plant, compared to the sap alone. A 6-fold increase in chymotrypsin

activity was observed in the residual gut tissues from grasshoppers fed wheat sap+SBTI

compared to wheat sap, but activity of this enzyme was lower in grasshoppers fed oat

sap+SBTI compared to sap alone.

Hymenoptera

Malone et al. (1995) studied the effect of SBTI on three endopeptidases (trypsin-

like, chymotrypsin-like and Elastase-like) and an exopeptidase, Leucine aminopeptidase

from adult honey bees, A. mellifera. In vitro tests using control bee midgut extracts showed

that SBTI had high affinity for Elastase-like and trypsin-like activity and less affinity for

chymotrypsin. Again in 1998, Malone et al. studied the effect of potato protease inhibitors,

POT1 and POT2 on the in vivo activity of endopeptidases (trypsin, chymotrypsin and

Elastase) and an exopeptidase, LAP of A. mellifera after an exposure of 3 and 8 days. They

reported that enzyme activities were significantly lower at day 8 than at day 3, except for

Elastase, which did not change. POT2 significantly reduced the activity of all

endopeptidases at both time points; regardless of the dose level. There was no consistent

trend in changes in LAP activity. Burgess et al. (1996) studied in vivo activity levels of gut

proteases in bees fed with SBTI at1.0, 0.3 and 0.1% (w:v) at two time points: the 8th day

after emergence and when 75% bees had died. LAP activity levels increased significantly

in bees fed with either inhibitor at all concentrations. At both time points, only bees fed

SBTI at the highest concentration had lowered trypsin, chymotrypsin and Elastase

activities. In vitro titration of proteinases by SBTI and BBI (Bowman-Birk Type Soybean

inhibitor) showed that both PIs are potent inhibitors of the honeybee (Pham-Delegue et al.,

2000). Ingestion of BBI or SBTI at 0.01 mg/ml increased the level of trypsin,

chymotrypsin and LAP activities relative to the controls, but not the level of Elastase-like

activity.

Azzouz et al. (2005) studied the effects of Soybean Bowman-Birk inhibitor (SBBI)

on A. abdominalis digestive system. The assays performed with different concentrations of

this PI showed that it induced significantly higher inhibitory effect as at 100µg/ml it

inhibited 76 and 77% of larvae and adult protease activity, respectively.

2.2.2.2 Effect of Cysteine PIs on Gut Enzymes

Coleoptera

In 1991, Liang et al. reported that Oryzacystatin purified from Newbonnet rice

fully inhibited the midgut proteolytic activities from T. castaneum and S. oryzae. The IC50

values were estimated to be 4x10-7 and 2x10

-6 M for rice weevil and red flour beetle

extracts, respectively.

Orr et al. (1994) monitored the changes in gut cysteine proteolytic activity of

southern corn rootworm Diabrotica larvae fed on various inhibitors and reported 72%

reduction in cysteine proteolytic activity with PMC while tryptic fragments of PMC (T-

PMC) and potato carboxypeptidase inhibitor (PCI) did not cause a significant reduction.

However, combining T-PMC with PCI caused 45% reduction in protease activity.

In 1995, Bolter and Jongsma studied the effect of methyl jasmonate (MJ) induced

cysteine PIs in potato leaves (4% of total protein) on larvae of Colorado potato beetle, L.

decemlineata. They reported that general protienase activity was significantly reduced

(42%) in insects reared on the high inhibitor diet, while protienase activity that was

insensitive to induced inhibitors in juice from MJ-treated leaves had increased two-fold.

Activities towards the specific cysteine protienase substrate p-Glu-Phe-Leu-pNA were the

same in guts from insects reared on the three leaf types. However, juice from MJ-treated

leaves inhibited as much as 67% of this activity in guts of insects reared on the low

inhibitor diet compared to only 27% of the activity in gut extracts from insects reared on

MJ-treated leaves indicating a 2.5-fold induction of cysteine protienase activity insensitive

to potato protienase inhibitors. None of the activities towards another specific cysteine

protienase substrate L-Arg-pNA were sensitive to inhibitors from MJ-treated leaves, but

guts of insects fed these leaves had a 3.5-fold induction of this protienase activity

compared to those reared on plants containing low papain inhibitor levels. Bolter and

Latoszek-Green (1997) observed that the general protease activity in control extracts of

Colorado potato beetle was 6.5 units/min/mg gut which decreased to 1.9 in guts of insects

reared on 1µg E-64/cm2 treated leaf surface. Zhao et al. (1996) isolated three soybean

cysteine PIs (L1, N2 and R1) and reported that N2 and R1 had substantially greater

inhibitory activities than L1 against gut cysteine proteinases of the third instar larvae of

Colorado potato beetle. Cysteine proteinases were the predominant digestive proteolytic

enzymes in the gut of this beetle at this developmental stage.

Cystatin CsC, a cysteine PI from chestnut (Castanea sativa) seeds, was purified by

Pernas et al. (1998). They isolated its full-length cDNA clone from an immature chestnut

cotyledon library, expressed in Escherichia coli and purified from bacterial extracts. In

vitro tests were carried out with extracts from various insects. Among the insects tested, a

strong inhibition of proteolytic activity was observed for T. castaneum (>50% inhibition

with 1µM cystatin), similarly to E-64. By contrast, the enzymes from L. decemlineata, also

susceptible to E-64, were not inhibited by CsC. Oppert et al. (2003) reported an inhibitory

effect of potato cysteine proteinase inhibitor on the activity of gut proteinases of red flour

beetle, T. castaneum which was evaluated in vitro with the general substrate casein in pH

4.2 buffer. The IC50 values ranged from 0.2-0.8 µM. They further observed that Soybean

Kunitz trypsin inhibitor (STI) only weakly inhibited T. castaneum gut proteolytic activity;

with IC50 value of 58 µM.

Zhu-Salzman et al. (2003) reported that total gut proteolytic capacity of C.

maculatus larvae significantly increased in the soyacystatin N (scN) adapted insects and

the elevated enzymatic activity was attributed to a differential expression of insect gut

cysteine proteases, and of aspartic proteases.

Hemiptera

Ashouri et al. (1998) studied the effect of oryzacystatin I (OCI) on two-spotted

stinkbug, P. bioculatus, predator of Colorado potato beetle, L. decemlineata by chronic

feeding for 18 days on prey loaded with 1-16µg OCI/day. They found that the general

digestive protease activity was significantly higher in stinkbugs submitted to the OCI

treatment. Adult females supplied for three days with OCI (16µg/day) exhibited a total

azocaseinase activity almost twice the activity noted for the non-treated females. Although

the effect of OCI against the insect proteases was lower in OCI-fed females, OCI-sensitive

proteinases were still present in the extracts.

Thysanoptera

Proteolytic activity in whole insect extracts of the flower thrips, F. occidentalis,

was found to belong predominantly to the class of cysteine proteases (Annadana et al.,

2002).The pH optima of the general proteolytic activity was determined to be 3.5. The

proteinaceous cysteine PI, potato cystatin inhibited in vitro more than 90% of the protease

activity.

2.2.3 EFFECT OF TRANSGENIC PLANTS EXPRESSING PIS ON INSECTS

Lepidoptera

A sweet potato (Ipomea batatas cv. Taionong 57) trypsin inhibitor gene was

introduced into tobacco plants (Nicotiana tabaccum cv. W38) by Agrobacterium

tumefaciens-mediated transformation by Yeh et al. (1997). In insecticidal bioassays of

transgenic tobacco plants, they reported that larval growth of S. litura was severely

retarded as compared to their growth on control plants. McManus et al. (1999) introduced

the coding region of the Tia allelic form of the soybean trypsin inhibitor, as a

transcriptional fusion protein with the CAMV 35S promoter, into tobacco. Inhibition

assays revealed that the protein could inhibit trypsin like activity extracted from S. litura

digestive tracts. Insect feeding trials, using neonate larvae of cutworm established that,

when compared with insects fed on non-transformed leaf tissue, larvae fed on transgenic

leaf tissue demonstrated significantly greater mortality, and the survivors grew more

slowly in terms of weight gain over time.

The cowpea trypsin inhibitor (CPTI) gene offers resistance against a wide array of

insects, and has been used for developing insect-resistant plants. Sane et al. (1997) cloned

the CpTI gene in a plant expression vector under the control of the CaMV 35S promoter

and the NOS terminator, and introduced into model plant tobacco by Agrobacterium-

mediated transformation. The efficacy of the expressed CpTI protein against S. litura was

tested by feeding trial larvae under laboratory conditions. Reduction to the extent of 50%

was observed in the biomass of S. litura larvae fed on transgenic leaves, expressing 3-5µg

CpTI/g fresh tissue. Novel types of protienase inhibitors with multi-inhibitory activities

were generated by replacement of phytocystatin domains in sunflower multi-cystatin

(SMC) by the serine protienase inhibitor BGIT from bitter gourd seeds which produced

two chimeric inhibitors; SMC-T3 and SMC-T23. They found that when the beat

armyworm, Spodoptera exigua (Hubner) second instar larvae were reared on a diet

containing SMC-T3 and SMC-T23 for ten days, a significant reduction in weight gain was

observed. In contrast, BGIT had little effect on the growth of the S. exigua larvae.

Marchetti et al. (2000) isolated three soybean genes (KTi3, C-II and PI-IV) coding

for serine protienase inhibitors and transferred to tobacco leaf and potato tuber discs by

Agrobacterium tumefaciens EHA 105. The level of insect resistance, tested with Egyptian

cotton worm, S. littoralis was particularly high in tobacco, where many plants caused the

death of all larvae. In potatoes, larval mortality was much less frequently achieved, but the

results were still encouraging in that larval weight gain was reduced by 50% in the

presence of adequate amounts of inhibitor. When 8-day old larvae were fed different KTi3-

expressing tobacco plants, a highly significant (p<0.01) correlation was observed between

inhibitor content and larval live weight.

Abdeen et al. (2005) showed that leaf–specific over-expression of the potato PI-II

and carboxypeptidase inhibitors (PCI) resulted in increased resistance to corn earworm,

Heliothis obsolete (Fab.) and American serpentine leafminer, Liriomyza trifolii (Burgess)

larvae in homozygote tomato lines expressing high levels of the transgenes. Leaf damage

in hemizygous lines for these transformants was, however, more severe than in the

controls, thus evidencing a compensatory response of the larvae to the lower PI

concentrations in these plants. Development of comparable adaptive response in both

insects suggested that insect adaptation did not entail specific recognition of the transgene,

but rather represented a general adaptive mechanism triggered in response to the nutritional

stress imposed by sublethal concentrations of the inhibitors.

Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis transgenic plants

expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained by De-Leo et

al. (1998). First instar larvae of S. littoralis were fed on detached leaves of these plants.

The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing

mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf

surface eaten. However larvae fed leaves from plants expressing MTI-2 at the low

expression level did not show increased mortality, but a net gain in weight and a faster

development compared with control larvae. The low MTI-2 expression level also resulted

in increased leaf damage. These results were in correlation with the differential expression

of digestive proteinases in the larval gut; over expression of existing proteinases on low

MTI-2 expression level plants and induction of new proteinases on high MTI-2 expression

level plants.

The defensive effect of endogenous trypsin protienase inhibitors (NaTPIs) on the

herbivore Tobacco hornworm, Manduca sexta (L.) was demonstrated by genetically

altering NaTPI production in M. sexta’s host plant, Nicotiana attenuate (Zavala et al.,

2008). The second and third larval instars that fed on NaTPI-producing genotypes were

lighter and had less gut protienase activity compared to those that fed on genotypes with

either little or no NaTPI activity. NaPTI activity in vitro assays not only inhibited the

trypsin sensitive fraction of the gut protienase activity but also halved the NaTPI-

insensitive fraction in third instar larvae. Unable to degrade NaTPI, larvae apparently

lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least

part of their gut protienase activity when they were transferred from NaTPI-producing host

plants to NaTPI-free host plants.

Altpeter et al. (1999) introduced the barley trypsin inhibitor CMe (BTI-CMe) into

wheat (T. aestivum) by biolistic bombardment of cultured immature embryos. The

significant reduction of the survival rate of the Angoumois grain moth (S. cerealella),

reared on transgenic wheat expressing the trypsin inhibitor BTI-CMe, compared to the

untransformed control confirmed the potential of BTI-CMe for the increase of insect

resistance. However, only early instar larvae were inhibited in transgenic seeds and

expression of BTI-CMe protein in transgenic leaves did not have a significant protective

effect against leaf-feeding insects.

In 2001, Pulliam et al. isolated a cDNA clone for a serine PI from Brassica

oleracea, cabbage (BoPI). Subcloned into a plant expression vector under the control of

the CaMV 35S promoter, and transgenic Nicotiana tabacum (tobacco) cv Xanthi were

produced to test the ability of BoPI to enhance resistance against insects in a heterologous

system. These plants were compared with transgenic plants containing different insect

resistance transgenes (PIs and Bt cry1Ac). The transgenic plants containing BoPI gene

outperformed over other transgenic plants produced with different PI genes and compared

favorably with Bt cry1Ac transgenic plants in a bioassay with H. virescens, tobacco

budworm.

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were formed with a

gene, KTi3, derived from soybean (G. max) coding for a Kunitz trypsin inhibitor by

Confalonieri et al. (1998). They reported that the trypsin like activity of the polyphagous

moth, L. dispar and poplar tip moth, Clostera anastomosis (L.) were inhibited in vitro by

Kunitz PIs from selected transgenic plants. Two insect bioassays were performed on P.

nigra transgenic plant lines and it was found that larval mortality and growth as well as

pupal weight were not significantly affected when the insects were fed on transgenic leaves

and control leaves.

Maheswaran et al. (2007) studied the generation and analysis of the apple cultivar

‘Royal Gala’ transgenic for N. alata PI and the impact of this PI on the growth and

development of the light-brown apple moth, Epiphyas postvittiana (Walker). Light-brown

apple moth larvae fed with apple leaves expressing Na-PI had significantly reduced body

weight after 7days of feeding and female pupae were 19-28% smaller than controls. In

addition, morphological changes such as pupal cases attached to the wing, deformed

wings, deformed body shape and curled wings attached to a deformed body were observed

in adults that developed from larvae fed with transgenic apple leaves when compared to

larvae fed with the non-transformed apple leaves.

Coleoptera

In the year 2000, Cloutier et al. examined the effects of feeding young females of

Colorado potato beetle, L. decemlineata with foliage from a cultivar of the ‘Kennebec’

potato line (K52) transformed with a gene encoding oryzacystatin I (OCI), a specific

cysteine proteinase inhibitor with proven activity against cathepsin H-like enzymes of

larvae and adults of the potato beetle. To evaluate the insect’s performance, they collected

data over a 16-d post emergence period on survival, diapause incidence, foliage

consumption, weight gain, and oviposition of females. The OCI-expressing foliage did not

affect female survival, incidence of diapause, relative growth rate during post emergence

growth or maximum weight reached, neither did it affect female reproductive fitness.

However, nutritional stress to females feeding on OCI foliage was evident, as reflected in

their lower efficiency of conversion of ingested foliage during post emergence growth,

increased foliage consumed per egg laid and adaptation of their digestive proteolytic

system to the inhibitory effect of OCI. Beetles fed foliage expressing the highest level of

OCI reacted rapidly to the presence of OCI by producing OCI-insensitive proteases, and

exhibiting strong hypertrophic behavior by ingestion of 2.4-2.5 times more OCI rich

foliage apparently as a compensatory response for nutritional stress due to the protease

inhibitor in their diet. Ninkovik et al. (2007) introduced a wound-inducible oryzacystatin II

(OCII) gene to alfalfa to evaluate its effect on survival of Lucerne leaf beetle, Phytodecta

fornicate Bruggemann larvae. After conducting feeding bioassays with second, third and

fourth instars, they reported that second and third instars were the most sensitive to the

ingestion of OCII, whereas, no effects were observed with fourth instars. About 80% of the

second and third instars died after 2 days of feeding on the transgenic plants as compared

to 0-40% on the controls.

Transgenic white poplar (Populus alba L.) plants expressing a novel Arbidopsis

thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using A.

tumefaciens-mediated gene transfer by Delledonne et al. (2001) Papain inhibitory activity

was detected in poplar transgenic tissues by means of a specific in vitro assay. Such

activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid

red poplar leaf beetle (Chrysomela populi L.) and confer resistance to the beetle larvae on

selected transgenic plants. A close relationship between the inhibition of papain and

resistance to poplar leaf beetle was observed in all tested transgenic lines. Similar results

were reported by Leple et al. in 1995 as they observed that transgenic poplar plants,

Populus tremula expressing OCI were toxic to the beetle, Chrysomela tremulae (Paykull).

Girard et al. (1998) assessed the potential effect of a transgenic line of oilseed rape

expressing oryzacystatin I (OCI) on two strains of cabbage seed weevil, Ceutorhynchus

assimilis Payk. and found that despite inhibition of digestive proteases in vitro by OCI in

both the strains, one strain showed an increased growth rate when fed with the transgenic

seeds, while the other strain remained unaffected.

To investigate the role of SKTI in a plant’s defense system against insect predation,

a recombinant plasmid containing the full-length cDNA of SBTI under control of CaMV

35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer

method and a large number of transgenic rice plants were regenerated by Lee et al. (1999).

Bioassay with transgenic plants revealed that they are more resistant to destructive insect

pest of rice, brown plant hopper (Nilaparvata lugens Stal), than the control plants

2.3 MELON FRUIT FLY, BACTROCERA CUCURBITAE (COQUILLETT):

2.3.1 INTRODUCTION

Tephritid flies (Diptera: Tephritidae) are among the most diverse groups of insects,

comprising over 4000 species in 481 genera (Thompson, 1998). They have a global

distribution, covering tropical, subtropical, and temperate regions and occupy habitats

ranging from rainforests to open savanna (Drew 1989, Michaux and White 1999). Nearly

all tephritid larvae are herbivorous, with diverse feeding habits such as flower feeding,

galling, stem boring and fruit feeding, however, the adults are free living in the

environment. With adult traits like high dispersive powers, high fecundity and in some

species extreme polyphagy, dacines are well documented invaders and rank high on

quarantine target lists (Clarke et al., 2005). Many species of tephritidae inflict heavy losses

on fruit and vegetable crops. Economic effects of pest species include not only direct loss

of yield, fruit drop and increased control costs, but also the loss of export markets and the

cost of constructing and maintaining fruit treatment and eradication facilities.

Over 700 species of dacines fruit flies are recognized, and the rate of discovery of

new species suggests there may be upwards of a thousand species in total (Fletcher, 1987).

The genus Bactrocera is the economically significant genus, with about 40 species

considered to be important pests (White and Elson-Harris, 1992). Bactrocera is native to

the Old World Tropics, and most of the major pests are from the Oriental and Australasian

regions. Drew and Hancock (1994) reported that the melon fruit fly (B. cucurbitae) and

other related species like oriental fruit fly (Bactrocera dorsalis Hendel), the olive fruit fly

(Bactrocera oleae Rossi), the Queensland fruit fly (Bactrocera tryoni Frogatt) and the

peach fruit fly (Bactrocera zonata Saunders) are among the most important species. Within

its range, the melon fly, B. cucurbitae, is one of the most notorious pests with which

vegetable growers have to contend. Over 125 species of host plants have been recorded,

the preferred host plants being cucurbits and solanaceous fruits. It has synonyms like

Chaetodacus cucurbitae (Coquillett), Dacus cucurbitae Coquillett, Strumeta cucurbitae

(Coquillett) and Zeugodacus cucurbitae (Coquillett).

2.3.2 GEOGRAPHICAL DISTRIBUTION

The melon fruit fly is well distributed over most of India, which is considered its native

home, and most of southeastern Asia, the Mariana Islands.

Asia: Afghanistan, Bangladesh, Brunei, Cambodia, China (Guangdong, Guangxi, Hainan,

Jiangsu, Yunnan), Christmas Island, Hong Kong, India, Indonesia, Japan (Iwahashi, 1977;

Anon., 1987), the Yaeyama group (1993), the Miyako group (1987), Kumejima (1978), the

rest of the Okinawa group (1990), the Amami group (1989); (Anon., 1993), Lao, Malaysia,

Myanmar, Nepal, Oman, Pakistan, Philippines, Saudi Arabia, Singapore, Sri Lanka,

Taiwan, Thailand, United Arab Emirates and Viet Nam.

Africa: Adventive populations in Egypt, Kenya, Mauritius, Réunion, Tanzania.

North America: USA, trapped in the wild in California (Carey & Dowell, 1989), but

eradicated (Spaugy, 1988); adventive populations in Hawaii, since the 1980s and

eradicated again.

Oceania: Australia, Guam (adventive populations), Kiribati, Nauru, Northern Mariana

Islands (Cunningham, 1989), Papua New Guinea (including New Britain, New Ireland,

Bougainville and Lihir Islands), Solomon Islands

2.3.3 HOSTS

B. cucurbitae occurs mainly on cucurbits, but tomatoes (Lycopersicon esculentum)

and pawpaws (Carica papaya) have also been recorded as its hosts. Nearly all cucurbits

may be attacked but B. cucurbitae has been reported mainly on courgettes (C. pepo),

cucumbers (C. sativus), melons (Cucumis melo) and other Cucurbita spp. such as

pumpkins. It also has a wide range of wild cucurbitaceous hosts. The other families on

which melon fruit fly is a serious pest are Anacardiaceae, Annonaceae, Arecaceae,

Brassicaceae, Bromeliaceae, Caricaceae, Clusiaceae, Fabaceae, Juglandaceae, Lauraceae,

Liliaceae, Loganiaceae, Malvaceae, Moraceae, Musaceae, Myrtaceae, Oxalidaceae,

Passifloraceae, Rosaceae, Rutaceae, Sapindaceae, Sapotaceae and Solanaceae (Allwood et

al.,1999).

2.3.4 MORPHOLOGICAL FEATURES

Adults: Adult melon flies measure 6 to 8 mm in length with a wingspan of 1/2 to 3/5

inch. The head and eyes are dark brown. Their bodies are yellowish brown with a yellow

spot above the base of the first pair of legs. A yellow stripe, with curved lines on either

side, is present down the center of the back. The tip of the body furthermost from the head

is yellow. Wings are patterned with a thick brown band extending along the leading edge,

ending in a larger brown spot at the tip. Another thin band extends from the wing base just

inside the trailing edge of each wing (Weems, 1964; Weems and Heppner, 2008). A brown

spot occurs near the wing margin. Abdomens are reddish yellow with darker bands on the

second and third abdominal segments. Legs are yellowish.

Fig. 2.1 (A) Adult fly abdominal features, (B) Adult fly thoracic features

Fig 2.2 Wing of adult fly showing banding pattern

Fig. 2.3 (A) Adult female of B. cucurbitae, (B) Adult male of B. cucurbitae

Eggs: The egg is pure white, about 2 mm long, elliptical, nearly flat on the ventral

surface, more convex on the dorsal. Eggs often are somewhat curved.

Larvae: The larva has three instars and they are entirely whitish to yellowish;

slender, elongate, tapering anteriorly, somewhat curved ventrally, with anterior mouth

hooks. Last instar larvae range from 7.5 to 11.8 mm in length. Integument is unsclerotized,

or partially sclerotized (mature larvae); sclerotization forming a transverse line beneath

posterior spiracles (mature larvae); sclerotized process on caudal segment absent. Mature

larvae are able to jump. Head of normal shape; cephalic lobes well developed.

Fig. 2.4 (A) Eggs of melon fruit fly (magnification 20x), (B) Pupae of melon fruit fly

Fig. 2.5 (A) Second instar larvae (64-72h), (B) First, second and third instar larvae of

fruit fly

Pupa: The puparium is about 5 to 6 mm in length and varies in color from dull red

or brownish yellow to dull white, according to host.

2.3.5 LIFE HISTORY

Oviposition occurs about 10 days after emergence and continues at intervals. One

female may deposit up to 1,000 eggs, although 300 eggs total are estimated in natural

conditions. Females prefer to oviposit in new plant growth such as young seedlings,

growing tips, and developing ovaries of all cucurbits except young cucumbers. Because of

their high egg laying capacity and mobility, each female is capable of destroying large

number of fruits in her lifespan.

Females lay eggs under the skin of host fruit, averaging about 15 eggs per day. The

eggs take 1-3 days to hatch depending on temperature. The developing larvae go through

three instars which may take from four to 17 days. At maturity, the larvae drop from the

fruit and burrow two to three cm beneath the soil to pupate. In nine to 18 days, the adults

emerge from these puparia. The newly emerged adults normally require 11 to 12 days to

become sexually mature depending on their diet. Breeding is continuous, with several

annual generations. Adults live from one to five months and feed on a diverse array of food

sources including honeydew, plant exudates, fermenting fruit, and animal feces.

Completion of the life cycle normally requires one to two months, but may be completed

in 15 days under optimal conditions.

2.3.6 DAMAGE

In the Indo-Malayan region, the melon fly, is considered the most destructive pest of

melons and related crops, and it has greatly curtailed the production of melons, cucumbers,

and tomatoes in Hawaii. In common with some other species in Bactrocera, the melon fly

can attack flowers, fruit, stem and root tissue. The damage is mostly caused by the larvae.

The female fly lays eggs into fruits, which hatch into larvae that feed inside the fruits.

Generally, the females prefer to lay the eggs in soft tender fruit tissues by piercing them

with the ovipositor. A watery fluid oozes out from the puncture, which becomes slightly

concave with seepage of fluid, and transforms into a brown resinous deposit (Dhillon et al.

2005a). Sometimes pseudo-punctures (punctures without eggs) have also been observed on

the fruit skin which reduces the market value of the fruits. In addition to eggs, the female’s

ovipositor introduces bacteria from the fruit surface. These bacteria cause the fruits to rot,

providing a food source for the developing larvae. Young larvae leave the necrotic region

and move to healthy tissue, where they often introduce various pathogens and hasten fruit

decomposition. The severe damage occurs mostly after the first shower of the monsoon,

which sometimes reaches up to hundred percent of the harvest. Fruit infestation by melon

fruit fly in bitter gourd has been reported to vary from 41 to 89% (Lall and Sinha, 1959;

Narayanan and Batra, 1960; Kushwaha et al., 1973; Gupta and Verma, 1978; Rabindranath

and Pillai, 1986). The melon fruit fly has been reported to infest 95% of bitter gourd fruits

in Papua (New Guinea), and 90% snake gourd and 60 to 87% pumpkin fruits in Solomon

Islands (Hollingsworth et al., 1997). Singh et al. (2000) reported 31.27% damage on bitter

gourd and 28.55% on watermelon in India. In India alone, B. cucurbitae destroys 60

percent vegetables partially or completely (Kapoor, 1993).

2.3.6 CONTROL

Various management strategies have been exercised to reduce the extent of damage

done by B. cucurbitae such as chemical control, male sterile technique, male annihilation,

biological control and host plant resistance. So far the most effective control measure cited

in the literature is the use of traditional organic insecticides with various formulations such

as organochlorines, organophosphates, carbamates and chemosterilants (Dhillon et al.,

2005).

Sterile male technique has also been used to eradicate this fruit fly. Sterilization is

accomplished through irradiation, chemo-sterilization, or by genetic manipulation. Chemo-

sterilization (by exposing the flies to 0.5g tepa in drinking water for 24h) and gamma

irradiation are the only widely tested and accepted male-sterile techniques (Gojrati and

Keiser, 1974; Odani et al., 1991). Male annihilation technique has been practiced to

eradicate B. cucurbitae by using the chemical attractants such as Cue-lure, Methyl-

Eugenol, Willson’s lure, Standarlure, Trimedlure, Plumelure, Dosalure and their

formulations (Lall & Singh, 1969; Ramsamy et al., 1987; Taniguchi et al., 1988;

Cunninggham, 1989; Li , 1990; Wong et al., 1991; Shelly & Villalobos, 1995).

The biological control involving the use of hymenopterous parasitoids like Psyttalia

fletcheri Silv., Dirhinus giffardii Silv., Dirhinus auratus ashm., Pachycrepoideus

vindeminae Rondani, Spalangia endius Walker and Diachasmimorpha tryoni Cameroon

etc. has been employed for controlling this fly ( Narayanan & Chawla, 1962; Messing et

al., 1995). A Mexican strain of the entomopathogenic nematode, Steinernema carpocapsae

Weiser (Neoaplectana carpocapsae), has been reported to cause 0 to 86% mortality to

melon fruit fly after an exposure of 6 days to 5000 to 5,000,000 nematodes/cup in the

laboratory, and an average of 87.1% mortality under field conditions when applied at 500

infective juveniles/cm2 soil (Lindegren, 1990). Sinha (1997) and Sinha and Saxena (1999)

reported that culture filterate of the fungus, Rhizoctonia solani Kuhn, Trichoderma viridae

Pers. and Gliocladium virens Origen to be an effective bio-agent against B. cucurbitae

larvae.

Since host plant resistance is an important component in integrated pest management

programs, so efforts have also been made in past to make resistant plant varieties such as

the resistant varieties of wild melon (Cucumis callosus) banana, avocados, pineapple etc.

(Chelliah and Sambandam, 1974a & b; Khandelwal and Nath, 1979; Armstrong, 1983;

Armstrong and Vargas, 1982) and wild bitter gourd (Dhillon et al., 2005a & b). More

recently, a number of plant Lectins have been screened for their anti-insect potential

against B. cucurbitae. Plant lectins from G. max, Ricinus communis, Galanthus nivalis,

Canavalia ensiformis, Arisaema curvatum, Erythrina indica, Arisaema helleborifolium and

Arisaema jacquemontii showed a good potential against growth and development of melon

fly larvae by prolonging the larval and development period, reducing the pupation and

emergence and decreasing reproductive potential (Singh et al., 2006a, 2006b, 2007,

2008,2009 ; Kaur et al. 2006a, 2006b) They also affected the activity of detoxification

enzymes.

It is quite clear from the present review of literature that the effect of plant PIs have

been studied against a good number of lepidopteran and coleopteran insects. The

researchers have attained some success and developed transgenic crops to manage insect

pests belonging to these orders, but diptera which contains a number of economically

important insect pests remained unexplored, as only two insects (C. capitata and S.

calcitrans) have been screened for bioefficacy of plant PIs against them. Also management

of fruit flies is a matter of concern as these flies cannot be controlled by spraying a

pesticide in the crop fields because the eggs are deposited inside the fruit and the larvae

(maggots) of the fruit fly bore into the fruit and can easily escape from the pesticide spray.

Other methods such as male sterile and male annihilation techniques are not very effective

in controlling the fruit flies in the fields. As a result fruit flies still retain the status of

economically important pests and needs exploration of alternative methods for better

management strategies.