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2. REVIEW OF LITERATURE

Helicoverpa armigera commonly called as old-world bollworm, is also known as

cotton bollworm, African cotton bollworm, corn earworm, tobacco budworm, legume pod

borer, and gram pod borer. In India it is called as Dendu chedak illi, Channe ki illi, and

Hari Sundi in Hindi, Kayatuluchu purugu in Telgu, and Kayi karevakulla in Kannada.

2.1 About pest status of Helicoverpa armigera

Being a polyphagus, H. armigera is a major pest of several crops including cotton,

tomato, pigeonpea, chickpea, peas, cowpea, sunflower, sorghum, pearl millet and other

crops. Other important host includes ground nut, okra, field beans, soybean, Lucerne, and

other Leguminosae, tobacco, potato, maize, linseed, and a number of fruits (Prunus,

Citrus), forest trees, and a range of vegetable crops. A wide range of wild plant species

support larval development of the pest and the important species in India include Hibiscus

sp., Acanthospermum sp., Datura sp. It is widely distributed in Asia, Africa, Oceania, and

the Europe. H. armigera like its close relatives H. zea and Heliothis virescens, is a pest of

major importance in most area wherever it occurs, damaging a wide variety of horticultural

and agricultural crops. H. armigera exhibits a facultative diapauses and astivation, which

enables it to survive the adverse weather conditions in both winters as well as in summer.

Its significance as pest is based on the peculiarities of its biology: mobility, polyphagy,

rapid and high reproductive rate, and diapause. Its preference for flowering/fruiting parts

of high value crops including cotton, tomato, corn and pulses confers a high socio-

economic cost to its depredations in subsistence agriculture in the tropics. Agronomic

factors, such as high yielding varieties, increased use of irrigation and fertilizers, and large-

scale production and planting of alternate crop host contribute towards greater prevalence

and increased severity. However, regional and local differences in host can give rise to

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differences in the pest status on particular crops, e.g., in northern and southern India, its

severe infestations on cotton and chickpea have been recorded only in the recent past. On

cotton, 2 to 3 larvae per plant can destroy all the balls within 15 days and on tomato, they

can invade flowers and fruit drop. Extensive damage has been reported in India on cotton,

chickpea and avoidable losses have been estimated to be 55.7%. On the other hand

Tomato (Lycopesicon esculentum Mill.) which is one of the most important vegetables

grown in the world and a good source of vitamins is attacked by a wide range of insects

and forms major limiting factor in its successful cultivation and improvement in yield.

Among them fruit borer, H. armigera is the most destructive insect pest and causes

considerable loss in quantity as well as quality of the tomato fruits (Tewari and

Krishnamoorthy, 1984; Reddy and Zehrm, 2004). The monetary loss due to this pest in

India has been estimated over rupees one thousand crores per year (Jayaraj et al., 1994)

and causing the loss in tomato yield to the tune of 50 to 80 per cent (Tewari and

Krishnamoorthy, 1984).

2.1.1 Nature of damage on cotton and tomato plants

On cotton, round holes made by the larvae are visible at the base of the flower

buds, flowers, and the bolls. Leaves and shoots may also be damaged. Large larvae bore

into maturing green bolls. Young bolls drop down following larval damage. Eggs are laid

on shoot tips, squares, flowers or young bolls, and at times on the leaves. On tomato,

larvae damage flowers and young fruits, which fall down following insect attack. Larger

larvae bore into the maturing fruits and secondary infections by other organisms lead to

rotting of the fruits.

2.1.2 About Trichogrammatids

Trichogramma (Hymenoptera: Trichogrammatidae) are distributed throughout the

world parasitizing eggs of over 200 insect species belonging to 70 families and 8 orders in

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diverse habitats. In India T. chilonis Ishii, T. japonicum Ashmead, T. achaeae Nagaraja

and Nagakatti and Trichogrammatoidea bactrae Nagaraja are widely distributed and are

key natural enemies of many crop pests (Singh and Jalali, 1994). These parasitoids attack

eggs of many lepidopterous pests such as sugarcane borers, Chilo sp. and Scirpophaga

excerptalis Walker; paddy stem borer Scirpophaga incertulas (Walker); tomato fruit

borer, Helicoverpa armigera (Hubner); cutworms Agrotis sp.; pink bollworms,

Pectinophora gossypiella Saunders and Earias sp.; Maize stem borer, Chilo partellus

(Swinhoe) and diamond back moth Plutella xylostella (L.). Trichogramma plays an

important role in parasitizing eggs of more than 400 hosts (Silva, 1999), mostly

lepidopterous pests. Trichogramma species are among the most commonly reared and

used natural enemies in the world (Hoffmann and Frodsham, 1993). This genus comprises

more than 210 nominal species (Pinto, 1998) found from temperate to tropical areas.

Trichogramma differs in the progeny produced, fitness attributes depending upon

the host insects. Since Trichogramma parasitize the eggs of the pest, hatching is prevented

thus reduce crop damage by availability of lesser larvae on crop canopy. Since many

lepidopteran pests have developed resistance to major groups of insecticides, use of

alternative methods is advocated to overcome sustainability within this perspective.

Trichogrammatids have been used in pest management programmes for the suppression of

pests like the tissue borers of sugarcane, maize, rice, cotton bollworms and vegetables like

tomato, cabbage, etc., particularly against lepidopterans like Chilo sp., Scirpophaga

excerptalis Walker, Scirpophaga incertulas Walker, Helicoverpa armigera (Hübner),

Plutella xylostella (Linnaeus).

Trichogrammatids are used in more than 30 countries and it was reported that an

average 32 million hectares of agricultural and forest land were covered with it for the

biological control of insect pests (Li, 1994). In most biological control programmes,

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Trichogramma are mass-produced for inundative and augmentative releases in the field.

However, it is in inundative release programmes that Trichogramma have been used more

than any other natural enemy (Li, 1994). Different species of Trichogramma are released

on a larger scale against lepidopteran pests on grapes, tomato (green house and field

conditions), sugarcane, sweet corn, maize, cotton, store grains and forests trees (Naranjo,

1993; Andow et al., 1995; Mertz et al., 1995; Bai et al., 1995; Scholler et al., 1996; Glenn

and Hoffmann, 1997; Shipp and Wang, 1998; Consoli et al., 1998; Greenberg et al., 1998;

Scholz et al., 1998) with varying degree of control. Augmentative releases of

Trichogramma ostriniae Pang and Chen alone have been found to be effective in the

suppression of Ostrinia nubilalis Hübner (Wang et al., 1999). In India releases of

Trichogramma chilonis in sugarcane and early rice ecosystems have proven effective in

decreasing the populations of the shoot borer Chilo infuscatellus (Snellen) in sugarcane

and leaf folder, Cnaphalocrocis medinalis (Guenée) in rice (Thirumurugan et al., 2006

and Mahal et al., 2006). Use of insecticides to control multiple pest problems in these

crops has reduced the action of Trichogramma. Trichogramma have generally been found

to be sensitive to insecticides (Franz et al. 1980). Susceptibility of trichogrammatids to a

broad spectrum of insecticides and the consequent reduction in parasitism levels on the

eggs of Helicoverpa sp. have been reported in insecticide treated fields (Stinner et al.,

1974; Bull and House, 1983; Jalali and Singh, 1993).

2.1.3 Life cycle of Trichogramma sp.

Trichogramma sp. has a short life cycle of 8-12 days, depending upon the

temperature. Trichogramma eggs hatch in ca. 24 h after oviposition and the parasitoid

larvae develop very quickly. There are 3 larval instars in Trichogramma. Larvae transform

to the inactive pupal stage. The parasitoids pupate within the host eggs. The host egg turns

black during the third instar (may be 5 days after parasitism) as a result of dark melanin

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granules deposited on the inner surface of the egg chorion. The black layer inside the

chorion and the exit hole are evidence of parasitism by Trichogramma. The adult wasps

emerge (after ca. 4 to 5 days) and escape from the host egg by chewing a circular hole in

the egg-shell (Strand, 1986). A few hours after emergence and mating, Trichogramma

females begin to oviposit (Pak and Oatman, 1982; Waage and Ming; 1984, Knutson,

1998). In India the detailed biology was worked out on Corcyra eggs (Singh and Jalali,

1994).

2.2 Insecticide resistance in Helioverpa armigera

In recent years H. armigera has emerged as a dominant pest of various crops,

including cotton, pulses and vegetables in the country. It has been claimed that annual

losses due to this pest on red gram and bengal gram alone amounts to Rs. 3000 million and

if losses on cotton, vegetables and other cereals are taken into consideration the total losses

due to this pest are colossal (Mehrotra, 1992).

2.2.1 Status of insecticide resistance in H. armigera in India

Among the pest complex invading the crop and limiting the productivity and

quality of cotton, bollworms take a major toll, particularly H. armigera takes a lions share

(Lingappa et al. 1993)). According past data cultivated cotton occupies only in 5% of the

total cultivable area in India, it consumes more than 55% of the total insecticides used in

the country (Armes et al., 1996). It commonly destroys more than half the yield with an

estimated annual loss of over US $ 500 million in cotton and pulse crops. Among the

options available to control H. armigera, insecticidal spray is considered as the most

practical option by the farmers in India, presently. The indiscriminate use of insecticides,

particularly during 1980s and 1990s, has contributed to the emergence of H. armigera as a

primary pest of cotton in recent years. In India, resistance in the H. armigera to almost

every class of insecticides has been documented (Singh et al., 1994). This phenomenon is

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complex and reported to vary in time and space including mechanisms responsible for

resistance development (Jadhav and Armes, 1996). Possible change in cropping pattern,

insecticide resistance and suppression of natural enemies have made H. armigera a

dominant pest in south India cotton ecosystem (Reynolds and Armes, 1994).

Organophosphates and organochlorines were first molecules to have recorded

resistance in bollworm (Sparks, 1981; Reddy, 1984). Monocrotophos and quinalphos

constitute 75% insecticide market in India, where, about 85% of quinalphos and 68% of

monocrotophos are used solely on cotton (Gunning, 1993). In India, high level of

pyrethroid resistance was reported from cotton growing region of Andhra pradesh in H.

armigera during 1980's (MaCaffery et al., 1989; Armes et al., 1994a). Subsequently it has

been shown that H. armigera developed resistance to virtually every insecticide group. In

1980's and early 1990's to combat the unprecedented pest pressure, farmers resorted to

application of heavy doses of insecticides and often used combination of two or three

insecticides, thereby creating high selection pressure on the insect to develop resistance.

McCaffery et al. (1989) have reported that H. armigera collected during October 1987

from the coastal cotton growing areas of Krishna in Andhra Pradesh were highly resistance

to cypermethrin and fenvalerate and moderately resistance to endosulfan. Armes et al.

(1994a) reported that insecticide resistance and concomitant field failures to control the

cotton bollworm, H. armigera were first recorded in south India in 1987. In their studies

very high levels of resistance to pyrethroids and significant organophosphate and

endosulfan resistance were recorded.

Kranthi et al. (2001a) reported that pyrethroid resistance was found in 54 field

strains of H. armigera collected during 1995 to 1999 from 24 districts in several states of

India. Resistance was high in the regions where pyrethroid use was frequent (four to eight

applications per season). Resistance to deltamethrin was exceptionally high. Resistance to

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cypermethrin, fenvalerate and cyhalothrin was also high in strains collected from central

and southern India. Pyrethroid resistance was high in strains collected from the districts of

Andhra Pradesh where a majority of cotton farmer’s suicide cases have been reported.

Resistance to pyrethroids appears to have increased over 1995-1996 in most of the areas

surveyed. On the other hand studies of Kranthi et al. (2002) also reported that insecticide

resistance was tested in representative commonly used insecticide groups (pyrethroids-

cypermethrin; organophosphates-chlorpyriphos; cyclodienes-endosulfan) in five major

insect pest of cotton from the main cotton growing regions of India with emphasis on

Andhra Pradesh and Maharashtra. The cotton bollworm H. armigera exhibited wide

spread resistance to cypermethrin with 23-8202-fold resistance recorded in field strains.

Resistance to endosulfan and chlorpyriphos was moderate

Ramasubramanian and Raghupathy (2004) surveyed on insecticide resistance

monitoring for a period of one year indicated that the H. armigera population of Tamil

Nadu developed very high resistance to synthetic pyrethroids, medium resistance to

chlorpyriphos and quinalphos, low level of resistance to endosulfan, thiodicarb and

profenofos and cent percent susceptibility to new chemicals spinosad. Duraimurugan and

Raghupathy (2005) diagnosed resistance to synthetic pyrethroids in the field populations of

American bollworm H. armigera from Coimbatore, Tamil Nadu, during 2003-2004

cropping seasons. The resistance levels to various synthetic pyrethroids to DDs varied

from 80 to 96.4% and the extent of resistance to percentage survival varied for

cypermethrin, fenvalerate, deltamethrin, lambda-cyhalothrin, beta-cyfluthrin respectively.

In several states of India particularly in Central and South India region including

Karnataka, higher levels of resistance to cypermethrin and chlorpyripos have been reported

during the cropping seasons of 2001-2005 (Chaturvedi, 2007). Nimbalkar et al. (2009)

studied insecticide resistance management (IRM) of H. armigera to five commonly used

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insecticides on cotton in different parts of Maharashtra and showed that resistance to

chlorpyriphos was higher in Aurangabad district (3.65-8.25 fold) followed by Prabhani

district (2.15-7.55 fold). Resistance to quinalphos was higher in Jalna (2.50-7.0 fold)

followed by Aurangabad (3.0-5.0 fold) and Prabhani (3.7-4.0 fold). Resistance to

cypermethrin increased from the same throughout the three districts over the time.

Resistance to endosulfan showed an increasing trend as the seasons progressed and was

greatest in Jalna (3.10-9.15 fold) followed by Aurangabad (3.92-8.25 fold) and Prabhani

(1.93-6.93 fold).

2.2.2 Status of insecticide resistance in H. armigera in other parts of the world

Henri et al. (1993) have assessed the level of resistance to synthetic pyrethroids

insecticides with the populations of H. armigera collected from cotton fields in

Kanchanaburi, Thailand, in comparison with that of susceptible populations, field

populations showed high levels of resistance to fevalarate. Pyrethroid resistance studies by

Gunning (1994a) in H. armigera collected from NS Wales cotton growing areas confirmed

a gradual loss of the pyrethroid susceptibility in the unsprayed populations and their survey

concluded that frequency of resistant H. armigera were greater than 50% and these

resistant frequencies are very similar to those found in the sprayed H. armigera

populations from cotton areas. Gunning (1994a) also reported that H. armigera larvae and

adults collected from North South Wales and Queensland during 1974 to 1990 along with

laboratory cultures showed 64-fold increase in the resistance to endosulfan when compared

with that of laboratory cultivars. Cameron et al. (1995) initiated a programme to monitor

H. armigera for resistance to fenvalerate in 1991 collected from the processing tomato and

sweet corn in New Zealand. Comparison of LD50 showed significant increase in the

resistance of 69-fold for adults and 47-fold for third instar larvae within a season.

Comparison with Australian data and lack of control failure suggested that New Zealand

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populations are still widely susceptible to insecticide, but the frequency of resistance to

pyrethroids was increasing.

Torres-Vila et al. (2002) have investigated the pyrethroid resistance status in field

populations of H. armigera collected from processing tomatoes in Spain during 5 years

period (1995-1999), toxicological bioassays on seven pyrethroids- cypermethrin,

bifenthrin, cyfluthrin, lambda-cyhalothrin, delamethrin, permethrin and fenvalerate were

tested showed high resistance (RF = 31 – 100) to cypermethrin and deltamethrin and very

high resistance (RF ˃ 100) to lambda-cyhalothrin and deltamethrin.

Torres-Vila et al. (2002) investigated the resistance status in field population of H.

armigera collected from processing tomatoe in Spain during 1995 – 1999, on eleven

chemicals including endosulfan, carbamates (carbaryl, methomyl, thiodicarb) and

organophosphates (chlorpyriphos, fentrothion, methamidophos, azinphos-methyl,

trichlorophon, aceptate, methomyl) showed a moderate resistance (RF= 11-18).

Zahid and Hamed, (2003) studied the efficacy of the insecticides Larvin 80 DF, Lannate

40SP, Loeshan 40 EC, Fastac 5 EC, Decis 10 ECand Fury-F181EC against 3rd

instar

larvae of American bollworm H. armigera in Pakistan under the controlled laboratory

conditions and showed that high level of resistance to carbamate and pyrethroid group of

insecticide.

Sohil et al. (2004) carried out a study of chemical control of H. armigera on cotton

and chick pea in the field and laboratory populations in Pakistan on insecticides

cypermethrin 10 EC, methomyl 40 SP, spinosad 240 SC and indoxacarb 150 SC at

intervals of 15 days with three applications showed spinosad was most effective

indoxacarb, methomyl, and cypermethrin was least effective. In the laboratory bioassays

on plant treatment with different concentrations of insecticides also presented that spinosad

was toxic to 2nd

instar larvae of the pest and cypermethrin was least effective. The

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presence of tolerance to cypermethrin in the field indicated a wide spread genetic factor or

possibly a common mechanism of resistance to cypermethrin.

Rashid et al. (2006) showed that toxicity of lambda-cyclothrin to H. armigera

collected from cotton at Faisalabad was more with a LC50 71.31 ppm followed by alpha-

cypermethrin with an LC50 of 287.87 ppm chlorpyriphos with 464.85 ppm respectively.

Their results showed that increased resistance of H. armigra to cypermethrin in F1

generation under laboratory conditions.

Brevault et al. (2008) conducted the resistance monitoring studies from 2002 to

2004 in regional and local cotton fields of Central Africa among the major host plants of

the bollworm. They reported that from 2002 pyrethroid resistance increased within and

across cotton growing seasons to reach a worrying situation at the end of 2005 growing

seasons. Cotton plants played a fundamental role in increase in the resistance, even if the

intensive use of the insecticides on the local tomato crops strongly concentrated resistance

alleles in the residual populations throughout the off-season.

Mironidis et al. (2012) conducted a 4-year survey (2007–2010) and examined the

insecticide resistance status of H. armigera populations from two major and representative

cotton production areas in northern Greece against seven insecticides (chlorpyriphos ,

diazinon, methomyl, alpha-cypermethrin, cypermethrin, gamma-cyhalothrin and

endosulfan) and showed by comparing with susceptible laboratory reference strain,

resistance rose to 46- and 81-fold for chlorpyriphos and alpha-cypermethrin, respectively

in 2010, when the resurgence of the pest was observed.

2.2.3 Insecticide resistance in Trichogrammatids

Information of the effect of the conventional chemical insecticides

(Organophosphates, Carbamate, Pyrethroids, etc.) used in vegetable crops is not extensive,

but the available studies suggest that although the parasitoids are sensitive to insecticides

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use, the long term effects are variable. Jacobs et al. (1984) examined the compatibility

between 2 insecticides and T. pretiosum released for the control of H. zea on fresh market

tomato and found that the residues of endosulfan had measurable negative effects on the

adult survivorship and egg parasitism. Similar results were found for the methomyl

(Oatman et al., 1983), where the negative effect was restricted primarily to those immature

and adults of T. pretiosum present at the time of application. In contrast residues of the

pyrethroids remained active against Trichogramma sp. for least 21 d on tomatoes (Jacobs

et al., 1984) and more than 7 d on cotton (Bull and House, 1983). Fenvelarate, also caused

extensive reduction in the percentage parasitism by T. exiguum Pinto & Platner and T.

pretiosum followed by application on tomatoes (Campbell et al., 1991). Of the other

compounds tested by these authors’ methomyl and methyl-parathion were found to reduce

parasitism rate significantly (70-100%) following repeated applications. Repeated (weekly)

applications of methomyl plus carbaryl (Roltsch and Mayse, 1983) or methomyl plus

fenvelarate (Hoffmann et al., 1990) have also been shown to substantially reduce the

percentage parasitism of several Trichogramma sp. on various lepidopteron pests of

tomatoes. Limited additional information is available on the other insecticides in the

literature. A variety of growth regulators (mostly chitin inhibitor) and other insecticides

has been studied for interactions with different Trichogramma sp.

Charles et al. (2000) studied the effect of insecticide lambda cyhalothrin,

cypermethrin, thiodicarb, profenophos, spinosad, methoxyfenozide, and tebufenozide on T.

exiguum Pinto & Platner collected from cotton fields of Raleigh, NC and showed that all

insecticides, with the exception of methoxyfenozide and tebufenozide, adversely affected

Trichogramma emergence from H. zea host eggs. Based on LC50 values, spinosad and

prophenofos were the most toxic compounds to female T. exiguum adults, followed by

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lambda cyhalothrin, cypermethrin, and thiodicarb while studying the toxicities of

insecticides.

Takada et al. (2001) while studying the toxicities of insecticides on T. dendrolimi

collected from cabbage and tomato fields in Japan showed ethofenpros had highest toxicity

and cartap showed relatively highest toxicity compared with the other insecticides. The

development of the parasitoid treated with these two insecticides was normal, similar to

that of the control group. Only the emergence of the adult wasp from the host egg was

disturbed.

Zhang et al. (2002) treated T. chilonis with insecticide in its egg, larvae; prepupae,

pupae and adults and found that the wasps are susceptible on all tested pesticide besides Bt

in adults. Geraldo et al. (2003) reported that when Trichogramma pretiosum Riley

collected from tomato fields, when tested for different insecticides, abamectin was the only

insecticide to affect parasitoid emergence and sex ratio, regardless of the developmental

stage and parasitoid generation exposed. abamectin, lufenuron and pirimicarb also

decreased the lifetime of F1 females exposed during the egg-larva stage.

Moura et al. (2006) found that when insecticides were sprayed on the immature

stages of the parasitoid T. pretiosum collected from tomato fields, cartap and chlorpyriphos

proved to be most harmful insecticide, affecting both emergence success and parasitism

capacity of this parasitoid. Abamectin was harmful to adults, slightly harmful to larvae

and moderately harmful to the pupae. Actamiprid were slightly harmful to adults, harmful

to the pupae and moderately harmful to the larvae. Cartap was harmful to adults,

moderately harmful to larvae and harmful to pupae. Chlorpyriphos was harmful to adults,

harmless to larvae and harmful to pupae.

Garcia et al. (2006) studied the influence of deltamethrin on the reproduction of T.

cordubensis, and found that offspring emergence was significantly influenced by the

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insecticide treatments experienced on their progenitors, decreasing significantly at 48 and

72 h for the highest tested concentration of deltamethrin (23.6 mg [a.i.]/L).

Hongbol et al. (2002) studied the insecticide resistance of 5 different geographic

populations of T. chilonis and T. dendrolimi to methomyl, deltamethrin and phoxim.

Analysis of LC50 data of these insecticides revealed that the parasitoids are susceptible to

the tested insecticides.

Preetha et al. (2009) found that thiamethoxam showed the highest toxicity to T.

chilonis with an LC50 of 0.0014 mg a.i. l−1

, followed by imidacloprid (0.0027 mg a.i. l−1

).

The LC50 values of aceptate and endosulfan were 4.4703 and 1.8501 mg a.i. l−1

, exhibiting

low toxicity when compared with other insecticides tested. Chlorantraniliprole was found

to be harmless to T. chilonis based on the risk quotients. The insecticides thiamethoxam,

imidacloprid, Virtako®, ethofenprox and BPMC were found to be dangerous to the

parasitoid.

2.3 Isolation, identification of insect gut microorganisms

Over the last century, the discovery of microbial endosymbionts in a wide variety of

arthropods has been a significant finding

in arthropod biology. For example, the

recognition that prokaryotic Rickettsial endosymbionts were widespread among arthropods

and may induce sterility in their hosts brought a new perspective

to studies of arthropod

speciation (Shoemaker, 1999). Bacteria are associated with a number of different insect

species across all major orders of the insects (Buchner 1965; Dillon and Dillon, 2004).

The insect gut provides a suitable habitat for bacteria (Bignell et al., 1984). In many insect

species the gut possess different types of bacteria, which are transient and do not remain in

the gut during all life stages. However in some cases, a variety of permanent

microorganisms are present that supply essential nutrients to their host and some posses’s

obligate microbial endosymbionts that benefit the insects (Bridges, 1981). Although

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cultivation based biochemical techniques have been used for analysis of the specific groups

of bacteria, several limitations are associated with such approaches, particularly for

surveying intestinal bacterial ecosystem. The introduction of high resolution molecular

techniques has improved the analysis of diverse microbial populations (Muyzer, 1999).

The important advance has been the use of 16S rRNA as a molecular fingerprint to identify

and classify organisms (Ohkuma and Kudo, 1996). Until recently little was known about

the bacteria associated with Lepidoptera, those studies on lepidopteron gut microbiota

suggested the possibility that microorganisms provided essential nutrients or assisted in

important biochemical function related to host food ingestion (Broderick et al., 2004).

H. armigera is a polyphagous lepidopteron pest that infests important crops like

cotton, tomato, sunflower and corn all over the world. The 5th

and 6th

instar larva oft feeds

voraciously and damages agricultural crops and hence reduces the yield (Sarode, 1999).

Control measures are difficult because the larvae feed inside the host plant and are difficult

to kill with insecticides and also have gained resistance to variety of insecticides (Kranthi

et al., 2001a). Knowledge of the gut microbiota of the cotton bollworm or tomato fruit

borer and the roles it might play in the larval biology may lead to new target for the

management of the pest. Many groups of microorganisms have been isolated from insect

groups using molecular methods.

2.3.1 Indian work on insect gut microorganisms in H. armigera and other insect

species

Mishra and Tandon, (2003) isolated 25 bacterial isolates representing eleven

different genera belonging to diverse families from different parts of the gut of H.

armigera. Bacillus species dominated the bacterial flora. Total viable count and heat

stable count showed highest incidence of the spore forming bacteria in hindgut rather than

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the foregut and midgut. Bacterial species identified in their study were Enterobacter,

Pseudomonas, Klebsiella, Staphylococcus, Bacillus, Proteus and Salmonella.

Gayathri Priya et al. (2012) using culturable techniques isolated and identified

members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas

variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and

Enterococcus cassiliflavus in field collected H. armigera insect samples from host plants

including cotton and tomato grown in different parts of India and also found that

Enterobacter and Enterococcus were universally present in all our Helicoverpa samples

collected from different crops and in different parts of India.

Thakur et al. (2005) dissected whole gut from the rice hispa, Dicladispa armigera

(Olivler) and the bacteria belonging to genera Bacillus, Proteus, Micrococcus,

Pseudomonas and Klebsiella were isolated and identified. All the bacteria were found to

be resistant to penicillin G, ampicillin and cephaloxin but susceptible to streptomycin,

ciprofloxin, rifampicin. The bacteria isolated also resisted 1000 ppm endosulfan and to

some extent chlorpyriphos and quinalphos.

Vyankatesh et al. (2002) isolated three noval bacterial strains (MTCC 3249, SH

and SLH) from the midgut of the female Culex quinquefaciatus and Aedes aegyptli

mosquitoes. 16S rRNA gene sequence analysis of these novel strains showed that they

were highly homogenous to the strains of Aeromonas. DNA-DNA hybridization studies

showed that the DNA of the strain MTCC 3249 was 96 and 88% similar to that of the

strains SH and SLH, respectively and showed 54% relatedness to Aeromonas jandaei and

61% relatedness to Aeromonas sobira.

Ramesh et al. (2009) characterized gram negative microbes Escherichia coli,

Yerainia enterocolitica, Klebseilla, Pneumonia sp. from the gut of the silk worm using

rapid identification biochemical tool kit. Thangamalar et al., (2009) reported that the gut

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microflora are associated with several important physiological systems in lepidopteran

insects including silkworm. Enterobacter isolates identified and characterized from the

silkworm breeds included Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia

odorifera.

2.3.2 International work on insect gut microorganisms in H. armigera and other

insect species

Xiang et al. (2006) compared the bacterial communities in the laboratory and field

populations of H. armigera using denatured gradient gel electrophoresis of amplified 16S

rRNA sequences. The laboratory populations harbored rather a simple gut microflora

containing mostly of the phylotypes belonging to Enterococcus. From the field

populations, phylotypes belonging to Enterococcus, Lactococcus, Flavobacterium,

Acineatobacter and Stenotrophomonas were dominant members.

Gebbardi et al. (2001) isolated the endosymbiotic bacteria from the genus Bacillus

including B. pantothenicus, B. subtilis, B. pumilis, Bacillus sp., from different

compartments of the gut of the various members of the insects (Hexapoda) and multipoda

(Diplopoda), which were grown in the submerged cultures and investigated by the

biological assays and HPLC-diode array analysis regarding their production of the

bioactive metabolites. In their analysis known compounds and yet unknown derivatives

from the primary metabolites were detected.

Zacchi and Vaughan-Martini, (2002) investigated the association of some of the

yeast species with insects (Dermaptera, Rynchota, Diptera, Hymenoptera) collected around

Perugia, Italy. Whole or specific body contents (gut, hemolymph and fat body) of over

450 insects was studied, isolated and identified by conventional and molecular analysis

represented ascomycetous (64%) and basidiomycetus (34%) strains. While Pichia

guillieramondii and Rhodotorella muciloginosa were the most commonly isolated species

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from the bodies of the host insects, they also reported that several other species were

consistently associated with the insects.

Kazim et al. (2004) investigated the gut bacterial flora of the alder leaf beetles

Agelastica aini (Coleoptera: Chrysomelidae) which is one serious pest of hazelnut and

alder trees throughout the world. Based on the morphological, physiological and

biochemical tests bacterial flora were identified as Pseudomonas chlororaphia,

Enterobacter agglomerans, Listeria sp., Pseudomonas flurosecens.

Jenny et al. (2005) while screening for the mid gut bacterial content in the field

collected mosquitoes of the two main malaria vectors in Africa, A. gambiae and A.

funestus. The identified bacterial species in these mosquitoes were found to be the close

relative of Stenotrophomonas, Aeromnas, Mycoplasma, Anapelasma ovi and Ehrlichia sp.

Using 16S rRNA analysis.

Claudia et al. (2005) reported that the Formosan subterranean termite, Coptotermes

formosanus is a highly destructive invasive pest species in many tropical and sub tropical

regions. The survival of this termite is dependent on the gut microbes. Therefore

alternative strategy may be devised in the future using the gut microbes of the termites as a

tool and target for ecologically sound termite control. They isolated 12 different bacterial

strains from four different groups (Bacteroides, Troponema, Spirochetae, Clostridiaceae)

using 16S rRNA analysis. Bacteroides were found to be dominant. Till date they cultured

25 strains of termite gut bacteria belonging to Enterobacteriacea, Bacteriodales,

Lactobacillus.

Shinzato et al. (2007) constructed a bacterial 16S rRNA gene clone library from the

gut microbial communities of a termite, Odontotermes formosanus and phylogenetically

analyzed it in order to contribute to the evolutionary study of the digestive symbiosis and

method development for the termite control. After screening by RFLP 56 clones with

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unique RFLP pattern were sequenced and phylogenetically analyzed. The representative

phyloptypes werr affiliated to four phylogeneic groups, Proteobacteria, Firmicates,

Chlorobi group and Actinobacteria of the domine Bacteria.

Park et al. (2007) investigated the bacterial communities within the guts of several

longicorn beetles by culture-dependent method and from 16S rRNA gene based analysis

the bacterial communities were identified as Gammaproteobacter, Actinobacter,

Fimricutes, Alphaproteobacter, Acidobacter and Betaproteobacter.

Zahner et al. (2008) profield bacterial species associated with different

developmental stages of the pine false webworm, Acantholyda erythrocepala and reported

that Pseudomonas sp. along with Bacillus sphaericus and Arthrobacter sp. were the

predominant components based on 16S rRNA analysis. PCR-DGGE also confirmed the

predominance of Pseudomonas sp. and Bacillus sphaericus.

Changmann et al. (2010) investigated the diversity of bacteria in acaricide resistant

and susceptible populations from the whole mite extracts through 16S rRNA gene

sequence analysis. The pyridaben resistant population was diversified with six genera

including Chryseobacterium, Sphingomonas, Acidovorax, Herbaspirillum,

Janthinobacterium, and Xenophilus, whereas the acequinocyl resistant population was

associated with Bacillus, Pasteurella, Staphylococcus, Enterobacter, and Pseudomonas.

The fenpropathrin resistant population harbored only two phylotypes (Pantoea and

Pseudomonas). Citrobacter, Enterobacter, and Pantoea were recovered from the

susceptible population. This study suggests that knowledge of the diversity of bacterial

phylotypes present in host insect pest species may be useful for developing biological

approaches in insect-microbe interaction.

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2.4 Insect gut microflora and its role in insecticide degradation

The functional role of the insect microbiota echoes that of higher vertebrates. The

intestinal tracts of mammals contain an extremely complex biota and the relationships can

be characterized as commensal or moderately mutualistic. Intestinal microbes may

contribute to food digestion, produce essential vitamins for the host, and keep out

potentially harmful microbes. In contrast to higher animals the adaptation of insects to their

environment is rapid. However, bacterial division can occur as often as every 20 min and

viable bacterial mutations are generated during every cycle, allowing the indigenous

microbiota to adapt rapidly to changes in the gut environment. This adaptation and its

consequences for the insect host are almost completely unknown.

Microorganisms play a key role in both host physiology and nutrition (Dillon and

Charnley 1995; Nardi et al., 2002). Bacteria and insects have evolved a diverse array of

symbiotic interactions, which play a role in insect nutrition (Bernays and Klien 2002;

Bracho et al., 1995; Douglas 1988; Douglas and Prosser 1992; Lal et al., 1994; Wicker

1983), defence (Ferrari et al. 2004; Kellner and Dettner 1996; Oliver et al. 2003),

reproduction and development (Caspari and Watson 1959; Gherna et al., 1991; Hurst et al.,

1999). Past studies have overwhelmingly focused on the contribution of endosymbionts

and gut microbiota to the nutrition of the host and these have been reviewed extensively

(Douglas, 1992). Nutritional contributions may take several forms: improved ability to live

on suboptimal diets, improved digestion efficiency, acquisition of digestive enzymes, and

provision of vitamins. These nutritional contributions are well established for

endosymbionts such as Buchnera sp. (Douglas, 1998)), but in many cases the indigenous

gut bacterial community could provide similar benefits. Plant material is low in nitrogen,

specific amino acids, sterols, and B vitamins, and in many cases microorganisms

synthesize these components (Cruden and Markovertz, 1987; Douglas, 1998). For

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example, aphids feeding on plants with phloem sap that contains a low concentration of

essential amino acids rely on bacterial endosymbionts to provide the required amino acids

(Douglas, 1998; Adams and Douglas, 1997). The bacteria in the hindgut of the house

cricket, Acheta domesticus, increase the efficacy of the utilization of soluble plant

polysaccharides in the insect (Kaufman and Klug, 1991). Spirochetes provide the carbon,

nitrogen, and energy requirements of termite nutrition via acetogenesis and nitrogen

fixation (Breznak, 2002). In fact, microbial nitrogen fixation accounts for 60% of the

nitrogen in some termite colonies (Tayasu et al. 1994). The gut microorganisms have the

ability to adapt rapidly to changes in the insect diet by induction of enzymes and

population changes in the microbial community (Santo et al., 1998; Kaufman and Klug,

1991). When cockroaches switch to a low-protein, high-fiber diet, there is a decrease in the

number of streptococci and lactobacilli inhabiting the foregut with a concomitant decrease

in production of lactate and acetate (Kane and Breznak, 1991). Similarly, a diet rich in

cellulose induces an increase in the protozoal population in the hindgut of the American

cockroach, Periplaneta americana (Gijzen et al., 1994). When crickets were fed on cricket

chow, the hindgut microbiota was dominated by bacteria with a GC content of 32%–57%

(Santo et al., 1998). A beet pulp or protein-based diet resulted in a microbiota with a low

GC content and an associated reduction in hydrogen and carbon dioxide production (Santo

et al., 1998). Microorganisms possess metabolic properties that are absent in insects and in

this way act as “microbial brokers,” enabling phytophagous insects to overcome

biochemical barriers to herbivory (Berenbaum, 1998; Douglas, 1992; Jones, 1984).

Microorganisms detoxify plant allelochemicals such as flavonoids, tannins, and alkaloids

(Douglas, 1992; Bhat et al., 1998). Microbial degradation of plant aromatic compounds

can occur in termite guts and may contribute to the carbon and energy requirement of the

host (Brune et al., 1995). Digestive enzymes of some insects might be derived from the

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microbiota, but there are few studies that show an unambiguous contribution of microbial

hydrolases (Terra et al., 1996). However, interaction between gut microbe and insect host

should not be simply regarded as helping nutritional balance or overcoming the insect

pathogens.

2.4.1 Indian work on role of insect gut microorganisms.

Thakur et al. (2005) dissected whole gut from the rice hispa, Dicladispa armigera

(Olivler) and the bacteria belonging to genera Bacillus, Proteus, Micrococcus,

Pseudomonas and Klebsiella were isolated and identified. All the bacteria were found to

be resistant to penicillin G, ampicillin and cephaloxin but susceptible to streptomycin,

ciprofloxin, rifampicin. The bacteria isolated also resisted 1000 ppm endosulfan and to

some extent chlorpyriphos and quinalphos. The GLC analysis showed that the isolated gut

bacteria had the potentiality to degrade endosulfan.

Indiragandhi et al. (2007) evaluated the gut bacteria of insecticide-resistant,

insecticide-susceptible and field-caught populations of the lepidopteran insect pest

diamondback moth (DBM) Plutella xylostella for their variability and their role in host

protection and nutrition and reported that the gut bacterial populations of the three DBM

larvae populations were found to be significantly different, irrespective of the

developmental stage. The 16S rRNA gene sequence analysis of the DBM gut bacteria

revealed that the bacterial population from the prothiofos-resistant larval gut was more

diversified with Pseudomonas sp., Stenotrophomonas sp., Acinetobacter sp., and Serratia

marcescens. Meanwhile, the susceptible larvae were associated with Brachybacterium sp.,

Acinetobacter sp. and S. marcescens and the field-caught population harboured a rather

simple gut microflora of phylotypes belonging to Serratia.

Anand et al. (2010) reported that Bombyx mori L. (Lepidoptera: Bombycidae) fed

with mulberry leaves which is mainly composed of pectin, xylan, cellulose and starch

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required digestive enzymes that degrade these carbohydrates might be produced by gut

bacteria. Eleven isolates were obtained from the digestive tract of B. mori, including the

Gram positive Bacillus circulans and Gram negative Proteus vulgaris, Klebsiella

pneumoniae, Escherichia coli, Citrobacter freundii, Serratia liquefaciens, Enterobacter

sp., Pseudomonas fluorescens, P. aeruginosa, Aeromonas sp., and Erwinia sp. Three of

these isolates, P. vulgaris, K. pneumoniae, C. freundii, were cellulolytic and xylanolytic, P.

fluorescens and Erwinia sp., were pectinolytic and K. pneumoniae degraded starch.

Aeromonas sp. was able to utilize the cellulose and xylan. S. liquefaciens was able to

utilize three polysaccharides including cellulose, xylan and pectin. B. circulans was able to

utilize all four polysaccharides with different efficacy. The gut of B. mori has an alkaline

pH and all of the isolated bacterial strains were found to grow and degrade polysaccharides

at alkaline pH. The number of cellulolytic bacteria increases with each instar.

2.4.2 International work on role of insect gut microorganisms.

Carol et al. (2003) reported that Dihydrochalcone Phloridzin, a plant derived

compound toxic to Rhagoletis pomonella (Walsh), was degraded and detoxified by the

bacterium Enterobacter agglomerans associated with the apple pest. All apple maggot

flies that fed on three different concentrations of phloridzin solution died within 24 h.

Incubation of E. agglomerans in 0.001 and 0.01 m aqueous phloridzin for 3 d eliminated

the toxicity for apple maggot flies but most died after fed with 1 M solution.

Vesta et al. (2006) reported that bark beetles lps typographus (Coleoptera: Scolytidae) fed

on conifers which produce myrcene (MR) among some other defensive compounds. Six

bacterial isolates were most resistant to the bactericidal compound myrcene. Based on 16S

rRNA analysis the bacteria were related to Enterobacteriaceae.

Studies conducted by Genta et al., (2006) on antibiotic treated and non-treated larvae of

Tenebrio molitor suggested that microbial products play subtle roles in the life of the

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insect, they are involved in the digestion of refractory food, detoxification of secondary

plant compounds and modify the volatile profiles of the insect host.

Visotto et al. (2009) reported that isolated bacteria colonies from gut homogenates

of fifth instar velvetbean caterpillars (Lepidoptera: Noctuidae) when subjected to antibiotic

sensitivity found that the bacterial colonies were highly susceptible to tetracycline

Tetracycline also provided higher inhibition of colony forming units than chloramphenicol

and was therefore provided to the caterpillars in increasing diet concentrations to assess the

contribution of gut bacteria to their digestion and development. The activity of proteases

(general), serine-proteinases and lipases were significantly suppressed by tetracycline. The

antibiotic was effective in suppressing them, particularly serine-proteinases, suggesting

that gut bacteria may significantly contribute with lipid- and mainly protein-digestion in

velvetbean caterpillars. Therefore, the gut bacteria inhibited by tetracycline does not seem

to play a crucial role in the survival and development of the velvetbean caterpillar, but may

be important in the adaptation of this pest species to hosts rich in protease inhibitors, such

as soybean.

Changmann et al. (2010) Investigated the diversity of bacteria in acaricide resistant

and susceptible populations from the whole mite extracts of two spotted spider mite-

Tetranychus urticae were made using 8 different bacterial growth media and identified

through 16S rRNA gene sequence analysis. The pyridaben resistant population was

diversified with six genera including Chryseobacterium, Sphingomonas, Acidovorax,

Herbaspirillum, Janthinobacterium, and Xenophilus, whereas the acequinocyl resistant

population was associated with Bacillus, Pasteurella, Staphylococcus, Enterobacter, and

Pseudomonas. The fenpropathrin resistant population harbored only two phylotypes

(Pantoea and Pseudomonas). Citrobacter, Enterobacter, and Pantoea were recovered from

the susceptible population. This study suggests that knowledge of the diversity of bacterial

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phylotypes present in host insect pest species may be useful for developing biological

approaches in insect-microbe interaction.

Kikuchi et al. (2012) reported a previously unknown mechanism of insecticide

resistance in the beanbug Riptortus pedestris and allied stinkbugs harboring mutualistic gut

symbiotic bacteria of the genus Burkholderia, which are acquired by nymphal insects from

environmental soil every generation in a Japanese island where fenitrothion has been

constantly applied to sugarcane fields. Their studies demonstrated that the fenitrothion-

degrading Burkholderia strains establish a specific and beneficial symbiosis with the

stinkbugs and confer a resistance of the host insects against fenitrothion. Experimental

applications of fenitrothion to field soils drastically enriched fenitrothion-degrading

bacteria from undetectable levels to >80% of total culturable bacterial counts in the field

soils, and >90% of stinkbugs reared with the enriched soil established symbiosis with

fenitrothion-degrading Burkholderia.

A more complicated polytrophic interaction between the insect or plant or animal

host were taken into consideration by Dillon and Dillon (2004), who analysed that diverse

group of microorganism inhabit gut of H. armigera in the field environment, but their role

in the host interaction is unclear. However, if they have functional significance with regard

to the detoxifying any toxic compounds, physiology and nutrition of the cotton bollworm

or tomato fruit borer H. armigera is to be studied.