2. REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/36405/5...on shoot...
Transcript of 2. REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/36405/5...on shoot...
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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.