Chapter 1Chapter 1 Review of LiteratureReview of...

Chapter 1Chapter 1Chapter 1Chapter 1

Review of LiteratureReview of LiteratureReview of LiteratureReview of Literature

Chapter 1 Review of Literature

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All organisms face the onslaught of unpredictable of weather, risks from their natural

enemies and competition from siblings for space, food and mate. The population of any

species is the function of severity of these factors. Usually, these interacting forces

maintain the population equilibrium by and large steady. In agroecosystems, the

components of modern intensive agriculture such as high yielding varieties, sumptuous

fertilization, irrigation etc. alter the crop physiology, morphology and phenology so as

to render the crop attractive for insect pests and diseases. With intensive agricultural

practices to meet the increase demand for food, particularly in the developing countries

of Asia, Africa and Latin America, this increase in food demand has to be met from

increased yields from major crops grown on existing cultivable land. As a consequence

insects feeding on the crops have also inadvertently obtained better opportunities to

multiply.

One practical means of achieving greater yields is to minimize the pest associated

losses, which are estimated at 14% of the total agricultural production (Oerke et al.,

1994). Insects not only cause direct loss to the agricultural produce, but also indirectly

due to their role as vectors of various plant pathogens. Insect control is essential in the

production of crop and animal produce. Hence, plant protection has assumed more

importance, and several methods are being tested and applied in pest control.

The continuous and injudicious application of pesticides results in adverse effects on

the beneficial organisms, development of insecticidal resistance (Gould et al., 1992,

Tabashnik 1994, Tabashnik et al., 1997), leaves pesticide residues in the food, and

results in environmental pollution. In fact, Brown (1971) and Brown and Pal (1971)

showed that among arthropods, about 130 species of agricultural and veterinary

importance and 102 species of medical importance had developed resistance to the

insecticides. In India, Spodoptera litura Fab. (Lepidoptera: Noctuidae) is a serious

economically important insect pest of cosmopolitan distribution. It has been reported to

attack more than 112 different species of cultivated crop plants throughout the world

(Moussa, M. A., et al 1960). This has necessitated the use of target specific compounds

with low persistence and an increase in emphasis on devising methods other than

pesticide use to control insect pests. In view of the limitations associated with chemical

control, scientific community across the world has been directed to seek some

alternative methods of insect control, which must be ecologically compatible.

i.exe


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The concept of integrated pest management (IPM) has received a greater acceptance in

the present scenario of environment degradation and food contamination. Promoting

natural enemies of key pests of important crops is one of the most important and

indispensable components of IPM.

1.1. Insect control techniques

1.1.1. Physical control

Physical controls are those that can be carried out by the farmer to alter environmental

factors in a way that reduces pest populations. A simple and common example of this is

crop rotation. Another physical control method sometimes called ‘mating disruption’

involves the use of sex pheromones. Female insects to attract males for mating produce

sex pheromones. For many insects, scientists have been able to analyze the chemistry of

sex pheromones and reproduce them synthetically in the laboratory. Quantities of the

chemical placed around an orchard can disrupt mating male insects become confused

and are less likely to find a mate (Witzgall, 2001).

1.1.2. Chemical control

The use of chemical pesticides often forms part of an integrated pest management

strategy. The key is to use pesticides in a way that complements rather than hinders

other elements in the strategy and which also limits negative environmental effects. It is

important to understand the life cycle of a pest so that the pesticide can be applied when

the pest is at its most vulnerable stage, as the aim is to achieve maximum effect at

minimum levels of pesticide.

1.1.3. Genetic control

Various radio-genetic phenomena have been proposed, for the management of the

insect pests, including sterile insect technique (SIT) and F1 sterility technique (Knipling,

1955 and 1959) SIT is a species specific and environmentally non-polluting method of

insect control that relies on the mass rearing, sterilization and release of large number of

insects. Mating of released sterile males with native females leads to decrease in their

reproductive potential and ultimately; if males are released in sufficient number over a

period of time and it may lead to the local eradication of the pest population. The


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genetic manipulation may also allow the removal of females from the release

population overcoming the difficult and often impractical need to physically removed

females before release technique known as RIDL TM (release of insects carrying the

dominant lethal). The technology is already developed and proven in the Drosophila

model system, and is being applied to a range of insect pests, this technique is known as

genetic sexing (Thomas et al., 2000; Alphey and Andreasen, 2002; Alphey, 2000).

Genetic technique for removing females from the release population have been

developed for several insect species using conventional mutagenesis methods, state of

the art being the 1st strain of Med fly develop by the United Nations IAEA/FAO labs

based in Austria (Coleman and Alphey, 2004). Since, high doses of gamma irradiation

(200-350 Gy) are required to induce complete sterility in Lepidoptera (North and Holt

1971) and exposure to high radiation levels also adversely affects male mating

behaviour and competence, therefore, one approach to reduce the negative effects of

radio resistance in Lepidoptera has been the use of inherited or F1 sterility (Carpenter et

al., 2005; North 1975). The feasibility of using induced F1 sterility as a genetic control

method has been studied for several species of Lepidoptera (Knipling, 1970).

1.1.4. Biological control

Biological control focuses on the manipulation of interactions like competition,

herbivory, predation and parasitism to reduce pest members and limit crop damage. De

Bach’s defined biological control as “action of parasites, predators, or pathogens in

maintaining another organisms’ population density at a lower average than would occur

in their absence” (De Bach, 1964). Biological control does not cause immediate

reduction in target population, but only achieves partial suppression of the target pest,

as a residual pest population is necessary to maintain natural enemies. It is not a new

concept. The ancient Chinese encouraged ants in citrus orchards because they attacked

many citrus pests.

There are three general approaches to biological pest control.

(a) Importation of natural enemies,

(b) Augmentative release of natural enemies, and

(c) Conservation of natural enemies.


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1.1.4.1. (a) Importation (classical biological control)

Biological control focuses primarily on the importation of natural enemies for

introduced pests that, after invading new area, have escaped from the regulation action

of their enemies in their native environment. The highly successful, biological control

of the cassava mealy bug in Africa provides a valuable case history of classical

biological control (Herren and Neuenschwander, 1991).

1.1.4.1. (b) Augmentative releases (Inoculation/Inundative release)

Augmentative releases of indigenous or naturalized (exotic species that have been

previously released and have become established) biological control agents have long

been used to suppress various pest species (Bellows et al., 1992). ‘Augmentation’ falls

in two categories: (a) Inoculative approaches, in which natural enemies are released in

relatively low numbers to established local population of resident natural enemies for

their long-or-short term suppression of a target pest or pest complex and (b) Inundative

approaches, in which large numbers of natural enemies are released to obtain rapid pest

suppression e.g. egg parasitoids of the hymenopterans genus Trichogramma have been

widely used worldwide for inundative releases against key pests (Li, 1994).

1.1.4.1. (c) Conservation of natural enemies

Conservation of biological control agents refers to any environmental modification that

either reduces or eliminates conditions that are unfavourable to natural enemies or that

provides resources that promote population growth, recruitment or performance of

biocontrol agents.

1.1.4.2. Agents of biological control

The diverse natural enemies of insects include protozoa, arthropods, nematodes and

microorganisms. The natural enemies may group as parasites, predators or pathogens.

(i) Parasites: A parasite is an organism that lives on the body of its host, which may

or may not be killed after it has completed development. Parasites with the

greatest impact on insect populations are mostly insects.


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(ii) Parasitoids: It completes its life on one host only. It is parasitic in its immature

stages but its adult (one) stage is free living. Parasitoids can attack all host stages:

eggs, larvae, pupae and adults. A parasitoid is “an organism which develops on or

in another single organism, extracts nourishment from it, and kills it as a direct or

indirect result of that development” (Eggleton and Gaston, 1990). Therefore,

opposite to a parasite, the parasitoid uses only one host and necessarily kills it.

(iii) Predators: A predator is a free-living organism that feeds on its prey. Major

category of predators of insects includes vertebrates and arthropods.

(iv) Pathogens: Pathogens are those microorganisms that cause diseases in the

insects. The most common pathogens of insects are bacteria, viruses, protozoan,

fungi and rickettsiae.

The most successful biocontrol agents have narrow host ranges and many feed

exclusively on the target pest organisms (Greathead, 1995). However, many potentially

useful biological control agents are oligophagus, feeding on target pests and closely

related species. It has been shown that biocontrol is distinctly safe.

1.1.4.3. History of biological control

Use of natural enemies in suppressing pest populations has been recognized for many

centuries (Hagen and Franz, 1973). Efforts to manage pest populations by manipulating

natural enemy populations culminated in the highly successful biological control of the

cotton cushion scale in California (and subsequently other areas of the world) by a

predaceous ladybeetle, the vedalia beetle (Rodolia cardianalis), and a parasitoid (the

cryptochetid fly, Cryptochetun iceryea) imported from Australia (Doutt, 1958;

Caltagirone and Doutt, 1989).

1.1.4.4. Tools of biological control

Biocontrol can be implemented with biopesticides, which encompass plant-derived

pesticides, microbials, entomopathogenic nematodes, secondary metabolites, pheromones

and genes used to express resistance to pests (Copping and Menn, 2000). Broadly, they

can be classified into 3 main classes:


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(a) Microbial pesticides consist of a microorganism (e.g. a bacterium, fungus,

nematode, protozoan and virus) as the active ingredient. Microbial pesticides can

control many different kinds of pests, although each separate active ingredient is

relatively specific for its target pests(s). For example, there are fungi that control

certain weeds, and other fungi that kill specific insects. Bacillus thuringiensis-

based insecticides are often applied as liquid sprays on crop plants, where the

insecticide must be ingested to be effective. It is thought that the solubilized

toxins form pores in the midgut epithelium of susceptible larvae. Recent research

has suggested that the midgut bacteria of susceptible larvae are required for B.

thuringiensis insecticidal activity (Broderick et al., 2006)

(b) Plant-incorporated-protectants (PIPS) are pesticidal substances that plants

produce from genetic material that has been incorporated in the plant through

Genetic Engineering. For example, gene for the Bacillus thuringiensis ( Bt)

pesticidal protein, when introduced into a plant’s genetic material results in the

plant manufacturing the substance (instead of the Bt bacterium), that destroys the

pest. Spores and crystalline insecticidal proteins produced by B. thuringiensis have

been used to control insect pests since the 1920s. The Belgian company ’Plant

Genetic Systems‘ was the first company (in 1985) to develop genetically

engineered (tobacco) plants with insect tolerance by expressing cry genes from B.

thuringiensis.

(c) Biochemical pesticides are naturally occurring substances that control pests by

non-toxic mechanisms. Conventional pesticides, by contrast, are generally synthetic

materials that directly kill or inactivate the pest. Biochemical pesticides include

substances of biological origin, such as insect sex pheromones that interfere with

mating and various scented plant extracts that attract insect pests to traps.

1.1.4.5. Benefits and risks of biological control

Many conservation biologists have what might be called a “green light - yellow light”

attitude towards the use of biological control. On the one hand, biological control gets

a ‘green light’ or ‘go ahead’ since it has the potential to be one of the most selective,


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powerful and cost-efficient tools available for control of invasive insect pests. It is an

attractive option in natural areas particularly because of its potential for specificity and

its ability to act over huge areas for the long term with little or no cost after the initial

research and release(s) of agents. Biocontrol may be the only affordable option capable

of bringing certain widespread insect pests under control over large areas. Natural and

applied biological control tactics are important in successful management of pest

populations. It is well known that natural enemies of insect pests play a key role in

biotic balance, reducing levels of pest population below those causing economic injury.

Most of the synthetic chemicals decimate the beneficial parasitoids and predators. The

value of biocontrol is now well recognized particularly in the context of environmental

protection as well as stable pest management strategy. The role of biopesticides

particularly microbial pathogens in biorational pest management has been well

documented and the efficacy of microbial pathogens can be enhanced further by genetic

improvement (Hokkanen and Lynch, 1995). On the other hand, Biological Control can

be ‘yellow light’. While parasitoid is supposed to manage one pest, there is always the

possibility that, predator/parasitoid will switch to a different target - they might decide

eating crops instead of the insects infesting them is a better plan! Not only that, but in

introducing a new species to an environment, there runs the risk of disrupting the

natural food chain. It’s a slow process. It takes a lot of time and patience for the

biological agents to work their magic on a pest population. For completely wipe out a

pest, biological control is not the right choice. Predators can only survive if there is

something to eat, so destroying their food population would risk their own safety.

Therefore, they can only reduce the number of harmful pests (Wither et al., 2000).

1.2. Trichogramma (Hymenoptera: Trichogrammatidae) as a pest management

tool

The egg parasitoid, Trichogramma (Hymenoptera: Trichogrammatidae), is used

extensively around the world as a biological control agent for the control of

lepidopterous pests. Wasps are either released to augment an existing population

“inoculative release,” or they are released in large numbers to coincide with maximum


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pest pressure “inundative release.” Field releases however, have had variable success.

These parasitoids may be released as adults or the parasitised egg cards may be stapled

to the underside of the leaves of the crop plant, so that the parasitoids may emerge and

attack the host eggs. At desired intervals, the inundative releases of parasitoids are

carried out to control the target insect pests.

The success of Trichogramma field releases has been attributed to wasp quality and

issues relating to the release and integration of wasps into an agricultural setting.

Release and integration issues that are considered important for the development and

maintenance of a successful IPM approach include host/parasitoid synchrony, pesticide

choice and timing of application as well as weather conditions at the time of release in

relation to the development and maintenance of Trichogramma as an effective

biological control agent (Smith, 1996).

1.2.1. Present status of Trichogramma in India

In India, 12 indigenous species of Trichogramma and 2 species of Trichogrammatoidea

have been recorded. Among these T. chilonis Ishii, T. japonicum Ashmead and T.

achaeae Nagaraja and Nagarkatti are widely distributed. A few exotic species have also

been introduced out of which T. brasiliensis Ashmead and T. exigum Pinto and Platner

were proved effective under Indian conditions.

Trichogramma species so far recorded from India include T. acheae, T. agriae, T.

australicum, T. brevifringiata, T. chilonis, T. chilotraeae, T. embryophagum, T. flandersi,

T. hesperidis, T. japonicum, T. minutum, T. pallidiventris, T. plasseyensis, T. poliae, T.

pretiosum, T. raoi and T. semblidis. These were recorded from different States including

Andhra Pradesh, Bihar, Gujarat, Himachal Pradesh, Jammu and Kashmir, Karnataka,

Orissa, Punjab, West Bengal, Uttar Pradesh and Uttaranchal parasitizing upon variety of

insect pests of forestry, agriculture, and vegetable crops (Ahmad et al., 2002).

1.2.2. Description to Indian Trichogrammatidae

After the microscopical observation, it was measured that the size of Trichogrammatids

varies in length from 0.4 to 0.70 mm, and in width across head from 0.15 to 0.25 mm.


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This group can be identified by tarsi, which is three segmented without a strigil on the

fore tarsus, fore wing broad, pubescens in rows or lines- marginal and stigmal veins

forming a sigmoid curve. The genus Trichogramma was erected by Westwood with T.

evanescens designated as the type species. The best known genera of the

Trichogammatidae are Trichogramma Westwood and Trichogrammatoidea Girault.

Both Trichogramma and Trichogrammatoidea are true representatives if the

Trichogrammatidae in that they are exclusively egg parasitoids. The two genera can be

differentiated by the longer wing fringes in Trichogrammatoidea, the presence of a two

segmented funicle in the antennae of both male and female and a three segmented

antennal club in males, the long stigmal veins in the absence of vein tracks RS1 or

(Radial sector vein-1st abscissa), in the forewing (Nagarkatti and Nagaraja, 1977).

It has been suggested that genus Trichogramma westwood contains 130 species of

which 20 species have been reported from India. Morphological and / or biological

characters can differentiate these species. On the basis of the male genitalia, Nagarkatti

and Nagaraja (1977) grouped them broadly as follows.

1. The australicum group: This group includes T. australicum and T. poliae Nagaraja.

2. The minutum group: This group includes T. chilotraeae Nagaraja and Nagarkatti, T.

semblidis (Aurivillius), T. pretiosum Riley and T. chilonis Ishii.

3. The flandersi group: This group includes T. flandersi Nagaraja and Nagarkatti and

T. hesperidis Nagaraja.

4. The japonicum group: This group includes T. japonicum Ashmead and T.

pallidiventris Nagaraja.

5. The agriae group: This group includes T. agriae Nagaraja and T. plasseyensis

Nagaraja.

6. The achaeae group: This group includes T. achaeae Nagaraja and Nagarkatti and T.

raoi Nagaraja.

Females of Trichogramma sps. show few interspecific differences and relative length of

hind tibia and ovipositor frequently overlap. Positive identification using these

characters is not possible.


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1.2.3. Phylogenetic studies on Trichogramma

Trichogramma was described by Westwood (1833) with T. evanescens as type species.

There were several additions in the late 19th and early 20th centuries, and by 1940

approximately 50 nominal species had been added, the majority from Europe and North

America. These were based largely on morphological differences such as colour and

setation, traits eventually found to be correlated with body size, seasonality and host

(Flanders, 1931; Oldroyd and Ribbands, 1936; Salt, 1937; Quednau, 1960). Because of

morphological homogeneity, it was proposed relatively early that certain problems in

Trichogramma systematics could be resolved only with biological and reproductive

characters.

A species catalogue for Trichogramma was published by Zerova and Fursov (1989).

Features separating the genus were summarized by Pinto (1992). Briefly,

Trichogramma is distinguished by the characteristics ‘sigmoid’ venation and RS1setal

track on the forewing, and the dorsal lamina associated with the male genitalia by

Nagarkatti and Nagaraja (1968, 1971).

1.2.4. Distribution and hosts

Egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) can be

found worldwide, in a diversity of crops and hosts, preferentially in the order

Lepidoptera, including a great number of agriculturally important pests (Hassan, 1994;

1997), such as Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae),

which is considered the main pest of corn in Brazil. The presence of Trichogramma

atopovirilia (Oatman and Platner, 1983) parasitizing eggs of that pest in corn crops

demonstrated the potential of use of this parasitoid for the control of S. frugiperda. The

presence of scales over Spodoptera exigua egg masses constitutes a barrier against

parasitism by Trichogramma spp. (Greenberg et al., 1998). However, a study conducted

by those authors in a cotton field artificially infested with S. exigua egg masses,

followed by release of T. pretiosum and Trichogramma minutum (Riley, 1879), resulted

in parasitism of 36.8% and 32.5%, respectively, demonstrating the potential of use of


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these parasitoids in controlling the pest. The difficulty in finding S. frugiperda egg

masses parasitized by Trichogramma species can be overcome by using a more

aggressive parasitoid, capable of breaking the physical barrier imposed by the presence

of layers and scales on the eggs of this pest, as, for example, T. atopovirilia (Rueda and

Victoria, 1993). According to these authors, from a total of 1,305 S. frugiperda

collected eggs, 42.1% were parasitized by this species, and among them four masses

had a parasitism rate higher than 80%.

1.2.5. Impact of egg parasitoid on non-target organisms and environment

Biological control is generally viewed as an environmentally sound method of pest

management. However, concerns have been expressed about potential risks for non-target

species including endangered butterflies (Boettner et al., 2000; Howarth, 1991; Samways,

1997). Lockwood (1996) correctly noted that the continued utilization of biological control

technology requires the assessment of potential detrimental effects on the environment.

There is both an urgent need to carry out pre- and post-release studies in order to assess the

interaction between non-target species and biological control agents and to evaluate the

environmental risks (Sands, 1997). The host range of a natural enemy is a central issue in

any biological control program and it is generally believed that especially polyphagous

agents have the potential for non-target effects (Howarth, 1991). However, this is

questioned by Sands (1997), who stated that the use of highly effective specialists may

also have detrimental effects. The vast majority of Trichogramma species are known to be

fairly polyphagous attacking a wide range of lepidopterans and even species of other insect

orders (e.g., Pinto and Stouthamer, 1994; Thomson and Stinner, 1989).

1.2.6. Biology and Life cycle of Trichogramma

Trichogramma wasps primarily parasitize eggs of moths and butterflies (Lepidoptera).

However, certain species of Trichogramma also parasitize eggs of beetles (Coeleoptera),

flies (Diptera), true bugs (Heteroptera), other wasps (Hymenoptera), and lacewings and

their relatives (Neuroptera).


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The adult female wasp uses chemical and visual cues to locate a host. The chemical

cues, called kairomones, are on the moth scales left near the egg by the female moth

during oviposition (Nordlund et al., 1981). Some of these same chemicals are also host

sex pheromones. Egg shape and colour also may be visual clues to the wasp (Ruberson

and Kring, 1993). Once a female finds a host egg, she drills a hole through the chorion

(egg shell) inserts two to three eggs into the host egg. The internal pressure of the host

egg forces a small drop of yolk out of the oviposition hole. Females feed on this yolk,

which increases their longevity.

Development of Trichogramma Wasp

Day1, Trichogramma Days 1-3 after Days 4-8 after Days 8-9 newly

wasp parasitizes egg parasitism parasitism. adult wasps emerge.

Figure 1.1. Life Cycle of Trichogramma

The yolk and embryo of the parasitized bollworm egg are digested before the

Trichogramma egg hatches. Venom injected by the female at the time of oviposition is

believed to cause this predigestion of the egg’s contents. Eggs hatch in about 24 hours

and the parasite larvae develop very quickly.

The larvae of parasitoid develop through three instars. During the 3rd instar (3 to 4 days

after the host egg was parasitized) dark melanin granules are deposited on the inner

surface of the egg chorion, causing the bollworm egg to turn black. Larvae then

transform to the inactive pupal stage. After 4-5 days, the adult wasps emerge from the

pupae and escape the bollworm egg by chewing a circular hole in the egg shell

(Figure 1.1). The black layer inside the chorion and the exit hole are evidence of

parasitism by Trichogramma. The life cycle from egg to adult requires about 9 days, but

varies from 8 days when mid-summer temperatures are high (90 0 F) to as many as 17

days at 60 0 F. Adults are most active at 75 to 85 0F.


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An average of two Trichogramma pretiosum adults will emerge from a single bollworm

egg. A single bollworm egg can yield wasps of the same or opposite sex. Trichogramma

adults emerge first and remain at the host egg to mate with emerging females if they are

present. Mated females produce male and female offspring. Unmated females produce

only males. Females begin egg laying within a few hours of emergence.

Trichogramma overwinter as immature forms in host eggs. Some species enter a state

of diapuase which allows them to tolerate long periods of subfreezing temperatures.

Other species, such as T. pretiosum, slow their rate of development and may be active

as adults during warm days.

1.2.7. Application technology

No matter how well suited an egg parasitoid is to a targeted pest, the application will

fail if the agent is not delivered in a manner that enables access to and oviposition on

the host. Nonetheless, the technical aspects of biopesticide application in the field are

often neglected. Effective and efficient delivery of egg parasitoid can only be achieved

with careful consideration of available application technology coupled with an

understanding of the attributes and limitations of the biocontrol agent (Shapiro Ilan et

al., 2006).

(i) Application methods: Trichogramma species are released worldwide as pupae in

parasitized host eggs against a number of lepidopterous pests. The different

techniques used can be separated broadly according to the distribution of the

parasitized material; either from point sources or in a broadcast application. Point

sources include parasitoids released from aircraft and from the ground in cards or

different types of containers. Broadcast applications have been conducted with

backpack sprayers or blowers from the ground as well as aerial applications of

parasitoid material in water, attached to carriers such as bran or simply as

unattached parasitized host eggs (Smith, 1994).

Methods of Field Release: Trichocards, Trichocapsules, Plastic release, packing in

bored corks and Aerial application (NCIPM, 2005)


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(ii) Application time: The timing of release of parasitoids should be considered for

the successful biological control strategy. The timing of Trichogramma releases

has varied according to factors such as host eggs availability and acceptability,

synchronization with the host, weather, use of other control measures, and host

population dynamics. Early morning and early evening or night application of

parasitoids is recommended to avoid solar radiation (Gaugler and Boush 1978)

and high temperatures.

1.3. Effect of host quality on parasitization ability of Trichogramma species

Taylor and Stern (1971) investigated the host preferences of the egg parasitoid,

Trichogramma semifumatum (Perkins) maintained on eggs of Trichoplusia ni (Hübner)

to seven species of lepidoptera, viz., Trichoplusia ni, Sitotroga cerealella (Olivier),

Colias eurytheme Boisduval, Estigmene acrea (Drury), Helicoverpa zea (Boddie),

Heliothis virescens (F.) and Baucculatrix thurberiella Busck. Size and relative

penetrability of chorion along with the age of eggs appeared to be the most important

factors, deterring the preference shown by the parasitoid for C. eurytheme eggs over T.

ni and other eggs. They also reported that oviposition by the parasitoid, in smaller, older

or otherwise less suitable eggs always resulted in a higher female: male ratio.

Biever (1972) reported that females of Trichogramma minutum Riley reared in eggs of

Trichoplusia ni traveled about 40% and 30% further at 30ºc and 35ºc, respectively, then

did those reared on S. cerealella. However, females of Trichogramma evanescens

Westwood, T. semifumatum and a species of Trichogramma known as Mexican black

did not differ significantly in their rate of search when reared on the eggs of S.

cerealella.

Laboratory and field studies to determine the degree of successful parasitization by

Trichogramma on the eggs of H. zea and Trichoplusia ni were conducted by Ashley et

al. (1974). Laboratory studies showed significant increase in female longevity, number

of eggs successfully parasitized and progeny production when H. zea eggs were used as

host. Field cage experiments indicated that Trichogramma successfully parasitized a

higher proportion of H. zea eggs even when T. ni eggs were more abundant.


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A comparative study was carried out to study the fecundity, fertility and adult life-span

of a Laboratory population of Trichogramma confusum Viggiani that had been reared

for over 200 generations eggs of Corcyra cephalonica and a wild population reared

from eggs of Agrius convolvuli (L.) by Nagarkatti and Nagaraja (1978). They found that

the laboratory reared females showed a significantly higher degree of sterility than wild

type females. Under laboratory conditions, laboratory reared females lived longer than

wild-type females, but reduced fewer progeny suggesting that their effective life was

shorter. They also suggested the use of suitable hosts with larger eggs in any mass

rearing programme, as the size of the host may influence the fecundity and behaviour of

laboratory reared Trichogramma.

Paul et al. (1981) studied the influence of different hosts, viz., Cadra cautella walker, S.

cerealella, C. cephalonica and H. armigera on parasitism by Trichogramma chilonis

Ishii and Trichogramma exiguum Pinto, Platner and Oatman. They found the C.

cephalonica was preferred by both the species of parasitoids for parasitization over

others. The percentage of female emerged was more when these parasitoids were reared

on H. armigera and C. cephalonica. The parasitoid progenies reared from these hosts

were also observed to be robust having higher longevity and fecundity. Biological

efficiency of the parasitoids reared from S. cerealella was observed to be poor as

compared to the other hosts. Zaslavskii and Kvi (1982) reported that no progressive

decline in fecundity was detected in a long sequence of generations of T. evanescens,

Trichogramma euproctidis Girault and T. chilonis Ishii reared on eggs of S. cerealella.

Transfer of a colony of T. evanescens to its preferred host eggs, Mamestra brassicae (L.)

caused immediate sharp increase in fecundity but did not result in an increase in

fecundity of following generations after the colony was transferred back to S. cerealella.

Stein and Parra (1987) indicated that the mean number of Trichogramma sp. emerging

from eggs of E. kuehniella (=A. kuehniella) was 1.16 while it was 1.0 for eggs of

P. interpunctella and S. cerealella.They also reported that the period of development

was longer in E. kuehniella whose eggs were larger. The longevity of the parasitoid was

not affected by the hosts, but it was longer for females which parasitized hosts than for

those which had no opportunity of doing so.


22

Somchoudhury and Dutt (1988) studied the effect of host on the biology of T. perkinsi

and Trichogramma australicum (=T. chilonis) Girault by utilizing eggs of seven crop

pests, viz., H. armigera, C. partellus, Achaea janata (L.), Exelastes atomosa

Walsingham, Euproctis fraterna Moore, Spodoptera litura (Fab.) and Spilarctia obliqua

(= Diacrisia) (Walker). Maximum longevity was observed in both the parasitoids when

they were supplied with eggs of H. armigera as host, whereas minimum longevity of

parental females was observed on S. oblique in both the parasitoid species.

A new rearing method was developed for Trichogramma chilonis and T. ostriniae, egg

parasitoids of the diamondback moth. Eggs of Ephestia kuehnialla and E. cautella were

treated by the ultraviolet light and these were found to be suitable for mass culture of

the two parasitoids (Hirashima, Y. et al., 1990).

Influence of nine species of lepidopteran host on the quality of Trichogramma semblidis

(Auriv.) was investigated by Sengonca et al., (1990). They observed that the mean body

length was more for parasitoids emerging from Aglais urticae (L.). The lowest mean

adult female life span of eight days was observed when reared on Anthophila fabricana,

Notocelia uddmanniana (L.) (Epiblema uddmanniana) and Argyroplace lacunana

schiiff. (Olethreutes lacunana), while those reared on A. urticae lived for 12.5 days.

They also reported that the females obtained from Haritala ruralis sc. (Pleuroptya

ruralis), Hypaena probascidalis (L.) and M. brassicae parasitized more than twice as

many eggs of Eupoecilia ambiguella, Hübner as those hatching from A. fabricana or E.

uddmanniana. A strong linear relationship existed between some of the quality

parameters and parasitization ability.

1.4. Effect of host age on Trichogramma

The age of the host insect as an important bearing on the parasitization, development

and multiplication of parasitoids. The relationship of host as to acceptance of host for

oviposition and to host suitability, defined as the probability of yielding viable,

reproductive progeny (Salt, 1938; Vinson and Iwantsch, 1980) for different,

Trichogramma sp. have been reviewed.


23

Parker and Pinnell (1973) studied effect of host egg age on Trichoplusia ni and

P. rapae. They reported that parsitisatism of T. ni by both the parasitoids was high in

young eggs which declined as the host egg age increased and then parasitism rise again.

Parasitism on P. rapae by both the parasitoids declined steadily with host egg age.

Nearly all the eggs of Papilio xuthus ( L.) that were less than 12 hr old, but hardly any

of those over 18 hr. old were parasitized by T. papilionis Nagarkatti, T. dendrolimi

Matsmura, and T. australicum (Hiehata et al., 1976).

Paul (1979) studied influence the host eggs age on parasitism by T. australicum, T.

chilonis and T. japonicum Ashmead on the eggs of C. cephalonica up to 78 hr. after

oviposition. No significant relationship was observed between the age of the host eggs

and the % parasitism by T. australicum, but in T. japonicum a definite relationship was

observed. The percentage parasitism by T. japonicum in eggs upto 18 hrs. old was high

but there after a decline trend was observed with the increase in age of the host egg.

Houseweart et al. (1982) studied the acceptability of C. funiforrana eggs of different

ages for parasitization by T. minutum. They reported that two different temperatures

210C and 27 0C with respect to acceptability of parasitization where significantly

superior to 1-3 days old eggs with respect to 2-6 days old eggs. Major deduction in host

eggs acceptability occurred after fifth day at 210C and after fourth day at 27 0C.

Sivapragasm and Ahmad (1986) noticed that attractiveness of T. australicum (T.

chilonis) decreased with increasing age of C. cephalonica eggs. Highest and lowest rate

of parasitism occured in 4 hrs and 72 hrs old eggs, respectively. However, C.

cephalonica eggs of all ages proved suitable for the development of the parasitoids with

no significant differences in the developmental time or sex ratio of the progeny.

Host age selection by T. nubilale for different aged egg masses of O. nubilasis was

examined by Hintz and Andow (1990). Attraction parasitization and successful

emergence of the parasitoids were greater for younger eggs than older eggs, although

emergence was better after one-day-old eggs than from fresh eggs. They found that

oviposition on younger eggs masses occurred for longer periods of time than on older

egg masses.


24

Number of attempts has been made to study the effect of host egg and size on

Trichogrammatids. Ram and Sharma (1977) used different sized sieves to obtain size of

eggs of Corcyra. But they did not obtain any significant difference in the fecundity of T.

fasicatum. Paul (1979) also did not observe any difference between age and %

paristization of eggs upto 18 hrs. The percentage parasitization of host eggs upto 18 hrs

old was high but a decline was observed thereafter with increase in age of host eggs.

Higher parasitism by different Trichogramma sp. was recorded on Earias vitella than

on Heliothis armigera or Pectinophora gossypiella (Hanumana, 1984). 0 and 1 day old

eggs of Plutella xylostella were suitable for T. bactrae parasitization with 89.6 % and

92.8% parasitism (Anonymous, 1993). DBM (Plutella xylostella) is an important pest

of cruciferous crops and particularly cabbage and cauliflower. For controlling such

noxious insect, Yadav et al. (2001) observed that T. chilonis Ishii was also a major

parasitoids for suppressing DBM population.

1.5. Effect of temperature on host eggs as well as parasitoids

Pak et al. (1985) studied the behavioral variations among strains of Trichgramma

species and their adaptability to field temperature conditions. In order to select a

candidate strain of Trichogramma sp. for inundative releases against lepidopterous pests

in cabbage in the Netherlands, the parasitization activity of a collection of 60 different

Trichogramma sp. strains was studied during 2 and 24 hrs exposures at 12 0C. The

activity and parasitism varied significantly among strains and the two characteristics

were not correlated, suggesting the action of two differentially temperature dependent

mechanisms influencing both characteristics. Native strains were characterized by a low

activity at 12 0C, making their usefulness for inundative releases doubtful. In 3 strains

tested at 12 0C, 17 0C, 20 0C, 25 0C and 30 0C, the activity increased linearly with

temperature. Parasitism increased with increase in temperature to a maximum at 20-25 0C

and decline at 30 0C. Handling time decreased asymptotically with increasing

temperature. The effect of temperature on the development of two egg parasitoids,

T. chilonis and T. ostriniae, which attack the diamondback moth, were observed in the

laboratory under the controlled conditions of constant temperatures (20 0C, 24 0C, and


25

28 0C). The longevity of the female adult of T. chilonis was significantly longer at 20 0C

as compared with 24 0C and 28 0C. The fecundity of each parasitoid was significantly

also higher at 20 0C (Hirashima, Y. et al., 1990).

1.6. Effect of humidity on development of parasitoid, Trichogramma spp.

Maintenance of proper humidity conditions appear important factor for the developmental

process of Trichogramma in Mamestra brassicae, Pieris brassicae eggs (Park, 1988).

At high humidity (80% RH), parasitized eggs of Pieris brassicae were more sensitive to

desiccation than unparasitized eggs. This suggests that, beside manipulative physical

damage, embryogenesis of the parasitoids also may interfere with the permeability of

the egg shell in this species. The embryonic effect is probably different from the effect

of physical damage, since the difference in desiccation between parasitized and

unparasitized eggs did not occur at lower humidities. Increased mortality of immature

Trichogramma with decreasing humidity has been reported by Calvin, et al., (1984) and

Lund (1934).

1.7. Effect of sex ratio on parasitism by T. chilonis

The effect of sex ratio on parasitism and reproduction by Trichogramma chilonis in the

eggs of Sitotroga cerealella was studied with male: female ratios of 10:1 to 1:10. The

parasitism rate decreased significantly as the number of males per female was increased.

The percentage survival of parasitoids in host eggs was high with ratios of 4:1 and 1:4.

The greatest number of female progeny (77.4%) was produced with a ratio of 1:5.

1.8. Effect of ovipositional experience on host discrimination of Trichogrmma

Females of Trichogramma chilonis that had never oviposited in any host after

emergence supposedly do not distinguish between parasitized and unparasitized host

eggs. However, experienced females distinctly discriminated parasitized host eggs from

unparasitized eggs. Acceptability of unparasitized host eggs was 90% or greater

irrespective of the host type that females had experienced. On the other hand, acceptance


26

of parasitized host egg decreased to 15% with females that had one prior experience with

a parasitized host egg. Females that experienced oviposition on an unparasitized host egg

began to accept parasitized host eggs more frequently 24 h after the experience.

Inexperienced females inserted their ovipositors into hosts parasitized 3 days earlier, but

they did not oviposit in hosts parasitized 5, 7 or 9 days earlier. The oviposition behaviour

of T. chilonis can be explained by the assumption that the wasp behaves to maximize her

own inclusive fitness through her lifetime (Miura et al., 1994).

1.9. Superparasitism

Superparasitism occurs in Trichogramma, especially when less number of eggs were

offered for parasitization. Chack (1953) has studied superparasitsm in T.chilonis.

Superparasitism is known to occur frequently in both the laboratory and the field (e.g.,

Van Alphen and Nell, 1982; Hubbard et al., 1987). In mass culture of parasitoids the

problem of superparasitism may lead to vary expensive rearing procedures in order to

prevent high parasitoid mortalities, development of small and weak adults, as well as

strongly male-biased sex ratios (Waage, 1986; Van Lenteren, 1986). Narayanan and

Chacko (1957) also worked with same species and reported that though, one to three

adult parasitoids may emerge from the egg of Corcyra cephalonica, and they are often

defective, had ill developed wings and were inactive. They mated with forms of about

their own size, if they show any tendency to mate at all. The reason they attributed for

the improper development was sharing of the limited amount of food in the host eggs

between the developing parasitoids. Chacko (1969a,b) studied phenomenon and causes

of superparasitsm in T. chilonis. He reported that when superparasitism occurred, there

was a reduction in fecundity, longevity and size of the female was less than that of

females that emerge from egg. It appeared that the male had a quicker rate of absorption

of nourishment in the larval stages and this coupled with its lesser food requirements

enables it to complete the development, leaving very little food for the females thus

accounting for the preponderance of males. When defective forms were produced as a

result of superparasitism, there was a further reduction in fecundity and longevity.

However, very little superparasitism occurred if sufficient number of host eggs were


27

available. Trichogramma has a well developed discriminative ability. Ram and Sharma

(1977) have studied that superparasitism increased as exposure period to C. cephalonica

eggs was increased, reaching a maximum of 71.4% with a 24 hr exposure.

1.10. Kairomones effect in Trichogramma

Many scientists have been working on Kairomone mediated interaction in Trichogramma

sp. since 1971 among which, Ananthakrishnan, et al. (1991) identified kairomones of H.

armigera (Hub.) and C. cephalonica Stainton and their influence on the parasitic potential

of T. chilonis. Hexatriacontane, pentacosane, heptadecane, docosane and 2,6,10-

dodecatrienal 3,7,11-trimethyl were identified from a kairomonally active extract on the

scales of H. armigera and C. cephalonica e.g. when scale wash from the abdomen of

Helicoverpa armigera females was applied to the eggs of C. cephalonica, such eggs

when placed in the field attracted more Trichogrammatids females, the percent parasitism

increased by 9 times even in host summer months in cotton ecosystem at Nagpur.

Grenier et al. (1993) reported some factors stimulating oviposition by the oophagous

parasitoid T. brassicae in artificial host eggs. O.nubilalis scale extract significantly

increased the number of eggs laid. A blend of saturated hydrocarbons stimulated

oviposition in a similar way to the extract. If solutions were deposited on part of the egg

plate, 77 to 88% of eggs were laid in the treated capsules. So it was concluded that the

extracts acted as contact kairomones which could be useful to improve the oviposition

of T.brassicae, permitting an increase in adult yield under artificial rearing conditions.

1.11. Trichogramma host and its preferred host plants

As shown in the table 1.1, there is an association with parasitoid host and host plant,

and host plant may play an important role in determining the efficacy of particular

species or strain of Trichogramma. A particular host plant may either attract or repel

Trichogrammatids. In one of the recent studies showed, T. chilonis parasitised more

number of Spodoptera litura eggs on cotton (93%) that of cauliflower (75%)

(Anonymous, 1992). On other host plants very few eggs were parasitized. Most of the

eggs parasitized were in top layer only. Percent parasitism indicated that on plants like


28

cauliflower, castor, cotton, beetroot and cabbage egg masses were parasitized from

66.6-100%.

Table 1.1. Trichogramma host and its preferred host plants

Parasitoid Hosts Host plant

Trichogramma chilonis Ishii Achaea janata (L.)

Agrius convolvuli (L.)

Chilo indicus (Kapur)

C.infuscatellus(Snellen)

C.partellus (Swinhoe)

C.suppressalis (Walker)

Helicoverpa armigera(Hb.)

Emmalocera depresella (Swinhoe)

Psara sp.

Scirpophaga incertulas (Walker)

Spodoptera litura (Fab.)

Tiracola plagiata (Walker)

Trichoplusia ni (Hb.)

Unidentified Anerastiinae

Earias insulana (Biosduval)

E. vittella (Fabricius).

Castor

Jasmine

Sugarcane

Sugarcane

Maize,sorghum

Sugarcane

Cicer arietinum, tomato

Sugarcane

Alternanthera sessilis

Rice

Tobacco

Castor

Castor

Vetiveria zizanio

Cotton

Cotton

On most of the host plants, that parasitoids were able to search eggs and parasitized

them. However, egg wise parasitism was very poor. On cotton, a parasitism of 9.3%

was recorded followed by cauliflower (7.5%), castor (4.1%), beetroot (3.6%), cabbage

(2.4%), tobacco (2.1%), kholkhol (1.1%) and radish (1.0%). T. chilonis is therefore,

able to search its host S. litura on most of the host plants but it may not be suitable for

field release because it is unable to parasitize multilayered eggs. Similarly, T. chilonis

Ishii was observed to parasitize the diamondback moth; 42% on cabbage, 4% on

cauliflower and 77.1% to 94.9% on Indian mustard (Yadav et al., 2001).

1.12. Effect of various diets on Trichogrammatids

Adult parasitoids do not feed on host (most cases) as they generally feed on nectar and

pollen from the flowers in nature. Studies on the effect of nutrition on T. evanescens

minutum (Narayanan and Mookherjee, 1956) showed that adults lived for 1-17 days


29

with no food or when fed only on Corcyra egg extract; 2.3-2.6 days on water only; 4-6

and 9-10 days on different sugar solutions and for 3-4 and 3-8 days on glucose and

protein respectively without mating. However in mated adults reduction in longevity

was observed. Female parasitised more when they were fed on sugar solution than on

glucose and protein or water or Corcyra egg extract. In studies on suitability of different

rearing media for C. cephalonica and their effect on fecundity, longevity and sex-ratio

of T. evanescens minutum, best results were obtained when C.cephalonica was reared

on sorghum +8% yeast (Katiyar, 1962). It was observed that nutrition of both adults of

Trichogramma and larvae of Corcyra affected the efficiency of the parasitoids (Paul,

1980).

The effect of host egg size on quality attributes of T. pretiosum using three hosts, viz.

T.ni, P. interpunctella and S. cerealella was studied by Bai, et al. (1992). They reported

that females from natural host, T. ni. were larger, more fecund and longer lived than

those from factitious host, P. interpunctella and S. cerealella. Compared to small

females, large females were substantially more fecund when honey was available but

marginally more fecund when honey was unavailable.

It was experimented that out of various food grains, sorghum and bajra were found

suitable for more production of Trichogramma. The varieties of sorghum which are

preferred for human consumption produced better stock of C. cephalonica which in turn

increased the efficiency and productivity of Trichogrammatids (Anonymous, 1993).

1.12.1. In vitro culture of Trichogramma spp. on artificial diets

Currently, however, mass rearing of Trichogramma requires the use of mass reared host

material, the eggs of C. cephalonica, Sitotroga cerealella, for example. Thus, mass

rearing of Trichogramma is expensive and automated rearing systems are difficult to

develop. Xie Zhong-Neng et al. (1996) developed artificial diets that contain no insect

components but containing Yeast extract and ultracentrifuged chicken egg yolk, which

supported the development of Trichogramma to the adult stage. These findings may

contribute to the development of automated artificial diet based rearing systems for

these important parasitoids. The development of artificial eggs would have significant

impact on the future approach to releasing Trichogramma.


30

1.13. Genetic progress

1.13.1. Genetic improvement

At PDBC, (Project Directorate of Biological Control (ICAR), Bengaluru) strains of T.

chilonis tolerant to high temperatures and pesticides have been selected and found to

perform better than conventional populations in the field. A strain tolerant to endosulfan

has been transferred to a private company for mass production and is being marketed as

“Endogram”, the first of its kind in the world (Kambrekar et al., 2008).

1.13.2. Hybridisation

The processes of random inbreed and outbreed within the genus of parasitoid resulted

Hybrid populations at different localities. Nagarkatti and Nagaraja, (1968) reported

reciprocal crosses between T. australicum, T. minutum and T. evanescens by crossing

male and females of each species. However no female population in F1 progeny was

obtained thus providing a strong indication that the three species are reproductively

isolated. The results also revealed that T. australicum males readily courted and

attempted copulation. However, such ‘inseminated’ females usually died within 15 to

30 minutes and in no case lived longer than 60 minutes. Nagaraja (1973) conducted

reciprocal crosses between 6 new species of Trichogramma which helped in

discovering sibling species of T. japonicum. Another experiments were conducted by

the hybridisation between species of Trichogrammatoidea viz., T. armigera, T. lutea,

T. prabhakeri, T. robusta and T. bactrae. All these are arrehenotokous species. T. lutea

is of African origin while the rest are indigenous to India. Reciprocal interspecific

crosses using the five species showed the existence of ethological, mechanical, genetic

isolations and hybrid sterility. Limited numbers of sterile/fertile hybrids were obtained

in some of the crosses. T. lutea and T. prabhakeri proved to be sibling species.

Although there was limited compatibility of the hybrid but the percentage of female

progeny obtained from the cross between the progeny [Tr. lutea (F) × (M) Tr.

prabhakeri i.e., T .LUT prab.] with T. prabhakeri was higher. Therefore T. LUT prab.

was considered a true introgressive hybrid (Nagaraja, 1978b).


31

1.14. Commercialization

To rear Trichogramma on a commercial scale, it is necessary to use a factitious rearing

host, such as Ephestia kuehniella or Sitotroga cerealella, Generally, the choice of

factitious host is often dictated by the ease of rearing and not necessarily by any factors

related to the likely success of the wasps being produced. Factitious hosts are selected

on the simplicity of their mass production, mechanisation of rearing processes and cost

of production compared with that of using the target pest (Greenberg, et al., 1998b).

1.15. Problems associated with Trichogramma use and their conservation

1.15.1. Survival and storage technique

The insects need to be reared on an industrial scale and cold storage makes possible the

management of large quantities of living material for intensive periods of use. It is

necessary for rearing facilities to have reliable systems for storing eggs, pupae or adults

(Chang et al., 1996). Trichogramma can be best stored at 10 0C for short-term storage

up to 49 days, whereas after storage at 2 0C and 5 0C for 14 days the fecundity and

longevity of the parasitoid declined drastically (Jalali and Singh, 1992). The host eggs

used for mass production of Trichogramma are usually stored at low temperature to

prevent egg hatching without affecting the nutritive value (Singh, 1969; Calderon and

Navarro, 1971; Voegle et al., 1974). Parasitized eggs after 6-8 days of parasitization can

be safely stored in refrigerator at 10 ± 10C for seven days without affecting the efficacy

of the parasites.

Diapause and quiescence constitute major physiological adaptations for sustaining

survival during environmental extremes and both adaptations can have practical

applications in storage during mass rearing (Chang et al., 1996; Zaslavski and Umarova,

1990). Unfortunately, cold storage may impact on the success or efficiency of the

organisms to be released. These effects include reduced fecundity (Chang et al., 1996;

Frei and Bigler, 1993), poor flight activity (Dutton and Bigler, 1995), reduced longevity

(Jalali and Singh, 1992) and reduced emergence rate (Cerutti and Bigler, 1995; Jalali and

Singh, 1992).


32

1.15.2. Transportation

Parasitoid egg cards can be easily transported in the pupal stage (3 days after

parasitisation) by folding the cards in such a way that the parasitized eggs do not get

damaged and stapled. These cards can be kept in polythene bags which are in turn

packed in cardboard boxes. ‘Trichocards’ can also be packed in thermocol or iceboxes

with a coolant. Alternatively, water frozen in polythene bags with frozen water can also

be kept at the base of the container (NCIPM, 2005).

1.16. Quality issues: genetic and environmental components

Phenotypic variation can have genetic and environmental components. Environmental

variation is often mediated via the size of the host egg, which in turn affects the size of

the Trichogramma (e.g. Bennett and Hoffmann 1998, Glenn and Hoffmann 1997). In

addition, there are genetic factors that can act independent of size. For example, Bigler

et al. (1988) and Dutton et al. (1996) found that walking speed was affected by genetic

factors independent of size. For successful control of insect pests with their natural

enemies, it has been emphasized (Bigler, 1989, 1991; van Lenteren, 1998, 2003a,

2003b) that quality of mass produced bio-control agents should be assessed periodically

for such attributes that would determine their control efficiency.

To ensure a high quality product is delivered to the grower, optimum conditions for

rearing, storage and shipment are imperative. It is important that producers become

aware of the negative effects of poor handling of Trichogramma (Dutton and Bigler

1995). Bigler et al. (1993) tested the quality of commercially available Trichogramma

and concluded that more elaborate product control systems were necessary to increase

reliability of the product. Product quality is recognised as one of the most important

reasons for failure of the biological control agent T. chilonis against H. armigera

(Romeis et al., 1998). O’Neil et al. (1998) evaluated the quality of four commercially

available natural enemies including T. pretiosum. The post shipment quality from ten

companies was assessed for emergence rates, sex ratio, survivorship, species identity,

reproduction and parasitism. Considerable differences in the number received,

survivorship and emergence rates were found. Field studies using commercially


33

available T. brassicae against O. nubilalis in sweet corn differed in emergence profiles

in two different years (Mertz et al., 1995). When several commercially available species

of Trichogramma used against Plutella xylostella were compared, inconsistent

responses were observed within most of the products indicating potential problems with

quality control (Vasquez et al., 1997).

Trichogramma have been used in inundative releases more than any other natural enemy

(Stinner, 1977). Situations where Trichogramma are used to control lepidopterous pests

include grapes (Glenn and Hoffmann, 1997), tomatoes in greenhouses (Shipp and Wang,

1998), tomatoes in the field (Consoli et al., 1998) and sugar cane (Greenberg et al.,

1998a). Trichogramma are also used against Helicoverpa armigera on a variety of crops

in India (Romeis and Shanower, 1996) and on sweet corn in Australia (Scholz et al.,

1998). Trichogramma is an effective biological control agent against the European corn

borer, Ostrinia nubilalis (Lepidoptera: Pyralidae), throughout Europe (e.g. Mertz et al.,

1995) and North America (Andow et al., 1995). They are used in nonfood crops such as

cotton (Naranjo, 1993) and to provide foliage protection in forests (Bai et al., 1995).

Trichogramma are even used against Lepidoptera in stored grain, where Trichogramma

evanescans and T. embryophaga attack Ephestia kuehniella and E. elutella (Scholler et

al., 1996).

Procedures for testing various quality indicators of these agents need to be simple,

economical and short duration, preferably conductible in a laboratory. Dispersal and

foraging activities of the parasitoids are, in turn, determined by their locomotor

characteristics which are of two main types: walking and flying. Walking enables the

parasitoids to move over contiguous substrates e.g., leaves and twigs of a plant, whereas

flying enables them to move across non-contiguous substrates e.g., from one plant to

another, thereby enlarging the area of their dispersal. Both walking and flying

characteristics of Trichogramma are thus as important indicators of its quality (Bigler et

al., 1988; Dutton and Bigler, 1995) as some other of its attributes (Bigler, 1989, 1994)

that contribute to its bio-control efficiency. Procedures followed by various workers

(Forsse et al., 1992; Dutton and Bigler, 1995; Prezotti et al., 2002; van Lenteren et al.,

2003) to test flight activity of these parasitoids differ in details but have more or less a


34

similar basic plan: Tests are conducted in a clear glass or plastic cylinder whose top is

covered with a clear glass or plastic lid coated on its under surface with a non-drying

adhesive. Alternatively, the lid may bear an inverted glass vial (Forsse et al., 1992).

1.17. Role of Nuclear energy in entomological studies: Indian perspectives

The contribution of nuclear technology in solving research problems related to plant

protection is well known. In India nuclear research related to Entomology and associated

fields is being intensively carried out at Bhaba Atomic Research Centre (BARC),

Mumbai; Nuclear Research Laboratory (NRL), IARI, New Delhi; University of Delhi,

Delhi and certain other premier institutes of India. Literature on the use of nuclear

techniques in various disciplines of Entomology has been reviewed earlier (Pradhan and

Sethi, 1971; Chatrath and Sethi, 1973; Sethi and Bhatia, 1979; Seth and Sethi, 1996). In

the present review, the possible uses and perspectives of the utilization of radiations in

entomological research studies and pest control tactics have also been high lightened.

Everything in the universe – soils, plants, animals, the air we breathe and the food we eat

– is made up of elements. For any given element, the number of neutrons in the atom’s

nucleus may vary, giving us different forms or “isotopes” of the same element. Some are

heavier than others, some are stable, some undergo decay and give off energy as

radiation. Scientists have learned to use these nuclear properties of isotopes to measure

and track many processes in the production of food and other agricultural products and

development of radio-genetic pest control methods. Isotopic tracers are particularly

useful, for example, in optimizing fertilizer and irrigation efficiency, as signals for

detecting disease agents and pursuing ecological and toxicological studies on insects.

1.17.1. Types of radiation and their use in insect control

Radiation can be broadly divided into two categories on the basis of its ionizing

capacity (Figure 1.2):

(a) Ionizing radiation

(b) Non-ionizing radiation


35

1.17.1. (a) Ionizing radiation

Ionizing radiation is a type of particle or electromagnetic radiation in which an

individual particle (for example, a photon, electron, or helium nucleus) carries enough

energy to ionize an atom or molecule (i.e. to completely remove from its orbit). An

atom can be ionized by a radiation whose photon has sufficient energy, such as x-rays,

gamma rays. The energy of ionization required to extract an electron depends on the

atom in question: It is different for each species. The effect of ionizing radiation on a

living organism can be very complex. If the extracted electron belongs to a molecule,

ionization involves the destruction of a chemical bond.

Ionizing radiation includes x-rays, and gamma rays. These are used for microbial and

insect sterilization, besides insect lethality. These are highly penetrating radiation which

can produce ionization in biological tissues, causing severe consequences if permitted

doses are exceeded. In the food industry, ionizing radiations are used to kill insects on

fruit and many spices (black pepper, coriander, ginger and marjoram).

(i) Gamma rays: Gamma ray photons are so energetic that they interact at the

electronic and atomic level of matter. They offer the advantage of being selective.

They are absorbed by the insects while penetrating deeply inside food material.

The potential of application for gamma rays is therefore considerable in the food

industry. Irradiation increases the storage time (Thayer, 1985) because it destroys

the harmful organism inside the food. In 1963, the United States Department of

Agriculture (USDA) authorised the use of gamma radiation for irradiation of corn

and corn flour (Van Kooij, 1982). Gamma rays are also used to eliminate insects

from spices such as black pepper, ginger, etc. Gamma rays are emitted by

radioactive elements (Co60and Cs137).

(ii) X-rays: Like gamma rays, X-rays are used for post-harvested processing to

selectively kill insects (or to make them sterile) without interacting with the

surrounding medium. Banks (1956) reported a great variability of the results on

the lethal doses of radiation for various insects. As for gamma rays, production of

X-rays is not very effective and is expensive. Their use remains therefore

relatively limited. X-rays are emitted when electrons collide on a metal plate.


36

All ionizing radiations have similar effects on the irradiated materials (since they have

similar relative biological effectiveness), and in particular on the irradiated insects

(Bakri, Mehta, and Lance 2005b). In a biological organism composed of differentiated

and undifferentiated cells, mitotically active cells such as stem cells and germ cells are

the most radiation-sensitive elements. Thus, radiation can make an insect reproductively

sterile by damaging the DNA of gonial cells. For certain insect life stages, several

studies found no significant difference in the lethal effects between electrons and

gamma rays (Adem, Watters, Uribe-Rendo´n, and de la Piedad 1978; Watters 1979;

Dohino, Tanabe, and Hayashi 1994).

Figure 1.2. Electromagnetic Spectrum

1.17.1. (b) Non-ionizing radiation

Non-ionizing radiations are low frequency electromagnetic radiation which does not

have enough energy to extract an electron or modify the chemical bond. For these non-

ionizing radiations, the electric field of the electromagnetic wave makes the charged

particles vibrate within the matter, without dissociating them from their chemicals bond.

These radiations include microwave, radio-wave, infrared, visible light and ultra violet.

(i) UV rays are non-ionizing radiations that lies between 0.32-0.4µm. Their penetration

depth is very small and the interaction with materials occurs at the surface.

(ii) Visible rays are those which originates when the electrons of excited atoms

undergo transition from one orbital energy level to a lower energy level. This lies

between 0.4-0.8 µm of the electromagnetic region.


37

(iii) Infrared rays occupy the region between 0.8-14 µm of electromagnetic

spectrum. It is usually associated with heat in that it is radiated by hot bodies and

absorption of IR produces heating in the absorbing body. Radiation of IR energy

arises from vibration and rotation phenomena associated with molecules at the

surface of the radiation bodies.

(iv) Microwave radiation involves frequencies starting above 500MHz with application

frequencies of 869, 915 and 2450 MHz. This causes molecules of the medium to

oscillate and vibrate and part of the radiation is thus transformed into heat.

(v) Radio and audio wave involves frequencies between 1 to 100 MHz. This causes

dielectric heating of biological materials, specially wood, stored grains and

foodstuff and this dielectric heating characteristic has been studied as a potential

for insect control.

1.17.2. Applications of ionizing radiation in entomology

There are a number of applications of ionizing radiation in entomology (Bakri, Heather,

Hendrichs, and Ferris 2005a), including disinfestation of commodities for quarantine

and phytosanitary purposes, and reproductive sterilisation of insects for pest

management programmes using the Sterile Insect Technique (SIT) (Dyck, Hendrichs,

and Robinson 2005). Radiation can also be applied in various ways to facilitate the use

of biological agents for control of arthropod pests and weeds (Carpenter 1997, 2000;

Greany and Carpenter 2000). These authors cite a number of potential advantages of

nuclear techniques for biological control: (i) avoidance of the emergence of pest insects

from non-parasitised hosts, allowing earlier transport and facilitating trans-boundary

shipment, (ii) improvements in rearing media (either artificial diets or natural

hosts/prey), (iii) provision of sterilised natural prey to be used as food during predator

shipment, to ameliorate concerns relating to the incidental presence of hitch-hiking

pests, (iv) provision of supplemental food or hosts in the field, to increase the initial

survival and build-up of released natural enemies, and (iv) reproductive sterilisation of

weed-feeding insects that are candidates for biological control allowing their risk-free

field assessment of host specificity.


38

Exposure to ionizing radiation is now the principal method for inducing reproductive

sterility in mass-reared insects. Irradiation of insects is a relatively straightforward

process with reliable quality control procedures (FAO/IAEA/USDA 2003). The key

parameter is the absorbed dose of radiation; efficacy of the irradiation process is

guaranteed as long as the dose is correctly delivered (Bakri et al., 2005b). Other

advantages of using radiation (gamma rays, X-rays and electrons) include (1)

Insignificant increase in temperature during the process, (2) Treated insects can be used

immediately after processing, (3) Irradiation does not add residues that could be

harmful to human health or the environment, and (4) Radiation can pass through

packaging material, thus allowing the insects to be irradiated after packaging.

1.17.2.1. Radiation dose

The absorbed dose, D, is radiation energy absorbed in unit mass of a material, and is

mathematically expressed as the quotient of ‘do’ by ‘dm’, where ‘do’ is the mean

energy imparted to matter of mass ‘dm’; thus, D=do/dm (ICRU 1998). The unit is J/kg.

The special name for the unit is gray (Gy); thus, 1 Gy=1 J/kg. The unit of absorbed dose

used earlier was rad (1 Gy =100 rad). Quite often, ‘absorbed dose’ is simply referred to

as ‘dose’ (Mehta, 2008).

1.17.2.2. Radiation doses for arthropod sterilization

Arthropods are more radioresistant than human and other higher vertebrates but

resistant than viruses, protozoa and bacteria (Ravera, 1967; Rice and Baptist, 1974;

Whicker and Schultz, 1982; Blaylock et al., 1996; Harrison and Anderson, 1996). One

of the main reasons for the higher radio-resistance is that arthropods have a

discontinuous growth during immature stages, and cells become active only during the

moulting process. This is encoded in Dyar’s Rule, i.e, insects double their weight at

each moult and thus their cells need to divide only once per moulting cycle (Hutchinson

et al., 1997; Behera et al., 1999). The high resistance of most adult insects to radiation

is attributed to the fact that they are composed of differentiated cells, which do not

undergo replacement (Sullivan and Grosch, 1953). Such cells are much more resistant

to death or damage induced by irradiation than are dividing or undifferentiated cells.

Group insects dose 30 Gy-1500 Gy (Whicker and Schultz, 1982).


39

1.17.3. Non-ionizing radiations: Their application in entomology

Photons from low frequency electromagnetic radiations (visible light, IR, microwaves

and radio wave), do not have enough energy to extract an electron or modify the

chemical bond. IR waves, microwaves, and radio wave produce a great quantity of heat

whereas visible light is used to dry many foods like fish or grapes. Microwaves are

specially used in the food industry to thaw frozen products for cooking.

1.17.3.1. Uses of UV-radiation for biological control of insect pest management

(i) Sterilization of artificial diet: UV radiation also used in sterilization of artificial

eggs to remove unwanted micro-organisms in the rearing of biological agents.

Sterilization of artificial diets by nuclear techniques may provide the following

advantages. (i) It may eliminate the need for other preservatives, (ii) It may

simplify procedures in diet preparing (e.g. autoclaving) (iii) It allows for ‘terminal

stage’ sterilization (e.g. after packaging)

(ii) Reproductive sterilization of host/or prey: UV radiations can also be used to

reproductively sterilize hosts, factitious host or prey thereby inhibiting further

development and preventing the emergence of unused individual. This application

would: (i) It allows for the earlier shipping of hosts together with natural enemies

without the need to wait for emergence of unused hosts; (ii) It reduces the

handling procedures required during rearing of natural enemies, thereby

increasing the cost effectiveness of the rearing process and the quality of the

natural enemy product; (iii) It facilitates the preservation of purity of host, prey

and/or natural enemy strains; (iv) It provides a cleaner product for customers

purchasing/using natural enemies produce in this fashion.

1.17.4. Effect of radiation for sterilization of host eggs

The host eggs used for mass production of Trichogramma are usually exposed to UV or

gamma radiation or X-rays radiation to prevent egg hatching without affecting the

nutritive value (Singh, 1969; Calderon and Navarro, 1971; Voegle et al., 1974)

Manjunath (1988) recommended an exposure period of 45 min. with a 15 V. UV-tube

at a distance of 12.5 to 15cm. for complete sterilization of loose eggs of C. cephalonica


40

and S.cerealella to UV-rays (15V lamp) for six minutes at a distance of 15cm. was

sufficient to cause complete sterilization without affecting the quality of the eggs for

parasitization by Trichogramma.

Elbadry (1965) was the first to suggest that moth eggs exposed to 3 to 9 krad of gamma

radiation could serve as a food resource for Trichogramma development. Marston and

Ertle (1969) also tested the acceptability of irradiated (23.3 krad) moth eggs to

Trichogramma minutum Riley and reported that irradiated eggs were as suitable as

control eggs for parasite development. Breniere (1965a,b) is the only worker who made

such an attempt. He stated that exposing C.cephalonica eggs for 15 minutes to UV-

radiation killed the embryos and that such eggs could be used for the multiplication of

T.australicum Girault. Eggs of Ephestia kuehniella (Zeller) killed with UV-radiation

were also found suitable for mass rearing of Trichogramma sp. (Vregele et al., 1974).

The hatching ratio of the Ephestia moth eggs was 0 per cent after exposure of the UV

light (NEC, GL-15 lamp) for 8 minutes (Hirashima, Y. et al., 1990).

1.17.5. Radiation sources

Only certain radiation sources can be used in irradiation of insect or artificial diet.

These are:

1. Accelerated electron machines having a maximum energy of 10 MeV;

2. Gamma rays using the radio nuclides cobalt-60 (used commonly) or cesium-137

(used very rarely);

3. X-ray machines having a maximum energy of 5 million electron volts (MeV).

1.17.6. Labeling of insects with radioactive compounds

The radioactive isotopes act as very versatile tool and are being increasingly used all

over the world in the field of insect ecology (Sethi et al., 1981). Radioactive isotopes,

when used as marker for insect pests, parasitoids and predators, have helped in

obtaining valuable information on their flight range, dispersal pattern, population build-up

etc. This information is very vital for rationalizing control options for integrated pest

management.


41

Prasad et al. (1970) reported that the solution of radioactive phosphorus in dil.

Hydrochloric acid was added to sorghum grains that were thoroughly mixed and dried

under IR lamp. In this 5th instar larvae of Corcyra cephalonica were reared for the

radioactivity and were offered to T. chilonis (=minutum) adults for parasitization. The

1st radioactive generation of T. minutum was then offered on radioactive C. cephalonica

eggs for parasitization. The parasitised eggs thus obtained were found to possess

detectable radioactivity. Labelling of Corcyra eggs (obtained by feeding predecessor

larvae on 32P- labeled diet) with no external contamination and with no significant

differences in fecundity, adult emergence, female emergence, adult longevity and

developmental period of the parasitoids, as compared to the control, is the suitable

method for labeling T. chilonis for various entomological investigations with radiotracer

technique (Bhattacharyya et al., 2004). Sealed sources containing radiation-emitting

isotopes are used to change the genetic make-up of plants, insects and micro-organisms

in ways that lead to better crops, more environmentally friendly pest control and safer

foods with longer shelf life. Therefore, nuclear techniques can be an efficient, effective

and inexpensive option for certain agricultural problems.

1.17.7. Sterile insect technique (SIT)

Muller (1950) in his report stated that ionizing radiations could cause male sterility by

inducing dominant lethal mutations in the sperm. Thus, encouraged by this, the tests

were begun with X-rays (Bushland and Hopkins, 1951) and gamma radiation (Bushland

and Hopkins, 1953). Knipling (1955) for the first time gave the idea of using sterile

insects to control and eradicate insect population. The origin of the idea and the

development of the technique were intimately related to his research on the screwworm

fly, C. hominivorax (Coquerel). Knipling observed that the female screwworm flies

mate only once, he speculated that in case of females mating once, if the males could be

sterilized without impairing their mating activity, then it might be feasible to use these

sterilized males to eradicate the isolated population of screwworms.

From its beginning almost 50 years ago the sterile insect technique (SIT) has slowly

evolved during the past quarter into an effective and widely acceptable method for

modern insect control and eradication (Krafsur et al., 1987). This technique has been a


42

potentially useful method for the eradication or containment of certain major insect

pests including the screwworm fly, Mediterranean fruit fly, melon fly, Mexican fruit fly

and pink bollworm. The technique has also shown to achieve suppression of several

other major pests including the boll weevil, codling moth, tsetse flies, certain

mosquitoes, horn fly and stable fly (Knipling, 1982). The area-wide concept, implicit in

the SIT appeared to be the rationale solution to many insect related problems

(FAO/IAEA, 1999). The power of the technique lies in the simplicity of the biological

principles on which it is founded (Knipling, 1955) and the lack of any negative

environmental effect following its application.

This technique relies on the use of radiation to induce dominant lethal mutations and

chromosome rearrangements in the sperm of adult male insects (Robinson and Franz

2000). Following release of the males and their subsequent mating with the wild

females these sperm are transferred to wild females. If the required proportion of wild

females is mated with sterile males at subsequent generations, then the population will

collapse and eventually disappear. The proportion of wild females required to be mated

by sterile males to initiate a population collapse will depend on the reproductive

potential of the target species and the degree of density dependence which is operating.

However an over flowing ratio with sterile insects will always be needed, and this will

require the release of large numbers of mass reared sterile insects. It is essential that the

ratio of wild to released insects is monitored during the programme, and it is calculated

by trapping insects in the field and identifying the released insects as they carry a

fluorescent dye that is added to the pupae before emergence and release. The accurate

and simple identification of released insects is critical to the evaluation of a SIT

programme.

Steady progress is being made by the scientists in various countries on several aspects

of SIT or alternative genetic manipulations that can eventually lead to more extensive

and more practical use of autocidal approach to the management or elimination of

major pests. Some of the steps in this direction include,

1. the discovery of various genetic mechanisms that when introduced into natural pest

population will adversely affect the normal development of the pest population;


43

2. improvements in the mass production of insects both from the standpoint of cost

and quality of the reared pests;

3. a better understanding of the ecology, dynamics and behavio

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