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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.
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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.
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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.
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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
Chapter 1 Review of Literature
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
Chapter 1 Review of Literature
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
Chapter 1 Review of Literature
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
Chapter 1 Review of Literature
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
Chapter 1 Review of Literature
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.
Chapter 1 Review of Literature
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).
Chapter 1 Review of Literature
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).
Chapter 1 Review of Literature
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
Chapter 1 Review of Literature
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
Chapter 1 Review of Literature
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
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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.
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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.
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(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.
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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).
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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
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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.
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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
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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;
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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