Chapter 4 ASSESSMENT, MASS PRODUCTION AND FIELD RELEASES

A. Assessment of Effectiveness of a Natural Enemy

Assessment of effectiveness in augmentative biological control programs is probably as

important as releasing the natural enemies. The tools in assessing effectiveness of natural enemies
include:

1. Comprehensive life table studies which show the extent of indispensible mortality attributable to a
specific natural enemy, and can be used to measure the expected impact on the target pest.

2. An understanding of expected yield gains attributable to natural enemies is also a useful measure that
may be used in deciding whether to employ augmentative biological control. This approach will also
allow the development of a meaningful measure of effectiveness, viz. to what extent a natural enemy
reduces dependence on chemical or other pest management options.

B. Nutrition and Mass Production of Biological Control Agents

1. Nutrition of Natural Enemies

The nutrition of entomophagous arthropods was originally discussed in detain bu Doutt

(1964) and Hagen (1964). Slansky (1982, 1986) and Thompson & Hagen (1999) illustrates the complex
interactions of behavioral, physiological and nutritional factors in arthropod nutrition. Nutrition is thus
the action or processes of transforming substances found in foods into body materials and energy to do
all the things attributed to life. Nutritional requirements are dependent on the synthetic abilities of the
organism, which is controlled genetically. House (1977) stated that “… through nutrition we have a
direct and essential connection between an environmental facto, foodstuff and the vital processes of
the insect organism. “Most nutrition research with insects has been aimed at improving rearing and not
developing a basic understanding of their nutrition. Research has emphasized feeding and the
development of artificial diets, which are concerned with dietetics (Beck 1972). Although critical to
insect rearing, such research has given only a little understanding of insect nutrition per se.

Qualitative nutritional requirements of all insects are very similar in spite of a great
diversity of feeding habits (Beck 1972, Dadd 1973, Hagen 1986b). Although knowledge of dietetics and
nutrition das advanced, practical application of principles to insect rearing to biological control is lacking.
Rearing each insect species is a unique challenge as there is meager knowledge of nutrition principals
that might provide a broad and sound basis for approaching insect species husbandry (house 1777).
With entomophaga, foodstuff is in a continuous state of qualitative and quantitative change, and very
little is known of the quantitative nutritional requirements for various life stages and physiological
functions of these insects. The requirements for many nutrients are often dependent on the presence
and concentration of others and correct nutrient balance may be critical for successful nutrition. Those
parasitoids and predators for which artificial diets have been may developed may serve as models for in
vitro investigation on quantitative requirements for specific nutrients.

Thompson (1976, 1082) used a defined artificial medium to examine the quantitative
requirements for supporting larval growth of Exerister roborator. Parasitoid development is intimately
associated with host physiology. Changes in the host’s physiology following parasitism are adaptive for
the parasitoid, which insures successful development (Vinson & Iwansch 1980, Thompson 1986).
Changes in composition of the host’s internal milieu may have significant nutritional consequences for a
parasitoid 9Grenier 1986, Thompson 1989). Endocrine interactions seem critical to successful parasitoid
development. Synchrony in development between many larval indoparasitoids and their host occurs
(Beckage 1985), and this suggests that the host’s hormones and endocrine physiology influence
parasitoid development (Lawrence 1986a). The physiological basis of developmental synchrony is not
well understood and knowledge is restricted to investigation of the relationship of Biosteres
longicaudatus with its host Anastrepha suspensa (Lawrence 1982, 1986b). Some studies have tested the
effects of hormones on the development of parasitoids in vitro with little success. The potential of using
insect hormone supplements in artificial media to achieve successful growth and development of
parasitoids in vitro deserves research emphasis.
 The importance of ecological considerations in the nutrition of insects was discussed by
Slansky (1982). It was emphasized that behavior and regulatory physiology of insects are in a state of
continuous flux in response to food supply, and that nutrition can be fully understood only by
considering the insects “nutritional ecology.” With entomophaga both the ecology of the entomophage
as well as that of the host or prey needs to be known.

Dietary and nutritional requirements are genetically based and genetic manipulation holds
promise as a way to modify the nutrition of entomophages. Chabora (1970) suggested that nutritional
content varies between strains on insects when he demonstrated that yields of two parasitoids,
Nasoniau vitripennis (Walker) and M. raptor Girault & Sanders were significantly increased when they
were reared on a hybrid of two strains of the host, Musca domestica L. The selection of desired traits for
insect rearing was discussed by Collins (1984). The potential for genetic improvement of entomophages
was outlined by Rousch (1979) and Hoy (1979, 1986). Most genetic selection has been directed to
increase field effectiveness of entomophages, such as improving sex ratio, host finding ability, host
preference, pesticides bresistance and improved climatic tolerance. However, genetic improvement
must also guarantee the preservation of vigor and vitality of the entomophage. Because these are
intimately associated with nutrition, genetic programs may involve selection for nutritionally related
traits.

Advances in recombinant DNA technology indicate a possibility for genetic manipulation

of the nutrition of entomophages (Thompson 1989). The incorporation of foreign or in vitro altered
genes for the expression of desirable traits by an organism, is rapidly advancing (Beckendorf & Hoy
1985), but is still not suited for practical application as of 1991.

History of Parasitoid Nutrition – Salt (1941) probably was the first to emphasize the
complexity of parasitoid nutrition in studies that demonstrated that the host influences growth and
survival of the developing parasitoid as well as sex ratio, fecundity, longevity and vigor of the adult wasp
(Clausen 1939, Salt (1941). Such complexities were demonstrated in work by Arthur & Wylie (1959),
Wylie (1967), Nozato (1969) Sandlan (1979a) and others (Vinson & Iwantsch 1980). It has long been
known that there is a relationship between host biomass and size of solitary parasotoids, larger
parasitoids developing from larger hosts. This relationship exists for parasitoids which attack every host
developmental stage, but applies more generally to parasitoids of host eggs and pupae where host size
is fixed (Sandlan 1982).The relationship applies when a parasitoid is reared on different host species of
variable size as well as when reared on different sized individuals of a single host species (Salt 1940,
Jowyk & Smilowitz 1978, Mellini & Campadelli 1981, Sandlan 1982, Mellini & Beccari 1984). It does not
seem to hold with ectophagous parasitoids, however (Legner 1969). The size of adult Trichogramma
pretiosum Riley reared on the eggs of five hosts showed a direct correlation between parasitoid size and
the volume of the host egg from which it emerged (Bai et al. 1989). A correlation also exists between
total parasitoid biomass and/ or numbers with host size in the case of gregarious larval parasitoids
(Wylie 1965, Bouletreau 1971, Thurston & Fox 1972). The means by which gregarious organisms
moderate their development relative to host size has been shown (Beckage & Riddiford 1983).

The relationship between size oh host and parasitoid is closely associated with food
quality and quantity (Arthur & Wylie 1959, Sandlan 1982). Salt (1940) found that adult Trichogramma
avanescens Westwood display behavioral dimorphism related to host size. Large females obtained from
large hosts failed to oviposit on small hosts, whereas small females accepted hosts of all sizes. Male
wing development was influence by host size, and this was also found in Geliscorruptor (Foerster) by
Salt (1952). Adult female Coccygomimus (= Pimpla) turionellae L. did not show morphological and
behavioral polymorphism, but larger females found it difficult to oviposit in small host. On the other
hand small females were more efficient in attacking small hosts. Fecundity was influenced by longevity
with the greatest longevity reported for longer individuals reared from large hosts (Sandlan 1982).

The success of parasitoids in parasitizing activity is directly related to nutritional factors.

Smith (1957) found differences in larval mortality and adult size, sex ratio and reproductive rate of
several species when reared on Aonidiellaaurantii (Maskell) and Comperiella bifasciata Howard
maintained on different food plants. Habrolepis rouxi Compere displayed limited mortality on A. aurantii
when feeding on Acyrthosiphon pisum (Harris) and Rhopalosiphum maidis (Fitch) than on Aphis fabae
Scopoli. Coccinellaseptempunctata L. gained more weight when feeding on Lipaphis erysimi
(Kaltenbach) than on two other aphid species, and it was demonstrated that L. erysimi had higher
protein levels (Atwal & Sethi 1963).

Utilization of Food

Parasitoids have been thought to show high efficiencies in food utilization. Larvae
consume food of high nutritional content and are mostly inactive within the host which offers a limited
food supply, which points to selection for high food efficiency (Fisher 1971, 1981; Slansky & Scriber
1985, Wiegert & Petersen 1983). Parasitoids examined for food utilization include Coccygomimum (=
Pimpla) instigator (F.), Pteromalus puparum (L.) (Chlodny 1968), Gelis macrurus (Thompson), Hidryta
frater (Cresson) (= sordidus) (Edgar 1971), Brachymeria intermedia (Nees) and C. turionellae (Greenblatt
et. al. 1982) Diadromusu pulchellus Wesmael (Rojas-Rousse & Kalmes 1978) and Trypatgilum
(=Trypoxylon) politum (Say) (Cross et al 1978), Phanerotoma flavitestacea Fischer (Hawlitzky & Mainguet
1976), Ventura (=Nemeritis) canescens (Gravenhorst) (Fisher 1968), Cidaphus alarius glomerata (L.)
(Slansky 1978). In these species, the mean net conversion efficiency, (= proportion of assimilated food
converted to body mass (Petrusewics 1967, Calow 1977, Hagen et al. 1984) varied broadly (11-62%),
with a mean of 37% that was ≤ than for many groups of insect herbivores and detritivores. Cameron &
Redfern (1974) of two studied parasiotids, Eurytoma tibialis Boheman and Habrocytus elevates (Walker),
were at the high end of this range. Net conversion efficiencies may not be very high because selection
might have been for rapid rather than efficient growth (Slansky 1986). Possibly the well known inverse
relationship between growth efficiency and assimilation (Welch 1968) may also be important. In
contrast to net conversion efficiency, the above parasitoids had relatively high percentages of
assimilation (= percentage of ingested food that is assimilated) ranging from 55-94%, with mean of 67%,
compared with means of 40-50% for most herbivores and detritivores.

Howell & Fisher (1977) reported the highest nutritional efficiencies for a parasitoid in
the ichneumonid V. canescens. Larvae had 65% net conversion efficiently and 95% assimilation when
maintained on the host Anagasta (=Ephestia) (Zeller); net conversion efficiency to the adult was 20%.

The proportion of food/host available that is consumed by the parasitoid and converted
to parasitoid biomass was calculated by Slansky (1986) and Howell & Fisher (1977). Calculated
exploitation indices varied among species from 3-80%, and V. canescens larvae consumed 90% of its
host’s biomass and converted 55%, but there was no clear correlation between host size and parasitoid
size nor biomass conversion.

Food utilization by predators has also been thought to be highly efficient, for reasons
similar to that for parasitoids. This is especially true when predators spend much time waiting for their
food (Lawton 1971), thus avoiding metabolic expenditure. Studies on food utilization of 11 predators
was reviewed by Slansky & Scriber (1985). All had similar net conversion efficiencies (4-64%, mean =
34%), but higher assimilation efficiencies (37-98%, mean = 86%) than those of parasitoids. Cohen (1984,
1989) in studies of food utilization by Geocoris punctipes (Say) when reared from 1st instar nymphs to
adults on eggs of Heliothis virescens (F.), found an assimilation efficiency of ca. 95%, gross conversion
efficiency of 53% and net conversion efficiency of 55%.

Nutritional Requirements in Development

Qualitative nutritional requirements of insects, determined by use of defined and

deficient artificial diets, were presented by several authors (Dadd 1973, 1977, 1985; Friend & Dadd
1982, Hagen et. al. 1984). All insects have similar requirements for ca. 30 chemicals that include protein
and/ or 10 essential amino acids 9arginine, histidine, isoleucine, lysine, methionine, phenylalanine,
threonine, tryptophan and valine), the B-vitamin complex (biotin, folic acid, nicotinic acid, panthothenic
acid, pyridoxine, riboflavin and thiamin), as well as other water soluble growth factors, including choline
and inositol, some fat soluble vitamins , cholesterol or a structurally similar phytosterol, a
polyunsaturated fatty acid, minerals and an energy source usually provided by simple or complex
carbohydrates and/ or lipids.

Nutritional requirements of entomophagous insects are similar, and similar to those of

nonentomophagous species. House (1977) referred to this common characteristics of insect nutrition as
the “rule of sameness” (House 1966a, 1974). The rule has been confirmed by recent studies with
parasitic and predaceous insects. In assessing the need for nutrients, it is important to consider that
rearing a single generation on a synthetic or semi-synthetic diet did most studies. Some investigations
overlooked the potential contribution of nutrients stored within the egg. Stored nutrients may support
limited development and, in the case of trace nutrients, supply a sufficient quantity to ensure
development of at least one generation. Studies with Itoplectis conquisitor (Say) (Yazgan 1972) and
Exeristes roborator (F.) (Thompson 1981a) demonstrated partial larval development on diets lacking
various essential amino acids and B-complex vitamins. Other studies have demonstrated that
entomophagous insects have no unusual qualitative nutritional requirements. A requirement for
asparagine by Eucelatoria bryani Sabrosky (Nettles 1986a) and the absence of a requirement for a
polyunsaturated fatty acid by A. housei (House & Barlow 1960) were consistent with finding for
nonparasitic Diptera (Dadd 1977).

The quantitative balance of different nutrients is a critical and dominating factor

determining dietary acceptability and suitability (House 1969, 1974). The predominant foods of both
parasitic and predaceous insects are of animal origin and, thus, are generally high in protein content and
low in carbohydrate and fat (House 1977). Thompson (1986a) found a high requirement for protein and/
or amino acids in parasitoids. Exeristes roborator at the 6% amino acid level completed larval
development without glucose and/or fatty acids (Thompson 1976a). However, glucose markedly
improved survival when the amino acid level was reduced to 3% and at 1% amino acid, no development
occurred with the carbohydrate. Similar effects of amino cid level on larval development were reported
by Yazgan (1972) for I. conquisitor. Adult eclosion was reduced by dietary amino acid levels of < 6% and
by deletion of glucose. Fatty acids were only marginally beneficial in enhancing growth and development
rates of both species. A polyunsaturated fatty acid, however, was required in small amounts. Adult I.
conquisitor (Yazgan 1972) and E. roborator (Thompson 1981a) displayed crumpled wings and/or bent
ovipositors without a polyunsaturated fatty acid in the larval diet. Linolenic acid alleviated these
deformities in I. conquisitor, and linoleic and linolenic acids were provided together in the case of E.
roborator.

Thompson (1983a) described the effect of nutritional balance on larval growth of Brachymeria lasus
(Walker). Media containing 0-10% glucose with 2% amino acids, and 1-8% amino acids with or without
2% glucose were tested. All media contained 15% albumin and 2.5% lipids. Weight gain increased on
diets containing 2% glucose when the amino acid level was increased from 1-4%, but was reduced at the
higher amino acid levels. Similar effects of varying the amino acid level were obtained with diets lacking
glucose, but the overall weight gain was less than observed with the diets containing glucose. On diets
containing 2% amino acids, weight gain increased dramatically when glucose was increased from 0.5-4%,
but decreased at higher glucose levels. Growth rates on the above diets were generally in the range of
15-200 mg/g/day. The maximal rate, 260 mg/g/day, was obtained on a medium containing 2% glucose
and 2% amino acids. The effects of nutrient balance were closely related to the osmolality of the
artificial medium (Thompson 1983b).

House (1966b) demonstrated similar quantitative requirements to those of

hymenopterous parasitoid in the dipteran Agria housie Shewell. Maximal growth and survival were
achieved when all nutrients were increased proportionately over the levels in a basal diet that contained
2.25% amino acids, 0.05% salts, 1.16% lipids and 2.25% other ingredients, including glucose, ribonucleic
acid, vitamins and agar. When amino acid level alone was increased, survival was reduced. On a diet
containing nutrient levels equivalent to pork liver (= 20% amino acids, 4% glucose, 3.5% lipids, 2% salts
and 0.75% ribonucleic acid), survival was >80%. House (1967, 1970) showed that the relative balance of
amino acids and glucose was critical in determining growth or development and that A. housie larvae
selected diets for feeding on the basis of nutrient balance.

Quantitative nutritional studies with parasitoids have generally evaluated the effects of
nutritional balance by univariate or monofactorial analysis. Grenier et al. (1986) thought that such an
approach had servere limitations because it ignored potential interactions between nutrients, including
“…additivity, competitivity, antagonism or synergy.” Thus, interpretation of effects of nutrient variation
aimed at medium optimization was difficult, and it was suggested that nutritional studies be designed
and analyzed in a multidimensional manner that accounted for interactions between all nutrients and
biological criteria.

Canonical correlation analysis, which constructs maximum correlation between all linear
combinations of variables within sets, such as between growth and development, and dietary
parameters, were reported with Lixophagadiatraeae (Townsend) by Bonnot (1986, 1988). Because
biologically meaningless correlations may be generated, accurate interpretation requires knowledge of
biological correspondence between variables. Bonnot varied the concentrations of 30 medium
components and determined the effects on 9 developmental criteria. Nine linear correlations were
obtained and three had correlation coefficients of >0.95.
 There is little information about the effects of developmental nutrition on the behavior
of parasitoid larvae apart from measurements related to growth and development rate. However,
Veerman et al (1985) reported that a photoperiodic response by C. glomerata was influenced by the
carotenoid content of its host’s diet. Vitamin A was essential for photoperiodic induction of diapause
and it was suggested that this vitamin or a derivative may function as a photoreceptor pigment.

Optimal nutritional balance can be influenced by environmental factors, as was shown

by House (1966b) with A. housie that the effects of dietary glucose level on larval survival and
development could be modulated by temperature. The nutritive value of a basal medium 9House 1966a)
was increased by increasing the temperature from 20 to 25 and 30ᵒ C at glucose levels between 0-1.5%.
At higher glucose levels larval survival and development were reduced with increasing temperature.
Two media of different composition were formulated whose superiority for larval growth and
development of A. housie was reversed at two different temperatures (15 & 30ᵒ C (House (1972). Such
nutritional effects might have ecological significance in affecting insect host range (House 1966b). It was
thought that in establishing host range, an insect might be affected differently if the nutrient
composition of its food were uniform but the temperature varied within the range rather than if the
temperature were uniform but the composition of food was variable. On the other hand, the insect
might not be affected if variation in food composition was accompanied by compensatory changes in
temperature. Therefore, an insect species that attacks a particular foodstuff in a region with a specific
temperature might, if introduced into another area with a different temperature, adapt to a different
food source whose nutrient composition is favored at the new temperature.

Non-nutritional factors are intimately and intrinsically involved in food acceptance and
ingestion. These include physical properties such as form, texture, etc., but also non-nutritive chemicals
that elicit specific behavioral and/or physiological responses essential for finding and accepting foodstuff
and in some cases for initiating behaviors associated with the feeding process itself (Bernays & Simpson
1982), Bernays 1985). Although such factors have been best shown on phytophagous insects, they also
play a role in the biology of entomophaga and will likely be of importance in the development of
continuous in vitro culture.

Predator Culture In Vitro

The artificial rearing of predators has stressed maintenance of the adult stage for
maximizing egg production rather than complete in vitro culture. Predator larvae are the preferred
biological control agent, and eggs and larvae produced by adults are placed directly in the field.
However, some effort has been aimed at complete artificial culture of predators. Among the first reared
artificially from egg to adult was the coccinellid Coleomegilla maculata maculata (DeGeer) by
Szumkowski (1952). Adults fed on raw liver or meat being kept for months on these food in the absence
of prey. However, survival of larvae was poor on meat products alone and only 38% reached the adult
stage. Supplementing vitamins resulted in ca. 86% of the larvae reaching adults. Oviposition and egg
viability were increased by addition of vitamin E to the adult diet. The culture methods were refined and
a diet of fresh yeast and glucose supported larval development (Szumkowski 1961a, b). Smith (1965,
1966) reared several coccinellid species including C. maculata lengi on dried aphids supplemented with
pollen. Success also was achieved on a diet of 40% brewer’s yeast, 55% sucrose, inorganic salts,
cholesterol, RNA, wheat germ oil and vitamins. Adults were fed the same diet supplemented with
powdered liver. Attallah & Newsom (1966) reared 8 generations of this coccinellid on a defined diet of
casein, sucrose, wheat germ, soybean hydrolysate, glycogen, butter fat, corn oil, a liver factor dextrose,
cotton leaf extract (with carotenoids and steroids), brewer’s yeast, ascorbate, inorganic salts, vitamins
and agar. Adults reared in vitro were fecund and mating was stimulated by addition of vitamin E to the
diet. The medium failed to support growth of Coccinella novemnotata Herbst, Cycloneda spp.,
Hippodamia convergens Guérin and Olla v-nigrum (=abdominalis) (Mulsant). The last species was
cultured in vitro by Bashir (1973). Optimum egg production required inclusion of vitamin E in the larval
diet, which was in contrast to the results of Szumkowski (1952) where supplementation of the adult diet
alone was insufficient for maximum egg production.

Several coccinellid species were reported to be successfully cultured in vitro by Smirnoff

(1958). These included Psyllobora (=Thea) virgintiduopunctata (L.), C. septempunctata, Oenopia
(=Harmonia) doublieri (Mulsant), O. (=Harmonia) conglobata (L.), Rhizobius lophantae (Blaisedell), R.
litura (F.), Rodolia cardinalis (Mulsant), Exochomusanchorifer Allard, E. quadripustulatus (L.) E.
nigromaculatus Erhorn, Scymnus suturalis Thunberg, S. pallidivestis Mulsant, S. kiesenwetteri Mulsant,
Stethorus punctillum Weise, Chilocorus bipustulatus (L.), Clitostethus arcuatus Rossi, Pharoscymnus
numidicus Pie, P. ovoideus Sicard and Mycetaea tafilaletica Smirnoff (Endomychidae). The diet
contained sucrose, honey, alfalfa flour, yeast, royal jelly and agar supplemented with dried pulverized
prey. Larval rearing in a few species was improved by adding beef jelly. All species developed more
rapidly and lived longer on the artificial diet compared with insects reared under natural conditions, and
the adults were very healthy. Harmonia axyridis (Pallas), C. septempunctata and Chilocorus kuwanae
Silvestri were reared on Smirnoff’s (1958) diet and other artificial media by Tanaka & Maeta (1965).
Successful culture of all species was obtained but adults failed to lay eggs. Chumakova (1962) reared
Crytolaemus montrouzieri Mulsant on similar crude diets supplemented with dried prey.

Okada et al (1971a, 1972) and Matsuka et al. (1972) successfully reared H. axyridis on
diets containing powdered larvae and pupae of drone honeybees (Aphis mellifera L.). Sixteen
generations of H. axyridis and three generations of Menochilus sexmaculatus (F.) were cultured in vitro.
Okada & Matsuka (1973) and Matsuka et al. (1982) later improved the rearing method for maintaining
adult Rodolia cardinalis. Chilocorus rubidus Hope, Scymnus hilaris Motschulsky, S. otohime Kamiya,
Vibidia duodecimguttata Poda and S. hilaris Motschulsky, C. septempunctata, Coccinula crotchi (Lewis),
Eocaria muiri, H. axyridis, Harmonia yedoensis Takizawa, H. convergens, Hippodamia tredecimpunctata
L., Lemnia beplagiata (Swartz), M. sexmaculatus, Propylea japonica, S. hilaris and S. otohime, Variable
results were obtained, but 11, 16 and 25 successive generations of E. muiri, H. axyridis and M.
sexmaculatus respectively were cultured from the egg to adult stage. Larval development, adult
longevity and fecundity were satisfactory.

The fractionation of honeybee powder was described by Matsuka & Okada (1975) who
found that the active factor stimulating predator growth was unstable but nonproteinaceous. Expanded
attempts to analyze bee powder was described by Niijima et al. (1977). Niijimi et al. (1986) then
formulated several chemically defined diets for rearing H. axyridis. Larvae developed from the 1-3rd
instar on a diet containing 18 amino acids, sucrose, cholesterol, 10 vitamins and 6 minerals.

Kariluoto et al. (1976) described rearing of A. bipunctata. About 60 variations of seven

artificial diets were tested. These contained varying amounts of wheat germ, brewer’s yeast, casein,
cotton-leaf extract, egg yolk, sucrose, liver fractions, honey, glycogen, soybean hydrolysate, butter fat,
corn oil, amino acids, dextrose, ascorbate, choline, inorganic salts, vitamin E and antibiotics. The best
diets yielded 60-80 % of larvae that became adults, but development time was slowed and adult weight
lowered. Kariluoto (1978) modified the medium, and Kariluoto (1980) obtained fecund adult of A.
bipunctata, C. septempunctata and others reared in vitro.

In vitro culture attempts with Chrysopa species did not succeed until Hagen & Tassan
(1965) got a complete culture of Chrysoperla carnea (Stephens) on an encapsulated liquid medium (in
paraffin droplets). The diet consisted of enzymatic yeast, protein hydrolysate, ascorbate, fructose,
choline and casein hydrolysate. Adults were fecund but development time from the egg stage was ca. 2X
that of insects reared on aphids. Vanderzant (1969) then successful cultured C. carnea for 7 generations
on pieces of cellulose sponge soaked in enzymatic casein and soy hydrolsates, fructose, inorganic salt,
lecithin, cholesterol, choline, ascorbate, vitamins and inositol. Development on this diet was slow, but
50-60% of larvae reached the adult stage compared with 85% when reared on natural insect eggs.
Hassan & Hagen (1978) reported obtaining three generations of C. carnea on an artificial diet of honey,
yeast flakes, sucrose, casein, yeast enzymatic hydrolysates and egg yolk. Developmental time and pupal
weights were similar to those of insects on eggs of Sitotroga cerealella (Olivier). Chrysoperla sinica
(Tjeder) was cultured for 10 generations on a diet of egg, brewer’s yeast, sucrose, honey and ascorbate
(Ye et al. 1979). Adults were fed powdered liver, honey and brewer’s yeast. Cai et al (1983) reared this
species on an encapsulated medium of soybean and beef hydrolysates, egg yolk, sucrose, honey,
brewer’s yeast, ascorbate and linoleic acid, with similar success reported by Zhou & Zhang (1983).
 The hemipteran predator, Geocoris punctipes may be reared on several diets (Dunbar &
Bacon 1972). Media were nevertheless supplemented with insects. Cohen (1981) reported in vitro
culture of G. punctipes from 1st stage nymph to adult on encapsulated semidefined diets. Six media
containing casein hydrolysates, yeast, sucrose, cholesterol, corn oil, lecithin, agar, inorganic salts,
phenylalanine and a vitamin mixture were formulated and encapsulated in different forms. The latter
included mixtures of polybutene 32, dental impression wax, Vaseline, epoline C-16, candelilla wax,
Sunoco, and Paraplast. Best results were with vitamin-enriched medium encapsulated in a mixture of 5%
polybutene 32 and 95% dental impression wax. Development of G. punctipes in vitro was better than
when reared on Spodoptera exigua (Hubner). The percent of nymphs that reached adults and survival of
the in vitro reared predators were significantly greater on the artificial diet. Cohen (1983) then described
modifications of media content, preparation and encapsulation and could rear two generations of G.
punctipes, Geocoris pallens Stal, H. convergens, H. axyridis and Nabis spp. also successfully fed on the
encapsulated medium. In all cases superior results were obtained on medium encapsulated with 30%
polybutene 32 and 70% dental wax. A diet composed of equal parts of fresh ground beef and beef liver
supplemented with sucrose for continuous rearing of G. punctipes was produced (Cohen 1985). The
ingredients were blended into a paste and small aliquots wrapped in stretched Parafilm presented to
developing nymphs for feeding. Twelve generations were successfully cultured, and artificially reared
predators displayed greater fecundity and adult weight than individuals reared on insect eggs and
coddled larvae (Cohen & Urias 1986). Nevertheless, development was slower on the artificial diet.

Parasitoid Cultures In Vitro

In vitro culture offers a simple alternative for mass culture (Mellini 1978, Greany et al. 1984) and also
enable dietary and nutritional mainioulations for fundamental studies of nutrition and biochemistry.
Some benefits of in vitro culture were given by Greany et al. (1984). However, the physiological and
metabolic adaptations exhibited by insect parasitoids in relation to their parasitic way of life are of
critical importance for successful in vitro culture (Mellini 1975a, Thompson 1981a, Grenier et al. 1986,
Campadelli & Dindo 1987). Parasitoid/host relationships are often incorrectly thought to lack the
complex physiological interactions typical of the host associations of other Metazoa (Thompson 1985,
1986a, Dindo 1987). The immature stages of many parasitoids are truly parasitic and such
parasitoid/host relationships are characterized by extensive physiological and biochemical interaction
(Beckage 1985, Thompson 1985, 1986a; Lawrence 1986a). Such interactions are often intimately
associated with nutrition and successful development of the parasitoid in the host (Beckage & Riddiford
1983, Thompson 1983a, 1986a). The potential importance of the host endocrine system and of
hormonal interaction in in vitro culture was discussed by Mellini (1975b, 1978, 1983) and Grenier et al.
hymenopterous larval endoparasitoids. The extent that parasitoid/host physiological interactions need
to be considered in the successful development of in vitro culture must still be determined but will
undoubtedly vary with the parasitoid species.

Diptera. - - A variety of natural foodstuffs, including fish and liver products, were utilized
in early rearing attempts with parasitoids. House & Traer (1948) reared the sarcophagid A. housei for
many generations on a diet of salmon and liver. Contrasted to 38% pupation among larvae reared on the
host, Choritoneura fumiferana (Clemens), 88% pupated when reared on the artificial medium. A related
species, Sarcophaga aldrici Parker was reared on the same medium and on liver alone (Arthur & Coppel
1953) and subsequently Coppel et al. (1958) maintained A. housei in the laboratory on fresh pork liver.
About 1,000 A. housei larvae were reared on ½ lb. of sliced liver and were not affected by putrefaction
of the tissue. Smith (1958) maintained Kellymyia kellyi (Aldrich) for 40 generations on pork liver and was
also able to rare larvae on a mixture of powdered milk, powdered egg and brewer’s yeast moistened
with water to form a thick paste.

House (1954) developed the first chemically defined medium for rearing a parasitoid,
using A. housei. The diet contained 19 amino acids, ribonucleic acid, dextrose, inorganic salts (U.S.P. XII),
B vitamins, choline and inositol. It was prepared aseptically and gelled with agar. About 84% of the
larvae reached the 3rd instar, 60% of those pupated and 32% of the pupae emerged as adults. The
medium was later refined and many of the developmental nutritional requirements of A. housei were
determined (House 1977). Vitamin E was necessary for reproduction (House 1966c).

Other dipterous parasitoids have been more difficult to culture outside the host. Many of these species
have specialized physiological adaptations associated with parasitism that are lacking in sarcophagids.
Tachinids, for example, have relatively high respiratory rates (Ziser & Nettles 1979, Bonnot et al. 1984)
and during or immediately following the first stadium form a direct connection to the host’s tracheal
system (Kellen 1944, Fisher 1971). First instar larvae of the parasitoid E. bryani attach to the host’s
tracheal system 12 hrs after hatching, and respiratory considerations were critical for the development
of in vitro cultures (Nettles et al. 1980). During initial studies, first instar larvae dissected from the host
were placed directly in a liquid artificial diet. They were then transferred to diets gelled with agar,
thereby exposing larvae directly to atmospheric oxygen. Improvements in the methods allowed
development without transfer. Powdered artificial diet containing 1.5% agar was preconditioned by
maintaining it at 5% RH for 24 hrs. The diet was then poured into petri dishes and held at 90% RH. Young
larvae dissected from the host 18-24 hrs after larviposition fed on the liquid diet covering the surface of
the gelled medium, and this was consistent with the normal feeding habit of first instar larvae that feed
on and develop in the host’s hemolymph. As the liquid was slowly absorbed by the agar gel, the surface
of the gelled medium dried and larvae were exposed to the atmosphere. The artificial medium for
rearing E. bryani was composed of mixtures of organic acids, amino acids, nucleic acid bases, B and fat
soluble vitamins, phospholipids and derivatives as well as ATP, lactalbumin hyydrolysate, bactopeptone,
yeastolate, albumin, cholesterol, triolein, glucose and trehalose. When thus reared, larvae developed at
an equivalent rate as when reared on the host, H. virescens, and 13% developed into adults with a sex
ratio of ca. 66% females. Adults were fecund but produced fewer progeny than host reared insects. The
medium was later refined and simplified and some of the basic developmental nutritional requirements
of E. bryani were determined (Nettles 1986a). The nutritive values adding albumin or soy flower to the
medium was tested, which greatly increased adult yields and fecundity (Nettles 1986b).

Other tachinid parasitoids have been successfully reared on artificial media. Larval
development of Phryxecaudata Rondani to the 3rd instar was obtained with a liquid artificial diet
(Grenier et al. 1974). However, in contrast to the results of Nettles et al. (1980) with E. bryani,
development of P. caudata

was not improved by rearing larvae on gelled diets (Grenier et al. 1975). It was suggested that this may
have resulted from the slower development rate and respiratory requirements of the latter when reared
in vitro (Nettles et al. 1980). Bonnot (1986) discussed the importance of respiratory requirements in the
in vitro culture of P. caudate. The first tachinid that was successfully cultured in vitro on artificial media
from the first instar larvae to the adult was Lixophaga diatraeae (Townsend) (Grenier et al. 1978). This
medium contained organic acids, amino acids, B and fat soluble vitamins, gelatin, enzymatic
hydrolysates of casein, soy protein, lactalbumin, ovalbumin, ATP, cholesterol, lecithin and gelled with
agarose. Adults were fecund and their progeny developed normally on Galleria mellonella. One critical
factor for successful development of both P. caudate and L. diatraeae was osmolality, which could not
exceed 450 mOs/Kg (Grenier et al. 1986).

Grenier (1979) investigated the embryonic development of P. caudate and L. diatraeae

on artificial media. Newly fertilized eggs were removed from adult females and placed on an agarose-
gelled medium similar to that for the larvae. Larval yield was equal to that observed in vivo and was
mush greater than when reared on a liquid diet. Again, respiratory requirements seemed critical for
success.

Hymenoptera. - - Simmonds (1944) made the first attempt to rear hymenopterous

parasitoids in vitro. There species of ichneumonid ectoparasitoids were maintained as larvae for
extended periods on raw beef and gelatin. Although some growth was observed, none could complete
their development. Bronskill & House (1957) did succeed in rearing C. turionellae on a slurry of pork liver
in 0.8% saline. An autoclaved homogenate of the liver was dispensed into sterile test tubes and surface
sterilized eggs were dissected from host pupae and transferred to this medium. Mature larvae were
placed in gelatin capsules for pupation and 7% of the eggs developed to adults. When reared naturally
on G. mellonella, 50% parasitoid adults were obtained. Culture of ichneumonid I. conquisitor. The diet
was a mixture of amino acids, fatty acids, fat soluble vitamins B vitamins and lipogenic growth factors,
and glucose, NA and gelled with agar. It was ground into a viscous slurry. Parasitoid eggs dissected from
the host were placed directly on this medium, and development from egg to fecund adult was obtained
with a development time twice that observed on the natural host, G. mellonella. Exeristes roborator was
reared on a diet with a similar nutrient composition (Thompson 1975), but unlike I. conquisitor, larvae of
this parasitoid would not tolerate direct contact with gelled media. Direct exposure to atmospheric
oxygen was important for successful in vitro culture of E. roborator and success was achieved by
retaining suspensions of the liquid diet in lipipholic Sephadex LH-20 gel filtration medium. Mortality, size
and development time of the parasitoid reared in vitro were similar to those of individuals reared on
Pectinophora gossypiella (Saunders). Many of the developmental nutritional requirements of I.
conquisitor and E. roborator were determined by Yazgan (1972) and Thompson (1976a, b).

Thompson (1989, 1981d) described the various chemically defined diets for rearing
various chalcids of the genus Brachymeria. Complete development of B. lasus from egg to adult at rates
approximating those observed in G. mellonella were obtained on diets containing heat-denatured
albumin, amino acids, glucose, B vitamins, inorganic salts, lipogenic growth factors and Intralipid. The
latter, a phospholipid emulsion of soybean oil, was necessary for complete development. Larvae were
reared from eggs dissected from host pupae immediately following oviposition and parasitoids were
cultured individually in the wells of micro tissue culture plates. Development of larvae was ca. 2X as long
on the synthetic medium compared to the insect host, and ca. 80% reached the active adult stage.
Interestingly, the yellow coloration of the femur did not develop if vitamin A was lacking.

A critical factor in formulating the artificial media for B. lasus was osmotic pressure
(Thompson 1983b). The effect of both carbohydrate and amino acid levels was similar and appeared
closely related to osmalality. Optimum osmotic pressure in the artificial diets ranged from 550-700
mOs.Kg which was much greater than the 350-450 mOs/Kg of host hemolymph and tissues.

Complete development of the pteromalid Pachycrepoideus vendemiae (Rondani) was

not obtained on an artificial medium similar to that used successfully for in vitro cultureof Brachymeria
(Thompson 1981c). When the amino acid component was replaced with a mixture of the corresponding
polymerized amino acids and the osmolality was reduced to ca. 390 Mos/Kg, development from egg to
adult was obtained (Thompson et al. 1983c).

These studies demonstrate that the importance of osmotic pressure varies with the
parasitoid species. Parasitoids such as I. conquisitor and E. roborator are very tolerant of osmotic
pressures. Artificial diets that supported in vitro culture of these species had osmolalities of ca. 2,000
mOs/Kg. On the other hand, the tachinids, P. caudata and L. diatraeae (Grenier et al. 1986), and the
pteromalid P. vindemiae did not develop at osmolalities of >450 mOs/Kg.

Pteromalus puparum was cultured in vitro by Bouleteau (1968, 1972). Complete

development on host hemolymph in hanging drop slide mounts was obtained. Similar results were
reported by Hoffman et al. (1973). Hoffman & Ignoffo (1974) had limited success with an artificial
medium containing yeast hydrolysate, fetal bovine serum and Grace’s tissue culture medium.
Tetrastichus schoenobii Ferriere was reared on modified Gardiner’s tissue culture medium
supplemented with egg yolk, milk and hemolymph from Anteraea pernyi Guérin (Ding et al. 1980a).
About 60% of the parasiotids completed development to the adult stage with no deformities nor
abnormal fecundities. Greany (1980, 1981) described studies on the in vitro embryonic development of
the barconid Cotesia (= Apanteles) marginiventris (Cresson), maintained in Grace’s tissue culture
medium supplemented with fetal bovine serum, bovine serum albumin and whole egg ultrafiltrate.
Insects were reared from the embryonic germ band stage to mature first instar larvae on this diet
cocultured with host fat body tissue. Greany (1986) obtained similar results with Microplitis croceipes.
Emphasis was placed on the importance of protein nutrition for success and protein secretion by the fat
body was considered a factor to explain the need for this tissue for successful embryonic development.

Vinson & Iwantsch (1980) found that teratocytes (cells derived from the embryonic
membrane of the parasitoid egg) are released into the host homocoel at the time of egg hatching. It was
suggested that the teratocytes may play a role in parasitoid nutrition. Sluss (1968) demonstrated that
the teratocytes of Perilitus coccinellae (Shrank) increased in volume several times in the coccinellid host
and where then subsequently eaten by the developing parasitoid larvae. Graeny (1980) found that
teratocytes present in artificial culture medium for C. marginiventris caused dissociation of cocultured
fat body and suggested that the teratocytes might facilitate larval growth. Rotundo et al (1988) obtained
complete larval development of the braconid Lysiphelebus fabarum (Marshall) on a similar artificial diet
that was lacking in fat body and teratocytes.

Strand et al. (1988) demonstrated a role for teratocytes in the successful in vitro culture
of the egg parasitoid Telenomus heliothidis Ashmead. Embryonic development of T. heliothidis was
obtained in Hinks TNH-FH medium containing 30% w/v M. sexta hemolymph. Mature embryos were
transferred to a medium containing 40% M. sexta hemolymph, chicken egg yolk, trehalose and milk.
Development of the adult stage required one day more than on the host H. virescens and 42% of the
larvae became adults. The sex ratio was ca. 50% females. The presence of teratocytes had no effect on
larval development to the third instar. However, when teratocytes were removed from the medium
during larval development, pupation was greatly reduced and the development time of parasitoids that
completed development increased. The authors concluded that the teratocytes aided larval feeding by
dispersing the particulate material in the medium and solubilizing nutrients. It was suggested by Strand
et al. (1986) that teratocytes of T. heliothidis aided in decomposition and necrosis of host tissue partially
due to release of lytic enzymes. Therefore their function in vitro might be the same that occurs during
the normal development of the parasitoid in the host, Heliothis virescens.

Culture of Trichogramma pretiosum in vitro was first attained by Hoffman et al. (1975)
following unsuccessful attempts by Rajendram (1978) with T. californicum Nagaraja & Nagarkatti.
Trichogramma pretiosum completed development on filter paper disc soaked in sterile H. zea
hemolymph. In vitro culture to the adult stage required ca. 25% more time than observed on the host,
Trichoplusia ni (Hübner). Even though most adults did not fully expand their wings, they mated and laid
eggs without difficulty. Progeny from eggs of in vitro cultured parasitoids had a sex ratio of 1.2:1
males/females when reared on host eggs. Hoffman et al. (1975) reported development to the prepupal
stage on a semisynthetic artificial diet similar to that described by Hoffman & Ignoffo (1974) for P.
puparum,but supplemented with wheat germ oil. Strand & Vinson (1985) obtained complete in vitro
culture of T. pretiosum on an artificial medium similar to that used by Thompson (1981d) for B. lasus but
supplemented with ca. 40% M. sexta hemolymph, the latter being required to induce pupation. Survival
to the adult stage was 70s% and the sex ratio ca. 1:2 males/females. Xie et al. (1986a) also reported that
host hemolymph was required for pupation of T. pretiosum and that factors in the host egg influenced
adult emergence. Irie et al. (1987) reported that the requirement of host hemolymph for the complete
in vitro development was due to the presence of specific factors that could be extracted in 76% ethanol.
Purification of the pupation factor by chromatographic methods showed the presence of two active
carbohydrate containing factors.

Trichogramma dendrolimi Matsumura was cultured in vitro in hanging drop amounts of

hemoplymph from A. pernyi Guan et al. (1978). Liu et al (1979) reported success in hanging drop mounts
containing media with A. pernyi or Attacuscynthia (Drury) hemoplymh and chicken egg yolk, bovine milk,
organic acids and pocine serum. The extent of development of Trichogramma japonicum Ashmead, T.
australicum Girault and T. evanescens was not reported, however, Wu et al. (1980, 1982) and Wu & Qin
(1982a) obtained successful culture of T. dendrolimi to the adult stage on media without host
hemolymph but containing chicken egg yolk, chicken embryo fluid, bovine milk, and amino acid mixture
and peptone. However, only 16% of the eggs completed development, and most adults were females of
poor vitality. The results did suggest that in contrast to T. pretiosum, the in vitro culture of T. dendrolimi
on a medium of yeast hydrolysate, fetal calf serum, Grace’s tissue culture medium, chicken embryo
extract, bovine milk and chicken egg yolk. However, adults were less viable than normal and displayed
abnormal wing development. The cooperative Research Group of Hubei Province, China (CRGHP 1979)
has carried out extensive studies on the complete in vitro culture of T. dendrolimi in artificial media
encapsulated in artificial eggs into which the adult females oviposited. Gao et al. (1982) reported rearing
35 continuous generations of this species hanging drop mounts of the artificial medium.
 Some studies have tried to determine the effects of hormone supplementation on
parasitoid development in vitro, with generally negative results. The tachinid Gonia cinerascens Rondani
depend on its host’s endocrine system for growth and development, but was not induced to mold from
the 1-2nd instar by addition of 20-hydroxy (b) ecdysone to an artificial medium of host tissue
homogenate and Grace’s tissue culture medium. Development from the 2nd instar to adult was
reported on artificial medium in the absence of hormones, indicating that some hormones may be
necessary for the 1-2nd instar molt in vitro. The 20-Hydorxy ecdysone failed to stimulate development
of B. intermedia in vitro (Thompson 1980); however, Greany (1980, 1981) reported that this hormone
inhibited egg hatching in C. marginiventris and ecdysone, 20-hydorxy ecdysone and the juvenile
hormone analog hydropene had no effect on larval growth or development. The deleterious effect of
this hormone could be overcome by simultaneous application of hydroprene.

Nenon (1972a,b) demonstrated that hormones greatly increased in vitro survival of

developing embryos and larvae of the encyrtid Ageniaspis fuscicollis (Dalman). The parasitoid was
maintained on a diet of chicken embryo extract, beef peptone and equine serum. Ecdysteroid or juvenile
hormone added in the medium had little effect, but when included together, resulted in nearly 100%
survival to the 2nd instar. Further study of the effects of host hormones in vitro culture systems must
require careful and detailed experimental design. Hormones act in a complex and often synergistic way,
and the timing of their application as well as the method of exposure may prove critical to assessing
their potential. There is no doubt as to the importance of hormonal interaction to the successful
development of parasitoids in vivo, particularly with regard to synchronizing parasitoid development to
the host’s life cycle.

Adult Entomophage Nutrition

Adults of many entomophaga must feed, and although adult parasitoids and predators
are usually fed in the laboratory, early workers had largely ignored the significance of such feeding in
nature. Bierne (1962) considered that many biological control attempts failed as a result. Leius (1967a)
gave one of the first field demonstrations of the importance of adult feeding when he reported a
relationship between the natural abundance and variety of wild flowers in apple orchards and the
incidence of parasitism of Malacosoma americanum (F.) and Laspeyresia (= Carpocapsa) pomonella (L.)
by the parasitoids, I. conquisitor, Apophua (= Glypta) simplicipes (Cresson), Scambus hispae Harris,
Telonomus sp., Ooencyrtusclisiocampae (Ashmead), and Eupelmus spongipartus Foerster. Eighteen
times as many L. pomonella eggs were parasitized in orchards with an undergrowth of wild flowers
when compared with other orchards lacking such flora.
 The early literature describing how adult parasitoids feed from flowers and other plant
parts was reviewed by Leius (1960). Generally insects fed on floral and extrafloral nectars as well as
pollens. Although knowledge of the specific nutritional requirements of adult entomophagous insects is
limited, much data are available on the chemical and nutritional requirements of adult entomophaga is
limited, much is available on the chemical and nutritional composition of these plant products. Floral
nectars contain up to 75% by weight of simple sugars, mainly sucrose, fructose and glucose (Baker &
Baker 1983), but considerable qualitative and quantitative differences exist between plant species. Free
amino acids are also abundant in nectars although most nectars do not contain all 10 essential amino
acids. Small amounts of protein, lipids, dextrins and vitamins that are nutritionally beneficial are also
found. The composition of extrafloral nectars is also complex (Baker et al. 1978). Pollens have a complex
composition of small molecular nutrients and many pollens have high levels of free amino acids (Barbier
1970, Stanley & Linsken 1974). By comparison, pollens generally have higher levels of protein, lipid and
polysaccharides. Pollens and nectars together can provide a complete diet sir successful growth,
development and reproduction. The predator Coleomegilla maculate lengi Timberlake can complete
larval development on pollen alone (Smith 1961); therefore, when prey are scarce, plant products may
play a critical role in maintaining predators (Hodek 1973). Hagen (1986a) discussed the complex
ecological and evolutionary interactions between plant flowers, nectars and pollens and several insect
groups.

Leius (1960) examained the plant feeding habits of I. conquisitor, Scambus boulianae
(Hartig) and Orgilusobscurator (Nees). The attractiveness of the flowers of wild mustard, white
sweetclover, wild parsnip, silky milkweed and annual sowthistle were tested. Except for annual
sowthistle, I. conquisitor was attracted to and fed from all flowers tested, but was most attracted to wild
parsnip. Similar results were shown with S. boulianae. Orgilus obscurator was attracted to and fed on
wild parsnip only, but further tests revealed that this parasitoid also fed on other umbelliferous plant
flowers, including those of wild carrot and water hemlock. The nutritive value of various pollens for
fecundity and longevity of S. boulianae was reported by Leius (1963). Itoplectis conquisitor and
S. boulianae accepted various semi-natural foods also, including honey, sucrose solution with or without
plant pollens and raisins. Plant feeding behavior of O. obscurator examined by Syme (1975) showed a
broad range of food plants, including species from five families. Adult parasitoids may emerge prior to
the availability of the insect host, and Syme (1977) suggested that a variety of plant species be provided
as foods to ensure sufficient longevity of the adult female.

Lingren & Lukefar (1977) demonstrated that adult Campoletis sonorensis (Cameron), a
parasitoid feeding on the extrafloral nectar of cotton, lives when exposed to extraforal nectaried cotton
than nectariless cotton. Parasitism of hosts was higher on the nectaried form. Adejei-Maafo & Wilson
(1983) showed that 15 categories of entomophaga, including the predators Deraeocoris signatus
(Distant), Geocoris lubra (Kirkaldy), Nabis capsiformis Germar, Chrysopa spp., Laius bellalus Guérin,
Coccinella repanda (Thunberg) and Verania frenata Erichson, were present at densities of 2-3 tomes
higher on nectaried versus nonnectaried cotton. Although semiochemicals contribute to attraction for
plants in these insects, the nutrition provided by nectars and pollens seems to be important. Hemptinne
& Desprets (1986) reported that following hibernation Adalia bipunctata (L.) fed on pollens as an
alternate food which allows the predators to lay eggs as soon as prey become available.

As was discussed in an earlier section, in addition to feeding plants and plant products
many parasitoids are host-feeders. Adult female Hymenoptera puncture or damage host larvae or pupae
and feed on the hemolymph and/ or internal tissues. Kidd & Jervis (1989) estimated that as much as 1/3
of the world’s parasitoid fauna (>100,000 species) host feed. Some parasitoids may kill more host
individuals by host feeding including ovipositor probing followed by host rejection, than by parasitization
(Johnston 1915, DeBach 1943, 1954). Legner (1979) emphasized that consideration of a parasitoid’s host
destructive capacity was important to correctly evaluate the impact of periodic inundative field releases
on pest populations, and Greathead (1986) and Yamamura & Yano (1988) suggested that host-feeding
behavior was important for assessing the potential of a biological control agent. Kidd & Jervis (1989)
recently discussed the significance of host-feeding on parasitoid-host population dynamics.

Bartlett (1964) in examining host-feeding in the encyrtid, Microterys flavus Howard, was
among the first to correlated host-feeding behavior with nutrition. He hypothesized that host feeding
developed coincidentally with depletion of eggs and suggested that host mutilation was a reflection of
“frustrated” host feeding when the host failed to bleed readily. Host feeding by M. flavus was usually
displayed following egg-laying, and oviposition resumed after host feeding. Reviewing this predatory
habit for adults from 20 families of Hymenoptera, Bartlett concluded that the behavior was indicative of
the necessity for dietary supplementation of some ubiquitous substances required by many diverse
species. He reported that a food supplement of enzymatic yeast and soy hydrolysate with honey
satisfied the nutrient requirements for sustaining reproductive activity in M. flavus, and suggested that a
protein nutrient source may be necessary.

The difference between proovigenic and synovigenic Hymenoptera was discussed

earlier, categories proposed by S. E. Flanders (1950). Females of proovigenic parasitoids complete
oogenesis prior to or shortly after emergence and lay eggs over a relatively short period of time
principally on larval stages of their host. Host feeding is important for ensuring that the female lives long
enough to deposit all eggs. In contrast, females of synovigenic species eclose with a minor fraction to
their total egg complement as mature eggs. Synovigenic parasitoids attacks primarily host eggs and
pupae, are longer lived than proovigenic species and produce eggs throughout their adult lives. To
sustain oogenesis the females of many synovigenic species require additional nutrients. Based on the
egg type, Dowell (1978) described two types of synovigenic parasitoids: (1) those producing large
anhydropic or yolk-rich eggs that contain sufficient nutrient for completion of embryonic development
prior to oviposition. Parasitoids that produce anhydropic eggs obtain nutrition for sustaining egg
production by host-feeding; (2) those producing hydropic or yolk-deficient eggs. Embryonic
development in hydropic eggs occurs in the host following oviposition, in which case the adult does not
require additional nutrient to support egg development and has no requirement to host feed. Legner &
Gerling (1967) showed the importance of early host feeding and oviposition to pteromalids of the first
type, as was previously discussed. Leius (1962, 1967b) demonstrated the importance of feeding habits of
fecundity of S. buolianae. Egg production was reduced to 1/3rd and longevity to 2/3rds, when females
were permitted to host-feed intermittently or were deprived after 15 days of age. No eggs were laid if
females were deprived for 290 days. The effects of feeding host body fluids, in conjunction with honey,
pollen and raisins on fecundity and longevity of S. buolianae and I. conquisitoir were examined by Leius
(1961a,b). Maximum fecundity and longevity of both species were obtained when host fluids and
seminatural foods were provided together. Host feeding was nevertheless essential, and S. buolianae
did not lay eggs when deprived of host hemolymph or tissues.

The feeding behavior of 140 hymenopterous parasitoids was also reviewed by Jervis &
Kidd (1986), who concluded that host feeding was important for maintenance and longevity. Four types
of host feeding distinguished were (1) concurrent feeding where the female used different host
individuals for feeding and oviposition, (3) the feeding habit may be nondestructive or destructive (the
host may survive or may die), and (4) destructive feeding which generally resulted in a host that was
most likely when hosts were readily available and that destructive feeding was advantageous when host
density was low.

Jervis & Kidd (1986) also gave several models to assess how the energetic demands and
constraints on parasitoid affect its host-feeding strategy. One model predicted the feeding strategy for
maximizing egg production of a single synovigenic female (see Thompson & Hagen 1999, for formulae).

Host feeding also occurs among dipterous parasitoids but is not as common as in
Hymenoptera (Clausen 1940). Host feeding by tachinid parasitoids may affect longevity and fecundity
(Shahjahan 1968). Nettles (1987b) demonstrated that fecundity was prolonged by feeding E. bryani host
hemoplymph compared with feeding a sucrose solution. The effect of host feeding on fecundity could
not be simulated by substituting a solution of free amino acids or bovine serum albumin.

The excretion of various Homoptera, such as honeydew, may serve as a food for many
adult entomophaga. Neuropterans of the genus Chrysoperla and other genera with nonpredaceous
adults feed actively on honeydew as well as on nectar and pollen (Principi & Canard 1984). Although
honeydew does not contain all the essential amino acids in some nonpredaceous species (Hagen &
Tassan 1972). Neuropteran predatory adults also feed on honeydew, but reproductive activity ensues
only after prey are eaten (Haggen 1986a). Hagen (1962) found that honeydew alone will not stimulate
egg production in coccinellid predators. Dipterous and hymenopterous parasitoids also have been found
to feed on honeydew (Clausen 1940, Zoebelein 1956). The importance of honeydew as a supplementary
food was suggested by Clausen et al. (1933) in work with Tiphia matura Allen & Jaynes. Female adults
traveled long distances from the location of their host to feed on honeydew, which migration occurred
annually. Ichneumonids of the genus Rhyssa appeared dependent on honeydew for maintaining the
longevity necessary to parasitize and regulate populations of Sirex (Hocking 1967). The nutritional value
of honeydew for parasitoids varies with the homopteran source, as Wilbert (1977) showed considerable
differences in longevity of several Hymenoptera when fed aphid or coccid honeydew.

Nutritional requirements of adult entomophagous insects are obscure. Bracken (1965,

1966, 1969) examined some requirements of the parasitoids Exeristes comstockii (Cresson), finding that
adult females fed an artificial medium containing amino acids, sucrose, fatty acids, cholesterol, vitamins
and inorganic salts produced eggs at an equivalent rate as individuals fed Galleria mellonella (L.) larvae
and sucrose. Egg production was reduced or eliminated when amino acids, sucrose, vitamins or salts
were deleted. Sucrose, pantothenic acid, folic acid and thiamine were all essential for egg-laying.
Nutritional requirements of adult predators similarly are not well known. Numerous semi-natural diets
have been successfully developed for maintaining chrysopid predators and various adults coccinellids. It
seems that predators require a complete and well balanced diet to ensure maximum longevity and
reproductive potential. The effects of various diets on fecundity of some chrysopids was summarized by
Hagen (1986b), and nutritional data for adults of several other species by Roussett (1984).

Continuous Culture on Artificial Media

The ultimate goal of studies on in vitro culture of entomophagous insects is continuous

artificial culture without the host insect. In order to achieve this goal, careful scrutiny of factors that
otherwise would not be considered of direct important to nutrition must be made. Commercial
parasitoid culturing requires the direct deposition of eggs or larvae onto an artificial substrate. Artificial
food must be acceptable for feeding by all stages of a predator. Behavioral considerations may be
critical for the successful continuous culture of many entomophaga. Success with in vitro culture thus far
reflects the level of complexity of behavioral interactions between parasitoid and host or predator and
prey. The first succeed with parasitoids was achieved with Sarcophagidae, many of which readily
oviposit and develop on carrion. Sarcophaga aldrici and K. kellyi were reared for many generations on
fish and liver, respectively (Arthur & Coppel 1953, Smith 1958). Agria housei was reared continuously for
756 generations on pork liver. However, the behavioral interaction between many parasitoids and their
hosts are complex, involving numerous physical and chemical cues that initiate specific behavior which
leads to oviposition. Host selection and successful parasitism is a multistep process which involves host
habitat location, host location, host acceptance, host suitability and host regulation, as was discussed in
previous sections (Doutt 1959, Vinson 1976, 1984). Factors that influence host acceptance in particular
are critical for continuous culture. The different events which lead to successful oviposition, including
examination of the host, probing with the ovipositor, insertion and oviposition (Schmidt 1974) may each
be stimulated by different chemical as well as physical cues (Arthur 1981, Vinson 1984). These cues may
be associated with the host species, the plant or other food source of the host, or may result from
interactions, involving both the host and its food (Vinson 1975). Physical factors associated with the
host’s food plant are essential for successful oviposition and parasitism by G. cinerascens (Mellini et. al.
1980). This tachinid deposits microtype eggs on the leaves of certain plants, and host larvae become
infected by ingesting the eggs. Leaf color, shape, thickness, size and reflectivity are among the several
factors which influence oviposition in this species. Mellini et al (1980) constructed polished, thin, yellow
oval-pointed bee’s wax leaves, 2-7 cm2, on which large numbers of parasitized eggs were laid. This
parasitoid readily developed in G. mellonella after host feeding on the artificial leaves. Complex
combinations of physical cues, including size, shape, color, texture and movement have been
demonstrated to have influence on oviposition behavior in paarsitoids 9Arthur 1981, Jones 1981,
Nordlund et al. 1981).

Important roles are played by chemicals in both parasitoid-host and predator-prey

interactions (Arthur 1981, Greany and Hagen 1981, Vinson 1984, Hagen 1986a). The involvement of
chemicals in host acceptance and oviposition by parasitoids is well documented. During predator-prey
relationships, kairomones produced by the prey may serve as attractants, arrestants and/or
phagostimulants. Chrysopa carnea adults, e.g.. are attracted to a variety of chemicals such as tryptophan
byproducts (Hagen et. al. 1976). Although studies with numerous predaceous insects have
demonstrated the role of semiochemicals in prey finding and recognition, their role in feeding is not well
established.

Deployment of behavior modifying chemicals in continuous artificial culture has

involved only a few species. Itoplectis conquisitor accepts a host and oviposits following detection of
specific components of host hemolymph during ovipositor probing (Arthur et al. 1969). This parasitoid
even oviposited into host hemolymph that was placed on paraffin tubes. The active fraction was
colorless, water soluble and gave a strong reaction to ninhydrin and folinphenol reagents. It had a
molecular weight of ca. 7,000, was heat stable and nondializable. Arthur et al (1973) concluded that the
stimulant was proteinaceous and they were successful in stimulating similar oviposition activity with a
variety of amino acid mixture containing trehalose and/or MgCl2. The best results were with a mixture
of serine (0.5M), leucine (0.065M), arginine (0.05 M) and MgCl2 (0.025 M). The ovipositional activity
observed greatly exceeded that stimulated by the host hemolymph. House 91978) then developed a
synthetic artificial host comprised of an artificial diet encapsulated in paraffin. The diet was based on
that described by Yazgan (1972) and contained gelatin, casein, inorganic salts, amino acids, glycogen,
lipids, trehalose, glucose, water and fat soluble vitamins and agar. Female parasitoids readily accepted
and oviposited into the artificial host, and the first successful complete artificial culture of a
hymenopterous parasitoid was realized. However, only one single adult male was obtained.
 There have been considerable studies to determine how chemical and other factors
influence adult reproductive capacity in in vitro cultures. Larviposition by E. bryani is stimulated by
kairomones which emanate from the host’s cuticle, and female adults examine artificial hosts coated
with cuticular extracts with great care (Burks & Nettles 1978). Tucker & Leonard (1977) extracted a
kairomone from the pupae of Lymantria dispar that appeared responsible for ovipositional behavior by
Brachymeria intermedia. Tetrastichus schoenobii was stimulated to oviposit in artificial eggs coated with
host scales (Ding et al. 1980b).

The parasitoid group, which has received the most attention, is the Trichogrammatidae.
There have been more extensive efforts to develop continuous artificial culture with Trichogramma spp.
than with other parasitoids. Many aspects of the ovipositional behavior of this genus were described by
Salt (1934, 1940) in studies on T. avanescens (Fisher 1986). Recent studies demonstrate the importance
of physical (Rajendram & Hagen 1974) and chemical factors, including kairomones (Nordlund et al. 1985)
for eliciting oviposition. Rajendram (1978a, b) obtained artificial oviposition by T. californicum into
physiological saline or Neisheimer’s salt solution encapsulated in paraffin. Nettles et al (1982, 1983)
reported that a dilute solution of KCl and MgSO4 induced oviposition by T. pretiosum artificial was eggs
(Nettles et al. 1984). Leucine, Phenylalanine and/or isoleucine stimulated oviposition by T. dentrolimi in
artificial eggs (Wu & Quin 1982b). Adult females laid more eggs than when insect hemolymph was used
then employing a complete mixture of all three amino acids, 600, 400 and 320 mg/100ml. A synthetic
membrane was developed as an alternative for paraffin through which T. pretiosum would oviposit
(Morrison et al. 1983). The silicon-polycarbonate copolymer was clear, highly elastic and adult females
oviposited through the surface into an ovipositional stimulants at rates that were comparable to host
eggs. The use of polyethylene as an ovipositional stimulants at rates that were comparable to host eggs.
The use of polyethylene as an alternative to wax for producing artificial eggs for oviposition by T.
dentrolimi was decribed by the Chinese CRGHT (1985).

Xie et al. (1986b) demonstrated the potential for large scale continuous artificial culture
of T. pretiosum. Three in vitro culture methods were developed as a follow up to earlier work by Xie et
al. (1986a) and Nettles et al. 91985). These utilized microtiter tissue culture plates, multiple drop rearing
in petri plates and flooded petri plat rearing. The basic diet was 50% heat treated insect hemolymph,
25% egg yolk, 15 g/100 ml dried milk suspension and 0.15% gentamycin. Each method supported large
populations of parasitoids larvae. Microbial contamination and subsequent loss of entire petri plats was
a major obstacle but several antibiotics were available for reducing losses.

Field trials with in vitro reared Trichogramma have been made. Continuous artificial
mass culture of T. dentrolimi was described by Li 91982) and Gao et al. (1982), who reported that field
release of in vitro reared parasitoids resulted in 93% parasitism of Heliothis armigera (Hubner) eggs in
cotton. Artificial mass culture of Chrysopa carnea was described by Yazlovetskij & Nepomnyashchaya
(1981) after the development of a suitable artificial medium for supporting larval development
(Nepomnyashchaya et al. 1979). The medium was microencapsulated and composed of casein
hydrolysate, brewer’s yeast extract, soybean oil, wheat germ extract, sucrose, lecithin, choline,
cholesterol and ascorbate. The effectiveness of the artificially reared larvae against Myzus persicae was
equal to that of insects reared on eggs of S. cerealella. A microencapsulated technique for mass
producing artificial eggs for C. carnea was also described by Morisson et al. (1975).

Contemporary Applications

Considerations of how nutrition currently applies in biological control programs, focuses

on its purpose as being restricted to use of food and food supplements to enhance the activity and
effectiveness of entomophagous insects in the field as suggested earlier (Hagen & Hale 1974, Hagen &
Bishop 1979, Greenblatt & Lewis 1983, Hagen 1986a, Gross 1987). Such use is dictated by a lack of
synchrony between natural enemies and their hosts and/or isolation of entomophagous insects from
the natural environment that normally supplies alternate food sources such as nectars and honeydews
(Hagen 1986a). These factors occurring in crop monoculture may intensify following pesticide
application. The importance of nutritional supplements for adult parasitoids and predators is well
known, and recent studies with Trichogramma demonstrated that fecundity and longevity could be
increased be feeding adult insects (Anunciada & Voegele 1982, Bai et al. 1988). The future use of feeding
prior to or following release in the field may have a significant effect on biological control successes. Few
studies on the effects of feeding parasitoids on the field performance are available, however. Temerak
(1976) reported spraying honey solution on sorghum stalks during winter to provide supplementary
food to Bracon brevicornisi Wesmael in the absence of pollen, honeydew and nectars. Parasitoid
cocoons significantly increased after spraying and the prevalence of hosts decreased. Despite field trials
employing kairomones for attracting and stimulating host searching by Trichogramma sp. (Lewis et al.
1979, 1982), no attempt has been to use kairomones in combination with supplemental foods to
maintain parasitoid populations when host numbers are low.

Supplementary food sprays have been successfully deployed with predaceous insects.
Ewert & Chiang (1966) sprayed sucrose solutions on corn to aggregate coccinellid and chrysopid adults.
Increased predator density and reproductive activity significantly lowered aphid populations. The
numbers of Chysopa sp. And Glischiochilus quadrisignatus (Say) were increased in corn sprayed with
sugar or molasses solutions (Carlson & Chiang 1973), with resultant increased predation resulting in
significant reductions of Ostrinia nubilalis (Hubner). Hagen et al. (1976) working in sugar sprayed alfalfa
plots were able to retain larger numbers of C. carnea and Hippodamia sp. in the field during periods of
low host density. Within 24 hrs the population of coccinellid adults increased 20X and that of C. carnea
200X. Populations of Lygus spp. also increased after application of sugar sprays (Lindquist & Sorenson
1970), and Hagen et al. (1971) concluded that sucrose was an arrestant for adult Lygus were attracted to
potato plants sprayed with honey, which suggested a critical role for volatile components (Ben Saad &
Bishop 1976a, b).

Adding semiochemicals to supplemental foods for C. carnea is useful. The complex

interactions of semiochemicals and food in influencing the behavior of C. carnea was described by
Hagen & Bishop (1979). The adult responds to a volatile signal, a synomone, from plant habitats in which
prey are located and is then attracted to the prey by tryptophan breakdown products from the
honeydew (Van Emden & Hagen 1976). Specific behavioral and flight patterns shown by C. carnea in
response to these interactions were discussed by Duelli (1980). The habitat synomone affecting the
behavior of C. carnea in cotton was shown by Flint et al. (1979) to b e caryophyllene, but several
chemicals from other plants also displayed synomone activity for this species (Hagen 1986b).

Chrysopa carnea adults were successfully attracted to alfalfa fields by applying artificial
honeydews composed of various yeast (Wheast) products and sugar (Hagen et al. 1971). Although the
specific synomone of alfalfa is unknown, application of caryphyllene with the kairomone from
tryptophan greatly improved attraction of C. carnea during the beginning of flowering (Hagen 1986a).
Application of artificial honeydew was also successful for aggregating Hippodamia spp. as well as
coccinellids and other predators. Subsequent trials employing the yeast mixture in combination with
sucrose and honey or molasses applied to various crops were successful in manipulating C. carnea
populations (Hagen & Hale 1974). The sugar was essential for retaining C. carnea adults in the field after
attraction. Butler & Ritchie (1971) reported that C. carnea adults were attached to the yeast/sugar
mixtures sprayed on cotton, but no increase in egg deposition was noted. Similar studies demonstrated
inconsistent oviposition in grape culture (White & Jubb 1980). There was no attraction of chrysopid
adults in treated people orchards (Hagley & Simpson 1981) nor in potato fields when only the yeast was
applied. Dean & Satasak (1983) gave reasons why food sprays might not be practical in control programs
for cereal aphids in England, which included the variable abundance of univoltine C. carnea populations,
low plant growth form and the development of sooty mold on plant where food sprays containing sugar
were applied. Duelli (1987) did not find an increase in oviposition by chrysopids when artificial
honeydews were applied to alfalfa, corn, sunflowers and in prune orchards. It was suggested that the
different responses of sibling species of C. carnea in Europe and North America may be related to
behavioral differences.

2. Mass Prodcution of Biological Control Agents

 Mass-production of parasites and predators is often necessary or desirable in
connection with many biological control projects, particularly where attempts are made to increase
parasitism or predation buy mass releases of entomophages over a wide area at a time in the season
when these natural enemies are few or absent. However, the term “mass-production” is vague and
individual differ in their concepts of large numbers. Moreover, the actual numbers needed in connection
with different biological control problems vary considerably. Some successful parasites introductions
have been made with only a dozen or so individuals; others have required very large numbers before
establishment has been obtained. There are two main types of biological control, each of which has
different requirements with regard to mass-production. As mentioned above, there is that in which large
numbers of entomophages are released at critical times in the season in order to increase the
destruction by natural enemies. In such cases it is obvious that mass-production methods are essential,
the scale depending on the size and duration of the operation. The main difficulties here are to produce
the required enormous numbers of entomophages at exactly the correct critical period of the season,
particularly when this period is dependent on weather and may vary considerably and somewhat
unpredictably from year to year. A thorough survey of the results obtained from this method of control
in the number of instances in which it has been used would be very interesting. The second, and far
more desirable, procedure is to established a suitable exotic entomophage against a pest which will
thrive in the new environment and give perennial reduction in the pest population. This has, of course,
been accomplished successfully many times, but where failure has occurred the exact reasons for it are
often obscure and it is some Director, Commonwealth Institute of Biological Control, Curepe, Trinidad,
West Indies times attributed to the introduction of too few individuals. In such cases it is claimed that
mass-production and release might yield better results.

Mass-production-weather of parasites or predators, fungi, protozoa, bacteria, viruses,

sterile males, or genetically incompatible individuals-has become an integral part of biological control
programmes. Large-scale field collection is often the simplest and most economical method of obtaining
large numbers of a given species. Often this entails feeding the host or immature predators in the
laboratory, where considerable care may be necessary to eliminate hyperparasites and diseases. Mass-
production methods usually produce large numbers of entomophages, free from contaminants, which
can be released immediately in the field. However, there are virtually no general rules that can be laid
down as to optimum conditions of cage-size, environment, feeding and illumination for breeding these.
Each problem demands different treatment, depending on the requirements of individual species and
numbers to be produced, and each scheme is usually based on a technique developed in the laboratory
and then modified for cheap, large-scale production. However, there are a number of general aspects of
mass-production of entomophages that should be considered. In many cases it is necessary to provide
large numbers of a host species. These may possibly be collected in the field and, if necessary, stored for
future use. Such collections may, however, be impossible or impracticable and it may be necessary to
propagate host in large numbers, often throughout the year, in heated insectaries. Great care is then
necessary to produce normal, healthy adults, rather than small individuals susceptible to disease. In
some cases, too, precautions have to be taken to avoid cannibalism. When normal plants are
unavailable or unsuitable for mass-production methods, it may be possible to find a more suitable
alternate host-plant, or even to provide an artificial, not necessarily synthetic, diet in place of the host.
Although the mass production of host-plants does nit actually concern insect vectors of medical
importance, it is mentioned here on general grounds. Perhaps the normal host cannot be mass-
production. It is then often possible to find an alternative, but unnatural, host on which the
entomophage can develop, and which can be produced in large numbers. Sometimes it may be
necessary to “condition” such alternative hosts with extracts of normal hosts before oviposition by
entomophages can be induced.

The logical development from this is research into producing artificial, and even purely
synthetic, diets on which entomophages may be mass-produced. The use of unnatural or artificial hosts
may possibly produce abnormalities in the progeny, and this must be guarded against. The diet of both
the preceding larval generation and also of the entomophages themselves may have considerable
influence on their longevity, reproductive rate, fertility of eggs, and possibly on the proportion of
progeny entering diapause-all of which are of major importance in mass-production schemes. There is
the possibility also that the use of unnatural hosts may affect the host preferences of the progeny, a
point which should be investigated where necessary.

The genetical aspect of breeding and mass-propagation of entomophages is important.

In most instances laboratory stocks, or those for mass-production schemes, are derived from a small
amount of field material and they are often further inbred during the course of many generations.
Genetic variability of the stock will almost certainly be reduced and with it, possibly, the adaptability of
the entomophage to different environmental conditions. Hence every effort should be made to maintain
or increase genetic variability in mass-propagation programmes.

The determination of optimum mating conditions to provide maximum numbers of

progeny with an adequate sex ratio is also essential. In mass-production schemes it is usually easy to
eliminate hyperparasites in the case of parasites, or parasites in the case of predators. Disease and
mites, however, may often cause losses in crowded mass-production conditions, and if this occurs
rearing methods must obviously be suitably altered. Disease transmitted through the eggs are
particularly difficult to eradicate.

In conclusion it should be stressed that not only does each problem in mass-production
of an entomophage present its own difficulties, but also the actual need for, or size of, a mass-
production programme should be carefully considered, since once such a programme is initiated there is
sometimes a tendency to increase production out of proportion to real needs.
Chapter 5 Methods and Approaches to Biological Control

Quarantine and Exclusion

The primary function of a biological control quarantine facility is to provide a secure

area where the identity of all incoming biological control candidates can be confirmed and undesirable
organisms, especially hyperparasitoids, parasitoids of predators, and extraneous host or host plant
material, can be eliminated. In fact the quarantine laboratory often represents the last chance to study
and evaluate potential biological control agents in the sequence of collection, importation and
liberation.

The number of quarantine in the United States which are certified to handle incoming
shipments of beneficial organisms has increased from four to 26 over the past 40 years. In addition to 24
listed by Coulson & Hagen (1985), new quarantine facilities have been constructed at Montana States
University, Bozeman for phytophagous insects and the University of California, Riversides for
nematodes. New or expanded quarantine facilities have been constructed in a number of other
countries (e.g., Australia, Great Britain, Mexico, Germany and Thailand).

The steady increase in quarantine need and capacity is due on part to an increased
interest in biological and non-polluting methods of pest control, and the desire to expand on the many
successes already achieved through the importation of exotic natural enemies. Also, there is an increase
in new pests that are transported throughout the world and which are amenable to biological as well as
the stricter prerelease information requirements on behavior and safety of biological control candidates.
Lengthy studies on candidates often ties up quarantine areas thereby increasing the need for greater
quarantine capacity to avoid limiting the amount of materials that can be handled. For example, in the
United States to prove the environmental safety of plant-feeding arthropods for the biological control of
weeds can include studies with as many as 10-20 North American native plant species related to the
target weed. When such studies are not permitted or feasible in the country of origin of the biological
control phytophage, these test must be conducted in a domestic quarantine facility. Similarly, testing
parasitoids against indigenous insect species which have been declared legally threatened or
endangered and which may be present in areas near or contagious to insect pest infested agricultural
crops that are targeted for parasitoid release, may not only require more quarantine space but also
delay or prevent the colonization of newly imported organisms. The longer the imported organisms
remain in quarantine before these tests can be conducted, the greater the risk that subtle genetic
changes occur, altering the potential fitness of the organism.

Increasing concern over the quality of the environment is also causing a proliferation of
regulations governing the importation and liberation of beneficial organisms. Explorer collectors,
quarantine officers and project scientists must spend increasing time to study and comply with
domestics and foreign regulations that cover importation, exportation and liberation of biological
control agents. Air travel has reduced the amount of time required to move biological control agents
from one continent to another, but the proliferation of international airports has spawned a logistical
confusion of unpredictable package routing, delayed agricultural and customs inspection and
unscheduled reloading and shipment to the final destination. Frequently material arrives dead or in a
weakened state and on occasion may never arrive.

Use of Resistant Host Plants

Plants have two ways of defense against herbivores. One is direct defense, which affects
herbivores directly through physical or chemical means, such as thorns, toxins, digestibility reducers.
Then other is indirect defense, which promotes the effectiveness of carnivores (Dicke, 1999). A direct
defense is to employ toxins such as alkaloids, phenolics, terpenoids, which are lethal for herbivores in
some cases. While another direct defense is to produce substances such as tannins and phenolics as
digestibility reducers to delay herbivore development. A plant’s indirect defense can promote the
performance of carnivores in several ways. For example plant morphological character plant substances
prolonging herbivore development which results in the herbivore remaining in a stage that is susceptible
to carnivores for a longer period of time, or induced plant volatiles that help carnivores to find their
herbivores prey (Dicke, 1999; Groot & Dicke, 2001).
 It has been recognized that many of the plant traits and processes that negatively affect
herbivores change following attack. Karban & Baldwin (1997) refer to changes in plants following
damage or stress as induced responses. Those induced responses that decrease the negative fitness
consequences of herbivore attack on plants are termed induced defenses, including induced direct and
indirect defenses. Induced direct defense normally acts through preventing herbivores from converting
a plant’s tissues into their own tissues after damage or stress. In induced indirect defense, two types can
be distinguished. One is wound-induced change in the production of extrafloral nectar. The other acts
through the emission of induced volatile compounds in response to herbivory that attracts predators
and parasitoids of herbivores. Many induced response to wounding are systemic. In such cases the
damage plant tissue may produce a signal that is transmitted systematically throughout undamaged
parts of the plant, causing the induction of new morphological or physiological states, the induced
response. Several different signal transduction pathways are involved, including chemical and electrical
signaling. Signalling compounds include 1) oligosaccharide fragments of plant cell walls; 2) systemin (an
oligopeptide); 3) salicylic acid; 4) ethylene, which mediates induced defenses such as induced volatile
production; 5) abscisic acid; 6) jasmonic acid and methyl jasmonate; and 7) electrical signals (Karban &
Baldwin, 1997; Leon et al., 2001; Lorenzo et al., 2004; Adie et al., 2007).

The volatile compounds that plants emit when they are damaged typically are mixtures
of C6-alcohols, -aldehydes, and –esters produced by the oxidation of membrane-derived fatty acids, and
terpenoids and aromatic compounds such as methyl salicylate and indole. These herbivore \-indiced
volatiles released by plants were found to increase the foraging efficiency of carnivores, and carnivores
may learn to associate the volatiles with actively feeding herbivores (Vet & Dicke, 1992; Karban &
Baldwin, 1997; D cke & van Loon 2000; Turling & Ton, 2006).

Plant defenses have been exploited by humans to develop two methods of

environmentally-benign pest control: plant resistance based on direct defense and biological control
based on indirect defense.

Host plant resistance (HPR) can be defined as the inherited property that enables a plant
to avoid, tolerate, or recover from injury by insect populations. For crop production, host plant
resistance represents the inherent ability of crop plants to restrict, retard or overcome pest infestations
and thereby to improve yield or quality of the harvestable product.

Three mechanisms of plant resistance to insects are commonly recognized: antixenosis

(non-preference), antibiosis, and tolerance. Antixenosis is defined as a relatively low acceptability of a
plant as a host to an insect herbivore. Plants that exhibit antixenotic resistance would be expected to
have reduced initial infestation or a higher emigration rate of the insect than susceptible plants. The
basis of this resistance mechanism can be morphological or chemical. Antibiosis is defined as the
mechanism leading to negative effects of a resistant plant on the biology of an insect which has
colonized the plant. Both chemical and morphological plant traits can have antibiotic effects. The
consequences of antibiotic ristics that can support the activities of natural enemy resistance may vary
from mild effects that influence fecundity, development times and body size, through to acute direct
toxic effects resulting in increased mortality. Tolerance is the degree to which a plant can support an
insect population that under similar conditions would severly damage a susceptible plant. That is, when
two cultivars are equally infested the less tolerant one has a smaller yield (Thomas & Waage, 1996).

Biological control is the suppression of a pest population using predators, parasitoids

and pathogens. There are three main forms of biological control: 1) conservation of natural enemies
already present in the environment; 2) augmentation and dissemination of natural enemies of pests,
such as microorganisms, nematodes, and arthropods. 3) classical biological control-to suppress a pest
population permanently through a single introduction of a natural enemy (Thomas & Waage, 1996). A
parasitoid is an insect that as a juvenile, lives at the expense of another one (host) and is usually does
not kill its host immediately but at the end of juvenile development. Parasitoids may parasitize eggs,
larvae, pupae or adult hosts and each species is usually highly specific in this. A predator is an insect that
eats more than one other organism during its life and kills its prey immediately. Predators are normally
larger than their prey and are generally much more generalistic than parasitoids. In a successful
biological control programme the pest kill rate of effective natural enemies should be always higher than
the potential maximum rate of population increase of the pest species (Dicke, 1996; Hawkins & Cornell,
1999).

The natural enemies of herbivores can be influenced by plant characteristics

independently of the herbivore or mediated through herbivore activities. The relevant plant
characteristics include the following aspects: i) plant tissues and products that are used as a source of
nutrition by natural enemies; ii) plant morphology; iii) visual and vibrational cues; iv) secondary plant
chemicals; v) plant volatiles (Thomas & Waage, 1996). Many pest management practitioners have found
that host plant resistance is fundamentally compatible with biological control (Verkerk et al, 1998).
Positive interactions result where natural enemies use herbivore-induced synomones emitted by plants
as cues to find prey (Vet & Dicke, 1992); Dicke et al., 2003; Arimura et al., 2005; D’Alessandro et al.,
2006). Partial plant resistance may also provide benefits for the third trophic level by reducing the
growth rate of prey which in turn increases the duration of their availability to natural enemies (Feeny,
1976; Price et al., 1980). However, in some cases, negative interactions between plant resistance and
biological control are caused by toxic secondary plant compounds which can be passed on through
herbivores to their natural enemies (Hare, 1992; Harvey et al., 2003). They may also be caused by plant
factors which can impede natural enemy effectiveness (e.g. leaf toughness, cuticle thickness, trichomes
(Price,1986). Therefore it is important to integrate HPR and BC dexterously.

In the future, fundamental and applied pest control research will have be more closely
integrated and coordinated to increase efficiency. In this project, the integration of host plant resistance
and biological control will be studied.

Destruction by Cultural Management or Direct Removal of Infected/Diseased Host

Crop termination and alternate host elimination are cultural management methods that
increase mortality of insect pests and decrease subsequent damage through destruction of food sources
and overwintering habitats. Destroying the crop soon after harvest and eliminating volunteer sorghum
plants suppresses insect pest abundance the following year. Destroying alternate host plants eliminates
sources of insect pests that infest sorghum. Tillage methods for sorghum vary regionally and have
evolved from plowing with a moldboard plow, followed by several secondary tillage and planting into
one trip over the field. Whatever tillage is used, sorghum stalks should be destroyed soon after harvest
to expose and kill insect pests and eliminate their food supply. It is equally important to destroy
volunteer sorghum and alternate host plants where reduced tillage is pests. Herbicides can be used to
kill sorghum and alternate host plants where reduced tillage is used. However, failure to destroy
overwintering sites mechanically may contribute to an increase in the survival of certain insect pests.

Destroying food sources and overwintering habitats reduce abundance of cutworms;

sorghum midge; sorghum webworm, Nola sorghiella Riley; sugarcane borer, Diatraea saccharalis
(Fabricius); and sugarcane rootstock weevil, Anacentrinus deplanatus (Casey). Johnsongrass, S.
halepense (L.) Pers., is a noncultivated host of many sorghum insect pests, including yellow sugarcane
aphid, Sipha flava (Forbes); greenbug; and sorghum midge. Destroying this weed is difficult but highly
beneficial to efficient sorghum insect pest management and crop production.

Crop rotation
 Crop rotation is a cultural management method that involves alternate use of host and
nonhost crops in a field to reduce insect pest abundance and damage. Sorghum benefits most when
rotated with a broadleaf or taprooted crop such as cotton, Gossypium hirsutum L., or soybean, Glycine
max (L.) Merr. Growing sorghum in a field planted to a different, nonhost crop the previous year
significantly reduces the abundance of some insect pests, as well as some diseases and weeds.

Crop rotation is most effective against insect pests with limited host range, long life
cycle (one or fewer generations a year), and limited ability to move from one field to another. For
example, several species of wireworms, white grubs, and some cutworms have only one generation a
year, must have a grass-type crop on which to develop and reproduce, and because they live
underground cannot during the damaging larval stage move from one field to another. Thus, growing a
nongrass crop such as cotton or soybean the year before growing sorghum reduces abundance of soil-
inhibiting insect pest in sorghum fields. Sorghum should be rotated annually with other crops.

Variety selection, seedbed preparation, and seed treatment

Variety selection, seedbed preparation, and seed treatment are important for managing sorghum insect
pests. Only sorghum varieties with seed germination of eighty percent or greater should be used. Poor
seed germination results in reduced stands and less competitive plants more susceptible to damage by
insects. A sorghum variety adapted to the locale, and preferably one least vulnerable to insect pests and
diseases, should be used. Sorghum hybrids selected should be resistant to greenbug. Sorghum midge-
resistant hybrids, if available, are useful in more southern regions of the United States. Larvae of corn
earworm; fall armyworm, Spodoptera frugiperda (J. E. Smith); and sorghum webworm that consume
developing kernels infest sorghum hybrids with open panicles less than those with compact panicles.
Also, kernels of open-panicle sorghum varieties are less likely to deteriorate from the combined effects
of weather and damage by panicle-infesting bugs and pathogens. Sorghum varieties should be selected
that mature as early and uniformly as practical in a locale. These varieties escape infestation by sorghum
midge, corn earworm, fall armyworm, sorghum webworm, and sugarcane borer. Sorghum varieties
resistant to pathogens and lodging also lessen detrimental effects from insect pests. Insect pests stress
sorghum plants, and this stress combined with pathogen infection increases the chance of plant lodging.
Greenbug and corn leaf aphid, Rhopalosiphum maidis (Fitch), transmit maize dwarf mosaic virus to
sorghum. This problem is best dealt with by using virus-resistant varieties. Iron-tolerant sorghum
varieties should be used in areas where iron deficiency is a problem.

Well-prepared seedbeds speed seed germination and seedling growth. The trend in
planting sorghum is less tillage and fewer trips over the field using appropriate planting equipment.
Tillage for seedbed preparation should modify the soil to allow desired control of seed placement,
weeds, water infiltration, water evaporation, and erosion.
 Rapid seed germination is essential to avoiding damage be seed-feeding insects such as
wireworms and red imported fire ant, Solenopsis invicta Buren. Rapidly growing and larger plants better
tolerate damage by yellow sugarcane aphid, greenbug, and chinch bug, Blissus leucopterus leucopterus
(Say).

Insecticide can be used to protect planted seed from insect pests. Recently, sorghum
seed treated commercially with a systematic insecticide is a available to protect against seed-feeding
insects and some seedling inslect pests. Also, insecticides can be applied to seed in the planter box or in-
furrow at planting. Systemic in-furrow insecticide protects seedling sorghum against some insect pests.
Insects controlled by systemic seed or in-furrow treatment include wireworms, red imported fire ant,
cutworms, southern corn rootworm, Diabrotica undecimpunctata howardi Barber, aphids, and chinch
bug.

Planting time

Planting time of sorghum in most locales should be as early as practical but not when
soil is too cool for rapid seed germination and seedling growth. In many areas, early planting takes
advantage of seasonal rainfall. Planting early avoids infestation and damage because the sorghum plant
is beyond the vulnerable stage when some insect pests are abundant enough to cause damage, or at the
very least, the crop is susceptible for a shorter period of time.

Early uniform planting of sorghum to avoid a damaging sorghum midge infestation is an

excellent example of the benefit of planting early. Planting sorghum early avoids high numbers of corn
earworm, fall armyworm, sorghum webworm, stalks borers, and panicle-feeding bugs.

Fertilizer and water

Fertilizer and water applied to sorghum either can be beneficial or detrimental to insect
pests. Using too much fertilizer and irrigation can cause sorghum plants to be especially succulent and
attractive to insect pests, amd may extend the time to maturity, increasing the duration of vulnerability.
On the other hand, healthy, vigorously growing sorghum plants better tolerate insect pest infestation
and other stresses. Chinch bug and Banks grass mite are favored by hot, dry conditions and moisture-
stressed plants. Yield of healthy plants is less reduced by most lea-feeding insect pests. In some areas,
application of iron is important for production of healthy sorghum plants.

Conservation and Augmentation

The conservation of natural enemies is probably the most important and readily
available biological control practice available to homeowners and gardeners. Natural enemies occur in
all areas, from the private garden to the open field. They are adapted to the habitat and to the target
pest, and their conservation generally simple and cost-effective. Lacewings, lady beetles, hover fly
larvae, and parasitized aphid mummies are almost always present in aphid colonies. Fungus-infected
adult flies are often common following periods of high humidity. These natural occurring biological
controls are often susceptible to the same pesticides used to target their pest host. Preventing the
accidental eradication of natural enemies is termed “simple conservation”.

To conserve and encourage pest insect eating birds, including native plants and
ornamentals plants that supply berries, acorns, nuts, seeds, nectar, and other vegetative foods, and also
bird nest building materials, encourages their presence, health, and new generations. These qualities
can also increase the visible population to enjoy in a garden. Using companion planting and the birds’
insect cuisine habits is a traditional method for biological control agent pest control in an organic garden
and any landscape, and in organic farming and sustainable agriculture. Installing specified nest boxes for
mosquito-eating bats reduces a pest and increases endangered species conservation.

Augmentation

This third type of biological control involves the supplemental release of natural
enemies. Relatively few natural enemies may be released at a critical time of the season (inoculative
release) or literally millions may be released (inundative release). Additionally, the cropping system may
be modified to favor or augment the natural enemies. This latter practice is frequently referred to as
habitat manipulation. An example of inoculative release occurs in greenhouse production of several
crops. Periodic releases of the parasitoid, Encarsia Formosa, are used to control greenhouse whitefly,
and the predaceous mite, Phytoseiulus persimilis, is used for control of the two-spotted spider mite.

Lady beetle, lacewings, or parasitoids such as those from the genus Trichogramma are
frequently released in large numbers (inundative release). Recommended release rates for
Trichogramma in vegetable or field crops range from 5,000 to 200,000 per acre (1 to 50 per square
metre) per week depending on level of pest infestation. Similarly, entomopathegic nematodes are
released at rates of millions and even billions per acre for control of certain soil-dwelling insect pests.

The spraying of octopamine analogues (such as 3-FMC) has been suggested as a way to
boost the effectiveness of augmentation. Octopamine, regarded as the invertebrate counterpart of
dopamine plays a role in activating the insects’ flight-or-fight response. The idea behind using
octopamine analogues to augment biological control is that natural enemies will be more effective in
their eradication of the pest, since the pest will be behaving in an unnatural way because its flight-or-
fight mechanism has been activated. Octopamine analogues are purported to have two desirable
characteristics for this type of application: (1) they affect insects at very low dosages (2) they do not
have a physiological effect in humans (or other vertebrates).

Figure 20 A turnaround flowerpot, filled with straw to attract Dermaptera species

Habitat or environmental manipulation is another form of augmentation. This tactic

involves altering the cropping system to augment or enhance the effectiveness of natural enemy. Many
adult parasitoids and predators benefit from sources of nectar and the protection provided by refuges
such as hedgerows, cover crops, and weedy borders. Also, the provisioning of natural shelters in the
form of wooden caskets, boxes or (turnaround) flowerpots is a form of this. For example, the stimulation
of the natural predator Dermaptera is done in gardens by hanging up turnaround flowerpots with straw
or wood wool.

Mixed planting and the provision of flowering borders can increase the diversity of
habitats and provide shelter and alternative food sources. They are easily incorporated into home
gardens and even small-scale commercial plantings, but are more difficult to accommodate in large-
scale crop production. There may also be some conflict with pest control for the large producer because
of the difficulty of targeting the pest species and the use of refuges by the pest insects as well as natural
enemies. Examples of habitat manipulation include growing flowering plants (pollen and nectar sources)
such as Buckwheat near crops to attract and maintain populations of natural enemies. For example,
hover fly adults can be attracted to umbelliferous plants in bloom.

Biological control experts in California have demonstrated that planting prune trees in
grape vineyards provides an improved overwintering habitat or refuge for a key grape pest parasitoid.
The prune trees harbor an alternate host for the parasitoid, which could previously overwinter only at
great distances from host vineyards. Caution should be used with this tactics because some plants
attractive to natural enemies may also be hosts for certain plant diseases, especially plant viruses that
could be vectored by insect pests to the crop. Although the tactic appears to hold much promise, only a
few examples have been adequately researched and developed.

Integrated with other Control Tactic for Pest Management

IPM system is designed around six basic components:

Acceptable pest levels: The emphasis is on control, not eradication. IPM holds that wiping an entire pest
population is often impossible, and the attempt can be expensive and environmentally unsafe. IPM
programmes first work to establish acceptable pest levels, called action thresholds, and apply controls if
those thresholds are crossed. These thresholds are pest and site specific, meaning that it may not be
acceptable at one site to have a weed such as white clover, but at another site it may not be acceptable.
By allowing a pest population to survive at a reasonable threshold, selection pressure is reduced. This
stops the pest gaining resistance to chemicals produced by the plant or applied to the crops. If many of
the pests are killed then any that have resistance to the chemical will form the genetic basis of the
future, more resistant, population. By not killing all the pests there are some un-resistant pests left that
will dilute any resistant genes that appear.

Preventive cultural practices: Selecting varieties best for local growing conditions, and maintaining
healthy crops, is the first line of defense, together with plant quarantine and ‘cultural techniques’ such
as crop sanitation (e.g. removal of diseased plants to prevent spread of infection).
Monitoring: Regular observation is the cornerstone of IPM. Observation is broken into two steps first;
inspection and second, identification. Visual inspection, insect and spore traps, and other measurement
methods and monitoring tools are used to monitor pest levels. Accurate pest identification is critical to
a successful IPM program. Records-keeping is essential, as is a thorough knowledge of the behavior and
reproductive cycles of target pests. Since insects are cold-blooded, their physical development cycles
modeled in terms of degree days. Monitor the degree days of environment to determine when is the
optimal time for specific insect’s outbreak.

Mechanical controls: Should a pest reach an unacceptable level, mechanical methods are the first
options to consider. They include simple hand-picking, erecting insect barriers, using traps, vacuuming
and tillage to disrupt breeding.

Biological controls: Natural biological processes and materials can provide control, with minimal
environmental impact, and often of low cost. The main focus here is promoting beneficial insects that
eat target pests. Biological insecticides, derived from naturally occurring microorganisms (e.g.: nicotine,
pyrethrum and insect juvenile hormone analogues), but the toxophore or active component may be
altered to provide increased biological activity or stability. Further ‘biology-based’ or ‘ecological’
techniques are under evaluation.

An IPM regime can be quite simple or sophisticated. Historically, the main focus of IPM programmes was
on agricultural insect pests. Although originally developed for agricultural pest management, IPM
programmes are now developed to encompass diseases, weeds, and other pests that interfere with the
management objectives of sites such as residential and commercial structures, lawn and turf areas, and
home and community gardens.

Process

IPM is applicable to all types of agriculture and sites such as residential and commercial
structures, lawn and turf areas, and home and community gardens. Reliance on knowledge, experience,
observation, and integration of multiple techniques makes IPM a perfect fit for organic farming (sans
artificial pesticide application). For large-scale, chemical-based farms, IPM can reduce human and
environmental exposure to hazardous chemicals, and potentially lower overall costs of pesticide
application material and labor.

Proper identification of pest – What is it? Cases of mistaken identity may result in ineffective actions. If
plant damage to over-watering are mistaken for fungal infection, spray costs can be incurred, and the
plant is no better off.

Learn pest and host life cycle and biology. At the time you see a pest, it may be too late to do much
about it except maybe spray with a pesticide. Often, there is another stage of the life cycle that is
susceptible to preventative actions. For example, weeds producing from last year’s seed can be
prevented with mulches. Also, learning what pest needs to survive allows you to remove these.

Monitor or sample environment for pest population – How many are here? Preventative actions must
be taken at the correct time if they are to be effective. For this reason, once the pest is correctly
identified, monitoring must begin before it becomes a problem. For example, in school cafeterias where
roaches may be expected to appear, sticky traps are set out before school starts. Traps are checked at
regular intervals so populations can be monitored and controlled before they get out of hand. Some
factors to consider and monitor include: Is the pest present/absent? What is the distribution – all over
or only in certain spots? Is the pest population increasing or decreasing.

Established action threshold (economic, health or aesthetic) – How many are too many? In some cases,
a certain number of pests can be tolerated. Soybeans are quite tolerant of defoliation, so if there are
few caterpillars in the field and their population is not increasing dramatically, there is not necessarily
any action necessary. Conversely, there is a point at which action must be taken to control cost. For the
farmer, that point is the one at which the cost of damage by the pest is more than the cost of control.
This is an economic threshold. Tolerance of pests varies also by whether or not they are a health hazard
(low tolerance) or merely a cosmetic damage (high tolerance in a non-commercial situation).

Different sites may also have varying requirements based on specific areas. White clover may be
perfectly acceptable on the sides of a tee box on a golf course, but unacceptable in the fairway where it
could cause confusion in the field of play.

Choose an appropriate combination of management tactics for any pest situation, there will be several
options to consider. Options include mechanical or physical control, cultural controls, biological controls
and chemicals controls. Mechanical or physical controls include picking pests off plant, or using netting
or other material to exclude pests such as birds from grapes or rodents from structures. Cultural
controlsinclude keeping an area free of conductive conditions by removing or storing waste properly,
removing diseased areas of plants properly. Biological controls can be support either through
conservation of natural predators or augmentation of natural predators.

Augmentative control includes the introduction of naturally occurring predators at either an inundative
or inoculative level. An inundative release would be one that seeks to inundative a site with a pest’s
predator to impact the pest population. An inoculative release would be a smaller number of pest
predators to supplement the natural population and provide ongoing control. Chemical controls would
include horticultural oils or the application of pesticides such as insecticides and herbicides. A Green
Pest Management IPM program would use pesticides derived from plants, such as botanicals, or other
naturally occurring materials.

Evaluate results – How did it work? Evaluation is often one of the most important steps. This is the
process to review an IPM program and the results it generated. Asking the following questions is useful:
Did actions have the desired effect? Was the pest prevented or managed to farmer satisfaction? Was
the method itself satisfactory? Were there any unintended side effects? What can be done in the future
for this pest situation? Understanding the effectiveness of the IPM program allows the site manager to
make modifications to the IPM plan prior to pests reaching the action threshold and requiring action
again.