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The culture of entomophagous arthropods
for biological control involves many of the same factors as those for
producing host organisms. However, there are a number of special
considerations necessary. Rarely do parasitoids of predaceous insects or
hyperparasitoids of primary parasitoids cause impediments. Such difficulties
usually occur only when a parasitoid or predator colony is first started from
field collected material. Chianese (1985) recommended that a colony of Cotesia
(Apanteles) melanoscelus (Ratzeburg), a braconid parasitoid
of gypsy moth larvae, be established from the first seasonal field generation
to minimize hyperparasitism problems. Field collected cocoons should be
isolated individually so that any hyperparasitoids present will not attack
other cocoons. His advice refers equally to aphid parasitoids which
frequently suffer from increasing hyperparasitism as the growing season
progresses. Entomophages may also be affected by microorganisms.
Goodwin (1984) remarked that parasitoids which develop in microbially diseased
hosts may or may not contract the disease, but nonetheless would suffer
physiologically and be less fit. The bacterium Serratia marcescens
causes mortality in culture of the tachinid Lixophaga diatraeae
on the sugarcane borer (King & Hartlay 1985a,c). Disease control is
obtained by soaking the maggots in 0.7% formalin solution for five minutes
prior to their parasitization of the borers and by disinfecting the puparia
with 1% sodium hypochlorite solution for three minutes (King & Hartley
1985c). Goodwin (1984) listed some rickettsiae
and closely related forms found to be pathogenic for entomophages. Chlamydiae
causing stunting have been found in the nerve tissue of the predaceous
coccinellid Coccinella septempunctata (L.) and in the
ichneumonid alfalfa weevil parasitoid Bathyplectes sp. Also Enterella
stethorae causes an acute disease in the predaceous coccinellid Stethorus
sp. by destruction of the midgut epithelium. Brooks (1974) reported that protozoans
had been found in parasitic Hymenoptera. Own & Brooks (1986) showed that Pediobius
foveolatus, and egg parasitoid of the Mexican bean beetle, is
highly susceptible to two protozoans. Since P. foveolatus
is commonly mass produced in the eastern United States for annual inoculative
releases, the protozoans can be a serious limiting factor in parasitoid
production. Although some microorganisms are
detrimental to entomophages, Goodwin (1984) noted that entomophagous
parasitoids may have symbiotic microorganisms enabling them to successfully
attack hosts. For example, Stoltz & Vinson (1979) showed that viruses
present in the oviducts of braconids and ichneumonids suppressed the
defensive hemocoelic encapsulation process in their hosts. Microorganisms also seem to be involved
directly in reproductive processes of parasitoids, as recently suggested for
pteromalids (Legner 1987b, d ) and trichogrammatids (Stouthamer et
al. 1990). Whether only chromosomal inheritance is involved in the
acquisition of thelytoky in Hymenoptera is uncertain, and there is mounting
evidence to suggest that process may also include extrachromosomal phenomena
such as infection by microorganisms (e.g., spiroplasmas) in the
reproductive tract (Legner 1987a , 1987b, 1987c ). Recent experimental data
further points out the probability of microorganisms being involved in thelytoky.
This work with Trichogramma employed three kinds of antibiotics
and high temperature to cure populations of their thelytoky (Stouthamer
et al. 1990). Pathogens are more a problem with
weed-feeding insects used for biological control than with parasitoid and
predators. Etzel et al. (1981) eliminated a Nosema disease interfering
with production of the weed-feeding chrysomelid Galeruca rufa,
and Bucher & Harris (1961) noted other cases. It is well to bear in mind that some
microbial contaminants can be hazardous because of pathogenicity and
allergenicity to rearing personnel (Sikorowski 1984). For example,
respiratory and intestinal allergic reactions have been associated with
yeasts present in the cocoons of the parasitoids Nasonia vitripennis
and Muscidifurax spp. (H. G. Wylie pers. commun., Legner
unpub.). Because of this and because of the problems caused by microorganisms
in insect rearing, Sikorowski (1984) emphasized that basic sanitation must be
an essential feature of insect mass-rearing programs. Genetic Changes Waage et al. (1985) noted that
laboratory genetic changes resulting from long-term culturing of entomophages
could unfavorably affect environmental fitness and behavioral characteristics
such as host finding, host acceptance and host preference. The genetic
constitution of biological control agents and changes therein, have been
considered by Legner & Warkentin 1985).as well as by
others, with regard to the potential for successful introduction of
parasitoids and predators into new areas. Considerable controversy exists as
to the influences of homozygosity and heterozygosity on the fitness and
capacity of biological control agents to be effective. There are theoretical
considerations that do not entirely support the requirement of heterozygosity
(Remington 1968, Legner 1979a, Legner &
Warkentin 1985). The fact that only small numbers of
insect parasitoids or predators are introduced into a new area does not mean
that they will not become successfully established and effective in
controlling a pest. There are examples of small numbers of pests, even single
inseminated females, invading new areas and establishing successful populations
(Bartlett 1985, Remington 1968). With respect to biological control agents,
Clausen (1977) noted the example of the predaceous coccinellid Scymnus
smithianus Clausen & Berry, imported from Sumatra to Cuba in
1930 for citrus blackfly, Aleurocanthus woglumi Ashby,
control. Insectary stock of the coccinellid declined to a single female, but
then increased to the point that releases at a number of locations in 1931
resulted in establishment. Fitness and genetic plasticity in insects
reared for field release are essential for them to exist in the environment
(Bartlett 1985, Joslyn 1984). These workers both noted that insects in
laboratory colonization will unavoidably become domesticated and lose
genotypes (variability), depending on a variety of factors. Bartlett (1984)
indicated that decreased fitness is often observed when homozygosity
increases in a culture (i.e., genetic plasticity decreases). Such insects
will be less likely to adapt to a natural environment than those with a high
fitness and high heterozygosity. it is important to observe that the total
amount of genetic variability in a laboratory culture may not change greatly
over time. Rather the important changes that can alter fitness are in
distribution of the alleles and in their arrangement (Bartlett 1985). Myers
& Sabath (1980) and Remington (1968) offer a number of interesting
theoretical viewpoints on colonizing insects and hetero- and homozygosity, as
noted in the previous section on Genetic Considerations (ENT229.3). Inbreeding & Out
Breeding--Inbreeding in beneficial insects that are typically out
crossing can have harmful consequences because of increased homozygosity of
recessive lethal and semi-lethal alleles.
This effect is often referred to as inbreeding depression and is well
known for diploid animals. In
haplodiploid animals, such as parasitic insects, the negative effects of
inbreeding seem to be reduced due to the elimination of deleterious recessive
alleles via haploid males and the generation of female-biased sex ratios
(Hamilton 1967, Roush 1990, Werren 1993).
It has recently been suggested that chronic inbreeding might be
responsible for the evolution of haplodiploidy (Smith 2000). Although there has been a
lot of discussion of the probable impact of inbreeding on parasitic insects
with respect to biological control (Waage et al. 1985, Roush 1990, Unruh
& Messing 1993, Etzel & Legner 1999), the hypothesis that parasitic Hymenoptera may not
suffer inbreeding depression has been tested in only a very few species of
non aculeate Hymenoptera (e.g., Muscidifurax species by Legner (1972), Trichogramma
species by Sorati et al. (1996), and Cothonaspis boulardi by Biemont
& Bouletreau (1980). As suggested
by the theory, significant inbreeding depression was not found in any of
these species. It should be
emphasized that within the non-aculeate parasitic Hymenoptera, sex
determination in the Ichneumonoidea dep4ends on heterozygosity at a single
locus, single locus complementary sex determination (CDS, often referred to
as the Whiting scheme), so members of this group are known to suffer
inbreeding depression via the development of nonfunctional diploid males
(Whiting 1943, Hedderwick et al. 1988).
CSD has been demonstrated or implicated in the Ichneumonidae,
Braconidae, Apidae, Diprionidae and Megachilidae, but is not known to occur
in other groups of parasitic Hymenoptera (Unruh & Messing 1993). Cook (1993) testged for inbreeding
depression and CSD in the bethylid Goniozus nephantidis (an aculeate)
and found no evidence for inbreeding depression or for either single-locus or
multiple-locus CSD after 22 generations of inbreeding.
However, outbreeding depression has rarely been studied in parasitic
insects. Chronic inbreeding can result in co-adapted gene complexes that may
be disrupted by outbreeding (Dobzhansky 1948). Outbreeding depression is known to occur in some plants
(Schneller 1996, Schierup & Christiansen 1996, Waser 1993, Waser et al.
2000), invertebrates (Chen 1993, De Meester 1993, Palmer & Edmands 2000)
and vertebrates (Coulson et al. 1999, Bross 2000). In insects a study of Drosophila Montana strains
revealed that out breeding resulted in an alteration in male courtship and a
reduction in fitness (Aspi 2000).
With parasitoids and biological control, Force (1967) suggested that
the crossing of geographic strains could in some cases reduce the fitness of
locally adapted parasitoid populations, and Unruh & Messing (1993) argued
that out breeding depression may be a problem in biological control
efforts. However, because out
breeding depression has never been examined in any species of parasitic
Hymenoptera, it is presently impossible to address the various concerns that
have been raised. Types of
Changes in Cultures.--Inbreeding
depression, producing a reduction of physiological vigor and reproductive
capacity, is likely to occur in small laboratory populations over time
(Collins 1984). Inbreeding and genetic deterioration of insectary stocks of
parasitoids and predators is always a concern, particularly when cultures are
initiated from very few individuals or are maintained as small colonies for
extended periods. In a laboratory study Legner (1979a) studied the influences of
inbreeding and extended culturing on the reproductive potential of the
pteromalids Muscidifurax raptor Girault & sanders
and Muscidifurax zaraptor Kogan & Legner, finding
that with M. raptor, the reproductive capacity of two inbred
lines established from an old laboratory culture was not reduced. There was
also no difference in longevity and progeny production between the three
cultures. With M. zaraptor, two of seven inbred lines taken from a
standard laboratory culture demonstrated significantly greater intrinsic
rates of increase (rm) in three comparisons with domestic culture.
The other inbred lines sometimes exhibited rm values significantly
lower than that of the standard culture. However, the average of the rm
values of the seven inbred lines was very close to that of the standard
colony. This indicated that the inbred lines represented a sample of
genotypes from the standard colony, and that considerable genetic
heterogeneity was maintained. Legner (1979a) also compared
a Danish strain of M. raptor with an American wild strain, an
old domestic laboratory culture, and two inbred lines started from the
domestic culture. The Danish strain had markedly lower reproductive rates
than any of the other cultures. This finding emphasizes the importance of the
initial genetic composition of a laboratory culture of parasitoids or
predators. Certain inbred long-cultured parasitoids or predators may be
better candidates for field releases than are recently collected wild
strains, or than specimens from the culture from which the inbred lines were
initiated (Legner 1979a). However,
generally speaking, laboratory inbreeding is usually considered to be
deleterious to field establishment. Sex Ratios.--Waage et al.
(1985) noted that inbreeding can have a pronounced effect on the sex ratios
of parasitic wasps and predaceous mites, which seems related to the
haplo-diploid system of sex determination. Femaleness evidently is determined
by heterozygosity at a number of chromosomal loci that collectively govern
sex. A haploid individual is hemizygous (has only one of every pair of
chromosomes found in a diploid individual) and therefore is effectively
"homozygous" (the single allele at each genetic locus has the same
effect as two identical alleles at the same locus in a diploid individual).
With inbreeding, homozygosity will increase and may result in formation of
diploid males, with a consequential male bias in the sex ratio. However, the
effect of inbreeding on sex determination also probably depends on the degree
of natural inbreeding (Waage et al. 1985), so that gregarious wasps with a
natural sex ratio biased toward females will likely suffer less from
laboratory inbreeding than will solitary wasps with a 1:1 natural sex ratio
(Waage 1982). The extensive inbreeding performed with solitary species of the
genus Muscidifurax (Legner 1979a) did not skew
the sex ratio towards males, however. Causes of Genetic
Change Founder Effect
(Field Sampling).--The loss of genotypes in laboratory
culture is considered especially dependent on the founder effect (Bartlett 1984),
a random event where there is initially a very restricted gene pool resulting
from the selection of few founder individuals (Joslyn 1984). The initial
variation in the new laboratory culture will depend on the size of the field
sample, in terms of both the number of individuals collected, and the number
of localities from which the specimens are collected, since "the larger
the original sample, the smaller the deviations of the sample from the
original gene frequencies; the smaller the sample, the greater the observed
deviation" (Bartlett 1985). Natural Selection
in the Laboratory.--Bartlett
(1984) noted that the variability and quality of the laboratory environment
is another important cause of loss of genetic variability in culture.
Provision of a constant favorable laboratory environment changes the criteria
that determine fitness and might eventually drastically change the capability
of laboratory reared insects to persist in nature when field released. He
further observed that space restrictions in laboratory rearing units could
affect insect behavior, including mating, oviposition and dispersal, and
might result in a genetically selective impact on that behavior. Once the
culture is begun, there will be a natural selection for individuals that are
most fit for the artificial environmental conditions of the laboratory, with
a resulting decrease of genotypes that are not favored in the laboratory, but
may be favored in nature (Bartlett 1985). Directional selection in the laboratory
culture, whether planned or unplanned, can be expected to cause a loss of
alleles that contribute to fitness in nature, particularly if environmental
conditions in the insectary are not varied (Joslyn 1984). Genetic Drift.--Part of the
loss of genetic variability in the laboratory is the result of genetic drift,
a random event that occurs as the laboratory population fluctuates in size
(Joslyn 1984). Alleles are lost at rates proportional to decreasing
population size. Mating Type.--According to
Bartlett (1985) the type of mating system will not change the gene
frequencies in the laboratory population, but will change the genotypic frequencies, i.e., the relative degrees
of homozygosity and heterozygosity. Random mating in a large laboratory
population can be expected to maintain existing genotypic frequencies, but
inbreeding will lead to increased homozygosity, which could result in
decreased fitness. Outbreeding can effect increased heterozygosity and the
accompanying hybrid vigor or degeneration, depending on the degree of integration
or disparity between the gene pools of the two populations. Joslyn (1984) described inbreeding as a
directional event which increases homozygosity across all loci, resulting in
genetic decay because a decrease in heterozygosity may cause lower fitness
through loss of hybrid vigor, and through inbreeding depression produced by
any harmful homozygous recessive genes. Experimental data with Hymenoptera,
however, is grossly lacking, most of the knowledge of genetics and probable outcomes
in culture being derived from other animals. In arrhenotokous parasitoids
where males issuing in the F1 female generation carry the P1
female genome, there is a high potential for the production of a greater
proportion of parental genotypes in succeeding generations, which in turn
sets up conditions for additional F1 hybrids and their
accompanying heterosis (Legner 1988b, 1988c). Most laboratory environmental
conditions, being generally uniform and favoring maximum reproduction, do not
expose the animals to selection for many characteristics that are required
for the species to persist in nature. Allele loss in culture would be due
almost exclusively to founder effects. Therefore, varying insectary
conditions, as suggested by Joslyn (1984) may actually result in a further
loss of alleles that otherwise might have remained in the culture. Thelytokous parthenogenetic
entomophages are a special case in terms of genetic selection in the
laboratory. Selection might be expected to occur very quickly as with aphids.
Forbes et al. (1985) cautioned that laboratory selection of aphids adapted to
artificial rearing occurs rapidly as a result of parthenogenesis, since no
sexual genetic recombination occurs under the usual rearing conditions for
aphids. However, genetic recombination may
occur in thelytokous Hymenoptera. In the aphelinid Aphytis mytilaspidis
(LeBaron) the greatest barrier to interbreeding seems to be the precopulation
period, where arrhenotokous males spend a greater length of time in courtship
with thelytokous females (Rossler & DeBach 1972). There is a tendency for
the thelytokous form to be replaced eventually by arrhenotokous forms; and
the persistence of thelytoky seems dependent on the hybrids finding suitable
environmental conditions, such as host type (Rossler & DeBach 1972). As
previously discussed, the question of whether only chromosomal inheritance is
involved in the acquisition of thelytoky is uncertain, since evidence is
accumulating that the process obviously includes extrachromosomal phenomena
(Legner 1987a, 1987b, 1987c). Recombination.--If sampling
bias or selection has reduced the number of alleles present at any locus, the
role of genetic recombination in increasing the number of genotypes will be
correspondingly less because of the fewer alleles that can be recombined.
(Bartlett 1985). The recently discovered complication in understanding
genetic events in the parasitic Hymenoptera shown by Legner (1988c, 1988d), which involves gene expression
in a cautionary manner (wary genes), and a stepwise pathway to
inheritance termed accretive inheritance, points to unique genetic
processes in Hymenoptera. Much parasitic Hymenoptera behavior is controlled
by polygenes, with quantitative inheritance involving both extranuclear
and chromosomal changes. In Muscidifurax raptorellus, a
South American species parasitizing synanthropic Diptera, male polygenes
coded for fecundity, gregarious oviposition, and other reproductive behavior,
are first partially expressed in the inseminated female by an
extranuclear phase of inheritance. Such genes acquired from the male and
partially expressed in mated females before subsequent incorporation into
progeny genomes have been called wary genes (Legner 1988c,d). Mated
female behavioral changes are permanent, with no switchback following a
second mating with a male possessing a different genotype. In hybridization,
wary genes may serve to quicken evolution by allowing natural selection for
both nonlethal undesirable and desirable characteristics to begin action in
the parental generation. Wary genes detrimental to the hybrid population
might thus be more prone to elimination, and beneficial ones may be expressed
in the mother before the appearance of her active progeny (Legner 1988a, 1988c, 1988d). Mutation.--The effect of
mutation on increasing the genetic complement and variability of a laboratory
culture is very low because natural mutation rates are also low and
beneficial mutants spread slowly through a population (Bartlett 1985). Retaining Genetic Diversity Bartlett (1984, 1985) and Joslyn (1984)
basically suggested the same three most important methods for retaining
genetic variability in laboratory cultures of insects. Bartlett (1984)
recommended to (1) begin the culture with as many founding individuals as
possible, (2) use an environment set to maintain the fittest genotype by
using appropriate fluctuating temperatures and photoperiods throughout the
life cycle and (3) maintain separate culture strains under unique conditions
and cross these systematically to increase F1 variability.
Considerations of periodic culture infusions and monitoring of colony quality
should also be included. Culture Initiation.--The
collection of large numbers of pest insects to initiate a laboratory culture
is not difficult, the main restriction being seasonal. However, the
collection of large numbers of entomophages for classical biological control
introduction work is the exception rather than the rule. An effective
entomophage which has reduced its host population to low subeconomic levels
will be difficult to find. Thus, foreign exploration trips frequently yield very
few specimens and it is not unusual for laboratory cultures to be initiated
from less than 10 individuals. From the practical point of view the best
procedure is to process the agents through quarantine promptly, and to begin
field releases quickly to minimize the genetic loss of alleles. Naturally,
cultures will have to be maintained in the insectary for further releases. Obviously field collecting the largest
number of individuals that can be accommodated in the laboratory will
maximize the number of feral population alleles present in the founder
laboratory culture and will consequently minimize the magnitude of genetic
drift and inbreeding (Bartlett 1985). However, the individuals from the
different areas must be reproductively compatible (Joslyn 1984). Bartlett
(1985) illustrated the mathematical procedure for estimating the number of
field specimens that should be collected to obtain a rare allele in the
founder laboratory culture. Culture Maintenance
& Size.--Unruh et al (1983) believed that the
best way to retain heterozygosity and prevent genetic drift in laboratory
cultures is to maintain relatively large population sizes (>100
individuals) in the laboratory. After studying heterozygosity and effective
size in laboratory populations of the aphidiid Aphidius ervi
Haliday, Unruh et al. (1983) warned that genetic drift and loss of
heterozygosity is more severe than would be expected from the number of
individuals used to maintain cultures. Discussed were factors that make
effective population sizes much smaller than apparent, including generation
fluctuations, haplodiploidy, sex linkage, high variation in parental progeny
production and highly skewed sex ratios. Wheeler (1984) suggested that in order
to start a laboratory insect culture, 300-500 individuals should be
collected, and that collecting only a single developmental stage should
increase the chances of obtaining maximum homozygosity, if that is the goal.
Conversely, collecting a variety of stages at the same time should increase
the chances of establishing a high heterozygosity in a new colony. Culture Maintenance
& Inbred Lines.--Another
procedure for maintaining genetic variability in cultures recommended by
Bartlett (1985) and Joslyn (1984) is to develop and maintain a number of
inbred subcultures. Joslyn (1984) suggested that the subcultures be exposed
to different variable rearing environments on a rotating schedule.
Individuals from these subcultures are systematically outbred to achieve
hybrid vigor in progeny that are to be field released. It is necessary,
however, to periodically outcross and reisolate the strains. It is
recommended that this be done every 4-6 generations to prevent development of
isolating mechanisms that could result in hybrid degeneration if inbred lines
were held too long before being outcrossed. Joslyn (1984) also indicated the
importance of maintaining a high effective population size (e.g., 500) in
each subculture in order to reduce the possible decay or variability caused
by random drift and inbreeding. Legner & Warkentin (1985).have also suggested that one
way to keep a broader range of genetic variability is to culture several
separate, noninterbreeding lines from an explorer's initial acquisitions, especially
as severe founder effects, reducing genetic heterogeneity, occur in the first
few generations of culture (Legner 1979a, Unruh et al.
1983). Although each culture might assume great homozygosity in time, different
cultures would be homozygous for different characteristics through random
founder effects. Specimens from the lines could then, if desired, be combined
prior to field release in order to increase heterozygosity. However, there is
insufficient data to decide whether homo- or heterozygosity is preferred in
establishing beneficial species (Legner & Warkentin 1985).). This procedure of maintaining many
inbred lines with periodic attempted restoration of variability by mixing was
thought be an uncertain technique by Unruh et al. (1983), who preferred the
maintenance of large cultures. Unfortunately, since exotic collections of
entomophages might yield very few founder individuals, the maintenance of
subcultures might be necessitated in an attempt to sustain genetic
variability. Culture Maintenance
& Periodic Infusions.--It has been
suggested that native individuals should periodically be introduced into
laboratory cultures to reduce loss of genetic variability from drift,
selection and inbreeding. Joslyn (1984) commented that wild specimens may
occasionally be added to the laboratory culture to simulate
"migration," with the addition of new alleles. This introduction
must be proportionately large enough so that new alleles can be fixed in the
population. The possibility of introducing an unwanted insect disease into
the culture must be considered with this method. Bartlett (1984) noted that
the effectiveness of such a procedure depends on regular introductions of
relatively large numbers of individuals, preferably obtained from the
geographical area where the laboratory culture was originally collected,
because of possible incompatibility in intraspecific crosses of individuals
from spatially and/or temporally separated populations. This idea is
supported by recent data on Muscidifurax parasitoids (Legner 1988c). Bartlett
(1984) also remarked that if native alleles are not introduced regularly,
selection will reestablish the original laboratory gene frequencies. Further,
the combined processes of selection and inbreeding will have definite rapid
effects on changing gene frequencies. King & Morrison (1984), feeling
that periodic replacement of a culture with field-collected material is
costly, recommended routine monitoring of behavioral traits coupled with
techniques to maintain essential characteristics and to even select for
desirable traits in mass produced entomophages. However, in the mass
production of Trichogramma spp., they stressed the need for
annual culture replacement. They further recommended that culture replacement
should only be from the target host on the affected crop, and in large enough
numbers (>2,000) to insure a broad genetic base. Culture Maintenance
& Environmental Conditioning.--Once the
founder individuals have been collected, Bartlett (1985) recommended that
they be reared in the laboratory as nearly as possible in natural
environmental regimes and densities. Joslyn (1984) indicated that, "A
static environment leads to a static genotype and ultimately to less fit
insects." This laboratory population which is exposed to variable
"natural" environmental regimes must be large enough to allow
random mating to preserve genotypic variability. In the situation where few
entomophages can be field collected, at least the laboratory environment can
be varied to maintain a pressure for adaptive fitness. Culture Maintenance
& Monitoring.--In obtaining and maintaining
genetic variability in the laboratory, it is first important to study the
biology and behavior of a species very well so that information is available
to compare attributes of the wild and domesticated populations, which will
thereby enable detection of genetic differences (Bartlett 1985). Singh &
Ashby (1985) observed that in establishing a new laboratory culture, genetic
selection of developmental traits such as a shortened life cycle begins first
in the early laboratory generations. Behavioral, physiological and
biochemical selection follows. Therefore, standards and tolerances for insect
quality testing should be established when newly colonized insects are still
genetically close to the wild population. Hsin & Getz (1988) suggested
that monitoring developmental variation in insectary cultures with an
appropriate life table might be very useful in maintaining genetic viability. Culture Maintenance
Following Field Establishment.--As soon as a
beneficial organism is field established following releases, it is desirable
to collect as many individuals as often as possible to use for further
laboratory culturing and/or of translocation to other localities. A
successfully field-established organism is oftentimes more vigorous after
confronting the hazards of nature during reproduction and development than is
one raised under continuous insectary conditions. Waage et al (1985) advocated the use of
this procedure, believing that the preservation of genetic variability and
insect quality required that entomophages should be reared in the insectary
as little as possible before being field liberated. Field collections of
established entomophages should serve as sources for initiating new
laboratory cultures. They further indicated that release areas for the
entomophages should be matched climatically as closely as possible to the
source areas for greater ease of establishment. Following the establishment of the
braconid Microctonus aethiopoides (Nees) on the Egyptian
alfalfa weevil in California, parasitized weevils were collected from
aestivation sites on trees and used as sources for further field distribution
of the parasitoids. Two primary benefits of this technique were that the
vigor and fitness of the new individuals to be distribution was high in
comparison to individuals removed from an old insectary culture; and costs
were reduced by using nature as an insectary (Etzel & Legner 1999.). Also, the Animal and Plant
Health Inspection Service (APHIS) of the United States Department of
Agriculture similarly distributed the ichneumonid Bathyplectes anurus
(Thoms.) for alfalfa weevil biological control. This parasitoid has only one
generation per year in nature, with two diapauses in its life cycle, and is
thus very difficult to culture. Field establishment in the eastern United
States required direct field releases annually of imported wild stock from
Europe. After several years B. anurus finally increased to
sufficient abundance at some sites to enable the collection of large numbers
which could be redistributed to other areas. A larger program was subsequently
developed by APHIS to effect a more general dispersal of this and other
species. An important consideration in relying
on the field redistribution procedure is that if the entomophage is
eventually successful in controlling a pest population below the economic
threshold, there may be only a few years when the pest population is still
large enough for the entomophage to be collected with ease. Culture & Synthetic
Diet Singh (1984) reviewed recent work on
rearing entomophagous parasitoids and predators on synthetic diet. Some
success has been obtained with two ichneumonids, Itoplactis conquisitor
(Say) and Exeristes roborator (F.), one trichogrammatid,
Trichogramma pretiosum (Riley); and Pteromalus
puparum. Nettles (1986) was able to obtain relatively good yields
of the tachinid Eucelatoria bryani, a specific
parasitoid of Heliothis spp., by raising it first in Heliothis
virescens for 20-28 hours and then on an artificial diet for the
common green lacewing. Waage et al (1985) noted that the greatest success in
the development of artificial diets for entomophagous parasitoids has been
with polyphagous parasitic wasps. Although some progress has been made on
rearing predaceous entomophages on artificial diets, production for field
release is still performed on living or dead hosts. Entomophage / Host Interactions (Host
Type & Quality) It is apparent that entomophage quality
can be dependent on host quality and in turn on host food quality.
Interactions between plants, insect herbivores and natural enemies were
reviewed by Price et al. (1980), which are similar interactions between
prepared diets, herbivores and natural enemies. Singh (1984) referred to two
such examples. The braconid Apanteles chilonus Munakata
was adversely affected when its host, the Asiatic rice borer, was raised on
artificial diet. Similarly the tachinid Lixophaga diatraeae
declined in quality when reared on greater was moth larvae, Galleria
mellonella (L.) unless the larval beeswax/pollen diet was
supplemented with vitamin E or wheat germ. O'Dell et al. (1984) reported that of
three artificial diets suitable for gypsy moth, only one was acceptable when
the braconid Rogas lymantriae Watanabe was to be
produced on gypsy moth larvae. On the other two diets the host larvae died
shortly after parasitoid oviposition. Moore et al. (1985) reported that the
same parasitoid had significantly higher female weights when reared on gypsy
moth larvae grown on a high wheat germ diet than on a commercial diet. With Brachymeria
intermedia, Greenblatt & Barbosa (1981) discovered that the
largest and heaviest individuals were obtained when gypsy moth host larvae
were fed on red oak foliage rather than on three other tree species. Artificial diet for the host insect can
sometimes result in greater parasitoid production. Beach & Todd (1986)
found that parasitized soybean looker larvae, Pseudoplusia includens
(Walker), fed on a susceptible soybean variety yielded 2.5 times more parasitoids
than on a resistant variety. When the host was fed an artificial diet
parasitoid production increased twofold. Other studies involving the effects
of resistant plant varieties on hosts and parasitoids are Grant & Shepard
(1985), Obrycki & Tauber (1984), Obrycki et al. (1985), Orr & Boethel
(1985), Orr et al. (1985), Powell & Lambert (1984), Yanes & Boethel
(1983). Although an artificial diet may result
in larger, more fecund phytophages, there may be disadvantages if the
phytophages are to be released for weed control. Frick & Wilson (1982)
mass reared the weed-feeding tortricid Bactra verutana
Zeller on a prepared diet and obtained adults that were 60% larger and twice
as fecund as those reared on nutsedge plants. However, there were indications
that the field flight capabilities were not as great with the diet-reared
insects. Flanders (1984) evaluated five
varieties of bean plants for rearing the Mexican bean beetle, which was
required for producing the parasitoid Pediobius foveolatus.
Of the two varieties that were essentially equivalent in providing the
highest net reproductive rate for the bean beetle, the preferred one had
superior growth characteristics and was consequently the most economical to
produce. Jalali et al. (1988) demonstrated the preference of the braconid Cotesia
kazak Telenga to attack Heliothis armigera
Hübner) larvae on cotton, tomato or okra, than on dolichos, pigeon pea, cow
pea or chick pea. The fecundity of the parasitoid also was statistically
greater on the first group of plants than on the second group. Similarly,
Kumar et al. (1988) showed that the aphidiid Trioxys indicus
Subba Rao & Sharma produced more progeny when its host aphid, Aphis
craccivora Koch, was reared on the plant Cajanus cajan
Millsp., than on Dolichos lablab or Solanum melongena. Host plants can also affect the
suitability of phytophages for predators such as coccinellids (Hodek 1973),
the lygaeid Geocoris punctipes (Say) (Rogers & Sullivan 1986), and
Scolothrips longicornis Priesner (Thysanoptera: Thripidae),
a thrips predaceous on tetranychid mites (Sengorca & Gerlack 1984).
Predaceous mites can be affected as well. deMoraes & McMurtry (1987)
found an indication that adult female Phytoseiulus persimillis
Athias-Henriot gained more weight when fed adult female two-spotted spider
mites, Tetranychus urticae Koch reared on lima bean, Phaseolus
vulgaris L., than on nightshade, Solanum douglasii
Dunal. Simmonds (1944) found that the encyrtid Comperiella bifasciata
Howard effected two to three times more parasitism when its host, the
California red scale, was reared on oranges rather than lemons. Chemicals in the host derived from its
food can affect an entomophage. Barbosa et al. (1982) noted that survival of Cotesia
(Apanteles) congregata (Say), a parasitoid of the
tobacco hornworm, was affected by the larval host nicotine level. Nutrition can affect susceptibility of
insects to pathogens and thereby the production of pathogens for microbial
control (Singh 1984). Shapiro et al. (1978) found that virus production was
most economical on gypsy moth larvae reared on a diet with high
concentrations of wheat germ. Although some adult entomophagous
insects require special food, most parasitic Hymenoptera that do not host-feed
can naturally produce mature eggs with a source of carbohydrate such as honey
(Waage et al. 1985). Morrison (1985a) used plump raisins or honey to feed
adult Trichogramma females. Munstermann & Leiser (1985) fed
adult predatory Toxorhynchites mosquitoes with diluted honey
absorbed onto strips of cellucotton or with raisins, apple slices or 15%
sucrose solution. Mendel (1988) showed that the longevities of one pteromalid
and two braconid parasitoids of scolytid bark beetles were directly related
to the provision of water and honey, and inversely related to temperature;
and that longevities of parasitoids given only water were directly related to
body size. More complex diets may be required for
adults of some predators. Morrison (1985b) used a diet of equal parts of
sucrose and yeast flakes moistened with enough water to make a thick paste,
for adults of the common green lacewing. This yeast was commercially cultured
on whey and therefore contained about 65% animal protein, which is necessary
for high fecundity. Other ingredients may occasionally be important in the
adult food used to raise entomophages. Moore (1985) reported that Nettles et
al. (1982) showed a synergistic effect of potassium chloride and magnesium
sulfate on oviposition by an insect parasitoid. Rearing can sometimes be simplified by
using factitious or unnatural hosts. Similarly,
a beneficial insect may have more than one kind of natural host, and one of
these may be easiest to raise in the laboratory. The quantity and quality of
parasitoid or predator progeny on different hosts seem to vary in the
insectary according to evolutionary contact, as mentioned by Legner &
Thompson (1977). Their study compared the
suitability of the potato tuberworm and the pink bollworm, the original
source host, as hosts for a braconid, Chelonus sp. nr. curvimaculatus
Cameron. It was found that after being reared for many generations on the
potato tuberworm, and then for one generation on pink bollworm, the
parasitoid was stimulated to increase its destruction of and fecundity on the
factitious host. This group of Chelonus parasitoids responds to
kairomones in the body scales of several lepidopterans (Chiri & Legner 1986), and might be characterized as
generalists. Fedde et al. (1982) reviewed guidelines
for choosing factitious or unnatural hosts to be used for rearing
hymenopterous parasitoids. They listed 43 examples of such hosts and
emphasized that ease of rearing was the most important consideration and that
potential factitious hosts should be tested and not prejudged as to possible
utility. Factitious hosts are ordinarily used
for laboratory rearing of Trichogramma spp., including the
Angoumois grain moth, Sitotroga cerealella (Olivier),
the Chinese oak silkworm, Antheraea perniyi
Guérin-Méneville, another type of silkworm, Samia cynthia ricini
(Boisduval), the Mediterranean flour moth and the rice moth, Corcyra
cephalonica (Stainton) (King & Morrison 1984). Additionally
the eggs of common noctuids and lepidopteran stored grain insects can often
be used to rear Trichogramma spp., although a few may be host
specific (Morrison 1985a). However, Trichogramma hosts must be
chosen with some care. For example, it was shown in testing the suitability
of pyralid species for Trichogramma evanescens Westwood,
Brower (1983) that the Mediterranean flour moth was by far a less suitable
host than the tobacco moth, Ephestia elutella (Hübner), the
almond moth, E. cautella (Walker), the raisin moth, Cadra
(Ephestia) figulilella (Gregson) or the Indian meal moth, Plodia
interpunctella (Hübner). Only 59% of exposed eggs yielded emerged
parasitoids. Ten percent of these were runts, whereas 88% of exposed almond
moth eggs produced parasitoids only 0.4% of them were runts. Trichogramma reared for long
periods on a factitious host can still maintain a natural host preference. Yu
et al. (1984) collected a strain of the egg parasitoid Trichogramma
minutum Riley from the codling moth, and then reared it for about
22 generations on eggs of the Mediterranean flour moth. Even after that time
period, the parasitoid still preferred eggs of the codling moth to eggs of
the Mediterranean flour moth. The greater wax moth also has been used
as a factitious host in the mass production of Lixophaga diatraea,
a tachinid fly which parasitizes sugarcane borer (King & Hartley 1985b,
Hartley et al. 1977, King et al. 1979). Mass rearing the wax moth was more
economical than rearing the natural host. Brachymeria intermedia, a parasitoid
of the gypsy moth, is also more easily reared in the insectary on the greater
wax moth (Palmer 1985). On the other hand, Rotheray et al. (1984) determined
that the gypsy moth was a better host for B. intermedia
because parasitoids produced from the wax moth were smaller and less able to
oviposit in gypsy moth pupae. Palmer (1985) found that even rearing Brachymeria
for 119 generations on the wax moth did not shift the host preference of the
parasitoid, since gypsy moth pupae were still readily attacked. However, B.
intermedia is apparently a generalist, and such a host preference
shift is not expected. King & Morrison (1984) noted that although it has
been shown that rearing a parasitoid on a factitious host eventually
increases its acceptance of that host, a parasitoid reared for only a few
generations on an unnatural host can still react strongly to its natural
host. Generalist parasitoids and predators
should of course be tested in the laboratory for potential field
effectiveness against a target pest. Drummond et al. (1984) reported that the
best host for rearing the spined soldier bug, Podisus maculiventris
(Say), was the greater wax moth, in contrast to the Mexican bean beetle, the
eastern tent caterpillar, Malacosoma americanum (F.), or
the Colorado potato beetle, Leptinotarsa decemlineata
(Say). The Colorado potato beetle in fact was a suboptimal host in comparison
to the other three prey species; therefore, the spined soldier bug apparently
has little potential as a field predator of this pest. Dead Hosts.--Many predators
and a few parasitoids can be reared in dead hosts, which simplifies
production (Waage et al. 1985). Etzel (1985) described the processing of
potato tuberworms used as food for producing certain coccinellids and
neuropterans. Sometimes insect eggs are
refrigerated for an extended period or frozen and then used to rear egg
parasitoids or predators. This prevents the hatching of larvae that might
interfere with entomophage production as well as enables the stockpiling of
host material. This technique is used with eggs of the Mediterranean flour
moth for rearing Trichogramma spp. (Etzel & Legner 1999). Similarly, Drooz & Weems
(1982) used freeze-killed eggs of Eutrapela clemataria (J.E.Smith)
to rear the encyrtid egg parasitoid Ooencyrtus ennomophagus
Yoshimoto. Prefrozen eggs of the southern green stink bug were utilized to
propagate the scelionid egg parasitoid Trissolcus basalis
(Wollaston) (Powell & Shepard 1982, Powell et al. 1981). As an alternative to prefreezing host
eggs, Gross (1988) irradiated eggs of the corn earworm with 25 krad of CO60
to prevent hatching, so they could be used to produce Trichogramma pretiosum.
Thorpe & Dively (1985) likewise irradiated tobacco budworm eggs for Trichogramma
production. Harwalkar et al. (1987) treated female potato tuberworms with
gamma irradiation, using the sterile eggs to rear Trichogramma brasiliensis
(Ashmead) with no ill effects. No significant differences were found between
the Trichogramma on irradiated or pre-frozen eggs. Kfir & Hamburg
(1988) used ultraviolet light to irradiate Heliothis armigera
eggs for two hours prior to parasitization by the egg parasitoids Telenomus
ullyetti Nixon and Trichogrammatoidea lutea
Girault. Morrison (1985b) froze eggs at -10°C for >24 hours in air-tight containers before using them as
food for rearing the predaceous common green lacewing. Likewise, Baumhover
(1985) used frozen tobacco hornworm eggs to rear the predaceous stilt bug, Jalysus
wickhami (VanDuzee). He found that these eggs can be stored for
two years at -23°C and still be suitable predator food. Prefrozen insect pupae
can be used for parasitoid production. Grant & Shepard (1987) raised the
chalcidid parasitoid Brachymeria ovata (Say) on
prefrozen pupae of seven species of Noctuidae. The velvet bean caterpillar
was the best host in the sense that acceptable pupae could be held in a
frozen state for a much longer period of time (up to 256 days versus 30-90
days for the other species). Prefrozen house fly pupae were used for
producing two pteromalid parasitoids, Muscidifurax zaraptor
(Petersen & Matthews 1984) and Pachycrepoideus vindemiae
(Rondani) (Pikens & Miller 1978). Spider mites have also been prefrozen
before use as food in phytoseiid mite production. In a biological control
program against the cassava green mite, Mononychellus tanajoa
(Bondar), a primary reason for prefreezing mass-produced spider mites for 18
hours was to eliminate contaminating predators, including phytoseiids, from
food used to produce desirable phytoseiids (Friese et al. 1987, Yaninek &
Aderoba 1986). Diapause
in the life cycle often interferes with entomophage production. For example,
Eskafi & Legner (1974) showed that
certain temperature and photoperiod combinations would induce adults and
progeny of females of the eye gnat parasitoid Hexacola sp. nr. websteri
(Crawford) to enter diapause. When larval parasitoids within their larval
hosts were exposed to a long photophase of 16 hours combined with a high
temperature of 32)C, the parasitoid prepupae entered a diapause state that
could be terminated by contact of the host puparia with moisture for a few
hours. However, this type of easily terminated diapause only occurred
following a parental generation that had been reared at 27°C with 14-hr light. If the parasitoid parental generation had
been reared at 32°c with 16-hr light, and the progeny
were held at 27°C with 14-hr light, then >90% of the
prepupal progeny entered diapause and could not be induced to terminate it by
exposure to moisture. When another set of progeny from the same parents were
reared at 32°C with 16-hr light, only 35% entered
diapause. This illustrates the great complexities
involved in determining which combinations of environmental regimes in the
insectary will prevent, induce or terminate diapause. Other examples include
the alfalfa weevil parasitoid system, in which both host and parasitoids have
complex diapauses. Chelonus spp. parasitoids of the pink
bollworm terminate diapause at different intervals (Legner 1979c ), and navel orangeworm
parasitoids where diapause seems triggered by hormonal changes in the host
situated at different latitudes (Legner 1983). Diapause in parasitoids is influenced
not only by environmental conditions but also by conditions of the host and
the host food plant. Diapause in the aphidiid Praon palitans
Muesebeck, is induced by its host, the spotted alfalfa aphid, Therioaphis
trifolii (Monell), which is in turn regulated by the
physiological state of the alfalfa plant (Clausen 1977). Diapause can often be manipulated by
appropriate combinations of environmental conditions, particularly relating
to light and temperature (Singh & Ashby 1985). Waage et al. (1985) noted
that one factor to consider is that entomophagous insects and their hosts may
have different optimum rearing temperatures. Rearing conditions vary with the
entomophage reared. For example, maggots of the parasitic tachinid Lixophaga
diatraeae are reared inside their host larvae, the sugarcane
borer, in complete darkness at 26-28°C and 80% RH
(King & Hartley 1985c), whereas Trichogramma spp. are
usually reared under constant light (20-25 ft-c, 26.7"1°C and 80"5% RH) (Morrison
1985a). Optimum conditions might even vary in
the same genus. The predaceous common green lacewing can be reared under
constant light (Morrison 1985b). However, Chrysoperla rufilabris
Burnmeister requires a 14 hour photophase for high fecundity (Nguyen et al.
1976). Singh & Ashby (1985) observed that light quality and photoperiod
are important factors in insect mating and oviposition. Waage et al. (1985) reviewed mating
problems in general and possible solutions with regard to entomophages.
Chianese (1985) noted that mating conditions are critical in the laboratory
production of the gypsy moth parasitoid Cotesia melanoscelus.
Cocoons of the parasitoid must be isolated in gelatin capsules before adult
emergence to ensure virgins. Then both sexes must be combined in a screen
cage under natural light. It is better to feed males before placement in the
mating cage, while females are fed in the cage itself to reduce activity when
males mate with them. Such females mate only once and refrigeration of adults
must not occur until after mating (24-48 hours). Rappaport & Page (1985) were
successful in maintaining a year-round culture of the ichneumonid Glypta
fumiferanae Vierek, a parasitoid of the western spruce budworm,
and attributed part of their success to the mating procedure, which was to
introduce a freshly emerged female into a 0.25 liter carton with mesh
screened ends and with three 2-4 day old males. Galichet et al. (1985) also noted
rather fastidious requirements for laboratory mating of the tachinid Lydella
thompsoni Hertig. Requirements included high humidity, high light
intensity (at least 8,000-10,000 lux) and food of casein hydrolysate and
honey. Godfray (1985) discovered extremely
precise mating requirements for Argyrophylax basifulva
(Bezzi), a tachinid parasitoid of the greater coconut spike moth, Tirathaba
complexa Butler. He had to place the flies in a 1.00 x 0.75 x 0.75
m outdoor cage in bright morning sunshine at 28°C and 90% RH,
with a strong breeze provided by an electric fan just to obtain 50% mating
success. Further complications were a 3-day premating period followed by an
8-day preoviposition period. In contrast, King & Hartley (1985c)
found that mating by the tachinid Lixophaga diatraeae
was easily obtained without any exacting requirements. Although some light
was necessary, the type and intensity was not critical. Mating occurred
readily when about 200 adult L. diatraea were placed in
a small screened cage under conditions of 26°C and 80% RH
with a 14-hr photophase. The proportion of males placed together
with females for mating can be quite low, and in fact may be preferable in
order to avoid problems caused by overmating. Palmer (1985) found that adding
25 males and 300 females of the parasitoid Brachymeria intermedia
to a 4-liter jar would insure complete mating. Females mate only once and
unmated females produce males. Mating takes place one to two days after
emergence in bright artificial light at 24-27°C, with resulting
progeny being 60-85% females. Sex-Ratio Changes In prolonged
culture of parasitoids, sex ratio changes can be a complication, as was
previously discussed in the section on Arrhenotoky and Thelytoky (ENT229.11).
There are, however, rearing conditions that can sometimes be modified to
ameliorate this problem. For example, a culture of the thelytokous pteromalid
Muscidifurax uniraptor, maintained for 16 years,
gradually began producing predominantly male progeny despite no apparent changes
in culturing techniques or the host insect (Legner 1985). Production of females could be
improved through allowing oviposition by only young mothers when temperatures
were moderate and when hosts were provided on alternate days. Nonetheless,
the original proportion of >95% females could no longer be duplicated. It
was interesting that insectary production of two freshly collected cultures
of M. uniraptor resembled that of the changed long-term
culture. The possible involvement of microorganisms in thelytoky (Legner 1987a , 1987b, 1987c ) as we noted earlier,
complicates interpretation of sex ratios in such species. Waage et al. (1985) reviewed the
factors related to rearing conditions which can significantly affect the sex
ratio of parasitic wasps. These factors include degree of mating (including
overmating), host size, crowding and high temperatures. Parasitic female
wasps have the capacity to regulate whether male or female eggs are laid,
depending on external conditions. This regulation is governed by host size
and crowding, females tending to lay more male eggs on small hosts if adapted
to such, in already parasitized hosts using physical and chemical cues. Males
larvae tend to be competitively superior, so there is the differential
survival of males in superparasitized hosts. Laboratory male-biased sex
ratios can be partially alleviated by provision of abundant food and space
(Waage et al. 1985). Hoffman & Kennett (1985)
demonstrated that prolonged exposure of the aphelinid Aphytis melinus
DeBach, to winter temperatures caused a male bias in F1 sex
ratios, and they briefly reviewed published reports of similar effects of low
temperatures on parasitoids. Rearing Density The provision of adequate food and
space is important for optimizing progeny production. Papacek & Smith
(1985) recommended a uniform rearing density of 30-50 oleander scales/cm2
on the surface of butternut pumpkins, when the scales are used to rear the
red scale parasitoid, Aphytis lingnanensis. Munstermann
& Leiser (1985) cautioned that in rearing the predaceous mosquito Toxorhynchites
amboinensis (Doleschall), the ratio of predaceous larvae to prey
larvae is critical. Too few prey can result in cannibalism among the
predaceous larvae, and too many prey can result in adult prey emergence in
the Toxorhynchites pans. Proff & Morgan (1983)
stressed the importance of using suitable parasitoid/host ratios to prevent
superparasitism in mass production of the pteromalid, Spalangia endius
Walker, on the house fly. Raupp & Thorpe (1985) noted that increasing
parasitoid/host ratios may result in multiple stinging and increased larval
mortality. Chianese (1985) used the ratio of 40 gypsy moth larvae to 8 mated
females of the parasitoid Cotesia melanoscelus for three
hours to allow oviposition. With this technique, 70-80% parasitism was
achieved. Maximum fecundity of Trichogramma spp. was obtained
with a ratio of 100 host eggs per female parasitoid (Morrison 1985a).
Morrison (1985b) also used a modified ice cream carton as an oviposition unit
for the common green lacewing. With 500 adults, ca. 79,000 eggs could be
obtained in 21 days. Morrison (1985b) emphasized that each adult needed 2.5
cm2 of resting space to prevent a reduction of longevity
and fecundity caused by overcrowding. Legner (1967 ) noted also
that female progeny production can decline with increasing parasitoid/host
ratios. Adequate space and abundant hosts for
cultures of entomophagous parasitoids and predators will usually prevent
cannibalism, mortality, lowered longevity and fecundity, and reduced fitness
(Waage et al. 1985). An exception was reported by Wajnberg et al. (1985) who
found that Drosophila melanogaster Meigen suitability
for the eucoilid endoparasitoid Leptopilina boulardi
(Barbotin, Cartin & Kelner-Pillault) increased from 50% in laboratory
conditions optimal for Drosophila to 90% when it was reared in
crowded conditions. Isenhour (1985) found 3rd-instar fall
armyworm larvae, Spodoptera frugiperda (Smith) to be
preferred for parasitization by the ichneumonid Campoletis sonorensis
(Cameron), and that at high host densities, significantly more larvae were
parasitized at 25°C than at 30°C. An understanding of entomophage
behavior can be an important component to entomophage culture. For example,
the positive phototaxis and negative geotaxis of Trichogramma spp.
provides greater ease of manipulation (Morrison 1985a). In fact, most
entomophagous parasitoids exhibit positive phototaxis, facilitating their
collection. Waage et al (1985) recommended rearing
entomophagous insects on their natural hosts on natural food sources to
provide all necessary behavioral stimuli. While this is desirable, it may not
be possible in mass production. King & Hartley (1985c) noted that Lixophaga
diatraeae, which parasitizes the sugarcane borer, is attracted by
volatile substances from feeding borers, and larviposits when it contacts
borer frass. Yet, they developed a mass production scheme for this tachinid
by rearing it on the greater wax moth, a factitious host whose use did not
simulate field conditions but did greatly facilitate production. Badgley
& Legner (unpubl.) successfully mass reared the encyrtid, Tachinaephagus zealandicus Ashmead, by
having late instar Musca domestica larvae roll down plastic sheeting
as they exited from larval media containers. Parasitization stimulus was
greatly enhanced in the presence of moving larvae, that was accelerated by
adding excess water to the larval rearing media. Feeding behavior is important. Although
a short life cycle of 15 days positively influences production of large numbers
of the red scale parasitoid Aphytis lingnanensis
(Papacek & Smith 1985), host feeding is a negative influence, with each
female destroying an average of 46 scales, while laying an average of 57 eggs
(Rosen & DeBach 1979). Pteromlaid parasitoids of Diptera that reproduce
by arrhenotoky require host feeding early in their adult life for maximum
fecundity, whereas fecundity of thelytokous populations is reduced by early
host feeding (Legner & Gerling 1967). In propagation, insect parasitoids that
host feed require special attention. Lasota & Kok (1986a) determined that
for optimum production of Pteromalus puparum, a
gregarious endoparasitoid of the imported cabbageworm, Pieris rapae
(L.), one parasitoid pair should be exposed to 10 freshly formed host pupae
for six days in order to provide sufficient hosts for host feeding and
sufficient time for egg formation and oviposition before the pupae became too
old. Lasota & Kok (1986b) concluded that balanced host/parasitoid ratios
are important in the mass production of gregarious parasitoids to optimize
host resource utilization while maintaining parasitoid quality. Host feeding in adult tachinids was
shown to be important by Nettles(1987) who found that fecundity of adult Eucelatoria
bryani was significantly increased by exposing them to their
host, the corn earworm, or to host haemolmyph. Feeding by the host is
sometimes important. Chianese (1985) noted that if gypsy moth larvae were not
well fed, they would cannibalize other larvae and eat cocoons of their own
parasitoid, Cotesia melanoscelus. Special Techniques A variety of special techniques have
been developed for producing parasitoids and predators. The handling of host
eggs depends on their use. If the eggs are laid on a natural substrate, the
deposition sites can be cut out and batched for exposure to egg-attacking
parasitoids (Morrison 1985a). Clair et al. (1987) used a cork borer to cut
out clusters of elm leaf beetle eggs from elm leaves so that they could be
grouped together in a small petri dish for efficient exposure to attack by
the eulophid egg parasitoid Tetrastichus gallerucae
(Fonscolombe). To prevent larval cannibalism in green
lacewings, Morrison (1985b) sealed larvae in separate chambers and fed them
through an organdy cloth with pre-frozen lepidopteran eggs. In China mass production of Trichogramma
spp. involves the grinding of freshly emerged oak silkworm female moths
to extract infertile eggs (King & Morrison 1984). Grinding is followed by
cleaning and drying the eggs, after which they are stored at low temperatures
for several weeks prior to use. A similar technique was used by King &
Hartley (1985c) who extracted parasitoid maggots from adult females of the
tachinid Lixophaga diatraeae with a small blender. After
chemical treatment, collection, rinsing and suspension in 0.15% agar
solution, the maggots were dispensed by using a special machine (Gantt et al
1978, King & Hartley 1985c). To prevent damage and mortality in the
parasitoid Cotesia melanoscelus, cocoons were hardened
before handling (Chianese 1985), yet they must be gathered regularly to
reduce predation by host gypsy moth larvae. Kairomones and pheromones may
concentrate in insect rearing cages where there is little air movement, so
that normal insect response is not stimulated by an odor gradient. Chiri
& Legner (1982 ) showed that
the parasitoid Chelonus sp. nr. curvimaculatus
responded to kairomones emitted by body scales not only from its natural
host, the pink bollworm, but also from unnatural hosts such as the beet
armyworm. It was speculated and later field demonstrated (Chiri & Legner 1983
) that high populations of beet armyworms in cotton fields would reduce
pink bollworm parasitization because of kairomonal distractions. Cossentine & Lewis (1986) used host
odor to induce larviposition by a parasitic tachinid, Bonnetia comta
(Fallén) on filter paper. They moistened the paper with water in which black
cutworm fecal material had been soaked. Rubink & Clement (1982) found
that fecal pellets from late instar larvae of the black cutworm provided the
greatest intensity of larviposition by the tachinid. Waage et ala. (1985) remarked that host
defense reactions are important in rearing some parasitoids. Thus, it seems
generally inadvisable to produce parasitoids on hosts exhibiting defense
reactions such as encapsulation of parasitoid eggs or larvae by host blood
cells, nor to field release them on such hosts. However, Strand & Vinson
(1982) noted that eggs lack such defense mechanisms and may be useful
factitious hosts. A recognition hormone from a normal host stimulated
oviposition by the scelionid parasitoid Telenomus heliothidis
Ashmead, in eggs of non-hosts. Such recognition hormones could thus be used
to produce specific egg parasitoids in nutritionally acceptable non-hosts. Extended storage of entomophagous
insects is often desirable. Morrison & King (1977) reviewed various
techniques and concluded that in almost all, low temperatures were used to
reduce developmental rates. Entomophages can be stored at low temperatures
for varying periods depending on the species. Palmer (1985) reported that the
gypsy moth parasitoid Brachymeria intermedia
could be stored at least five months at 10°C and 50% RH,
with a mortality of 30-50%, depending on sex ratio (males are unable to
survive prolonged storage). However, it was best not to store parasitoids
that were intended for field release for longer than 48 hours at 16°C. For maximum survival parasitoids should be field released
within 72 hours of collection. Morrison (1985b) found that eggs of the common
green lacewing could be stored at 13-14°C and 70-80% RH
for only 10 days before measurable viability reduction occurred. Papacek
& Smith (1985) noted that the California red scale parasitoid Aphytis
lingnanensis can only be stored for up to three days at 16°C. Various factors should be considered
and tested before cold storing entomophages. For example, Clausen (1977) reported
that prolonged storage of adult Aphidius smithi Sharma &
Subba Rao, a parasitoid of the pea aphid, inactivated sperm in males or mated
females; and Chianese (1985) found that refrigeration of Cotesia melanoscelus
adults must not occur until after mating. Life cycles of entomophages in the
laboratory usually vary between eight and 42 days, depending on temperature
and excluding the effect of diapause. However, it is common for most species
to develop in 14-30 days. At 26.7°C and constant
light, the egg parasitoid Trichogramma praetiosum Riley
averages 9.5 days, and T. minutum, eight days (Morrison
1985a). Brachymeria intermedia develops in 15-30
days at 24°C (Palmer 1985) and the common green
lacewing requires about 30 days at 26.7°C and ca. 75%
RH, with constant room light (Morrison 1985b). Since the scales of emerging greater
wax moth moths may interfere with Brachymeria intermedia
parasitoids ovipositing in pupae, the oviposition units must be cleaned of
moths and scales. If Brachymeria appear inactive in the
oviposition jars, the light intensity and/or temperature is increased. If
parasitoids are active but many moths are emerging and the parasitism rate is
low, the following factors should be checked: the wax moth pupae may be too
old when presented for parasitization, the ratio of parasitoids to hosts may
be too low (optimum = 25 females/300 cocoons), or the photophase may be too
short (optimum = 10 hours) (Palmer 1985). A high mortality of ovipositing Brachymeria
females may be caused by overheating oviposition jars (optimum = 24-26°C). In mass production technology notable
systems have been under development for the Africa-wide Biological Control
Programme of the International Institute of Tropical Agriculture (IITA)
(Herren 1987, Haug et al. 1987). Hydroponic culture techniques have been
devised for producing cassava, Manihot esculenta Crantz
(Herren 1987), and semiautomated systems are in use for producing organisms
at three trophic levels [cassava, the cassava mealybug, Phenacoccus
manihoti Matile-Ferrero, and its encyrtid parasitoid, Epidinocarsis
lopezi (DeSantis) (Haug et al. 1987)]. Quality Assessment The quality of entomophages is
dependent on their genotype, nutrition and rearing environment and is
obviously critical to a biological control program. Moore et al. (1985)
discussed quality of laboratory produced insects. Quality assessment tests
were categorized by Moore et al (1985) into three groups, relating to
production, process and performance. They stated that performance tests
measure field, behavioral and clinical variables. Field variables include
degree of pest population control and recoveries of a released species.
Behavioral variables encompass characteristics relating to mobility (flight
propensity and capacity, locomotion), sexual activity and reproduction and
habitat adaptability (such as circadian rhythms). King & Morrison (1984)
also included host selection as a quality component for entomophages.
Enzymatic, biochemical, electrophysiological and pheromone tests are clinical
in nature (Moore et al. 1985). It was also noted that enzyme tests can be
qualitative (isoenzyme electrophoresis to detect genetic diversity) or
quantitative (to indicate physiological state). A biochemical profile in
which measurements of insect chemicals (cholesterol, lipids, proteins, uric
acid, lactic dehydrogenase, etc.) can be used as a quality assessment tool to
indicate physiological state. Of special interest are pheromone production
tests in which gas liquid chromatography is the assessment technique used.
Other sophisticated methods being pioneered for quality assessment include
electrophysiological techniques: electroretinograms for evaluating insect eye
response, and electroantennograms for determining the response of insects to
pheromones and other chemicals. Singh & Ashby (1985) felt that the
standard life history measurements of fecundity, fertility and adult and
pupal weights are usually adequate as quality indicators. For entomophage
release programs, however, Moore et al. (1985) believed that methods for
measuring insect production of behavioral chemicals and response to them
should be important additions to quality testing. Chambers & Ashley (1984) defined
industrial quality control concepts and evaluated their applicability to insect
rearing. A review of quality assessment and control procedures used in mass
production of several insect parasitoids revealed the following: It was noted
that quality control monitoring of Trichogramma spp. typically
consists of keeping records on numbers reared, parasitization rate and sex
ratio (King & Morrison 1984). Quality assessment tests used by Morrison
(1985a) for Trichogramma production were percent parasitized
eggs, percent emergence from parasitized eggs and sex ratio. At least three
samples of about 200 eggs were taken from each oviposition unit. Accepted
quality assessment standards and limits were 80"10% parasitism
of 48-hr-old eggs, 90"5% adult emergence and sex ratio
of 1.2 females / 1.5 males. In terms of quality control procedures, vigor was
maintained in laboratory cultures of Trichogramma spp. in China
by forcing adult females to fly several feet in search of host eggs, to
eliminate weaker individuals (King & Morrison 1984). O'Dell et al. (1985) used parasitoid size, longevity and fecundity parameters to check quality of gypsy moth parasitoids. A variety of factors affected these parameters, including host diet, host density, microbial infection of the host and environmental changes. Quality assessment in production of Cotesia melanoscelus, a parasitoid of gypsy moth larvae, consisted of determining fecundity, defined as the number of progeny produced rather than the number of eggs laid (Chianese 1985). Palmer (1985) used the following quality assessment parameters in producing the gypsy moth parasitoid Brachymeria intermedia: (1) production totals per cage, per week and per month, (2) percent successful parasitism, (3) female/male ratio and (4) percent recovery from storage |