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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. Microorganisms may also affect entomophages. 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). It is well known that wild parasitoid
populations exhibit seasonal and geographical differences in behavior and
morphology. Therefore, collections
meant for importation should optimally include isolates from diverse areas
and different times of the year. Differences include aggressiveness, heat and cold tolerance,
uniparentalism, gregarious versus solitary development, the number of eggs
deposited into a single host, larval cannibalism intensity and parasitoid
size. Detailed studies on Muscidifurax
uniraptor, M. raptor and M. raptorellus demonstrate the
great amount of diversity that can be found within one genus (fly-par.htm). When only small
numbers of insect parasitoids or predators are introduced into a new area it
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 out bred 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 could 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. The
standard and minimum acceptable values for percentage successful parasitism
were 70-75% and 60%, respectively. Finally, King
& Hartley (1985c) used the following standards in assessing the quality
of mass produced Lixophaga diatraeae: (1)
puparial weight (male = 14 mg, female = 20 mg), (2) percent parasitism (90%),
(3) number maggots/female (70) and (4) maximum adult longevity (male = 29
days, female = 24 days). Exercise
29.1--In culturing
entomophages, what principal biological characteristics does a researcher strive
to maintain? Give a few procedural
examples of how such traits might be maintained? Exercise
29.2--What operational
procedure must be routinely and rigorously followed to guarantee healthy
cultures of hosts and entomophages? Exercise
29.4--How would you
practically counteract the trend toward homozygosity in cultures of
entomophages? Exercise
29.4--Name some ways to
favor the successful mating of females in arrhenotokous cultures. REFERENCES: Please refer to <bc-30.ref.htm> [Additional references may
be found at
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