CULTURE OF NATURAL ENEMIES
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Entomophaga / Host Interactions (Host Type & Quality)
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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.
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 ,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 ). 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.
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.
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.
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)].
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.