Archives of Insect Biochemistryand Physiology 13:167-I 75 (1990) In Vitro Rearing of Parasitoids: Role of Host Factors in Nutrition William C. Nettles, Jr. Subtropical Agricultural Research Laboratoy,Biological Pest Control Research Unit, Weslaco, Texas Evidence for the role of host components in the growth and development of parasitoids i s briefly reviewed, and implications concerning the development of in vitro culture media are discussed. I n vitro development of the entomophagous parasitoids Eucelataria bryani Sabrosky (Diptera: Tachinidae) and Trichograrnma pretiosum Riley (Hymenoptera: Trichogrammatidae) i s dependent o n low-molecular-weight host chemicals. Asparagine, but not aspartic acid, and several other free amino acids are essential dietary ingredients for E. bryani. From insect hemolymph, small, unidentified polar molecules with characteristics similar to sugars are needed for pupation of T. pretiosum. Both adaptive and nonadaptive changes likely are responsible for the dependence of parasitoids o n host factors. The genetic changes have no apparent adverse effect unless the parasitoids are fed artificial media devoid of certain chemicals that are obtained in vivo from host insects. The rearing of at least several species of important entomophages to control pest insects of great economic importance is dependent on the identification of constituents from host insects and inclusion of these chemicals in artificial diets. Key words: artificial media, parasitoid growth factors, Tachinidae, Trichogrammatidae INTRODUCTION The use of parasitoids in augmentation research and in large-scale biocontrol programs is limited by labor costs and by the technical problems and expense associated with rearing both the host and parasitoid when only the parasitoid is needed. A promising solution to the problems associated with in vivo production is to rear parasitoids in vitro. The first in vitro rearing of sarcophagids was on diets composed of salmon and liver, the subsequent extensive investigations [l]revealed that sarcophagids are dependent on nutrients that today are well known to be needed by many species of insects. Pioneering research on other species of parasitoids demonstrated that they also do not appear to be dependent on any unknown or unusual Received October 17,1988; accepted July 7,1989. Address reprint requests to W.C. Nettles, Jr., USDA, ARS, 2413 East Highway 83, Weslaco, TX 78596. 0 1990 Wiley-Liss, Inc. 168 Nettles dietary chemicals [2,3]. However, as a wider range of species is studied, more and more difficulties are being encountered in the in vitro rearing of parasitoids. Many of the economically important parasitoids appear to be especially difficult to rear on artificial media. At least 33 species of parasitoids representing two orders (Diptera and Hymenoptera) and at least 10 families have been reared with varying degrees of success on artificial diets (Table 1, based on , and Table 2). For many species of parasitoids growth and development are poor at best in the absence of host components. Only 14 species of parasitoids have been reared to the adult stage in the absence of host components (Table l),and these include sarcophagids (five species), ichneumonids (three species), tachinids (three species), and a single trichogrammatid species (Table 1).For the tachinid and trichogrammatid species reared to the adult stage on artificial diets without host components, yields and parasitoid quality have been low. This brief review describes research approaches and the current status of investigations on the role of host factors for Eucelatoria b a n i (Diptera:Tachinidae) and Trichogrurnmapretiosurn (Hymenoptera:Trichogrammatidae).Also presented is evidence supporting the role of host metabolites for growth and development of parasitoids and speculation about the reasons that parasitoids are dependent on host factors. Problems that may affect the successful and economical mass pr.oduction of parasitoids on artificial media are discussed. CURRENT STATUS OF THCHOGRAMMA AND EUCELATORIA RESEARCH The poor growth and development of Trzchogramrna spp. (Hymenoptera: Trichogrammatidae) and E . bryani on rich artificial diets containing most of TABLE 1. Number of Species of Parasitoids Reared on Artificial Diets According to Order and Family* No. of Species Order and family Diptera Sarcophagidae Tachinidaea Hymenoptera Ichneumonidae Trichogrammatidae Chalcididae Pteromalidae Scelionidae Tetrastichidae Braconidae Encyrtidae Total Attempted Some growth and development 5 8 5 4 4 6 3 2 1 1 2 1 33 6 3 2 1 1 2 1 33 8 Reared to adult stage On all On diets without diets insect components 5 4 5 3 3 3 1 2 1 3 1 1 1 0 0 1 0 0 20 “Includes unreported data for two species (Nettles; Bratti and Nettles). *Data are summarized from Thompson and Hagen . 0 0 14 Role of Host Factors for Parasitoids In Vitro 169 TABLE 2. Species of Parasitoids That Grow and Develop on Artificial Diets Containing Insect ComDonents Such as Hemolvmuh Order, family, and species Diptera Tachinidae Gonia cinerascens Archytas marmoratus Palexorista laxa" Hymenoptera Trichogrammat idae Trichogramma australicum Trichogramrna confusum Trichogramma dendrolimi Trichogramma tmanescens Trichogrammajaponicum Trichogramma pretiosurn Pteromalidae Dibrachys cams Pteromalus puparum Scelionidae Telenomusheliothidis Tetrastichidae Tetrastichusschoenobii Braconidae Apanteles marginiventris Microplitis croceipes Reference Nos. 25 Bratti and Nettles, unreported data Bratti and Nettles, unreported data 26 26 26-29,31 26 26 5,6,10,30-32 33 19,34-36 37 38 39-41 39 "Also has been reared on artificial diet without insect components. the substances known to be essential for insects suggests that important host components are missing from artificial diets. Host Factors for Trichogrammapretiosum Important host constituents for T. pretiosurn development recently were extracted from hemolymph of Munduca sexta (L.) (Lepidoptera: Sphinigidae) . A diet composed of M. sextu hernolymph, chicken egg yolk, and bovine milk (there is no difference between whole or skim milk) supports excellent growth to the pupal stage, but poor development to the adult stage . Addition of the supernatant from M. sextu egg homogenates to M. sexta hemolymph-based diet produced yields as high as 75.9% of adults . However, a nonhemolymph diet composed of chicken egg yolk, bovine milk (there is no differencebetween whole or skim milk), and a modified Nettles  diet does not allow T. pretiosurn to develop beyond the larval stage . Bioassays for host factors were performed by allowing 7'. pretiosurn females to oviposit in a dilute solution of potassium chloride and magnesium sulfate encapsulated in an artificial wax egg [8,9]. Following removal of the salt solution, the eggs and subsequent larvae  were incubated in a mixture composed of each purified fraction of hemolymph being tested, chicken egg yolk, bovine milk, and a modification of the artificial diet of Nettles  for E . bryani. The salt solution used as an ovipositional stimulant is important because it allows the collection of eggs in the absence of hemolymph or other host components. The insects were reared in the wells of microtiter plates [lo]. 170 Nettles M . sextu hemolymph was extracted with 76% ethanol and several chromatographic techniques (a Sephadex G-10 column, a (2-18 reversed-phase cartridge, and HPLC on DEAE and amino columns) were used to purify possible host factors. There was a loss of activity with purification, and, for the purest extracts, activity was completely lost until certain fractions were recombined. This indicates that two or more chemicals are responsible for pupation activity. The factors have molecular weights of less than 1,000 and are polar chemicals similar to sugars or low-molecular-weight peptides . Host Factors for E. b y m i E. bryuni is an important and highly host-specific parasitoid of Heliothis spp. Research on identification of host constituents needed by E. bryuni began about 10 years ago and illustrates a different approach from that used for 7'.pretiosurn factors. The techniques and diets were those of Nettles et al. [ l l ] and Nettles 171. The diets contain organic acids, minerals, carbohydrates, ATP, glutathione, free amino acids, soy flour, vitamins, antibiotics, lipids, and agar. The composition of the diets is based on research of Grenier et al. [12,13], numerous tissue culture media, and analyses of hosts, other insects, and the parasitoids themselves. E. bryuni maggots, deposited in last instar Heliothis spp. larvae, developed in the host for 24 h before they were removed by dissection and transferred to artificial diets that did not contain insect components. Development of maggots in the host for the first 24 h prior to transfer to artificial diet produced approximately 50% late first-instar and 50%early second-instar larvae, which, when reared in vitro for the remainder of the larval stage, gave higher yields of pupae and adults than when maggots were placed on artificial diets immediately after larviposition [ l l ] . All values given for yields are based on the numbers of E . bryuni larvae collected from the hosts and placed on artificial media. E. bryani grew clearly for the first time on artificial diet after aspartic acid was replaced with asparagine. There were several reasons for doing so, but the most important was that asparagine is more water soluble than aspartic acid. Asparagine is an essential component of artificial diets for E. bryuni larvae, host hemolymph contains greater quantities of asparagine than of aspartic acid (Nettles and Holman, unreported data), and free amino acids have striking effects on growth of E. bryuni [14,15]. When free arnino acids were deleted from the artificial diet and the soy flour concentration was increased from 2.6 to 10.4%,yields of third-instar larvae increased slightly; but all of the maggots died in the third instar . In the presence of 1.83%total free amino acids and 2.6%soy flour, yields were much higher, and 50% and 28%of E. bryani developed to the pupal and adult stages, respectively . When each of the 10 essential amino acids and asparagine were deleted from diets containing 1.83%total free amino acids and 2.6% soy flour, most of the E. brydni larvae died in the second instart and very few insects developed beyond the third instar [14,15]. Yields (based on numbers of l-day-old host-reared maggots placed on artificial diets) of 45% pupae and 28% adults of E. bryuni were obtained with a soy flour-based artificial diet containing asparagine and other amino acids. Role of Host Factors for Parasitoids In Vitro 171 Current yields are approximately 70% pupae and 50% adults on the best diets (Nettles, unreported data and diets). Occasionally adult yields reach 70% or higher (Nettles, unreported data). When aspartic acid replaced asparagine in the amino acid mixture, no pupae and no adults were obtained . These data demonstrate that E . bryani larvae are unable to convert aspartic acid to asparagine at a significant rate and that asparagine synthetase either is missing or is present at exceptionally low levels in the parasitoids. DISCUSSION Importance of Host Factors The beneficial effects of rearing several species of parasitoids in the presence of host components such as hemolymph and the generally poor results of rearing in the absence of host components are strong evidence that at least some species of parasitoids are dependent on certain host chemicals. Although there had been no attempt to identify host factors until recently, it is becoming increasingly clear that identification of host chemicals and inclusion of essential and other active synthetic chemicals in artificial diets likely is necessary for successful, economical in vitro rearing of several parasitoid species of economic importance. Not all parasitoids are dependent on insect host factors for their growth and development; a few species of parasitoids that first were reared on host components such as hemolymph later were reared on artificial diets devoid of host components. These include several Trichogramma species [16-181, Pteromalus puparum (Pteromalidae) , and Palexorisfa l a m (Diptera: Tachinidae; Bratti and Nettles, unreported data, and Nettles, unreported data). However, for these species the low yields and inferior quality of the parasitoids reared in vitro indicate that, although certain noninsect components were present and beneficial, other chemicals were missing from the diets. Host Factors and Genetic Changes In E . bryani and probably in several other species of parasitoids, certain host chemicals have become essential dietary components as a result of mutations and other genetic changes. In vivo the genetic changes are harmless to the parasitoids; host metabolites are either essential or needed for optimal growth and development. When certain species of parasitoids are reared on artificial diets devoid of the required insect components, the genetic changes are harmful or lethal. Host Factors for E. bryani The dependence of E . bryani on dietary free amino acids likely is an adaptation to the high concentration of free amino acids known to occur in the hemolymph of host insects. Because most insects are able to digest proteins and are not dependent on free amino acids, it is especially interesting that deletion of even one of the 10 essential (for rats) amino acids from a proteincontaining diet results in the death of €. bryani larvae reared in vitro on a proteincontaining diet .€. bryani larvae apparently digest proteins slowly because growth is extremely poor and death results when free amino acids are deleted 172 Nettles from a protein-rich medium [14,15]. Although the amino acids essential for E. bryuni are obtained from plant proteins ingested by the host, free amino acids in a sense are host factors for E . bryani larvae because they are metabolic products of the host. The best evidence that a parasitoid species is dependent on a host factor comes from E . bryuni research. Asparagine is an essential dietary ingredient for E. bryuni larvae, and, if aspartic acid is converted to asparagine at all, it obviously is not metabolized at a rate adequate to meet the demands of the rapidly developing larva . Because sizable quantities of asparagine are present in host hemolymph (Nettles and Holman, unreported data), the genetic change that resulted in the loss or in a lower level of asparagine synthetase was harmless in vivo and was retained because there presumably was no selection against this trait. As asparagine is a constituent of many proteins and E. bryani larvae grow rapidly, the absence or removal of asparagine from the diet has a disastrous and immediate effect. Consequently, there is little, if any, reason to expect the loss or attenuation of asparagine synthetase to be advantageous or of adaptive value to the parasitoid. Asparagine is an essential dietary component for two species of mosquito larvae, Culex pipiens (L.) and CuEisetu incidens Thompson [20,21], and the dependence on asparagine of another dipteran species such as E. bryuni might seem to be of little significance. However, it is remarkable that dietary asparagine is a common requirement for insects with such dissimilar environments. Mosquito larvae presumably are not dependent on free asparagine; however, proteins from decomposing plants, etc., undoubtedly are a major source of asparagine in mosquito diets. As with E. bryuni, nonadaptive genetic changes resulting in low or in sigruficantly reduced levels of asparagine synthetase doubtless are harmless because adequate quantities of asparagine are present in the natural diets of mosquito larvae. The dependence ot T. pretiosum on host factors present in insect eggs and hemolymph also may have resulted from nonadaptive evolution. Host Factors for 1 pretiosum The unidentified polar host components needed by T. pretiosum are not trehalose, glucose, common amino acids, or most of the chemicals known to be needed for insect nutrition, because sizeable quantities of trehalose, glucose, at least 18 free amino acids, and proteins and other materials from chicken egg yolk and bovine milk were present in complex diets tested by Irie et al. 151. However, low-molecular-weightcarbohydratescannot be ruled out as growth factors, because the unidentified chemicals from M . sextu hemolymph have solubilities and polarities similar to those of sugars, and the addition of certain carbohydrates (fructose, a-glycerophosphate, N-acetylglucosamine, sorbitol, and xylose) to complex artificial diets containing trehalose and glucose produced weak and inconsistent pupation of T. pretiosum . Costs and Other Considerations Relative to In Vitro Production of Parasitoids For the large-scale production of parasitoids, the cost of artificial diets is an important consideration. Artificial media will be expensive if, to overcome the effects of high osmotic pressure, it is necessary to use poly-amino acids sim- Role of Host Factors for Parasitoids In Vitro 173 ilar to those used by Thompson et al.  to rear the pteromalid Puchycrepoideus vinderniue to the adult stage. However, for rearing E. bryuni and Pal. luxu, polyamino acids are not needed (Nettles, unreported data). Fetal bovine serum is rather expensive, and, although hemolymph is available and may be used in countries with silk industries, hemolymph currently cannot be produced economically for large-scale in vitro rearing of parasitoids in countries that do not rear large numbers of silk-producing insects. The expense of formulating artificial media couId be severly affected by the numbers of host factors required for parasitoids. If large numbers of genetic changes have occurred in parasitoids, sizable numbers of host chemicals will have to be identified and added to diets. The difficulty and expense of producing parasitoids in vitro would be greater for the more specific diets. The identification and synthesis of certain low-molecular-weighthost chemicals present in hemolymph, and which also may be present in fetal bovine serum and other materials, is expected to result in more economical means of in vitro rearing of certain species of parasitoids. It is highly encouraging that the chemicals, both identified and unidentified, needed by E . bryuni, Pal. luxu, and T. pretiosum have molecular weights of less than 1,000. Thus far there is no strong evidence that expensive or unusual chemicals with high molecular weights are needed by these three species. Parasitoids with requirements similar to those of E. bryuni, Pal. luxu, and T. pretiosum likely can be produced by adding low-molecular-weightchemicals to their artificialdiets. Such an approach is feasible because current technology is suitable for the identification and synthesis of small molecules. The cost of chemicals at the concentrations known to be needed by E. bryani and Pal. laxu is surprisingly low (Nettles, unreported data) and also probably will be low for Trichogrumrnu spp. While milk used in the artificial media for Trichogrurnma spp. is inexpensive, reducing the cost of chicken egg yolk in the medium will be useful for large-scale production. Identification of the important nutrient(s) in egg yolk may help reach this goal. Complex research requirements cause in vitro rearing of parasitoids to be much more difficult to achieve than in vivo rearing. However, for large-scale production of parasitoids, in vitro culture will be more efficient (because of ease of automation) and more economical and dependable (large-scale production of host insects will not be necessary). The most difficult and timeconsuming task now appears to be the identification of host factors needed by parasitoids for nutrition or possibly for other functions. For obtaining eggs economically and in large numbers, several ovipositional stimulants have been identified and the methods of Xie et al. [lo], Li et al. , and Liu et al.  for in vitro rearing of T. pretiosurn and other species of Trichogrummu demonstrate that in vitro production of Trichogrurnm spp. is surprisingly well suited to automation. Potential Role of Host Factors for Other Species of Parasitoids Because of the considerable importance of asparagine to E. bryuni and of low-molecular-weight chemicals to 7'. pretiosum, several other species of parasitoids also may be dependent on host factors. 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