MXPA97007034A - Synthetic diet for cultivating the ectoparasitoidehimenoptero, catolaccus gran - Google Patents

Abstract

An artificial diet or improved growth medium is described for cultivating the insect parasitoid Catolaccus grandis, the growth medium is composed of fractions of amino acids, minerals, vitamins, lipids and carbohydrates that are present in amounts and proportions effective to support the growth of Catolaccus grandis, and the amino acid fraction includes alanine, glutamic acid, histidine and proline at concentrations of about 3.0-5.0%, 8.4-9.4%, 10.0-15.1% and 11.7-12.8% by weight, respectively, the growth medium is appropriate to mass propagate to C. grandis from the egg-to-adult stages its subsequent release as a biological control agent, alternatively, the growth medium can be used to support the growth of wasps adult strands of C. grandis that are used as reproductive material for mass propagation contin

Description

SYNTHETIC DIET FOR CULTIVATING THE ECTOPARASITOID HII1ENOPTERO, CATOLACCUS TRflNDIS BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to an improved artificial diet for growing Catolaccus grandis, an ectoparasite of cotton weevil.

DESCRIPTION OF THE PREVIOUS TECHNIQUE In recent years, the potential of the ectoparasitoid Catolaccus grandis (Burks) as a biological control agent against cotton boll weevil, Anthonorous grandis Boheman, has been established by several investigators, including Johnson et al. (1973, Environ. Entomol., 2 : 112-118), Morales-Ramos and King (1991, Evaluation of Catolaccus grandis (Burks) as a Biological Control Agent Against the Cotton Ueevil Boil, page 724, In D. 3. Herber and DA Richter Ceds.], Proc. Beltwide Cotton Conferences 199.1, Vol.2, Proc. National Cotton Council of America, Memphis, TN), Morales-Ramos and Cate (1992, Ann. Entomol. Soc. A., 85: 469-476), Sumrny and others ( 1992, Southwest, Environment!, 17: 279-288) and Morales-Ramos et al. (1994, Suppression of the Boeing First Generation by Aug entative Releases of Catolaccus grandis in Aliceville, Alaba a, pp. 958-964, in D. 3. Herber and DA Richter [eds.3, Proc. Beltwide Cotton Conferences 1994, Vol. 2, National Cotton Council of America, Memphis, TN). Several biological attributes of C. grandis have been reported that improve its activity as a biological control agent. These attributes include greater fecundity than that of the cotton weevil (Morales-Ramos and Cate, ibid); adaptability to various environments, including Mississippi (Johnson et al., ibid), Central Texas (Cate et al., 1990, Pests of Cotton, pp. 17-29, In DH Habeck, FD Bennett and 3. H. Frank Ceds.3, Classical Biological Control in the Southern United States, South, Coop. Ser. Bull. 355), the Rio Grande Valley (Sumrny and others, ibid) and North Alabama (Morales-Ramos et al., 1994, ibid.) 7 high efficiency research under low densities of the host population (Morales-Ramos and King, 1991, ibid) 7 ability to search for hosts in the terrain where the susceptible stages of the cotton weevil exist (Su rny et al., 1992, ibid) 7 synchronization with the life cycle of the cotton weevil (Morales-Ramos and Cate, 1993, Environ, Entomol., 22: 226-233); and adaptability for temperature scales similar to those tolerated by the cotton boll weevil (Morales-Ramos and Cate 1992, Environ. Entomol., 21: 620-627). The augmentative releases of C. grandis have been successfully used to control populations of cotton boll weevil in experimental fields in the Rio Grande Valley as described by Surnrny et al. (1993, Suppression of Boil Ueevil Infestations by Augmentative Releases of Catolaccus grandis , pp. 908-909, in DJ Herber and DA Richter Ceds.], Proc. Beltwide Cotton Conferencee Vol. 2, National Cotton Council of America, Memphis, TN) and in commercial cotton fields in Aliceville, Alabama, as described by Morales-Ramos et al. (1994, ibid). However, because C. grandis lacks the ability to survive winter in the United States (Johnson et al., Ibid), populations of this ectoparasitoid must be released again every year. The use of C. grandis as a biological control agent against cotton weevils depends, therefore, on the development of technology for mass propagation. The current method of mass propagation consists in encapsulating the larvae of the cotton weevil in Parafilm * (Cate, 1987, Southwest, Entomol., 12: 211-215). This method of encapsulation has been modified by Morales-Ramos et al. (1992, Feasibility of Mass Reaction of Catolaccus grandis, to Parasitoid of the Boeing Ueevil, pp. 723-726, in D. 3. Herber and DA Richter Ceds.3 , Proc. Beltwide Cotton Conferences 1992, Vol. 2, National Cotton Council of America, Memphis, TN) and mechanized by Roberson and Harsh (1993, Mechanized Production Processes to Encapsulate Boe Uevil Larvae (Anthono us grandis) for Mass Production of Catolaccus grandis (Burks), pp. 922-923, in DJ Herber and DA Richter Ceds.], Proc. Beltwide Cotton Conferences, Vol. 2, National Cotton Council of America, Memphis, TN) but, the high costs of using these methods, they cause the price of augmentative releases of C. grandis to be 5 to 10 times greater than other control methods of the cotton weevil. Despite the success of the augmentative releases of C. grandis to control cotton weevil populations in experimental fields, commercial application of this technology is largely limited by the high costs of mass propagation of this parasitoid. Mass propagation has been identified as a critical constraint to commercialize the augmentative releases of natural enemies (King and Morrison, 1984, So e Systems for Production of Eight Entomophagous Arthropods, pp. 206-222.

N. C. Leppla Ceds.], Advances and Challenges in Insect Rearing, USDA- Agrie. Research Serv., New Orleans, LA, p. 306). The development of artificial diets to cultivate in vitro the parasitoids is considered as the scientific advance needed to open the way to commercially apply biological control by increasing natural enemies (King, 1993, Augmentation of Parasite and Predators for Suppression of Arthropod Pests, pp. 90-100, in RD Lumsden and JL Vaughn Ceds.], Pest Management: Biologically Based Technologies, Proceedings of Beltsville Symposium XVIII, USDA-ARS, Beltsville, MD, American Chemical Society, Washington, DC). Artificial diets have been described to cultivate in vitro the ectoparasitoids hyophoptera. Thompson (1975, Ann.

Entomol Soc. Am., 68: 220-226) described an artificial diet for Ex eristes roborator (Fabricius), while Guerra et al (1993, Entomol. Exp. Appl., 68: 303-307) described an artificial diet for both Bracon ellitor Say as for C_¡_ grandis. Both diets lacked insect components and were composed of fractions of amino acids, minerals, vitamins, lipids and carbohydrates. The main differences between these two diets are found in the proportions and amounts of amino acids in the amino acid fraction. In addition, Thompson used albumin co or complement, and Guerra and others used fresh egg yolk. Guerra and others subsequently modified the artificial diet described in the 1993 publication, substituting the antimicrobial agent, and adding an antirnicotic, and describing an improved technique for oviposition (1994, Etornol. Exp. Appl., 72: 11-16). . Both Thompson and Guerra reported the successful development of parasitoids feeding on their respective diets. However, no extensive evaluation of the biological characteristics of the parasitoids cultured in vitro was presented; and none of the parasitoids was released in the field. The difficulty in producing large numbers of parasitoids of sufficient quality cultured in vitro has perhaps been the most important factor limiting the evaluation of their biological attributes. The production of uniformly high quality parasitoids is critical to the success of the augmentative release approach. The release of low quality parasitoids can greatly reduce the degree of success of augmentative release practices.

BRIEF DESCRIPTION OF THE INVENTION An artificial diet or improved growth medium has now been discovered to cultivate the insect paras.itoi.de Catolaccus grandis. The growth medium is composed of fractions of amino acids, minerals, vitamins, lipids and carbohydrates that are present in amounts and proportions effective to support the growth of Catolaccus, grandis. The amino acid fraction, which comprises a mixture of at least 19 amino acids, includes alanine, glutamic acid, histidine and proline at concentrations of about 3.0-5.0%, 8.4-9.4%, 10.0-15. IX and 11.7-12.8% by weight, respectively. The growth medium is suitable for mass propagation of C. grandis from egg to adult stages for subsequent release with biological control agents. Alternatively, the growth medium can be used to support the growth of adult female wasps of C. grandis that are used as an ovipositor element for continuous mass propagation. The use of this medium to cultivate C. grandis yields ectoparasitoids of unexpectedly superior quality than that which is achieved using the artificial diets described above.

In accordance with this finding, it is an object of this invention to provide an improved artificial diet for cultivating C. grandis ectoparasitoids. Another objective of this invention is to provide an artificial diet to cultivate C. grandis that yields high quality ectoparasites. A further object of this invention is to provide an artificial diet for growing C. grandis that is free of insect components. Other objects and advantages of this invention will be readily apparent from the following description.

DETAILED DESCRIPTION OF THE INVENTION The artificial diet or growth medium of this invention is formulated to support the growth of larvae of Catolaccus grandis Burks from the incubation period to pupation, and / or to support the growth of adult female wasps. The medium lacks insect components such as hemolymph, and is suitable for producing C. grandis ectoparasitoids on a commercial scale at a relatively low cost. In addition, the ectoparasitoids are of sufficient quality to function effectively as biological control agents against the cotton weevil after being released in the field. The growth medium is composed of a fraction of amino acids, a fraction of minerals, a fraction of vitamins, a fraction of lipids and a fraction of carbohydrates essential for the growth of larvae and / or adults of C. grandis. The medium preferably includes other optional components, such as an anti-microbial agent, an antioxidant and / or a protein source, as described in more detail below. The composition of the amino acid fraction is critical to produce high quality ectoparasitoids. This amino acid fraction is composed of a mixture of amino acids including but not limited to, alanine, arginine, asparagine, cysteine, glutaric acid, glutamine, glycine, histidine, isoleucine, leucine, Usin, methionine, phenylalanine, proline, serine. , threonine, tryptophan, tyrosine and valine. Aspartic acid may optionally be included. Although these amino acids have previously been identified as co-components in an artificial diet for a plurality of ectoparasitoids including C. grandis, as described both by Guerra et al. (1993, Entomol. Exp. Appl., 68: 303-307) and by Thompson (1975, Ann. Entomol. Soc. A., 68: 220-226), it has been unexpectedly discovered that when the concentration of alanine, glutamic acid, histidine and proline within the amino acid fraction is between approximately 3.0-5.0 %, 8.4-9.4%, 10.0-15.1% and 11.7-12.8% by weight, respectively, produce ectoparasitoids that are significantly superior to those produced using previously available artificial diets. Specifically, C. grandis cultivated in this medium exhibits substantially greater growth of pupae, fecundity (number of eggs produced by adult female), progeny females (number of female pupae produced by adult female), and net reproductive rate (R0, defined more forward), comparatively with those produced based on the artificial diets of the prior art. The concentrations of the remaining amino acids are variable, but they must be effective to promote the growth of C. grandis. The actual concentrations selected can be determined by the person skilled in the art. Without being limited in the present invention, the preferred amino acids and their concentrations within this fraction are shown in Table 1. The mineral and vitamin fractions are similar to those described in the Diets of War and others (1993, ibid) and Thompson (1975, ibid), the contents of each of which are incorporated by reference in the present invention, and only vary in the concentrations of the inorganic salts and vitamins therein. In summary, the mineral fraction comprises CaCl2, C0CI2, FeCl3, ZnCl2, K2HPO4, a2HP? 4, MnS0 «, MgSO * and CuS0«. As indicated by Thompson, these inorganic salts can be prepared as three separate aqueous supply solutions to avoid precipitation. The vitamin fraction comprises an aqueous mixture of biotm, calcium pantothenate (hemi-Ca ~ d-pan + or + eic acid), choline chloride, folic acid, io-inositol, nicotine to ida, pyridoxal-HCl, riboflavin and tia i a. The inclusion of vitamin B12 (cyanocobala ina) is optional. The vitamin fraction can also be prepared as aqueous supply solutions in the manner described by Thompson (1975, ibid), according to which riboflavin dissolves in water, followed by the remaining vitamins. The lipids in the growth medium include, but are not limited to, cholesterol in combination with free fatty acids, including linoleic acid, linolenic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid. To ensure proper distribution of the lipid within the medium, the lipids must be formulated with an emulsifier to form an emulsion. The emulsifier and the concentration thereof used in the medium must be non-toxic for the subject ectoparasitoid. Although it is envisaged that a variety of emulsifiers may be used, dilute aqueous solutions of lauryl sulfate, added at a concentration of less than, or equal to, about 160 mg lauryl sulfate per 100 ml of growth medium, particularly a approximately 100 mg / 100 ml. Techniques for preparing the lipid fraction were described by Thompson (1975, ibid) and Guerra et al. (1993, ibid). However, the formulation of the lipid fraction has been modified to reduce the amounts of the potentially toxic agents, ie, solvents, emulsifiers and bases, present therein. In a first preferred embodiment, the lipid fraction can be prepared by homogenizing cholesterol and the fatty acids with a small amount of an appropriate organic solvent, such as acetone, and adding enough emulsifier to form an emulsion. A Lewie base, such as KOH, can be added to raise the pH of the emulsion to about 7.0. Even when the lipid fraction can be prepared from cholesterol and fatty acids in pure or substantially pure form (eg, reagent grade), in the alternative these components can be provided in an impure source that occurs naturally or in vegetable oil. For example, in accordance with another preferred embodiment, olive oil can be used in place of pure fatty acids. Surprisingly, olive oil also works as a preservative, avoiding the need to incorporate antimicrobials. Likewise, egg yolk (fresh or dried) can be used as a source of cholesterol, to reduce or replace the pure cholesterol incorporated in the diet. The egg yolk also provides the double benefit of also functioning as a complementary protein source. Glucose is the preferred carbohydrate source used in the growth medium. However, it is envisaged that other carbohydrates, such as trehalose, may also be used.

Other adjuvants or supplements may also be incorporated into the medium to increase the growth of C. grandis, prevent the growth of microbial contaminants, or prevent the decomposition of the aforementioned fractions. The addition of a complementary source of proteins to the medium is particularly preferred to increase the growth of larvae of C. grandis. While it is contemplated that a plurality of protein sources is suitable for use in the present invention, including hydrolyzed proteins, such as yeast extract or peptone, it is preferred to use egg yolk (fresh or dried). As mentioned above, egg yolk not only provides a complementary source of protein, but can also supplement or replace coleeterol in the lipid fraction. When the medium is being used to support the growth of adult wasps rather than larvae, the yolk may be omitted from the medium. Several antimicrobials and preservatives can be incorporated into the growth medium to prevent contamination by bacteria and fungi. Suitable antimicrobials include anti-icotic or broad spectrum antibacterial agents as are known in the art, such as gentamicin, or preferably antifungal and phosphoric acid. Antioxidants can also be incorporated into the growth medium to increase stability and shelf life. Without being limited to that, propionic acid is preferred. As mentioned above, olive oil can also be used instead of antimicrobials. Agar, carrageenan or other solidifying agents as are conventional in the art can optionally be incorporated into the growth medium when a solid or semi-solid formulation is desired. The choice between a solid or liquid formulation of the growth medium will depend, in effect, on the manner of presentation to the ectoparasite as described above. The concentration and amount of all the components added to the medium must be effective to support the growth of C. grandis. The absolute amount of the amino acid fraction provided in the diet, that is, the combined amount of all the amino acids in the amino acid fraction, must be between approximately 5.8 to 6.5 g per 100 rnl of growth medium (exclusive of the yerna volume) of egg and added agar, if any is added). The concentrations and amounts of the remaining components, including minerals, vitamins, lipids, carbohydrates, proteins and other adjuvants are a little more variable, and can be determined by those skilled in the art. Without being limited to that, the preferred concentrations and amounts of these components are shown in Table 1. The amounts of the amino acid, mineral, vitamin, lipid, carbohydrate, antimicrobial and antioxidant fractions listed in Table 1 are reported as mg per 100 ml of growth medium exclusive of the volume of protein (egg yolk) or aga. Techniques for cultivating C. grandis larvae in vitro based on artificial diets were previously described by Guerra et al. (1993, ibid; and 1994, Entomol. Exp. Appl., 72: 11-16), the contents of each one of which is incorporated by reference in the present invention, and can be used with the growth medium of this invention. Hoffman (U.S. Patent 4,418,647), the contents of which are also incorporated by reference in the present invention, described artificial host eggs for culturing ectoparasitoids that are suitable for use in the present invention. In accordance with the preferred embodiment, solid or liquid growth medium is deposited in an appropriate container such as a petri dish or multiple well plate, and covered with a permeable membrane. When liquid medium is used, an absorbent or other solid material may be placed, such as a gel, cotton, or particularly polyester pad, within the container to retain the medium. In addition, due to the mobility of the larvae of the first and second pupae and their predisposition for cannibalism, multiple well plates are preferred? other compartmentalized vessels for use with solid media to segregate developing larvae. Cannibalism has not been observed using liquid medium that has been distributed in polyester pads due to the reduced mobility of the larvae on it. The permeable membrane covers can be formed from a variety of polymeric materials including, but not limited to, paraffin, calcium alginate, polyethylene, polypropylene and Parafilm. The use of Parafilm as a cover for the plates is particularly advantageous, since this membrane functions as an additional stimulator of oviposition for adult ovipositor females of C. grandis. Although treatment of the membrane with other oviposition stimulants, such as hexane or other low molecular weight alkanes, has been reported by Guerra et al. (1994, ibid) to further increase the oviposition of adult females, the use of these compounds are not preferred because of the potential toxicity to female wasps, particularly if large amounts are used. The growth medium per se of this invention functions as a stimulator of the oviposition of C. grandis females, but oviposition can be further increased by the use of these additional stimulants. The membrane can also be molded with dome-shaped convex or round projections, as described by Hoffman (4,418,647) to further stimulate oviposition. Coated plates containing growth medium are then exposed to adult gravid C. grandis females for a period sufficient to allow the ectoparasitoids to oviposit directly through the membrane cover, generally about 4 to 8 hours.

Any egg that adheres to the inner surface of the membrane can be easily removed by sprinkling the outer surface of the membrane with water and gently draining the plates until the eggs fall on the medium, as described by Guerra et al. (1994, ibid) . The plates containing the parasitoid eggs are then incubated at approximately 27 ° C to allow the eggs to hatch, and the larvae feed on the diets, and pupate therein. Emerging adult wasps are then collected for later use. Depending on the direct oviposition in the environment by the female wasps, the aforementioned technique allows mass cultivation of C. grandis with a minimum of handling by laboratory personnel. In an alternative modality, although more intensive in work, the eggs are first collected using larvae of the cotton weevil that have been encapsulated in a sheet of Parafilm, as described by Cate (1987, ibid), the contents of which are incorporated by reference in the present invention. The females of C_¡_ grandis are exposed to the encapsulated larvae of the cotton weevil for a period sufficient to allow oviposition. The Parafilm sheet can be removed later, and the eggs that adhere to its surface and / or the larvae are collected manually by brushing. After transferring them into the growth medium, the eggs are incubated until adult emergence, as described above.

Although newly emerged wasps of C. grandis can be released directly into a target field infested with cotton weevils, the effectiveness of these artificially cultivated wasps can be reduced as biological control agents. To achieve maximum efficacy, before release in the field, emerging adults of C. grandis are collected and exposed to cotton weevils inside environmental chambers in the production facility for a period sufficient to "train" the wasps and stimulate the production of eggs by females. After this exposure, usually around 2 to 4 days, wasps can be released in the field and exhibit a substantially greater efficacy as biological control agents than wasps that have not been trained in this way. Formulations of this growth medium can also be effective as a diet to support the growth of adults of C. grandis, and particularly of the female ovipositor wasps used as reproductive material for the mass propagation of this ectoparasitoid. The same formulation can be used as shown in Table 1, although egg yolk and agar are preferably omitted. In accordance with this modality, the adult wasps will feed on the growth medium when they are exposed to it. Oviposition can be achieved later as described, generally when female wasps are approximately 8 to 12 days old. In a preferred embodiment, it has been found that if adult females are exposed to larvae of the cotton weevil for a short period prior to oviposition, egg production increases substantially. During this exposure, the wasps do not need to feed, but only touch or come into contact with the larvae of the cotton weevil to stimulate maximum egg production. The following examples are only intended to further illustrate the invention, and are not intended to limit the scope thereof, which is defined by the claims.

EXAMPLE 1 Artificial diets 4 growth media of this invention, designated as a-1, a-2, β and t, were prepared and evaluated, in comparison with the diets described by Thompson (1975, ibid) and Guerra et al. (1993, ibid). The composition of the amino acid, vitamin, lipid and mineral fractions of the media a-1, a-2, ß and t is shown in tables 2 to 5, respectively, while the remaining components are shown in Table 6. Media a-1, a-2, ß and t were prepared as follows: Mixture of amino acids: Dissolve 6g (6.3g in media a-2) of the mixture of amino acids from table 2 in 70 ml (means a-1, ß and t) or 74 rnl (means a-1) of milli-Q water in a 250 ml screw cap bottle. Stir with a 5 crn long Teflon coated stir bar. Place the bottle in a 900-watt microwave oven and set the temperature as a maximum for 1.5 minutes. Shake the bottle manually for 10 seconds, and place it back in the microwave for another 30 seconds. Place the bottle on a magnetic plate and stir at medium speed until the mixture cools to 30 ° C. Fraction of vitamins: The vitamins included in table 3 were dissolved in 5 rnl (for media a) or 4 ml (for media ß and t) of water. Mix of 1ipids: Weigh 50 mg of cholesterol and place it in a 25 ml glass homogenizer, add 0.5 mL of acetone, mix, and dry with a current of 2 until it has a paste consistency. Pipette 3 ml of the fatty acid mixture from table 4 into acetone in the homogenizer. Dry the mixture as previously done until a pasty consistency is achieved by evaporating the acetone solvent. Add 5 ml of 2% lauryl sulfate solution (100 mg of lauryl sulfate) and homogenize until the mixture is uniform. Fraction of minerals: 3 supply solutions designated A, B and C were prepared by dissolving the salts shown in table 5 in 2.5 ml (for media a) or 2.0 nm (for media ß and t) of water ( volumes are per supply solution). Final mix of the diet: Pipet the lipids into the amino acids. Rinse the homogenizer and the plunger with 7 rnl of 2M KOH for diets a and 4 ml of 3M KOH for the ß and t diets. Autoclave the mixture for 20 minutes at 121 ° C and then allow the mixture to cool to room temperature. Under aseptic conditions, add 2.5 ml (for diets a) and 2 ml (for ß and t diets) of each of the saline solutions (A, B, and O). Add 5 ml of 50% aqueous glucose solution, and 5 ml (for diets a) or 4 rnl (for diets ß and t) of the vitamin solution, 1.5 ml of antibiotic (Sigma A 7292 antinicotic), and propionic acid solutions at 150 μl (41.8 ml in 8.2 ml of water per milliliter) -Q) and phosphoric acid (4.2 ml in 45.8 ml of water) All diets included egg yolk as a complement, portions of each previous basic diet were transferred (80 ml of the diets a and ß and 86.65 g of the diet r) by filtration (sterile organza polyester fabric) to a 150 ml bottle of sterile serum 20 ml of fresh egg yolk was added with a sterile syringe to the a and b diets, and 13.35 g of dried egg yolk was added to the diet t (Sig to E-0625) The jars were sealed with a rubber plug of the sleeve type, marked, and soul They dined at 2 ° C for up to 2 weeks. The unused portion of the diets, without egg yolk, could be stored at -20 ° C for 30 days. Diets a and ß were used in liquid form, while diet t was solid. A portion of the diet t (87.22 g) was transferred with a sterile syringe to a 50 ml sterile glass beaker. The beaker was placed in a 30 ° C water bath for 1 minute and 10.78 g of 7% ultra sterile pure agar solution was added (USB-product number 10907) (maintained in a water bath at 80 ° C). ° C). The Thompson diet was prepared as described in the 1975 publication, and the Guerra diet and others were prepared as described in the 1993 publication, except that it was modified to ensure the successful development of immature parasitoids. Lauryl sulfate at 200 mg / 100 ml H2O was toxic to immature parasitoids; thus, it was reduced to 100 mg / 100 ml of H2O. The concentration of the KOH solution was changed from 1N to 2M (7 rnl) to avoid dilution. Genital icine was substituted with anti-icotic and a solution of propionic acid-phosphoric acid to provide better fungal control.

EXAMPLE 2 Growth media a-1, a-2, ß and t of Example 1 were compared with the Diets of War and others and by pson to evaluate the quality of C. grandis grown therein. The parasitoids used in this study were cultured on larvae of the cotton weevil as it was first 00 described by Cate (1987, ibid). Larvae of the cotton boll weevil were produced at the R. T. Gast Insect Rearing Laboratory, Mississippi State, Mississippi. The adult wasps were kept in plexiglass cages and exposed to the weevils encapsulated within these cages, and developed at constant temperature. The time of exposure of the host to the wasps and the environmental conditions of the cultivation area varied between 25 to 28 ° C and a RH of 50 to 70%. Presentation of the diet Three mi of the liquid diets a, ß and of pson in sterile petri dishes coated with p > Sterile olieester (pellón, sewn interlining 910) and covered firmly with Parafilm to provide a barrier to stimulate the oviposition of female parasitoids. The diet t was distributed (220 μl per well) in disposable plastic trays of 128 wells for biensay (Bio-Ba-128, CD International, Pitman, NJ) while it was still molten, and the agar was allowed to solidify for 1 hour. minute. The diet of Guerra and others was placed in dishes of 24 wells for tissue culture, as described by the aus (Guerra et al., 1994, ibid). Algh Guerra and others reported that n-pentane, n-hexane, n-heptane, and isooctane acted as oviposition stimulants, none of these alkanes was used to stimulate oviposition in this study because of its toxicity to female wasps.

Oviposition Two mes were used to place the parasitoid eggs in the artificial diet. The first me was direct oviposition by wasps over the media. This was achieved by covering the petri dishes (containing the artificial diets) with Parafilm as explained above. The Parafilm cover was molded with round dome-shaped projections to stimulate the female parasitoids. The peri boxes covered with Parafilm were then exposed for 4 to 6 hours to the female parasitoids from 5 to 10 days old, which had previously oviposited on the larvae of the cotton weevil. The petri dishes, containing the eggs of the parasitoid, were placed in an environmental chamber at 27 ° C for 24 hours to allow incubation of the eggs. Afterwards, the Parafilm was removed and the petri dishes were covered with their covers, and the edges were sealed with Parafilrn to prevent the intrusion of microbial contaminants. The parasitoids were allowed to develop inside the petri dishes at 27 ° C, and each larva was fed daily with 20 μl of the corresponding liquid diet (9 days). This me did not require handling the parasitoid eggs. However, this me was substantially more effective with liquid diets (a-1, a-2, and ß) than solid diets (t and Guerra and others). There were high rates of cannibalism by the first pupae of the paraeitoid when the solid diet was used.

After ovipositing in the artificial diet, the females of the parasitoid were allowed to feed daily on 25 encapsulated larvae of the cotton weevil. After feeding on the host and coming into direct contact with it, the larvae were essential to maintain a high level of egg production in the females of C. grandis. The second me consisted of manually collecting the eggs of C. grandis from cells that contained encapsulated larvae of the weevil. A sheet of 25 encapsulated larvae of the cotton weevil was exposed to the parasitoid females for 14 hours. The Parafilm sheet that covered the larvae was removed manually, and the eggs adhered to its surface were brushed with a fine brush (No. 0) on a sheet of waxed paper. The eggs were washed in a 0.1% bleach solution for 20 minutes and then rinsed with sterile distilled water on a polyester cloth filter (autoclaved). The eggs were manually transferred to the aforementioned media using a fine brush (10/0 Robert Simn ons CP51). An egg was placed in each piozo; Afterwards, all the wells were closed with the plastic caps provided. Parasitoid fitness assessment The parasitoids developing in the test diets, as well as those developing on the natural host (cotton weevils) as controls, were maintained at 27 ± 1 ° C in a Percival environmental chamber. The development of 200 eggs until the third chrysalis in each diet and control to evaluate the survival from cannibalism was evaluated. 100 members of the third pupae of the parasitoid were observed developing in each diet and control until the adult stage to evaluate the survival.

Weight of pupae The female pupae of the parasitoid cultured in the different artificial diets (and controls) were collected 2 to 3 days after pupation and weighed in groups of variable numbers on a Mettler PM.100 scale. The weights of the different groups were analyzed in a regression model using the GLM software SAS procedure (SAS Institute 1988). Fecundity / sex ratio. Seventy-two females emerging from each artificial diet and controls were individually cultured in plastic petri dishes at a constant temperature of 27 ± 1 ° C. The sample size was selected to estimate the population average (μ) of eggs / female and eggs / female / day within a confidence interval (E) of 20 and 1.5, respectively, with a = 0.05 using the equation: n = ((Za /) 2 s2) / E2 where n is the sample size, Za / 2 = l-96 (from the tables), s is the standard deviation of the population (estimated from the sample 's'), and E is the confidence interval (Ott, 1984, An introduction to statistical methods and data analysis, second edition, Duxbury press, Boston, Massachusetts, 775 pp).

Each female was provided with only one male, water and honey. Three days after the emergence, each female parasitoid was exposed to 12 encapsulated larvae of the cotton weevil every 24 hours for 15 days. The dead females were not replaced, but the dead males were replaced during the first 13 days. Each day, Parafilm capsules including parasitized weevils were opened to count the number of oviposited eggs per female. Afterwards, they were resealed and returned to the environmental chamber for the development of the parasitoid. Nine days later, the Parafilm capsules were reopened to count and determine the sex of the parasitoid pupae. The number of oviposited eggs per female per day and the number and sex of the developing offspring during the 15-day period was recorded. The total number of oviposited eggs per female during a period of 15 days and the average oviposited eggs per day were used to compare the fecundity of developing females in the different diets against the controls. The sex of each member of the progeny of the hens was recorded, and a sex ratio was established for the total progeny. The GLM procedure of SAS software was used to analyze fertility and sex ratio data. Life tables were calculated for each group (Developed in each of the artificial diets and controls) of 72 females for the experimental period of 18 days (3 days before oviposition and 15 days of oviposition).

The 'm? '(progeny of females produced by female) multiplying the average number of eggs produced per female of age' x 'by the average proportion of developing females (= l- (l / sex ratio)) at age' x '. The '? X' (survival ratio from birth to age 'x') was compared to age 18 between the different treatments and controls. The net reproductive rate (R0) was calculated as: n R0 = S i? rn * x = 0 where n is the oldest age (18 in this study) (Krebs, Ecology: The Experimental Analysis of Distribution and Abundance, 3a. ed., Harper »Row, New York, 1985). Results and Discussion The Thompson diet presented in liquid form did not produce adults of C. grandis. Of a total of 200 eggs deposited in the Thompson diet, no second pupae were recorded; all C. grándis larvae died during the first stage. Adults of C. grandis were successfully obtained from all the other diets studied. The survival of larvae of C. grandis from the first to the third pupae was greatly affected by cannibalism. There were differences in the incidence of cannibalism between the different diets. The use of the polyester pad prevented cannibalism by reducing the ability of the first and second pupae to move. Thus, a liquid diet petri dish (a or ß) coated with a polyester pad produced between 15 to 25 adult parasitoids. The mode of presentation of the diet of Guerra and others reduced cannibalism by isolating groups of larvae within the wells. The yield of adult parasitoids in the modified diet of Guerra and others was between 10 to 14 per 24-well dish. The cannibalism in the diet was prevented by isolating the larvae of the parasitoid in individual wells in the disposable plastic plates for bioassay used. After the parasitoids moved to the third chrysalis, cannibalism ceased. The average survival of C. grandis from the third chrysalis to the adult was 95 ± 5% in diets a, ß, t and Guerra and others. Weight of pupae The weight of the female parasitoid pupae cultured on the a-1, a-2, ß and t diets was significantly lower than that of the controls (F = 51.15, d_f 1, 62, P = 0.0001) grown on cotton weevils (Picture 7). However, the weight of the pupae of females grown on these diets was significantly greater than the weight of the pupae of the females of the parasitoid cultured in the diet of Guerra and others (F = 57.7, df 1, 62, P = 0.0001). Fertility. The differences in fecundity observed among the females of C. grandis cultivated in the different diets and the controls, showed the same pattern as the weight of the pupae (Table 8). The females cultured in diets a-1, a-2, b and t produced a significantly lower number of eggs compared to females cultured in vivo (F = 79.0, df 1, 292, P = 0.0001). Likewise, the daily oviposition rate of the females cultivated in these four diets was lower than that of females cultured in cotton weevil larvae (F = 79.0, df 1, 292, P = 0.0001). However, the females cultured in these four diets (a-1, a-2, ß and t) showed a greater fecundity and a daily oviposition rate than those cultivated in the diet of Guerra and others (F = 139.3, df 1, 292, P = 0.0001). Progeny / sex relationship Significant differences were not observed in the number of members of the developing progeny of the females or in the progeny / sex ratio (in females per male) among the females cultured in the diets (a-1, a-2, ß and t ) (Table 9). The females cultured in the a-2 diet did not show significant differences in the progeny / sex ratio or in the survival of members of the progeny of females when compared with the females cultured in vivo. However, a significantly smaller number of females developed in the progeny of the females cultured in the a-1, ß and t diets than in the females cultured in vivo. The progeny / sex ratio of the females cultured in the a-2 and t diets was not significantly different from that of the females cultured in vivo, p >However, the progeny / sex ratio of the females cultured in the ß diet was significantly lower than that of the controls (F = 4.73, df 1, 287, P = 0.000.l). Females cultured in the diet of Guerra and others produced significantly fewer adult females in the progeny than females cultured i_n vivo (F = 44.28, df 1, 287, P = 0.0001) and in diets a-1, a-2, ß and t (F = 30.91, df 1, 287, P = 0.0001). The progeny / sex ratio of the females cultured in the War diet and others was significantly lower than that of the females cultured in vivo (£ = 13.94, _df 1, 287, P = 0.0002), in the diet a-1 (F = 4.58, df 1, 287, P = 0.033), in the diet a-2 (F = 10.94, df 1, 287, P = 0.00.11), and in the diet t (F = 5.46, df 1, 287 , P = 0.0202). There was no significant difference in the progeny / sex relationship between the females cultured in the War diet and others and those cultured in the ß diet. Because the females cultured in all treatments were provided with only 12 larvae of the cotton weevil per day, the occurrence of hyperparasitism due to host limitation was common. Therefore, the mortality of immature C. grandis observed in this study was mainly due to cannibalism among the first pupae. This is perhaps the reason for the lack of significance of the difference between the controls and diets a-1, a-2, ß and t in the number of females of the progeny. Net reproductive rate. The life table analysis showed a net reproductive rate (R0) of 138.29 for females cultured in vivo (Table 10). The highest value of R0 obtained from females cultured in vitro was 116.4 from the diet t. Diets a-1, a-2 and ß had a value of R0 of 77.9, 85.1 and 93.7, respectively. The lowest value of or was 29.9 obtained at p > Artir of the Diet of War and others. Diets produced females with fecundity slightly higher than the ß diet; however, females cultured in the ß diet had a higher survival rate (l?) than those cultured in the a diets (Table 10). Females cultured in the War diet and others had survival rates comparable to those of females cultured in vivo; However, their fertility was considerably lower (approximately 75%).

EXAMPLE 3 C. grandis grown in the growth medium t described in Examples 1 and 2 was evaluated for efficacy against cotton weevils under field conditions.

Materials and methods A 1/2 hectare cotton lot located at Rio Farms Inc. in Monte Alto, Texas was selected as the experimental field for testing C. grandis grown in vitro. This field was cultivated with Stoneville 132 cotton. No insecticide or other chemical was used in this field, and mechanical cultivation was restricted to the first 30 days after sowing. A control field was not necessary at this stage because no comparisons of the damage caused to the fruits and the yield of cotton were planned, and only the mortality of the cotton weevil was studied. Cultivation and releases of the parasitoid. During this experiment, 10 releases were made twice a week from 400 females of C. grandis. The parasitoids were cultured in the diet t as described in examples 1 and 2, except that the parasitoid eggs were initially obtained using the gamma diet as an oviposition stimulant, rather than encapsulated larvae of the cotton weevil. Approximately 5 ml of the diet was placed in a disposable plastic petri dish that was covered with Parafilm, and then exposed to a colony of 100 C. grandis females for a period of 4 hours. The parasitoid eggs were collected from the inner wall of the Parafilm cover. Once placed in the multiple well vessels, the parasitoid eggs were allowed to develop to the pupa stage at a constant temperature of 27 ° C. The pupae of the parasitoid were then collected and placed in an emergency cage until they were fully developed. Paraeitoid females were kept in the laboratory for 5 to 7 days after emergence before being released into the experimental lot. During this time, the females of the parasitoid were exposed to cotton weevils encapsulated for 2 to 4 days to stimulate egg production (preparation). Once ready, the females of the parasitoid were collected by aspiration in 11 boxes of paper filled with shredded paper. Two boxes were prepared, each containing 200 females, twice a week (Tuesday and Friday). The boxes were taken to the field on the same day and opened at two previously marked release points. The two selected release points consisted of the central cotton groove 40 from the perimeter of the field and 70 m separated from each other. Field samples 15 random samples of 1 rn were taken weekly from the experimental batch. The samples consisted of all the fructification material separated by abscission found within sample points of 1 ra2 and all the fruits present in a plant selected at random within each sample point of 1 2. All the fructification structures collected were dissected then in the laboratory under a microscope. All the immature stages of the cotton weevil and C. grandis present were recorded in each sample. The densities of the different immature stages of the cotton weevil were estimated from the random samples. The percentage of apparent parasitism of the second and third pupae and pupae of the cotton weevil was calculated as the quotient of parasitized weevils and the sum of healthy and parasitized weevils. The percentage of unexplained mortality was calculated as the quotient of dead weevils (for unexplained reasons) and the total weevils (dead, alive and parasitized) multiplied by 100. The percentage of mortality induced by parasitism was calculated as the quotient of parasitized cotton weevils and total weevils multiplied by 100. Results The observed densities of the different stages of live immature cotton weevils (in insects per rn2) are presented in Table 11. The females of C. grandis cultivated vitro imposed a significant percentage of parasitism on the second (36.4-100%) and third (18.4-57.1%) pupae and pupae (0-75%) of cotton weevils (Table 12). This parasitism resulted in a substantial mortality of these three stages of the cotton weevil (63.2-100%, 31.9-60% and 0-75% in the second and third pupae, and pupae, respectively) (Table 13). It is understood that the above detailed description is given by way of illustration only, and that modifications and variations may be made therein without departing from the spirit and scope of the present invention.

TABLE 1 Scale of proportions of the ingredients to prepare the merid diet (mg / 100 ml) Percentages of amino acids in the amino acid mixture Min. - Max. Vitamins Min. Max.

Alanine 3.1 - 4.83% Biotin 0.036 - 0.06 Pan ot nato Arginine 1.77 - 3.0% of Ca 1.2 - 2.0 Chloride of Asparagi at 4.0 - 6.0% choline 120.0 - 200.0 Acid Aspartic acid 0.0 - 0.33% folic acid 0.06 - 0.1 Cysteine 1.83 - 4.77% Myo-inositol 9.0 - 15.0 Glutaric acid 8.5 - 9.33% Nicotine ida 3.0 - 5.0 Glutamine 0.78 - 5.17% Pyridoxal-HCl 0.18 - 0.3 Glycine 2.9 - 3.0% Riboflavin 0.6 - 1.0 Histidine 10.05 - 15.0% Thiamine 0.12 - 2.0 Isoleucine 1.83 - 2.35% Vitamin B12 0.0 - 0.02 Leucine 1.83 - 2.57% Lysine 3.63 - 6.00% Lipids Methionine 1.22 - 1.67% Phenylalanine 3.0 - 3.42% Cholesterol 0.0 - 70.0 Proline acid 11.83 - 12.68% linoleic 18.53 - 30.88 Seriña acid 3.18 - 5.17 X linolenic 17.63 - 29.38 Threonine 3.67 - 7.07% Oleic acid 41.78 - 69.63 Aci or Tryptopf 6.0 - 6.1% palmitic 46.5 - 77.5 Tyrosine 6.0 acid 16.53% palmitoleic 9.15 15.25 Valine acid 4.43 - 5.17% stearic 9.38 15.63 Lauryl sulfate 80.0 160.0 Mixture of amino acids 5800.0 6500.0 Carbohydrates Minerals Glucose 240.0 - 280.0 Strain A CaCl2 18.0 - 30.0 Antimicrobials C0CI2 • 6H20 3.0 ~ 5.0 FeCl3 6H20 12.0 - 20.0 Antifungal 0.0 - 1.05 Acid ZnCl2 3.0 - 5.0 phosphoric 0.0 - 1.02 Strain B Antioxidant K2HPO4 44.25 - 74.0 Na2HP0 acid <; 7H20 6.0 - 10.0 Propionic 0.0 - 62.07 Strain C Protein source Egg yolk MnSO «H20 0.6 - 1.0 (dry) 140.00 - 200.0 MgSO * 7H20 72.0 - 120.0 CuSO «3.0 5.0 TABLE 2 Amounts (raq / 100 ml of the diet) of amino acids in four artificial diets to cultivate in vitro Catolaccus grandis Amino acid a-1 a-2 ß and t Alanina 186 195 290 Argi ina 106 111 180 Asparagine 240 252 360 Aspartic acid 7 8 20 Cysteine 286 300 110 Glutamine 47 50 310 Glutaric acid 560 588 510 Glycine 174 182 180 Histidine 603 633 900 Hydroxyproline 0 0 0 Isoleucine 141 148 110 Leucina 154 162 110 Lisina 218 229 360 Methionine 73 77 110 Phenylalanine 205 216 180 Proline 761 800 710 Seri to 19.1 201 310 Threonine 424 445 220 Tryptophan 366 384 360 Ti osina 992 1041 360 Valina 266 279 310 Total 6000 6300 6000 TABLE 3 Quantities (mg / 100 ml of the diet) of vitamins in four artificial diets to cultivate in vitro Catolaccus grandis Vitamin a-1 and a-2 ß and t Biotin 0.06 0.048 D-pantothenic acid (herni-Ca!> 2.00 1.60 Hill Chloride 200.00 160.00 0.10 0.08 folic acid Myo-inositol 15.00 12.00 Niacinamide 5.00 4.00 Pyridoxal-HCl 0.30 0.24 Riboflavin 1.00 0.80 Thiamin-HCl 0.20 0.16 Vitamin B12 0.02 0.016 Total 223.68 178.944 TABLE 4 Amounts (mg / 100 ml of the diet) of lipids in four artificial diets to cultivate in vitro Catolaccus grandis Lipido a-1, a-2, ß and t Cholesterol 50.0 Linoleic acid 24.7 Linolenic acid 23.5 Oleic acid 55.7 Palrnitic acid 62.0 Palrnitolic acid 12.2 Stearic acid 12.5 Other chemicals Acetone »(rnl) 3.0 Lauryl b Sulfate 100.0 a-1 and a-2 ß t KOH * 673.4 757.0 588.0 • As solvent of lipids (evaporated after mixing). * > As a homogenizer. c To neutra the pH to 7.0.

TABLE 5 Amounts (mg / 100 ml of the diet) of inerals in four artificial diets to cultivate in vitro Catolaccus grandis Inorganic salts a - 1? ? a - 2 ß and t Strain A CaCl2 30.0 24.0 C0CI2.6H2O 5.0 4.0 FeCl3.6H2? 20.0 16.0 ZnCl2 5.0 4.0 Strain B K2HPO4 dibasic 74.0 59.0 a? HPO; dibasic 10.0 8.0 Strain C MgSo «.7H20 120.0 96.0 CuS04.5H20 5.0 4.0 MnSO «.H2? 1.0 0.8 Total 270.0 215.8 TABLE 6 Amounts (mg / 100 ml of the diet) of other ingredients in four artificial diets for in vitro cultivation Catolaccus grandis Compound a-1, a-2, ß, t Carbohydrate 10 d-glucose 250.00 Antibiotic Antirnicotic 1.05 15 Fungicide Phosphoric acid 1.02 Antioxidant Propionic acid 62.07 20 aa ß Water 92175.18 91875.18 92190.52 92359.52 Complement a-1, a-2, ß t OK Egg yerna 20 * »13.35 Agar 0% 0.755« 20 ml of fresh egg yolk in 80 rnl of the diet added 30 after preparing the diet. ? »13.35 g of dried egg yolk in 86.65 g of basic diet to produce the supplemented diet. 35 c 10.78 g of 7% agar solution in 89.22 g of the supplemented diet.

TABLE 7 Comparison of the weight (mg) of female pupae of Catolaccuß grandis grown in five different artificial diets and in live* Diet n Sh Control 224 5.5 + 0.52a ß 125 4.4 + 0.23b a-2 186 4.2 + 0.49b t 204 4.1 + 0.34b a-1 180 4.0 + 0.19b War and others »1 116 2.3 + 0.32c «Insects maintained at 27 ± 1 ° C, 60 ± 5% RH, 14:10 L: D. ° Averages with a different type are significantly different in a «grown in cotton weevils encapsulated from the third chrysalis. or "Modified, see text." n = sample size TABLE 8 Comparison of the fecundity of Catolaccus grandis females grown in five different artificial diets and in vivo, at a constant temperature of 27 ° C Oviposition Eggs / Females »daily» Diet nx ± Se X ±? C Control * 1 49 294.2 ± 53.4a 19.6 ± 3.56at 62 217.2 ± 49.1b 14.5 ± 3.27b a-1 47 214.3 ± 63.7b 14.3 ± 4.25b a- 2 39 210.6 ± 65.9b 14.0 ± 4.39b ß 52 208.5 ± 60.0b 13.9 ± 4.00b War and others »49 104.4 ± 50.6c 7.0 ± 3.37c «During a period of 15 days. "Eggs per female / day Averages with different types are significantly different at 0.05." • Cultivated in cotton weevils encapsulated from the third chrysalis • Modified, see text n = sample size TABLE 9 Comparison of the progeny / sex ratio and females produced by Catolaccus grandis female cultured in five different artificial diets and in vivo, at a constant temperature of 27 ° C Pupa? S females Relationship by female »progeny / sex» Diet n X ± S x ± Se Control "1 49 33.6 ± 19.0a 4.84 ± 5.88a a-2 39 27.4 ± 12.8ab 4.65 ± 3.67ab t 62 27.4 ± 17.3 b 3.61 ± 3.65ab a-1 46 25.8 ± 14.5 b 3.58 ± 3.08ab ß 52 25.6 ± 14.4 b 3.15 ± 3.54bc War and others »45 12.2 ± 12.1 c 1.83 ± 2.30c • During a period of 15 days. "In females per male, averages with different types are significantly different in 0.05." * Cultivated in cotton weevils encapsulated in the third chrysalis. • Modified, see text n = sample size.

TABLE 10 Analysis of the life table of Catolaccus grandis females grown on five different artificial diets and in vivo, at 27 ± 1 ° C and 60 ± 5% RH Live females lx «R0h Control 79 0.620 143.66 a-1 97 0.485 77.92 a-2 78 0.500 85.09 ß 75 0.693 93.70 and 73 0.849 116.36 War and others0 79 0.620 29.85 • During a period of 17 days. o Proportion of times of average rate of oviposition of females in the offspring, c Modified, see text TABLE 11 Densities »of different live immature stages of the cotton weevil observed in an experimental cotton batch in Monte Alto, Texas, and calculated from 15 random samples Date Eggs Ll L2 L3 Pupa MAY 16 0.00 0.60 0.00 0.00 0.00 MAY 23 1.79 1.85 0.00 0.40 0.13 MAY 30 4.76 1.79 0.60 0.53 0.07 JUNE 6 4.76 4.17 0.66 1.80 0.07 JUNE 13 11.31 13.16 4.63 5.32 1.87 lIn insects by rn2 TABLE 12 Percentage of apparent parasitism by Catolaccus grandis of susceptible immature stages of the cotton boll weevil on the spot DATE Second Third Pupa All: Pupa pupa stages MAY IB MAY 23 100. .00 57, .14 0. .00 55 .56 MAY 30 100. .00 46, .67 75., 00 57 .14 JUNE 6 50, .00 40. .00 0., 00 39 .39 JUNE 13 36. .36 18. .42 20., 00 20 .49 Unexplained mortality was excluded from these calculations.

TABLE 13 Percentage of apparent unexplained mortality induced by parasitism by Catolaccus grandis observed in different immature stages of the cotton boll weevil in the field Date Second pupae Pupa pupae third Inexp. Ind. Inexp. Ind. Inexp. Ind.

MAY 16 15 MAY 23 33.3 66.7 6.7 53.3 0.0 0.0 MAY 30 60.0 40.0 6.3 43.8 0.0 75.0 JUNE 6 90.5 4.8 23.1 30.8 66.7 0.0 JUNE .1.3 42.1 21.1 16.5 15.4 2.8 19.4 ,twenty

Claims (20)

NOVELTY OF THE INVENTION CLAIMS

1. - A means of growth p > to cultivate insect ectoparasitoids comprising a fraction of amino acids, a fraction of minerals, a fraction of vitamins, a fraction of lipids and a fraction of carbohydrates; wherein the improvement comprises said fraction of amino acids, fraction of minerals, fraction of vitamins, fraction of lipids and fraction of carbohydrates being present in amounts and proportions effective to support the growth of Catolaccus grandis, and in addition where said fraction of amino acids comprises alanine , glutamic acid, histidine and proline at a concentration of approximately 3.1-4.83%, 8.5-9.33X, 10.05-15.0% and 11.83-12.68% by weight, respectively.

2. The growth medium of claim 1, wherein said amino acid fraction comprises alanine, arginine, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

3. The growth medium of claim 2, wherein said amino acid fraction further comprises aspartic acid.

4. - The growth medium of claim 2, wherein said amino acid fraction comprises 3.1-4.83% of alanine, 1.77-3.0% of arginine, 4.0-6.0% of asparagine, 1.83-4.77% of cysteine, 8.5-9.33% of glutamic acid, 0.78-5.17% glutamine, 2.9-3.0% glycine, 10.05-15.0% histidine, 1.83-2.35% isoleucine, 1.83-2.57% leucine, 3.63-6.00% plant, 1.22-1.67% ethionine, 3.0-3.42% phenylalanine, 11.83-12.68% proline, 3.18-5.17% serine, 3.67-7.07% threonine, 6.0-6.1% tryptophan, 6.0-16.53% tyrosine and 4.43-5.17% valine , and also comprises 0-0.33% aspartic acid.

5. The growth medium of claim 4, comprising between about 5800-6500 mg of said amino acid fraction per 100 rnl of said medium.

6. The growth medium of claim 1, further comprising a protein source.

7. The growth medium of claim 6, wherein said protein source comprises egg yolk.

8. The growth medium of claim 1, wherein said lipid fraction comprises linoleic acid, linolenic acid, oleic acid, palmitic acid, palmitinic acid, stearic acid, and an emulsifier.

9. The growth medium of claim 8, wherein said lipid fraction further comprises cholesterol or egg yolk, or both.

10. The growth medium of claim 1, wherein said lipid fraction comprises olive oil and a surfactant.

11. The growth medium of claim 10, wherein said lipid fraction further comprises cholesterol or ye-a-egg, or both.

12. The growth medium of claim 1, wherein said carbohydrate fraction comprises glucose or t slab.

13. The growth medium of claim 1, further comprising one or more antimicrobial agents.

14. The growth medium of claim 13, wherein said antimicrobial agents are selected from the group consisting of an antifungal agent and phosphoric acid.

15. The growth medium of claim 1, further comprising an antioxidant.

16. The growth medium of claim 1, wherein said action of minerals comprises CaCl2, C0Cl2, FeCl3, ZnCl2, K2HPO ?, Na2HP4, MnSO *, MgSO and CuS0".

17. The growth medium of claim 1, wherein said fraction of vitamins comprises biotin, Ca pantothenate, choline chloride, folic acid, myo-inositol, nicotinamide, pyridoxal-HCl, riboflavin and thiamine.

18. The growth medium of claim 17, wherein said vitamin fraction further comprises vitamin B12.

19. - A method for cultivating the ectoparasitoid Catolaccus grandis comprising p > contacting the eggs of C. grandis with the growth medium of claim 1, and incubating under conditions and for an effective period to allow said eggs to mature in adults.

20. A method for cultivating the ectoparasitoid Catolaccus grandis, comprising providing adults of C. grandis with the growth medium of claim 1.