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In Vitro Rearing of Muscidifurax zaraptor (Hymenoptera: Pteromalidae) on Artificial Diets with and Devoid of Insect Material
Biological Control 18, 49–54 (2000) doi:10.1006/bcon.1999.0807, available online at http://www.idealibrary.com on
In Vitro Rearing of Muscidifurax zaraptor (Hymenoptera: Pteromalidae) on Artificial Diets with and Devoid of Insect Material Paolo Fanti*,1 and S. Bradleigh Vinson† *Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Universita` della Basilicata, via Nazario Sauro 85, 85100 Potenza, Italy; and †Department of Entomology, Texas A&M University, College Station, Texas 77843 E-mail: [email protected] Received June 22, 1999; accepted November 28, 1999
INTRODUCTION We present the first report on in vitro rearing of Muscidifurax zaraptor Kogan and Legner, a pupal parasitoid of Musca domestica L. and other muscoid flies. We first tested seven artificial diets composed of different amounts (0, 25, and 50%) of a Mu. domestica pupal extract, 25% fresh chicken egg yolk, and 25% powdered milk solution. Then we compared one of the previously tested diets (25% of pupal extract) with three other diets in which either the egg yolk or milk or both were removed. We tested these four diets using insect material either from Mu. domestica or a nonhost insect source, which was Heliothis virescens (F.). We observed no significant differences in the rates of development to adult in the diets with 50 or 25% pupal extract. Almost no adults emerged from two diets without insect material, but in a third one about 8% of parasitoids completed development. Egg yolk significantly improved the yield of adults, while the addition of milk was not beneficial. Using the H. virescens pupal extract similar results were observed, indicating that insect material from a nonhost source is suitable for the development of M. zaraptor. The emerged adults mated and parasitized house fly pupae, producing viable offspring. The developmental time in vitro from egg to adult was slightly longer than that reported in vivo. The results we observed suggest that the highest yields of parasitoid adults from diets with insect material can be explained in terms of a better nutritional balance rather than advocating ‘‘host factors.’’ r 2000
Many species in the family Pteromalidae (Hymenoptera: Chalcidoidea) are important biological control agents against muscoid flies (Rueda and Axtell, 1985). Several species have been mass reared and released to reduce filth fly populations in poultry and livestock facilities (Morgan, 1986; Petersen, 1993). Hunter (1994) listed 40 commercial suppliers of filth fly parasitoids in the United States and Canada. Data supporting the efficacy of parasitoid releases to control filth flies conflict, and successes as well as failures have been reported. Some problems affecting the effectiveness of this approach are linked to the quality of released parasitoids, the high release rates necessary to achieve a major fly reduction, and the economical feasibility of this control tactic (Petersen, 1993; Catangui et al., 1997; Greene et al., 1998). It has long been theorized that artificial diets and in vitro rearing techniques could improve the opportunities for biological control through augmentative releases by lowering the cost of production of entomophagous insects (Greany et al., 1984; Grenier et al., 1993; Vinson, 1994). In this paper we report the preliminary results from in vitro development of the pteromalid Muscidifurax zaraptor Kogan and Legner, a pupal parasitoid of muscoid flies, on some diets with or devoid of insect material. In previous trials (Fanti, unpublished results), the development of M. zaraptor from egg to adult was achieved on a diet consisting of 50% insect material (i.e., a pupal extract of the natural host Musca domestica L.). The objectives of the present work were to assess (1) if M. zaraptor could develop on diets devoid of or with an amount of insect material lower than 50%; (2) if an insect source other than a natural host could replace the house fly pupal extract; and (3) the nutritional value of chicken egg yolk and reconstituted milk as components of artificial diets.
Academic Press
Key Words: hemolymph-based diets; filth fly parasitoids; Muscidifurax zaraptor; Hymenoptera; Pteromalidae; egg yolk; artificial diets; in vitro rearing; biological control.
1 To whom correspondence should be addressed. E-mail: [email protected]. Fax: ⫹⫹39 0971 55748.
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1049-9644/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS
Biological Material The parasitoids used to start the colony were obtained from a commercial M. zaraptor strain from Kunafin Trichogramma, Quemado, Texas. The species identity was verified with the morphological keys given by Rueda and Axtell (1985). The adult wasps were kept in small plastic cages at 26.5°C, 60% RH, and 16:8 (L:D) h. The house fly pupae were provided by F. W. Plapp from colonies maintained at the Department of Entomology, Texas A&M University (College Station, TX). Because M. zaraptor can develop on freeze-killed hosts (Petersen and Matthews, 1984), we froze the host pupae for practical purposes and kept them stored at ⫺5°C before exposure to the wasps. Diet Preparation The host pupal extract (PE) was prepared using the following procedure: 48-h-old house fly pupae were coddled at 60°C for 12 min and then squeezed using a 20-ml syringe. The homogenate was collected in tubes and centrifuged at 1000 rpm for 15 min. The supernatant was collected and filter sterilized using a 0.22-µm Millex filter (Millipore). All diets tested were retained with agar as 1% of final diet volume. Agar was dissolved in either dry bovine milk reconstituted with water (RM) or a 7% v/v maltose solution (MS) and autoclaved at 120°C for 20 min. We let the autoclaved solution cool down before mixing it with the other ingredients, all of them filter sterilized with 0.22-µm Millex filters (Millipore), except fresh chicken egg yolk (EY). We poured each diet into 60 cells of a Multiwell 96 Falcon plate. The 36 external cells of the plate were half filled with sterile water to guarantee a high RH in the plate. After pouring the diet, the rearing plates were left for 1 day at room temperature before transferring the parasitoid eggs to the diet. In all the diets tested, gentamicin sulfate (Sigma Chemical, Co., St. Louis, MO) was added as 0.1% of the final diet volume.
Experimental Design, Data Collection, and Statistical Analysis Influence of pupal extract. We tested seven oligidic diets, each with a different PE content (Table 1). We replaced the PE partially or completely with two insect cell culture media (TCM) (i.e., TNM-FH by Sigma or SF-900 by Gibco) or by a modified version of a diet developed by Nettles for the rearing of a dipteran parasitoid, Eucelatoria bryani Sabrosky (Nettles, 1986a). The amount of the other two components (i.e., fresh chicken EY and dry milk (RM) reconstituted with water as described in Xie et al. (1986)) was the same in all the diets tested. The number of replicates (60 transferred M. zaraptor eggs in each replicate) was n ⫽ 5 for diet PE50, PE-25/TNM-25, and PE-00/TNM-50 and n ⫽ 4 for all the other diets. Influence of egg yolk and reconstituted milk. We tested another four diets (see Table 2) to assess the value of the EY and RM. When EY was not added, we correspondingly increased PE and TCM. When we removed the RM, we replaced it with a 7% MS. The four diets were tested using a PE from the natural host Mu. domestica (four replicates), or from a non-natural host source, Heliothis virescens (F.) (five replicates). The latter PE was prepared following the same procedure used with Mu. domestica. After the transfer of the eggs, the Multiwell plates containing the diets were kept at 25°C and 75% RH. The development was monitored visually. The egg hatching was evaluated by counting the larvae and the empty egg chorions. After pupation, we transferred each new parasitoid pupae into a cell of a new Microwell plate until the emergence of the adult. We measured the developmental time and the sex ratio of the emerging adults only in the second part of the experiment TABLE 1 Percentages of Composition (v/v) of the Diets Tested Relative to Variations in Host Pupal Extract (PE), Insect Cell Culture Media (TNM-FH or SF-900), and Nettles’ Diet for Eucelatoria bryani (Nettles, 1986) Modified by Deleting All Lipids (NDM) a
Egg Collection Previously freeze-killed house fly pupae were exposed to parasitoid females for as long as 15–18 h to obtain superparasitization. Then the pupae were surface disinfected in a 70% ethanol solution and rinsed with distilled water. The puparium was cut with a scalpel and removed to expose the naked pupa and eggs were collected using a sterile needle attached to a wooden handle. The collected eggs were surface disinfected twice with a 0.4% formaldehyde solution and then rinsed with sterile water before transferring them to diet.
Diet
PE
TNM-FH (Sigma)
SF-900 (Gibco)
NDM
PE-50 PE-25/TNM-25 PE-00/TNM-50 PE-25/SF-25 PE-00/SF-50 PE-25/NDM-25 PE-00/NDM-50
50 25 — 25 — 25 —
— 25 50 — — — —
— — — 25 50 — —
— — — — — 25 50
a All diets included 25% each of fresh chicken egg yolk and dry milk reconstituted with water. All diets were gelled with agar at 1% of final diet volume. Gentamicin sulfate (Sigma) was added as 0.1% of the final diet volume.
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(influence of the EY and RM). All the operational steps, included visual monitoring, were done under a laminarflow hood. When more than 10 adults were collected from a diet, they were exposed to house fly pupae to determine if in vitro reared adults still recognized fly pupae as an oviposition site. The emerging parasitoids of the next generation were tested again in the same way. We did not determine the parasitoid fertility or make any comparison of parasitoids reared in vitro with those reared in vivo. Data collected in the first part of the experiment were analyzed by one-way ANOVA. Data collected in the second part of the experiment were analyzed by a 4 ⫻ 2 factorial design (Diet vs. PE source), except for developmental times, analyzed by a factorial design (Diet vs. PE source vs sex of the parasitoid). Mean comparison (Tukey’s test) was performed when statistical significance (␣ ⫽ 0.05) occurred. All percentages were arcsine transformed before analysis (Zar, 1984). Mean percentages presented in tables were transformed back into proportions after analysis. Because the confidence limits are not symmetrical about the means when expressed again in proportions, we present in tables the mean value and the mean value plus and minus the SE. All data analyses were performed with the statistical package Systat ver. 6.01 for Windows (Systat, 1996). RESULTS
Influence of Pupal Extract Eggs hatched 24–36 h after being transferred onto the diets. The hatching rate was about 90% or more (Table 3) and was unaffected by the diets tested (F ⫽ 0.95; df ⫽ 6, 21; P ⫽ 0.48). The pupation rate was significantly affected by the rearing media (F ⫽ 5.8; df ⫽ 6, 21; P ⫽ 0.001), with no statistical difference among the diets with 50 or 25% PE. In the diets with no PE, most of the larvae reached the last instar, but their
TABLE 2 Percentages of Composition (v/v) of the Diets Tested Relative to Variations in Host Pupal Extract (PE), Insect Cell Culture Media (SF-900), Fresh Chicken Egg Yolk (EY); Dry Milk Reconstituted with Water (RM), and Maltose Solution (7% w/v) a Diet
PE
SF-900 (Gibco)
EY
RM
MS
1 2 3 4
25 25 37.5 37.5
25 25 37.5 37.5
25 25 — —
25 — 25 —
— 25 — 25
a All diets were gelled with agar at 1% of final diet volume. Gentamicin sulfate (Sigma) was added as 0.1% of the final diet volume.
TABLE 3 Developmental Parameters for M. zaraptor as Percentages of Transferred Eggs, Hatched Eggs, and Pupae with Respect to the Influence of Pupal Extract a,b Mean percentage c (⫹SE to ⫺SE) Diet PE-50 PE-25/TNM-25 PE-00/TNM-50 PE-25/SF-25 PE-00/SF-50 PE-25/NDM-25 PE-00/NDM-50
Hatching
Pupation
91.9a (93.3–90.4) 96.9a (98.0–95.7) 94.4a (95.9–92.6) 93.2a (97.3–87.5) 92.2a (93.4–90.9) 90.1a (94.3–85.0) 89.0a (91.6–86.1)
47.3a (60.5–34.4) 31.4ab (45.3–19.0) 1.7b (4.1–0.3) 49.5a (64.8–34.2) 1.0b (3.9–0.0) 45.4a (57.6–33.5) 11.1ab (20.0–4.5)
Emergence n 85.3a (90.4–79.4) 79.7a (87.5–70.5) 0.0b (0.0–0.0) 75.5a (83.6–66.5) 25.0ab
4 4 2 4 1
79.2a 4 (86.0–71.5) 68.5a 3 (72.8–64.0)
Adults 40.2a (52.6–28.2) 26.2ab (39.3–14.9) 0c (0.0–0.0) 38.1a (52.6–24.7) 0.2bc (0.9–0.0) 36.1a (47.7–25.3) 7.6abc (14.2–3.0)
Number of replicates ⫽ 4 except for Emergence, where indicated by n. Means followed by the same letter do not differ significantly (Tukey’s test, ␣ ⫽ 0.05). c See Materials and Methods for explanation relative to SE. a b
size was smaller and they failed to pupate, without voiding the meconium. When the PE was completely replaced with TNM-FH or SF-900 either no or a very few pupae were obtained, and almost all of the pupae failed to emerge as adults (Table 3). When the PE was completely replaced with the modified Nettles’ diet, the yield in adults was lower, but about 8% of parasitoids still completed the development and emerged as adults (Table 3). Adults that emerged from diets with 50 or 25% PE were allowed to mate and females were able to parasitize house fly pupae, producing viable offspring. Influence of Egg Yolk and Reconstituted Milk In this experiment, the hatching rate was about 90% (Table 4) and it was unaffected by the diets tested (F ⫽ 0.16; df ⫽ 3, 28; P ⫽ 0.93) or the PE source (F ⫽ 0.004; df ⫽ 1, 28; P ⫽ 0.95). The diet composition influenced the parasitoid larval development in a highly significant way, in terms of both pupae (F ⫽ 24.95; df ⫽ 3, 28; P ⬍ 0.0005) and emerged adults (F ⫽ 22.1; df ⫽ 3, 28; P ⬍ 0.0005). Because neither the PE source (P ⫽ 0.98 for the pupae and P ⫽ 0.31 for the adults) nor the interaction between the factors (P ⫽ 0.35 for the pupae and P ⫽ 0.60 for the adults) was significant, the multiple comparisons of pupae and adult mean rates were performed pooling the data from diets with a different PE source (Table 4). The percentages of developed parasitoids were significantly higher in the diets containing EY and the lowest in diet 4, with no EY and RM (Tukey’s test, ␣ ⫽ 0.05). RM was not a beneficial
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TABLE 4 Developmental Parameters of M. zaraptor as Percentages of Transferred Eggs, of Adults from Pupae, and of Females of Emerged Adults with Respect to the Influence of Egg Yolk and Reconstituted Milk a,b Mean percentage (⫹SE to ⫺SE) Diet
PE source Md
1 Hv Md 2 Hv Md 3 Hv Md 4 Hv
Hatching
Pupation
89.2a (90.5–87.8) 91.4a (94.5–87.7) 89.6a (91.1–88.0) 89.2a (91.1–87.0) 88.1a (89.8–86.4) 89.8a (92.0–87.3) 90.1a (92.4–87.4) 87.0a (90.9–82.5)
Emergence 85.2a (93.4–74.4) 71.9ab (81.4–61.2) 91.1a (92.5–89.6) 79.2ab (88.2–68.5) 58.4ab (73.8–42.2) 28.1b (39.2–18.2) 50.0ab (85.4–14.6) 27.0ab (50.0–9.22)
59.0a (65.8–52.1)
79.2a (83.5–74.5)
27.5b (36.6–19.2)
3.4c (6.8–1.1)
n 4 5 4 5 3 5 2 2
Adults
46.7a (55.4–38.0)
63.8a (70.0–57.0)
11.7b (18.3–6.4)
1.7b (3.3–0.6)
Sex ratio 46.4a (55.2–37.7) 44.3a (46.3–42.2) 45.4a (48.1–42.7) 52.3a (58.2–46.4) 46.2a (50.9–41.6) 51.2a (69.6–32.7)
n 4 5 4 5 3 5
85.4
1
70.0
1
Note. Md, Musca domestica, and Hv, Heliothis virescens. Number of replicates ⫽ 4 with Md and 5 with Hv except for Emergence and Sex Ratio where indicated by n. a Means followed by the same letter do not differ significantly (Tukey’s test, ␣ ⫽ 0.05). b See Materials and Methods for explanation relative to SE.
component when EY was present in the diet. In the media with no EY, the addition of milk resulted in a significantly higher percentage of pupae, but not of adults (Table 4). H. virescens proved to be an alternative source of insect material in terms of pupae and adults, even if the emergence rate (Adults from Pupae) was negatively affected (F ⫽ 4.30; df ⫽ 1, 22; P ⫽ 0.05). Because of the low number of adults obtained on diet 4, we did not consider those data in the analysis of developmental times and sex ratio. The rate of development (as days from egg to adult) was influenced in a significant way by the diet composition (F ⫽ 17.56; df ⫽ 2, 37; P ⬍ 0.0001) and the parasitoid sex (F ⫽ 17.55; df ⫽ 1, 37; P ⬍ 0.0001), (i.e., male adults emerged earlier than females) (Table 5). Neither the PE source (P ⫽ 0, 94) nor the interactions among factors TABLE 5 Egg–Adult Development (Days) of M. zaraptor with Respect to the Influence of Egg Yolk and Reconstituted Milk a Mean ⫾ SE Diet
Males
n
Females
n
1 2 3
25.8 ⫾ 1.4a 23.8 ⫾ 0.5a 31.6 ⫾ 1.1b
9 9 7
31.1 ⫾ 1.6ab 27.8 ⫾ 0.6a 36.1 ⫾ 1.9b
9 9 6
a Number of replicates indicated by n. Means followed by the same letter do not differ significantly (Tukey’s test, ␣ ⫽ 0.05).
were significant. The sex ratio of emerged adults (expressed as a percentage of females) was not affected by the composition of the diet (P ⫽ 0.25) nor by the PE source (P ⫽ 0.83). The mean values ranged from 44.3% in Diet 1 (Hv Pe source) to 52.3% in Diet 2 (Hv PE source) (Table 4). Adults that emerged, except those from diet 4 because of the low number, were allowed to mate and females were able to parasitize house fly pupae, producing viable offspring. DISCUSSION
Although some parasitoid species have been reared on a diet devoid of insect material (reviewed by Bratti, 1990; Grenier et al., 1993), a recurring theme in the development of in vitro rearing techniques is the apparent need to include either hemolymph or hemolymph derivatives in artificial media. Nettles (1990) suggested that host metabolites (‘‘host factors’’) are likely to be necessary components for successful in vitro rearing of several parasitoid species. Nettles provides as examples of host factors the role of asparagine for the development of the tachinid Eucelatoria bryani Sabrosky (Nettles, 1986b) and polar chemicals with a molecular weight less than 1000 as pupation factors for Trichogramma pretiosum Riley (Irie et al., 1987). Nakahara et al. (1997) report as host factors for the ichneumonid Venturia canescens (Gravenhorst) protein-like molecules with a molecular weight over 100,000, obtained by fractionation of a Galleria mellonella L. pupal
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extract. Bratti (1991) extends the concept of host factors to the host hormones, which have proved either essential, as in Nenon (1972), Lawrence (1988), and Fanti (1990), or at least beneficial (Hu and Vinson, 1997) by addition to the artificial diet. The concept of necessary host factors can be a useful one which can lead to the identification and synthesis of low-molecular-weight chemicals present in hemolymph. It should be stressed, however, that poor development of a parasitoid species on artificial diets with low amounts or devoid of insect material does not necessarily mean that host factors are involved or essential. It may simply reflect an inadequate knowledge of the optimal balance among the nutrients. For example, Bratti and Coulibaly (1995), observing a drop in adult yield of Exorista larvarum (L.) when grown on diets with an amount of insect material lower than 2.5%, concluded that one or more host factors were involved. However, several diets devoid of insect material yet providing successful growth and development of E. larvarum were later developed (Bratti et al., 1995; Mellini and Campadelli, 1995; Coulibaly, personal communication). The results we observed in the first part of our experiment could lead to the conclusion that host factors are involved in the successful growth of M. zaraptor. Although using a diet with no insect material (diet PE-00/NDM-50 of the first set of tested diets) we observed the complete development of the parasitoid, but in a lower percentage. In the second trial we obtained a significantly higher rate of adults in diets 1 and 2, with a PE content lower than that in diets 3 and 4 (Table 2). Also, the lepidopteran H. virescens, which is not a host, proved to be a suitable source of PE. We believe that the better results we obtained with diet 2 can be explained in terms of a better balance of nutrients, even if our present data do not exclude the presence of host factors. The lower percentage of pupae and adults obtained in diets 3 and 4 cannot be related to an increase in PE because, in the first experiment, diet PE50 gave results similar to those with the diets with a 25% PE content. Also, we know that the increase in SF-900 is not responsible for the poor performance of diets 3 and 4 because we later developed media with no PE and an amount of tissue culture media as high as 50%, on which we obtained pupation and adult rates at least as good as those in the best diet (diet 2) described in this paper (Fanti and Vinson, unpublished data). EY is a good source of proteins, amino acids, phospholipids, many minerals, and vitamins (Stadelman, 1992) and proved to be a valuable component of artificial diets for M. zaraptor. It has been used successfully in the in vitro rearing of many species (reviewed in Bratti, 1990; Thompson, 1999). In some instances egg yolk has proven beneficial only in small amounts (Hu and Vin-
son, 1997; Bratti and Nettles, 1992). Raising the amount of EY, Hu and Vinson (1997) reported physical more than nutritional problems in the medium, as the respiration and movement of the larvae appeared restricted. A similar problem was reported by Consoli and Parra (1997) and House (1954), which was reduced larval respiration when high concentrations of EY increased the media viscosity. Xie et al. (1997) successfully used the sediment obtained by ultracentrifugation of chicken EY instead of whole EY. They obtained a precipitate with an increased protein content and lower amounts of triacylglicerols. They credited the good results obtained by adding this component to a better balance of protein/ lipids, resembling the ratio present in the lepidopteran host eggs. In the present study we did not observe any problem using a concentration of EY as high as 25%. Our media were gelled with agar and the larvae were found to feed mainly on the surface of the diet, as they normally do in vivo on the host pupa. Thus they avoid any problem of respiration and movements due to a high lipid content. The use of RM did not prove beneficial, although we observed a higher percentage of developed pupae when it was added in diet 3, compared to diet 4. By contrast, in the diets with EY, M. zaraptor development was not improved by addition of RM. A detrimental effect of milk in artificial diets has been reported in Trissolcus basalis (Wollaston) by Volkoff et al. (1992) and in Trichogramma galloi Zucchi and T. pretiosum by Consoli and Parra (1997). Xie et al. (1997) concluded that milk has no beneficial effect on the in vitro development of Trichogramma minutum Riley and T. brassicae Bezdenko. As for the biological parameters of the M. zaraptor adults developed in vitro on our best medium (diet 2), the results are encouraging. The mean developmental times, 23.8 and 27.8 days for males and females, respectively, are slower, but comparable with the developmental time (23.4 days for the species) in vivo reported by Mann et al. (1990). The sex ratio was close to 1:1 and the females were able to oviposit on house fly pupae, producing viable offspring. No gross morphological malformations were observed in the adults that developed in vitro. This paper is the first to report rearing the larval stages of M. zaraptor on artificial diets. Our results show that this pteromalid wasp is a promising candidate for developing in vitro rearing techniques. Although the best medium described in this paper cannot be considered economically feasible, it can be used as an experimental tool for biological and physiological studies on M. zaraptor. ACKNOWLEDGMENT Support from the Advanced Technology Program of the Texas Coordinating Board for Project 999902-050 is acknowledged.
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Mellini, E., and Campadelli, G. 1995. Further simplifications in the composition of oligidic diets for the parasitoid Exorista larvarum (L.). Boll. Ist. Ent. Univ. Bologna 49, 211–223. Morgan, P. B. 1986. Mass culturing microhymenopteran pupal parasites (Hymenoptera: Pteromalidae) of filth breeding flies. In ‘‘Biological Control of Muscoid Flies’’ (R. S. Patterson, and D. A. Rutz, Eds.), Vol. 61, pp. 77–87. Miscellaneous Publications of the Entomological Society of America. Nakahara, Y., Iwabuchi, K., and Mitsuhashi, J. 1997. In vitro rearing of the larval endoparasitoid, Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae). III. Growth promoting ability of extract of Galleria mellonella pupae. Appl. Entomol. Zool. 32(1), 91–99. Nenon, J. P. 1972. Culture in vitro des larves d’un Hymenopte`re endoparasite polyembryonnaire: Ageniaspis fuscicollis. Role des hormons de synthe`se. C. R. Acad. Sci. Paris 274, 3409–3412. Nettles, W. C. Jr. 1986a. Effects of soy flour, bovine serum albumin, and three amino acid mixtures on growth and development of Eucelatoria bryani (Diptera: Tachinidae) reared on artificial diets. Environ. Entomol. 15, 1111–1115. Nettles, W. C. Jr. 1986b. Asparagine: a host chemical essential for the growth and development of Eucelatoria bryani, a tachinid parasitoid of Heliothis spp. Comp. Biochem. Physiol. 85A, 697–701. Nettles, W. C. Jr. 1990. In vitro rearing of parasitoids—Role of host factors in nutrition. Arch. Insect Biochem. Physiol. 13(3–4), 167– 175. Petersen, J. J. 1993. Biocontrol for insect pests of livestock and poultry. In ‘‘Pest Management: Biologically Based Technologies’’ (R. D. Lumsden and J. L. Vaughn, Eds.), pp. 124–132. Am. Chem. Soc., Washington, DC. Petersen, J. J., and Matthews, J. R. 1984. Effects of freezing of host pupae on the production of progeny by the filth fly parasite Muscidifurax zaraptor (Hymenoptera: Pteromalidae). J. Kansas Entomol. Soc. 57, 387–393. Rueda, L. M., and Axtell, R. C. 1985. ‘‘Guide to Common Species of Pupal Parasites (Hymenoptera: Pteromalidae) of the House Fly and other Muscoid Flies Associated with Poultry and Livestock Manure.’’ Technical Bulletin 278, North Carolina Agricultural Research Service, North Carolina State University, Raleigh, NC. Stadelman, W. J. 1992. Eggs and products. In ‘‘Encyclopedia of Food Science and Technology’’ (Y. H. Hui, Ed.), pp. 671–678. Wiley, New York. Systat, 1996. ‘‘Systat 6.01 for Windows: Statistics.’’ SPSS, Chicago, IL. Thompson, S. N. 1999. Nutrition and culture of entomophagous insects. Annu. Rev. Entomol. 44, 561–592. Vinson, S. B. 1994. Parasitoids in vitro rearing: Successes and challenges. In ‘‘Techniques of Insect Rearing for the Development of Integrated Pest and Vector Management Strategies’’ (J. P. R. Ochieng’-Odero, Ed.), pp. 49–104. ICIPE Sci. Press, Nairobi. Volkoff, N., Vinson, S. B., Xu, Z.-X., and Nettles, W. C. Jr. 1992. In vitro rearing of Trissolcus basalis (Hym., Scelionidae) an egg parasitoid of Nezara viridula (Hem., Pentatomidae). Entomophaga 37, 141–148. Xie, Z.-N., Nettles, W. C. Jr., Morrison, R. K., Irie, K., and Vinson, S. B. 1986. Three methods for the in vitro culture of Trichogramma pretiosum Riley. J. Entomol. Sci. 21, 133–138. Xie, Z.-N., Wu, Z.-X., Nettles, W. C. Jr., Saldana, G., and Nordlund, D. A. 1997. In vitro culture of Trichogramma spp. on artificial diets containing yeast extract and ultracentrifuged chicken egg yolk but devoid of insect components. Biol. Control 8, 107–110. Zar, J. H. 1984. ‘‘Biostatistical Analysis,’’ 2nd ed. Prentice Hall, Englewood Cliffs, NJ.
In Vitro Rearing of Muscidifurax zaraptor (Hymenoptera: Pteromalidae) on Artificial Diets with and Devoid of Insect Material Paolo Fanti*,1 and S. Bradleigh Vinson† *Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Universita` della Basilicata, via Nazario Sauro 85, 85100 Potenza, Italy; and †Department of Entomology, Texas A&M University, College Station, Texas 77843 E-mail: [email protected] Received June 22, 1999; accepted November 28, 1999
INTRODUCTION We present the first report on in vitro rearing of Muscidifurax zaraptor Kogan and Legner, a pupal parasitoid of Musca domestica L. and other muscoid flies. We first tested seven artificial diets composed of different amounts (0, 25, and 50%) of a Mu. domestica pupal extract, 25% fresh chicken egg yolk, and 25% powdered milk solution. Then we compared one of the previously tested diets (25% of pupal extract) with three other diets in which either the egg yolk or milk or both were removed. We tested these four diets using insect material either from Mu. domestica or a nonhost insect source, which was Heliothis virescens (F.). We observed no significant differences in the rates of development to adult in the diets with 50 or 25% pupal extract. Almost no adults emerged from two diets without insect material, but in a third one about 8% of parasitoids completed development. Egg yolk significantly improved the yield of adults, while the addition of milk was not beneficial. Using the H. virescens pupal extract similar results were observed, indicating that insect material from a nonhost source is suitable for the development of M. zaraptor. The emerged adults mated and parasitized house fly pupae, producing viable offspring. The developmental time in vitro from egg to adult was slightly longer than that reported in vivo. The results we observed suggest that the highest yields of parasitoid adults from diets with insect material can be explained in terms of a better nutritional balance rather than advocating ‘‘host factors.’’ r 2000
Many species in the family Pteromalidae (Hymenoptera: Chalcidoidea) are important biological control agents against muscoid flies (Rueda and Axtell, 1985). Several species have been mass reared and released to reduce filth fly populations in poultry and livestock facilities (Morgan, 1986; Petersen, 1993). Hunter (1994) listed 40 commercial suppliers of filth fly parasitoids in the United States and Canada. Data supporting the efficacy of parasitoid releases to control filth flies conflict, and successes as well as failures have been reported. Some problems affecting the effectiveness of this approach are linked to the quality of released parasitoids, the high release rates necessary to achieve a major fly reduction, and the economical feasibility of this control tactic (Petersen, 1993; Catangui et al., 1997; Greene et al., 1998). It has long been theorized that artificial diets and in vitro rearing techniques could improve the opportunities for biological control through augmentative releases by lowering the cost of production of entomophagous insects (Greany et al., 1984; Grenier et al., 1993; Vinson, 1994). In this paper we report the preliminary results from in vitro development of the pteromalid Muscidifurax zaraptor Kogan and Legner, a pupal parasitoid of muscoid flies, on some diets with or devoid of insect material. In previous trials (Fanti, unpublished results), the development of M. zaraptor from egg to adult was achieved on a diet consisting of 50% insect material (i.e., a pupal extract of the natural host Musca domestica L.). The objectives of the present work were to assess (1) if M. zaraptor could develop on diets devoid of or with an amount of insect material lower than 50%; (2) if an insect source other than a natural host could replace the house fly pupal extract; and (3) the nutritional value of chicken egg yolk and reconstituted milk as components of artificial diets.
Academic Press
Key Words: hemolymph-based diets; filth fly parasitoids; Muscidifurax zaraptor; Hymenoptera; Pteromalidae; egg yolk; artificial diets; in vitro rearing; biological control.
1 To whom correspondence should be addressed. E-mail: [email protected]. Fax: ⫹⫹39 0971 55748.
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1049-9644/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.
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FANTI AND VINSON
MATERIALS AND METHODS
Biological Material The parasitoids used to start the colony were obtained from a commercial M. zaraptor strain from Kunafin Trichogramma, Quemado, Texas. The species identity was verified with the morphological keys given by Rueda and Axtell (1985). The adult wasps were kept in small plastic cages at 26.5°C, 60% RH, and 16:8 (L:D) h. The house fly pupae were provided by F. W. Plapp from colonies maintained at the Department of Entomology, Texas A&M University (College Station, TX). Because M. zaraptor can develop on freeze-killed hosts (Petersen and Matthews, 1984), we froze the host pupae for practical purposes and kept them stored at ⫺5°C before exposure to the wasps. Diet Preparation The host pupal extract (PE) was prepared using the following procedure: 48-h-old house fly pupae were coddled at 60°C for 12 min and then squeezed using a 20-ml syringe. The homogenate was collected in tubes and centrifuged at 1000 rpm for 15 min. The supernatant was collected and filter sterilized using a 0.22-µm Millex filter (Millipore). All diets tested were retained with agar as 1% of final diet volume. Agar was dissolved in either dry bovine milk reconstituted with water (RM) or a 7% v/v maltose solution (MS) and autoclaved at 120°C for 20 min. We let the autoclaved solution cool down before mixing it with the other ingredients, all of them filter sterilized with 0.22-µm Millex filters (Millipore), except fresh chicken egg yolk (EY). We poured each diet into 60 cells of a Multiwell 96 Falcon plate. The 36 external cells of the plate were half filled with sterile water to guarantee a high RH in the plate. After pouring the diet, the rearing plates were left for 1 day at room temperature before transferring the parasitoid eggs to the diet. In all the diets tested, gentamicin sulfate (Sigma Chemical, Co., St. Louis, MO) was added as 0.1% of the final diet volume.
Experimental Design, Data Collection, and Statistical Analysis Influence of pupal extract. We tested seven oligidic diets, each with a different PE content (Table 1). We replaced the PE partially or completely with two insect cell culture media (TCM) (i.e., TNM-FH by Sigma or SF-900 by Gibco) or by a modified version of a diet developed by Nettles for the rearing of a dipteran parasitoid, Eucelatoria bryani Sabrosky (Nettles, 1986a). The amount of the other two components (i.e., fresh chicken EY and dry milk (RM) reconstituted with water as described in Xie et al. (1986)) was the same in all the diets tested. The number of replicates (60 transferred M. zaraptor eggs in each replicate) was n ⫽ 5 for diet PE50, PE-25/TNM-25, and PE-00/TNM-50 and n ⫽ 4 for all the other diets. Influence of egg yolk and reconstituted milk. We tested another four diets (see Table 2) to assess the value of the EY and RM. When EY was not added, we correspondingly increased PE and TCM. When we removed the RM, we replaced it with a 7% MS. The four diets were tested using a PE from the natural host Mu. domestica (four replicates), or from a non-natural host source, Heliothis virescens (F.) (five replicates). The latter PE was prepared following the same procedure used with Mu. domestica. After the transfer of the eggs, the Multiwell plates containing the diets were kept at 25°C and 75% RH. The development was monitored visually. The egg hatching was evaluated by counting the larvae and the empty egg chorions. After pupation, we transferred each new parasitoid pupae into a cell of a new Microwell plate until the emergence of the adult. We measured the developmental time and the sex ratio of the emerging adults only in the second part of the experiment TABLE 1 Percentages of Composition (v/v) of the Diets Tested Relative to Variations in Host Pupal Extract (PE), Insect Cell Culture Media (TNM-FH or SF-900), and Nettles’ Diet for Eucelatoria bryani (Nettles, 1986) Modified by Deleting All Lipids (NDM) a
Egg Collection Previously freeze-killed house fly pupae were exposed to parasitoid females for as long as 15–18 h to obtain superparasitization. Then the pupae were surface disinfected in a 70% ethanol solution and rinsed with distilled water. The puparium was cut with a scalpel and removed to expose the naked pupa and eggs were collected using a sterile needle attached to a wooden handle. The collected eggs were surface disinfected twice with a 0.4% formaldehyde solution and then rinsed with sterile water before transferring them to diet.
Diet
PE
TNM-FH (Sigma)
SF-900 (Gibco)
NDM
PE-50 PE-25/TNM-25 PE-00/TNM-50 PE-25/SF-25 PE-00/SF-50 PE-25/NDM-25 PE-00/NDM-50
50 25 — 25 — 25 —
— 25 50 — — — —
— — — 25 50 — —
— — — — — 25 50
a All diets included 25% each of fresh chicken egg yolk and dry milk reconstituted with water. All diets were gelled with agar at 1% of final diet volume. Gentamicin sulfate (Sigma) was added as 0.1% of the final diet volume.
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In vitro REARING OF Muscidifurax zaraptor
(influence of the EY and RM). All the operational steps, included visual monitoring, were done under a laminarflow hood. When more than 10 adults were collected from a diet, they were exposed to house fly pupae to determine if in vitro reared adults still recognized fly pupae as an oviposition site. The emerging parasitoids of the next generation were tested again in the same way. We did not determine the parasitoid fertility or make any comparison of parasitoids reared in vitro with those reared in vivo. Data collected in the first part of the experiment were analyzed by one-way ANOVA. Data collected in the second part of the experiment were analyzed by a 4 ⫻ 2 factorial design (Diet vs. PE source), except for developmental times, analyzed by a factorial design (Diet vs. PE source vs sex of the parasitoid). Mean comparison (Tukey’s test) was performed when statistical significance (␣ ⫽ 0.05) occurred. All percentages were arcsine transformed before analysis (Zar, 1984). Mean percentages presented in tables were transformed back into proportions after analysis. Because the confidence limits are not symmetrical about the means when expressed again in proportions, we present in tables the mean value and the mean value plus and minus the SE. All data analyses were performed with the statistical package Systat ver. 6.01 for Windows (Systat, 1996). RESULTS
Influence of Pupal Extract Eggs hatched 24–36 h after being transferred onto the diets. The hatching rate was about 90% or more (Table 3) and was unaffected by the diets tested (F ⫽ 0.95; df ⫽ 6, 21; P ⫽ 0.48). The pupation rate was significantly affected by the rearing media (F ⫽ 5.8; df ⫽ 6, 21; P ⫽ 0.001), with no statistical difference among the diets with 50 or 25% PE. In the diets with no PE, most of the larvae reached the last instar, but their
TABLE 2 Percentages of Composition (v/v) of the Diets Tested Relative to Variations in Host Pupal Extract (PE), Insect Cell Culture Media (SF-900), Fresh Chicken Egg Yolk (EY); Dry Milk Reconstituted with Water (RM), and Maltose Solution (7% w/v) a Diet
PE
SF-900 (Gibco)
EY
RM
MS
1 2 3 4
25 25 37.5 37.5
25 25 37.5 37.5
25 25 — —
25 — 25 —
— 25 — 25
a All diets were gelled with agar at 1% of final diet volume. Gentamicin sulfate (Sigma) was added as 0.1% of the final diet volume.
TABLE 3 Developmental Parameters for M. zaraptor as Percentages of Transferred Eggs, Hatched Eggs, and Pupae with Respect to the Influence of Pupal Extract a,b Mean percentage c (⫹SE to ⫺SE) Diet PE-50 PE-25/TNM-25 PE-00/TNM-50 PE-25/SF-25 PE-00/SF-50 PE-25/NDM-25 PE-00/NDM-50
Hatching
Pupation
91.9a (93.3–90.4) 96.9a (98.0–95.7) 94.4a (95.9–92.6) 93.2a (97.3–87.5) 92.2a (93.4–90.9) 90.1a (94.3–85.0) 89.0a (91.6–86.1)
47.3a (60.5–34.4) 31.4ab (45.3–19.0) 1.7b (4.1–0.3) 49.5a (64.8–34.2) 1.0b (3.9–0.0) 45.4a (57.6–33.5) 11.1ab (20.0–4.5)
Emergence n 85.3a (90.4–79.4) 79.7a (87.5–70.5) 0.0b (0.0–0.0) 75.5a (83.6–66.5) 25.0ab
4 4 2 4 1
79.2a 4 (86.0–71.5) 68.5a 3 (72.8–64.0)
Adults 40.2a (52.6–28.2) 26.2ab (39.3–14.9) 0c (0.0–0.0) 38.1a (52.6–24.7) 0.2bc (0.9–0.0) 36.1a (47.7–25.3) 7.6abc (14.2–3.0)
Number of replicates ⫽ 4 except for Emergence, where indicated by n. Means followed by the same letter do not differ significantly (Tukey’s test, ␣ ⫽ 0.05). c See Materials and Methods for explanation relative to SE. a b
size was smaller and they failed to pupate, without voiding the meconium. When the PE was completely replaced with TNM-FH or SF-900 either no or a very few pupae were obtained, and almost all of the pupae failed to emerge as adults (Table 3). When the PE was completely replaced with the modified Nettles’ diet, the yield in adults was lower, but about 8% of parasitoids still completed the development and emerged as adults (Table 3). Adults that emerged from diets with 50 or 25% PE were allowed to mate and females were able to parasitize house fly pupae, producing viable offspring. Influence of Egg Yolk and Reconstituted Milk In this experiment, the hatching rate was about 90% (Table 4) and it was unaffected by the diets tested (F ⫽ 0.16; df ⫽ 3, 28; P ⫽ 0.93) or the PE source (F ⫽ 0.004; df ⫽ 1, 28; P ⫽ 0.95). The diet composition influenced the parasitoid larval development in a highly significant way, in terms of both pupae (F ⫽ 24.95; df ⫽ 3, 28; P ⬍ 0.0005) and emerged adults (F ⫽ 22.1; df ⫽ 3, 28; P ⬍ 0.0005). Because neither the PE source (P ⫽ 0.98 for the pupae and P ⫽ 0.31 for the adults) nor the interaction between the factors (P ⫽ 0.35 for the pupae and P ⫽ 0.60 for the adults) was significant, the multiple comparisons of pupae and adult mean rates were performed pooling the data from diets with a different PE source (Table 4). The percentages of developed parasitoids were significantly higher in the diets containing EY and the lowest in diet 4, with no EY and RM (Tukey’s test, ␣ ⫽ 0.05). RM was not a beneficial
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TABLE 4 Developmental Parameters of M. zaraptor as Percentages of Transferred Eggs, of Adults from Pupae, and of Females of Emerged Adults with Respect to the Influence of Egg Yolk and Reconstituted Milk a,b Mean percentage (⫹SE to ⫺SE) Diet
PE source Md
1 Hv Md 2 Hv Md 3 Hv Md 4 Hv
Hatching
Pupation
89.2a (90.5–87.8) 91.4a (94.5–87.7) 89.6a (91.1–88.0) 89.2a (91.1–87.0) 88.1a (89.8–86.4) 89.8a (92.0–87.3) 90.1a (92.4–87.4) 87.0a (90.9–82.5)
Emergence 85.2a (93.4–74.4) 71.9ab (81.4–61.2) 91.1a (92.5–89.6) 79.2ab (88.2–68.5) 58.4ab (73.8–42.2) 28.1b (39.2–18.2) 50.0ab (85.4–14.6) 27.0ab (50.0–9.22)
59.0a (65.8–52.1)
79.2a (83.5–74.5)
27.5b (36.6–19.2)
3.4c (6.8–1.1)
n 4 5 4 5 3 5 2 2
Adults
46.7a (55.4–38.0)
63.8a (70.0–57.0)
11.7b (18.3–6.4)
1.7b (3.3–0.6)
Sex ratio 46.4a (55.2–37.7) 44.3a (46.3–42.2) 45.4a (48.1–42.7) 52.3a (58.2–46.4) 46.2a (50.9–41.6) 51.2a (69.6–32.7)
n 4 5 4 5 3 5
85.4
1
70.0
1
Note. Md, Musca domestica, and Hv, Heliothis virescens. Number of replicates ⫽ 4 with Md and 5 with Hv except for Emergence and Sex Ratio where indicated by n. a Means followed by the same letter do not differ significantly (Tukey’s test, ␣ ⫽ 0.05). b See Materials and Methods for explanation relative to SE.
component when EY was present in the diet. In the media with no EY, the addition of milk resulted in a significantly higher percentage of pupae, but not of adults (Table 4). H. virescens proved to be an alternative source of insect material in terms of pupae and adults, even if the emergence rate (Adults from Pupae) was negatively affected (F ⫽ 4.30; df ⫽ 1, 22; P ⫽ 0.05). Because of the low number of adults obtained on diet 4, we did not consider those data in the analysis of developmental times and sex ratio. The rate of development (as days from egg to adult) was influenced in a significant way by the diet composition (F ⫽ 17.56; df ⫽ 2, 37; P ⬍ 0.0001) and the parasitoid sex (F ⫽ 17.55; df ⫽ 1, 37; P ⬍ 0.0001), (i.e., male adults emerged earlier than females) (Table 5). Neither the PE source (P ⫽ 0, 94) nor the interactions among factors TABLE 5 Egg–Adult Development (Days) of M. zaraptor with Respect to the Influence of Egg Yolk and Reconstituted Milk a Mean ⫾ SE Diet
Males
n
Females
n
1 2 3
25.8 ⫾ 1.4a 23.8 ⫾ 0.5a 31.6 ⫾ 1.1b
9 9 7
31.1 ⫾ 1.6ab 27.8 ⫾ 0.6a 36.1 ⫾ 1.9b
9 9 6
a Number of replicates indicated by n. Means followed by the same letter do not differ significantly (Tukey’s test, ␣ ⫽ 0.05).
were significant. The sex ratio of emerged adults (expressed as a percentage of females) was not affected by the composition of the diet (P ⫽ 0.25) nor by the PE source (P ⫽ 0.83). The mean values ranged from 44.3% in Diet 1 (Hv Pe source) to 52.3% in Diet 2 (Hv PE source) (Table 4). Adults that emerged, except those from diet 4 because of the low number, were allowed to mate and females were able to parasitize house fly pupae, producing viable offspring. DISCUSSION
Although some parasitoid species have been reared on a diet devoid of insect material (reviewed by Bratti, 1990; Grenier et al., 1993), a recurring theme in the development of in vitro rearing techniques is the apparent need to include either hemolymph or hemolymph derivatives in artificial media. Nettles (1990) suggested that host metabolites (‘‘host factors’’) are likely to be necessary components for successful in vitro rearing of several parasitoid species. Nettles provides as examples of host factors the role of asparagine for the development of the tachinid Eucelatoria bryani Sabrosky (Nettles, 1986b) and polar chemicals with a molecular weight less than 1000 as pupation factors for Trichogramma pretiosum Riley (Irie et al., 1987). Nakahara et al. (1997) report as host factors for the ichneumonid Venturia canescens (Gravenhorst) protein-like molecules with a molecular weight over 100,000, obtained by fractionation of a Galleria mellonella L. pupal
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extract. Bratti (1991) extends the concept of host factors to the host hormones, which have proved either essential, as in Nenon (1972), Lawrence (1988), and Fanti (1990), or at least beneficial (Hu and Vinson, 1997) by addition to the artificial diet. The concept of necessary host factors can be a useful one which can lead to the identification and synthesis of low-molecular-weight chemicals present in hemolymph. It should be stressed, however, that poor development of a parasitoid species on artificial diets with low amounts or devoid of insect material does not necessarily mean that host factors are involved or essential. It may simply reflect an inadequate knowledge of the optimal balance among the nutrients. For example, Bratti and Coulibaly (1995), observing a drop in adult yield of Exorista larvarum (L.) when grown on diets with an amount of insect material lower than 2.5%, concluded that one or more host factors were involved. However, several diets devoid of insect material yet providing successful growth and development of E. larvarum were later developed (Bratti et al., 1995; Mellini and Campadelli, 1995; Coulibaly, personal communication). The results we observed in the first part of our experiment could lead to the conclusion that host factors are involved in the successful growth of M. zaraptor. Although using a diet with no insect material (diet PE-00/NDM-50 of the first set of tested diets) we observed the complete development of the parasitoid, but in a lower percentage. In the second trial we obtained a significantly higher rate of adults in diets 1 and 2, with a PE content lower than that in diets 3 and 4 (Table 2). Also, the lepidopteran H. virescens, which is not a host, proved to be a suitable source of PE. We believe that the better results we obtained with diet 2 can be explained in terms of a better balance of nutrients, even if our present data do not exclude the presence of host factors. The lower percentage of pupae and adults obtained in diets 3 and 4 cannot be related to an increase in PE because, in the first experiment, diet PE50 gave results similar to those with the diets with a 25% PE content. Also, we know that the increase in SF-900 is not responsible for the poor performance of diets 3 and 4 because we later developed media with no PE and an amount of tissue culture media as high as 50%, on which we obtained pupation and adult rates at least as good as those in the best diet (diet 2) described in this paper (Fanti and Vinson, unpublished data). EY is a good source of proteins, amino acids, phospholipids, many minerals, and vitamins (Stadelman, 1992) and proved to be a valuable component of artificial diets for M. zaraptor. It has been used successfully in the in vitro rearing of many species (reviewed in Bratti, 1990; Thompson, 1999). In some instances egg yolk has proven beneficial only in small amounts (Hu and Vin-
son, 1997; Bratti and Nettles, 1992). Raising the amount of EY, Hu and Vinson (1997) reported physical more than nutritional problems in the medium, as the respiration and movement of the larvae appeared restricted. A similar problem was reported by Consoli and Parra (1997) and House (1954), which was reduced larval respiration when high concentrations of EY increased the media viscosity. Xie et al. (1997) successfully used the sediment obtained by ultracentrifugation of chicken EY instead of whole EY. They obtained a precipitate with an increased protein content and lower amounts of triacylglicerols. They credited the good results obtained by adding this component to a better balance of protein/ lipids, resembling the ratio present in the lepidopteran host eggs. In the present study we did not observe any problem using a concentration of EY as high as 25%. Our media were gelled with agar and the larvae were found to feed mainly on the surface of the diet, as they normally do in vivo on the host pupa. Thus they avoid any problem of respiration and movements due to a high lipid content. The use of RM did not prove beneficial, although we observed a higher percentage of developed pupae when it was added in diet 3, compared to diet 4. By contrast, in the diets with EY, M. zaraptor development was not improved by addition of RM. A detrimental effect of milk in artificial diets has been reported in Trissolcus basalis (Wollaston) by Volkoff et al. (1992) and in Trichogramma galloi Zucchi and T. pretiosum by Consoli and Parra (1997). Xie et al. (1997) concluded that milk has no beneficial effect on the in vitro development of Trichogramma minutum Riley and T. brassicae Bezdenko. As for the biological parameters of the M. zaraptor adults developed in vitro on our best medium (diet 2), the results are encouraging. The mean developmental times, 23.8 and 27.8 days for males and females, respectively, are slower, but comparable with the developmental time (23.4 days for the species) in vivo reported by Mann et al. (1990). The sex ratio was close to 1:1 and the females were able to oviposit on house fly pupae, producing viable offspring. No gross morphological malformations were observed in the adults that developed in vitro. This paper is the first to report rearing the larval stages of M. zaraptor on artificial diets. Our results show that this pteromalid wasp is a promising candidate for developing in vitro rearing techniques. Although the best medium described in this paper cannot be considered economically feasible, it can be used as an experimental tool for biological and physiological studies on M. zaraptor. ACKNOWLEDGMENT Support from the Advanced Technology Program of the Texas Coordinating Board for Project 999902-050 is acknowledged.
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