(Hymenoptera: Ichneumonidae) Reared on Artificial Diets: Effects of a Lipid Extract from Host Pupae and Culture Media Conditioned with an Insect Cell Line
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Armyworm Symposium 2000: Carpenter et al. 43 FECUNDITY AND LONGEVITY OF DIAPETIMORPHA INTROITA (CRESSON) (HYMENOPTERA: ICHNEUMONIDAE) REARED ON ARTIFICIAL DIETS: EFFECTS OF A LIPID EXTRACT FROM HOST PUPAE AND CULTURE MEDIA CONDITIONED WITH AN INSECT CELL LINE J. E. CARPENTER, S. M. FERKOVICH2 AND P. D. GREANY3 1Crop Protection and Management Research Unit Agricultural Research Service, U. S. Department of Agriculture, Tifton, GA 31793-0748 2Center for Medical, Agricultural, and Veterinary Entomology, USDA, ARS 1700 SW 23rd Drive, PO Box 14565, Gainesville, FL 32604 3University of Florida, Gainesville, FL ABSTRACT Diapetimorpha introita (Cresson) (Hymenoptera: Ichneumonidae) is a native ectoparasitoid of Spodoptera spp. pupae. This parasitoid has been reared in the laboratory on an artificial diet devoid of any insect host components. However, wasps reared on this artificial diet had reduced fecundity. Efforts to increase fecundity included supplementing the diet with cell culture media conditioned with a cell line from ovaries of the fall armyworm, S. frugiperda, in one experiment and fortifying the diet with lipids extracted from pupae of S. frugiperda in a second experiment. In the first experiment, differences in mean oviposition and mean longevity among females reared on the artificial control diet (artificial diet), cell line-supple- mented diet (Sf9Cell), and natural host (Host) were not significant. However, during the first 10 days of oviposition, Sf9Cell-reared females oviposited at a rate similar to the Host-reared parasitoids and at a rate faster than artificial-diet reared females. In the second experiment, females reared on the diet with added host lipid (host lipid) laid significantly more eggs than females on the artificial diet, however, longevity was not significantly affected by diet treat- ment. We conclude that total egg production by D. introita was improved on artificial diet supplemented with lipids from the natural host but was not increased by the addition of ma- terials produced by an ovarial cell line derived from S. frugiperda. Future research efforts should focus on increasing fecundity of wasps reared on the artificial diet by identifying the lipid(s) or lipid-soluble material in the host pupal extract that is responsible for enhancing egg production in D. introita females. Key Words: Diapetimorpha introita, Spodoptera, parasitoid, artificial diet, fecundity, host lipids, insect cell line RESUMEN Diapetimorpha introita (Cresson) (Himenóptera: Ichneumonidae) es un ectoparásito nativo en pupas de especies de Spodoptera. Este parásito ha sido criado en el laboratorio en una dieta artificial desprovista de componentes de insecto hospedero. Sin embargo, avispas cria- das en esta dieta artificial tuvieron fecundidad reducida. Esfuerzos para incrementar la fe- cundidad incluyeron: suplir la dieta con medio de cultivo de células acondicionadas con una línea de células de ovarios de S. frugiperda en un experimento, y fortificando la dieta con lí- pidos extraídos de pupas de S. frugiperda en un secundo experimento. En el primer experi- mento, diferencias en oviposición promedio y longevidad promedio entre hembras criadas bajo la dieta artificial de control (artificial diet), la dieta complementada con línea de células (Sf9Cell), y hospedero natural (Host) no fueron significantes. Sin embargo, durante los pri- meros 10 días de oviposición, hembras criadas con Sf9Cell ovipositaron a una velocidad si- milar a los parásitos criados con Host y a una velocidad más rápida que hembras criadas con artificial diet. En el segundo experimento, hembras criadas con la dieta complementada con lípidos de hospedero (host lipid) pusieron significativamente mas huevos que hembras con artificial diet, sin embargo, la longevidad no fue afectada significativamente por el trata- miento de dieta. Concluimos que producción total de huevos por D. introita fue mejorada por la dieta artificial complementada con lípidos del hospedero natural pero no fue incremen- tada por la adición de materiales producidos por la línea de células de ovario derivada de S. frugiperda. Futuros esfuerzos de estudio deberán enfocarse en incrementar la fecundidad de avispas criadas con la dieta artificial al identificar el lípido (s) o material soluble en lípidos en el extracto pupal de hospedero que es responsable por aumentar la producción de huevos en hembras de D. introita. 44 Florida Entomologist 84(1) March 2001 Diapetimorpha introita (Cresson) (Hymen- and was prepared according to Carpenter and optera: Ichneumonidae) is a native ectoparasitoid Greany (1998) under aseptic conditions in a clean of Spodoptera spp. (Pair & Gross 1984) that has room as described by Ferkovich et al. (1999). All been reared in the laboratory on an artificial diet the ingredients were added to 25 ml of serum-free devoid of any insect components (Carpenter & SF-900 II cell culture medium. The diet was en- Greany 1998; Greany & Carpenter 1996). Female capsulated in Parafilm® using a diet encapsula- parasitoids that are reared on this artificial diet tion apparatus (Greany & Carpenter 1996). Diet are able to search for and parasitize natural hosts was dispensed at 0.5 ml of diet/dome with 24 in the field (Carpenter and Greany 1998). How- domes/sheet. Each diet sheet was covered with a ever, survival rate, fecundity, and weight are less modified (bottomless) Falcon® tissue culture for diet-reared D. introita than for host-reared D. plate (Sigma, St. Louis, MO) so that each dome introita. Also, developmental time is significantly (one larva/dome) was situated within a well. The longer for wasps reared on the artificial diet than entire culture plate was covered with a Plexiglas® for wasps reared on host pupae (Carpenter & Gre- plate to prevent escape of the larvae. Diet was any 1998). Efforts to increase wasp weight and re- changed during larval development four days af- duce developmental time have included the ter the neonates were initially placed on the diet. addition of commercial nutrients, the use of cul- ture media conditioned by insect cell lines, and supplementing the diet with lipid extracts from Preparation of Cell line-supplemented Diet host pupae (Ferkovich et al. 1999; Ferkovich et. The Sf9 cell line was an embryonic line origi- al., in press). One of the cell lines, Sf, derived from nally derived from ovaries of the fall armyworm, ovaries of S. frugiperda resulted in some improve- S. frugiperda, and purchased from ATCC, Rock- ment in wasp weight (Ferkovich et al. 1999), ville, MD. The cells were cultured in Grace’s me- whereas, the use of a lipid extract from S. fru- dium with 10% fetal bovine serum (FBS), 1% giperda not only enhanced the average weight of bovine serum albumin (BSA) and 0.33% lactalbu- the males and females but also reduced their min enzymatic hydrolysate (Sigma, St. Louis, developmental time. Other parameters such as MO). For larger-scale culture of the cell lines, cells cocoon production or adult emergence were unal- were grown in 250 ml magnetic spinner flasks tered. Molting hormone titers of diet-reared and (Bellco Glass, Vineland, NJ) at 29°C and were host-reared D. introita were examined and it was grown to densities of 1.3 × 105 to 2 × 105 cells/ml 10 concluded that insufficient ecdysteroid in the days post inoculation. For the experiments, 25 ml hemolymph during metamorphosis may contrib- of cell suspension were centrifuged at 250× g for ute to the lowered emergence in wasps reared on 2 min at room temperature. The resultant cell- the artificial diet (Gelman et al. 1999). conditioned supernatant then was substituted for In view of some of the positive effects on the SF-900-II medium in preparing the artificial growth and development of D. introita with di- treatment diets. Two cell line control diets were etary supplements of extracted host lipids and also tested to measure the effects of Grace’s cul- cell line-conditioned media (Ferkovich et al. 1999, ture medium and the additives FBS, 1% BSA and Ferkovich et al., in press), we decided to examine 0.33% lactalbumin enzymatic hydrolysate, addi- their effects on fecundity and longevity of D. in- tives that were required for optimal cell growth. troita females. Preparation of Diet With Extracted Host Pupal Lipids MATERIALS AND METHODS Insect Rearing Lipids were extracted using a modified method of Folch et al. (1957) as described by Ferkovich et Insects used in this study were obtained from al. (in press). Briefly, twenty-four 4 day-old pupae laboratory colonies at the Crop Protection and of Spodoptera frugiperda pupae were homoge- Management Research Unit, Tifton, GA. D. in- nized in 12.5 ml of Ringers solution (Ephrussi & troita were reared according to the methods de- Beadle 1936); the homogenate was filtered through scribed by Pair (1995), unless noted otherwise. S. glass wool to remove cuticular debris and the fil- frugiperda larvae were reared in plastic cups (30 trate saved. The filtrate was then extracted with ml) containing meridic diet (Burton 1969) at a a chloroform:methanol (2:1) mixture and the chlo- photoperiod of 14:10 (L:D) h and temperature of roform phase was dried down. 28 ± 1 and 25 ± 1°C, respectively. Twenty-five ml of diet were added to the dried chloroform extract and the flask was rotated for 5 Diet Preparation and Encapsulation of Diet min to dissolve the residue. The chloroform ex- tract of freshly homogenized pupae of S. fru- The original artificial diet (control diet) con- giperda was added to the artificial diet so that tained ground beef liver, chicken egg yolk, and the each diet dome contained one pupal equivalent of amino acid L-glutamine (Sigma, St. Louis, MO) lipid per D. introita larva. Armyworm Symposium 2000: Carpenter et al. 45 Treatment Diets velop to adults (described below) at 29.1 ± 1°C and 70% RH. Diet was replaced four days after The treatment diets used in this study were as the neonates were initially placed on the diet follows: 1) Host, S.