Mass Rearing of Aphidius gifuensis (Hymenoptera: Aphidiidae) for Biological Control of Myzus persicae (Homoptera: Aphididae)

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1 Biocontrol Science and Technology (2003) 13, 87/97 Mass Rearing of Aphidius gifuensis (Hymenoptera: Aphidiidae) for Biological Control of Myzus persicae (Homoptera: Aphididae) JIANING WEI 1, TIANFEI LI 2, RONGPING KUANG 3, YI WANG 4, TIANSHUI YIN 4, XINFU WU 2, LI ZOU 1,5, WANYUAN ZHAO 1, JUN CAO 1 AND JIANHUA DENG 2 1 Department of Entomology, Kunming Institute of Zoology and Graduate School of Chinese Academy of Sciences, Chinese Academy of Sciences, 32 Jiaochang Eastern Road, Kunming, Yunnan , PR China; 2 Agriculture Institute of Yunnan, Academy of Tobacco Science, Mid Hongta Road, Yuxi city, Yunnan , China; 3 Southwestern University of Forestry, Bailong Shi, Kunming, Yunnan , China; 4 Yuxi Institute of Tobacco, Yunnan Hongta Tobacco Ltd. Co., Yujiang Road, Yuxi City, Yunnan , China; 5 Department of Entomology, Agricultural Center, Louisiana State University, Baton Rouge, LA 70803, USA (Received for publication 13 August 2001; accepted 10 September 2002) The green peach aphid, Myzus persicae, is a major pest of tobacco, Nicotiana tabacum, in Yunnan province, China, where its control still depends on the use of insecticides. In recent years, the local government and farmers have sought to improve the biological control of this tobacco pest. In this paper, we present methods for mass rearing Aphidius gifuensis, a dominant endoparasitoid of M. persicae on tobacco plants in this region. The tobacco cultivar K326 (N. tabacum) was used as the host plant and M. persicae as the host insect. In the greenhouse, we collected tobacco seedlings for about 35 days (i.e., until the six-true-leaf stage), transferred them to 7.5-cm diameter pots, and kept these plants in the greenhouse for another 18 days. These pots were then transferred to an insectary-greenhouse, where the tobacco seedlings were inoculated with five to seven wingless adult M. persicae per pot. After 3 days, the infested seedlings were moved to a second greenhouse to allow the aphid population to increase, and after an additional 49/1 days when 1829/4.25 aphid adults and nymphs were produced per pot, they were inoculated with A. gifuensis. With this rearing system, we were able to produce 2569/ 8.8 aphid mummies per pot, with an emergence rate of 95.69/2.45%; 69% were females. The Correspondence to: Rongping Kuang, 32 Jiaochang Eastern Road, Kunming, Yunnan , People s Republic of China. Tel.: / ; Fax: / ; rongping@public.km.yn.cn ISSN (print)/issn (online)/03/ DOI: / # 2003 Taylor & Francis Ltd

2 88 WEI ET AL. daily cost of parasite production (recurring costs only) was US$ 0.06 per 1000 aphid mummies. With this technique, we released parasitoids in 1998, in 1999, in 2000, and in 2001 during a 2-month period each year. This production method is discussed with respect to countrywide usage in biological control and integrated control of M. persicae. Keywords: mass rearing, Aphidius gifuensis, biological control, Myzus persicae, rearing cost INTRODUCTION The economic importance of the green peach aphid, Myzus persicae (Sulzer), is largely a result of its ability to transmit over 100 viral diseases among its over 400 host plants (Mackauer & Way, 1976). In addition, its feeding and deposition of honeydew can directly injure the host plant causing decreased yield (Kulash, 1949; Stary, 1970). M. persicae has been known to occur in China for many years as a pest of a wide variety of vegetables, fruits, and ornamental plants (Li et al., 1966; Zhao, 1981; Yang et al., 1999). In Yunnan, which is the biggest tobacco growing and flue-cured tobacco manufacturing area of China, tobacco fields are commonly infested by M. persicae and these infestations cause serious economic losses (Zhao, 1981). In Yunnan, control of this pest is still based largely on insecticides (Zhao et al., 1980), but resistance has become widespread. Repeated applications, incorrect application rates and inappropriate application methods have all contributed to this resistance (Han et al., 1989; Gao et al., 1992). Furthermore, several environmental and social problems resulting from overuse of insecticides are receiving more attention by government officials and local farmers in Yunnan. Thus, development of alternative control methods, especially those involving biological control, is essential. Species in the genus Aphidius constitute one of the more important groups of parasitoids attacking the green peach aphid (Stary, 1970). Aphidius gifuensis Ashmead is one of the most important primary endoparasitoids and has been found attacking the aphid in various habitats, including fields of vegetable, tobacco, and ornamental plants in China (Chen, 1979; Lu & Shi, 1994). The distribution of this parasitoid is almost countrywide, from northern China, i.e., Jilin, Hebei, Shannxi and Henan provinces (Bi & Ji, 1993; Wang & Li, 1996), to southern China, i.e., Hunan, Fujian and Yunnan provinces (Zhao et al., 1980; Tang, 1984). Unfortunately, it was not until the mid-1970s that its potential for biological control of M. persicae attracted the interest of researchers (Chen, 1979). Since then, many biological and ecological studies of this insect have been conducted (Zhao et al., 1980; Bi & Ji, 1993; Lu et al., 1993). Consequently, A. gifuensis was widely applied to control vegetable aphids such as Brevicoryne brassicae (L.), Lipaphis erysimi (Kaltenbach), M. persicae, and Macrosiphum avenae (Fabricius) in greenhouses, and the tobacco aphid, M. persicae, in greenhouses and fields with satisfactory results (Zhao et al., 1980; Xin, 1986). Coincidentally, different massrearing methods were proposed (Zhao et al., 1980; Xin, 1986), but few of the resulting publications provided sufficient details for developing a large scale rearing program for this parasitoid. Furthermore, cost analyses of the rearing procedures were unavailable. Thus, in our study, we sought to develop a mass-rearing program to produce A. gifuensis for use in augmentative biological control of M. persicae and to provide a cost analysis of the rearing procedure. MATERIALS AND METHODS Rearing Procedure The overall rearing procedure was divided into two parts according to the time schedules of the overall mass-rearing procedure (See Table 1 and Figure 1). The rearing system consists of three sections as follows:

3 MASS REARING OF APHIDIUS GIFUENSIS 89 TABLE 1. Procedure Labor (h) required per day for the mass production of A. gifuensis using tobacco seedling as a host plant and M. persicae as the host insect Time required/( x9s:e:)/ a Title Step Days Per pot (s) Per aphid mummies (h: min: s) Part I. Aphid and parasite population multiplication Floating culture of tobacco seedlings 1 1/ /0.12 0:00:24 Potting and maintaining plants in large pots /0.21 0:01:12 Augmentation of overwintering aphid population 4,4a /0.3 0:01:44 Augmentation of overwintering parasite population /0.9 0:06:48 Parasites from aphid augmentation greenhouses /0.14 0:01:32 hyperparasites from parasite augmentation greenhouse /0.2 0:00:50 Part II. Mass rearing of parasites Floating culture of tobacco seedlings /0.2 0:08:40 Potting and maintaining plants in small pots /2.3 1:37:30 Exposing pots to aphids /0.7 0:45:15 Exposing pots to parasites /0.2 0:50:50 Plant and aphid and parasite maintenance while parasites are developing /1.07 1:14:10 Aspirating hyperparasites from parasite developmental greenhouse b / b / Moving pots into the field /1.4 1:17:30 Total 6:06:25 a Based on three replicates of the entire procedure. For aphid mummies, 20 large pots are required in the aphid and parasite multiplication procedure and 500 small pots with 250 aphid mummies per small pot are required in the mass-rearing procedure (see text). b Negligible. Plant Production We used tobacco seedlings to rear the aphid, M. persicae, to be used for the production of the parasitoid, A. gifuensis. The tobacco plants (cultivars K326) were grown from seeds donated by the Hongta Tobacco Company, Yuxi City, Yunnan province, China. The seeds were sown in a tray, which was made from cellular plastic (70/35 cm in size and 4 cm deep), containing 200 compartments (10 in row and 20 in column). Each compartment had the form of an inverted pyramid with four sides and a bottom (with a 0.5-cm diameter hole) filled with a standard potting mixture (see Figure 2). Two seeds were planted in each of the 200 compartments. After planting, the trays were floated in a basin, 2.2/1 m in size and 40 cm deep, containing a 15-cm deep mixture of nutrient solution of liquid fertilizer (a special compound fertilizer for planting tobacco that is composed of 60% nitrate nitrogen and 40% amino nitrogen with some microelements, which contain N 20%, P 2 O5 10%, and K 2 O 20% and N:P:K (1:0.5:1)) and water in a ratio of 1:1000. The basin was placed in a greenhouse and the seedlings were grown in long-day conditions (photoperiod L14:D10) at 22/288C. After approximately 20 days, tobacco seedlings were pinched or redistributed in order to maintain one seedling per compartment. The plants were then allowed to grow for an additional 15 days until they reached a height of approximately 7 cm (six-true-leaf stage). They were then either transplanted into 25-cm diameter plastic pots for overwintering of the aphids and parasite population multiplication (Figure 1, from S1 to S3), or were transplanted into 7.5-cm diameter pots for mass rearing of the aphids and parasitoids (Figure 1, from S2 to S5). All pots were moved into developmental greenhouses and allowed to grow for 18 days. At this stage, most tobacco seedlings in the big pots typically had 13 true

4 90 WEI ET AL. FIGURE 1. Schematic figure of the overall rearing procedure for A. gifuensis using tobacco seedling as a host plant and M. persicae as the host insect. The rearing consists of two parts divided into 13 steps according to the time of the onset of each step (S1 to S13). In first part, aphid and parasitoid population multiplication, S1 represents the place of rearing greenhouse of floating culture; S3, seedlings developmental greenhouses (planting in large pots); S4, aphid augmentation greenhouses; S4a, aphid developmental greenhouses; S6, parasitoid augmentation greenhouses; S7, aspirating parasitoid from aphid developmental greenhouse; S9, aspirating hyperparasites from parasitoid developmental greenhouse. In second part, mass rearing A. gifuensis population, S2, rearing greenhouse of floating culture; S5, tobacco seedling development greenhouse; S8, M. persicae developmental greenhouse; S10, A. gifuensis sting greenhouse; S11, parasitoid development greenhouse; S12, aspirating hyperparasites from parasitoid developmental greenhouse; S13, field; NB, nylon bags. TM indicates the large pots with tobacco seedlings for the multiplication and maintenance of M. persicae colony; TA, the large pots of tobacco seedling for the production of Aphidius; T, the small pots of tobacco seedling for mass rearing A. gifuensis population; M, aphid mummies; A, adult A. gifuensis; H, hyperparasites of A. gifuensis. A vertical drawn line and two horizontal broken lines indicate the greenhouses for tobacco seedlings, aphids and parasite rearing. The lines with arrowheads represent the step-by-step progress of the procedures. leaves and were 20 cm tall, whereas those in small pots had nine true leaves and were 12 cm tall; they were then ready to be used for mass production of the aphids and parasitoids. Green Peach Aphid Production Two M. persicae populations were used for mass rearing of the parasitoid: (1) a population collected each spring from the field on Brassica species in the Yuxi District, Yunnan Province from 1998 to 2001; the aphids were then acclimatized on tobacco seedlings for two to three generations; and (2) a population that had been maintained in a growth chamber at 209/

5 MASS REARING OF APHIDIUS GIFUENSIS 91 FIGURE 2. Schematic figure of floating culture tray. The tray (left) was made from plastic, containing 200 compartments (10/20), 70/35 cm in size and 4 cm deep. Each compartment (right) has the form of an inverted pyramid with four plastic sides and a bottom (with a 0.5-cm diameter hole) filled with potting mixture. 0.58C on tobacco seedlings (cultivars K326) in the insectary of Yuxi Institute of Tobacco for 5 months. Overwintering Aphid Population Multiplication To multiply aphid populations, individual aphids, winged and wingless, were placed on 60- day-old, 20-cm high tobacco plants that were placed in a 1.7/3/3-m high, glass-topped greenhouse (50 pots (25 cm diameter) per greenhouse) and at a photoperiod of L14:D10. A 4/4-m black net was hung over the greenhouse to maintain conditions at 259/48C and 50 / 70% r.h. Mass Rearing Aphids After 2 weeks of multiplication, the wingless aphids were transferred to 500, 12-cm high, nine-true-leafed tobacco seedlings with a moistened fine horse hair brush, after which the seedlings were placed in a 1.7/3/5-m glass-topped insectary-greenhouse. Two 4/6-m black nets were hung over the roof of the greenhouse. They were opened from 09:00 to 17:00 h each day and closed for the remainder of the time to maintain the greenhouse temperature at 22/288C and 50/70% r.h. Two fluorescent lamps of 30 W each were fastened on the sidewalls and three incandescent lights of 60 W each on the ceiling to obtain a L16:D8 photoperiod. Five to seven adult green peach aphid individuals were inoculated on each tobacco seedling to ensure synchronization of larviposition and an even age distribution and developmental times. To estimate the number of aphids produced, the number of aphids per pot was counted every third day on three replicates of 10 large and 30 small randomly selected pots. When aphid numbers per small pot exceeded 150 individuals (after 5/7 days), the pots were transferred to an empty greenhouse for parasitoid oviposition. Parasite Production Adult A. gifuensis were obtained from field-collected mummies or from laboratory cultures that had been maintained in growth chambers for two to five generations after field collection. Cultures were maintained in a manner similar to that described for M. persicae. The aphid mummies were collected and put in glass vials (25/95 mm) by using a horse hair brush, with 50 mummies per vial. Newly emerged (B/24 h old) males and females were moved into separate glass vials, provided with honey water and held for an additional 24 h at 229/28C for mating.

6 92 WEI ET AL. Overwintering Parasitoid Population Multiplication Adult A. gifuensis were used to inoculate tobacco seedlings infested with green peach aphids for population augmentation in a 1.7/3/3-m, glass-topped greenhouse (50 pots (25 cm diameter) per greenhouse) (Figure 1, from S4a to S6). Uniform numbers of parasites were maintained in the greenhouses by adjusting the number of adult parasitoids released daily into the greenhouse (approximately 500, sex ratio 1:1), thus keeping the parasitoid population in the greenhouse at about 2000/3000. After 25 days (about two generations), the parasitization rate was approximately 90%, and the ratio of aphid mummies to aphids was 1:2. At this point the parasitoid population was ready for inoculation. Mass Rearing Procedure for Parasitoids At day 85 (Figure 1, from S6 to S10) with 20 big pots of tobacco seedlings containing a mixture of aphid mummies, parasitized aphids, and a few adult parasitoids, an aspirator was used to transfer most adult parasitoids into a second greenhouse, named the inoculation (or sting) room. This greenhouse contained 500 tobacco seedlings (Figure 1, from S8 to S10) each containing approximately 150 aphids, thus creating a ratio of one parasitoid to every 10 aphids. The higher ratio (1:10) of parasites to aphids and short exposure time to adult parasites would result in synchronous egg deposition and larval development of parasites. The adult parasites that emerged in the small greenhouse were collected by hand-held aspirators and released in the inoculation room daily to supplement the population. These greenhouses were maintained under the conditions described previously. After a 2-day exposure, tobacco seedlings were moved to another greenhouse for parasite development (Figure 1, from S10 to S11). After 8 days (just at the time parasites developed to the mummy stage and before parasite emergence from mummies), these pots were used in field releases or were returned to the inoculation room for parasite production. To estimate parasite production, three replicates of 10 big pots and 30 small pots were monitored through this rearing process at 3-day intervals. Analysis of the Rearing Procedure To analyze the rearing procedure, pots were held individually to determine the number of aphids surviving parasite exposure and the number and sex of the parasites produced. A total of 120 pots was used to determine production statistics. On a per-pot basis, each step (Table 1 and Figure 1) was timed during parasite production, and the time required to produce aphid mummies of A. gifuensis per day was calculated. At the same time, leaf area, newly larviposited nymphs, aphid mummies emerged, and aphid parasitization were recorded at 3-day intervals. The parasitization rate was determined by dissecting living aphids that were randomly collected on the tobacco leaves. Then, nymphs surviving exposure to the parasites, and the sex ratio, were calculated. The production costs were divided into non-recurring costs (e.g., greenhouses, pots, etc.) and recurring costs (e.g., plants, compost, fertilizer, labor) using the price level during 1999 in China and the cost of Renminbi (Chinese dollar) was converted into U.S. dollar by the foreign exchange rate 1:8. The cost of space (greenhouse rooms) and utilities (water, electricity, etc.) was not included in the calculations. In addition to production figures on a per-pot basis, the production of aphid mummies and adult A. gifuensis could be expressed on a per unit of leaf-area basis. A group of 350 tobacco leaves (dependent variable) that varied in shape and size were divided into a small leaf group (SLG, leaf length/widthb/120 cm 2 or leaf lengthb/13 cm) and a large leaf group (LLG, leaf length/width]/120 cm 2 or leaf length]/13 cm) and were measured with a leaf area meter. Leaf areas were regressed on leaf length/width or length only (independent variable) (SAS Institute, 1985). These relationships were used to calculate total leaf area of each pot used in the production study.

7 MASS REARING OF APHIDIUS GIFUENSIS 93 RESULTS Significant relationships between leaf area and leaf length/width, and between leaf area and leaf length, were found in both the small leaf group and large leaf group. SLG: y(leaf area)0:6507x(lengthwidth) (R 2 0:9977); y(leaf area)4:2149x(leaf length)14:04 (R 2 0:9355): LLG: y(leaf area)0:6315x(lengthwidth) (R 2 0:9205); y(leaf area)10:619x(leaf length)91:365 (R 2 0:8942): These relationships allowed rapid estimation of leaf area per pot by the use of a nondestructive method. After 90 days (from first day of whole procedure) of plant growth and before field release of mummified aphids, leaf area per small pot was /12.6 cm 2 (/ x9s:e:; n/90). Fewer than 100 aphids per leaf were usually present in this study, so intraspecific competition did not appear to be a problem. In the procedure for aphid mass production, the number of aphids per small pot, each inoculated originally with five to seven adult aphids, yielded 1829/4.25 (/ x9s:e:; n/90) nymphs after about 7 days. After 2 days exposure of small pots in the greenhouse containing adult A. gifuensis (6749/16.7 (/ x9s:e:; n/90) aphid mummies per big pot giving a parasite: aphid ratio :/1:10 in this greenhouse), and 3 days development in the developmental room, 809/3.5% ( x9s:d:) of living aphids were parasitized and over 90% of living aphids were parasitized within another 5 days (Figure 1, S10 to S11). Aphid mummy production per small pot was 2569/8.8 (/ x9s:e:; n/90); the emergence rate of these was 95.69/2.45% ( x9s:e:); 69% of these were females. Production was equivalent to 0.34 aphid mummies per cm 2 of tobacco seedling leaf. Approximately 6.1 h day 1 were required to produce aphid mummies (Table 1). Therefore, under full-time conditions (8 h day 1 ), there was ample time to deal with all the rearing procedures. In the whole production procedure, more than 95% of this time was spent on mass rearing of A. gifuensis, with potting and maintaining plants, maintenance of aphid and parasite and plant, and field releasing being the most time-consuming steps. The total cost of producing aphid mummies of A. gifuensis per day was estimated to be $ (Table 2). When production was underway, the recurring costs that needed to be considered were labor, compost, fungicide, liquid fertilizer, and plants. Therefore, the recurring cost of production of aphid mummies per day was nearly $7 (approximately $0.06 per 1000 aphid mummies). DISCUSSION Although some studies on mass rearing of aphidiid parasites (Finney et al., 1960; Stary, 1970) have been conducted, none of the previous studies included a cost analysis of the rearing procedure. We are confident that the cost of $0.06 per 1000 mummified aphids (recurring costs only) is relatively low for the production of A. gifuensis. With this technique, we have released parasitoids in 1998, in 1999, in 2000, and in 2001 during a 2-month period each year and achieved satisfactory results (Wu et al., 2000). It has been shown above that 69% of the parasites produced were females. Similarly, during the late spring and early summer from 1998 to 2001, the percentage of females produced ranged between 65 and 75, which suggests that A. gifuensis has a female-biased sex ratio. Further studies are needed to clarify this phenomenon.

8 94 WEI ET AL. TABLE 2. Cost estimates for the production of 125,000 aphid mummies of A. gifuensis per day using tobacco as the host plant and M. persicae as the host insect Quantity Unit price a ($) Total a ($) Nonrecurring costs Material Small pots (7.5-cm diameter) Large pots (25-cm diameter) Aspirator Vials Horse-hair brush Floating tray Watering pot Small greenhouses Large greenhouses Total Recurring costs b Labor: two technicians (full-time) 8 h 0.25/h 4 Material Soil mix 425 cm Liquid fertilizer 6 L Tobacco seeds one package (5 g) Fungicide three packages (300 ml) Total a The cost of Chinese Renmingbi was converted into US dollar ($) by the foreign exchange ratio 1:8 in year b The total recurring costs of the production of aphid mummies is $6.915:/$7 per day. However, the result in Table 1 showed that time required for aphid mummies is 6.1 h day 1. Therefore, under fulltime conditions (8 h day 1 ), there is ample time to deal with each procedure. The recurring cost of production of 1000 aphid mummies per day is about $0.06. Two species of hyperparasitoids, Pachyneuron aphidis (Bouché) and Asaphes suspensus (Nees), were identified during this study. The former is a dominant species which can form more than 85% of hyperparasitoid population (Z.-Q. Yang, Institute of Forest Protection, Chinese Academy of Forestry, pers. comm.). Hyperparasites were accidentally introduced along with some predators of M. persicae, so the parasitization by hyperparasites was 0.87% in parasite augmentation greenhouses during the terminal days of propagation of parasites. But, because the duration of this step of the rearing procedure was not longer than 30 days, these undesirable natural enemies usually did not reproduce too much. Hyperparasitization in the parasite developmental greenhouse was nearly zero, for it took about 8 days for living parasitized aphids to develop to the mummified stage in this developmental greenhouse before mummified aphids were released in the field. However, on the withdrawn tobacco plants, which had been placed in a tobacco field for about 6 days, parasitization by hyperparasites was 0.1%. This result reveals that a few aphid mummies were attacked by the hyperparasites in the field. Sullivan (1987) suggested that hyperparasitoids are normally detrimental to biological control programs. It was found in this study that hyperparasitoids represented one of the most important factors lowering the production of parasites in the rearing procedure and parasite effectiveness in the field. In spite of this, we are confident that we have developed an effective method of aspiration to remove hyperparasites in the A. gifuensis rearing greenhouse as well as in the tobacco fields (Figure 1). Mass rearing of A. gifuensis is part of a chain of procedures, which consists of the mass growing of N. tobaccum, mass rearing of M. persicae on this plant, and mass rearing of A. gifuensis on this aphid. Obviously, if any one of the three is unsuccessful, it will lead to a complete failure of mass production. Fleschner (1959) emphasized that, in the mass culture

9 MASS REARING OF APHIDIUS GIFUENSIS 95 of any organism, the basic problem is usually mass production of its food, and in the culture of beneficial insects, the production of suitable quantities of host insects. Therefore, the selection of a suitable host plant was the first step of the mass rearing program. We found that tobacco plants are relatively slow-growing compared with other potential host plants of M. persicae, such as Brassica species, radish, etc., with approximately 50 days required before tobacco seedlings are suitable for parasite production (Table 1, Figure 1). Tao et al. (1995) found that the rate of increase of population of M. persicae reared on tobacco seedling is higher than that of M. persicae reared on Brassica species. Heathcote (1962) found that M. persicae developed better on Brassica species than on spinach, sugar beet, or lettuce. It seems that N. tabacum is the most appropriate host plant for mass rearing of M. persicae in spite of its slow growth rate. In addition, N. tobaccum is the crop into which A. gifuensis will be liberated in our program, and use of this plant in the rearing process would reduce the possibility of conditioning of the parasite to another host plant. Rearing records from five aphid species revealed that M. persicae is the most common and most important host of A. gifuensis (Zhao et al., 1980; Bi & Ji, 1993). M. persicae developed faster at lower temperature regimes (below 188C) than radish aphid, Lipaphis erysimi (Liu & Meng, 1990), cabbage aphid, Brevicoryne brassicae (Deloach, 1974), cotton aphid, Aphis gossypii (Wyatt & Brown, 1977), and potato aphid, Macrosiphum euphorbiae (Barlow, 1962), which are the host aphids of A. gifuensis. Though the optimal temperature for M. persicae for development is 258C (Deloach, 1974), the ability of M. persicae to develop at lower temperature can facilitate maintenance and mass rearing of M. persicae population under lower temperature conditions in unheated greenhouses. All these studies implied that M. persicae is the most suitable host aphid for mass rearing of A. gifuensis. In addition, use of M. persicae as the host aphid that they will likely encounter after release, and rearing parasites under greenhouse conditions should enhance their potential success. Although alternative or unnatural hosts or even artificial media have been used successfully in the mass culture of host insects, parasites or predators (Fleschner, 1959), very few successful cases of breeding hymenopterous parasites on artificial diet are known (Stary, 1970). Direct culturing of parasites on artificial diets does not seem to be suitable for the aphidiids because of their unique and complex biological and ecological characteristics (Stary, 1970). Mittler & Dadd (1962) have mentioned the rearing of M. persicae on artificial diet. Thus, it may be possible to culture this parasite on artificial diet, but it is questionable whether the use M. persicae from artificial diet is more advantageous than from its natural host plant. This question will be addressed in future studies. Early and quantitative release of A. gifuensis is the strategy that we have taken for control of M. persicae in tobacco planting fields. The purpose of this strategy was to establish the parasite population in aphid-infested fields earlier than they could disperse and occur in the fields under natural conditions. For instance, A. gifuensis could be mass-released in M. persicae-infested seedbeds of tobacco seedling before these seedlings were transplanted into the field. Alternatively, after seedlings were transplanted into the tobacco fields, A. gifuensis could be mass-released to control M. persica populations. To sum up, the method of mass rearing of A. gifuensis was devised on the basis of a strategy of early mass release. In general, we can release all the developmental stages of parasites, but each stage has certain negative and (or) positive features (Stary, 1970). The release of living parasitized aphids is not appropriate for control because aphid populations were never completely parasitized and a certain number of aphids escape parasitization, and even a parasitized aphid may be able to produce a number of unparasitized progeny. The adult parasites seem to be the best material for release, but collection and maintenance of adult parasites in greenhouse and insectary is relatively tedious and laborious work. In order to do that, we aspirated and counted the adult parasites from the greenhouse, and transferred them into a glass tube where they were fed with honeydew before releasing. Alternatively, we had to mass-collect them from host plants and rear them in the insectary.

10 96 WEI ET AL. Furthermore, we maintained and fed the emerged adult parasites, and waited for other parasites that had not yet emerged from mummies. If this period was too long, the mortality of early parasites increased and the vigor of these parasites would decrease. Under practical conditions, it is impossible to collect and release the adult parasites in sufficient quantity and quality. We could avoid these unfavorable conditions by using tobacco plants bearing unemerged mummified aphids, which attached firmly to the leaves of tobacco, and can be easy transported. Parasites thus transported were more vigorous and had greater longevity than those collected (J.N. Wei, unpubl.), and could be shipped to the field for releasing. The problem of synchronization of adult emergence could be resolved as a result of this study (see Material and Methods). We are confident that mass rearing and release of mummified aphids is the most suitable method in our study. Our general policy of mass rearing and mass releasing A. gifuensis for biological control of M. persicae is to release a combination of mummified aphids and adult parasites. However, mummified aphids were more suitable for use than adult parasites, although release of adult parasites can be used as a complementary method for field aphid control. ACKNOWLEDGEMENTS We wish to express our sincere thanks to Dr M.J. Stout, Department of Entomology, Louisiana State University Agricultural Centre, and to Dr. A.K. Minks for their valuable advice and numerous corrections on this manuscript. Many thanks to Prof. Zhongqi Yang for his valuable suggestions and identification of hyperparasitoids, and to Ms. Ruifeng Su for her generous help during this study. Authors thank Huishu Li, Kaifu Peng and KaiLiang Peng for their competent technical assistance. This study was supported by grant from the Tobacco Company of Yunnan and Hongta Tobacco Co. Ltd.. REFERENCES BARLOW, C.A. (1962) The influence of temperature on the growth of experimental populations of Myzus pericae (Sulz.) and Macrosiphum euphorbiae (Thomas) (Aphididae). Canadian Journal of Zoology 40, 145/156. BI, Z.B. & JI, Z.D. (1993) Bionomics of Aphidius gifuensis Ashmead. I. Development stages and morphology of larval stage. Journal of Hebei Agricultural University 16, 1/8. CHEN, J.H. (1979) The fundamental knowledge of taxonomy of Aphidiidae in China. Entomological Knowledge 16, 265/268 [in Chinese]. DELOACH, C.J. (1974) Rate of increase of population of cabbage, green peach, and turnip aphids at constant temperatures. Annals of the Entomological Society of America 67, 332/340. FINNEY, G.L., PUTTLER, B. & DAWSON, L. (1960) Rearing of three spotted alfalfa aphid hymenopterous parasites for mass release. Journal of Economic Entomology 53, 655/659. FLESCHNER, C.A. (1959) Biological control of insect pests. Science 129, 537/544. GAO, X.W., ZHENG, B.Z. & CAO, B.J. (1992) Resistance in Myzus persicae to organophosphorus and carbamate insecticides in China. Acta Phytophylacica Sinica 19, 365/371 [in Chinese]. HAN, Q.F., ZHUANG, P.J, TANG, Z.H., XU, X.Z. & DENG, X.-Q. (1989) The preliminary study on resistance of Myzus persicae to insecticides. Contributions of Shanghai Insititue of Entomology 9, 19/27 [in Chinese]. HEATHCOTE, G.D. (1962) The suitability of some plant hosts for the development of the peach-potato aphid, Myzus persicae (Sulzer). Entomologia Experimentalis et Applicata 5, 114/118. KULASH, W.M. (1949) The green peach aphid as a pest of tobacco. Journal of Economic Entomology 42, 677/ 680. LI, X.R., CHANG, G.X. & CHU, H.F. (1966) Bionomics of Myzus persicae Sulzer on tobacco. Acta Phytophylacica Sinica 2, 297/308 [in Chinese]. LIU, S.S. & MENG, X.D. (1990) The relationships between temperature and development rates of Myzus persicae and Lipaphis erysimi. Acta Phytophylacica Sinica 17, 169/175 [in Chinese]. LU, H., SHI, B.C. & ZHANG, Z.L. (1993) Studies on fecundity of Aphidius gifuensis Ashmead. Acta Agriculturae Boreali-Sinicae 8, 76/79 [in Chinese]. LU, H. & SHI, B.C. (1994) Development thresholds and thermal constants of Aphidius gifuensis and Diaeretiella rapae. Acta Agriculturae Boreali-Sinica 9, 72/75 [in Chinese]. MACKAUER, M. & WAY, M.J. (1976) Myzus persicae Sulzer, an aphid of world importance, in Studies in Biological Control (DELUCCHI, V.L., Ed.). Cambridge University Press, Cambridge, pp. 51/119.

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