In Vitro Rearing of the Parasitoid Exorista larvarum (L.) (Diptera: Tachinidae) on Meat Homogenate-Based Diets

Biological Control 16, 258–266 (1999) Article ID bcon.1999.0772, available online at http://www.idealibrary.com on

In Vitro Rearing of the Parasitoid Exorista larvarum (L.) (Diptera: Tachinidae) on Meat Homogenate-Based Diets M. L. Dindo, R. Farneti, M. Scapolatempo and G. Gardenghi Istituto di Entomologia ‘‘G. Grandi,’’ via Filippo Re, 6, 40126 Bologna, Italy Received May 4, 1998; accepted August 6, 1999

The tachinid Exorista larvarum (L.), a polyphagous gregarious larval endoparasitoid of Lepidoptera, was reared from egg to fecund adult on media containing commercial meat homogenates for babies as the main ingredient. Four media, each containing a diverse homogenate supplemented with extract of Galleria mellonella L. pupae, were tested first. Despite the difference in nutrient content, the kind of homogenate did not significantly affect the adult yields (30.2 to 40.7%) or puparial weights. Two other diets free of host materials (I and II) were then tested. Both were based on Gerber veal homogenate combined with different amounts of yeast extract and chicken egg yolk and were supplemented with wheat germ (I) or saccharose (II). Adult yields (28.7 to 32.7%) and puparial weights did not differ significantly between the two diets. Fly longevity and fecundity of the females obtained on diet I were comparable to those of the females emerged from puparia formed in G. mellonella larvae. Male and female puparial weights were, however, higher and development times longer on the diet than in the host. r 1999 Academic Press Key Words: Exorista larvarum; parasitoid rearing; artificial diets; meat homogenates.

INTRODUCTION

In recent years, interest in artificial rearing of parasitoids has increased worldwide. Artificial rearing may not only prove useful for investigating parasitoid larval biology and behavior but may also be suitable for mass producing parasitoids more easily and less expensively (Greany et al., 1984; Thompson, 1986, 1999; Vinson, 1986, 1994). To date, about 40 parasitoid species have been reared on artificial diets to the adult stage and, although in vitro rearing techniques cannot yet compete economically with conventional rearing, considerable progress has been made in reducing costs (Grenier et al., 1994; Grenier, 1997). One of the most promising species for in vitro mass production is Exorista larvarum (L.), a polyphagous 1049-9644/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

gregarious larval endoparasitoid of Lepidoptera that is well distributed throughout Europe, northern Africa, and several Asian regions (Herting, 1960). Its biology was studied by Hafez (1953) in the noctuid host Prodenia litura F. In cork-oak forests in Sardinia (Italy), this tachinid is very important as a natural antagonist of Lymantria dispar (L.), causing mortality upwards of 50% (Luciano and Prota, 1984). Herting (1960) reported that E. larvarum is the second most important dipteran parasitoid of L. dispar in Europe. Yet, to date it has been used as a biological control agent against this defoliator only in inoculative releases in the northern United States (Sabrosky and Reardon, 1976). The possibility of mass culturing this entomophage on artificial diets may simplify production, reduce labor costs, and eventually make large-scale biocontrol strategies feasible. Several characteristics make E. larvarum particularly suitable for in vitro rearing. They include nonsynchronized development with the host, gregariousness, polyphagy, and the fact that both in the host and in the diet the larvae remain in contact with atmospheric oxygen from the beginning of their development (Bratti et al., 1995; Mellini et al., 1996). Complete development of this tachinid was obtained on various artificial diets both including and devoid of host components (Mellini et al., 1993a; Mellini and Campadelli, 1995a,b; Bratti and Coulibaly, 1995; Bratti et al., 1995). These results stimulated research on new diets for E. larvarum, with the aim of discovering more efficient and easier to prepare media. This paper describes the results obtained by rearing E. larvarum from egg to adult on media containing commercial meat homogenates for babies as the main component. Efficient artificial meat-based homogenate diets for parasitoids (Bronskill and House, 1957) and predators (Cohen, 1985; Cohen and Urias, 1986; De Clercq and Degheele, 1992) have previously been developed. Commercial products, readily available and easy to use, have already been successfully employed in artificial diets for the chalcidid wasp Brachymeria intermedia (Nees) by Dindo et al. (1994, 1997a,b).

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MATERIALS AND METHODS

Biological Materials A colony of E. larvarum was maintained in the laboratory using Galleria mellonella L. as a factitious host. G. mellonella larvae were reared on a diet developed by Campadelli (1973), at 30 ⫾ 1°C, 65 ⫾ 5% RH, and in complete darkness. E. larvarum adults were kept in plexiglass cages (40 ⫻ 30 ⫻ 30 cm) in a rearing chamber at 26 ⫾ 1°C, 75 ⫾ 5% RH, and a 16:8 L:D photoperiod. The flies were fed on lump sugar and cotton balls soaked in a honey and water solution. The colony was established in 1992 from adults which had emerged from L. dispar and Hyphantria cunea Drury larvae collected in the field. Preparation of Diets Test 1. Four diets were tested, each containing a different commercial meat homogenate. Diets (A) and (B) were based on Plasmon (by Plasmon Dietetici Alimentari Co., Italy) veal homogenates, both intended for babies at the beginning of weaning but dissimilar in their nutrient and calorie content. Diets (C) and (D) were respectively based on Gerber (by Gerber Products Co., USA) beef homogenate for babies at the beginning of weaning (C) and Gerber veal homogenate for babies well on in weaning (D). According to the ingredients reported on the label, in addition to meat, all homogenates contain water, rice starch, and maize oil. Furthermore, the four homogenates respectively contain rice meal (A and B) and milk proteins (C and D). The B homogenate also contains maize starch. No information is available as to ingredient proportion. The D homogenate has sodium chloride added (0.25%). The homogenates were analyzed in the Laboratory of Animal Husbandry and Nutrition of the University of Bologna and some data on their nutrient content are reported in Table 1. Feed analyses were performed according to AOAC (1990). All diets had a composition (v/v) of 75% homogenate, 8.3% G. mellonella pupal extract prepared as in Bratti

TABLE 1 Nutrient Content (g/100 g) and Calories of the Commercial Meat Homogenates for Babies Used for the Preparation of the Artificial Diets for Exorista larvarum Homogenate

Dry residue

Proteins

Lipids

Carbohydrates

Minerals

kcal a

A B C D

19.5 18.7 21 22.1

8.8 8.2 9 12.5

4.4 5.5 5.5 5.2

5.8 4.7 6 3.5

0.5 0.3 0.5 0.9

118 101 109 112

a

Values taken from those reported on the homogenate labels.

259

and Coulibaly (1995), and 16.7% agar–water suspension (12% of agar) to give a 2% agar final concentration in the medium. Each diet was prepared by placing the homogenate in a water bath at 50°C for about 30 min in order to soften it. Using sterile syringes, 9 ml homogenate and 1 ml pupal extract were then placed in a 25-ml sterile beaker. Separately, a 10-mg/ml solution of Gentamicin (Sigma Chemical Co., USA) was mixed into the beaker’s contents at the rate of 0.01 ml/ml using a 1-ml sterile syringe. After being stirred, the beaker was put into a water bath at 50°C for about 10 min. In a 50-ml flask, 3 g agar was dissolved in 25 ml bidistilled water, sterilized at 120°C for 15 min, and left to cool for about 5 min at room temperature. About 2 ml agar suspension was then removed using a 5-ml syringe and added to the beaker’s content. The diet was stirred with a sterile spatula and pipetted into the wells of a 24-well plastic rearing plate (Nunclon, Denmark) (0.4–0.5 ml per well). The plates were left for 4–5 h at room temperature and RH before infestation with parasitoid eggs. Each replicate consisted of 1 plate per diet. Diet pH was 6.4 for A, B, and C and 6.6 for D. The osmotic pressures of the media, measured by a cryoscopic osmometer (DIC, Japan), were (mOsm/Kg) 237 (A), 207 (B), 241 (C), and 345 (D). Test 2. Two diets free of host material, based on the D homogenate, were tested. Both were supplemented with yeast extract and chicken egg yolk, two key components of the media developed for several parasitoids (Bratti, 1990), including E. larvarum (Mellini and Campadelli, 1995a,b). The first diet (I) also contained wheat germ, a common protein source used in insect media (Reinecke, 1985). In diet (I) the amounts of the ingredients were the same as those in the medium developed for B. intermedia by Dindo et al. (1997). Ingredients included (v/v) 74.1% homogenate, 9.2% chicken egg yolk, and 16.7% agar–yeast extract and wheat germ–water suspension (12% agar, 9.5% yeast extract, and 9.5% wheat germ) to give a 2% agar 1.6% yeast extract and 1.6% wheat germ final concentration in the diet. In the second diet (II), yeast extract, a good source of amino acids and other nutrients (Reinecke, 1985), was used instead of wheat germ. Diet (II) therefore contained twice as much yeast extract than diet (I) and also included saccharose (after Mellini and Campadelli, 1995b), which partially replaced homogenate. Diet (II) thus contained (v/v) 65.8% homogenate, 9.2% chicken egg yolk, 8.3% yeast extract and saccharose solution (38% yeast extract and 20% saccharose), and 16.7% agar–water suspension (12% agar) to give a 3.2% yeast extract, 1.7% saccharose, and 2% agar final concentration in the medium. Diet (I) was prepared by mixing 8.9 ml homogenate and 1.1 ml chicken egg yolk in a 25-ml sterile beaker,

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which was then placed into a water bath at 50°C for about 10 min. The egg yolk was obtained by carefully opening one end of an egg, which had been surface sterilized with 70% ethanol, and removing the yolk with a 1-ml sterile syringe. Gentamicin solution was then added, using the same method and at the same rate described for Test 1. Separately, in a 10-ml beaker, 0.19 g yeast extract (Sigma Chemical Co.) and 0.19 g wheat germ (Sigma Chemical Co.) were put in 2 ml agar–water suspension, obtained as described for Test 1 and heated at 100°C for 10 min. The resulting suspension was sterilized at 120°C for 15 min, allowed to cool for about 5 min at room temperature, and added to the contents of the 25-ml beaker. Diet (II) was prepared by mixing 7.9 ml homogenate and 1.1 ml chicken egg yolk in a 25-ml sterile beaker. As in diet (I) gentamicin solution was then added. Separately, in a 10-ml beaker, 0.38 g yeast extract and 0.2 g saccharose were dissolved in 1 ml bidistilled water. The solution was sterilized at 120°C for 15 min, left to cool for about 5 min at room temperature, and mixed into the contents of the beaker. About 2 ml agar suspension, prepared and sterilized as described for Test 1, was added to the medium. After stirring, both diets (I) and (II) were distributed into 5-cm-diameter glass petri dishes using sterile syringes. Each replicate consisted of 1 petri dish per diet. The pH was 6.2 in diet (I) and 6 in diet (II). The osmotic pressures of the media, measured as above, were 326 mOsm/kg in diet (I) and 691 in diet (II). General considerations. The parasitoid eggs were collected from superparasitized G. mellonella larvae and transferred onto the diet by the method described by Bratti and Coulibaly (1995). In Test 1, the eggs were placed singly into the plastic plate wells. In Test 2, 15 eggs were placed per petri dish. Both the plates and the petri dishes were sealed with Parafilm and kept in darkness at 26 ⫾ 1°C and 70% RH throughout the experiment, except when they were removed for daily inspection. In Test 1, the 2nd, 3rd, and 4th replicates were performed by placing on each diet the eggs laid by the in vitro-reared females obtained in the previous replicate from the same diet. The parasitoids were therefore cultured in vitro for three generations. Instruments and glassware were sterilized by autoclaving for 20 min at 120°C and 1 bar. All operations, including visual examinations, were performed in a laminar flow hood. When puparia formed, they were placed singly into glass tubes and left there until adult emergence. Adult sex was recorded at emergence. The adults obtained in vitro were placed in 20 ⫻ 20 ⫻ 20 Plexiglass cages (one cage per diet) and supplied with G. mellonella larvae to verify if females were capable of parasitizing.

Fly Longevity and Fecundity Test Female parasitoids obtained in vivo from G. mellonella (treatment a) and in vitro on diet (I) (treatment b) were compared. In treatment (a), 90–100 G. mellonella mature larvae (weight range 250–300 mg) were exposed to 70–80 parasitoids in a rearing cage and removed 5–10 min later when 3–4 eggs per host had been laid. As at 26°C the eggs hatch in 3 days (Hafez, 1953), on the 4th day the penetration holes made by the maggots in the host integument were examined and the monoparasitized larvae, with one penetration hole, were selected and kept in the E. larvarum rearing chamber until puparium formation. In treatment (b), about 40 ml diet was prepared and distributed into the wells of three 24-well plates and the parasitoid eggs were placed singly into the wells according to the methods described above. The plates were kept in the dark at 26°C ⫾ 1°C and 70% ⫾ 5% RH until puparium formation. In both treatments, after forming, the puparia were rinsed, weighed, and placed singly into glass tubes. Parasitoid sex was determined upon adult emergence. Females which had emerged on the same day were placed in the E. larvarum rearing chamber inside a 20 ⫻ 20 ⫻ 20 cm plexiglass cage with an equal number of males and supplied with food and water. As the preoviposition period lasts about 3 days (Hafez, 1953), on the 4th day the males were removed and G. mellonella mature larvae (two per female; after Bratti and Coulibaly, 1995) were daily exposed to parasitoids until death. Similarly to the standard in vivo rearing procedure of E. larvarum, the larvae were removed from the cage after about 30 min and the eggs which had been laid on their body were counted. The larvae were then placed in the rearing chamber inside a plastic box until puparium formation. Dead females were removed daily. Experimental Design and Statistical Analysis Four and six replicates were performed in Test 1 and 2, respectively. Each replicate consisted of 24 eggs (Test 1) and 15 eggs (Test 2). In Test 1, the data were analyzed in terms of percentages of live first-instar larvae observed on the diet (⫽ live first-instar larvae/ eggs originally placed on the media ⫻ 100) (LI), pupation rates (⫽ puparia/live first-instar larvae ⫻ 100) (P), adult emergence (⫽ adults/puparia ⫻ 100) (AE), and adult yields (⫽ adults/eggs originally placed on the media ⫻ 100) (AY). Mellini et al. (1993a) showed that, also on the diet, at 26°C E. larvarum eggs hatch in 3 days. The larvae may immediately penetrate into the diet or crawl for a few hours on the diet surface before sinking into it. Thus, in the present study, the number of live first-instar larvae was determined by visually counting either the live parasitoid maggots on the diet surface or their penetra-

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tion holes, 3–4 days after placing the eggs on the medium. In Test 2, only P, AE, and AY were calculated, as it was extremely difficult to visually detect the small first-instar larvae and their penetration holes spread over the media contained in petri dishes. Male and female puparial weights 24 h after formation and development times from egg to adult were also recorded. The data were analyzed by one-way analysis of variance (STATISTICA for WINDOWS, 1994). The percentage values were transformed for the analysis using an arcsine transformation (Mosteller and Youtz, 1961). In the fly longevity and fecundity test, seven (treatment a) and eight (treatment b) replicates were performed, 4–8 females being used for each replicate. The puparial weights and development times from egg to adult of the females and males used for the test as well as female longevity from emergence (L) were recorded. To evaluate fecundity, the eggs/female laid on host larvae (e) (⫽ number of eggs/number of alive females) was calculated daily. The single e values were then added to determine the mean number of eggs laid on the larvae throughout female lifespan (E). Puparia yields (PY) based on the total number of eggs oviposited on host larvae were also calculated. The data for puparial weights, development times, L, and E were analyzed by one-way analysis of variance, while the Kruskall–Wallis nonparametric procedure (STATISTICA for WINDOWS 1994) was adopted for PY analysis. The e values were also grouped in 12 3-day time intervals from the 4th day after emergence. Separately for each treatment, the relationship between female age, expressed in days from emergence, and the eggs/female/time interval was then analyzed by curvilinear regression using the model y ⫽ A (e⫺cx ) (Snedecor and Cochran, 1980). The e values were also analyzed by a factorial analysis of variance (Zar, 1984) (2 ⫻ 12 factors tested for the rearing technique and time effect).

RESULTS

Test 1 Given that the actual egg hatching rate of E. larvarum is very difficult to evaluate on the diet, the percentages of live first-instar larvae (LI) were calculated instead of the percentages of hatched eggs. In fact, empty egg shells are difficult to distinguish from nonhatched eggs by visual observation, as this tachinid oviposits macrotype, dehiscent eggs (Mellini, 1990). In a preliminary test, we observed that actual hatching could be detected only by opening the rearing container and removing the egg, a procedure deemed to be unsuitable in the present study, which was aimed at observing the complete parasitoid development on the diet. Though not perfect, numbers of LI reflect the actual hatching rate. In the present study, no significant difference was found among the four diets (Table 2). On the media, the larvae fed and moved normally, as on the host and as seen in previous in vitro rearing studies of E. larvarum (Mellini et al., 1993a, 1996). The puparia formed next to the diet remains. Occasionally, third-instar larvae moved from one well to another, before pupating. Pupation rates did not differ significantly among the diets but the highest mean values were found for diets B and D (Table 2). For both males and females, the puparial weight did not differ significantly among the media, although the highest mean values (Table 2) were recorded for diet (D). Adult emergence percentages were above 80% for all diets (A, B, C, D) and no significant differences were recorded among the treatments. Adult yields ranged from 30 to 33% (diet A and C) to 41 to 42% (diet B and D). Differences, however, were not significant. The development times for both males and females did not reveal any marked differences among the four media, ranging from 23 to 25 days (Table 2).

TABLE 2 Percentages of First-Instar Larvae (LI), Pupation Rates (P), Percentages of Adult Emergence (AE), Adult Yields (AY), Male and Female Development Times in Days (Tm; Tf ), and Puparial Weights in mg (Wm; Wf ) of Exorista larvarum Reared on Artificial Media Based on Commercial Meat Homogenates Combined with 10% Host Pupal Extract a Diet A B C D Fb P a b

LI

P

AE

AY

Tm

Tf

Wm

Wf

70.8 ⫾ 6.1a 76 ⫾ 5.7a 85.4 ⫾ 4.3a 83.3 ⫾ 6.2a 1.4510 0.2769

32.3 ⫾ 4.3a 46.8 ⫾ 12a 37.5 ⫾ 7.4a 47.9 ⫾ 10a 0.8798 0.4790

94.7 ⫾ 3.1a 87.2 ⫾ 2.7a 89.7 ⫾ 3.8a 81.3 ⫾ 6a 1.5357 0.2559

30.2 ⫾ 3.1a 41.6 ⫾ 12a 33.3 ⫾ 6.1a 40.7 ⫾ 10.7a 0.3776 0.7708

24.3 ⫾ 1.7a 24.2 ⫾ 2.6a 23.4 ⫾ 3.5a 23.3 ⫾ 1a 0.1996 0.8941

25.4 ⫾ 1.4a 24.6 ⫾ 2.3a 23.7 ⫾ 2.7a 23.9 ⫾ 0.7a 0.5949 0.6303

44.1 ⫾ 8.01a 45.5 ⫾ 6.8a 48.8 ⫾ 6.6a 60.9 ⫾ 20.5a 1.6119 0.2384

39.9 ⫾ 2.6a 44.6 ⫾ 7.8a 42.8 ⫾ 7.2a 48.7 ⫾ 16.5a 0.5582 0.6526

(Means ⫾ S.E.); means followed by the same letter are not significantly different (␣ ⫽ 0.05). df 3, 12.

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TABLE 3 Pupation Rates (P), Adult Emergence (AE), Adult Yields (AY), Male and Female Development Times in Days (Tm; Tf ), and Puparial Weights in mg (Wm; Wf ) of Exorista larvarum Reared on Two Host Material-Free Artificial Media Based on Commercial Veal Homogenate a Diet

P

AE

AY

Tm

Tf

Wm

Wf

I II Fb P

41.5 ⫾ 5.4a 46.1 ⫾ 6.9a 0.2399 0.6348

70.7 ⫾ 6.4a 72.4 ⫾ 5.1a 0.5198 0.8242

28.7 ⫾ 4.2a 32.7 ⫾ 4.3a 0.3467 0.5691

24 ⫾ 1.6a 23.8 ⫾ 1.8a 0.0228 0.8829

24.3 ⫾ 2.4a 23.4 ⫾ 1.9a 0.4876 0.5009

60.8 ⫾ 20.7a 58 ⫾ 15.1a 0.2709 0.614

54.2 ⫾ 12.6a 48.1 ⫾ 9.6a 0.7946 0.3937

a b

(Means ⫾ S.E.); means followed by the same letter are not significantly different (␣ ⫽ 0.05). df 1, 10.

Test 2 In this test, pupation rates ranged from 42 to 46%. No significant differences were found between the two diets. Also adult emergence and adult yield percentages did not differ significantly between the two diets (Table 3). Puparial weights and development times were comparable to those obtained in Test 1 on diet (D). No significant differences were found between diet (I) and diet (II) (Table 3). In both Test 1 and Test 2 the parasitoid adults obtained in all media mated and the females oviposited on G. mellonella larvae, producing a second generation in vivo. The puparia obtained from eggs laid by the in vitro-reared females were comparable in size to those usually obtained with standard in vivo rearing (Mellini and Campadelli, 1996). Fly Longevity and Fecundity Test In this test, in monoparasitized hosts, both male and female puparial weights were significantly lower, and the development times about 5 days shorter, than in those reared on diet I (Table 4). Female longevity (L) was about 21 days on average, and no significant difference was found between the in vivo- and the in vitro-reared females (Table 4). In both treatments,

however, some females lived much longer, up to 39 days. The mean number of eggs/female laid throughout female lifespan was slightly higher for the females produced on diet I (178.1) than for in vivo-reared females (172.4), but this difference was not significant (Table 4). In both treatments, the number of eggs/female decreased with female age (Fig. 1). The 2 ⫻ 12 factorial analysis of variance confirmed that the time effect was significant (F ⫽ 33.9; df ⫽ 11, 108; P ⫽ 0.0000001), whereas both the rearing technique effect (F ⫽ 2.3; df ⫽ 1, 108; P ⫽ 0.13) and the interaction (F ⫽ 0.2; df ⫽ 11, 108; P ⫽ 0.9) were not significant. The puparia yields did not differ significantly between the in vivo- and the in vitro-reared females (Table 4). DISCUSSION

The results confirmed the high degree of tolerance of E. larvarum to variations in diet composition. In fact, in both Tests 1 and 2, parasitoids developed to the adult stage at rates of 29–42% on different media, and no significant differences were found among diets in any of the parameters measured, including adult yield and puparial weight. In previous studies (Mellini and Campadelli 1993a,b, 1995a,b) the latter was calculated

TABLE 4 Male and Female Puparial Weights in mg (Wm; Wf ) and Development Times in Days (Tm; Tf ), Female Longevity from Emergence in Days (L), Eggs/Female Laid on Host Larvae (E), and Puparia Yields (PY) of Exorista larvarum Reared in Vivo on G. mellonella Larvae and in Vitro on Diet I a Rearing system In Vivo In Vitro

P a

Wm

Wf

Tm

Tf

L

E

PY

54.1 ⫾ 9.2a 72.1 ⫾ 12b df 1,13 F ⫽ 10.5 0.006*

44.9 ⫾ 3.5a 61.8 ⫾ 6.1b df 1,13 F ⫽ 41.5 0.00002*

15.4 ⫾ 1.4a 20.5 ⫾ 0.7b df 1,13 F ⫽ 84.2 0.0000001*

15.7 ⫾ 1.3a 21.2 ⫾ 0.6b df 1,13 F ⫽ 103.1 0.0000001*

21.5 ⫾ 4.5a 21.2 ⫾ 6.5a df 1,13 F ⫽ 0.008 0.93

172.4 ⫾ 44.3a 178.1 ⫾ 44.8a df 1,13 F ⫽ 0.06 0.8

16.9 ⫾ 5a 15.3 ⫾ 2.6a N ⫽ 15 H ⫽ 0.56 0.45

(Means ⫾ S.E.); means followed by the same letter are not significantly different (␣ ⫽ 0.05).

IN VITRO REARING OF Exorista larvarum

263

FIG. 1. Comparison between Exorista larvarum females obtained in vivo from G. mellonella larvae and in vitro on diet I. Relationship between female age, expressed in days from emergence, and the number of eggs/female laid on G. mellonella larvae. Equations: y ⫽ 69.79 (e⫺0.09x ), r ⫽ 0.88 (in vivo) and y ⫽ 72.66 (e⫺0.08x ), r ⫽ 0.85 (in vitro).

without considering sex. Nevertheless, Mellini et al. (1993a) observed that male puparia were heavier than female ones. The same trend was observed in the present study and it was therefore decided to consider male and female puparial weights separately. The ability of E. larvarum to grow on media based on diverse ingredients has already been shown by other authors. Mellini et al. (1993a) first reported the complete in vitro development of E. larvarum on a bovine serum-based diet supplemented with 20% extract of G. mellonella pupae. Adult yields of 36% were recorded. Host material in the diet was then replaced with different amounts of chicken egg yolk and yeast extract without any drop in adult yields or vitality (Mellini and Campadelli, 1995a). Subsequently, Mellini and Campadelli (1995b) developed a simple diet devoid of host material composed of skimmed milk, chicken egg yolk, yeast extract, and saccharose and obtained adult yields of 43–44%. According to Mellini and Campadelli (1996b) these yields were comparable to those usually obtained from the factitious host G. mellonella. Diets based on tissue culture media, both with (Bratti and Coulibaly, 1995) and without host components (the latter containing 10–15% chicken egg yolk [Bratti et al., 1995]) were

also utilized for E. larvarum with adult yields reaching as high as 55%. In previous studies (Bronskill and House, 1957; Cohen, 1985; Cohen and Urias, 1986; De Clercq and Degheele, 1992) meat homogenates were successfully utilized in artificial diets for entomophages. Commercial meat homogenate-based media with and without host components were developed for the hymenopterous parasitoid B. intermedia, giving parasitoid adult yields of 67 and 44%, respectively (Dindo et al., 1997a,b). These results led us to test commercial meat homogenates also in artificial media for E. larvarum. The meat homogenates utilized in Test 1 were selected among those available on the market and the test was performed in order to determine which was best for use in subsequent diets devoid of host components. As no parasitoid development was obtained on homogenates alone (Dindo and Farneti, unpubl.), the homogenates were supplemented with 10% host material. Though the differences, in tests reported here, among diets for all the parameters considered were not significant, the highest adult yields and puparial weights were obtained from the diet containing the D homogenate, which was therefore employed as basic ingredi-

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ent in the media assessed in Test 2. This test was performed using petri dishes, instead of plastic multiwell plates, after Mellini et al. (1993b), who showed that, being a gregarious parasitoid, E. larvarum can be reared in groups, instead of individually. In these studies, rearing conditions (individual vs in groups) did not affect either adult yield or puparial weights and resulted in qualitative improvements in parasitoid production. In our study, however, we found that visual observations of parasitoid development were more difficult to make in petri dishes than in multi-well plates, at least up to third larval instar. Multi-well plates are therefore to be preferred in research studies on E. larvarum in vitro culture aimed at investigating parasitoid larval biology and behavior on artificial diet, whereas petri dishes are better for artificial mass rearing. In Test 2, both diets I and II gave pupation rates comparable to those obtained in Test 1 on diet (D). Adult yields, however, were found to be lower, owing to the lower emergence rates, than those observed on diet (D). They were also lower than those obtained on diets free of host materials by Mellini and Campadelli (1995b) and Bratti et al. (1995). Moreover, the parasitoid development times observed in both Test 1 and Test 2 were 5–6 days longer than those observed at the same temperature by Mellini et al. (1993a) and Mellini and Campadelli (1995a,b). On the other hand, the puparia obtained from both diet (D) and the host material-free media tested in the present study were about 40–50% heavier than those formed in vitro on the diets developed by Mellini et al. (1993a) and Mellini and Campadelli (1995b) and in vivo in G. mellonella larvae (Mellini and Campadelli, 1996a). Grenier et al. (1994) emphasized the importance of the quality of artificially reared entomophages, and recent studies have addressed this topic (Rojas et al., 1996; Morales-Ramos et al., 1996; Nordlund et al., 1997). Rojas et al. (1996) showed that a correlation exists between the pupal weight of in vitro-reared females of the pteromalid parasitoid Catolaccus grandis (Burks) and their fecundity and postulated that parasitoid weight may be used as the first criterion to evaluate the quality of new artificial diets. Moreover, size may also be correlated with individual fitness for in vivo-reared parasitoids (Doutt et al., 1976) and, in particular, with female potential fecundity, as shown for both hymenopteran parasitoids (Wylie, 1966; van den Assem et al., 1989; Charnov et al., 1991; Croft and Copland, 1993) and tachinids (King et al., 1976; Bourchier, 1991; Reitz and Adler, 1995). A correlation between puparial weight and parasitoid fitness (especially female fecundity) has not, however, been demonstrated for E. larvarum. In the present study, fly longevity and fecundity were measured to determine whether the high weight of the

puparia obtained on host material-free media could indeed be taken as an indicator of good parasitoid quality rather than being an effect caused by a harmful retention of nondigestible diet components. Mellini and Campadelli (1996b) showed that gregarious E. larvarum puparia are smaller than those obtained from monoparasitized hosts. In this test, therefore, females emerged from puparia obtained on diet I (which gave the highest puparial weights in Test 2) were compared with females emerged from puparia formed in monoparasitized G. mellonella larvae, which had no more than one penetration hole made by a maggot, so as to obtain from the host the heaviest possible puparia. Nevertheless, male and female puparial weights were considerably higher and development times longer on the diet than in the host. No significant difference was found between in vivo- and in vitro-reared females for longevity, a factor, however, which may not be affected by size, as shown by Reitz and Adler (1995) for another tachinid, Eucelatoria bryani (Sab.). The mean number of eggs laid on G. mellonella larvae throughout the female lifespan was in agreement with that found by Hafez (1953) on the larvae of the natural host Prodenia litura F. and was only slightly higher for the in vitro- than for the in vivoreared females, although the former emerged from heavier puparia than the latter. Both in vivo- and in vitro-reared flies laid most eggs from the 4th to the 9th day after emergence but continued to oviposit until death. Puparial yields were lower than those normally observed in G. mellonella larvae, owing to high superparasitization level (Mellini and Campadelli, 1996b). No significant difference in yield was, however, found between in vivo- and in vitro-reared parasitoids. The fecundity of the females obtained in the diet was thus comparable to that of the females emerged from puparia formed in G. mellonella larvae. The greater puparial weights and longer development times of male and female E. larvarum obtained on the diet may have been related to the larger amounts of food available for the in vitro- than for the in vivoreared parasitoids, which were, respectively, cultured on 0.4–0.5 ml diet and in mature larvae weighing 250–300 mg. Because larger size did not lead to greater fecundity, the possibility of a disturbing effect of nondigestible diet components cannot be excluded. Additional work is therefore required to compare parasitoids reared in the host and on lower amounts of artificial diet so as to obtain, in vivo and in vitro, puparia of similar size. Further study is also needed to ascertain the correlation between puparial weight and fecundity of E. larvarum females. A comparison of the biological characteristics and the effectiveness in the field between the flies obtained on our diets and those reared on the other media free of host materials (Bratti et al., 1995; Mellini and Cam-

IN VITRO REARING OF Exorista larvarum

padelli, 1995a,b) also needs to be made, so as to determine the best diet for the mass production of E. larvarum. ACKNOWLEDGMENT This research was supported by Italian Research and University Ministry (MURST ex-40%)

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