Age-Related Effects of the Autographa californica Multiple Nucleopolyhedrovirus egt Gene in the Cabbage Looper (Trichoplusia ni)

Biological Control 19, 57– 63 (2000) doi:10.1006/bcon.2000.0841, available online at http://www.idealibrary.com on

Age-Related Effects of the Autographa californica Multiple Nucleopolyhedrovirus egt Gene in the Cabbage Looper (Trichoplusia ni) K. R. Wilson, D. R. O’Reilly,* R. S. Hails, and J. S. Cory 1 Ecology and Biocontrol Group, NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, United Kingdom; and *Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, United Kingdom Received December 21, 1999; accepted April 4, 2000

INTRODUCTION The effects of deleting the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egt gene on speed of kill and virus productivity were compared in second and fourth instar Trichoplusia ni (Hu ¨ bner) larvae. Time to death was significantly reduced in larvae infected with an egt deletion mutant compared to insects infected with the wild-type virus. Moreover, time to death was reduced by the same proportion (11%) in second and fourth instar larvae. Virus yield was also significantly lower in fourth instar larvae infected with the deletion mutant but no difference was apparent in second instar larvae. A comparison of cadaver weights showed that insects infected with the deletion mutant were lighter than those infected with the wildtype virus, suggesting that the decrease in virus yield resulted from a reduction in larval growth. An analysis of yield per unit body weight showed no evidence for differences in replication rate in the two viruses. To determine whether differences in larval growth rate were related to differences in feeding activity, frass production was monitored in fourth instar larvae. Larvae infected with the deletion mutant produced less frass than larvae infected with the wildtype virus. Whereas the average rate of feeding for fourth instars did not differ between virus treatments, the rate peaked and declined earlier for larvae infected with the deletion mutant than for those infected with wild-type virus, suggesting enhanced early feeding in the absence of egt expression. © 2000 Academic

Sequencing the baculovirus genome has revealed a number of genes whose functions act at the level of the host organism. The first gene of this type to be identified was the ecdysteroid UDP-glucosyltransferase, or egt gene (O’Reilly and Miller, 1989). The egt gene codes for an enzyme, ecdysteroid UDP-glucosyltransferase (EGT) (O’Reilly and Miller, 1990). EGT catalyzes the conjugation of insect molting hormones (ecdysteroids) with UDP-sugars, one effect of which is to suppress host molting and arrest development (O’Reilly and Miller, 1989). The egt gene was first identified in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) where it lies within the PstI-G region (O’Reilly and Miller, 1989). This region was found to be commonly deleted during serial passage in cell culture (Kumar and Miller, 1987), suggesting that it was not necessary for replication. Recent phylogenetic analysis has indicated that the egt gene was present in the ancestral baculovirus and has been retained in most wild-type isolates (Clarke et al., 1996). The egt gene of at least 13 different baculoviruses has now been sequenced (O’Reilly and Miller, 1990; Pearson et al., 1993; Riegel et al., 1994; Barrett et al., 1995; Faktor et al., 1995; Clarke et al., 1996; Chen et al., 1997; Hu et al., 1997; Popham et al., 1997; Smith and Goodale, 1998; Gomi et al., 1999; Vlak et al., unpublished). Only Xestia c-nigrum granulovirus has been definitively shown to lack an egt gene (Hayakawa et al., 1999). Since the egt gene is not required for viral replication but has been retained in most baculovirus genomes, it is likely to confer a selective advantage at the insect level. However, its precise function and how it confers a selective advantage are not clearly understood. O’Reilly and Miller (1991) showed that final instar Spodoptera frugiperda (Smith) larvae infected with AcMNPV lacking a functional egt gene yielded smaller amounts of virus than those infected with wild-type

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Key Words: baculovirus; nuclear polyhedrosis virus; deletion mutant; recombinant virus; microbial control; productivity; speed of kill.

1 To whom correspondence should be addressed. Fax: ⫹44 1865 281696. E-mail: [email protected].

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1049-9644/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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AcMNPV. This was attributed to the onset of prepupal behavior, especially cessation of feeding and associated weight loss, as would be expected to occur in uninfected insects. These insects were also found to die significantly faster than their wild type-infected counterparts. Therefore, the wild-type virus, by blocking molting or pupation, could be gaining a selective advantage by producing a greater number of progeny. This could be achieved by inducing the host to continue feeding and growing, so that there is a greater volume of insect tissue for virus replication. However, O’Reilly and Miller (1991) measured the effects of the egt gene only in final instar larvae. Among uninfected larvae, the earlier instars do not normally display such a dramatic weight loss prior to molting as do the older instars, so it is unclear whether this effect is general. Moreover, the insects used by O’Reilly and Miller (1991) were infected with very high doses of virus by injection rather than the normal oral route, and S. frugiperda is only semipermissive to the AcMNPV. Slavicek et al. (1999) showed recently that final instar Lymantria dispar (L.) larvae infected with LdMNPV lacking the egt gene (vEGT-) died significantly faster than larvae infected with wild-type LdMNPV and yielded less virus. They also ceased feeding earlier than those infected with the wild-type virus, which could account for the yield difference, but a reduction in body weight was detected only in female larvae. Fourth (penultimate) instar larvae infected with vEGT-, however, did not die significantly faster than those infected with the wildtype virus, although they were found to cease feeding earlier. Only female larvae infected with the deletion mutant showed a reduction in body weight and yielded less virus. To date, the effects of egt on yield and time to death have been measured simultaneously only in penultimate and final instar larvae (O’Reilly and Miller, 1991; Slavicek et al., 1999). However, in neither case were the two parameters analyzed together to determine the exact nature of the trade-off. Moreover, it is not known whether similar effects occur in earlier larval instars. In this study, we have quantified the effects of the egt gene on virus yield and speed of kill in second and fourth instar larvae of the permissive host Trichoplusia ni (Hu¨bner) to assess whether the effects of egt in the AcMNPV/T. ni system are age specific and to gather more evidence to explain the role that the egt gene plays in the baculovirus:host interaction. MATERIALS AND METHODS

Second and fourth instar T. ni larvae were obtained from a laboratory colony maintained at the NERC Institute of Virology and Environmental Microbiology, Oxford. Larvae were reared on a modification of Hoffman’s tobacco hornworm diet (Hunter-Fujita et al., 1998) at 22 ⫾ 2°C under a 16:8 h light:dark regime.

Viruses The wild-type isolate used in this study was the L1 clone of AcMNPV (Lee and Miller, 1978). The second virus, vEGTDEL, was an AcMNPV derivative with a deletion of approximately 1 kb within the egt gene in the PstI-G region of the genome (O’Reilly et al., 1991). Both viruses were propagated in T. ni larvae and purified from lysed cadavers by homogenization and density gradient centrifugation (King and Possee, 1992). The purified suspensions were counted in a 0.1-mm depth hemocytometer with improved Neubauer ruling. Insect Bioassay T. ni larvae were precisely staged by first separating first and third instar pharates and selecting only those that molted during the night. These larvae were inoculated as newly molted second or fourth instars by the diet plug method. Briefly, each larva was provided with a small plug of artificial diet to which 1 ␮l of inoculum had been applied. Only larvae which consumed the plug within 24 h were used in the experiment and transferred to fresh diet to be reared to death or pupation. The inoculum contained either water only (control), 85 occlusion bodies (OBs)/␮l AcMNPV/vEGTDEL for second instar larvae, or 360 OBs/␮l AcMNPV/ vEGTDEL for fourth instar larvae. In previous tests, a dose of 85 or 360 OBs had been shown to kill approximately 50% of second and fourth instar T. ni larvae, respectively. An LD 50 was chosen to reduce dose effects in the measurement of time to death (Van Beek et al., 1988). Between 70 and 100 larvae were inoculated per instar/virus treatment so that there were sufficient test subjects (virus-killed larvae) on which to carry out statistical analysis. Thirty-five second instar larvae and 45 fourth instar larvae received water only. All larvae were observed every 8 h until pupation or death, and the time to death was recorded in the latter case. All larvae were weighed at the point of death, prior to lysis by first weighing empty 1.5-ml microtubes and then transferring the cadavers to the tubes to be reweighed. The yields of virus produced by a subsample of 22 larvae/instar/virus treatment were quantified by hemocytometry using a 0.1-mm depth hemocytometer with improved Neubauer ruling. Each larva was homogenized in a known quantity of sterile deionized water, the suspension was thoroughly mixed, and two counts were made per larva. The average of the two counts was multiplied by the total volume of homogenate (ml) to provide a value for the total number of occlusion bodies per cadaver. Additionally, the frass produced between 0700 and 1500 h each day by 30 fourth instar larvae per treatment (these larvae subsequently died of virus infection) and 30 mock-infected larvae was also weighed as a measure of feeding behavior.

EFFECTS OF THE AcMNPV egt GENE IN T. ni

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Yield Analysis

FIG. 1. The mean time to death of second and fourth instar Trichoplusia ni larvae infected with AcMNPV (L1 clone) and vEGTDEL. Error bars were determined for each instar by least significant difference calculations. (n ⫽ 52 and 55 for second instar larvae infected with AcMNPV (L1) and vEGTDEL, respectively; n ⫽ 80 and 95 for fourth instar larvae infected with AcMNPV (L1) and vEGTDEL, respectively).

Statistical Analysis Statistical analysis was carried out using the statistical package GLIM 3.77 (Royal Statistical Society, 1985). Since the larvae received a LD 50 dose, not all larvae succumbed to infection; therefore, only those larvae that had died of virus infection under any treatment were included in each analysis. A Weibull distribution was fitted to the time to death data that allows the death rate to vary with time. Frass production, cadaver weights, and virus yields were analyzed using standard model simplification techniques. A Box–Cox analysis was carried out on the yield per unit body weight data to determine the correct power transformation, and an analysis of variance was carried out on the transformed data.

The yield data were best described by a square-root transformation. Time to death and cadaver weight were found to be confounding factors; i.e., the effects of each factor upon yield could not be separated. Therefore, time to death was chosen as an explanatory variable in the yield analysis since this was considered to be more central to the yield/time to death trade-off. The yields of both wild type and vEGTDEL virus in fourth instar larvae were found to rise with time to death. However, the yield rose by a far greater extent among wild type-infected larvae (time to death ⫻ virus treatment interaction: F (1,82) ⫽ 16.0; P ⫽ 1.39 ⫻ 10 ⫺4 ). Thus, in general, the yield of wild-type virus far outweighed that of vEGTDEL virus for corresponding times to death (Fig. 2a). For example, larvae which died 144 h postinfection (the last vEGTDEL death) yielded 1.42 ⫻ 10 9 OBs/larva AcMNPV (L1), whereas those infected with vEGTDEL yielded 5.40 ⫻ 10 8 OBs/ larva. Across all time points, the mean yield from wild type-infected larvae was 1.13 ⫻ 10 9 OBs/larva (95% C.I. (8.58 ⫻ 10 8, 1.44 ⫻ 10 9)), whereas the mean yield from larvae infected with vEGTDEL was 4.23 ⫻ 10 8 OBs/larva (95% C.I. (3.47 ⫻ 10 8, 5.07 ⫻ 10 8)).

RESULTS

Time to Death Second instar larvae were found to die significantly faster than fourth instar larvae ( ␹ 2 ⫽ 38.8; df ⫽ 1; P ⫽ 4.62 ⫻ 10 ⫺10 ) and larvae infected with the deletion mutant died significantly faster than those infected with the wild type virus ( ␹ 2 ⫽ 13.6; df ⫽ 1; P ⫽ 2.26 ⫻ 10 ⫺4 ) (Fig 1). The proportional difference in time to death between wild type- and vEGTDEL-infected larvae was the same in each instar, suggesting that the effect of the deletion was uniform across instars. On average, second instar larvae infected with the wild-type virus died after 130 h, whereas larvae infected with the deletion mutant died after 116 h, a reduction of 14 h. Fourth instar larvae died after 140 h if infected with the wild-type virus compared to 125 h if infected with the deletion mutant, a reduction of 15 h. In both cases, this is equivalent to a reduction in time to death of 11%.

FIG. 2. Yield of occlusion bodies from Trichoplusia ni larvae inoculated with AcMNPV (L1 clone) (•) or vEGTDEL (*), plotted against time to death. (a) Fourth instar T. ni; (b) second instar T. ni. The fitted lines showing the yields from larvae which died between 104 and 160 h overlap; so, vEGTDEL has been represented by a dashed line and AcMNPV (L1 clone) by a solid line. OBs, occlusion bodies.

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In contrast, the yields of both viruses in second instar larvae were constant, regardless of time to death (the slopes were not significantly different from zero: F (1,81) ⫽ 2.16; P ⫽ 0.145) (Fig. 2b). Moreover, the yields for the two virus treatments were not significantly different from one another (F (1,82) ⫽ 0.383; P ⫽ 0.537). The average yield of AcMNPV (L1) or vEGTDEL from second instar larvae was 3.02 ⫻ 10 7 OBs/ larva (95% C.I. (2.25 ⫻ 10 7, 3.91 ⫻ 10 7)). Yield/Cadaver Weight Analysis To determine whether the differences in virus yield observed in the previous analysis could be attributed to differences in replication rate in the wild-type and vEGTDEL viruses, an analysis of virus yield per unit cadaver weight was carried out. The data were best described by a cube root transformation. A comparison of the wild-type and vEGTDEL viruses showed that virus yield per unit body weight did not differ between virus treatments (F (1,85) ⫽ 2.25; P ⫽ 0.137), whereas a comparison of instars showed that fourth instars produced a greater amount of virus per unit body weight than second instars (F (1,85) ⫽ 5.34; P ⫽ 0.0233). Therefore, although the absence of the egt gene (and hence the presence of ecdysteroids) reduced the absolute yields of virus produced by fourth instar larvae (as shown in the previous analysis), there is no evidence that this is due to an alteration in the rate of virus replication. Cadaver Weight Analysis Cadaver weights were analyzed to determine whether insect growth could be responsible for the difference in virus yields. These data were best described by a logarithmic transformation. There was a three-way interaction between virus type, larval instar, and time to death (F (1,247) ⫽ 6.73; P ⫽ 0.010). In all cases, the longer the insects took to die the heavier they were at death, as might be expected. Second and fourth instar cadavers infected with wild type virus were significantly heavier than those infected with vEGTDEL (F (1,251) ⫽ 4.54; P ⫽ 0.034), although in second instars the difference between AcMNPV- and vEGTDEL-treated insects was less apparent than for fourth instars (a 24.4% weight increase compared to 33.7%). (AcMNPV-infected second instars: mean 0.00541 g, 95% C.I. (0.00447, 0.00655); vEGTDEL-infected second instars: mean 0.00409 g, 95% C.I. (0.00337, 0.00496); AcMNPV-infected fourth instars: mean 0.106 g, 95% C.I. (0.0949, 0.118); vEGTDELinfected fourth instars: mean 0.0730 g, 95% C.I. (0.0663, 0.0805)). Frass Analysis The frass produced by 30 fourth instar larvae per virus treatment during one 8-h period each day was

FIG. 3. Total frass produced by fourth instar Trichoplusia ni larvae inoculated with water (}), AcMNPV (L1 clone) (*), and vEGTDEL (Œ), plotted against time to death or pupation (for uninfected insects). r 2 ⫽ 68.9%.

weighed to give an indirect measure of changes in feeding (all these larvae went on to die of virus infection). Frass from the same larvae was weighed at each timepoint; so, the data were not independent and could not, therefore, be compared directly. Thus, analyses of total frass production, average rates of frass production, and peak frass production were carried out and compared to those of mock-infected control insects. In the first analysis, the total amounts of frass produced by individual larvae until death or pupation were compared. Uninfected larvae produced the greatest amount of frass (mean 0.220 g; 95% C.I. (0.207, 0.233)), followed by AcMNPV (L1)-infected larvae (mean 0.0553 g; 95% C.I. (0.0420, 0.0728)) and then vEGTDEL-infected larvae (mean 0.0390 g; 95% C.I. (0.0312, 0.0489)) (F (2,77) ⫽ 158; P ⫽ 5.36 ⫻ 10 ⫺28 ) (Fig. 3). These results could be explained solely by the differences in time to death or pupation (F (1,77) ⫽ 29.9; P ⫽ 5.49 ⫻ 10 ⫺7 ). In the second analysis, comparisons were made between the average amounts of frass produced by individual larvae in 8-h periods, as a measure of differences in feeding rates for larvae under each treatment. Virus-infected larvae were found to produce frass at a third the rate of uninfected larvae (F (2,77) ⫽ 53.1; P ⫽ 3.23 ⫻ 10 ⫺15 ). However, there was no significant difference between the two different virus treatments, i.e., the egt gene had no effect (F (1,77) ⫽ 2.19; P ⫽ 0.143). The average amounts of frass produced by larvae in 8-h periods were as follows: controls, 0.0314 g (95% C.I. (0.0265, 0.0373)) and AcMNPV (L1) and vEGTDEL, 0.0105 g (95% C.I. for AcMNPV (0.00876, 0.0127) and 95% C.I. for vEGTDEL (0.00888, 0.0125)). The frass production by each larva in 8-h stretches is shown in Fig. 4. Finally, to investigate whether frass production changed over time, the times of peak frass production (in hours) for all larvae were compared, with virus treatment and time to death as explanatory variables. The length of infection had no effect on the time of peak

EFFECTS OF THE AcMNPV egt GENE IN T. ni

FIG. 4. Frass production by individual larvae in 8-h periods plotted against time after infection. (Each datapoint represents frass production by a single larva in 8 h. Datapoints not independent.) (a) Uninfected larvae (E); (b) AcMNPV (L1)-infected larvae (⫻); (c) vEGTDEL-infected larvae (‚).

production rate (F (1,76) ⫽ 3.55; P ⫽ 0.0635). However, significant differences existed between virus treatments (F (2,76) ⫽ 4.45, P ⫽ 0.0148). On average, the rate of frass production by uninfected larvae peaked at 141 h post mock infection (95% C.I. (125, 160)), whereas larvae infected with wild-type virus produced frass at a maximum rate at 81.6 h post infection (95% C.I. (71.4, 93.3)), and vEGTDEL-infected larvae did so at 59.3 h (95% C.I. (52.4, 67.2)). DISCUSSION

The ecdysteroid UDP-glucosyltransferase or egt gene codes for an enzyme, EGT, that conjugates and inactivates the insect molting hormones. It is not necessary for viral replication (Kumar and Miller, 1987) but is believed to confer a selective advantage for the baculoviruses in nature. The exact nature of this advantage is unknown, although it would seem likely that promoting virus yield would increase the chances of sec-

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ondary transmission. In this study, we have explored the relationship between virus yield and speed of kill in second and fourth instar T. ni larvae infected with wild-type and egt minus AcMNPV. Yield of OBs, speed of kill, and larval feeding behavior were all found to be affected by the absence of the egt gene compared to the wild-type virus. Expression of the egt gene in the wild-type virus appeared to prolong the lifespan of larvae in both instars, but an increase in virus yield was detected only in older larvae. It is possible that the lack of difference in yields in second instar larvae infected with wild-type and egt minus viruses simply reflects difficulties in detecting small changes using a hemocytometer, which is perhaps operating near the limits of reliability. Cadaver weight was found to rise with time to death, with EGT expression apparently promoting larval growth in both instars. This suggests that, for fourth instar larvae, an increase in virus yield results from an increase in body size, which, if correct, reinforces the possibility that differences in virus yield in second instars also exist, but were not detected in this experiment. To determine whether the reduction in body size in egt minus-infected larvae was due to a cessation of feeding, frass production by fourth instar larvae was monitored as an indirect measure of feeding behavior. The average rate of frass production was not affected by the gene deletion, although the rate peaked earlier when the gene had been deleted. Larvae infected with the deletion mutant also produced less frass overall than those infected with the wild type, suggesting that these larvae may have ceased feeding owing to rising ecdysteroids, resulting in a cessation of growth. An increase in speed of kill seems to be a general effect of deleting the egt gene. Eldridge et al. (1992) found a 20-h reduction in time to death of neonate T. ni larvae fed an LD 100 dose of wild-type AcMNPV and vEGTDEL and a reduction of 1 day in fourth instars. Cory et al. (unpublished data) also found that deleting the egt gene caused a reduced time to death in fifth instar T. ni larvae. An increase in speed of kill has been noted in other lepidopteran species infected with egt minus AcMNPV. O’Reilly and Miller (1991), for example, found a reduction in the median survival time of 27.5 h when neonate Spodoptera frugiperda (J. E. Smith) larvae were infected with an LC 95 dose of vEGTDEL and wild-type AcMNPV. Therefore, the effect of deleting the egt gene appears to be widespread (although of different magnitude in different host/virus systems), suggesting that alteration in speed of kill has a physiological basis. Indeed, Flipsen et al. (1995) found that the Malpighian tubules, outgrowths of the alimentary canal that control osmoregulation and excretion, degenerated precociously in Spodoptera exigua (Hu¨bner) larvae infected with an egt deletion mutant. They suggested that this could account for the increased speed of kill in these insects. An alternative

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suggestion for the reduced time to death in egt minus infected larvae is that the metabolic demands of molting may not be well supported by an insect “fighting” viral infection (O’Reilly, 1995). However, Slavicek et al. (1999) recently reported a significant difference in speed of kill of fifth instar L. dispar larvae infected with wild-type LdMNPV and an egt deletion mutant, but no difference in first or fourth instar larvae. This was in contrast to earlier preliminary findings, where a difference was observed in first and fourth instar larvae (Riegel et al., 1994; Slavicek et al., 1999), emphasizing the fact that the action of baculoviral EGT is still poorly understood. In terms of biocontrol, deleting the egt gene appears to provide an advantage by reducing feeding. In greenhouse trials, Treacy et al. (1997) found a significant reduction in cotton flower bud damage by Heliothis virescens (F.) infected with vEGTDEL compared to those infected with wild-type AcMNPV. Moreover, O’Reilly and Miller (1991) recorded a 40% reduction in diet consumption by fifth instar S. frugiperda infected with vEGTDEL compared to those infected with AcMNPV. In this study, frass production was used as an indicator of larval feeding. The total amount of frass produced by larvae infected with vEGTDEL during the experimental period was found to be less than that produced by larvae infected with the wild-type virus. This was due solely to the fact that they died earlier. However, in this case, it was also found that the highest rates of frass production occurred earlier in those larvae infected with the deletion mutant, suggesting that feeding was enhanced earlier than for larvae infected with the wild-type virus and then declined earlier owing to rising levels of ecdysteroids. Slavicek et al. (1999) recommend using weight loss as an indicator of feeding cessation. They observed that fourth and fifth instar Lymantria dispar (L.) larvae infected with vEGT⫺ tended to gain weight earlier than larvae infected with wild-type LdMNPV, but stopped gaining weight or even lost weight before the latter group. Again, this suggests that feeding is enhanced earlier in larvae infected with the deletion mutant than larvae infected with wild-type LdMNPV. However, the cumulative weight gain in wild type and egt minus larvae differed only for female fourth and fifth instar larvae. Despite this, virus yields for female fourth and fifth instar larvae and male fifth instar larvae were significantly reduced for egt minus-infected larvae compared to wild type-infected larvae. O’Reilly and Miller (1991) observed an effect of feeding patterns on larval growth in fourth and fifth instar S. frugiperda larvae infected with vEGTZ, an egt deletion mutant containing the lacZ gene. These larvae fed for shorter periods after infection than those infected with wild-type AcMNPV and consequently gained less weight. To determine whether weight loss was related to OB yield, they also infected fifth instar S. frugiperda

with vEGTDEL and wild-type AcMNPV and found that those infected with vEGTDEL yielded 23% less progeny virus. In systems using genetically modified baculoviruses expressing insect-selective toxins, it has also been shown that an increased speed of kill results in a reduction in body mass and hence OB yield (Kunimi et al., 1996; Burden et al., 2000; Hernandez-Crespo et al., unpublished). Since these viruses do not have the additional benefit of inducing an ecdysteroid-dependent feeding cessation, it would be interesting to compare an egt deletion mutant with a toxin-expressing virus, to measure the effects on larval food consumption and OB yield. In summary, our results indicate that, in the absence of the egt gene, a decline in feeding causes a reduction in body weight which, in turn, leads to a diminished virus yield. Since yield is an important component of fitness (Anderson and May, 1981), the egt gene probably confers a selective advantage in field populations by indirectly promoting the virus yield and increasing the chance of secondary transmission to other larvae. However, field trials are required to examine the link between virus yield and secondary transmission. ACKNOWLEDGMENTS The authors thank Mr. Tim Carty for rearing the Trichoplusia ni larvae and preparing the diet used in the experiments. The work was supported by a Natural Environment Research Council non-thematic award GR3-A8967.

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