Effects of a protease-expressing recombinant baculovirus on nontarget insect predators of Heliothis virescens

Biological Control 28 (2003) 101–110 www.elsevier.com/locate/ybcon

Effects of a protease-expressing recombinant baculovirus on nontarget insect predators of Heliothis virescens Anthony J. Boughton,1 John J. Obrycki, and Bryony C. Bonning* Department of Entomology, Iowa State University, 407A Science II Building, Ames, IA 50011, USA Received 26 July 2002; accepted 23 January 2003

Abstract The baculovirus AcMLF9.ScathL expresses a basement membrane-degrading protease and represents a new class of recombinant baculovirus insecticides. Risk assessment studies were conducted according to the requirements of the US Environmental Protection Agency to investigate potential negative effects of consumption of Heliothis virescens larvae infected with AcMLF9.ScathL, on two common predators, the lacewing Chrysoperla carnea, and the ladybird beetle Coleomegilla maculata. Predators were reared on one of three feeding regimes consisting of H. virescens larvae that were uninfected or infected with AcMLF9.ScathL or AcMNPV C6. Control regimes consisted of Sitotroga cerealella eggs for C. carnea and Ostrinia nubilalis eggs and aphids for C. maculata. Survival of C. carnea fed Sitotroga eggs or AcMLF9.ScathL-infected H. virescens was significantly higher than for C. carnea fed H. virescens that were uninfected or infected with AcMNPV C6. There were no significant differences in development rates between C. carnea fed H. virescens infected with AcMNPV C6 or AcMLF9.ScathL. Baculoviruses ingested by C. carnea larvae remained viable within the digestive tract until adult emergence but had no detrimental effect on egg production. There was no evidence of adverse effects of AcMLF9.ScathL on C. maculata although this species exhibited low survival on diets composed exclusively of H. virescens. In choice tests, neither predator exhibited a preference between uninfected H. virescens and H. virescens infected with AcMNPV C6 or AcMLF9.ScathL. The data suggest that use of AcMLF9.ScathL in pest management would pose no greater risk to insect predators in the environment than use of the wild-type virus AcMNPV C6. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Recombinant baculovirus; Nucleopolyhedrovirus; Risk assessment; Nontarget effects; Insect predators; Coleomegilla maculata; Chrysoperla carnea

1. Introduction Advances in genetic engineering have made it possible to genetically manipulate wild-type baculoviruses to enhance their properties as insect pest management agents (Bonning et al., 2002; van Beek and Hughes, 1998). Genes encoding neurotoxins, enzymes, and insect peptide hormones have been engineered into baculovirus genomes to produce recombinant baculoviruses with increased speed of kill that reduce feeding damage caused by insect pests. Some of the most effective re-

* Corresponding author. Fax: 1-515-294-5957. E-mail address: [email protected] (B.C. Bonning). 1 Present address: Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA.

combinant baculoviruses express insect-specific neurotoxins (Smith et al., 2000a; Treacy et al., 2000; van Beek and Hughes, 1998). In susceptible hosts, these neurotoxin-expressing viruses reduce feeding damage and LT50 estimates by 25–50% relative to larvae infected with wild-type viruses (Cory et al., 1994; McCutchen et al., 1991; Stewart et al., 1991; Tomalski and Miller, 1992; Treacy and All, 1996). The United States Environmental Protection Agency (EPA) requires study of whether the release of genetically modified organisms, such as recombinant baculoviruses, will have detrimental impacts on nontarget organisms in the environment. For microbial pest control agents, nontarget predators are selected from two of the following groups: Hemiptera, Coleoptera, Neuroptera, and predaceous mites. For virus control agents, the species selected for testing must be known to attack the

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targeted host, or share the same ecological niche. If data generated by the maximum dose, single species hazard tests (Tier 1 testing) as described in this study indicate no adverse effects, the US EPA would not normally require further testing of nontarget arthropod predators. Owing to the host range of baculoviruses, which is restricted primarily to insects in the order Lepidoptera, adverse effects on insect predators, parasitoids, and pollinators will not result from virus infection of the nontarget organism (Huang et al., 1997; McNitt et al., 1995). However, it is possible that adverse effects could result from contact of the nontarget organism with the foreign protein within lepidopteran hosts infected with recombinant baculoviruses. To date, studies using hosts infected with recombinant baculoviruses expressing neurotoxins or juvenile hormone esterase have failed to detect any adverse effects on parasitoids (McCutchen et al., 1996; Smith et al., 2000b), scavengers (Lee and Fuxa, 2000), or predators (Heinz et al., 1995; Lee and Fuxa, 2000; Li et al., 1999; McNitt et al., 1995; Smith et al., 2000a) relative to impacts observed with hosts infected with wild-type viruses. Recently a new group of recombinant baculoviruses that express basement membrane-degrading enzymes has been developed (Harrison and Bonning, 2001). Basement membranes surround the tissues of all insects and vertebrates and constitute barriers to the dissemination of baculovirus infection within susceptible hosts (Hess and Falcon, 1987). By producing recombinant baculoviruses that express proteases that degrade basement membranes, it was hypothesized that systemic virus infections would be established more quickly within infected hosts, leading to increased speed of kill. The nucleopolyhedrovirus (NPV) of the alfalfa looper, Autographa californica (Speyer), (AcMNPV) was engineered to express a gene for a basement membrane-degrading cathepsin L protease (ScathL) derived from the flesh fly Sarcophaga peregrina Robineau-Desvoidy (Diptera: Sarcophagidae), to produce the virus AcMLF9.ScathL. Late instar Heliothis virescens F. (Lepidoptera: Noctuidae) larvae infected with AcMLF9.ScathL melanize prior to death. The lethal dose of AcMLF9.ScathL was not significantly different from that of the parental virus AcMNPV C6. In contrast, the survival time of AcMLF9.ScathL-infected larvae was reduced by 51%, and feeding damage caused by AcMLF9.ScathL-infected larvae was reduced by 80% when compared to those of AcMNPV C6-infected larvae (Harrison and Bonning, 2001). This paper examines the effects of consumption of H. virescens larvae infected with AcMLF9.ScathL or AcMNPV C6 on survival, development, and oviposition by two common insect predators, the green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), and the twelve-spotted lady beetle, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae) that feed

upon H. virescens in agricultural systems (Knutson and Ruberson, 1996; Lindgren et al., 1968). The question of whether predators exhibited a preference between H. virescens infected with AcMNPV C6, H. virescens infected with AcMLF9.ScathL, or uninfected larvae was also addressed.

2. Materials and methods 2.1. Viruses Construction of the recombinant baculovirus AcMLF9.ScathL has been described previously (Harrison and Bonning, 2001). AcMNPV clone C6 (Possee, 1986), the parental virus from which AcMLF9.ScathL was constructed, was used as the wild-type virus control. 2.2. Virus amplification and purification Virus was amplified by feeding 3-mm cubes of diet inoculated with 1
105 polyhedral occlusion bodies (POBs) of tissue-culture derived AcMLF9.ScathL to fifth instar H. virescens. Larvae that consumed the inoculated cube were provided with additional diet and maintained at 27 °C until death. POBs were then purified as described previously (Boughton et al., 1999). From this master stock of AcMLF9.ScathL, the amplification and purification procedures were repeated to produce a working stock of AcMLF9.ScathL that was used for all subsequent experiments. 2.3. Insects Green peach aphids, Myzus persicae (Sulzer) (Homoptera: Aphididae) were obtained from a colony maintained on Chinese cabbage plants in the lab at 28 °C, 80% RH. Eggs of the European corn borer, Ostrinia nubilalis (H€ ubner) (Lepidoptera: Crambidae) were obtained from USDA-ARS Corn Insects & Crop Genetics Research Unit, Ames, IA. Eggs of the tobacco budworm, H. virescens, were obtained from a colony maintained by Dr. T.C. Baker, Entomology Department, Iowa State University, Ames, Iowa and from USDA-ARS Southern Insect Management Laboratory, Stoneville, Mississippi. H. virescens were reared at 27 °C, 60% RH, on H. virescens soyflour diet (Southland Products, Lake Village, AR). Larvae of C. maculata and C. carnea were obtained from Rincon Vitova Insectaries (Ventura, CA) and were housed individually in 11-ml glass vials (1.5 cm diameter
9.0 cm) stoppered with clean cotton, and maintained at 28 °C, 80% RH. Prior to experiments, C. maculata were maintained on green peach aphids and O. nubilalis eggs, whereas C. carnea were maintained on eggs of the moth Sitotroga

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cerealella (Olivier) (Lepidoptera: Gelechiidae). All insects were maintained with a photoperiod of 14 h light, 10 h dark. 2.4. Survival studies Chrysoperla carnea larvae were reared to the third instar on Sitotroga eggs and were assigned to one of four feeding regimes: (1) Sitotroga eggs; (2) mock-inoculated H. virescens larvae; (3) H. virescens larvae infected with AcMNPV C6; or (4) H. virescens larvae infected with AcMLF9.ScathL. C. carnea larvae on the Sitotroga egg regime were fed every 2 days. C. carnea larvae reared on the H. virescens regimes were fed daily with two second instar H. virescens that had been inoculated 48 h previously using a diet cube inoculation technique. H. virescens larvae were infected with AcMNPV C6 or AcMLF9.ScathL or were mock-inoculated. Second instar H. virescens were transferred into individual wells of 48-well tissue-culture plates (Fisher Scientific) containing 3-mm cubes of soyflour diet onto which had been pipetted 1 ll aliquots of distilled water and 4% Schilling blue food coloring dye (McCormick, Hunt Valley, MD), containing 100
LC50 doses of AcMNPV C6 (5900 POBs/diet cube) or AcMLF9. ScathL (5900 POBs/diet cube). Mock inoculations were carried out with distilled water and food coloring dye alone. H. virescens larvae were confined to wells using Parafilm (American National Can, Menasha, WI). Plates were maintained for 48 h before H. virescens larvae were used in the feeding trials. Twenty-five C. carnea larvae were used per feeding regime for each replicate. Development and survival of C. carnea larvae in the different feeding regimes were monitored daily and trials were continued until all C. carnea larvae had either died or emerged as adults. Second instar C. maculata larvae were assigned to one of four feeding regimes: (1) aphids and O. nubilalis eggs; (2) mock-inoculated H. virescens larvae; (3) H. virescens larvae infected with AcMNPV C6; or (4) H. virescens larvae infected with AcMLF9.ScathL. C. maculata larvae on the aphid and O. nubilalis egg regime were fed every 3 days with two or three O. nubilalis egg masses and a small piece of Chinese cabbage leaf on which were 7–10 aphids. C. maculata larvae reared on the H. virescens regimes were fed daily with first instar H. virescens that had been inoculated previously using a droplet feeding technique (Hughes and Wood, 1981) with a mock solution of distilled water and 4% food coloring dye, or suspensions of distilled water and food dye containing 100
LC50 doses of AcMNPV C6 (11,200 POBs/ll) or AcMLF9.ScathL (13,300 POBs/ ll). Larvae that ingested the colored suspensions were transferred into 240-ml cups containing artificial diet, and maintained for 24 h before being used in the feeding studies. In the first replicate of the survival studies, 40

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C. maculata larvae were used per feeding regime, and C. maculata larvae were fed 2–4 mock-inoculated, AcMNPV C6-infected or AcMLF9.ScathL-infected H. virescens larvae per day. To increase prey availability in the second replicate of the survival studies, only 20 C. maculata larvae were used per feeding regime, and to compensate for differential H. virescens sizes, C. maculata larvae were fed 5–7 mock-inoculated, 6–8 AcMNPV C6-infected, or 7–9 AcMLF9.ScathL-infected H. virescens larvae per day. Development and survival of C. maculata larvae were monitored daily and trials were continued until all individuals had either died or emerged as adult beetles. 2.5. Virus transmission Studies were performed to determine if recombinant baculovirus ingested by predator larvae and subsequently excreted from adult predators would retain sufficient activity to initiate infections in susceptible hosts. Because significant numbers of C. maculata failed to survive to the adult stage, virus transmission studies were restricted to C. carnea. Larval lacewings do not defecate, but materials ingested during the larval stage are stored and subsequently voided shortly after adult emergence as a meconium (Castillejos et al., 2001). Meconial pellets were collected at the end of the survival studies from adult lacewings that emerged from larvae reared on the four different feeding regimes. Pellets selected at random from the different regimes were individually resuspended in 200 ll of distilled water containing 4% blue food coloring dye. Aliquots ð2 llÞ of these meconial suspensions were applied to 3-mm cubes of soyflour diet, which were subsequently fed to second instar H. virescens. Larvae that consumed more than two thirds of their diet cube were transferred onto fresh diet and were maintained for 7 days before virus-induced mortality was scored. Cuticular lysis of cadavers was used as an indicator of virus-induced mortality. Twenty H. virescens larvae were inoculated with aliquots from each meconial pellet. Bioassays were performed on three meconial pellets from each of the four feeding regimes in both replicates of the C. carnea survival studies. 2.6. Oviposition studies Adults that developed from lacewing larvae reared on the four different feeding regimes were used to establish mating pairs. Pairs were housed in 240-ml cups covered by pieces of fine mesh netting (Consoltex-Seatex, New York, NY) and provided with distilled water and a 1:1 mixture of honey and wheat (Qualcepts, Minneapolis, MN). Containers were examined daily. Following the onset of oviposition, eggs were counted and removed daily for 21 days.

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2.7. Choice tests Melanization of AcMLF9.ScathL-infected larvae prior to death raised the question of whether these larvae would be accepted as prey by predators. Choice tests were conducted to address whether uninfected or AcMNPV C6-infected H. virescens were preferred as prey by lacewing larvae or ladybeetle larvae relative to H. virescens infected with AcMLF9.ScathL. Experiments were performed in the laboratory at a temperature of 23–26 °C, with lighting provided by fluorescent ceiling lights. Experimental arenas consisted of 90-mm petri dishes that were screened from adjacent arenas and lateral light sources by white cardboard. Choice tests with C. carnea used third instar larvae and second instar H. virescens, while choice tests with C. maculata used third instar larvae and first instar H. virescens. The H. virescens prey used in the choice tests were mockinoculated or virus-inoculated as described above for survival studies. For choice tests, the H. virescens receiving the AcMNPV C6 or AcMLF9.ScathL treatments were placed onto diet containing 2% blue or 2% red food coloring dyes, respectively, following virus inoculation. After feeding on colored diets for 24 h (C. maculata studies) or 48 h (C. carnea studies), H. virescens from the mock-inoculated, AcMNPV C6-inoculated, or AcMLF9.ScathL-inoculated treatments were easily distinguished by gut content color. For the choice tests, a single predator was introduced into the center of a petri dish into which had previously been introduced three H. virescens larvae—one uninfected, one AcMNPV C6-infected, and one AcMLF9.ScathL-infected. Each arena was observed for 24 min and the prey type of the first H. virescens attacked was recorded and used as a measure of prey preference. Following attacks, additional prey larvae were added to arenas to maintain the same overall prey proportions. Predators, prey, and arenas were used only once. Experimental arenas containing prey larvae and a single predator were set up at 2 min intervals and were run in batches of 12. This was repeated four times for a total of 48 observations for each of the two predator species. In addition, to test for predator preferences for prey of particular colors, AcMNPV C6 and AcMLF9.ScathL were omitted from experimental treatments and the studies repeated using only distilled water and food coloring dyes. 2.8. Statistical analyses Differences in the response variables were examined between replicates and across feeding regimes using analysis of variance (GLM or ANOVA) (SAS-Institute, 1990). To normalize the data and stabilize the variance, the response variables were squareroot transformed before analysis. Reported means and standard errors of the mean (SE) were back-transformed following analy-

sis. Differences in mean response values between feeding regimes, were examined using TukeyÕs means separation test. Mean daily egg production was calculated for egg production during the 21 day observation period. Predator preference for prey of a particular color or virus-infection condition was assessed using v2 tests assuming a null hypothesis of no preference among categories.

3. Results 3.1. Survival studies The proportions of C. carnea larvae surviving to the adult stage on the different feeding regimes were not significantly different between replicates (F ¼ 0.00; df ¼ 1, 3; P ¼ 0.992); therefore, survival data from the two replicates were pooled. One-way ANOVA indicated that feeding regime explained a significant amount of the variation seen in survival (F ¼ 52.44; df ¼ 3, 4; P ¼ 0.001). TukeyÕs means separation test indicated that survival to the adult stage was significantly higher in C. carnea reared on Sitotroga eggs or AcMLF9.ScathLinfected H. virescens than C. carnea reared on mock-inoculated or AcMNPV C6-infected H. virescens (Fig. 1A). There were no significant differences in survival between C. carnea reared on Sitotroga eggs or AcMLF9.ScathLinfected H. virescens or between C. carnea reared on mock-inoculated or AcMNPV C6-infected H. virescens (Table 1). Significant replicate effects were observed in the time taken for C. carnea larvae to reach the pupal stage (F ¼ 209.69; df ¼ 1, 176; P ¼ 0.000) and the adult stage (F ¼ 186.58; df ¼ 1, 151; P ¼ 0.000), so data from the two replicates were analyzed separately (Table 1). C. carnea larvae in the second replicate of the feeding trials reached the pupal and adult stage more quickly than in the first replicate. Feeding regime accounted for a significant amount of the variation observed in time taken to reach the pupal stage (F ¼ 17.65; df ¼ 3, 89; P ¼ 0.000) and adult stage (F ¼ 10.68; df ¼ 3, 74; P ¼ 0.000) in the first replicate. Feeding regime also accounted for significant variation observed in time to pupation (F ¼ 8.51; df ¼ 3, 84; P ¼ 0.000) and time to adult (F ¼ 9.48; df ¼ 3, 74; P ¼ 0.000) in the second replicate. In general, C. carnea on the Sitotroga regime pupated and reached the adult stage significantly faster than C. carnea reared on the three H. virescens regimes. The proportions of C. maculata larvae surviving to the adult stage on the different feeding regimes were not significantly different between replicates (F ¼ 2.98; df ¼ 1, 3; P ¼ 0.183); therefore, survival data from the two replicates were pooled. Feeding regime had a significant effect on survival (F ¼ 24.99; df ¼ 3, 4; P ¼ 0.005). TukeyÕs means separation test indicated that survival to the adult

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Fig. 1. Survival over time of Chrysoperla carnea (A) and Coleomegilla maculata (B) fed on four different feeding regimes. Data are presented until the point of adult emergence, when feeding was discontinued. Mean survival proportions calculated from two replicates. Twenty-five C. carnea were used per feeding regime per replicate with 40 and 20 C. maculata per feeding regime in the first and second replicates, respectively. Bars show standard errors.

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Table 1 Developmental times and survival proportions for Chrysoperla carnea reared on AcMLF9.ScathL-infected Heliothis virescens larvae, or control feeding regimesa Feeding regime

Developmental timesb Replicate 1

Sitotroga eggs H. virescens Mock H. virescens AcMNPV C6 H. virescens AcMLF9.ScathL

Proportion survivingc  SE

Replicate 2

Pupation  SE

Adult  SE

Pupation  SE

Adult  SE

6:5  0:01 A 9:3  0:00 B 9:8  0:00 B

15:2  0:00 A 17:8  0:00 B 18:1  0:00 B

3:9  0:00 A 5:0  0:00 AB 6:2  0:01 B

12:2  0:00 A 13:4  0:00 AB 14:6  0:00 B

0:94  0:00 A 0:62  0:00 B 0:70  0:00 B

9:2  0:00 B

17:3  0:00 B

5:2  0:01 B

13:6  0:00 B

0:86  0:00 A

a

Means and SE are back-transformed from squareroot transformed data. Within columns, different letters indicate means that are significantly different by TukeyÕs means separation test at the 5% significance level. b Mean time (days) to pupation or adult. Significant differences were present between replicates, so data were analyzed separately. 25 C. carnea per feeding regime in both replicates. c Mean proportion of C. carnea larvae surviving to adult. Table 2 Survival times and survival proportions for Coleomegilla maculata reared on AcMLF9.ScathL-infected Heliothis virescens larvae, or control feeding regimes Feeding regime

Mean time to death  SE (days)

Mean proportion surviving to adult  SE

O. nubilalis eggs and aphids H. virescens Mock H. virescens AcMNPV C6 H. virescens AcMLF9.ScathL

—

18:8  0:01 A 13:3  0:01 B 12:3  0:01 B

0:75  0:02 0:01  0:01 0:00  0:00 0:00  0:00

A B B B

There were no significant differences between replicates, so data were pooled. Forty and 20 C. maculata per feeding regime in the first and second replicates, respectively. Means and SE are back-transformed from squareroot transformed data. Within columns, different letters indicate means that are significantly different at the 5% significance level by TukeyÕs means separation test.

stage was significantly higher in C. maculata larvae fed on O. nubilalis eggs and aphids than in C. maculata fed on the three H. virescens regimes (Table 2). There were no significant differences in survival between C. maculata reared on the three H. virescens regimes. The mean times to death for C. maculata reared on the three H. virescens regimes did not differ between replicates (F ¼ 2.27; df ¼ 1, 174; P ¼ 0.133); therefore the time to death data were pooled. Feeding regime had a significant effect on time to death (F ¼ 21.86; df ¼ 2, 175; P ¼ 0.000). TukeyÕs means separation test showed that C. maculata fed virus-infected H. virescens died significantly earlier than those fed mockinoculated H. virescens (Table 2, Fig. 1B). 3.2. Virus transmission No significant differences in virus-induced H. virescens mortality were observed between replicates (F ¼ 2.70; df ¼ 1, 19; P ¼ 0.116); therefore, data from the two replicates were pooled. One-way ANOVA revealed significant differences in virus-induced mortality attributable to the feeding regime of the larval lacewings from which the meconial pellets were obtained (F ¼ 504.83; df ¼ 3, 20; P ¼ 0.000) (Table 3). Suspensions of meconial pellets obtained from lacewing adults reared as larvae on Sitotroga eggs or mock-inoculated H. virescens, caused low levels (<1%) of virus-induced

Table 3 Mortality of Heliothis virescens larvae inoculated with suspensions of meconial pellets from lacewings fed on AcMLF9.ScathL-infected H. virescens larvae or control feeding regimesa Pellet sourceb

Percentage H. virescens mortalityc  SE

Sitotroga regime Mock regime AcMNPV C6 regime AcMLF9.ScathL regime

0:6  0:25 0:0  0:00 100:0  0:00 100:0  0:00

A A B B

a

Two replicates. Three meconial pellets from each feeding regime bioassayed in each replicate. Each pellet bioassayed in 20 H. virescens larvae. There were no significant differences between replicates and the data were pooled. Means and SE are back-transformed from squareroot transformed data. Within columns, different letters indicate means that are significantly different at the 5% significance level by TukeyÕs means separation test. b Meconial pellets obtained from lacewing adults reared as larvae on one of the four feeding regimes. c Virus-induced mortality observed in H. virescens larvae inoculated with meconial pellet suspensions.

mortality when bioassayed in H. virescens larvae (Table 3). Suspensions of meconial pellets obtained from lacewing adults reared as larvae on AcMNPV C6-infected or AcMLF9.ScathL-infected H. virescens larvae, caused virus-induced mortality in 100% of the H. virescens larvae used in bioassays.

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3.3. Oviposition studies Due to low numbers of C. carnea mating pairs established from certain larval feeding regimes, data from the two replicates were combined prior to analysis. Oneway ANOVA indicated that larval feeding regime did not have a significant effect on time to onset of ovipo-

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sition (F ¼ 2.31; df ¼ 3, 30; P ¼ 0.096) or mean daily egg production (F ¼ 0.37; df ¼ 3, 30; P ¼ 0.774) among lacewing females established from C. carnea larvae reared on the four different feeding regimes (Table 4). All females produced viable eggs, but percentage viability of eggs from females reared on the different larval feeding regimes was not determined.

Table 4 Time to onset of oviposition and mean daily egg production for Chrysoperla carnea maintained on AcMLF9.ScathL-infected Heliothis virescens or control feeding regimes Feeding regime

Number of mating pairs

Mean time to oviposition  SE (days)

Mean daily egg production  SE

Sitotroga eggs H. virescens Mock H. virescens AcMNPV C6 H. virescens AcMLF9.ScathL

13 8 7 6

5:2  0:00 6:2  0:00 6:5  0:04 4:8  0:02

8:6  0:01 9:0  0:07 10:3  0:38 11:1  0:03

A A A A

A A A A

Due to low numbers of mating pairs, data from two replicates were pooled. Means and SE are back-transformed from squareroot transformed data. Within columns, different letters indicate means that are significantly different at the 5% significance level by TukeyÕs means separation test.

Fig. 2. Histograms showing frequency of first attack in color preference (A, C) and virus preference (B, D) prey choice tests for Chrysoperla carnea (A, B) and Coleomegilla maculata (C, D), respectively. Histograms are based on observations of 48 C. carnea attacks for Figs. 1A and B; 35 and 30 C. maculata attacks for Figs. 1C and D, respectively. Columns topped with the same letter are not significantly different at the 5% significance level ðP > 0:05Þ by v2 test.

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3.4. Choice tests Data were collected on the first and subsequent prey items attacked during each 24 min observation period. Because analyses of total numbers of prey items attacked and the first prey item attacked both showed lack of preference, only data on first attacks are presented. All C. carnea larvae attacked at least one H. virescens prey larva during the observation period. No preference for H. virescens prey reared on the different colored diets was detected (v2 ¼ 0:13; df ¼ 2; P ¼ 0.94) (Fig. 2A). C. carnea larvae exhibited no preference for prey that were uninfected relative to those that were infected with AcMNPV C6 or AcMLF9.ScathL (v2 ¼ 0:37; df ¼ 2; P ¼ 0.83) (Fig. 2B). In tests for color preference, C. maculata larvae in 35 of the 48 arenas attacked H. virescens larvae during the observation period. No significant preference for H. virescens prey reared on the different colored diets was detected (v2 ¼ 4:34; df ¼ 2; P ¼ 0.114) (Fig. 2C). In tests for virus preference, C. maculata larvae in 30 of the 48 arenas attacked H. virescens larvae. C. maculata had no significant preference for prey that were uninfected relative to those that were infected with AcMNPV C6 or AcMLF9.ScathL (v2 ¼ 2:40; df ¼ 2; P ¼ 0.30) (Fig. 2D).

4. Discussion 4.1. Survival studies Chrysoperla carnea and C. maculata are two predators that contribute to regulation of populations of H. virescens larvae in agricultural systems, and consequently were chosen as nontarget organisms for evaluation in this study. As per US EPA guidelines, C. maculata and C. carnea were fed on pest individuals infected with 100
LC50 doses of the recombinant baculovirus AcMLF9.ScathL. At these dose levels, 50% of first instar H. virescens infected with AcMLF9.ScathL were dead by approximately 48 h postinfection (Harrison and Bonning, 2001) and 40% of infected second instars were dead by 72 h postinfection (data not shown). To ensure that prey infected with AcMLF9.ScathL would still be alive during the feeding studies, first instar H. virescens were used 24 h postinfection and second instars at 48 h postinfection. Use of H. virescens larvae infected with high doses of AcMLF9.ScathL in no-choice feeding studies, simulated a worst case scenario in which C. maculata and C. carnea were exposed to maximum possible levels of the recombinant protein ScathL, as per US EPA Tier 1 testing requirements. The survival rates to the adult stage for C. carnea reared on Sitotroga eggs or AcMLF9.ScathL-infected H. virescens were significantly higher than for C. carnea larvae reared on mock-inoculated or AcMNPV C6-in-

fected H. virescens. High survival of C. carnea reared on Sitotroga eggs was expected. However, elevated survival of C. carnea reared on H. virescens infected with AcMLF9.ScathL relative to C. carnea reared on the two other H. virescens regimes, was unexpected. The significantly higher survival of C. carnea larvae on the AcMLF9.ScathL regime may be due to a combination of basement membrane damage in infected H. virescens larvae, and the unusual feeding mechanism used by lacewing larvae. Lacewing larvae use extra-oral digestion in which prey are impaled by hollow, sickle-shaped mouthparts through which is then injected a mixture of digestive enzymes. These enzymes begin to digest the internal organs of the prey, and the resulting digestive soup is sucked up through the mandibles into the lacewing stomach (Cohen, 1998). Degradation of basement membranes by ScathL in AcMLF9.ScathL-infected H. virescens larvae may enhance the efficiency of extraoral digestion, leading to elevated survival of C. carnea reared on AcMLF9.ScathL-infected H. virescens larvae. Significant differences were apparent between replicates in times taken for lacewing larvae to reach the pupal and adult stages. However, despite these differences, the same trends in developmental times were seen within replicates, with lacewings reared on the three H. virescens regimes typically taking 2–3 days longer to pupate and subsequently emerge as adults than lacewings reared on Sitotroga eggs. The shorter developmental times observed in the second replicate of the C. carnea feeding trials may have resulted from the use of older lacewing larvae in the second replicate. Oviposition studies revealed no significant differences in time to onset of oviposition or mean daily egg production between lacewing pairs reared on the four different larval feeding regimes, and all mating pairs produced viable eggs. Heinz et al. (1995) studied C. carnea that were fed H. virescens infected with a recombinant baculovirus expressing an insect-specific neurotoxin (AcAaIT), and found development times and survival rates were not significantly different from those for C. carnea fed H. virescens infected with wild-type AcNPV. Parameter estimates for that study were similar to those documented here. Seventy-five percent of C. maculata larvae fed on O. nubilalis eggs and aphids completed development to the adult stage, whereas 99% of C. maculata larvae fed on the H. virescens regimes died as larvae. C. maculata larvae readily consumed H. virescens during the survival studies, and some C. maculata larvae survived for 4.5 weeks when fed H. virescens larvae. It is likely that the low survival of C. maculata fed an exclusive diet of H. virescens resulted from nutritional deficiencies. This suggestion is supported by data from a previous study (Li et al., 1999) in which adults of the ladybird beetle, Hippodamia convergens Guerin-Meneville were maintained on a diet composed of H. virescens larvae, and

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exhibited similar declines in survival over time to those seen in our study. Beetle larvae fed virus-infected H. virescens died sooner than those fed mock-inoculated H. virescens, possibly due to lower nutritional quality of virus-infected prey. Previous studies have documented adverse effects on survival, longevity, and fecundity of predators and scavengers fed baculovirus-infected lepidopteran larvae relative to those fed lepidopteran larvae that had been mock-inoculated (Lee and Fuxa, 2000; Ruberson et al., 1991). Nevertheless, there was no evidence of any adverse effects of AcMLF9.ScathL because C. maculata fed H. virescens infected with AcMLF9.ScathL survived for the same length of time as those fed H. virescens infected with wild-type AcMNPV C6. 4.2. Virus transmission In transmission studies, inoculation of susceptible lepidopteran larvae with suspensions of meconial pellets from adult lacewings fed as larvae on virus-infected H. virescens larvae, subsequently caused virus-induced mortality of 100% of the inoculated test larvae. In contrast, test larvae inoculated with suspensions of meconial pellets from adult lacewings reared as larvae on Sitotroga eggs or uninfected H. virescens exhibited less than 1% virus-induced mortality, which was likely due to viral cross contamination. These results clearly demonstrate that virus ingested by lacewing larvae remains active within the digestive tract at least until adult emergence. We did not address whether virus remained within the digestive tract of adult lacewings after voiding of meconial pellets. Castillejos et al. (2001) studied Chrysoperla rufilabris (Burmeister) to determine whether Spodoptera frugiperda, multicapsid nucleopolyhedrovirus (SfMNPV) was viable after passage through the lacewing gut but failed to detect the presence of viral occlusions in fecal meconial pellets either by microscopic examination or by bioassay of suspensions of meconial pellets in larvae of a susceptible host. However, in these studies, C. rufilabris were only fed virus-infected prey for a few days before being supplied with a diet of uninfected prey. As such the quantities of SfMNPV ingested by C. rufilabris would have been considerably lower than the quantities of AcMNPV C6 or AcMLF9.ScathL ingested by C. carnea in our study.

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subsequent timing of onset of oviposition in adult female lacewings or on the daily egg production observed during the first 21 days of oviposition. 4.4. Choice tests Late instar H. virescens infected with AcMLF9. ScathL undergo melanization prior to death (Harrison and Bonning, 2001). Since the products of melanization include quinones that are toxic to insect cells (Ashida and Brey, 1997), this raised the question of whether H. virescens larvae infected with AcMLF9.ScathL would be less palatable to predators than H. virescens larvae infected with AcMNPV C6 or larvae that were uninfected. First and second instar H. virescens infected with AcMLF9.ScathL did not melanize completely prior to death but did exhibit some dark mottling, indicating low levels of melanization. In prey choice tests, neither C. maculata nor C. carnea exhibited a preference for mock-inoculated or AcMNPV C6-infected H. virescens relative to H. virescens larvae infected with AcMLF9.ScathL. Lee and Fuxa (2000) examined food preference in two species of scavenging insects, the house cricket, Acheta domesticus (L.) and the fly, S. bullata, but found no preference for cadavers of lepidopteran larvae killed by low temperatures relative to cadavers killed by wild-type or recombinant baculoviruses. In summary, no detrimental impacts on mean survival rates or development times were detected between C. maculata and C. carnea fed AcMLF9.ScathL-infected prey relative to those fed AcMNPV C6-infected prey. Production and dissemination of AcMLF9.ScathL from infected hosts are lower than from hosts infected with AcMNPV C6 (Harrison, personal communication), and neither C. maculata nor C. carnea exhibited a preference for prey infected with AcMLF9.ScathL compared to AcMNPV C6. Thus, given that neither detrimental effects nor increased exposure are expected, we can conclude that the use of AcMLF9.ScathL as a biological insecticide within agricultural systems would pose no greater risk to C. maculata and C. carnea than would the use of the wild-type virus AcMNPV C6 (Richards et al., 1998). The use of recombinant baculoviruses for insect pest management would have significantly less impact on nontarget organisms than would the use of broad spectrum chemical insecticides.

4.3. Oviposition studies Acknowledgments A great deal of variation in daily egg production was seen among C. carnea females, but this variability in mean daily egg production was just as large among females reared on the same larval feeding regime as for females reared on different larval feeding regimes. Consumption of H. virescens infected with AcMLF9.ScathL by larval C. carnea had no effect on the

The authors thank Dr. Robert Harrison for helpful discussion. This material is based upon work supported by the Cooperative State Research, Education, and Extension Service, US Department of Agriculture, under Agreement No. 00-39210-9772 as well as Hatch Act and State of Iowa funds.

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