Interactions between the ParasitoidAmetadoria misella(Diptera: Tachinidae) and the Granulovirus ofHarrisina brillians(Lepidoptera: Zygaenidae)

Biological Control 14, 146–151 (1999) Article ID bcon.1998.0682, available online at http://www.idealibrary.com on

Interactions between the Parasitoid Ametadoria misella (Diptera: Tachinidae) and the Granulovirus of Harrisina brillians (Lepidoptera: Zygaenidae) Diane M. Stark, Nicholas J. Mills, and Alexander H. Purcell Division of Insect Biology, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112 E-mail: [email protected] Received April 22, 1998; accepted October 22, 1998

Interactions between a granulovirus (HbGV), a tachinid parasitoid Ametadoria misella, and their host, the western grapeleaf skeletonizer Harrisina brillians, were investigated. In field populations, the occurrence of A. misella in HbGV-infected H. brillians pupae was less frequent than would have been expected by random assortment of the virus and the parasitoid. Furthermore, enzyme-linked immunosorbent assay detected granulovirus less frequently and in lower concentrations in parasitized pupae than in nonparasitized pupae. Finally, in the host pupae that tested positive for virus, parasitoids were more likely to survive pupation than hosts. When laboratory-reared H. brillians larvae were exposed to naturally occurring A. misella in a field experiment, the parasitoid oviposited more often in older than in younger host larvae and more often in healthy than in HbGVinfected host larvae. These results are consistent with the hypothesis that selective oviposition by A. misella leads to reduced overlap of the parasitoid and HbGV in hosts, resulting in greater parasitoid survival. r 1999 Academic Press

Key Words: Harrisina brillians granulovirus; baculovirus; biological control; microbial control; host– parasitoid–pathogen interaction; interspecific competition; grape.

INTRODUCTION

Baculoviruses offer an attractive alternative to synthetic pesticides for pest suppression. Understanding the complex interactions that occur between viruses and other species in natural populations will be key to improving their efficacy as biological control agents and assessing their risks as genetically modified pesticides. Within individual hosts, the interaction between baculoviruses and parasitoids has been well explored. In general, the outcome of competition depends upon the 1049-9644/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

timing of parasitoid attack relative to baculovirus infection. Few parasitoids were able to develop successfully in hosts already infected at the time of parasitoid attack (reviewed by Brooks, 1993). Given the cost of competition, a number of parasitoids exhibit some degree of avoidance of virus-infected hosts (Versoi and Yendol, 1982; Caballero et al., 1991) although others appear not to (Beegle and Oatman, 1975; Levin et al., 1983). At the population level, investigations of host– parasitoid–virus interactions have been scant, but also suggest that patterns of host exploitation are potentially important determinants of host regulation. From a theoretical perspective, the coexistence of natural enemies may be enhanced by the balance between baculovirus superiority within hosts and parasitoid superiority in searching for and exploiting hosts (e.g., though foraging) in the environment (Hochberg et al., 1990). However, using a laboratory system, Begon et al. (1996) found that host populations were destabilized and frequently became extinct in the presence of both baculovirus and parasitoid. The destabilizations, in this simple system, resulted from the very different distributions of attack by the two natural enemies in relation to the age of the host, which combined to act as a single conglomerate natural enemy throughout the larval stage of the host. The western grapeleaf skeletonizer, Harrisina brillians (Barnes & McDunnough) (Lepidoptera: Zygaenidae), and its two main natural controls in California, a granulovirus (HbGV) and a tachinid parasitoid, Ametadoria misella (Wulp) (Diptera: Tachinidae), offer an interesting opportunity for studying host–parasitoid– pathogen interactions. HbGV is considered one of the best examples of successful classical biological control with an insect virus (Federici, 1993). Long-term suppression of H. brillians was observed where both natural enemies were established. This led to speculation that the natural enemies were complementary (Clausen, 1961). Unlike viruses previously considered in

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host–parasitoid–pathogen studies, replication of HbGV is apparently restricted to the host midgut epithelium, and infectious granules are continuously shed via diarrhea (Federici and Stern, 1990). HbGV is more infectious but less virulent (Stark, unpublished data) than most nucleopolyhedroviruses (NPVs). Infected larvae are slow to die and may survive to become infected adults (Stern and Federici, 1990; Stark et al., 1999). This pathology may provide a greater window of opportunity for parasitoid development because A. misella requires that its host reach the pupal stage in order to develop beyond the first instar (Smith et al., 1955). This study investigated the interaction of HbGV and A. misella in field populations. First, H. brillians pupae were sampled to determine the extent of overlap of A. misella and HbGV attack and the consequential survival of A. misella. Second, possible avoidance of competition based on selective oviposition of the parasitoid was investigated in a field experiment. We tested the hypothesis that A. misella parasitizes uninfected H. brillians larvae more often than HbGV-infected hosts and parasitizes older hosts more often than younger hosts. Unlike early-instar H. brillians larvae, lateinstar larvae are able to survive HbGV infection into the pupal and adult stage. Thus, the selection of late-instar larvae by A. misella would increase the probability of parasitoid larvae being able to complete their development in infected hosts. MATERIALS AND METHODS

Virus The original source of H. brillians granulovirus (HbGV) used in these studies was from a vial of lyophilized diseased larvae provided by V. Stern and B. Federici (University of California at Riverside, CA). HbGV was propagated in field-collected H. brillians larvae. Granules from infected insects were purified as described previously (Stark et al., 1999). Briefly, HbGV infected larvae were homogenized with a Polytron (Brinkman, Cottingham, UK) in 0.1% sodium dodecyl sulfate (SDS) or phosphate-buffered saline (PBS, pH 7.4), and the homogenate was filtered through Miracloth (Calbiochem, San Diego, CA). The filtrate was centrifuged at 400g for 5 min to remove debris, and the resulting supernatant was centrifuged at 10,000 g for 30 min to concentrate the granules. Granules were isolated from the pellet by centrifugation for 40 min at 12,000 g on a 30–80% (vol%) continuous glycerol gradient, followed by centrifugation for 60 min at 50,000 g on a 45–60% (wt%) continuous sucrose gradient. Purified granules were suspended in PBS (pH 7.0). The concentration of granules was determined by smearing 5 µl of virus preparation onto a 15-mm-diameter circle on a microscope slide, staining with Buffalo Black 12B, and counting the number of granules using a standard

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sampling regime according to the method of Wigley (1980). Harrisina brillians Attempts to rear H. brillians on artificial diet have been largely unsuccessful. Therefore, eggs laid by apparently healthy H. brillians adults (Stern and Federici, 1990) were collected from a commercial vineyard near the town of Del Rey, California, in the summer of 1995. They were surface sterilized in 0.1% sodium hypochlorite for 1–1.5 min and rinsed with sterile water. Cohorts were transferred to 1-liter plastic food containers. After 10 min exposure to UV light (Sterilamp No. 615TB; Westinghouse, Pittsburgh, PA) at a distance of 30 cm, grapeleaf bouquets from Thompson Seedless vines grown in a greenhouse isolated from virus were added to the containers which were subsequently sealed with Sterigauze (Kimberly–Clark, Dallas, TX). Larvae were reared under continuous light to prevent diapause and under temperatures ranging from 17 to 24°C as necessary to synchronize development of different cohorts. Individuals from each cohort at the appropriate stage of development were randomly assigned to treatments. Enzyme-Linked Immunosorbent Assay (ELISA) A double antibody sandwich ELISA employing polyclonal antibodies to HbGV was used to detect virus in field samples. The ELISA detected as little as 10 ng of purified HbGV, detected infections in H. brillians larvae as early as 72 h after infection, and reliably discriminated between healthy and HbGV-infected H. brillians larvae, pupae, and adults (Stark et al., 1999). The ELISA was performed as described by Clark and Adams (1977) with modifications (Stark et al., 1999). Samples were prepared for ELISA by homogenizing insects separately in 1.5-ml microcentrifuge tubes with plastic pestles after addition of 500 µl PBS-T-A buffer (phosphate-buffered saline, 0.05% Tween 20, 0.02% NaN3, pH 7.2). Standard controls, from frozen stock solutions, of (1) PBS-T-A, (2) purified HbGV in PBS-T-A (100 ng viral protein/well), and (3) healthy H. brillians larvae homogenated in PBS-T-A diluted as per unknowns were tested in duplicate wells on each plate. All samples from the same study were assayed together and, where possible, on the same plate. Twice the mean absorbance of the healthy H. brillians control was used as a threshold for positive readings. Prevalence of HbGV in Parasitized and Nonparasitized Host Pupae A localized outbreak of H. brillians occurred in a vineyard near Del Rey, California, in 1995. The outbreak spread from a small patch in the vineyard at the beginning of the season to cover one-quarter of the vineyard by the end of the season. Pupae were collected late in the season to determine (1) if parasitism by A.

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misella was independent of HbGV infection in H. brillians pupae, (2) if the frequency and severity of HbGV infection was greater in nonparasitized H. brillians than those parasitized by A. misella, and (3) if distance from the center of an outbreak of H. brillians affected the frequency of infection by HbGV. Corrugated cardboard bands were stapled to the trunks of eight vines at distances of 110–230 m from the center of the initial patch of the outbreak to trap host larvae searching for pupation sites on the trunk of the vines. After all larvae had pupated, the bands were collected and stored in separate paper bags at 4°C. Pupae were removed from bands individually, taking precaution to avoid cross contamination by virus. They were examined for signs of parasitism, as indicated by the presence of a parasitoid respiratory siphon, and assayed for presence and concentration of HbGV by ELISA. Comparative Survival of Parasitoids and Hosts Naturally occurring populations of H. brillians were sampled in 1992 to determine the relative survival rates of A. misella and H. brillians in a host population infected by HbGV. Third-generation H. brillians pupae were collected from a commercial vineyard near the town of Reedley, California, where H. brillians, A. misella, and HbGV were present. The vineyard was managed for organic table grape production and had been treated with Bacillus thuringiensis Berliner during the first, but not second or third generation of H. brillians. Pupating larvae were trapped in corrugated cardboard bands stapled to the trunks of 14 vines: 2 adjacent vines in seven systematically spaced locations. After collection from the field, the bands were stored in paper bags in a cold room (4°C) for approximately 3 months to allow completion of diapause. Preliminary studies had shown that virtually all healthy H. brillians pupae treated in this way emerged as adults. For each pair of adjacent vines, one was used to estimate the percentage of parasitism by A. misella, by destructive examination of host pupae for the presence of a parasitoid respiratory siphon, and the other to estimate the percentage of emergence of adult A. misella and H. brillians at 25°C and 16 h daylength after cold storage. It was assumed that the hosts on adjacent vines had the same percentages of parasitism and viral infection. The percentage of parasitoids surviving pupation in the presence of virus was estimated from the percentage of emergence of A. misella (No. emerged/total pupae) as determined from the one set of vines divided by the percentage of host pupae that were parasitized (No. parasitized pupae/total pupae) as determined from the other set of vines. The corresponding percentage of survival of host pupae that were not parasitized was estimated from the percentage of emergence of H. brillians (No. emerged/total pupae) from one set of vines divided by the percentage of host pupae

that were not parasitized (No. nonparasitized pupae/ total pupae) from the other set of vines. To estimate the prevalence of HbGV in the host population, a total of 90 H. brillians pupae was collected, as described above, from three sites within the vineyard and assayed by ELISA. Ovipositional Preference of Ametadoria misella H. brillians larvae were exposed to A. misella in a field experiment to determine if the parasitoids prefer to oviposit on (1) third-instar H. brillians larvae, (2) fourth-instar H. brillians larvae, (3) HbGV-infected third-instar H. brillians larvae, and (4) HbGV-infected fourth-instar H. brillians larvae. Prior to the field experiment, cohorts of 10–100 second- and third-instar H. brillians larvae were infected with HbGV by feeding them for 4 h on grape leaves, approx 1 cm2, onto which 0.025 µg (approx 1.5 ⫻ 106 granules) of purified HbGV in 0.1% SDS–PBS (pH 7.0) had been dried. Cohorts of the infected larvae, and the corresponding sets of noninfected larvae, were subsequently reared separately and fed on virus-free grape leaves until reaching the next step in development (approx 4 days). To control for viral infection, a subsample of 2 or 3 larvae from each cohort was transferred to separate clean containers, reared to pupation (to maximize the chance of viral replication and detection), and assayed by ELISA. The remainder of the larvae was transported to the Del Rey vineyard where both H. brillians larvae exhibiting symptoms of HbGV infection and A. misella had been observed. Larvae were placed on 1-m-high potted Thompson Seedless vines (positioned under trellises in the vineyard) and exposed to parasitism by A. misella for 4 days (the approximate instar duration). The experimental design was completely randomized, with one treatment per vine and three replicate vines per treatment. For each replicate, 85 larvae were randomly selected from cohorts of the same treatment, taking care to ensure a similar distribution of test larvae on each replicate vine as distribution has been shown to affect parasitism (Clausen, 1961). To prevent loss of test larvae falling to the ground, a plastic skirt was placed around each vine, which acted as a bridge back onto the vine. To prevent immigration of nonexperimental larvae, vegetation on the trellises above the potted vines was removed. The number of test larvae recovered at the end of the experiment was noted, and 30 randomly selected larvae from each replicate were dissected to determine the presence of A. misella. RESULTS

Prevalence of HbGV in Parasitized and Nonparasitized Host Pupae Table 1 shows the number of H. brillians pupae that were parasitized by A. misella and the percentage of

INTERACTIONS BETWEEN A PARASITOID AND GRANULOVIRUS

parasitized and nonparasitized H. brillians pupae that tested positive for HbGV by ELISA for each of the eight vines sampled in the Del Rey vineyard in 1995. The percentage of H. brillians testing positive for HbGV decreased with increasing distance from the initial center of the outbreak, as determined by regression analysis of arcsine-transformed data (replacing 0/n with 1/4n and n/n by 1 ⫺ 1/4n after Zar, 1984), accounting for 69% of the variance in infection rate (Fig. 1). The occurrence of A. misella in H. brillians pupae was not independent of HbGV infection when data from all vines were pooled (␹2 ⫽ 14.27, df ⫽ 1, P ⬍ 0.01). However, it is important to note that data from each of the eight vines are paired samples (i.e., parasitized and nonparasitized). Therefore, a better test of independence of parasitism and viral infection is obtained by restricting the data set to vines with both infected and healthy hosts (i.e., excluding vines with either 0 or 100% viral infection). When data from vines 4, 5, and 8 were pooled, the occurrence of A. misella in H. brillians pupae was similarly not independent of HbGV infection (␹2 ⫽ 4.32, df ⫽ 1, P ⫽ 0.04). Moreover, the mean percentage of parasitized pupae from vines 4, 5, and 8 testing positive for virus (29.5 ⫾ 19.5% SD) was significantly lower (t ⫽ 2.81, df ⫽ 2, P ⫽ 0.05, one-tailed paired t test of arcsine transformed data) than the mean percentage of nonparasitized pupae testing positive for virus (48.6 ⫾ 26.9% SD). Mean ELISA absorbance values of pupae from all vines were significantly lower in parasitized pupae (0.76 ⫾ 0.39 SD) than in nonparasitized pupae (1.27 ⫾ 0.45 SD), as determined by a Mann–Whitney test (U ⫽ 540.5, df ⫽ 64, P ⬍ 0.001). Higher absorbance values indicate higher concentrations of virus and are assumed to reflect greater severity of disease. Comparative Survival of Parasitoids and Hosts From the population of H. brillians that was sampled at Reedley in 1992, 69% of the pupae were infected with HbGV. The percent of A. misella surviving pupation in the presence of virus (19.8 ⫾ 14.3% SD) was significantly greater (t ⫽ 2.88, df ⫽ 6, P ⫽ 0.03, paired t-test TABLE 1 The Number and Percentage of Harrisina brillians Pupae, Either Parasitized or Nonparasitized by Ametadoria misella, Testing Positive for Granulovirus by ELISA Vine position in vineyard Parasitized pupae Percentage of viral infection Nonparasitized pupae Percentage of viral infection

1

2

3

4

5

6

7

8

3

0

0

7

13

8

5

14

100 22

— 4

— 2

43 21

38 15

0 9

0 5

7 15

100

100

100

52

73

0

0

20

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FIG. 1. Relationship between distance from the initial center of an outbreak of Harrisina brillians in a vineyard and prevalence of granulovirus in H. brillians pupae (y ⫽ 2.59 ⫺ 0.011x; F ⫽ 13.1, P ⫽ 0.01).

of arcsine transformed data) than the percent of nonparasitized H. brillians surviving pupation (10.6 ⫾ 7.3% SD). This again was consistent with the hypothesis that the prevalence of HbGV is lower in parasitized than nonparasitized host pupae. Ovipositional Preference of A. misella Approximately 97% of H. brillians sentinel larvae were recovered from each of the treatments except the third (HbGV-infected third instar). The majority of H. brillians larvae in the third treatment died and/or fell off the vines, and were not recovered. Therefore, in place of 2-way ANOVA (with host instar and virus infection as separate factors), the three remaining treatments were considered as separate treatments in a one-way ANOVA. The percentage of parasitism of sentinel H. brillians larvae was arcsine transformed before analysis (again replacing the extreme values 0/n with 1/4n and n/n with 1-1/4n after Zar, 1984). Parasitism was significantly influenced by treatment (F ⫽ 5.09, df ⫽ 2,6, P ⫽ 0.05), and a comparison of treatment means (Table 2) by LSD test (P ⬍ 0.05) revealed that A. misella oviposited in healthy fourth-instar H. brillians larvae more than six times as often as in healthy third-instar larvae and more than three times as often as in HbGV-infected fourth-instar larvae. All of the H. brillians from the control samples of non-virus treatment cohorts tested negative for granulovirus, whereas all larvae from the control samples of the HbGVinfected treatment cohorts tested positive for granulovirus by ELISA. DISCUSSION

This field study indicates that selective oviposition by A. misella leads to reduced overlap of the parasitoid

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TABLE 2 Percentage of Parasitism of Harrisina brillians by Ametadoria misella Larvae in Relation to Age and Infection with a Granulovirus H. brillians

Mean percentage of parasitism ⫾ SD a

Fourth instar Third instar Infected fourth instar

51 ⫾ 10.1 a 8 ⫾ 7.2 b 15 ⫾ 26.0 c

a Different letters indicate significant between-treatment differences (P ⬍ 0.05) by ANOVA and LSD of arcsine-transformed data. Each value represents the mean of three replicates and a total of 90 individuals examined.

and HbGV in hosts, resulting in greater parasitoid survival. We found that A. misella prefers to oviposit in older H. brillians larvae over younger hosts. This confirms previous casual observations of this parasitoid (Smith et al., 1955; Clausen, 1961). We also found that A. misella prefers to oviposit in healthy H. brillians larvae over HbGV-infected hosts of the same age. As HbGV-infected larvae are much smaller than their healthy counterparts, this choice may merely reflect their preference for larger (older) larvae. A. misella also occurred less frequently in HbGV-infected hosts than in healthy hosts and less frequently in severely infected hosts than in less severely infected hosts. This pattern would not be expected if host attack by the parasitoid and virus were randomly associated with respect to each other. Moreover, from host pupae which tested positive for virus, the parasitoid was more likely to survive and emerge than the nonparasitized host. It is important to note that the degree of overlap of attack that we observed was based on sampling the pupal stage of the host and did not take into account the occurrence of A. misella and HbGV in early-instar host larvae. Given our finding that younger instars are less frequently parasitized than older instars, the overlap we observed is probably an overestimation. Our results also indicate that the interaction of A. misella and HbGV is likely to vary with host population density. On vines closer to the initial point of development of an outbreak, where the host population density was presumably highest, 100% of the pupae were infected with HbGV. This indicates that transmission of virus between host larvae is very efficient and that parasitoid attack or survival is adversely affected at high host density. Conversely, we found that parasitoids had an advantage in the Reedley vineyard, where the prevalence of virus among host pupae was 69% and where both natural enemies had been established for more than 8 years. Host population density in this vineyard has rarely reached levels requiring intervention. The advantage conferred to parasitoids by their

preference for older host larvae may be applicable to other baculoviruses and parasitoids. For many species of Lepidoptera challenged with baculoviruses, host age is positively correlated with the time between viral infection and death (Smits and Vlak, 1988; Sait et al., 1994) and negatively correlated with susceptibility (reviewed by Briese, 1986). Therefore, parasitoids that oviposit in older hosts may benefit from a longer window of opportunity for development before host death. Given that premature death of the host is the most commonly reported cause of parasitoid death in baculovirus-infected hosts (reviewed by Brooks, 1993), the relationship between parasitoid–pathogen competition, age of host attacked, and relative development times warrants further study. The relative contributions of A. misella and HbGV to host population regulation are unclear. HbGV alone may be capable of regulating H. brillians. It was found to yield excellent seasonal control (Federici and Stern, 1990) and to decimate heavy field infestations (Stern and Federici, 1990). Furthermore, HbGV and its host share many of the characteristics (e.g., midgut-restricted viral replication and gregarious feeding behavior of host) that have been attributed to the success of the few viruses (e.g., Gilpinia hercyniae NPV and Oryctes rhinoceros virus) that have proven effective as classical biological control agents (Federici, 1993). Significantly, however, host suppression has also been observed where both A. misella and HbGV were established (Clausen, 1961). Moreover, an apparent decrease in the number of H. brillians outbreaks in Fresno county from 1990 to 1996 coincided with increased detection of A. misella (Stark et al., 1999), suggesting that A. misella is able to coexist with HbGV and that together they have a significant impact on H. brillians population abundance. The consequences of host–parasitoid–pathogen interactions on the dynamics of host populations have not been well studied. Two recent studies suggest that both the intensity and balance of mortality imposed by each natural enemy are potentially important determinants of host regulation (Hochberg et al., 1990; Begon et al., 1996). Our system appears to be characterized by consistently low levels of host abundance, as well as the coexistence of both virus and parasitoid. The differential use of hosts by A. misella and HbGV that we found suggests that they have different patterns of host exploitation both in time (early vs late instars) and space (center vs edge of outbreak). This later effect is likely to be promoted by the superior flight abilities of healthy H. brillians compared to HbGV-infected individuals (Stern et al., 1992) and the relatively strong flight ability of A. misella (Smith et al., 1955). Therefore, we favor the view that spatial heterogeneity is potentially important in this system and may explain the regional persistence of both natural enemies in a

INTERACTIONS BETWEEN A PARASITOID AND GRANULOVIRUS

way analogous to that shown for competing insect parasitoids in metapopulation models (Comins and Hassell, 1996). ACKNOWLEDGMENTS This study was funded by the California Raisin Advisory Board and the California Table Grape Commission.

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