Cydia Pomonella L.) Caused by Early Block of Virus Replication

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Virology 410 (2011) 360–367

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Virology

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Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication

Sabine Asser-Kaiser a, Pit Radtke a, Said El-Salamouny a,1, Doreen Winstanley b, Johannes A. Jehle a,c,⁎

a Laboratory of Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435 Neustadt a. d. Wstr., Germany b Warwick HRI, University of Warwick, Wellesbourne, England c Julius Kühn-Institut (JKI) Bundesforschungsinstitut für Kulturpﬂanzen, Heinrichstraße 243, 64287 Darmstadt, Germany

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Article history: An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was Received 8 October 2010 observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental Returned to author for revision approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune 29 October 2010 response could be excluded as possible resistance mechanisms. When CpGV replication was traced by Accepted 22 November 2010 quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic Available online 28 December 2010 infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic fl Keywords: block prior to viral DNA replication. This conclusion was corroborated by uorescence microscopy using a hsp-eGFP Cydia pomonella granulovirus modified CpGV (bacCpGV ) carrying enhanced green fluorescent gene (eGFP), which showed that CpGV infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV Fluorescent brightener can enter but not replicate in the cells of resistant codling moth larvae. Hemolymph injection © 2010 Elsevier Inc. All rights reserved. Budded virus Quantitative PCR Enhanced green fluorescent protein eGFP Fluorescent microscopy

Introduction individuals are selected rapidly against susceptible ones when CpGV is used in codling moth control due to the dominant, monogenic, and sex- The Cydia pomonella granulovirus (CpGV) is used worldwide for linked mode of inheritance of resistance (Asser-Kaiser et al., 2007). For biological control of codling moth (C. pomonella L.), a major insect pest of the development of successful resistance management strategies, it is apples,pears,andwalnuts(Huber, 1998). CpGV belongs to the genus necessary to understand the mechanism of resistance in addition to the Betabaculovirus of the family Baculoviridae (Jehle et al., 2006). Until inheritance of resistance. recently, baculoviruses were considered to be robust against develop- Insects have evolved many different ways to defend themselves ment of insect resistance (Moscardi, 1999; Cory and Meyers, 2003). against pathogens like fungi, bacteria, nematodes, and viruses. Previous laboratory selection experiments using different baculovirus- Differences in viral susceptibilities have been observed in both host systems suggest that insects may develop up to 10-fold resistance laboratory and field populations of insects (Fuxa, 1993; Fuxa and but that the resistance is not stable without selection pressure (Huber, Richter, 1990, 1998; Briese, 1982, 1986). Insects resist virus infection 1974; Fuxa, 1993). However, in 2005, the first codling moth populations through morphological barriers, behavioral resistance, developmental with up to 1000-fold reduced susceptibility to CpGV were reported in resistance, physiological, nutritional, biochemical, and molecular Germany and France (Fritsch et al., 2005; Sauphanor et al., 2006). To mechanisms (reviewed by Narayan, 2004). Many insects show date, about 35 codling moth orchard populations resistant to CpGV developmental resistance to baculovirus infection with decreasing products have been observed in several European countries (Fritsch susceptibilities at older larval stages (Briese, 1986; Kirkpatrik et al., et al., 2005; Asser-Kaiser et al., 2007; Jehle, unpublished). Resistant 1998; Teakle et al., 1986). However, resistance to CpGV in C. pomonella is prevalent in all five larval instars (Eberle et al., 2008). Resistance may occur at different steps during the infection process. Baculo- ⁎ Corresponding author. Julius Kühn-Institut (JKI) Bundesforschungsinstitut für viruses enter the larvae as viral occlusion bodies (OBs) by oral uptake. Kulturpflanzen, Heinrichstraße 243, 64287 Darmstadt, Germany. Fax: +49 6151 407 290. After OBs have dissolved in the alkaline milieu in the midgut, E-mail address: [email protected] (J.A. Jehle). 1 Permanent address: Department of Economic Entomology and Pesticides, Faculty of occlusion derived virions (ODVs) are released (Federici, 1997). Agriculture, Cairo University, 12613- Giza, Egypt. Virions have to pass through the peritrophic membrane (PM) lining

0042-6822/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2010.11.021 S. Asser-Kaiser et al. / Virology 410 (2011) 360–367 361 the midgut epithelium, a morphological barrier (Brandt et al., 1978; Codling moth larvae that are resistant to CpGV have developed a Granados, 1980), to start a primary infection in the larval midgut mechanism that prevents lethal virus infection. In order to identify epithelial cells (Federici, 1997). Optical brighteners, such as Calcoflour which part of the infection cycle this mechanism becomes effective and White M2R or Tinopal LPW, are used in baculovirus formulations to gain information about the mechanism, different experimental protect viral OBs from inactivation by UV light in the field (Shapiro, approaches were followed: First, an optical brightener was used to 1992). Furthermore, optical brighteners enhance the susceptibility of break down the PM in susceptible and in resistant codling moth larvae to insects to baculoviruses by breaking down the PM and preventing see whether the PM is involved in resistance. Second, budded virus was sloughing of infected midgut epithelial cells (Washburn et al., 1998; injected directly into the insect's hemocoel in order to investigate El-Salamouny, 2005, 2007). whether resistance is midgut based only. Third, susceptible and resistant The midgut is an important barrier that has to be passed by a fifth instar larvae were infected either per os or intra-hemocoelically, baculovirus to initiate the infection. Resistance of Agrotis segetum and the spread of virus replication was traced in midgut, hemolymph, larvae to oral infection by Spodoptera exigua multiple nucleopolyhe- and fat body using quantitative PCR. Fourth, the spread of CpGV drovirus (SeMNPV) is midgut based. In bypassing the midgut by intra- infection in susceptible and resistant codling moth was observed using a hemocoelic injection of budded virus (BV), the second viral recombinant virus, CpGV-bacmid (bacCpGVhsp-eGFP), expressing en- phenotype, SeMNPV is able to infect A. segetum (Jakubowska et al., hanced green fluorescent protein (eGFP). Fifth, CpGV induced mortality 2010). Different mechanisms of midgut based virus resistance have was determined after transfusion of hemolymph between resistant and been described. For example, Engelhard and Volkman (1995) showed susceptible codling moth larvae to determine if a humoral factor is that fourth instar larvae of Trichoplusia ni can completely clear involved in CpGV resistance. infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by sloughing off infected midgut cells whereas earlier Results instars are susceptible. Resistance of Bombyx mori to B. mori densovirus type 2 (BmDNV-2) is caused by a 6-kb deletion in a Bioassays with fluorescent brightener 28 gene that encodes a transmembrane protein, which is a functional receptor for the virus in the midgut (Ito et al., 2008). Larvae of the fall The fluorescent brightener 28 (FB28) was added to the diet at armyworm Spodoptera frugiperda are highly resistant to oral infection different concentrations in a bioassay using CpGV concentrations of by AcMNPV but are susceptible to infection by BVs when delivered 2×103 OB/ml for the susceptible codling moth strain CpS and 2×105 directly into the hemocoel (Haas-Stapleton et al., 2003). This OB/ml for the resistant strain CpR (Fig. 1). In previous experiments, it resistance is due to aberrant binding of ODVs to the midgut cells had been shown that CpR was about 100 less susceptible to CpGV than (Haas-Stapleton et al., 2005). Furthermore, baculovirus replication CpS (Eberle and Jehle, 2006; Asser-Kaiser et al., 2010). The average can be blocked or inhibited within the midgut epithelial cells. As a mortalities of CpS larvae increased from 67% without FB28 up to 91% result, no BVs are produced to cause fatal infection (Pinnock and Hess, at an FB28 concentration of 0.075% due to the addition of fluorescent 1977). brightener (Fig. 1). At higher FB28 concentrations, the mortality After the initial infection of the midgut, baculoviruses produce BVs. decreased to 57% at 0.3% FB28. The average mortalities of the resistant These spread the infection from cell to cell, often transported through strain CpR were much lower compared to the susceptible strain CpS the trachea and/or hemolymph within the host (Engelhard et al., and did not exceed a value of 27%. The observed differences between 1994; Flipsen et al., 1995). Eventually, most of the insect's tissues are the different treatments within CpS and CpR were not statistically infected and new OBs are produced (Federici, 1997). significant when the Kruskal–Wallis test and Dunn's Multiple Vertebrates have both innate and acquired immunity with an Comparison test were applied. As CpR still contained up to 30% of “immunological memory” to defend themselves against microbial susceptible individuals (Asser-Kaiser et al., 2007, 2010), a genetically infections. In contrast, insects only possess an innate immune system, homogenous strain CpRR1 derived from CpR was used in the which is characterized by more or less nonspecific cellular and following experiments. humoral immune reactions and lack the acquired immune system (reviewed by Cory and Meyers, 2003; Narayan, 2004). Phagocytosis, Intra-hemocoelic infection aggregation of hemocytes, and encapsulation are cellular defense mechanisms, whereas melanization and induction of immune A dose of 2×105 BV particles was injected into the hemocoel of proteins (lysozymes, lectins, and anti-bacterial and anti-fungal susceptible CpS and resistant CpRR1 fourth instar larvae and mortality proteins) belong to humoral reactions (Narayan, 2004). Increased concentrations of phenoloxidase in the insect's plasma affect 100 baculovirus infection (Wilson et al., 2001). Popham et al. (2004) CpS CpR showed that the plasma of Heliothis virescens exhibit virucidal activity against BVs of Helicoverpa zea single nucleopolyhedrosis virus 80 (HzSNPV) which is caused by phenoloxidase activity (Shelby and

Popham, 2006). In Lepidoptera, the hemolymph protein hemolin is 60 induced by bacterial and baculovirus infection (Faye et al., 1975; Hirai et al., 2004). It binds to bacterial lipopolysaccharides (LPS), lipid A, 40

and hemocytes (Sun et al., 1990; Daffre and Faye, 1997; Bettencourt Mortality [%] et al., 1999). Hemolin is thought to function as an opsonin or as a pattern recognition molecule and thus to be involved in antiviral 20 immune response (Faye and Kanost, 1998; Hirai et al., 2004). Lepidopteran larvae resist baculovirus infection by programmed 0 cell death, called selective apoptosis (reviewed by Clem, 2001). To Without FB28 0.05% FB28 0.075% FB28 0.1% FB28 0.3% FB28 overcome this defense strategy, baculoviruses have evolved inhibitors Concentration FB28 [%] of apoptosis, which block cell death (Clem et al., 1991). Infection of S. Fig. 1. Mortalities of susceptible (CpS) and resistant (CpR) larvae in a 10-day bioassay frugiperda cells with a mutant of AcMNPV lacking a functional anti- with ﬂuorescent brightener (FB28) added to virus concentrations of 2×103 OB/ml for apoptotic gene p35 leads to apoptosis and inhibition of OB formation CpS and 2×105 OB/ml for CpR. FB28 was added in concentrations of 0.0%, 0.05%, 0.075%, (Clem et al., 1991). 0.1%, and 0.3%. 362 S. Asser-Kaiser et al. / Virology 410 (2011) 360–367 data were collected every day. The mortality of CpS individuals caused l]

by CpGV infection reached 100% seven days post injection (Table 1). µ Larvae of CpRR1 did not show any signs of CpGV infection and all 108 survived the BV injection. The surviving CpRR1 individuals were further observed until pupation but their mortality did not increase 106 (data not shown). Mortality in the controls injected with hemolymph free of virus was 2.7% (CpS) and 0.0% (CpRR1). 104 emolymph [copies/7 emolymph Replication of CpGV in different tissues a H

102 Fifth instar larvae of CpS and CpRR1 were infected either orally 24 hpi 48 hpi 72 hpi 96 hpi with 103 OBs or intra-hemocoelically with a dose of 2×105 BVs. Virus replication in different tissues was determined 24, 48, 72, and 96 h p.i. using qPCR. After oral infection of CpS larvae, virus replication was t] detected first in the fat body after 48 h (Fig. 2). At 72 h p.i., virus u 108 replication could be observed also in the hemolymph and the midgut and increased until 96 h p.i. In contrast, virus replication was not detected in tissues of resistant CpRR1 larvae after oral inoculation, 106 independent of the time point. When budded virus was injected into t [copies/midg t CpS larvae, CpGV copies were first detected in the hemolymph at 24 h u p.i. and after 48 h also in the fat body (Fig. 3). In the hemolymph of 104 Midg CpRR1 larvae, there was no detection of the PCR product until 72 h p.i. Some weaker CpGV signals were detected in CpS and CpRR1 controls 102 as well as in CpGV-M infected CpRR1 at 96 h p.i. (Fig. 3). These weak 24 hpi 48 hpi 72 hpi 96 hpi signals, however, did not derive from the CpGV-M inoculum but from a background contamination with CpGV-I12 in the reared insects as substantiated by PCR using CpGV-I12 specific primers (data not shown). CpGV-I12 is a resistance overcoming isolate able to replicate 108 in CpRR1 (Eberle et al., 2008).

GFP expression tracking 106

The spread of virus infection in both CpS and CpRR1 was traced by t body [copies/mg] 4 fl a 10 uorescence microscopy. Fifth instar larvae were orally infected with F a dose of 1×10³ OBs of the eGFP expressing recombinant bacCpGVhsp- eGFP and fluorescence was determined after 24, 48, 72, and 96 h p.i. 102 Infected CpS showed replication in the fat body already after 48 h p.i., 24 hpi 48 hpi 72 hpi 96 hpi notably increasing until 96 h p.i., whereas virus replication in CpRR1 CpRR1 Control CpRR1 Inoculated CpS Control CpS Inoculated was barely detected (Fig. 4a and d). Some individuals of infected fl CpRR1 larvae showed very few single uorescent spots after 96 h p.i. Fig. 2. Tissue specific replication of CpGV in susceptible (CpS) and resistant (CpRR1) spread over different tissues (fat body, gut, and tracheal system). fifth instar larvae at 24, 48, 72, and 96 h post oral infection (h.p.i.) using 1×103 OB/ Infection of CpRR1, however, was never permissive (Fig. 4a and d) and larva. the larvae survived and pupated successfully. In CpS larvae, time course of fat body infection correlated to qPCR results whereas infection of intestinal tract and hemolymph was not traceable. In some CpS individuals, fluorescent spots at junction points between CpRR1 larvae, which can directly or indirectly convey resistance trachea and midgut could be observed at late stage of infection against CpGV (Fig. 5). As a control for hemolymph injection, (Fig. 4e). hemolymph from CpS larvae was injected into CpS larvae (experiment C). To see if resistance in CpRR1 larvae is consistently present or is induced by CpGV infection, hemolymph from uninfected CpRR1 Hemolymph transfusion (treatment D) and from CpGV infected CpRR1 individuals (treatments E and F) were injected into CpS larvae. As a control, cell culture Hemolymph from resistant larvae was injected into susceptible medium Sf900 was injected into CpS larvae which were afterwards larvae before they were orally infected with CpGV in order to either fed with virus free diet (treatment B) or a piece of diet investigate whether there is a humoral factor in the hemolymph of containing 1×103 OBs (treatment A). Mortality of CpS larvae nine days post injection (d.p.i.) with Sf900 medium and oral infection was 76.1% (treatment A). The group of CpS larvae that were injected with Table 1 Sf900 medium and not infected with occlusion bodies displayed 1.2% Average mortality of susceptible (CpS) and resistant (CpRR1) larvae at day 7 after intra- hemocoelic injection of hemolymph from uninfected larvae (control) or hemolymph mortality (treatment B). Treatments D and E, when CpS larvae were containing 2×105 budded virus (BV). Data are the mean of three replicates of 24–27 injected with CpRR1 hemolymph prior to oral inoculation, showed larvae per treatment. slightly lower mortalities compared to treatments A and C when CpS

Control BV injection larvae were injected with Sf900 medium or CpS hemolymph, respectively. However, these differences were not statistically CpS CpRR1 CpS CpRR1 signiﬁcant as indicated by the high standard deviation (SD). The Mean mortality [%] 2.7 0 100 0 mortality of treatment F was 2.5% which is similar to the control Standard deviation 4.6 0 0 0 (treatment B). S. Asser-Kaiser et al. / Virology 410 (2011) 360–367 363

Comparing inoculation of OBs per os and injection of BVs into the hemocoel can be used to differentiate between midgut based and l] µ 108 systemic resistance to a baculovirus. Fuxa and Richter (1990) investigated the resistance of S. frugiperda larvae against S. frugiperda multiple nucleopolyhedrovirus (SfMNPV). When SfMNPV was 106 injected into the hemocoel of resistant larvae, there was no signiﬁcant difference in mortality compared to susceptible ones. Thus, they concluded that resistance to the virus was associated with the gut of 4 emolymph [copies/7 10 the insect. There are several examples for midgut based resistance to a a

H virus (Watanabe, 1971; Fraser and Stairs, 1982; Engelhard and Volkman, 1995; Hoover et al., 2000; Haas-Stapleton et al., 2005). 102 24 hpi 48 hpi 72 hpi 96 hpi However, in the present study, injection of BVs of CpGV killed all susceptible larvae within seven days, whereas all larvae of the resistant strain CpRR1 survived (Table 1). Consequently, CpRR1 larvae are resistant to both infection of the midgut epithelium by ODVs and secondary infection induced by BVs. Therefore, resistance solely due 108 to an altered midgut receptor, as described by Ito et al. (2008), or a nonspeciﬁc binding of ODVs to midgut receptors, as reported by Haas- 106 Stapleton et al. (2005), can be excluded. In order to get a more detailed information about the location of resistance in the infection route, replication of the virus in susceptible and resistant larvae was t body [copies/mg]

a 4

F 10 traced after oral and intra-hemocoelar infection with CpGV using qPCR and fluorescence microscopy. The results of qPCR experiments clearly showed that replication of the virus was inhibited in midgut as 102 well as in hemocytes and fat body of CpRR1 but not in CpS, indicating 24 hpi 48 hpi 72 hpi 96 hpi that CpGV is unable to replicate in any cell type of CpRR1 (Figs. 2 CpRR1 Control CpRR1 Inoculated CpS Control CpS Inoculated and 3). These results were confirmed by visualization of eGFP expression after oral infection of CpS and CpRR1 with bacCpGVhsp-eGFP.Here,we Fig. 3. Tissue specific replication of CpGV in susceptible (CpS) and resistant (CpRR1) demonstrate that in CpRR1, oral infection was transmitted into fat fifth instar larvae at 24, 48, 72, and 96 h after intra-hemocoelar injection of 2×105 BV/larva. cells but infection was stopped and larvae survived infection by bacCpGVhsp-eGFP (Fig. 4). The role of tracheae for the systemic spread of baculoviruses has Discussion been previously discussed (Engelhard et al., 1994; Rahman and Gopinathan, 2004). The tracheal system was suggested to act as a Optical brighteners are reported to have an enhancing effect on the major conduit for the systemic spread of A. californica MNPV (AcMNPV) virulence of baculoviruses by disintegration of the PM (Shapiro and and B. mori NPV (BmNPV). Remarkably, some infected CpS larvae Robertson, 1992). The loss of this mechanical barrier results in more showed enhanced virus replication at junction points between tracheae efficient passage of virus to the midgut cells (Corsaro et al., 1993). and midgut (Fig. 4e). Since this occurred solely during a late phase of Addition of FB28 to A. segetum nucleopolyhedrovirus and A. segetum infection and in an unsteady manner, it is unlikely that the role of granulovirus increased the susceptibility of A. segetum (Lepidoptera: tracheae is crucial for the systemic spread of CpGV in C. pomonella larvae. Noctuidae) to both viruses (El-Salamouny, 2005, 2007). Furthermore, We further investigated whether an immune factor present in the optical brighteners affect sloughing of infected midgut epithelial cells, hemolymph of CpRR1 may trigger resistance to CpGV. Hemolymph of which is also a defense mechanism of insects to viral infection CpRR1 was transfused into CpS larvae in order to see whether a (Washburn et al., 1998). In the experiments shown here, no resistance inducing factor can be carried over with the hemolymph significant effect of FB28 on the efficacy of CpGV against CpS and (Fig. 5). Using this method, there was no evidence that transfusion of CpR larvae was observed (Fig. 1). In C. pomonella larvae, the PM is very hemolymph from CpRR1 larvae could reduce susceptibility of CpS thin and its functions as a physiological barrier of CpGV have not been larvae to oral infection with CpGV. Phenylthiourea (PTU) was added demonstrated (R. Kleespies, personal communication). When FB28 to the hemolymph during its preparation in order to prevent was added to the diet at a concentration of 0.3%, the mortality melanization. However, PTU is an inhibitor of the enzyme phenolox- decreased. However, this difference was statistically not significant. idase and eliminates its virucidal activity (Popham et al., 2004). If an The slight decrease may be explained by the acidic effect of the increased level of phenoloxidase in the plasma of CpRR1 was related compound which lowers the pH value in the midgut. Sheppard et al. to resistance, similar to the virucidal effect of plasma of H. virescens (1994) observed a significant reduction in midgut luminal pH due to larvae described by Popham et al. (2004), we would not have seen it the fluorescent brightener Tinopal. Solubilization of OBs in the midgut in experiments using PTU. Immune reactions like an increased activity could be affected by the lower pH value, resulting in a passage of OBs of the enzyme phenoloxidase in the plasma can reduce susceptibility through the midgut without a release of infectious virions from the of a host to its virus (Popham et al., 2004; Shelby and Popham, 2006). OBs. Nevertheless, for CpR, it is evident that addition of FB28 did not However, it is unlikely that a virus infection is completely blocked by increase the susceptibility to CpGV; therefore, it is unlikely that a this mechanism, as it is the case in CpRR1. Furthermore, the virucidal modified PM in CpR is involved in CpGV resistance. The mortality rates effect of phenoloxidase is nonspecific and would be expected to affect observed for CpR in this experiments correspond to the average both CpGV-M and CpGV-I12. However, CpGV-I12 is able to overcome mortality generally observed for strain CpR (Eberle and Jehle, 2006; resistance in CpRR1 very effectively (Eberle et al., 2008). Therefore, Asser-Kaiser et al., 2010). This mortality can be ascribed to the fraction and with regard to the results of the tissue specific virus replication of susceptible individuals present in the heterogeneous strain CpR experiment, we propose that resistance is caused by inhibition of which die from virus infection, whereas the resistant individuals virus replication in the cells rather than a humoral immune response. survive the CpGV concentration of 2×105 OBs/ml (Asser-Kaiser et al., Beyond this, there is an additional conclusion that can be drawn 2007, 2010). from the transfusion experiment. Mortality of CpS larvae injected with 364 S. Asser-Kaiser et al. / Virology 410 (2011) 360–367

Fig. 4. Replication of the eGFP expressing recombinant bacCpGVhsp-eGFP in susceptible (CpS) and resistant (CpRR1) fifth instar larvae at 24, 48, 72, and 96 h post oral infection with 1×10³ OBs obtained under fluorescence microscopy (12× magnification). (a) eGFP expression 96 h.p.i. (25× magnification) of upper midgut and fat body of (b) an uninfected larva (CpS), (c) an infected susceptible (Cps) larva, and (d) an infected resistant (CpRR1) larva; (e) midgut and adjacent tracheae of infected CpS larvae showing fluorescent spots at junction points. hemolymph from infected CpRR1 larvae (Fig. 5, F) did not differ epithelium of CpRR1 larvae. However, no further spread and increase significantly from the mortality of CpS larvae injected with Sf900 of fluorescence was determined. The different experiments under- medium (Fig. 5, B). Hence, hemolymph derived from infected CpRR1 taken to dissect the infection process of CpGV-M in susceptible and larvae did not contain active BVs that would infect susceptible insects. resistant C. pomonella larvae provided clear evidence that CpGV This is consistent with the observation that there is no virus resistance is caused by a blockage of CpGV replication in CpRR1 at an replication in the hemocytes or other cell types of CpRR1. early stage of infection and that it is specific to CpGV-M. In summary, the mechanism of resistance to CpGV in CpRR1 is prevalent in the whole insect. Furthermore, the mechanism must be Materials and methods very specific as it inhibits infection by CpGV-M but not by CpGV-I12 (Eberle et al., 2008). All mechanisms of virus resistance like a modified Insects PM, sloughing of infected cells, inactivation of BVs by phenoloxidase activity, and other nonspecific immune responses can be excluded. Codling moth larvae used for the experiments were reared at the Resistance occurred in BV and ODV infection with the same efficacy. DLR Rheinpfalz (Agricultural Service Center Palatinate), Neustadt/ As the envelopes of BVs and ODVs contain different attachment Weinstraße, where three different strains of C. pomonella are proteins binding to cellular receptors (Rohrmann, 2008), a modifica- maintained. Strain CpS is a CpGV susceptible strain and has been tion of the cellular receptors as a reason for resistance is also unlikely. reared in Neustadt for ten years. The strain CpR has been reared in As no virus DNA replication could be detected in the resistant insects, Neustadt since 2005 and was obtained from Andermatt Biocontrol AG it is obvious that the mode of resistance is located during an early (Switzerland). It was collected in 2003 and originated from an organic event in the replication process. Observation of the infection process orchard in South Baden, Germany, where CpGV application had failed using the eGFP expressing recombinant bacCpGVhsp-eGFP has shown to control an infestation with codling moth. This resistant strain is that the virions are able to enter the cells and to permeate the midgut identical to the resistant strain described by Fritsch et al. (2005), S. Asser-Kaiser et al. / Virology 410 (2011) 360–367 365

Fig. 5. Hemolymph transfusion experiment. Mean mortality rates [%] of CpS larvae are given in different experiments (experiments A–F) nine days post inoculation. In experiment A, CpS larvae were injected with Sf900 medium before they were orally infected with 1×103 CpGV occlusion bodies (OBs). Experiment B represents injection of CpS larvae with Sf900 medium without subsequent oral inoculation. In experiment C, CpS larvae were injected with hemolymph taken from uninfected CpS larvae and were orally infected with 1×103 OBs afterwards. In experiment D, larvae which were injected with hemolymph taken from uninfected CpRR1 larvae were orally infected with 1×103 OBs afterwards. Finally, hemolymph of CpRR1 larvae orally inoculated with 1×103 OBs was transferred to CpS followed by oral infection with 1×103 OBs (experiment E) or without oral infection (experiment F). n indicates number of treated CpS animals; SD, standard deviation of the mean. called “Suedbaden” or CpR (BW FI 03) (Asser-Kaiser et al., 2007). CpR supernatant was stored at −70 °C in aliquots of 250 μl. Concentration is about 100 times less susceptible to CpGV-M than CpS (Eberle and of BV was estimated by quantitative PCR described below. Jehle, 2006). This strain is genetically heterogeneous concerning its resistance to CpGV and contains a background of approximately 30% Bioassays with fluorescent brightener 28 susceptible individuals (Asser-Kaiser et al., 2007, 2010). The genetically homogeneous resistant C. pomonella strain CpRR1 was selected Diet incorporation bioassays were conducted with the strains CpS by single pair crosses from CpR and did not contain susceptible and CpR to investigate the effects of fluorescent brightener 28 (FB28) individuals (Asser-Kaiser et al., 2007). (Tinopal LPW, Sigma-Aldrich) on CpGV induced mortality. Bioassays The insects were reared at 26 °C, 60% relative humidity, and 16/8 h were performed in autoclavable 50-well plates containing 50 ml diet light/dark photoperiod. The same conditions were used for the in total. Artificial diet was prepared as described above but using 10% experiments. Larvae were kept in autoclavable 50-well plates on less water and half of the amount of agar in order to ensure that diet semi-artificial diet as described by Ivaldi-Sender (1974). It was could cool down to 40 °C without solidifying. This was necessary to composed of water and agar-agar, which were autoclaved for 20 min prevent thermal inactivation of the virus. Forty milliliters of diet was at 120 °C and then mixed with maize meal, wheat germ, brewer's mixed with five milliliters of the appropriate virus suspension and five yeast, ascorbic acid, and methyl 4-hydroxybenzonate dissolved in milliliters of FB28 diluted in distilled water. A constant CpGV ethanol. After accomplishing the last larval stage, the insects were concentration of 2×103 OB/ml diet was used for the susceptible allowed to pupate in corrugated cardboard stripes. strain CpS. Due to the resistance factor of 100, a CpGV concentration of 2×105 OB/ml diet was chosen for strain CpR. Five different Virus concentrations of FB28 were used for both strains as follows: 0.0%, 0.05%, 0.075%, 0.1 %, and 0.3%. For each FB28 concentration, one The CpGV isolate used for the bioassays was the “Mexican isolate” control was performed where the virus suspension was replaced by (CpGV-M), which was originally collected in Northern Mexico and 5 ml distilled water. Fifty larvae of the fourth instar were used for each described by Tanada (1964). It is the virus strain used in many concentration, and four replicates were performed. Mortality due to commercial CpGV based biocontrol products (Huber, 1998). This virus virus infection was scored 10 days after the larvae were exposed to was propagated in fourth instar larvae of the susceptible strain CpS. the virus and FB28 containing diet. Mortality data were corrected for The OBs were purified following the method described by Jehle et al. control mortality according to Abbott (1925). Data were statistically (1992). The number of OBs per μl of the stock solution was scored analyzed using ANOVA Kruskal–Wallis and Dunn's Multiple Compar- using a Petroff–Hauser counting chamber (depth, 0.02 mm) using the ison test. dark field optics of a light microscope (Leica DMRBE). Budded virus (BV) was produced in fifth instar larvae of the Intra-hemocoelic infection susceptible strain CpS. After starvation for eight hours, the larvae were fed with small pieces of diet (2 mm³) containing 1×103 OBs. Larvae Freshly molted fourth instar CpS and CpRR1 larvae were that did not ingest the diet during a period of 12 h were excluded from anesthetized in diethyl ether vapor for 2–3 min. Prior to injection, the experiment. Three days later, hemolymph was extracted: After each larva was ventrally disinfected with a 0.4% hyamine solution superficial disinfection with 70% ethanol the larvae were anesthetized using a sterile cotton bud. Two microliter of hemolymph containing with diethyl ether vapor for 2–3 min. The second proleg was cut off 2×105 BVs or hemolymph extracted from uninfected larvae were using micro-scissors. Hemolymph droplets were collected using a injected into the second proleg using a gas-proof twenty-five 0.5–10 μl Eppendorf pipette. Hemolymph was transferred into IZD04 microliter Hamilton syringe (0.21 mm in diameter). Twenty-five cell culture medium containing 50 μg/ml streptomycin, 100 U/ml larvae of each strain were injected with BV and the same number was penicillin, and a small crystal of phenylthiourea (Winstanley and injected with control hemolymph free of virus. After the larvae had Crook, 1993). After centrifugation at 1000g and 4 °C for 5 min, the recovered from treatment, they were transferred onto virus free 366 S. Asser-Kaiser et al. / Virology 410 (2011) 360–367 artificial diet. Larvae that died the next day were regarded as dead by CpS and CpRR1 fifth instar larvae were orally infected with handling and were excluded from the test. Mortality data were bacCpGVhsp-eGFP by feeding them a small piece of diet (2 mm3) collected seven days post injection. Three independent replicates of inoculated with 1×103 OBs of bacCpGVhsp-eGFP. Twenty-four, forty- each experiment were conducted. eight, seventy-two, and ninety-six hours p.i., two to four larvae of each variant were dissected. In order to dissect the larvae for fluorescence Tissue specific virus replication microscopy, the cuticles of the larvae were cut open dorsally and pulled aside and pinned down with needles. The dissecting dish was fl Newly emerged fifth instar larvae of CpS and CpRR1 were infected ooded with PBS buffer. Larvae were visualized under a Leica MZ16 FA fl fi either per os or intra-hemocoelically. For oral infection, larvae were uorescence stereomicroscope using a GFP plus lter set. Photos were starved for twelve hours before they were fed with small pieces of diet taken under consistent conditions with a Leica DFC320 CCD camera (2 mm³) containing 1×103 OBs. Intra-hemocoelic infection was per- controlled by Leica LAS 2.6 software. formed as described above, with a dose of 2×105 BVs. Twenty-four, forty-eight, seventy-two, and ninety-six hours post-infection (h.p.i.), Hemolymph transfusion larvae were dissected, and the midgut, 7 μl hemolymph, and segmental fat body tissue were transferred into individual 1.5 ml micro centrifuge Hemolymph from orally infected CpRR1 and noninfected CpS and tubes containing 180 μl PBS buffer and stored at −20 °C before DNA was CpRR1 larvae was prepared following the method described above. extracted. Tubes for the collection of fat body were weighed before and Larvae of the susceptible strain CpS were reared on virus free diet until after the fat bodies were added in order to determine the weight of the they reached the fourth instar. Larvae were injected intra-hemocoe- collected tissue. lically as described above with 2 μl of Sf900 cell culture medium or hemolymph from CpS larvae and from infected and noninfected CpRR1. After recovering from the injection, the larvae were fed with a DNA isolation and quantitative PCR piece of diet either contaminated with 1×103 OBs or free of virus. Mortality data were collected nine days later. Each variant consisted Prior to DNA isolation, midgut and fat body samples were ground of 20–25 larvae. Each experiment was replicated four times. with sterile plastic microtube pestles. In order to generate a standard curve for quantitative PCR (qPCR), the following dilutions of CpGV occlusion bodies were made using sterile deionized water: 1.25×106, Acknowledgments 3.75 ×105, 1.25 ×105, 3.75 ×104, 1.25 ×104, 3.75 ×103, 1.25 ×103, 3.75×102, and 1.25×102 OB/5 μl. For solubilization of OBs, 10 μlof This work was supported by a grant of the Federal Organic Farming Scheme (050EO23/1) of the Federal Agency for Agriculture and Food 1MNa2CO3 was added to each of the homogenized tissue samples as well as to the hemolymph samples and 100 μl of the serial dilutions. (BLE) of Germany and the Boehringer Ingelheim Fonds. After incubation at room temperature for 10 min, the pH was adjusted to 8.0 using 1 M HCl. DNA was isolated using DNeasy® Blood and References μ Tissue Kit (Qiagen). DNA was eluted in 400 l elution buffer (AE) Abbott, W.S., 1925. A method of computing effectiveness of an insecticide. J. Econ. provided with the kit. For the quantification of CpGV genomes in the Entomol. 18, 265–267. tissue samples, oligonucleotides located in the granulin gene were Asser-Kaiser, S., Fritsch, E., Undorf-Spahn, K., Kienzle, J., Eberle, K.E., Gund, N.A., used: nested_PRCP1 upper (5′GGC CCG GCA AGA ATG TAA GAA TCA Reineke, A., Zebitz, C.P.W., Heckel, D.G., Huber, J., Jehle, J.A., 2007. Rapid emergence of baculovirus resistance in codling moth due to dominant, sex-linked inheritance. 3′) and nested_PRCP1 lower (5′GTA GGG CCA CAG CAC ATC GTC AAA Science 317, 1916–1918. 3′). The PCR reaction generated a 422 bp fragment. The PCR reaction Asser-Kaiser, S., Heckel, D.G., Jehle, J.A., 2010. Sex linkage of CpGV resistance in a fl heterogeneous field strain of the codling moth Cydia pomonella (L.). J. Invertebr. and uorescence detection were performed using DNA engine – ™ Pathol. 103, 59 64. Opticon System (MJ Research, Biozym Diagnostic GmbH). For Bettencourt, R., Gunne, H., Gastinel, L., Steiner, H., Faye, I., 1999. 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