Effects of plant protease inhibitors, oryzacystatin I and soybean Bowman–Birk inhibitor, on the aphid Macrosiphum euphorbiae (Homoptera, Aphididae) and its parasitoid Aphelinus abdominalis (Hymenoptera, Aphelinidae)

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Journal of Insect Physiology 51 (2005) 75–86 www.elsevier.com/locate/jinsphys

Effects of plant protease inhibitors, oryzacystatin I and soybean Bowman–Birk inhibitor, on the aphid Macrosiphum euphorbiae (Homoptera, Aphididae) and its parasitoid Aphelinus abdominalis (Hymenoptera, Aphelinidae) H. Azzouza,, A. Cherquia, E.D.M. Campana, Y. Rahbe´b, G. Duportb, L. Jouaninc, L. Kaiserd, P. Giordanengoa a BPCIR, UPRES 2084-2085 Universite´ de Picardie Jules Verne, Somme, 33 rue Saint Leu, 80039 Amiens Cedex 1, France UMR INRA-INSA de Lyon, Biologie Fonctionnelle Insectes et Interactions, Bat. Louis-Pasteur, 20 av. A. Einstein, 69621 Villeurbanne Cedex, France c INRA-CNRS, Laboratoire de Biologie Cellulaire, route de St Cyr, 78026 Versailles Cedex, France d De´veloppement, Evolution et Plasticite´ du Syste`me Nerveux, Institut Alfred Fessard, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France

b

Received 25 August 2004; received in revised form 19 November 2004; accepted 19 November 2004

Abstract Transgenic plants expressing protease inhibitors (PIs) have emerged in recent years as an alternative strategy for pest control. Beneficial insects such as parasitoids may therefore be exposed to these entomotoxins either via the host or by direct exposure to the plant itself. With the objective of assessing the effects of PIs towards aphid parasitoids, bioassays using soybean Bowman–Birk inhibitor (SbBBI) or oryzacystatin I (OCI) on artificial diet were performed on Macrosiphum euphorbiae– Aphelinus abdominalis system. OCI significantly reduced nymphal survival of the potato aphid M. euphorbiae and prevented aphids from reproducing. This negative effect was much more pronounced than with other aphid species. On the contrary, SbBBI did not affect nymphal viability but significantly altered adult demographic parameters. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of Aphelinus abdominalis predominantly relies on serine proteases and especially on chymotrypsin-like activity. Immunoassays suggested that OCI bound to aphid proteins and accumulated in aphid tissues, whereas SbBBI remained unbound in the gut. Bioassays using M. euphorbiae reared on artificial diets supplemented with both OCI and SbBBI showed a fitness impairment of Aphelinus abdominalis that developed on intoxicated aphids. However, only SbBBI was detected in parasitoid larvae, while no PI could be detected in adult parasitoids that emerged from PI-intoxicated aphids. The potential impact of PI-expressing plants on aphid parasitoids and their combined efficiency for aphid control are discussed. r 2004 Elsevier Ltd. All rights reserved. Keywords: Aphid parasitoid; Protease inhibitors; Tritrophic interactions; Risk assessment; Transgenic plants

1. Introduction Present methods of crop protection rely mainly on the use of chemical pesticides. To limit the harmful effects of these synthetic molecules on environment and human Corresponding author. Tel./fax: +33 322 82 75 47.

E-mail address: [email protected] (H. Azzouz). 0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.11.010

health, plant genetic engineering was proposed as an alternative to create insect-resistant plants. Numerous transgenic plants expressing entomotoxic proteins of various origins have thus been engineered (Gatehouse and Gatehouse, 1998; Jouanin et al., 1998; Carlini and Grossi-de-Sa, 2002; Ranjekar et al., 2003). Among the proteins exhibiting insecticidal effects originating from plants, protease inhibitors (PIs) emerged as an

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interesting strategy for insect pest control (Reeck et al., 1997; Lawrence and Koundal, 2002) using genetic engineering. Plant PIs occur naturally in a wide range of plants as a part of their natural defence system against herbivores (Ryan, 1990). Through binding to digestive proteases of phytophagous insects, PIs impair protein digestion (Broadway and Duffey, 1986). Moreover, moulting and non-digestive enzyme regulation could also be affected (Faktor and Raviv, 1997). Insecticidal effects of PIs, especially serine and cysteine protease inhibitors, have been studied by diet incorporation assays or by in vitro inhibition studies. They induce delayed growth and development, reduced fecundity and sometimes increased mortality (Edmonds et al., 1996; Ortego et al., 1998; Gatehouse et al., 1999; Annadana et al., 2002; Oppert et al., 2003). Several serine and cysteine PIs have been expressed in transgenic plants belonging to different families to enhance their resistance against Lepidoptera (Hilder et al., 1987; De Leo et al., 2001; Falco and Silva-Filho, 2003) and Coleoptera (Lecardonnel et al., 1999; Alfonso-Rubı´ et al., 2003). PI-based strategies target especially leaf-feeding insects which use digestive proteases to degrade ingested proteins. Although sap-feeding homopterans have been considered to lack digestive proteolytic degradation (Terra et al., 1996), recent works pointed out some putative protease activity in the rice brown plant hopper Nilaparvata lugens (Foissac et al., 2002), in Myzus persicae (Cherqui et al., 2003; Rahbe´ et al., 2003a) and in Aphis gossypii (Deraison et al., 2004). Recently, the insecticidal effect of PIs against several aphid species was established. The pea and soybean trypsin-chymotrypsin inhibitors (PsTI-2, SbBBI) belonging to the Bowman–Birk family (Rahbe´ et al., 2003b) and the mustard-type trypsin-chymotrypsin variant Chy8 (Ceci et al., 2003) induced significant mortality and growth inhibition on the pea aphid Acyrthosiphon pisum. The phytocystatin oryzacystatin I (OCI) isolated from rice seeds (Abe et al., 1987) significantly reduced adult weight and fecundity of the aphid M. persicae (Rahbe´ et al., 2003a). Genetically engineered plants expressing PIs thus appear a promising strategy to control aphids. As aphid limitation by sublethal effects of PIs could be complemented by beneficial insects, the consequences of this new technology should be assessed on non-target insects. Parasitoids may indeed be exposed to these entomotoxins either via their hosts or by direct exposure to the plant itself. No deleterious effects of insecticidal recombinant PIs have been found on the aphid parasitoids Aphidius spp. (Ashouri et al., 2001; Cowgill et al., 2004) and Diaeretiella rapae (Schuler et al., 2001) parasitising aphids reared on OCI-expressing plants.

However, this lack of toxicity could be explained by a low and variable level of expression of the recombinant inhibitors in the plants. So, using an artificial diet, the objective of this study was to characterise and analyse the effects of OCI and SbBBI on the aphid Macrosiphum euphorbiae Thomas (Homoptera: Aphididae) and its parasitoid Aphelinus abdominalis Dalman (Hymenoptera: Aphelinidae) in conditions of controlled exposure. Aphelinus abdominalis is a solitary endoparasitoid commercially available as a biological control agent and commonly used for controlling several aphid species of economic importance (Mo¨lck and Wyss, 2001). Aphelinus abdominalis reduces aphid populations by parasitism and host feeding (Blu¨mel and Hausdorf, 1996). Effects of PIs enriched artificial diets were first investigated at the host level then on parasitoid development and adult longevity. Digestive protease activity of larval and adult stages and PI transfer through trophic levels were analysed to assess effects of direct exposure. The potential impact of PI-expressing plants on aphid parasitoids and their combined efficiency for aphid control are discussed.

2. Materials and methods 2.1. Reagents Trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E-64) and soybean trypsin-chymotrypsin Bowman–Birk Inhibitor (SbBBI) were obtained from Sigma. Pepstatin A and chymostatin were supplied by Alexis biochemicals. Ethylenediamine Tetra-Acetic Acid (EDTA) was obtained from Prolabo and pefablock from Interchim. The EnzChekR Protease Assay Kit (fluorescent protein BODIPYR-FL casein) was purchased from Molecular Probes. Oryzacystatin I (OCI) was obtained as previously described by Leple´ et al. (1995). The polyclonal anti-OCI (Leple´ et al., 1995) or anti-SbBBI (Bonade´-Bottino, unpublished results) were raised in rabbits and used as primary antibodies. The secondary antibody peroxidase-coupled goat anti-rabbit IgG (A 0545) was purchased from Sigma. ECL kit and nitrocellulose membrane HybondTM-ECLTM were obtained from Amersham Biosciences. 2.2. Artificial diets As a control and a basis for the protein dilution, a standard diet adapted for M. euphorbiae was prepared as described by Febvay et al. (1988) and modified by Down et al. (1996). OCI, SbBBI and bovine serum albumin (BSA) were incorporated to the standard diet to obtain the following diets: OCI-20, OCI-100, OCI250 and OCI-500 contained, respectively, 20, 100, 250

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and 500 mg/mL of OCI; SbBBI-20, SbBBI-100 and SbBBI-500, contained, respectively, 20, 100 and 500 mg/mL of SbBBI and BSA-500 contained 500 mg/ mL of BSA. The diet sachets were prepared as described in Couty et al. (2001) under aseptic conditions, sterilised by filtration through 0.45 mm filters (Millipore Corp., Bedford, MA) then kept frozen at
20 1C until used. To expose adult parasitoids to PIs through food intake, sugar solution of 70% (w/v) 1:1 glucose–fructose mixture was used as a sugar source for Aphelinus abdominalis female. BSA of 20 mg/mL was added to this sugar solution to stimulate the protease activity of this synovogenic parasitoid (control solution). This control solution was then supplemented with BSA, OCI or SbBBI at 100 mg/mL (BSA100, OCI100 and SbBBI100, respectively). 2.3. Insects The LB05 Sm clone of the potato aphid M. euphorbiae provided by Yvan Rahbe´ (INRA-INSA Villeurbanne, France) was reared in the laboratory on potato plants (Solanum tuberosum cv De´sire´e) in a controlled environment room (T ¼ 20  1 1C; RH ¼ 60  5%; 16L/8D). Neonate nymphs (12 h old) used in the experiments were issued from females synchronised on an excised potato leaf isolated in a plastic box. The mummies of Aphelinus abdominalis purchased from Biobest (Belgium) were placed in plastic boxes (17  11  7 cm) until adult emergence under the same conditions as described above. Emerging parasitoids were collected and fed ad libitum on cottonwool imbibed with sugar solution (glucose and fructose 70% (w/v)) during 48 h before infesting aphids. 2.4. Insect bioassays 2.4.1. Effects of protease inhibitors on M. euphorbiae development To study the effect of protease inhibitors on the development of M. euphorbiae, the following diets OCI20, OCI-100, OCI-500, SbBBI-20, SbBBI-100, SbBBI500, BSA-500 and the standard diet as control were tested. The protease inhibitors concentrations of these experimental diets used in this study are based on the quantities of gene products measured in plant tissues in which the gene is expressed. As emphasised by Couty et al. (2002), concentrations of 10 and 100 mg/mL protein delivered to aphids as food intake via artificial media are similar to the amounts potentially expressed into leaf tissue which seems to be close to the amount present in the phloem sap (Rahbe´ et al., 2003a). Five replicates of 15 neonate nymphs (0–24 h old) were set up for each diet. Aphids were checked daily to determine nymphal

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and adult mortality and pre-reproductive period (i.e. the period of time from birth until onset of reproduction). For each diet, aphids reaching the adult stage were collected and transferred on fresh diet of the same composition. Their offspring were counted and removed every day. Recorded values were divided by the number of living adults in the corresponding replicate to evaluate daily fecundity. Adults were checked during a period equal to their pre-reproductive period. This method reliably explains more than 98% of the final rm value (Le Roux et al., 2004). For each diet, the intrinsic rate of natural increase (rm) values were calculated according to the Lotka equation (Birch, P
r 1948 ): e m x l x mx ¼ 1; where x is the age, lx the agespecific survival and mx the age-specific fecundity. The Jackknife method (Meyer et al., 1986) was used to evaluate the variance of the rm with the ‘‘Petitr’’ program (J.S. Pierre, unpubl.). The finite rate of increase ðl ¼ erm Þ and the doubling time (DT ¼ ln 2=rm ) were evaluated according to DeLoach (1974). 2.4.2. Effects of protease inhibitors on Aphelinus abdominalis development Neonate aphids were fed for three days on OCI-100, SbBBI-100 and control diets. Fifteen replicates were set up for each treatment. Host infestation was performed by placing a single Aphelinus abdominalis female in a cylinder of artificial diet with ten 4-day-old nymphs for 24 h, then aphids were kept on their respective diet until mummification. Mummies were collected on the day of formation, individually weighed on a precision balance (Sartorius M 500P;72 mg) and then isolated into gelatin capsules until adult emergence. These parameters allowed to calculate the percentage of mummies formed (i.e. the ratio between the number of mummies formed and the number of aphid nymphs submitted to parasitisation) and the percentage of emergence (i.e. the ratio between the number of emerged parasitoids and the number of mummies formed). Larval and adult development time were defined as the period from host infestation to mummification or adult emergence, respectively. Newly emerged adult parasitoids were sexed. They were then isolated in Petri dishes (5.5 cm diameter) provided with a filter paper imbibed with water until death to evaluate their resistance to starvation. The tibia length was then measured under a binocular microscope to estimate the adult size. 2.4.3. Effects of protease inhibitors on Aphelinus abdominalis adult survival Fifteen 1-day-old females were placed in ventilated plastic boxes (17  11  7 cm) and allowed to feed for 4 h daily on a cotton-wool imbibed with sugar-control or BSA100 or OCI100 or SbBBI100. Dead insects were counted and removed every day. Three replicates

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were carried out under controlled conditions (T ¼ 20  0:5 1C; RH ¼ 60  5%; 16L/8D). 2.5. Determination of Aphelinus abdominalis digestive protease activity 2.5.1. Enzyme preparation Four-day-old neonate aphids issued from synchronised females were placed on cut potato leaves and parasitised by Aphelinus abdominalis females. Third instar parasitoid larvae were extracted by dissecting aphids in 0.15 M NaCl under a binocular microscope at 4 1C. Ten whole parasitoid larvae or 10 dissected larval or adult guts were placed in 30 mL 0.15 M NaCl buffer. Each sample was immediately homogenised on ice then centrifuged at 5000g at 4 1C for 15 min. Supernatants were aliquoted and stored at
20 1C. Adult guts were dissected out of cold-anaesthetised females and extracts made according to the above procedure. Total protein content of parasitoid extracts was determined by the Bradford (1976) method using BSA (1 mg/mL) as standard protein. 2.5.2. Inhibition assays Preliminary experiments (data not shown) showed that whole larva and larval gut extracts presented the same proteolytic profile. Thus, protease activity of both Aphelinus abdominalis adult guts and whole larvae was quantified using the fluorescent protein casein as substrate, at a final concentration of 0.15 mg/mL. Inhibition assays were carried out in 0.1 M Tris-HCl pH 8. An equivalent of 1.5 larvae or 8 adult guts per well was incubated for 15 min at ambient temperature with inhibitors, before adding the casein substrate. Fluorescence was measured every 2 min during 60 min at 37 1C on a LabSystems Fluoroskan II fluorimeter at 538 nm after an excitation at 485 nm. The class of chemical inhibitors and final assay concentrations tested are presented in Table 3. Larval and adult protease activities were also measured using a concentration range of two protein inhibitors OCI and SbBBI (0.1, 1, 10 and 100 mg/mL). All assays were performed in triplicate for larvae and duplicate for adults, with appropriate controls. The inhibition of the proteolytic activity was expressed relatively to the control. Inhibitors were dissolved in milli-Q water (E-64, pefablock, EDTA, SbBBI and OCI) or DMSO (pepstatin A and chymostatin). 2.6. Transfer of protease inhibitors through the three trophic levels 2.6.1. Aphids and parasitoids on artificial diet Three batches of aphids were raised on the following diets OCI-100, OCI-250, SbBBI-100, SbBBI-500 and control. The high mortality of aphids fed with OCI-500

diet led us to use a lower concentration (250 mg/mL). On the fourth day, the aphids of the first batch were parasitised as described before. At the beginning of the aphid cuticle melanisation (corresponding to the 3rd instar of the parasitoid larva), a group of aphids were dissected to collect parasitoid larvae. The remaining aphids were kept on their respective diet until mummification. Mummies were collected on the day of formation and placed in a Petri dish until adult emergence. The second aphid batch was reared for 12 days and then used for protein extraction, while the third batch was allowed to develop for 2 more days on the control diet. 2.6.2. Immunoassay blot Detection of SbBBI and OCI in protein extracts from aphids and parasitoids was performed by western analysis using anti-SbBBI and anti-OCI antibodies, respectively. Protein samples were extracted by homogenising aphids and parasitoids (larvae and adults) in 50 mM Tris-HCl, (pH 6.8) containing 1% SDS (3mL of buffer per individual). Laemmli buffer containing 2% bmercaptoethanol was then added and the samples were heated at 95 1C during 5 min. Samples were centrifuged for 15 min at 5000g at 4 1C. The supernatants were electrophoresed in 10% SDS-PAGE then blotted onto nitrocellulose membrane according to Towbin et al. (1979). OCI and SbBBI diluted in sample buffer were used as controls. After electrotransfer at 100 mA, the membrane was blocked for 1 h at room temperature with a solution of 5% (w/v) skimmed milk in TBS (10 mM Tris-HCl pH 7.5, 0.1 M NaCl) containing 0.25% (v/v) Tween-20. OCI or SbBBI antibodies (1:500) and the peroxidase-coupled goat antirabbit IgG (1:50 000) were used to hybridise the membrane. Immunostained bands were detected by chemiluminescence (ECL) according to the manufacturer’s instructions. 2.7. Statistical analysis Statistical analysis was done using STATISTICA 5.5 software (StatSofts, Tulsa, Oklahoma, USA). The Pearson’s w2 test was carried out to analyse the diet effect on aphid larvae survival. The effects of protease inhibitors on the demographic parameters of the aphids and the parasitism success and development of the parasitoid were analysed using one-way ANOVA, after data normalisation with arcsine transformation (Rao, 1951). The positive least significant difference test (PLSD) of Fisher was used to compare the means of each parameter when a significant effect was found. The acceptance level of statistical significance was Po0.05. The results are reported as mean7standard error (SE).

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SbBBI (Table 1). When compared to the insects reared on the control diet no demographic parameters were significantly affected for the aphids bred on the BSA-500 diet. Aphids reared on the SbBBI-100 and SbBBI-500 diets showed a significantly extended pre-reproductive period. With regard to daily fecundity, it was significantly reduced at the higher dose of SbBBI (SbBBI-500), but enhanced at the lower dose SbBBI-20. As a consequence of the alterations on post-embryonic development and fecundity, the intrinsic rate of natural increase (rm), and therefore the finite ratio of increase (l) (i.e. the number of aphids added to the population per adult and which will produce adults) were significantly reduced for M. euphorbiae reared on SbBBI-100 and SbBBI-500 diets. In contrast, the lowest SbBBI dose (20 mg/mL) did not lead to any alteration. Directly linked with the intrinsic rate of natural increase, the doubling time of populations fed on SbBBI-100 and SbBBI-500 diets were significantly lengthened.

3. Results 3.1. Effects of protease inhibitors on M. euphorbiae development 3.1.1. Nymphal survival The nymphal viability of M. euphorbiae (Fig. 1) was significantly reduced for the nymphs reared on OCI diets at all concentrations used (OCI-20, OCI-100 and OCI-500 diets, respectively, w2 ¼ 709:49; 499.95 and 2412.47 with df ¼ 33 and Po0:05). It is noteworthy that no aphid reached the adult stage when exposed to OCI. In contrast, no significant effect on nymphal survival was observed for the aphids fed on SbBBI or BSA diets (SbBBI-20, SbBBI100, SbBBI-500 and BSA-500 diets, respectively, w2 ¼ 2:99; 0.27, 12.73 and 1.23 with df ¼ 33 and P40:05). 3.1.2. Life history traits Since no adult was obtained with the OCI diets, effects of PI on life history traits were only measured for

OCI-20

100

OCI-100 OCI-500

90

Nymphal survival (%)

SbBBI-20 SbBBI-100

80

SbBBI-500 BSA-500

70

Control 60 50 40 30 1

3

5

7

9

11

13

15

17

19

21

23

25

27

29

31

Time (days) Fig. 1. Survival rate of Macrosiphum euphorbiae larvae reared on a control diet or on diets containing oryzacystatin I (OCI-20, OCI-100 and OCI500), soybean Bowman–Birk Inhibitor (SbBBI-20, SbBBI-100 and SbBBI-500) or bovin serum albumin (BSA-500). For control diet, OCI-20, OCI500 and SbBBI-100: n ¼ 78; for SbBBI-20 and OCI-100: n ¼ 79; for SbBBI-500: n ¼ 80; for BSA-500: n ¼ 65: n: number of aphid tested.

Table 1 Demographic parameters of Macrosiphum euphorbiae reared on diets containing soybean Bowman–Birk inhibitor (SbBBI-20, SbBBI-100 and SbBBI500) and bovin serum albumin (BSA-500)

Pre-reproductive period (days) Daily fecundity (larvae/female/day) rm (female/female/day) l (female/female/day) Doubling time (days)

Control diet n ¼ 78

BSA-500 n ¼ 47

SbBBI-20 n ¼ 74

SbBBI-100 n ¼ 73

SbBBI-500 n ¼ 72

F

P

14.670.1a 1.370.0a 0.1470.00a 1.1570.00a 5.070.1a

14.570.2a 1.370.0a 0.1470.00a 1.1570.00a 4.970.1a

14.870.2ab 1.570.0b 0.1470.00a 1.1570.00a 5.070.1a

15.270.2b 1.370.0ac 0.1370.00b 1.1470.00b 5.670.1b

15.970.2c 1.270.0c 0.1270.00c 1.1270.00c 6.270.2c

10.36 7.83 16.97 16.10 25.41

o o o o o

0.00 0.00 0.00 0.00 0.00

Results are expressed as mean7SE. rm: intrinsic rate of a natural increase; l: finite rate of increase; n: number of aphids tested. F: Fisher’s test value of one-way ANOVA analysis. P: P value of the Fisher’s test. Values in the same row followed by the same letter indicate no significant difference according to the PLSD test of Fisher (Po0.05). SE: standard error.

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3.2. Effects on Aphelinus abdominalis development These effects were investigated only for diets with 100 mg/mL of PIs because 500 mg/mL of OCI caused too much host mortality and 20 mg/mL could lead to underestimation of potential effects of PI plants on parasitoids. Larval development time was significantly extended for offspring developed in OCI-100 intoxicated aphids (Table 2). Mummification rate and nymphal development time were not affected while mummy weight was significantly lowered by about 10%. Adult emergence was also significantly decreased but without effect on the sex-ratio or on adult size. Finally, resistance to starvation was enhanced by 35%. When offspring developed in SbBBI-100 intoxicated aphids, the only observed effects were the reduced percentage of adult emergence and the enhanced resistance to starvation. In all groups, nymphal development time, adult size and resistance to starvation were analysed only for females due to the low number of emerged males. When females were directly exposed to PIs via the sugar solution diet, their longevity was not affected by OCI100 (32.571.9 days; n ¼ 43) or SbBBI100 (32.371.9

days; n ¼ 39) or BSA100 (31.871.8 days; n ¼ 42) in comparison to the parasitoids fed with the control glucose–fructose diet (33.372.1 days; n ¼ 40) (F ¼ 0:10; df ¼ 3; P40.05). 3.3. Determination of Aphelinus abdominalis digestive protease activity Chemical inhibitors were first used to determine classes of Aphelinus abdominalis digestive proteases. In whole larvae extract, four classes of protease inhibitor decreased the proteolytic activity (Table 3). With regard to serine protease inhibitors, both pefablock, a serine protease inhibitor, and chymostatin, targeting chymotrypsin as well as cysteine proteases, induced a significant inhibition of protease activity, which was stronger for chymostatin (88% of inhibition). However E-64, a highly specific cysteine protease inhibitor, caused only a slight inhibition (9%) compared to the inhibitors of the other classes. Intermediate proteolytic activity inhibition was measured with EDTA and pepstatin A. The four-inhibitor protease classes were also used to inhibit the proteolytic activities of adult gut extracts

Table 2 Different parameters (means7SE) of development and parasitism success of Aphelinus abdominalis on Macrosiphum euphorbiae fed either with or without protease inhibitors

Larval development time (days) Mummification rate (%) Nymphal development time (days) Mummy weight (mg) Emergence success (%) Sex ratio (% females) Adult size (mm) Resistance to starvation (days)

Control

n

OCI-100

7.770.1 (a) 54.676.2 15.6370.3 305.3710.6 (a) 58.877.9 (a) 80.578.8 37976 2.370.2 (a)

64 15 27 64 14 12 27 27

8.570.1 53.775.2 15.5370.4 272.879.9 42.076.8 71.279.8 38377 3.170.2

(b)

(b) (b)

(b)

n

SbBBI-100

n

Statistics

61 15 19 61 14 11 19 19

7.770.1 (a) 41.775.3 15.4370.4 302.3710.8 (a) 37.775.8 (b) 70.8 713.0 381713 3.370.3 (b)

52 15 14 52 14 12 14 14

F ¼ 15.02, Po0.05* F ¼ 0.83, P40.05 ns F ¼ 0.07, P40.05 ns F ¼ 3.04, Po0.05* F ¼ 3.23, Po0.05* F ¼ 0.19, P40.05 ns F ¼ 0.62, P40.05 ns F ¼ 5.06, Po0.05*

Different letters signify that treatment were significantly different at the 95% confidence level (PLSD test of Fisher). The acceptance level of statistical significance was Po0.05. *: indicates significant differences at Po0.05. ns: indicates no significant differences at Po0.05. For mummification rate, emergence success and sex ratio (n) correspond to the number of replicates and to the number of parasitoid offspring for the other parameters. SE: standard error.

Table 3 Effects of chemical inhibitors on the proteolytic activity of Aphelinus abdominalis Inhibitor

E-64 Pefablock EDTA Pepstatin A Chymostatin

Target proteases

Cysteine Trypsin and chymotrypsin Metallo-proteases Aspartate Chymotrypsin and cysteine

Final assay concentration

500 mM 10 mM 10 mM 100 mM 300 mM

Inhibition (% of control) Larvae

Adult

970 5371 3070 3970 8870

2070 2474
1376 3773 7070

Assays were performed in triplicate for whole larvae and in duplicate for adult gut at pH 8. Results are expressed as means7SE. SE: standard error. An equivalent of 1.5 larvae (9.7 mg of protein) or an equivalent of 8 adult guts (3.2 mg of protein) were incubated for 15 min at ambient temperature with inhibitors, before addition of substrate to start the reaction.

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Inhibition (% of control)

80 70

Adult-OCI

60

Adult-SbBBI Larvae-OCI

50

Larvae-SbBBI

40 30 20 10 0 -10

0.1

1

10

100

Inhibitor concentration (µg /mL)

Fig. 2. Effects of OCI and SbBBI concentration on the enzyme activity of Aphelinus abdominalis. Assays were performed in triplicate for whole larvae and in duplicate for adult gut at pH 8. An equivalent of 1.5 larvae (9.7 mg of protein) or an equivalent of 8 adult guts (3.2 mg of protein) were incubated for 15 min in ambient temperature with inhibitors, before addition of substrate to start the reaction. Results are expressed as means7SE. SE: standard error.

(Table 3). With regard to serine protease inhibitors, chymostatin induced 70% inhibition while pefablock inhibited only 24% of the proteolytic activity. The cysteine inhibitor, E-64, induced 20% inhibition of proteolytic activity. The aspartyl protease inhibitor, pepstatin A, induced 37% inhibition. The metallo-protease inhibitor EDTA enhanced adult gut proteolysis. The protein inhibitors of serine and cysteine proteases, SbBBI and OCI, respectively, were used against the protease activities of Aphelinus abdobminalis whole larvae and adult gut extracts. The assays performed with different concentrations of these PIs showed that SbBBI induced a significantly higher inhibitory effect compared to OCI (Fig. 2). The trypsin-chymotrypsin inhibitor SbBBI at 100 mg/mL inhibited 76% and 77% of larvae and adult protease activities, respectively. Nevertheless the cysteine protease inhibitor OCI (100 mg/mL), presented non negligible inhibition against digestive protease from whole larvae (38%) and adult gut extracts (39%). 3.4. Transfer of protease inhibitors through the three trophic levels Western blot analysis of the protein extracts of aphids reared for 12 days on SbBBI or OCI diets showed that the primary antibodies recognised the proteins ingested by aphids and the specific SbBBI (100 ng) and recombinant Eschericha coli OCI (10 ng). As expected, in extracts from aphids fed with control diet no hybridised band could be observed. Extracts of aphids fed with SbBBI diets showed hybridisation only for the highest (500 mg/mL) SbBBI dose (Fig. 3A). Extracts of aphids allowed to feed 2 days more on control diet did not present any hybridised band. In the positive control lane, the OCI protein appeared at 11.5 kDa as expected (Fig. 3B). In contrast, the hybridisation of the anti-OCI antibody with the electrophoresed extracts of aphids fed

Fig. 3. (A) Western blot analysis of aphids fed on diets containing SbBBI. Lanes 1 and 2 correspond to 10 and 100 ng of SbBBI, respectively. Extracts of aphids reared for 12 days on control diet (lane 3), on SbBBI-100 diet (lane 4) and on SbBBI-500 diet (lane 5). Extracts of aphids allowed to develop for 2 more days on the control diet after 12 days on SbBBI-100 diet (lane 6) or on SbBBI-500 diet (lane 7). Hundred micrograms of protein of aphid extracts were submitted to electrophoresis under reducing conditions. (B) Western blot analysis of aphids fed on diets containing OCI. Lanes 1 and 2 correspond to 0.1 and 10 ng of OCI, respectively. Extracts of aphids reared for 12 days on control diet (lane 3), on OCI-100 diet (lane 4) and on OCI-250 diet (lane 5). Extracts of aphids allowed to develop for 2 more days on the control diet after 12 days on OCI-100 diet (lane 6) or on OCI-250 diet (lane 7). 50 or 100 mg of protein of aphid extracts were submitted to electrophoresis under reducing conditions.

with OCI diets showed two major bands at 50 and 60 kDa and several proteins of lower molecular weights suggesting that ingested OCI bound to insect proteins. The presence of this OCI-protein complex was observed for both OCI-100 and OCI-250 diets, even when aphids were fed a control diet for 2 days after OCI intake. This analysis revealed that the OCI-protein complex accumulated in aphid tissues. Extracts of parasitoids developed in aphids fed with SbBBI-100 and SbBBI-500 exhibited a unique hybridisation band only for parasitoid larvae extracts dissected from aphids bred on SbBBI-500 diet (Fig. 4A). No hybridisation occurred in the adult extracts from SbBBI fed aphids, regardless of the dose. When host aphids were fed OCI diets, western blots did not reveal staining in larvae or adult parasitoids extracts (Fig. 4B).

4. Discussion The results presented here show an unexpected strong insecticidal effect of oryzacystatin I on Macrosiphum euphorbiae and a negative effect of soybean Bowman–Birk inhibitor (SbBBI) on aphid population growth. Both protease inhibitors influence the development of the aphid parasitoid Aphelinus abdominalis. In addition, our study of both indirect and direct risks of parasitoid exposure gives us clues to interpret the effects on the

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Fig. 4. (A) Western blot analysis of parasitoid larvae developed in aphids fed with SbBBI diets. Hundred nanograms of SbBBI (lane 1). Parasitoid larvae developed in aphids reared on control diet (lane 2), on SbBBI-100 diet (lane 4) and on SbBBI-500 diet (lane 6). Parasitoid adults emerged from aphids reared on control diet (lane 3), on SbBBI100 diet (lane 5) and on SbBBI-500 diet (lane 7). Thirty-five micrograms of protein of larvae and adults parasitoid extracts were submitted to electrophoresis under reducing conditions. (B) Western blot analysis of parasitoid larvae developed in aphids fed with OCI diets. Ten nanograms of OCI (lane 1). Parasitoid larvae developed in aphids reared on control diet (lane 2), on OCI-100 diet (lane 4) and on OCI-250 diet (lane 6). Parasitoid adults emerged from aphids reared on control diet (lane 3), on OCI-100 diet (lane 5) and on OCI-250 diet (lane 7). Thirty-five micrograms of protein of larvae and adults parasitoid extracts were submitted to electrophoresis under reducing conditions.

parasitoid and to discuss the potential of combining PI expressing plants and parasitoids to control aphids. M. euphorbiae fed with OCI supplemented diets exhibited strongly reduced nymphal survival regardless of the PI dose used. In addition, no offspring were observed for these aphids. Such drastic effects on M. euphorbiae could be explained since aphids digestive proteolytic activities seem to rely mainly on cysteine proteases (Cristofoletti et al., 2003; Rahbe´ et al., 2003a; Deraison et al., 2004). It is noteworthy that previously reported insecticidal effects of OCI delivered in vitro at comparable doses to other aphid species (Rahbe´ et al., 2003a) were significantly lower. This observation highlights a variable susceptibility of the different aphid species towards OCI. SbBBI intake did not affect nymphal viability of M. euphorbiae. In contrast, demographic parameters expressed through the synthetic rm value were significantly altered. As no difference occurred when the BSA protein was added to the standard diet, one could assume the inhibitory activity of SbBBI was responsible for the toxic effect observed. It should be noted that the variation of the intrinsic rate of natural increase (rm) was closely related to the concentration of SbBBI used. When fed with this PI, Acyrthosiphon pisum nymphal survival was significantly reduced (Rahbe´ et al., 2003b) while such effect was not observed on M. euphorbiae. This comparison underlines again the variable susceptibility of aphid species towards PIs. The influence of SbBBI could be the consequence of the disruption of non-digestive proteases. Immunologically active proteins have already been reported to cross through the intestinal barrier in various insect orders (Allingham et al., 1992; Powell et al., 1998; Cherqui et al., 2003). Internalisation of active SbBBI into mice enterocytes was reported by Billings et al. (1991). Whereas no study

was performed with SbBBI in insects, PsTI-2, another inhibitor of the BBI family, was shown to accumulate in various tissues of Acyrtosiphon pisum and Aphis gossypii (Deraison, 2002). This first part of our work predicts potential indirect effects of both PIs, through host mortality or reduced host quality. Our study of PIs direct effect on the parasitoid showed that the protease activities revealed in the whole larvae or adult gut extracts were highly inhibited by the serine protease inhibitor SbBBI. Aphelinus abdominalis proteases were also inhibited by chymostatin, which is a common inhibitor of both chymotrypsin and cysteine proteases. Nevertheless, E-64 did not inhibit the digestive proteolytic activity suggesting that the major A. abdominalis proteases belong to the serine protease class and are chymotrypsin-like. Such serine protease-based digestive metabolism was previously shown in the larval stage of the eulophid ectoparasitoid Eulophus pennicornis (Down et al., 1999) and in the honeybee (Burgess et al., 1996). However, OCI which is a specific protein inhibitor of cysteine proteases has a non-negligible inhibitory effect against digestive proteases of larvae and adult gut extracts. These effects could be induced by unpredicted allosteric interactions between this PI and the parasitoid proteases (Bode and Huber, 2000). An enhancement of adult gut proteolysis by the metallo-protease inhibitor EDTA has been observed. Such activation in response to the use of EDTA has been reported on the midgut cysteine protease of Acyrthosiphon pisum (Cristofoletti et al., 2003). Digestive proteases of the parasitoid and their sensitivity to OCI and SbBBI indicate a potential risk of toxicity of both PIs and particularly of SbBBI at the 100 mg/mL concentration, considered as a realistic concentration in plant tissues. The analysis of PI transfer from the artificial diet to tissues of aphids and parasitoids can indicate the risk of exposure during parasitic development. Immunoassays made it possible to detect both OCI and SbBBI in protein extracts of aphids exposed to the PIs. The high molecular weights of anti-OCI-hybridised proteins suggested that ingested OCI was bound to insect proteins. Moreover, the presence of this OCI-aphid protein complex in extracts of insects fed for 2 additional days on a control diet indicated its fixation to aphid tissues. As cysteine protease ensures major digestive proteolytic activity in aphids (Rahbe´ et al., 2003a; Deraison et al., 2004), the observed complex would be most probably an OCI-cysteine protease complex, either with the processed or proprotein forms, which are both found at comparable levels in aphid tissues (Deraison et al., 2004). Alternatively, nondigestive targets of OCI would explain the presence of this protein complex even 2 days after the last OCI ingestion (higher molecular weight bands in western analysis; Fig. 3B). This hypothesis is supported by

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immuno-histological studies performed on Myzus persicae showing that OCI crosses the midgut epithelium, reaches the hemolymph and therefore accumulates in oenocytes and bacteriocytes (Rahbe´ et al., 2003a). Such potential presence of OCI in aphid hemolymph implies a risk of exposure during host-feeding by A. abdominalis females. In addition, larvae of the Aphelinus genus ingest the whole aphid content prior to pupation (Viggiani, 1984). Because OCI accumulates in tissues of exposed aphids, it could therefore intoxicate the parasitoid larvae. However, OCI could not be detected in both parasitoid adult and larvae regardless of the OCI concentration used to feed the host aphids, indicating that remnant accumulation at the third trophic level is very low (less than 2 ng of OCI per parasitoid). Regarding SbBBI, it was observed at the expected molecular weight of 8 kDa in aphid extracts, so it remains free when ingested. The absence of any hybridisation band when aphids were allowed to develop 2 additional days on a control diet shows that ingested SbBBI does not accumulate in the aphid body. However, we cannot reject the hypothesis that low amount of SbBBI crossed the gut epithelia, because less than 20 ng of SbBBI per aphid was undetectable with the immunological method used. No band was observed in extracts from both aphids and parasitoids coming from the SbBBI-100 diet, indicating that less than 20 ng per individual was present in aphids or parasitoids. According to our analysis of parasitoid protease inhibition by SbBBI, such a small quantity should not cause protease inhibition in larval or adult parasitoids. The 8 kDa band was observed in aphids and parasitoid larvae fed the SbBBI-500 diet, but not in adult parasitoids. SbBBI ingested by parasitoid larvae could have been metabolised due to high proteolytic activity known in larval instars of endoparasitoids. As extracts were done with larvae dissected just prior to pupation, SbBBI could also have been excreted in the form of meconial pellets, as reported in the parasitoid Aphidius ervi developed on M. persicae reared on lectin-enriched artificial diet (Couty et al., 2001). Considering the results of the dose–response analysis of protease inhibition on the parasitoid and the lethal and sublethal effects of PIs on the host aphid, we anticipate that the effect observed is an indirect effect of the PIs on the parasitoid, via lower host quality, rather than a direct effect. In Aphelinus abdominalis, some parameters of development and parasitism success were affected, depending on the type of PIs in the diet of its aphid hosts. Some parameters were not altered regardless of the inhibitor: mummification rate, nymphal development time, sexratio and adult size. Mummification rate depends on the percentage of parasitised aphids (host-acceptance) and on successful larval development. Thus, in our nonchoice experimental procedure, parasitoid females ac-

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cepted PIs-intoxicated aphids as well as control ones. This is not always the case since a cowpea trypsin inhibitor supplemented diet delivered to the tomato moth altered its susceptibility to parasitism by Eulophus pennicornis (Bell et al., 2001). When delivered to aphids via artificial diet, OCI led to extended larval development, reduced mummy weight and emergence success, while exposure to SbBBI only affected this latter parameter. This loss of weight is in agreement with other results showing a 48% reduction of size of M. euphorbiae fed 100 mg/mL OCI (data not shown.). However, in our study the size of emerging adult parasitoids was not affected by OCI. This could be explained by the death of small-sized adults, leading to the observed reduced emergence success. Alternatively, protease activity is intense during metamorphosis of the parasitoid larvae and might be impaired even by very small amount of the PIs, leading to mortality. SbBBI was indeed reported to disrupt the larval molt of Spodoptera littoralis (Faktor and Raviv, 1997). As host size can be lowered by OCI and Aphelinus abdominalis lays male eggs in smaller hosts (Honek et al., 1998), effects were expected on the sex-ratio. However, no alteration of this parameter was observed in our experimental conditions. Surprisingly, parasitoid females emerged from aphids reared on both OCI and SbBBI supplemented diets showed a significantly enhanced resistance to starvation. As reported in M. persicae bred on OCI-expressing transgenic plants (Rahbe´ et al., 2003a), PIs could be present in honeydew and haemolymph that represent potential food sources for synovigenic parasitoids like Aphelinus abdominalis. However, direct exposure to PIs during adult food intake did not affect females longevity, while SbBBI and OCI (100 mg/mL) induced 77% and 39% inhibition of digestive protease activity, respectively. Nevertheless, possible effect on adult parasitoid could not be rejected as variation in haemolymph composition, and thus its quality as a food source, could occur in intoxicated aphids. The few data documenting effects of PIs-expressing plants focussed on OCI and indicate variable effects, depending on the host–parasitoid system. When developed on M. euphorbiae feeding on OCI-expressing potatoes Aphidius nigripes exhibited increased size and fecundity (Ashouri et al., 2001). In contrast, OCIexpressing oilseed rape did not induce any influence on Diaeretiella rapae bred on M. euphorbiae (Schuler et al., 2001). Neither the fitness of Aphidius ervi females nor the sex ratio of their offspring were affected when the parasitoids developed on aphids feeding on OCIpotatoes (Cowgill et al., 2004). With the objective of obtaining reliable effects on the parasitoid, controlled amounts of PIs were delivered via artificial diets while PI production in plants might vary according to transgenic line, plant physiology and tissue

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nature (Cloutier et al., 2000; De Leo et al., 2001; Cowgill et al., 2004). Our results allowed us to estimate the risk of exposure and the potential effects of plant PIs at two trophic levels, by combining immunological, biochemical and biological approaches. As serine protease inhibitors mainly target lepidopteran pests, no study focussed on the influence of such inhibitors on aphid parasitoids. Thus, our results constitute the first report of the influence of a serine protease inhibitor on an aphid parasitoid. Finally, our data provide relevant clues to predict potential efficiency of combining PI plants and parasitoids to control aphids. Indeed, in the case of plants expressing cysteine proteases such as OCI, aphids will not reproduce but will allow development of the parasitoid Aphelinus abdominalis, with fitness impairment. In the case of plants expressing serine protease such as SbBBI, which do not target aphids, aphid development and reproduction will be only slightly impaired, but production of adult parasitoids will be impaired. In such case where the plant protection method is more noxious to parasitoids than to secondary pest insects, demographic explosion of the latter could occur (Hardin et al., 1995; Longley et al., 1997).

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