parasitoid interactions: critical timing of parasitoid-derived products

Journal of Insect Physiology 44 (1998) 721–732

Host/parasitoid interactions: critical timing of parasitoid-derived products Darcy A. Reed a, John J. Brown b

b,*

a USDA-ARS, Western Cotton Research Laboratory, Phoenix, AZ 85040, USA Department of Entomology, Washington State University, Pullman, WA 99164-6382, USA

Received 1 October 1996; accepted 13 October 1997

Abstract Short-term in vitro incubations were used to examine the ability of endoparasitoid larvae to produce and release both ecdysteroids and proteins into their environment. Second instar larvae of both Chelonus near curvimaculatus and Ascogaster quadridentata were observed by SDS–PAGE to release temporally-similar polypeptides in the 20–30 kD Mr range. Peak occurrence of these polypeptides coincided with shedding of the anal vesicle, immediately prior to ecdysis to the third instar. Ecdysis also coincided with the switch from endoparasitic to ectoparasitic development in vivo. Polyclonal antibodies were generated against a particular 27 kD polypeptide of Chelonus, which was found to be species-specific and localized primarily within the anal vesicle during the latter part of the second stadium and whole body homogenates of third instars. In vitro incorporation studies using 35S-methionine indicated rapid changes in the synthetic abilities of second instar larvae shortly before ecdysis. The production and release of ecdysteroids, as measured by RIA, was found to precede the peak occurrence of the 27 kD polypeptide and ecdysteroid presence was undetectable following the molt. In contrast, the polypeptides were observed to gradually increase prior to the molt and slowly decrease after the molt. The Chelonus polypeptide was not detected in host tissues until after parasitoid egression.  1998 Elsevier Science Ltd. All rights reserved. Keywords: Ecdysteroids; In vitro culture; Developmental arrest; Endoparasitoids; Proteins; RIA; SDS–PAGE

1. Introduction The general scenario for insect host/endoparasitoid relationships is as follows: (i) the host is attacked by the adult female parasitoid; (ii) the host exhibits physiological distress due to parasitization, eventually resulting in cessation of activity; which, (iii) culminates in the egression of the parasitoid progeny from the host hemocoel resulting in host death. Parasitized lepidopteran larvae exhibit a variety of signs and symptoms after having been attacked by endoparasitic wasps. Among many host/parasitoid systems, the most easily recognizable event is a slowing or cessation of growth and development, also termed developmental arrest. Disruption of development is mainly attributed to abnormalities in various aspects of the host endocrine system. Lepidopteran hosts of egg-larval chelonine parasitoids

* Corresponding author. Tel.: 509-335-5505; Fax: 509-335-1009; E-mail: [email protected] 0022–1910 /98 /$19.00  1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 0 5 - 5

(Family Braconidae) differ from most host/parasitoid systems intensively studied thus far. Host development is first accelerated by skipping the last larval stadium and exhibiting prepupation behavior, wandering and spinning of cocoons, one larval stadium earlier than normal; and then, host activity and development ceases just prior to parasitoid egression (Jones et al., 1981; Brown et al., 1990; Grossniklaus-Bu¨rgin and Lanzrein, 1990). Metamorphosis and reproduction, as well as diapause, are governed by hormonal titers; specifically, juvenile hormones and ecdysteroids (Nijhout, 1994). In general, high levels of juvenile hormone accompanied by low levels of ecdysteroids results in maintainence of tissues in the immature state; whereas, low juvenile hormone levels and high levels of ecdysteroids promotes differentiation and metamorphic development. The first larval molt of chelonine parasitoids may coincide either directly with the host’s molt into the fourth stadium in the case of Chelonus (Soldevila and Jones, 1993) or several days after spinning activity in the case of Ascogaster (Brown et al., 1993). Host development is arrested after

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completion of the cocoon and is terminated by apolysis of the integument and the subsequent emergence of the third instar (L3) parasitoid. Compounding the complexity of host/parasitoid physiological relationships is the involvement of polydnaviruses (viruses associated with the calyx fluid of the female wasp and introduced into the host upon oviposition) and teratocytes (serosal cells of the parasitoid egg). These wasp-associated components have been implicated in causing many of the physiological abnormalities seen in parasitized larvae, particularly with regards to the disruption of the normal endocrine events (Fleming, 1992; Stoltz, 1993; Dahlman and Vinson, 1993). Recently, research has begun on products emanating from the developing parasitoid larvae themselves (Vinson et al., 1994; Pfister-Wilhelm and Lanzrein, 1996) which may influence host development. The timing of the endocrine malfunction involved in host developmental arrest is critical and in some model systems disruption appears exclusively limited to particular host stadia (Dover et al., 1995) and; therefore, associated with a particular developmental stage of the parasitoid larva (Pfister-Wilhelm and Lanzrein, 1996). In hosts of chelonine parasitoids there is a premature drop in JH which is presumed to initiate precocious metamorphosis (Jones, 1985). There is currently some debate as to whether this alteration in JH levels is due to the developing parasitoid larva (Grossniklaus-Bu¨rgin and Lanzrein, 1990; Pfister-Wilhelm and Lanzrein, 1996) or to maternally derived products other than the progeny (Jones, 1987). However, there is agreement that the corpora allata is the target of this regulation. Here we describe the timing of ecdysteroid production fluctuations in concert with proteinaceous contributions made by the developing parasitoid larva just hours before egression from the host. We observed a temporal correlation between the production and release of ecdysteroids and the appearance of a particular low molecular weight polypeptide during in vitro incubation of a chelonine parasitoid. Although the function of the parasitoid-derived protein is currently unknown, the careful timing of its appearance is suggestive of an involvement in host developmental arrest or subsequent events crucial to the successful emergence of the parasitoid from the host hemocoel.

2. Materials and methods 2.1. Insects Cabbage looper (Trichoplusia ni) and codling moth (Cydia pomonella) larvae were reared on artificial diet (Henneberry and Kishaba, 1966; Brown, 1980). T. ni is a host for Chelonus sp. near curvimaculatus and C. pomonella is a host for Ascogaster quadridentata. Both

wasps are egg-larval solitary endoparasitoids. T. ni adult mating and oviposition cages consisted of circular plastic containers (2.25 l capacity) lined with removable paper toweling as an oviposition substrate. Cages were sealed with plastic lids, modified with wire mesh screening for ventilation. Adult codling moth cages were constructed of wire mesh and lined with waxed paper. Moths were given 10% sucrose and 10% casein solutions via moistened cotton dental wicks for carbohydrate and protein nutrition, respectively. Toweling and waxed paper with adhering eggs (egg strips) were removed daily. Egg strips were placed in their respective parasitoid cages, rectangular glass enclosures (37.85 l capacity), for parasitization. Chelonus parasitoids were graciously provided by Davy Jones, Univ. of Kentucky (Lexington, KY) in 1990 and since that time have been continuously reared at Washington State University (Pullman, WA). Ascogaster parasitoids were collected from parasitized codling moth larvae obtained each autumn from abandoned apple orchards and continuously reared in the laboratory. Adult wasps were given moisture via wet cotton dental wicks and honey thinly smeared onto pieces of acetate served as a carbohydrate source. Parasitoids were allowed to oviposit into host eggs ( ⬍ 24 h old). Following parasitization, host eggs hatched ca. 4 days after oviposition (25°C, 70% R.H.) and neonates were given artificial diet (see Brown, 1980 for diet formulation and rearing procedures). To ensure the use of temporally similar hosts and parasitoids for experimental purposes, wandering host larvae were removed daily from diet containers and placed in smaller, transparent cups (35 cm3) (Fill-Rite) sealed with paper lids (Stanpak) and allowed to spin cocoons. Parasitoids emerged from hosts as third instars (L3) ca. 4–6 days post-wandering of the host at 25°C. Development of the Chelonus population was much more synchronous than that of Ascogaster. Chelonus larvae also developed within a shorter time period within the host’s ultimate stadium than did Ascogaster, with third instars emerging ca. 4 days following host initiation of wandering, rather than six or more days following wandering as was the case with Ascogaster. 2.2. In vitro incubation Data were collected regarding each parasitoid (instar, presence or absence of the anal vesicle) and its host (physiological age or behavior) prior to in vitro incubation of the parasitoid. Parasitoid larvae were excised from hosts and incubated in 2 ml conically shaped, capped polyethylene vials (Baxter) containing 100 ␮l of Grace’s media without hemolymph (GIBCO–BRL). Gentamycin (1 mg/ml) (Sigma) was added to the incubation medium to inhibit fungal contamination. Vials containing parasitoid larvae were incubated in a water

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bath (18L:6D, 25°C) with constant gentle agitation. The various instars of Chelonus and Ascogaster were incubated in vitro for 24 h. Second instar larvae (L2) were rinsed briefly prior to individual incubation. First stadium (L1) parasitoids were incubated in groups of 5 to 10 larvae, to faciliate the detection of any liberated proteins by silver staining and were not rinsed due to their small size and fragility. Following incubation and immediately prior to collection of the medium, it was noted whether: (i) the parasitoid was either submerged beneath or floating on the media surface; (ii) the outer surface of the anal vesicle (AV) was still attached to the body or had been shed; (iii) a parasitoid exuvium was present, indicating a molt; and (iv) the larva had spun silk. Only data from samples containing larvae which were alive after completion of the incubation period were retained. Samples containing damaged larvae or cloudy media indicating degradation or contamination were always discarded. Media were collected from incubation vials of individual or pooled larvae, placed in polypropylene microcentrifuge tubes, and either used immediately or stored at − 80°C. 2.3. Polyacrylamide gel electrophoresis of materials released in vitro by parasitoids Aliquots of media samples were collected, diluted 1:1 with SDS sample buffer [2.0 ml 10% SDS, 0.4 ml 2mercaptoethanol (2-ME), 1.0 ml glycerol, 0.63 ml 1.0 M Tris (pH 6.8), 6.0 ml distilled water (dH2O), and bromophenol blue dye (to color)], boiled for 4 min and centrifuged (4 s, 14,000g) to pellet debris. Samples were electrophoresed onto 12.5%, 0.75 mm thick discontinuous polyacrylamide gels (Laemmli, 1970), for 4 h at 15 mA constant current in a water-cooled Hoefer SE-600 vertical slab gel apparatus. A mixture of low Mr standards (14.2–66 kD) (Sigma) were run in parallel. The apparent molecular weight of the denatured form of the most dominant low Mr Chelonus protein was derived from its varying mobilities on an SDS–PAGE crosslinkage series (8, 9, 10 and 11% acrylamide) alongside the protein Mr standards and calculated using Ferguson plots (as described by Dunbar, 1987) and found to be ca. 27 kD (data not shown). Polyclonal antibodies were generated against this polypeptide of Chelonus (see below). Samples were also subjected to non-denaturing PAGE and subsequent Western blotting techniques to determine the approximate Mr of the native protein (data not shown). Upon completion of electrophoresis, gels were fixed in preparation for either silver staining (Wray et al., 1981) and/or staining with Coomassie Blue R-250 (Laemmli, 1970). Double-staining first with silver and then with Coomassie Blue allowed easy visualization of polypeptides which stained negatively with silver. Following staining, gels were dried on pieces of chromatog-

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raphy paper with a BioRad vacuum drier (BioRad Laboratories) for 1 h at 80°C or placed between two pieces of ultraclear cellophane (Research Products International Corp.) and allowed to air dry. 2.4. Polyclonal antibody production Polyclonal antisera was generated against the dominant low molecular weight protein (27 kD) produced by L2 Chelonus sp. larvae (Fig. 1A) because it was present in greater quantities, consistently and predictably produced, and appeared as a discrete band compared to the predominant Ascogaster proteins (Fig. 1B). The use of Chelonus was also preferred because of the amount of information already existing about the developmental physiology of the host (Jones et al., 1981) and of the parasitoid/host relationship (Jones, 1985; Jones et al., 1986). Immunodetection methods were necessary to determine if the dominant low Mr polypeptide was being produced in samples, at levels below the detection threshold of silver staining, and to localize the source of the protein. Aliquots, of in vitro media in which L2 Chelonus parasitoids had molted during incubation, were tested for the presence of the 27 kD peptide by SDS–PAGE and pooled if positive. Pooled samples were filtered through a Millipore Ultrafree-MC filter unit (Millipore Corporation) with a cutoff of 30 kD to eliminate extraneous higher Mr proteins. These filtrates were subjected to SDS–PAGE (12.5%) under denaturing conditions and resultant gels stained with Coomassie Blue. Four hundred ␮l of filtered in vitro fluid were loaded onto each gel and electrophoresed and stained with Coomassie Blue as described above. The procedure used for production of antisera was a modification of the protocols described by Dunbar (1987). The band corresponding to the 27 kD peptide was excised from gels which had been stained with Coomassie Blue and soaked in 95% ethanol for 1 h to fix the peptide within the gel slice. The slice was then emulsified in 5.0 ml of 1X phosphate buffered saline (pH 7.5) between two hypodermic syringes with 18 gauge needles connected by a piece of sterile polyethylene tubing (I.D. 1.19 mm, O.D. 1.70 mm). No additional type of adjuvant was used since the polyacrylamide alone acts as an adjuvant. Rabbits possessing subcutaneously implanted, perforated plastic balls specifically for antibody production (Hillam et al., 1974), were used. Preimmune sera from within the plastic balls were collected to provide a baseline of non-specific antibody production. The animals were injected with prepared antigen (400 ␮l equivalents) directly into the center of the ball. Rabbits received three booster injections at three week intervals. Immune sera was collected prior to each booster injection. One per cent sodium azide was added to all sera samples to pre-

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Fig. 1. SDS–PAGE (12.5%) gels of proteinaceous secretions from chelonine endoparasitoids which had been incubated in vitro in Grace’s medium for 24 h. Lanes represent 20 ␮l of incubation medium. (A) Polypeptides released by various developmental and behavioral stages of second stadium Chelonus parasitoids and double-stained with Coomassie Brilliant Blue R-250 and silver. Larvae which were sunken beneath the medium surface or floating on the surface released much less protein, based on intensity of staining of bands, than those which molted from second to third instar during incubation. Note the progression and increase in detectable protein in the 23–27 kD range. Arrows indicate the 23, 25, 26 and 27 kD polypeptides. (B) Polypeptides released by second stadium Ascogaster parasitoids and stained with silver. Lane 4 contains medium in which the larva molted during incubation. All other lanes contain medium in which larvae did not ecdyse. Arrow indicates the major low Mr range (ca. 26 kD) polypeptide associated with the molt.

vent microbial contamination. Freshly collected sera were retained and frozen in 1.0 ml aliquots at − 20°C. Only the final antiserum collected from the plastic ball was used for the experiments. 2.5. De novo synthesis (35S-methionine incorporation) L2 parasitoids, excised from hosts, were incubated in methionine-free Grace’s media without insect hemolymph (GIBCO-BRL, Grand Island, NY) to which 35Smethionine (ICN Biomedicals, Inc., Costa Mesa, CA) had been added (50 ␮Ci 100 ␮l−1 Grace’s) similar to the amount used by Baehrecke et al. (1992). Vials containing 35S-methionine-labeled Grace’s media but without parasitoids were incubated as controls. After a 48 h incubation period, under conditions given above, media was collected as before. Sample media were diluted with equal volumes of 20% TCA and placed on ice for 15 min. Samples were then microfuged for 5 min at 14,000g. The supernatant was placed in scintillation vials and the pellet was resuspended in 10% TCA and microfuged again. The pelleting, resuspension and collection of supernatant was repeated twice. The final pel-

let was suspended in 100 ␮l of sample buffer, boiled for 4 min and analysed by SDS–PAGE and autoradiography. Incubation media were processed for SDS–PAGE as described above. Samples were electrophoresed and gels were silver stained and dried on an Hoefer SE 540 gel dryer. Autoradiographs were made using X-ray film (X-Omat AR, Eastman Kodak Company, Rochester, NY) in contact with dried gels and intensifying screens (DuPont NEN Research Products, Boston, MA) and held within X-ray film cassettes (Kodak) sandwiched between transparent plastic plates at − 80°C for 4 days. Autoradiographs were observed for detection of de novo proteins. 2.6. Detection and localization of the 27 kD polypeptide of Chelonus by Western blotting Electrophoresed gels were blotted onto ImmobilonPVDF transfer membrane (Millipore Corp.) using a TE 42 Transphor Electrophoresis Unit (Hoefer Scientific) according to instructions provided by manufacturer of the membrane. Western blotting techniques were modified after Towbin et al. (1979). Membrane blots were

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placed in blocking solution for 30 min to reduce nonspecific binding; probed with primary antibody (1:1000) for 1 h; washed in blocking solution for 5 min (5X); incubated with secondary antibody (1:10,000), donkey anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Life Sciences), for 1 h; and washed in blocking solution 5X for 5 min. Immunoreactivity was visualized using an Enhanced Chemiluminescence Kit (Renaissance-ECL, Amersham; as described in kit literature). Permanent records of immunoblots were preserved on X-ray film (Kodak) and developed according to manufacturer instructions. Various body organs (e.g. midgut, salivary glands, anal vesicle) of L2 larvae of Chelonus were isolated, pooled, and homogenized. Samples were subjected to SDS–PAGE and Western blotting techniques in an effort to localize the source of the 27 kD polypeptide. To determine whether or not the proteins came from the AV, late L2 larvae having intact AVs were dissected from hosts and collected in 100 ␮l of Grace’s medium. Once the desired number of parasitoids had been isolated, they were individually removed from the pool and the AV severed and placed into fresh medium. AVs from eighteen individuals were collected in 100 ␮l Grace’s medium and homogenized. Both types of collected media were analysed by SDS–PAGE/silver staining. 2.7. Radioimmunoassay (RIA) for ecdysteroids Wandering Trichoplusia ni larvae parasitized by Chelonus were collected on three consecutive days. On day 3, all hosts were observed for wandering, spinning and spun behavior, and the individual parasitoids excised for in vitro incubation. It was consistently found that Chelonus parasitoids would egress from the host hemocoel, as third instars, four days after the host initiated wandering behavior. The media were collected and replenished at 12 h intervals, until 12 h after each parasitoid had molted. The collected medium from each sample was split for analysis between SDS–PAGE (20 ␮l), for qualitative protein content, and RIA (40 ␮l), for the presence of ecdysteroids. RIA procedures have been described previously (Brown and Reed-Larsen, 1991). 3 H-ecdysone (NEN; specific activity 89 Ci mmol−1) was used as a competitive substrate and antibodies were a gift of S. Takeda (National Institute of Sericultural and Entomological Science, Tsukuba, Japan). Late L2 parasitoids were incubated in in vitro media as mentioned earlier. Aliquots (40 ␮l) were frozen at −80°C and processed for radioimmunoassay (RIA) for the presence of ecdysteroids. Yields of picogram amounts of ecdysteroids per 40 ␮l aliquot were recorded and graphed as amounts per microliter. A minimum number of eight individually incubated larvae from a given set of larvae taken from same day hosts were sampled for the production of ecdysteroids and the

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yielded amounts were averaged for each 12 h incubation period.

3. Results 3.1. Temporal release of low molecular weight proteins Chelonine endoparasitoids were found to release a variety of proteins into the in vitro medium (Fig. 1). The number of distinct bands appeared to increase with increasing age within the stadium. The progression of premolt events during the incubation period are described as: (i) sunken or submerged beneath the surface, (ii) floating on the surface or (iii) molted to the third stadium. The behavior of each larva during incubation was dependent on its age and extent of development within the second stadium, with older larvae tending to float immediately upon excision from the host. Four major bands from Chelonus (Fig. 1A) and one major polypeptide band from Ascogaster (Fig. 1B) were found within a relatively low Mr range (23–27 kD). These bands stained intensely when the L2 parasitoids shed their anal vesicles (AVs) and molted during the 24 h incubation period. These same bands were not detectable by silver staining (Fig. 2A) or immunoblotting (Fig. 2B) in secretions from L1 larvae of either species. The antibody for the 27 kD protein of Chelonus was found to be highly specific and did not bind to the slightly lower Mr polypeptide (ca. 26 kD) found in A. quadridentata larvae which had released their AVs during incubation (Fig. 2); although, it did bind to secretions from Chelonus larvae which had intact AVs and floated, as well as to larvae which molted during incubation. The antibody also did not bind to any other polypeptides of Chelonus. The variety of protein bands observed in incubation media of L1 larvae (Fig. 2; Lanes A-L1 and CL1) is likely due to the presence of host hemolymph proteins since these lanes represent 5–10 pooled larvae that were not rinsed prior to incubation in vitro. A more precise Mr determination of the four bands of Chelonus was made using Ferguson plots. Both Ascogaster and Chelonus protein profiles revealed a 26 kD polypeptide. However, the 27 kD Chelonus peptide was considered a more suitable antigen because of its apparent greater abundance and predictability of production, as compared to the 26 kD Ascogaster protein in this region (compare intensity of staining of bands in Ascogaster lanes (A–AV release) with that of Chelonus (Cfloat or C-molt) in Fig. 2A), and thus facilitating the collection of protein in sufficient quantities to elicit an acceptable vertebrate immune response. The timing of the second larval molt and; therefore, the presence of the desired protein of Ascogaster were harder to anticipate than that of Chelonus since the molt of Ascogaster

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Fig. 2. (A) Silver-stained SDS–PAGE (12.5%) gel and (B) corresponding Western blot comparing the profiles of proteins released into Grace’s medium by Chelonus (C) and Ascogaster (A) parasitoids after 24 h in vitro incubation period. L1 = medium from first stadium larvae incubated in groups of five. EL2 = individually incubated early second instar larvae which were rinsed prior to incubation. The behavioral position of the larvae at the time of media collection was described as: sunken (larvae which were submerged beneath the medium surface), float (larvae which floated on the medium surface), molt (larvae which molted to the third instar during incubation) and AV release (larvae which released the outer layer of the anal vesicle but did not complete ecdysis during incubation). Numbers indicate molecular weight markers (in kilodaltons) and arrow indicates the position of the 27 kD protein of Chelonus. Note the appearance of host hemolymph storage proteins ( > 66 kD) in samples of incubation fluid from first instar parasitoids. The Western blot was probed with polyclonal serum (1:1000) against the 27 kD protein of Chelonus and visualized with chemiluminscence. Film was exposed for 10 min. Only media from Chelonus parasitoids that floated or molted during incubation were immunoreactive for the 27 kD protein. Twenty ␮l of incubation fluid were loaded per lane.

appeared to be keyed into some factor other than the host’s molt into the final stadium. A. quadridentata does not intiate its first larval molt in accordance with the host entering the precocious final stadium, as do various Chelonus sp. (Soldevila and Jones, 1993; GrossniklausBu¨rgin et al., 1994); but instead delays its development by several days (Brown et al., 1993). The Chelonus protein was chosen on the basis of abundance and developmental time of appearance rather than on similarity of electrophoretic mobility of protein bands of the two chelonine species. Western blotting analysis of in vitro secretions from both chelonine parasitoids (Fig. 2B) revealed that the antibody generated was specific to Chelonus and that only Chelonus specimens which were floating (late L2) or had molted during the incubation period generated the polypeptide of interest. The antibody did not cross-react to media from incubated Ascogaster parasitoids of any age nor did it react with proteins from L1 and early L2 Chelonus larvae. Antibodies generated against the 27 kD polypeptide obtained from denatured gel slices were used to identify the native protein by Western blotting techniques. Immunoreactivity was detected against a single broad band with an approx. Mr of 390 kD (data not shown). This native protein’s broad banding pattern suggests that it may be glycosylated and the appearance of several bands within the 23–27 kD range under denaturing conditions (Fig. 1A) may suggest various subunits or degradation products of the native form. However, the native

protein did not appear to be held together by disulfide bonds since comparison of treatments of samples with and without 2-mercaptoethanol showed little difference in banding patterns (data not shown). 3.2. Parasitoid-derived polypeptides secreted in vitro In an effort to determine if parasitoid larvae actively produced and secreted proteins into the incubation medium, they were incubated in methionine-free media to which 35S-methionine had been added. Both chelonines were capable of producing and secreting a multitude of polypeptides within a variety of Mr ranges and the number and variety of bands which were visible changed depending on the age and behavior of the individual parasitoids during incubation (Fig. 3). Many polypeptides which were prominent in the autoradiograph (Fig. 3A) were barely visible in the silver-stained SDS–PAGE gel (Fig. 3B). Both species appeared to produce a large number of heavily glycosylated polypeptides as evidenced by the broad banding patterns. First instar parasitoids (Lanes 7, C and F) did not synthesize many polypeptides even though incubated in groups of five larvae. Chelonus parasitoids which molted during the incubation period (Lanes 1 and 3) produced the greatest number of distinct bands and produced greater amounts of the 20– 27 kD polypeptides than younger L2 parasitoids (Lanes 2, 4–6). In particular, Chelonus parasitoids synthesized different proteins in each stage (Fig. 3A) despite nearly

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Fig. 3. (A) Autoradiograph and corresponding (B) silver-stained gel comparing in vitro protein secretions of first and second stadia Chelonus (Lanes 1–7) and Ascogaster (Lanes A–F) larvae run on SDS–PAGE (12.5%). Parasitoids were incubated in 100 ␮l methionine-free Grace’s medium with added 35S-methionine (50 mCi) for 48 h. Lanes contain 15 ␮l of in vitro fluid. Lanes 1 and 3 = Chelonus second instars molted during incubation; Lanes 2, 4, 6, A, D and E = second instars floated on the medium surface; Lanes 5, B, C and F = second instars submerged beneath the medium surface; and Lanes 7, C and F = medium from five submerged first stadia larvae. Film was exposed for 4 days. Arrow indicates 27 kD protein of Chelonus. (B) Corresponding silver-stained SDS–PAGE (12.5%) gel. Arrow indicates 27 kD protein of Chelonus and numbers on the left indicate Mr standards.

identical banding patterns visible by silver-stained SDS– PAGE (Fig. 3B, Lanes 5 and 6). 3.3. Tissue localization of the 27 kD polypeptide Various homogenized organs of late L2 Chelonus parasitoids, host hemolymph and host carcasses which remained after excision of the parasitoids were tested for the presence of the 27 kD polypeptide. All tissues tested showed a large number of bands detectable by silverstained SDS–PAGE gels (Fig. 4A); however, when sub-

jected to Western blotting analysis only the isolated anal vesicle of the parasitoid yielded cross-reactivity (Fig. 4B; arrowhead). This reaction was further tested by increasing the number of anal vesicles in the sample and by testing homogenized whole body tissues of late L2 and early L3 parasitoids and isolated AVs from late L2 parasitoids (Fig. 5). Each of these parasitoid tissues and incubation fluid showed cross-reactivity with the antibody to the 27 kD polypeptide. Despite the short incubation time of 30 min prior to severing their AVs, a pooled sample of 18 late L2 parasitoids secreted a large

Fig. 4. (A) Silver-stained SDS–PAGE (12.5%) gel and (B) corresponding Western blots comparing protein profiles from various Chelonus parasitoid and Trichoplusia ni parasitized host tissues. Salivary glands (Gl), gut tracts (Gt) and anal vesicles (AV) were isolated from five second stadium parasitoids, pooled and homogenized in Grace’s medium. The remaining parasitoid body tissues (PB) following the various organ dissections were also tested. One ␮l of host hemolymph (HH) and homogenized host tissues (HT) after parasitoid excision were also tested for the presence of the 27 kD protein of Chelonus. Filtered in vitro fluid from parasitoids that molted during incubation ( ⬍ 30 kD) was used as a positive control. Arrow indicates 27 kD protein of Chelonus. Arrowhead indicates slight cross-reactivity of antibodies with AV tissues from five parasitoids.

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Fig. 5. Western blot of SDS–PAGE (12.5%) gel of whole body homogenates of Chelonus parasitoids immediately upon excision from the host, isolated anal vesicles and incubation media from 30 min and 24 h incubation periods. AV intact = second stadium parasitoid. AV release = second stadium parasitoid prior to completion of the molt. L3 Par = third stadium parasitoid which had egressed from the host and fed on host tissues. Short incub. = 30 min incubation of 18 second stadium parasitoids prior to excision of the anal vesicles for the 18 AV lane. Filtered in vitro fluid from parasitoids that molted during incubation (Molt media) was used as a positive control. Film was exposed for 10 min. Note that all samples showed immunoreactivity with the anti − 27 kD antibody. The intense band in the short incubation sample indicates that the protein is readily secreted at high levels from intact parasitoids.

amount of 27 kD polypeptide into the medium. This indicates that the protein is readily secreted at high levels from intact parasitoids. However, the greatest concentration of 20–27 kD peptides coincided with the release of the AV by late L2 parasitoid larvae (Fig. 1A, Figs 2 and 3). The outer layer of the AV is lost just prior to the L2 to L3 molt and is one of the first outwardly noticeable events of an impending molt. Despite the absence of an AV in L3 parasitoids (the inner portion of the anal vesicle becomes the invaginated hindgut at this developmental state), homogenized tissues from these larvae showed a large amount of the 27 kD polypeptide. It should be noted that these L3 parasitoids were removed from hosts immediately upon egression and prior to any opportunity to feed on host tissues. 3.4. Temporal secretion of both ecdysteroid and 27 kD polypeptide in vitro Fig. 6 illustrates the sequence of secretion of both ecdysteroids (Fig. 6A) and the 27 kD polypeptide (Fig. 6B) by Chelonus L2 parasitoids. Parasitoids were readily secreting ecdysteroids at the time of wandering of the host and did so continually until ecdysis to the third stadium occurred and then secretion ceased entirely (Fig. 6A). The 27 kD polypeptide also was being produced at levels detectable by silver-staining at the time of host

wandering, although levels of protein secretion appeared to increase gradually until the parasitoid molted and then gradually decreased (Fig. 6B). The protein however was still being secreted at least 12 h after the molt occurred. Groups of parasitoid larvae isolated from hosts differing in age by 24 h molted within 12 h of each other, indicating that parasitoids which were held in vitro had an accelerated development compared to those which remained in vivo. If host/parasitoid units had been left intact, the groups of parasitoids would have molted within 24 h of each other. Only 20% (2 of 15) of parasitoids excised from hosts at the onset of wandering molted after 48 h of in vitro incubation (Fig. 6B, asterisk), compared to > 88% of parasitoids from spinning and spun hosts and these parasitoids removed from older hosts molted after just 36 h of incubation. The relative inability of younger parasitoids to molt may indicate nutritional or other biochemical deprivation due to in vitro conditions.

4. Discussion Chelonus sp. near curvimaculatus and Ascogaster quadridentata are egg-larval endoparasitic wasps of lepidopteran hosts. Parasitized host larvae initiate precocious metamorphosis by exhibiting cocoon-spinning activity in the fourth rather than the fifth stadium. The mechanisms which elicit this accelerated behavior and development in hosts of chelonine parasitoids remain unknown. Much research involving other host/parasitoid systems has concentrated on changes in the host physiology which result in developmental arrest. In particular, the effects of parasitoid-associated polydnaviruses on brain (Zitnan et al., 1995) and prothoracic gland function (Dover et al., 1995) and release of neuropeptides and hormones involved in metamorphosis have been implicated. However, previous in vitro incubations of late L2 larvae of chelonine parasitoids indicated the production and release of ecdysteroids into the medium (Brown and Reed-Larsen, 1991; Brown et al., 1993), which may be involved in host premetamorphic activity. Other braconid parasitoids have also recently been shown to produce ecdysteroids (Alleyne et al., 1997; Gelman et al., 1998). Therefore, the contribution of developing parasitoid larvae to the manipulation of host development deserves further attention. This study was undertaken to investigate proteinaceous materials and ecdysteroids released in vitro by these parasitoids shortly (up to three days) before egression of the parasitoid larva from the host. In vitro techniques were used to avoid confusion with host-derived macromolecules. Such proteins may prove important in the completion of the last stages of the host/parasitoid relationship. Here we report the presence of proteins in incubation media of L2 larvae of both species held under

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Fig. 6. Concentrations of (A) ecdysteroids and (B) the 27 kD polypeptide released by late second stadium Chelonus near curvimaculatus larvae during in vitro incubation as determined by radioimmunoassay (RIA) (for methods see Brown and Reed-Larsen, 1991) and SDS–PAGE (12.5%), respectively. In vitro medium was collected from incubated parasitoids which were dissected from hosts on three consecutive days following the initiation of host wandering. Medium containing released products was collected and fresh medium added at 12 h intervals. Samples collection was terminated 12 h after larvae exhibited a molt. Arrows indicate the event of the parasitoid molt to the third instar. Samples were split between RIA and SDS–PAGE analyses. (A) Picograms of ecdysteroids produced/secreted by individual parasitoid larvae per 100 ␮l of incubation medium. Note: RIA analyses were reported as pg ␮l−1. Columns represent the mean ecdysteroid production of each group (n > 8) during each incubation period. Bars indicate standard error of the mean. Parasitoid larvae which were excised from wandering hosts seldom ecdysed during in vitro incubation. The asterisk (*) indicates ecdysteroid production from remaining larvae after cohorts had molted. (B) Twenty ␮l samples of incubation medium containing the 27 kD protein of Chelonus were given a qualitative rating based on intensity of silver staining, 1 = low, 5 = high. Bars represent the average rating for at least eight individual parasitoids for each day tested. Following ecdysis, ecdysteroids were no longer secreted; however, the protein continued to be produced, but at lower levels.

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in vitro conditions. These polypeptides were particularly abundant in the media of late L2 larvae, especially if the parasitoids molted during the incubation period (Figs 1 and 2). These materials were continuously secreted several hours prior to and after the molt to the third stadium (Fig. 6) and were temporally associated with the precocious behavior and physiology of the host. In vitro incubation products from Chelonus and Ascogaster were analysed by SDS–PAGE (Figs 1 and 2). Incubation media from both parasitoids yielded a large variety of proteinaceous materials and the complexity of banding patterns increased with increasing age of the parasitoid, especially within the second stadium. A particular sequence of banding patterns developed, where the intensity and number of bands within an Mr range of 20– 30 kD increased with parasitoid age and was associated with the molt to the third stadium during incubation (Fig. 1A). Samples from both species yielded conspicuous polypeptides at the time of the in vitro molt (Fig. 1), but that of Chelonus appeared to be produced in greater quantities and its temporal appearance was more easily predictable than that of Ascogaster (Fig. 2A). Proteins emanating from parasitoids may have several origins, such as: (a) sequestration and subsequent release of host-derived material, (b) parasitoid metabolic waste products or (c) materials which are actively synthesized by the parasitoid. Parasitoid-derived proteins plausibly may function in regulation of the host. The 27 kD protein of Chelonus is not a host-derived material because immunoblots of host hemolymph and carcasses (hosts from which L2 parasitoids had been removed) were negative when tested with the anti − 27 kD protein antibody. However, antibodies did cross-react with whole host carcasses from which the parasitoid had egressed just minutes prior to tissue homogenization, processing and analysis. This is most likely due to the presence of the parasitoid AV or its contents within the hemocoel of the host following parasitoid egression. It is unlikely that the 27 kD polypeptide is a waste product because of the absence of the 27 kD polypeptide in samples from first and early second stadium parasitoids. However, if the host hemolymph composition should change drastically just prior to parasitoid emergence (e.g. the precocious accumulation of arylphorin, Kunkel et al., 1990) then the resultant by-products of parasitoid metabolism may also change at this time. It is generally accepted that parasitic wasp larvae retain waste products and void them within a meconium only upon pupation. Bidirectional movement of molecules across the anal vesicle (evaginated hindgut) has been suggested (Edson and Vinson, 1976, 1977) however the function of this organ has yet to be clearly defined. Further negation of the waste product theory is the continual release of the protein into replenished media (Fig. 6) and its detection in an L3 parasitoid, which had shed the anal vesicle prior to egression and was isolated

before feeding on host tissues (Fig. 5). Both pre-molt and post-molt protein production (Fig. 6) also argue against the possibility of it being an ecdysis product such as ecdysial fluid proteins. 35 S-methionine incorporation experiments (Fig. 3A) further suggest that the 27 kD polypeptide is not a waste product since it was being actively synthesized and released by the parasitoid and that this protein was produced primarily by late L2 larvae. Larvae which were sunken beneath the surface of the medium only occasionally synthesized this protein but this may be a reflection of a critically timed production of this protein by larvae of a particular physiological age. This result may also be a reflection of the relatively long incubation time of 48 h and shorter periods of incorporation may clarify the situation. The majority of synthesized proteins produced by sunken larvae were of a higher molecular weight (ca. 40–80 kD). At this time, it is unclear whether the protein of concern is higher in methionine residues or more abundantly synthesized than other polypeptides. Western blot analysis of fluid into which late L2 parasitoids had been placed prior to severance of the anal vesicles (Fig. 5—Short incub.) indicated a rapid release of the 27 kD polypeptide. These larvae were specifically selected because they were of a physiological age expected to produce the protein. The rapid release may be in response to air as would naturally occur when the parasitoid breaches the confinement of the host’s integument upon egression and the presence of the polypeptide would coincide with its limitation to late L2 and early L3 parasitoids. The 27 kD polypeptide was particularly abundant when larvae floated or molted during incubation. These events were found to be associated with larval maturity. Once a larva exhibited floating behavior it was impossible to submerge it beneath the medium surface. Parasitoid maturity could be estimated by observing host responses. If the host could no longer respond to probing by flexing the ventral abdominal muscles, then the parasitoid would egress within the next 12–24 h. Therefore, if host apolysis had begun, then the very late L2 parasitoids would be expected to float and molt during the 24 h incubation period. Early to mid-L2 parasitoids did not show recognizable signs that would indicate that they would float; therefore, floating may be an indication that a particular developmental or physiological event has occurred. Floating may also be due to the introduction of air into fully developed tracheae, necessary for ectoparasitic development, which are indicative of a maturing larva (Grossniklaus-Bu¨rgin et al., 1994) or to the production of epicuticle waxes in the more exposed L3 parasitoid. The premature introduction of air by dissection may mimic the eruption of the parasitoid’s head through the host integument during egression and therefore should not be considered artifactual. Brown et al. (1990) reported the survival of diapausing A. quadriden-

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tata larvae sunken below the surface of the medium for 14 days. Therefore, the in vitro condition should not be considered an entirely disruptive situation. The remaining hypothesis to be tested is the production of a proteinaceous material which has some effect on the host which may be beneficial to the parasitoid. Insect hemolymph typically coagulates and melanizes upon exposure to the air as part of a non-specific immune/healing response. It is plausible that the 27 kD protein may be involved in preventing oxidative processes associated with the host’s normal healing response. If left intact, this host tissue response could inhibit the successful egression of the L3 parasitoid. Inhibitory proteins (150–200 kD) with anti-microbial properties have been identified from the anal vesicles of ichneumonid parasitoids (Willers et al., 1982; Fu¨hrer and Willers, 1986). These inhibitory proteins may prevent degradation of host tissues and contamination prior to consumption by the parasitoid. The 27 kD protein of Chelonus has not been tested for its capability of inhibiting hemolymph melanization or for antibiotic properties. Previous studies by Brown and Reed-Larsen (1991) indicated that ecdysteroids were released by parasitoids held in vitro. Host apolysis appears to be initiated by the release of ecdysteroids by endoparasitoids (Brown et al., 1988; Brown and Reed-Larsen, 1991) as hosts of chelonines have been shown to have an impaired ability to synthesize ecdysone at this stage of parasitism (Grossniklaus-Bu¨rgin and Lanzrein, 1990; Jones et al., 1992). The parasitoid ecdysteroid form has been identified as the active form of the molting hormone, 20-OH ecdysone (Brown et al., 1993). The parallel study of protein and ecdysteroid release by parasitoids reported here indicates a peak of ecdysteroid activity prior to a molt. This peak also preceeds the peak of protein secretion. The hypothesis of parasitoid control over host apolysis is as follows: There is a rapid increase in ecdysteroid levels produced by the L2 parasitoid to aid in host apolysis, which also facilitates the egression of the third instar and there is a subsequent cessation of ecdysteroids after the molt. The appearance of the 27 kD polypeptide coincides with that of the ecdysteroids but is more gradual and continues after the molt. The protein may aid in the successful action of the parasitoid-derived ecdysteroids by either inhibiting host degradative enzymes or directing the ecdysteroids specifically to the host integument. Parasitism-specific host hemolymph proteins have been previously reported in several host/parasitoid relationships (Smilowitz, 1973; Beckage et al., 1989; Lawrence, 1990; Soldevila and Jones, 1993). However, most of these proteins have been found to be of host origin and are apparently induced by the parasitic relationship. Exceptions include: a polydnavirus translation product from the larval endoparasitoid Cotesia

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congregata (Harwood et al., 1994; Harwood and Beckage, 1994) and the 185 kD protein of Chelonus reported by Soldevila and Jones (1993). This Chelonus protein was found to be synthesized by late L2 parasitoids and appeared to be secreted into in vitro media rather than being sequestered in parasitoid tissues. This 185 kD protein, unlike the 27 kD polypeptide investigated in this study, was found to be persistent in host hemolymph. This suggests the possible rapid uptake of the 27 kD polypeptide by host tissues. The function of either of these proteins is currently unknown. Currently much attention has focused on the role of polydnaviruses in host/parasitoid relationships; whereas few studies have directed any attention to the developing parasitoid larvae. Clearly, parasitoid larvae are not passive occupants of the host hemocoel but are instead capable of actively monitoring and manipulating their host environment to suit their nutritional and developmental needs. Much more research needs to be conducted on this dynamic aspect of host/parasitoid interactions.

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