Disruption of pupariation and eclosion behavior in the flesh fly Sarcophaga bullata Parker DipteraSarcophagidae by venom from the ectoparasitic wasp Nasonia vitripennis Walker HymenopteraPteromalidae.код для вставкиСкачать
78 Rivers et al. Archives of Insect Biochemistry and Physiology 57:78–91 (2004) Disruption of Pupariation and Eclosion Behavior in the Flesh Fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by Venom From the Ectoparasitic Wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) David B. Rivers,1* Jan Zdarek,2 and David L. Denlinger3 The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone’s role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior. Arch. Insect Biochem. Physiol. 57:78–91, 2004. © 2004 Wiley-Liss, Inc. KEYWORDS: ectoparasitoid; wasp venom; mode of action; ecdysone; fly development INTRODUCTION Non-aculeate parasitic wasps are a tremendously diverse group, with over 600,000 described species and an estimated 400,000 more yet to be discovered (Whitfield, 1998). The venoms of these 1 Department of Biology, Loyola College in Maryland, Baltimore 2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Prague, Czech Republic 3 Department of Entomology, Ohio State University, Columbus parasitic wasps are known to have a wide range of effects on their hosts, including arrestment of development, alterations in growth and physiology, suppression of immune responses, paralysis, and death (Jervis and Copeland, 1996; Quicke, 1997). Such biological and physiological diversity suggests Abbreviations used: CNS = central nervous system; EtOH = ethanol; 20E = 20-hydroxyecdysone; LC = lethal concentration; LT = lethal time; VRE = venom reservoir equivalent. Contract grant sponsor: Czech Granting Agency; Contract grant number: 522/01/0501; Contract grant sponsor: USDA-NRI; Contract grant number: 98-353026659; Contract grant sponsor: USDA-NRICGP; Contract grant number: 2001-1005; Contract grant sponsor: Loyola College. *Correspondence to: Dr. David Rivers, Department of Biology, Loyola College in Maryland, 4501 North Charles Street, Baltimore, MD 21210. E-mail: firstname.lastname@example.org Received 14 January 2004; Accepted 12 May 2004 © 2004 Wiley-Liss, Inc. DOI: 10.1002/arch.20015 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology Action of Venom From N. vitripennis that these venoms contain a wealth of compounds with unique chemistries and modes of action (Jones and Coudron, 1993). Due primarily to limitations imposed by the small size of most parasitic wasps, research investigating the insecticidal properties of these venoms has been limited. Several species of wasps belonging to the family Pteromalidae produce venoms with high potency toward an array of pest insects. The most intensively studied member of this family, Nasonia vitripennis (Walker), has an elaborate venom system (Ratcliffe and King, 1967, 1969) that is central to successful parasitism (Rivers et al., 1999b). A fly accepted as a suitable host is always injected with venom prior to oviposition (Dawei and Dingxi, 1987), with posterior body regions usually serving as the sites for the sting and egg deposition (Rivers, 1996). The venom inhibits host cellular immune responses within 30 min after envenomation (Rivers et al, 2002), suppresses respiratory metabolism within a matter of hours (6–8 h) (Rivers and Denlinger, 1994a), elevates hemolymph and fat body lipid titers (Rivers and Denlinger, 1995; Rivers et al., 1998), and ultimately induces death. In muscoid flies, N. vitripennis venom can retard adult fly mobility within 1–2 h when injected artificially, and the flies will succumb to death in less than 24 h (Beard, 1964; Rivers et al., 1993). The venom has been shown to be highly active toward several life stages (wandering larvae, pupae, pharate adults, adults) of flies from at least 4 families (Muscidae, Drosophilidae, Calliphoridae, and Sarcophagidae) (Rivers et al., 1993). Additionally, in vitro assays using cultured insect cells have revealed high dipteran specificity in terms of LT50s and LC50s but virtually no mammalian cell sensitivity (Rivers et al., 1999a,b). Thus, venom from N. vitripennis appears to be a rich source of toxins displaying specificity toward dipterans. The precise host tissue targeted by the wasp venom and its mode of action has not been fully revealed and is the focus of this study. Tensiometric recordings of pupariation and eclosion behavior of the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae) have been used previously as tools for monitoring immediate and protracted effects of several drugs, venoms, and October 2004 79 insecticides (Zdarek and Fraenkel, 1987). An eclosing flesh fly performs stereotypic extrication behavior as long as it is confined in the puparium or in the substrate (soil) in which the larva pupariated (Reid et al., 1987a,b). This stereotypic behavior can be monitored barographically or tensiometrically (Zdarek et al., 1986; Zdarek and Denlinger, 1992). The techniques can also be exploited for monitoring the effects of various neurotropic agents (drugs, poisons, insecticides) on insect behavior (Zdarek and Denlinger, 1992). The latency period, lethal time, and type of action can be deduced from the records. In the present study, we used these techniques for the study of the effects of crude venom from N. vitripennis. Pupariation behavior is also a stereotypic activity that can be recorded barographically (Zdarek, 1985) and tensiometrically (Zdarek and Denlinger, 1991). As with eclosion, the barographic and tensiometric techniques can serve as tools for monitoring the pharmacological effects of noxious compounds (Zdarek et al., 1987). Venom from N. vitripennis has been shown to delay the onset of pupariation in S. bullata and disrupt puparial morphology (Rivers et al., 1993). Thus, we employed the barographic technique to probe the nature of the irregular pupariation behavior. We also explored the possibility that the venominduced delay in pupariation was a consequence of a deficiency in ecdysteroids, the insect growth hormone that is essential for puparium formation. Venom from N. vitripennis has been shown previously to suppress development of nondiapausing pupae and pharate adults of S. bullata in a manner consistent with an ecdysteroid deficiency, although, surprisingly, exogenous injections of 20-hydroxyecdysone (20E) did not restore fly development (Rivers and Denlinger, 1994a). Here we show that 20E can rescue some elements of pupariation in venom-injected larvae, but the effect is dose- and age-dependent. MATERIALS AND METHODS Insect Rearing N. vitripennis was maintained as a laboratory colony on pupae and pharate adults of S. bullata 80 Rivers et al. as previously described (Rivers and Denlinger, 1994a). Adults and larvae were reared under a LD 15:9 h light-dark cycle at 25°C. Twenty to thirty females (3–7 days after emergence from host puparia) were placed in a Petri dish (15 × 100 mm) with 50–75 nondiapausing pupae (4 days after pupariation) of S. bullata and a 50% (v/v) honey solution. After 24 h, the adult wasps were removed and parasitized pupae maintained at 25°C, LD 15:9 h. Under these conditions, N. vitripennis develops from egg to adult (eclosion) in 12 days. A colony of S. bullata was reared according to Denlinger (1972). Larvae and adults were fed pork liver throughout development at 25°C with a lightdark cycle of LD 15:9 h. To synchronize fly development for assessing host age, third instar larvae that had begun to wander from food (but prior to crop emptying) were collected and placed in a vented glass jar (1 liter) with 1–2 ml tap water. Larvae were held under these conditions for 3 days at 25°C with frequent (3–5 times/d) water changes. This “wet” treatment temporarily inhibits the release of ecdysteroids until the larvae are placed in dry conditions, thereby synchronizing pupariation (Ohtaki, 1966). Isolation of Crude Wasp Venom Crude venom from N. vitripennis was isolated from host-fed females in phosphate-isolation buffer [10 mM sodium phosphate (pH 8.0), 0.9% (w/v) NaCl, 15% (w/v) sucrose, 1 mM ethylenediamineteraacetic acid, and 1 mM phenylmethylsulfonyl fluoride] (Rivers et al., 1993) and stored frozen at –70°C. Venom activity was confirmed in vitro and in vivo with BTI-TN-5B1-4 cells and young pharate adults (5 days after pupariation at 25°C) of S. bullata, respectively, as described previously (Rivers et al., 1993). Venom Injection Wandering larvae of S. bullata were injected with venom in the pre-red spiracle (6 h prior to pupariation at 25°C), early-red spiracle (2 h prior to pupariation) or late-red spiracle (30–60 min prior to pupariation) stage of development. Larvae were immobilized on ice for 5-10 min prior to injections, and injections were accomplished by means of finely-drawn glass capillaries (Rivers et al., 1993). Volumes of injected solutions ranged from 1–6 µl. Newly emerged imagoes (≤5 min after emergence) were immobilized briefly (2–3 min) on ice and then injected (1 µl/fly) with crude venom lateral to the dorsal midline of the abdomen using finely pulled capillaries. Larvae and imagoes injected with phosphate isolation buffer served as controls. Effects on Puparium Morphology and Behavior The effects of crude wasp venom on pupariation morphology and behavior were investigated as described (Zdarek et al., 1987). The behavior of venom-injected larvae following recovery from being placed on ice prior to injections (termed immediate effects) until the completion of tanning was monitored continuously at 25°C. The size and shape of puparia were also recorded with the following morphological features being scored (according to the criteria of Zdarek et al., 1987): (1) ability to retract anterior segments (r), (2) ability to contract longitudinally to a barrel shape (c), (3) smoothing of the cuticle (s), and sclerotization and tanning of puparia (t). Effects on Denervated Musculature To test the importance of the central nervous system (CNS) on venom-induced changes in pupariation behavior, pre-red spiracle or early-red spiracle larvae were immobilized on ice and then tightly ligated behind the fused CNS (Zdarek et al., 1987). This procedure disconnected all nerves leading from the fused CNS to the abdomen (Zdarek and Fraenkel, 1972). Larvae were then removed from ice and after the anterior segments resumed activity, crude venom was injected into posterior body regions as described above. Ligated larvae were then kept at 25°C, LD 15:9 and observations of cuticular tanning made at 24-h intervals for 4 days. Archives of Insect Biochemistry and Physiology Action of Venom From N. vitripennis Injection of 20-Hydroxyecdysone 81 Preliminary observations revealed an age-dependent inhibition of puparial tanning that suggested a possible venom-evoked ecdysteroid deficiency. To examine whether development in venom-injected larvae could be rescued by exogenous ecdysteroids, pre-red spiracle larvae were injected with 20hydroxyecdysone (20E) [5 µg/larva in 10% (w/v) EtOH] using finely pulled glass capillaries. Since the effect of wasp venom on ecdysteroid responsiveness was likely also time-dependent, fly larvae were injected with 20E at different time intervals (0–120 min) post-venom injection. Larvae injected with 10% EtOH (1 µl/larva) were used as controls. All larvae were maintained at 25°C, LD 15:9 h following injections and checked at 24-h intervals for the puparium morphological features described above. siometric transducer (Grass Instruments, Quincy, MA) into signal outputs that were amplified (Universal Precision Amplifier CA 110, Peckel Instruments, Rotterdam, The Netherlands) and recorded (Line Recorder LZ 4620, Laboratorni pristroje, Prague, Czech Republic). To record the behavioral patterns characteristic of obstacle removal (Zdarek et al., 1986), a puparium containing a pharate adult just about ready to emerge was inserted into a plastic 1-ml pipette tip and positioned in front of the force transducer. Mechanical resistance of the sensor prevented the imago from extricating itself from the puparium, and a cotton plug positioned behind (posterior) the puparium prevented backward movement. Comparisons were made between saline-injected and venom-injected imagoes. Injections were made into the dorsal surface of the thorax using finely drawn capillaries. Effects on Hemolymph Pressure During Pupariation Effects on Post-Eclosion Behavior A barographic method of recording hemocoelic pressure changes that reflect muscular activity and cuticular transformation (Zdarek et al., 1979) was used to continuously monitor hemolymph pressure in red spiracle stage (1–2 h prior to pupariation) larvae of S. bullata. Larvae were immobilized on ice for 5–10 min and then injected with crude wasp venom [1–6 venom reservoir equivalents (VRE)/larva: 1 VRE = LC99 dose for larvae and pupae of S. bullata (Rivers et al., 1993)]. Immediately following injection, the immobilized larvae were catheterized with a hypodermic needle (23-G) connected to the hydraulic system of a pressure transducer. Three pressure records (barograms) were made for each venom dose tested. Comparisons were made to untreated and saline-injected larvae. All recordings were performed at room temperature. Effects on Extrication Behavior Extrication behavior was studied using the tensiometric technique described previously (Zdarek et al, 1986; Zdarek and Denlinger, 1992). Movement of the ptilinum was transformed by a tenOctober 2004 Flies ready to emerge from puparia were prepared as described by Zdarek et al. (1984). Immediately after emergence, adult flies were injected with either saline (1 µl/fly) or crude venom (1 or 3 VRE/fly) from N. vitripennis and then placed in Petri dishes (15 × 100 mm) lined with filter paper. The onset and duration of several aspects of posteclosion behavior were monitored continuously at room temperature and included: (1) walking (W), (2) abdominal expansion (AE), (3) wing expansion (WE), (4) wing smoothing (WS), (5) tanning (t), and (6) grooming (G) (Zdarek et al., 1984; Zdarek and Denlinger, 1987). Post-eclosion behaviors were followed until adult flies resumed walking behavior (W2). Statistical Analyses Means were compared using 1-way and 2-way analysis of variance (ANOVA) and Student-Newman-Keuls multiple comparisons tests (alpha = 0.05; Sokal and Rohlf, 1969). Percentage data were arcsine transformed prior to analysis. Data analyses were performed using StatView statistical software (v. 5.0.1, Abacus, Berkeley, CA). 82 Rivers et al. RESULTS Puparium Morphology and Behavior Saline injection into larvae of S. bullata (regardless of age tested) had no obvious effects on the onset of pupariation or puparium morphology (Fig. 1A–C). In contrast, crude venom from N. vitripennis induced a dose-dependent delay or inhibition of pupariation: the onset of pupariation in larvae injected with 1 VRE crude venom was delayed by 1–2 days at 25°C, whereas higher concentrations (3 VRE) completely inhibited pupariation in a high proportion of larvae injected in either the pre-red spiracle (87.5%, n = 24) or early-red spiracle (49.9%, n = 27) stage of development (Table 1). The timing of pupariation initiation in late-red spiracle larvae and the number of larvae that attempted to pupariate did not appear disrupted by crude venom, regardless of the dose tested. Larvae that were delayed in pupariation did eventually pupariate but they did not form normal puparia. Puparial shape ranged from larva-like to elongated and partially folded intersegmentally (Fig. 1E,F). None of the puparia formed by venomtreated larvae properly contracted nor were they smooth (Fig. 1D–F). Pupariation behavior is characterized by a stereotypic chain of events that include gradual immobilization, retraction of anterior segments, contraction longitudinally to a barrel shape, smoothing of the surface due to shrinkage of the cuticle, and sclerotization and tanning (Zdarek et al., 1986). Injection of saline into S. bullata in different stages of larval development did not alter these normal elements of pupariation behavior (Table 1, Fig. 2). The same was not true for larvae injected with crude venom as several aspects of pupariation behavior were observed to be altered or absent entirely, and larval age did influence the effects evoked by venom on pupariation behavior (Table 1). At low doses of venom (1 VRE/larva), most larvae tanned, but all behavioral events preceding sclerotization and tanning were altered in a manner dependent on larval age (Table 1). Likewise, high doses (3 VRE/larva) of venom suppressed or abol- ished all behavioral events preceding sclerotization and tanning. Only larvae injected in the late-red spiracle stage showed tanning comparable to saline-injected flies (Table 1). Pupariation behavior was also monitored by continuous measurements of changes in hemocoelic pressure as pupariation proceeded. The entire activity appeared on a barogram as a unique series of pressure pulses that reflect specific muscle movements over time within different body segments. The muscle movements, and hence pressure pulses, are specific for the different stages of the stereotypic behavior (Fig. 2A). Barographic records revealed that all elements of pupariation behavior of venom-injected larvae (early-red spiracle) were present, but were performed earlier in relation to the onset of tanning in comparison to saline-injected flies (Fig. 2). The immobilization period that precedes puparial contractions was also extended in duration (Fig. 2B). Puparial contractions in venom-injected larvae were patterned regularly, but the intensity of muscular efforts was subdued and tanning was greatly delayed (Fig. 2B). Denervated Musculature To test the importance of the CNS for venominduced changes in pupariation behavior, pre-red spiracle larvae were tightly ligated behind the fused CNS and then injected with wasp venom (1 VRE) in the posterior body half. Cuticular changes were monitored for 4 days after ligations. All larvae tanned in front of the ligation within 48 h. However, tanning posterior to the ligature did not occur in any larva ligated during the pre-red spiracle Fig. 1. Morphogenetic effects of saline and crude venom from Nasonia vitripennis on puparium morphology in 3 developmental stages of Sarcophaga bullata. Normal puparium formation occurred in S. bullata following saline injection (1 µl/larva) in (A) pre-red spiracle, (B) early-red spiracle, and (C) late-red spiracle larvae. Age-dependent effects on puparium formation were observed in larvae injected with crude wasp venom (1 VRE/larva = 1.35 µg venom protein/larva): (D) pre-red spiracle, (E) early-red spiracle, and (F) late-red spiracle larvae. Thirty larvae were injected per treatment using pulled glass capillaries. Archives of Insect Biochemistry and Physiology Action of Venom From N. vitripennis October 2004 Figure 1. 83 84 Rivers et al. TABLE 1. Venom-Induced Changes in Pupariation Behavior of Third Instar Larvae of Sarcophaga bullata* Percent response (X ± SEM) Experimental group Saline Pre-RS Early-RS Late-RS Venom-injected 1 VRE/larva Pre-RS Early-RS Late-RS 3 VRE/larva Pre-RS Early-RS Late-RS N Immediate effects Retraction of anterior segments (r) Contraction to barrel shape (c) Smoothing (s) Sclerotization and tanning (t) 60 60 60 None None None 100a 100a 96.7 ± 0.01a 100a 100a 96.7 ± 0.01a 100a 100a 96.7 ± 0.01a 100a 100a 96.7 ± 0.01a 25 23 20 Sluggish Sluggish Sluggish 100a 13.0 ± 4.5b 64.5 ± 4.6c 13.3 ± 1.2b 8.7 ± 2.5c 55.6 ± 3.7d 13.3 ± 1.2b 0c 0c 98.5 ± 2.3a 91.3 ± 1.7ab 100a 24 27 24 Immobilization Immobilization Immobilization 0d 7.4 ± 0.8e 64.3 ± 1.5c 0e 0e 7.1 ± 2.6c 0c 0c 0c 12.5 ± 2.4c 50.1 ± 4.2d 94.8 ± 3.8ab *Wandering 3rd instar larvae of Sarcophaga bullata were injected lateral to the dorsal midline in the posterior 1/3 of the body with crude wasp venom (either1 VRE- or 3 VRE/larva) from Nasonia vitripennis using pulled glass capillaries (5 µl). Fly larvae were wet treated for 48 h to synchronize development for injections at 3 developmental stages: pre-red spiracle (6 h prior to pupariation at 25°C), early-red spiracle (2 h prior to pupariation), and late-red spiracle (30–60 min prior to pupariation). Injected larvae were allowed to pupariate at room temperature, being monitored continuously until the end of tanning. Larvae injected with saline (1 µl/larva) and untreated larvae served as controls. Percentage data was arcsine transformed prior to statistical analysis. Values in the same column followed by the same letter do not differ significantly from each other at P = 0.05. stage regardless of treatment (Fig. 3). When the same experiment was repeated using early-red spiracle larvae, cuticular tanning was evident in front and behind the ligature by 96 h for all larvae except those injected with crude venom (Fig. 3). In those larvae, tanning never occurred and eventually (1–2 weeks) the posterior body regions slowly became necrotic and desiccated. Hormone Injections At high doses (3 VRE/larva), wasp venom triggered an age-dependent inhibition of puparial tanning (Table 1) that suggested a possible ecdysteroid deficiency. To examine whether tanning, as well as other aspects of pupariation, in venom-injected larvae could be rescued by exogenous ecdysteroids, pre-red spiracle larvae were injected with 20E at different time intervals post-venom injection. Larvae treated with saline-only or saline and 20E displayed the typical events of pupariation with nearly all becoming properly sclerotized and tanned (Table 2). A similar trend was not evident for prered spiracle larvae injected with crude venom only, as none of the flies (n = 55) retracted anterior segments, contracted longitudinally, or tanned (Table 2). Hormone treatment did restore some aspects of pupariation in venom-injected larvae in a timedependent manner, but the responses were not as fully expressed as in control flies. For example, though a high percentage of venom-injected larvae tanned following injection of 20E (up to 60 min post-venom injection, Table 2), the cuticle was not as intensely tanned as with control flies, tanning was incomplete or patchy over the surface of puparia, and in many cases the cuticle remained soft. By 15-min post-venom injection, 20E was not capable of rescuing longitudinal contraction, and by 2 h, >90% of the venom-treated larvae were entirely unresponsive to 20E (Table 2). Extrication Behavior In saline-injected flies, extrication behavior occurred with the same frequency and amplitude of contractions for several hours (Fig. 4A) and gradually disappeared only after 11 h (n= 3). Imagoes injected with 1 VRE of crude wasp venom at the time of emergence performed regular extrication behavior for 7.0 h (n = 5). Typically, only the last hour of behavioral performance was less regular, showing at first a decreased frequency of muscular contractions before their rather sudden disappearance. The effects of venom on extrication behavior Archives of Insect Biochemistry and Physiology Action of Venom From N. vitripennis 85 Fig. 2. Barographic recordings of pupariating larvae (red spiracle stage) of Sarcophaga bullata that were injected (A) with isolation buffer (1µl/larva) or (B) crude wasp venom (1 VRE/larva) using pulled capillaries. T: beginning of tanning. The X-axis indicates time after recovery from immo- bilization on ice (in minutes). Letters represent specific pupariation events: i, immobilization of the larva; r, retraction of the anterior segments; s, shrinkage of cuticle; t, tanning of the cuticle. became much more pronounced with higher (pharmacological) doses. For example, flies that received 3 VRE performed extrication efforts for only 1.2 h (n = 5) and typically the cycles were progressively slower with decreasing amplitude of pressure peaks (Fig. 4D). A further depression of muscular activity was detected when 4 VRE were injected (Fig. 4C) and 6 VRE completely immobilized adult flies within 15 min (Fig. 4B). then followed by expansion behavior in the newlyeclosed imagoes. This latter set of behaviors (posteclosion or expansion) is necessary to inflate the imago body for attainment of the final size and shape of the adult. Untreated or saline-injected, newly eclosed imagoes walked (W) rapidly for a short period of time (Table 3), and then stopped and remained stationary, unless disturbed, for the remainder of the expansion period before resuming walking (W2) 133–137 min later (Table 3). During the stationary period, flies pumped air into the gut, elevating internal pressure to facilitate abdominal expansion followed shortly (9–11 min) by wing expansion (Table 3). When the wings were Post-Eclosion Behavior The initial phase of eclosion in higher Diptera involves extrication from the puparium, which is October 2004 86 Rivers et al. Fig. 3. Importance of the CNS for the venom-induced changes in pupariation behavior of third instar larvae of Sarcophaga bullata. The ability to tan in posterior body regions was monitored in pre-red spiracle and early-red spiracle 3rd instar larvae of S. bullata that were ligated and then injected either with saline (1 µl/larva) or crude wasp venom (1 VRE/larva). Ligations were performed at midbody (behind the CNS). Cuticular tanning was assessed at 24, 48, and 96 h after manipulations. Twenty larvae were used for each treatment. completely spread, the fly used the hind legs, alternating between the left and right leg, to smooth the wings. Tanning of the adult cuticle occurred approximately 57–60 min after the onset of expansion behavior, and was followed by grooming of the entire adult body before walking resumed (Table 3). Low doses (1 VRE) of venom did not alter any of the expansion activities prior to the initiation of tanning, and induced only slight delays in the induction of tanning, grooming, and resumption of walking behavior in imagoes (Table 3). Higher concentrations, however, disrupted all elements of expansion behavior as flies injected with 3 VRE required significantly more time than controls before initiating each activity (Table 3). Despite these venom-induced disturbances, all flies, regardless of venom dose tested, were able to complete all aspects of post-eclosion behavior. DISCUSSION The mechanism of action of most parasitic wasp venoms has not been studied sufficiently to reveal the details of which tissues are targeted in the host and how those tissues are injured to alter normal functions (Piek and Spanjier, 1986; Quicke, 1997). Venom from N. vitripennis has been the focus of several recent studies (Rivers et al., 1999a,b, 2002) that have examined the intoxication pathways associated with cell death using an in vitro approach (Rivers et al., 1999a). In cultured cells, crude venom from this ectoparasitic wasp appears to evoke cell death by a colloid osmotic lytic mechaArchives of Insect Biochemistry and Physiology Action of Venom From N. vitripennis 87 TABLE 2. Injection of 20-Hydroxyecdysone Into Pre-Red Spiracle Larvae of Sarcophaga Bullata Following Venom Injection* Percent response (X ± SEM) Treatment N Retraction of anterior segments (r) Contraction to barrel shape (c) Sclerotization and tanning (t) Saline Saline + 20-E Time of injection (min) 0 15 30 60 120 Venom Venom + 20-E Time of injection (min) 0 15 30 60 120 60 96.7 ± 3.5a 95.8 ± 2.3a 100a 40 40 40 40 40 55 95.0 ± 1.4a 100a 100a 100a 91.9 ± 2.5a 0b 95.1 ± 1.4a 100a 100a 100a 91.9 ± 2.5a 0b 95.0 ± 1.4a 100a 100a 100a 93.6 ± 3.1a 0b 50 50 50 40 40 80.7 ± 4.1c 51.3 ± 3.6d 30.4 ± 1.6e 40.0 ± 2.7f 0b 78.1 ± 1.1c 0b 0b 0b 0b 100a 80.1 ± 2.2c 80.0 ± 3.4c 62.3 ± 1.4d 9.5 ± 2.0e *Pre-red spiracle 3rd instar larvae of S. bullata were injected with either saline (µl/larva) or crude wasp venom (1 VRE/larva) from Nasonia vitripennis. Larvae were then injected with 20-E (5 µg/larva) at various time intervals following saline or venom injection in an attempt to rescue development from the effects of venom. All injections were performed lateral to the dorsal midline in posterior body regions using a 5-µl Hamilton syringe. Treated larvae were kept at 25°C, L15:D9, and monitored at 24 h-intervals for 5 days. Percentage data was arcsine transformed prior to analysis by ANOVA and Student-Newman-Keul’s multiple comparisons tests. Values in the same column followed by the same letter do not differ significantly from each other at P = 0.05. nism (Knowles and Ellar, 1987) that involves the release of intracellular calcium stores via the activation of phospholipase C and subsequent phosphoinositol turnover (Rivers et al., 2002). While these investigations have provided details of the venom’s action at the cellular level, it is unclear whether the same venom effects are triggered in the intact insect. Complicating efforts to decipher this issue is a lack of understanding of which tissues are directly targeted by venom from N. vitripennis and difficulty in working with a fly pupa (and pharate adults), the stage of host development parasitized by the wasp. The pupal and pharate adult stages develop within a puparium, the walls of which result from the cuticle of the last larval instar. During formation of the puparium, a high hemocoelic pressure is established that results from longitudinal contraction of the body and shrinkage of the cuticle, events that are essential for proper morphology of the puparium (Zdarek et al, 1979). In this study, neuropharmacological evaluation of crude wasp venom relied on the pupariation asOctober 2004 say described by Zdarek et al. (1987). During pupariation, the fly larva performs specific temporal and spatial patterned movements of two muscular systems: (1) the segmental and intersegmental somatic musculature, and (2) the retractors of the anterior prespiracular segments. Longitudinal contraction of the body during pupariation is under the control of the muscles of system 1 whereas system 2 causes withdrawal of the frontal segments and eversion of the anterior spiracles (Zdarek et al., 1987). The combined actions of these two muscle systems comprise a stereotypic pupariation behavior (Zdarek et al., 1979) that, if altered by noxious compounds, can yield information about specific target sites and receptors. The use of this evaluation approach in the present study revealed that venom from N. vitripennis delays or inhibits the ability of fly larvae to contract into barrelshaped puparia and subsequently tan, but these effects are dependent on venom concentration and larval age. It has been hypothesized that formation of a normal-shaped puparium is dependent on patterned muscular contractions during the 88 Rivers et al. Fig. 4. Tensiometric recordings of ptilinum movements of eclosing imagoes of Sarcophaga bullata after injection of (A) isolation buffer (1 µl), (B) 6, (C) 4, and (D) 3 venom reservoir equivalents of crude wasp venom from Nasonia vitripennis at the beginning of extrication behavior. The vertical bar indicates 0.5-mm displacement of the tensiometric sensor and is a measure of ptilinum movement during extrication. contraction phase of pupariation (Zdarek et al., 1979) and that if the patterns are disrupted, distorted puparia are formed (Zdarek et al., 1987). If true, then the morphological and behavioral effects evoked by venom from N. vitripennis would be the direct result of disruption of the patterned muscular contractions of peripheral neuromuscular that are under the control of muscle system 1. This system is, in turn, under the control of the motor neurons of the CNS. There are several lines of evidence that suggest that venom alters pupariation both via the CNS and through somatic musculature: 1. Barograms of venom-injected larvae showed that all the normal elements of pupariation behavior were present, but the timing of events had been altered in relation to the onset of tanning, a feature consistent with modification of temporal organization of central motor programs (Zdarek et al., 1987); 2. Ligated larvae did not respond to venom injection into posterior body regions, suggesting no direct stimulatory effects on muscles or neuromuscular junctions (Zdarek et al., 1987; Zdarek and Denlinger, 1992); 3. Injection of exogenous ecdysteroid could restore Archives of Insect Biochemistry and Physiology Action of Venom From N. vitripennis 89 TABLE 3. Quantitative Aspects of Post-Eclosion Behavior in Venom Injected Adults of Sarcophaga bullata Time distribution (X min ± SEM) Experimental group N Walking (W) Abdominal Expansion (AE) Wing Expansion (WE) Wing Smoothing (WS) Tanning (T) Grooming (G) Resumed Walking (W2) Untreated 25 13.7 ± 2.1a 22.2 ± 3.4a 33.4 ± 2.6a 40.5 ± 3.6a 59.4 ± 0.9a 110.8 ± 3.1a 137.5 ± 2.7a Saline 21 15.0 ± 1.9a 25.6 ± 2.1a 34.6 ± 3.0a 36.9 ± 2.8a 57.8 ± 1.2a 106.4 ± 3.5a 133.6 ± 2.6a Venom-injected 1 VRE/fly 25 14.2 ± 2.4a 17.9 ± 3.4a 30.8 ± 1.1a 34.6 ± 2.9a 66.3 ± 1.5b 120.1 ± 2.8b 150.4 ± 3.9b 3 VRE/fly 24 25.0 ± 3.2b 30.5 ± 1.6b 55.0 ± 0.7b 55.9 ± 0.6b 58.8 ± 2.0a 127.9 ± 2.8c 147.5 ± 2.5b *Adult flies emerging from puparia were injected with either saline (1 µl/fly) or crude venom (1 or 3 VRE/fly) from Nasonia vitripennis. All injections were performed lateral to the dorsal midline in the posterior half of abdomens using pulled glass capillaries. Behavioral observations were made at room temperature (23–25°C) until adult flies initiated resumed walking (W2) behavior. Values in the same column followed by the same letter do not differ significantly from each other at P = 0.05. some elements of pupariation behavior in venom-injected larvae, consistent with ecdysone’s role in stimulating release of anterior retraction factor (ARF) and puparium tanning factor (PTF), two factors supposedly released from the CNS to regulate pupariation (Fraenkel et al., 1972; Seligman et al., 1977). Recent observations revealed that precocious shrinkage of the cuticle stimulated by a neuropeptide pyrokinin can cause a nearly perfect shape of the puparium even without the specific “contraction” patterns of muscular activity (Zdarek et al., 2000). Thus, the distorted puparia of venom-injected larvae may have been formed due to both depressed muscular function and inhibited ability of the cuticle to shrink. In fact, barograms revealed a delay in the onset of shrinkage of the cuticle that underlies the process of smoothing. This asynchrony of behavior and cuticular processes (i.e., shrinkage, loss of elasticity, phenolic tanning) can account for the abnormal shape of the puparium since only timely orchestration of neuromuscular and cuticular processes can produce morphologically normal puparia (Zdarek and Fraenkel, 1972; Zdarek et al., 1979). This does not preclude the possibility that other specific gene expression events required for puparium formation have been altered by the venom. The fact that exogenous ecdysteroid could rescue some aspects of pupariation in venom-injected larvae of S. bullata implies that venom may have blocked ARF and/or PTF release, which is October 2004 dependent on ecdysteroids (Fraenkel et al., 1972; Fraenkel, 1975), and can be inhibited by specific transcriptional and translational inhibitors (Seligman et al., 1977). Larvae treated with these inhibitors cannot undergo pupariation as retraction of anterior segments, longitudinal contraction, and sclerotization and tanning are blocked. Subsequent injection of fly larvae with ARF reverses inhibition of retraction but PTF treatment does not rescue tanning (Seligman et al., 1977). Similarly, in this study, injection of 20E into venom-injected prered spiracle larvae reversed the inhibition of retraction as well as tanning. Consistent with this scenario is the observation that a high percentage of late-red spiracle larvae, a stage of development in which ARF and PTF may have already been released prior to treatment, injected with venom retracted anterior segments, contracted longitudinally, and tanned. Development of envenomated pupae or pharate adults of S. bullata, however, does not resume following 20E treatment (Rivers and Denlinger, 1994b), arguing that venom is not inhibiting ecdysteroid release. Rather than triggering an ecdysteroid deficiency, it is more likely that venom alters the ability of target tissues in the CNS or elsewhere to bind hormone or suppresses tissue responsiveness by inhibiting transcription and/or translation. Supporting the latter scenario are the findings that the tanning response can be initiated by cAMP in fly larvae treated with transcriptional inhibitors but not in flies injected with translational blockers (Seligman et al., 1977), and that crude venom has been 90 Rivers et al. shown to elevate cAMP levels 4–5-fold in cultured cells within 1 h after exposure (Rivers, unpublished data). Collectively, these observations suggest that translational activity may have been reduced or abolished in certain tissues within the CNS of venom-injected larvae. This possibility is certainly not surprising since the venom has been shown to alter expression of genes encoding for specific heat shock proteins in envenomated pharate adults of S. crassipalpis (Rinehart et al., 2002). The altered behaviors of extrication and posteclosion can alternatively be explained by venom disturbing motor programs of the CNS, as argued for pupariation. For example, extrication is accomplished by cycles of stereotypic behaviors composed of anteriorly directed peristaltic muscular contractions and relaxations, resulting in abdominal and thoracic flexions and deformations (Reid et al., 1987a). Crude venom’s effect on shortening the duration of extrication behavior in an eclosing adult implies that the venom affects the motor neurons of the CNS that command the muscular system engaged in extrication, since the periodicity and coordination of patterned muscular movements are under the control of the CNS (Reid et al., 1987b). Fraenkel G. 1975. 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