TMOF-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 47:169–180 (2001) TMOF-Like Factor Controls the Biosynthesis of Serine Proteases in the Larval Gut of Heliothis virescens Ralf Nauen,1 Dorian Sorge,1 Andreas Sterner,2 and Dov Borovsky2* 1 Bayer AG, Agrochemicals Division, Research Insecticides, Leverkusen, Germany 2 University of Florida-IFAS, FMEL, Vero Beach Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity increases immediately after the molt from the 3rd to the 4th instar larvae and declines just before the molt into the 5th instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI) 4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosylL-phenylalanine chloromethylketone (TPCK) indicate that protease activity is mostly trypsinlike (80%) with a small amount of chymotrypsinlike activity (20%). Injections of late 3rd and 4th instar larval hemolymph into H. virescens larvae inhibited tryspin biosynthesis in the larval midgut. Similar results were obtained when highly purified 4th instar larval hemolymph that crossreacted with Aea-TMOF antisurm using ELISA was injected into 2nd instar larvae. Injections of AeaTMOF and its analogues into 2nd instar, and Aea-TMOF alone into 4th instar larvae stopped trypsin biosynthesis 24 and 48 h after the injections, respectively. Injections of 4th instar H. virescens larval hemolymph into female Aedes aegypti that took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsinlike enzymes in the midgut of a lepidoptera is modulated with a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch. Insect Biochem. Physiol. 47:169–180, 2001. © 2001 Wiley-Liss, Inc. Key words: trypsin; chymotrypsin; Aea-TMOF; proteases; midgut; Lepidoptera; Heliothis virescens INTRODUCTION Protein digestion in Heliothis virescens is mediated by endopeptidases and exopeptidases secreted from the midgut epithelium cells into the luminal fluid of the midgut (Terra, 1990). There is considerable interest in characterizing proteolytic enzymes from agricultural pest insects in order to develop genetically engineered insect crop resistant plants that carry factors that modulate serine proteases. © 2001 Wiley-Liss, Inc. Ralf Nauen and Dov Borovsky contributed equally to this work. Contract grant sponsor: NIH; Contract grant number: AI 41254, NATO CRG 940057; Contract grant sponsor: U.S. Israel Binational Science Foundation; Contract grant sponsor: Insect BioTechnology Inc.; Contract grant sponsor: Bayer A.G. *Correspondence to: Dov Borovsky, University of FloridaIFAS, FMEL, 200 9th St. SE, Vero Beach, FL 32962. E-mail: firstname.lastname@example.org Received 12 December 2000; Accepted 1 April 2001 170 Nauen et al. The use of genes encoding plant protein protease inhibitors (PI) has been tried (Hilder et al., 1987; Johnson et al., 1989; Ryan, 1990); however, no successful control of insects fed on these transgenic plants was achieved. An explanation to these results is that larvae of Helicoverpa zea (corn ear worm) and Lymantria dispar (gypsy moth) produce large quantities of trypsin-like enzymes that are resistant to natural and genetically engineered protease inhibitors that are expressed in plants (Broadway, 1995). These observations were also confirmed in other phytophagous insect pest species, i.e., Spodoptera exigua (beet army worm), which uses serine proteases as its major digestive enzyme, and the coleopteran Leptinotarsa decemlineata (Colorado potato beetle), which uses aspartate and cysteine proteases (Jongsma et al., 1995; Bolter and Jongsma, 1995). A second approach to control agricultural pest insects is to develop transgenic plants that produce a bacterial toxin protein such as Bacillus thuringiensis δ-endotoxins (Vaeck et al., 1987). However, the high production of these toxins in plants has caused rapid resistance in insects to these proteins (Alstad and Andow, 1996). TMOF, isolated from the dipteran ovary, was shown to be the physiological signal that terminates trypsin biosynthesis in mosquitoes and fleshflies (Borovsky et al., 1990, 1993, 1994, 1996; Bylemans et al., 1994). These observations indicate that insects control their digestion with natural hormones that could be used, if identified, to control digestion in many agricultural pest insects that use serine proteases as their main digestive enzymes (Lehane et al., 1995). In Neobellieria bullata, TMOF was shown to control trypsin biosynthesis through a translational control of the trypsin gene (Borovsky et al., 1996). We tested the effect of Aea-TMOF and a highly purified TMOFlike peptide from the hemolymph of H. virescens larvae, on trypsin biosynthesis in Heliothis and mosquitoes in order to determine if H. virescens, which uses serine proteases as the main digestive enzymes, controls these enzymes with a TMOF-like hormone(s). MATERIALS AND METHODS Insects Larvae of H. virescens (Lepidoptera: Noctuidae) were synchronized at head-capsule slippage from 3rd to 4th instar and raised individually in 24-well tissue culture plates (Falcon, Lincoln Park, NJ) on an artificial diet. Under these conditions, the first and second instar stages are 2 days each, and the 4th instar stage is 4–5 days. Chemicals Azocoll, general protease substrate, was purchased from Calbiochem (Bad Soden/Ts., Germany), bovine serum albumin (BSA), N-benzoyl-L-arginine p-nitroanilide (BApNA), Triton-X-100, SBTI (Soybean Trypsin Inhibitor), BBI (Bowman-BirkInhibitor), AEBSF (4-(2-Aminoethyl)benzensulfonylfluoride) and TPCK (N-Tosyl-L-Phenylalanine chloromethylketone) from Sigma (St. Louis, MO). Aea-TMOF H-Tyr-Asp-Pro-Ala-(Pro)6 -COOH, Mr 1047.18, 97% pure) was obtained from Insect Biotechnology Inc. (Durham, NC). All other chemicals were obtained from Serva (Heidelberg, Germany). Isolation of Midguts and Extraction of Larval Proteinases Midguts were excised from cold-anaesthetized or etherized larvae, washed and transferred into ice-cold 0.15 M NaCl, and homogenized in Eppendorf tubes with a hand homogenizer. The homogenates were centrifuged at 14,000g and the supernatants filtered by centrifugation (Millipore Ultrafree-MC, Millipore Corp., Bedford, MA) and stored at –20°C until used. Protein concentrations in the extracts were determined according to Bradford (1976) using bovine serum albumine (BSA) as a standard. Enzyme Assay Protease activity was determined using a proteinase substrate Azocoll 1% (w/v) in 0.1 M borate/NaOH buffer, pH 10.0. Trypsin activity was measured with N-benzoyl-L-arginine p-nitroanilide (BApNA) (Borovsky and Schlein, 1988). Reactions were incubated with 50 µl of midgut extracts containing either 40 µg protein or a known number of midgut-equivalents for 75 min (Azocoll) or 30 min (BApNA) at 30°C. Controls were incubated without midgut extracts, to determine nonenzymatic hydrolysis of substrates. After incubations, reactions containing Azocoll were chilled on ice for 5 min, centrifuged for 10 min at 16,000g in an Eppendorf microfuge, and the absorbance of the supernatant was measured Control of Serine Proteases in H. virescens With TMOF at 520 nm. To determine the effect of various inhibitors on proteolytic activity, midgut-extracts were pre-incubated for 20 min at room temperature with the inhibitors in assay-buffer before the addition of the substrate. Controls were pre-incubated without inhibitors with water for SBTI and AEBSF or water:ethanol (1:10) for TPCK. Polyacrylamide Gel Electrophoresis (PAGE) SDS PAGE was run using a vertical slab apparatus (Biometra Minigel, Biometra Gottingen, Germany) (Laemmli, 1970) without Dithiothreitol (DTT) and the samples were not heated to avoid denaturation of proteases. Aliquots of midgut extracts were mixed with an equal volume of sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2 mM EDTA, 0.02% bromophenol blue) and were run at 4°C on a 12% acrylamide gel at 14 mA. After electrophoresis, the gel was transferred to a glass plate. An indicator gel of 12% polyacrylamide containing 0.15% BSA was laid over it and a second glass plate was placed on the top of the gel. The resulting gel sandwich was held in place by two clamps and was incubated in 0.1 M Tris-HCl, pH 8.5, buffer at room temperature for 21 h to allow diffusion of proteins from the electrophoresis gel into the indicator gel. After the transfer, the indicator gel was incubated with 2.5% (w/v) Triton X-100 for 1 h at room temperature and washed two times for 10 min each with double distilled water and incubated with 0.1 M Tris-HCl, pH 8.5, for 1 h. The gel was then stained with 0.1% (w/v) Coomassie brilliant blue, dissolved in ethanol:acetic acid:water (45:10:45). Proteolytic activity appeared as clear zones on a dark blue background of non-digested BSA substrate after destaining the gel in 25% (v/v) ethanol and 8% (v/v) acetic acid. Larval Feeding Food consumption and intermoult development were observed using 4th instar larvae of known age. Insect growth was synchronized as follows: 3rd instar larvae showing head-capsule slippage were weighed at the time of collection and individually placed in 24-wells tissue culture plates, containing artificial diet of known weight. After ecdysis to the 4th instar (usually happens within 24 h) larval weight, developmental state, and diet weight were monitored daily until the 5th instar molt. 171 Food was provided ad libitum and if necessary changed every 2 days to reduce microbial contamination. To determine the time point of initial diet ingestion after ecdysis to the 4th instar, larvae and diet were weighed 1, 3, and 6 h after eclosion. Changes in larval weight were determined and food utilization was expressed as a percentage of weight gain vs. food intake. Feeding of Trypsin Inhibitors and TMOF AEBSF or SBTI (5 to 200 µg in 2 µl water were adsorbed onto food pellets (45–60 mg each). Control pellets were treated with 2 µl of water without inhibitors. Food pellets were distributed into 24-well tissue culture plates. Following eclosion, pre-weighed individual 4th instar larvae were placed into each well. Since larvae consumed about 45–60 mg of diet every 24 h, food was given to larvae every 24 h and weight increase of each larva was monitored at 24 and 48 h. Aea-TMOF (36 µg) in 3 µl water was applied to food pellets (4 × 4 mm; 35–45 mg) that were entirely consumed within 24 h. Control pellets were adsorbed with 3 µl water and the pellets were transferred into the wells of 24-wells tissue culture plates. Following eclosion, individual pre-weighed 4th instar larvae were placed in each well. New food pellets were given after 24 h and individual larvae, thus, consumed 72 µg of TMOF within 48 h. Injections Aea-TMOF and its analogues. Third instar larvae weighing 20–26 mg were injected with TMOF and several synthetic analogues (1 µg to 1 ng) in 0.5 µl insect Ringer (86 mM NaCl, 5.4 mM KCL, 3 mM CaCl2.2H2O) into the ventral abdomen using finely drawn glass capillary tubes. Newly molted 4th instar larvae were injected with Aea-TMOF (36 and 72 µg) in 3 µl insect Ringer solution with a 10-µl Hamilton-syringe into the ventral abdomen. Controls were injected with 0.5 or 3 µl Ringer without TMOF and its analogues. After injection, the larvae were weighed and placed individually in 24-well trays containing artificial diet. The weight development of each larva was monitored after 24 and 48 h. Trypsin activity in the midguts of 3rd and 4th instar larvae was measured at 24 and 48 h in 3 individual guts or 3 groups of 3–4 guts. 172 Nauen et al. Larval hemolymph. Hemolymph of 3rd instar larvae of H. virescens showing head capsule slippage and late 4th instar larvae was collected in chilled Eppendorf tubes by cutting off an abdominal leg. After centrifugation for 5 min at 16,300g, 5 µl hemolymph of 3rd instar larvae was injected into newly molted 4th instar larvae using a Hamilton syringe. Controls were injected using 5 µl insect Ringer solution. Both, hemolymph and insect Ringer contained very small amounts (ng) of N-phenylthiourea (PTU). The weight of injected larva was measured 24 and 48 h later. Hemolymph (0.5 µl) that was collected from late 4th instar larvae was injected with a finely drawn glass capillary into female Ae. aegypti. When smaller hemolymph volumes were used, i.e., 0.0625 to 0.25 µl appropriate dilutions of the hemolymph were done in insect Ringer and the final volume that was injected was 0.5 µl. Hemolymph from late 4th instar larvae was also highly purified using Sep Pak C18 reverse-phase cartridge and HPLC (Borovsky et al., 1990). TMOF active fractions were dried by Speed Vac at 40°C, and rehydrated in insect ringer and 0.5 µl were injected into 3rd instar H. virescens lar- vae. Twenty-four hours later, guts were dissected out and trypsin biosynthesis was determined (Borovsky and Schlein 1988). Fig. 1. Comparison between larval gut protease activity, food consumption and weight gain during the 4th instar development of H. virescens larvae. Results are expressed as the average of 3 determinations ± S.E.M. Proteolytic activity is expressed as 100% on day 3, and as a percentage of the maximal activity otherwise. Determination of TMOF Concentration in the Hemolymph Hemolymph of late 4th instar larvae was assayed for TMOF using ELISA (Borovsky et al., 1992). Statistical Analysis Data were analyzed using the Student’s t-test. RESULTS Food Consumption, Weight Gain, and Proteolytic Activity of 4th Instar Larvae Under the conditions of these trials, the 4th larval instar (L4) stage of H. virescens is 4 to 5 days. The relationship between an increase in insect weight, food intake, and proteolytic activity was studied (Fig. 1). Initial feeding started 1 h after the molt; however, food consumption, increase in weight, and proteolytic activity at 6 h after ecdysis were low (Fig. 1). The first fecal Control of Serine Proteases in H. virescens With TMOF droppings were observed 6 h after the molt in most of the larvae that were tested (data not shown), indicating that 5 h are needed for the food to pass through the digestive system. Six hours after the 3rd to 4th instar molt, the weight, food consumption, and proteolytic enzymes activity rapidly increased and reached a peak at day two. In preparation for the next molt, body weight decreased from day three onward, whereas food consumption and proteolytic enzymes activity remained high until day four (Fig. 1). The decrease in weight that occurred between the 3rd to the 4th larval stage and the 4th to the 5th larval stage is due mostly to the shedding of the cuticle (Fig. 1). Effect of Serine Protease Inhibitors In vitro. In order to find out if the major midgut proteolytic activity of the 4th larval instar is trypsin-like, several trypsin inhibitors SBTI, BBI, AEBSF, and chymotrypsin inhibitor TPCK were incubated with larval midgut extracts and general protease substrate Azocoll (Fig. 2). Proteolytic activity of days 1–3 of 4th instar larval gut extracts was 98 and 95% inhibited by 100 µM of trypsin inhibitors SBTI and BBI, respectively (Fig. 2). AEBSF (100 µM) inhibited 50% of the proteolytic activity in the larval midgut extract (Fig. 2). To inhibit 87% (day 1) to 92% (day 2) of total proteolytic activity, 1 mM of AEBSF was needed (data not shown), indicating that it is not Fig. 2. Effect of different serine protease inhibitors, in vitro, on midgut proteases of 4th instar H. virescens larvae. Protease activity is expressed as percentage of controls incubated without the inhibitors (100 µM). Results are expressed as an average of 3 determinations ± S.E.M. 173 as good an inhibitor as SBTI or BBI. TCPK, a specific chymotrypsin inhibitor, had little effect on midgut proteolytic activity activity at day 1 and 3 (22 and 14% inhibition, respectively) and no effect at day 2 (Fig. 2). These results indicate that the major larval proteolytic activity in the midgut of H. virescens is trypsin-like. In vivo. The in vitro incubations of larval gut extracts indicated that AEBSF and SBTI were effective in inhibiting trypsin-like activity. To find out if these inhibitors were equally effective in vivo, increasing amounts (5–200 µg) of the inhibitors were fed with the synthetic diet to larvae. Feeding 400 µg of AEBSF in 2 days (200 µg/day) to individual larvae caused 80% inhibition of total proteolytic activity (Fig. 3A) with a calculated IC50 of 60.5 µg/day. Higher quantities above 200 µg could not be tested because larvae refused to eat the artificial diet. SBTI was more effective earlier causing 44 and 80% inhibition of proteolytic activity within 24 h when 5 and 10 µg were fed, respectively (Fig. 3B). Higher doses up to 200 µg did not cause more inhibition of proteolytic activity (Fig. 3B). Feeding SBTI caused higher inhibition of proteolytic enzymes activity at 24 than at 48 h (Fig. 3B). These results are different than the AEBSF feeding in which 100 and 200 µg caused higher inhibition at 48 h than at 24 h (Fig. 3A). Feeding increasing amounts of AEBSF proportionally reduced larval growth. Mean weight gain of 4th instar larvae feeding on different amounts of AEBSF treated diet was at 48 h between 42 ± 2 (mg ± S.E.M.) and 19.7 ± 1 (mg ± S.E.M.) as compared to the weight of control larvae 62.6 ± 1.5 (mg ± S.E.M.) that were fed the untreated diet. When 5 µg of AESBF was fed daily to individual larvae, the weight gain decreased by 67% as compared with controls. After feeding larvae daily 20 µg of AEBSF, the weight gain at 48 h was 70% lower than in controls (Fig. 4). The weight gain of larvae that were fed SBTI for 48 h was 85 to 100% similar to controls that were fed on the untreated diet (Fig. 4). These results indicate that larvae that were fed SBTI for 48 h synthesized trypsin-like enzymes that were not affected by SBTI. Effect of Mosquito TMOF To find out if trypsin biosynthesis in H. virescens is regulated by a TMOF-like factor, 2nd 174 Nauen et al. Fig. 4. Effect of feeding trypsin inhibitors AEBSF and SBTI for 48 h on weight gain of 4th instar H. virescens larvae. Weight gain is expressed as percentage of controls that were fed artifical diet without the inhibitors. Results are the average of 3 determinations ± S.E.M. Fig. 3. Effect of feeding trypsin inhibitors (A) AEBSF and (B) SBTI on gut proteolytic activity of 4th instar H. virescens larvae. Inhibition is expressed as percentage of controls that were fed artificial diet without inhibitors. Results are expressed as an average of 3 determinations ± S.E.M. and 4th instar larvae were injected with AeaTMOF (Table 1, Fig. 5). Injections of TMOF and its analogues (1 µg to 1 ng) caused 50% inhibition of trypsin biosynthesis when 0.42 ± 0.026 (µM ± S.E.M.) TMOF was injected. When TMOF hexapeptide analogue (YDPAPP) was injected, 46 ± 2.8 (µM ± S.E.M.) caused 50% inhibition of trypsin biosynthesis in 2nd instar H. virescens larvae. This analogue was 109-fold less effective than the decapeptide. The penta-peptide analogue in which the Y was replaced with F was 1.3-fold less effective than the hexa-peptide (Table 1) and the tripeptide DPA caused 50% inhibition of trypsin biosynthesis at a concentration of 50 ± 2 (µM ± S.E.M.), which was similar to the effective concentrations of the hexa-peptide (Table 1). These results indicate that H. virescens TMOF is probably very similar to mosquito TMOF, and trypsin biosynthesis in 2nd instar larvae can be controlled by a TMOF-like molecule. To find out if TMOF can also inhibit trypsin biosynthesis in 4th instar larvae, TMOF (36 µg; 3 µl) was injected into larvae and 72 µg of TMOF (36 µg every 24 h) were fed to 3 groups of newly eclosed 4th instar larvae. Controls were injected with insect Ringer or were fed on a synthetic diet without TMOF. Forty-eight hours later, midguts were removed and analyzed for trypsin activity. In larvae that were injected with the hormone, 70% of trypsin activity and 61% of the weight gain were inhibited. On the other hand, in larvae that were fed the hormone, only 30% of trypsin biosynthesis and 26% of the weight gain were inhibited (Fig. 5). These results indicate that TMOF might be degraded in the gut, or it is not efficiently transported from the gut into the hemolymph. Effect of Hemolymph To find out if a TMOF-like molecule(s) is synthesized by H. virescens larvae and regulates trypsin biosynthesis, hemolymph (0.25–0.5 µl) from late 4th instar larvae was injected into blood Control of Serine Proteases in H. virescens With TMOF 175 TABLE 1. Effect of TMOF and Its Analogues on Trypsin Biosynthesis in H. virescens* Analogue N YDPA(P)6 YDPAPP FDPAP DPAP DPA 3 3 3 3 3 Inhibition of 50% trypsin biosynthesis (In50) (µM ± S.E.M.) 0.42 46 145 190 50 ± ± ± ± ± 0.026 2.8 8 46 2 *Twelve groups of H. virescens (10 larvae per group) were injected with 0.5 µl of water containing TMOF and its analogues (1 µg to 1 ng). Twenty-four hours later, guts were removed and analyzed with BApNA for trypsin biosynthesis. Controls were injected with water and compared with non-injected controls. Inhibition of trypsin activity was plotted against log concentrations of TMOF and its analogues and 50% inhibition was read from this curve. Results are expressed as an average of 3 determinations ± S.E.M. fed females Ae. aegypti and 24 h later midguts and ovaries were removed and analyzed for trypsin biosynthesis and egg development (Table 2). In intact females that were injected with 0.25 µl and 0.5 µl of hemolymph, trypsin biosynthesis was inhibited by 65 ± 18 (% ± S.E.M.) and 84 ± 10 (% ± S.E.M.), respectively, and egg development was inhibited by 44 ± 2 (% ± S.E.M.) and 100 ± 0 (% ± S.E.M.), respectively (Table 2). To find out if the TMOF-like factor from the hemolymph acts on the gut or affects the release of neuroendocrine factors from the brain or the thorax, female mosquitoes were fed a blood meal. One group was immediately decapitated and a second group was immediately ligated between the thorax and abdomen. Injection of H. virescens hemolymph into both groups caused 45.6 ± 14 (% ± S.E.M.) and 100 ± 0 (% ± S.E.M.) inhibition of trypsin biosynthesis (Table 2). The enhanced inhibition of trypsin biosynthesis in ligated abdomens indicates that the Heliothis TMOF might be structurally similar to mosquito TMOF, which is rapidly degraded in the thorax (Borovsky et al., 1993). To find out if the effect of the hemolymph and TMOF on trypsin biosynthesis in Heliothis can be sustained for 3 days, 2nd instar larvae of H. virescens (9 larvae/group) were injected with 4th instar larval hemolymph (0.25 mosquito TMOF (5 µg in 0.25 µl) and insect Ringer. Three days later, each group was analyzed for trypsin biosynthesis (Fig. 6). Trypsin biosynthesis 3 days after the injections of hemolymph Fig. 5. Effect of feeding and injecting Aea-TMOF on trypsin biosynthesis in the midgut of 4th instar H. virescens larvae. Larvae were fed 72 µg of TMOF (36 µg every 24 h) or were injected with 36 µg of TMOF dissolved in insect Ringer immediately after the molt to the 4th instar. Controls were fed artificial diet without TMOF or injected with insect Ringer. Forty-eight hours later, guts were removed and assayed for trypsin activity using BApNA. Results are expressed as percentage inhibition as compared to controls and are an average of 3 determinations ± S.E.M. and TMOF, was 70 and 49% inhibited in the midguts of injected larvae, respectively, as compared with controls that were injected with saline (Fig. 6). When hemolymph from late 3rd instar larvae was injected into 4th instar larvae, and midgut extracts were separated by PAGE (Fig. 7), 60 and 80% of the proteolytic activity in the midgut was lost 24 and 40 h, respectively, after the injections. (Fig. 7, lanes B and D) as compared with salineinjected controls (Fig. 7, lanes A and C). To find out if the inhibition was caused by a mosquito TMOF-like molecule, hemolymph of H. virescens was highly purified by HPLC (Borovsky et al., 1990). A fraction eluted between 60 to 70% acetonitrile 0.1% TFA that cross-reacted with TMOF antiserum by ELISA (Borovsky et al., 1992), was dried in a Speed Vac, rehydrated in insect Ringer, and 0.5 µl (equivalent to 0.25 µl of single 4th instar larval hemolymph) was injected into second 176 Nauen et al. TABLE 2. Effect of H. virescens Hemolymph on Trypsin Biosynthesis in Ae. aegypti* Female Ae aegypti were fed blood and immediately: N Trypsin synthesis (ng/gut ± S.E.M.) Inhibition (% ± S.E.M.) Yolk size (µm ± S.E.M.) Injected with 0.5 µl hemolymph 0.25 µl hemolymph 0.5 µl insect Ringer 3 3 3 93 ± 11 200 ± 55 661 ± 15 84 ± 10 65 ± 18 0 17 ± 5.6 111 ± 5.6 178 ± 6 Decapitated and injected with 0.25 µl hemolymph 0.25 µl insect Ringer 3 3 194 ± 29 425 ± 61 45.6 ± 14 0 Ligated and injected with 0.25 µl hemolymph 0.25 µl insect Ringer 3 3 0 400 ± 45 100 ± 0 0 Inhibition (% ± S.E.M.) 100 ± 0 44 ± 2 0 *Three groups of female Ae. aegypti (10 per group) were fed a blood meal, one group was kept intact, a second group was immediately decapitated, and a third group was immediately ligated between the thorax and abdomen. Females were injected with Heliothis hemolymph and controls were injected with insect Ringer. Nineteen hours after injections, midguts and ovaries were dissected out (3 per group) and analyzed for trypsin biosynthesis in midguts (Borovsky and Schlein, 1988) and yolk length in oocytes of intact females (Borovsky et al., 1990). Results are expressed as means of 3 determinations ± S.E.M. instar larvae (Table 3). Twenty-four hours later, midguts were assayed for trypsin biosynthesis. Trypsin biosynthesis was significantly inhibited 54 ± 5 (% ± S.E.M.) in larvae that were injected with purified hemolymph. When insect Ringer solution was injected, a significant increase of 2.2fold in trypsin activity was observed 24 h after the injection as compared with a control group that was assayed before the injections (Table 3). On the other hand, no significant difference was found between the control group and the hemolymph-injected larvae (Table 2). These results strongly indicate that H. virescens larvae regulate trypsin biosynthesis in the gut with TMOFlike factor(s). DISCUSSION Fig. 6. Effect of injections of 4th instar larval hemolymph and Aea-TMOF on trypsin biosynthesis in the midgut of 2nd instar H. virescens larva. Hemolymph (0.25 µl) of 4th instar larvae, TMOF (5 µg in 0.25 µl saline) and saline (0.25 µl) were injected into 2nd instar larvae and 3 days later guts were analyzed for trypsin biosynthesis (Borovsky and Schlein, 1988). A control group prior to the injections was also analyzed. Results are expressed as an average of 3 determinations ± S.E.M. Synthesis of trypsin in blood-fed insects that take a single protein meal to produce egg yolk proteins is cyclical. After the blood meal, trypsinlike enzymes are synthesized, reach a peak 24– 30 h after the blood meal, and then the synthesis declines, reaching a low point 56 to 72 h later (Borovsky, 1988). The down-regulation of trypsin biosynthesis in mosquitoes is controlled by TMOF (Borovsky, 1988, Borovsky et al., 1990, 1993, 1994, 1996). A similar mechanism has not been reported in lepidopteran larvae that continuously feed. When 4th instar larvae of H. virescens were fed a meal, proteolytic enzymes activity increased from a very low level immediately following Control of Serine Proteases in H. virescens With TMOF 177 TABLE 3. Effect of Purified H. virescens Hemolymph on Trypsin Biosynthesis† Second instar larvae Injected with purified hemolymph (0.5 µl) and assayed 24 h later Controls Injected with insect Ringer (0.5 µl) and assayed 24 h later Not injected, and assayed immediately N Trypsin (nmol/min/gut ± S.E.M.) Inhibition (% ± S.E.M.) 3 6.6 ± 0.6*,** 54 ± 5 3 9.4 ± 1.2*,*** 0 3 4.25 ± 1.4**,*** — † Hemolymph of late 4th instar larvae was partially purified on C18 Sep Pak column and HPLC (Borovsky et al., 1990), dried in a Speed Vac, and rehydrated in insect Ringer. Aliquots (0.5 µl) were injected into 2nd instar H. virescens larvae weighing 20 to 25 mg each. Midguts were removed 24 h later and analyzed for trypsin activity (Borovsky and Schlein, 1988). *Significant difference 0.025 < P < 0.05. **Not significant 0.1 < P < 0.375. ***Significant difference 0.01 < P < 0.025. Fig. 7. PAGE of midgut proteases of H. virescens 4th instar larvae that were injected with saline (5 µl) or 3rd instar larval hemolymph (5 µl) and analyzed 24 and 40 h later. Each lane was loaded with 0.02 midgut equivalents and the PAGE was analyzed for protease activity on an indicator gel. Light color bands indicate protease activity, 24 h after injections, (lane A) saline, (lane B) hemolymph, and 40 h after injections, (lane C) saline and (lane D) hemolymph. ecdysis, reached a peak in mid instar (days 1–3), and declined at the end of the instar. The increase and decrease in proteolytic enzymes biosynthesis followed food consumption, which was maximal on days 1–3 and very low before and after ecdysis (Fig. 1), and showed a similar pattern to that observed in mosquitoes (Borovsky, 1988). Similar results were obtained when gut extracts were incubated with the amidolytic substrate BApNA. Although BApNA can be hydrolyzed by other proteases that are not trypsin-like, there is now evidence that most midgut enzymes that hydrolyze BApNA are trypsin-like (Terra and Ferreira, 1994). To find out the relative amounts of trypsin and chymotrypsin-like enzymes in the midgut of H. virescens 4th instar larvae, trypsin inhibitors SBTI, BBI, AEBSF, and chymotrypsin inhibitor TPCK were incubated in vitro with a general proteinase substrate Azocoll. General proteolytic activity was almost completely inhibited by trypsin inhibitors SBTI and BBI but only 52% by AEBSF. Low inhibition was observed with TPCK, indicating that chymotryptic activity is low in H. virescens and the majority of the enzymes in the larval midgut on days 1–3 of the 4th instar are trypsin-like (Fig. 2). SBTI was reported to exhibit a high level of inhibition in vivo on H. virescens (Johnston et al., 1995) and on several other lepidopteran species (Johnston et al., 1991; Christeller et al., 1992; Purcell et al., 1992; Ferreira et al., 1994; McManus and Burgess, 1995; Novillo et al., 1999) and low level of inhibition on chymotrypsins from lepidopteran larvae (Christeller et al., 1992). Although there are several reports that chymotrypsins from several insect species are not sensitive to TPCK (Johnston et al., 1991; Gatehouse et al., 1999; Valaitis et al., 1999) and that TPCK is more stable at neutral pH (Lee and Anstee, 1995), our results indicate that chymotrypsin activity is no more than 20% of the total 178 Nauen et al. trypsin activity using BApNA, trypsin specific substrate, and BTpNA, chymotrypsin specific substrate (Borovsky, unpublished observations). Gene sequencing of both enzymes and Northern analysis also confirmed that the chymotrypsin transcript is less than 20% of the trypsin transcript (Borovsky, unpublished observations). These results contradict an earlier report that chymotrypsin activity accounts for 50% of total proteolytic activity in H. virescens (Johnston et al., 1995). In Helicoverpa armigera it is thought that trypsin and chymotrypsin activities play an equal role in proteolysis (Bown et al., 1997), whereas the chymotryptic activity of the tomato moth, Lacanobia oleracea, accounted for 30% of the total proteolytic activity (Gatehouse et al., 1999). The data presented by Johnston et al. (1995) are not comparable to our results because the larvae in the previous studies were not standardized as to age and phase within an instar. It is possible that enzyme levels may vary in different phases of larval development (Christeller et al., 1992; Keller et al., 1996; Novillo et al., 1999). It has been shown that several herbivorous insects adjust to the presence of protease inhibitors in their diet by producing novel inhibitor resistant trypsins in their midguts (Broadway, 1995; Jongsma et al., 1995; Wu et al., 1997; Bown et al., 1997; Gatehouse et al., 1997; Giri et al., 1998). Feeding of SBTI in a diet for 30 min stimulated the synthesis of SBTI resistant enzymes in newly molted 5th instar larvae of H. zea and Agrotis ipsilon and the enzymes persisted even if the insects were not fed proteinase inhibitor (Broadway, 1997). Our results from feeding larvae with a SBTI-containing diet suggest that a similar mechanism exists in H. virescens (Figs. 3,4). The higher levels of proteolytic activity observed 48 h after feeding SBTI are due to the induction of SBTI-insensitive serine proteases. This also explains why the increase in larval weight was almost unaffected by SBTI (Fig. 4). Failure to affect H. virescens larval growth was reported by Gatehouse et al. (1994) after feeding transgenic tobacco plants expressing SBTI, despite the fact that larval tryspsin was completely inhibited by SBTI in vitro. Bown et al. (1997) showed that feeding SBTI to H. armigera larvae caused preferential induction of trypsin mRNA transcripts that encoded trypsin insensitive SBTI. Thus, these results suggest that inhibitors that arrest enzyme activity might also stimulate over-production of the same enzymes or stimulate over-production of mRNA transcripts that are usually rare but, in the presence of an inhibitor, become the preferred transcripts. Borovsky (1988) showed that trypsin synthesis in mosquitoes is regulated by a factor that is synthesized by the mosquito ovary. A decapeptide was later purified and sequenced from the mosquito ovaries and was named TMOF (Borovsky et al., 1990, 1991). The hormone terminates the biosynthesis of trypsin-like enzymes in mosquitoes and several other insects (Borovsky et al., 1990, 1992). In the fleshfly, a hexapeptide named Neb-TMOF was isolated, sequenced, and shown to have a translational control of trypsin mRNA (Bylemans et al., 1994; Borovsky et al., 1996). Since the majority of the proteases in the midgut of H. virescens are trypsin-like, our results show that trypsin biosynthesis in Lepidoptera is regulated by TMOF like factor(s) circulating in the hemolymph. When Aea-TMOF and several of its analogues were injected into 2nd instar larvae, trypsin biosynthesis was inhibited 50% by 0.42 µM TMOF, whereas other analogues of shorter chain length were 100to 452-fold less effective, indicating that H. virescens TMOF might be structurally similar to Aea-TMOF. Indeed, injections of 4th instar larval hemolymph and highly purified hemolymph into blood-fed female A. aegypti stopped trypsin biosynthesis and egg development (Table 2). H. virescens hemolymph factor, like mosquito TMOF, acts directly on the midgut; ligation and decapitation did not prevent the factor from directly inhibiting trypsin biosynthesis in the midgut and indirectly inhibiting egg development (Borovsky et al., 1990, 1993). To prevent the possibility that the hemolymph carries toxic substances that indirectly may cause these effects, a highly purified hemolymph fraction that exhibited crossreactivity with Aedes TMOF antibodies by ELISA (Borovsky et al., 1992) was injected and significantly inhibited trypsin biosynthesis in 2nd instar larvae of H. virescens (Table 3). This is the first report that shows that A. aegypti TMOF like factor is circulating in the hemolymph of late 4th and 3rd instar H. virescens larvae and it has a role in regulating trypsin biosynthesis in the larval gut. Control of Serine Proteases in H. virescens With TMOF ACKNOWLEDGMENTS This work was partially supported by NIH AI 41254, NATO CRG 940057, U.S. Israel Binational Science Foundation, Insect BioTechnology Inc., Durham, N.C. and Bayer A.G. grants to professor Dov Borovsky. This paper is part of University of Florida, Florida Agricultural Experimental Station Journal Series No. R-07903 LITERATURE CITED Alstad DN, Andow DA. 1996. 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