Cause of mortality in insects under severe stress Hitoshi Matsumoto, Kohjiro Tanaka, Hirofumi Noguchi and Yoichi Hayakawa Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan Mortality in the host armyworm larvae Pseudaletia separata parasitized by the parasitic wasp Cotesia kariyai was dra- matically increased when they were simultaneously infected by the entomopathogen Serratia marcescens. Previous studies have shown that this strong insecticidal effect is due to a metalloprotease-like insecticide (MPLI) released from S. marcescens enterobacter. This study was conducted to elucidate the exact cause of the mortality resulting from MPLI.InjectionofMPLIcausedasharpincreasein hemolymph dopamine concentration followed by elevated levels of brain dopamine in armyworm larvae. [ 3 H]Dop- amine injected into the hemocoel, was incorporated into the brains of MPLI-injected larvae to a level eight times greater than in BSA-injected control larvae. Transmission electron microscopy showed an obvious decrease in thickness and density of the brain sheath in insects injected with MPLI. This was probably due to the MPLI-induced elevation of hemocyte metalloprotease activities. Further, electron microscopic and TUNEL staining analyses showed a signi- ficant increase in apoptotic cells in the brain 12 h after the injection. Injection of 3-iodotyrosine (a tyrosine hydroxylase inhibitor) before MPLI completely prevented the increase in hemolymph dopamine in test larvae and their following death. From these observations, we conclude that MPLI- injected larvae may have suffered mortal damage through increased apoptosis of brain cells caused by an influx of dopamine from the hemolymph. Keywords: apoptosis; brain; dopamine; insect; stress. Most insects are generally short-lived. They may die from a slight accident or injury. Intense external stress such as mechanical immobilization or enforced activity some- times triggers autointoxication, culminating in paralysis and death [1–3]. For example, fighting between pairs of male cockroaches (Nauphoeta cinerea) establishes a dominant– subordinate relationship. Such interactions often kill the subordinate insect without any visible external damage [4]. This is similar to the social stress found in mammals. It is well known that male rats in particular show pronounced dominant–subordinate behavior. Prolonged aggression produces stress in the subordinate, and ultimately a diseased state, characterized by a stress syndrome eventually leading to death. These deaths cannot be attributed to external damage [5]. Subordinate cockroaches may also die from physiological changes comparable to those accompanying stress syndromes in mammals. However, it is worth emphasizing some notable differences between insects and mammals: insects (cockroaches) die much more readily than mammals. This is probably not only due to the difference in body size but also to something unique in the physiological systems of insects; they must possess a mechanism that renders them particularly susceptible to intense stress. To clarify the mechanism that controls mortality in insects, we focused on dying parasitized host insects. Parasitoid wasps never kill their host insects before their larvae emerge from them. However, when host insects are infected with entomopathogens such as baculoviruses and microsporidia before or after parasitization, their premature deaths have been observed before the wasp larvae have completed their development [6]. In fact, most host Pseu- daletia separata larvae die within 3 days of parasitization by the wasp Cotesia kariyai when they are simultaneously infected by the enterobacter Serratia marcescens. Previous studies have shown that this mortality is mainly due to metalloprotease-like insecticide (MPLI) released by S. mar- cescens enterobacter [7]. Purified MPLI showed a strong insecticidal effect with a median lethal dosage (LD 50 ) of 13 pmol per larva. In preliminary experiments, we injected purified MPLI into mice with the same dose per weight to that used for the armyworm larvae, but we did not observe any symptoms or disorders. In this study, we tried to extended these experiments to elucidate the mechanism by which MPLI kills armyworm larvae within a few days of injection. Our results indicate that the dopamine concentration in the hemolymph was elevated by the injection of MPLI, resulting in influx of dopamine into the brain through the externally damaged sheath. At the same time, apoptosis of brain cells was observed in the test larvae. Materials and methods Animals P. separata larvae were reared on an artificial diet at 25 ± 1 °C with a photoperiod of 16-h light : 8-h dark. Correspondence to Y. Hayakawa, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan 060-0819. Fax: 011 706 7142, Tel.: 011 706 6880, E-mail: hayakawa@orange.lowtem.hokudai.ac.jp Abbreviations: MPLI, metalloprotease-like insecticide; NH 2 -Mec, 7-amino-4-methylcoumarin; A2pr, 2,3-diaminopropionyl; ECD, electrochemical detection; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. (Received 14 May 2003, revised 25 June 2003, accepted 7 July 2003) Eur. J. Biochem. 270, 3469–3476 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03745.x Penultimate-instar larvae undergoing ecdysis 2–2.5 h after lights on were designated as day-0 last-instar larvae. The weight difference between P. separata larvae used for bioassay was limited to within 0.02 g [8]. Chemicals 3,4-[7- 3 H]Dopamine (21.5 CiÆmmol )1 ) was purchased from Dupont-NEN. Dopamine hydrochloride and 3-iodotyro- sine were obtained from Nacalai Tesque Inc., Kyoto, Japan. Fluorescent substrates (NH 2 -Mec-Ac-substrates) were pur- chased from the Peptide Institute Inc., Minoh, Japan. Experimental injection MPLI used for injection experiments was purified as described previously [7]. MPLI and BSA were diluted with NaCl/P i (8 m M NaH 2 PO 4 ,1.5m M KH 2 PO 4 , 137 m M NaCl, 2.7 m M KCl, pH 7.2) to 0.1 lgÆlL )1 (final volume 10 lL) and injected into day-1 last-instar larvae of the armyworm 8 h after lights on. A 3-iodotyrosine-saturated solution was made by mixing  5 mg 3-iodotyrosine with 1 mL NaCl/P i . Before injection of MPLI, 10 lL of the 3-iodotyrosine solution was injected twice into day-2 penultimate-instar and day-0 last-instar larvae 6 h after lights on. MPLI was injected 2 h after the second injection of 3-iodotyrosine. Biogenic amine assay The hemolymph sample (20 lL) was collected into a 1.5-mL microtest tube containing 200 lLice-cold0.2 M perchloric acid and homogenized in an Artek Sonic Dismembrator (10 pulses at 40 W). The supernatant after centrifugation at 20 000 g for 10 min at 4 °C was collected, and a 1-lL aliquot was analyzed byHPLC–electrochemical detection (ECD) [9]. A dissected brain was placed in a 1.5-mL microtest tube containing 70 lL0.2 M perchloric acid, sonicated, and centrifuged at 20 000 g for 10 min. A 50-lL aliquot of the resulting supernatant was analysed by HPLC–ECD [9]. The HPLC–ECD system comprised an RP-HPLC C 18 column (Capcell Pak C 18 UG120; 4.6 · 150 mm; Shiseido Co., Tokyo, Japan) and a coulometric electrochemical detection system (ESA 5100 A, Bedford, MA, USA) [10]. Radiolabeled dopamine incorporation into brains 3,4-[7- 3 H]Dopamine was diluted with NaCl/P i to 10 lCiÆlL )1 , and injected into larvae 6 h after injection of BSAorMPLI.After1h,thebrainwasdissected,washed three times with NaCl/P i , and immediately homogenized with 100 lL0.2 M perchloric acid. Dopamine was separated by paper chromatography, and its radioactivity counted using a liquid-scintillation counter (Aloka LSC-5100). Microscopic observation Brains were dissected from test armyworm larvae and fixed with 2.5% glutaraldehyde and 1% paraformaldehyde in Pipes buffer (0.1 M Pipes, 0.05 M sucrose, pH 7.4) at 4 °C. Post-fixation and staining was performed in 2% aqueous OsO4 and 2% uranyl acetate, respectively. The tissue was embedded in Epon 812 (TAAB Laboratories Equipment Ltd, Aldermaston, Berkshire, UK) after dehydration. Thin sections were cut on an Ultracut (Reichert-Jung, Wien, Austria). For electron microscopy, thin sections were briefly stained in 2% aqueous uranyl acetate and 0.1% lead citrate [11]. Micrographs were taken with a JEM-1200EX (Jeol Ltd) electron microscope. Assay of metalloprotease activity Hemolymph was collected into a chilled microtest tube containing NaCl/P i with 0.05% phenylthiourea, and imme- diately centrifuged at 4 °C for 10 min at 500 g.The collected supernatant was used as a plasma sample. The remaining pellet was washed with NaCl/P i by gentle suspension and centrifugation. The pellet was then homo- genized, centrifuged at 20 000 g for 10 min at 4 °C, and washed three times with NaCl/P i containing 0.05% phenyl- thiourea. The precipitate after centrifugation was suspended in NaCl/P i and used as the hemocyte membrane sample. Dissected brains and fat body were homogenized in ice-cold NaCl/P i containing 0.05% phenylthiourea by sonication, and centrifuged at 20 000 g for 10 min at 4 °C. The pellets Fig. 1. Dopamine levels in hemolymph (A) and brains (B) of MPLI- injected larvae. Day-0 last-instar larvae of the armyworm were injected with 17.4 pmol per larva of MPLI (s)(n ¼ 5–7), or BSA as a control (d)(n ¼ 5–6), 6–7 h after ecdysis. *Significantly different from control larval value (P <0.01:Student’st-test). **Significantly different from control value (P <0.05:Student’st-test). Each point represents the mean ± SD from the number of determinations in parentheses. 3470 H. Matsumoto et al.(Eur. J. Biochem. 270) Ó FEBS 2003 were suspended in NaCl/P i andusedasbrainandfatbody samples, respectively. Three metalloprotease substrates (1,NH 2 -Mec-Ac- Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH 2 ; 2, NH 2 -Mec-Ac-Asp-Glu-Val-Asp-Ala-Pro-Lys(Dnp)-NH 2 ; 3, NH 2 -Mec-Ac-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH 2 ) were solubilized in dimethyl sulfoxide (final concentration 10 m M ) and used as stock solutions. The reaction mixture (total volume 190 lL) consisted of 50 m M Tris/HCl buffer (pH 7.5), 0.1 M NaCl, 10 m M CaCl 2 , 0.05% Brij35 and one of the substrates (10 l M ) [12,13]. The mixture, without the tissue samples, was equilibrated at 37 °C for 10 min, and the reaction was started by adding the tissue sample. The reaction was terminated after 30 min by adding 20 lLice- cold 50% (v/v) acetic acid. The release of fluorescent product was detected at k ex 328 nm and k em 393 nm using a fluorescence spectrophotometer (Shimadzu Co.) [14]. Analysis of DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) To detect apoptotic neural cells on sectioned preparations, brains dissected from test larvae were fixed with 4% paraformaldehyde in NaCl/P i and embedded in paraffin. Apoptotic cells were detected on sections with the In situ Cell Death Detection Kit POD (Boehringer-Mannheim) according to the manufacturer’s instructions. All sections were counterstained with hematoxylin [15]. Results Hemolymph and brain dopamine levels in MPLI-treated larval brains Dopamine is the most abundant catecholamine in the insect hemolymph and nervous tissues, where it may serve as a hormone, neuromodulator and neurotransmitter [16,17]. Fig. 3. Morphological changes of the neural sheath layer of MPLI-injected larval brains. Brains were dissected from day-0 last-instar larvae of the armyworm 12 h after injection of 17.4 pmol per larva of BSA (A,C) or MPLI (B,D), and observed at a magnification of ·5000 (A,B) and · 20 000 (C,D). Note that the neurilemma of MPLI-injected larva is thinner (indicated with bars shown in A and B) and less dense (indicated with stars as shown in C and D) than that of control BSA- injected larva. Further, a gap between the neurilemma and perineural cells is visible in MPLI-injected larval brain (as indicated in D with an arrow). Fig. 2. [ 3 H]Dopamine incorporation into MPLI-treated larval brains. Brains were dissected from day-0 last-instar larvae of the armyworm which had been injected with 17.4 pmol per larva of MPLI (closed bar)orBSA(openbar)andtheninjectedwith10lCi [ 3 H]dopamine per larva. Radioactive dopamine was quantified as described in Materials and methods. Each bar represents the mean ± SD from four independent determinations. Ó FEBS 2003 Death from stress in insects (Eur. J. Biochem. 270) 3471 Because it is also an essential intermediate of N-acetyldop- amine and N-b-alanyldopamine, which function as tanning agents for insect cuticle, the integument also contains dopamine in high concentration. Previous studies have shown that parasitization by C. kariyai wasps results in increased dopamine levels in the integument, and that this tissue secretes dopamine into the hemolymph, thereby raising dopamine levels there [18,19]. Based on these observations, we speculated that dopamine concentration may be increased in the dying armyworm larvae injected with MPLI. Dopamine levels were measured in the hemo- lymph and brains of the larvae after injection of MPLI (Fig. 1). As expected, hemolymph dopamine was increased within 3 h of injection and reached levels 20–23 times higher than those observed in control BSA-injected larvae. Brain dopamine levels were also elevated by the injection and reached the maximal level  9 h after the injection. Dopamine incorporation into MPLI-treated larval brains The delay in reaching maximal brain dopamine level compared with maximal hemolymph dopamine level sug- gested that dopamine flows into the brain from the hemolymph. This was confirmed by the influx of [ 3 H]dop- amine into the brain after its injection into the hemocoel of MPLI-injected larvae (Fig. 2). Radiolabeled dopamine incorporated into MPLI-injected larval brain was 7–8 times more abundant than that observed in control larvae. Structural changes in MPLI-treated larval brains The dopamine influx into the MPLI-injected larval brain suggested that brains in the larvae may be damaged by the MPLI treatment. To address this, brains of the MPLI- injected larvae were analyzed by transmission electron microscopy. Differences in the thickness and density of the neurilemma are evident in Fig. 3: the neurilemma of the MPLI-injected larvae is thinner and less dense than that of the control larvae. Further, the perineural cells, which are tightly attached to the neurilemma in the control brains, are slightly separated from the neurilemma in the MPLI-treated larval brains. MPLI-induced enhancement of metalloprotease activity in hemocytes The structural changes in the brain sheath caused by the MPLI treatment suggested that the neurilemma matrix may be degraded by MPLI-induced proteolysis. To test this, we first confirmed metalloprotease activity of MPLI using three commercially available fluorogenic peptide substrates [1,NH 2 -Mec-Ac-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met- Lys(Dnp)-NH 2 ; 2,NH 2 -Mec-Ac-Asp-Glu-Val-Asp-Ala- Pro-Lys(Dnp)-NH 2 ; 3,NH 2 -Mec-Ac-Pro-Leu-Gly-Leu-A2pr (Dnp)-Ala-Arg-NH 2 ] [12,13]. Although MPLI hydrolyzed all three, substrate 2 was hydrolyzed most rapidly (Fig. 4A). This substrate specificity is similar to that of matrix metalloproteinase stromelysin 1 [12]. As only 17.4 pmol MPLI was injected into each test armyworm larva, it is unreasonable to expect that the injected MPLI could directly degrade the neurilemmal matrix. Instead, we specu- lated that the injected MPLI activated other metallopro- teases. To confirm this, metalloprotease activities in the hemolymph (plasma and hemocytes), fat body and brain were determined after injection of MPLI. Unexpectedly, the hemocyte enzyme activity with substrate 2 was significantly elevated 6 h after injection of MPLI, but the enzyme Fig. 4. Metalloprotease activities of MPLI and extracts of various tis- sues. (A) Enzyme activities of MPLI for three substrates (n ¼ 6); (B) enzyme activities for substrate 2 in brains, fat body, hemocytes and plasma 6 h after injection of MPLI (n ¼ 4); (C) time course of enzyme activity with substrate 2 in hemocytes after injection of 17.4 pmol per larva of MPLI (s) or BSA (d) into test larvae (n ¼ 4). Each point and bar represents the mean ± SD for the number of determinations in parentheses. 3472 H. Matsumoto et al.(Eur. J. Biochem. 270) Ó FEBS 2003 activities in the other tissues were not changed (Fig. 4B). The substrate 2 hydrolyzing activity was increased approxi- mately twofold in hemocytes 9 h after injection of MPLI (Fig. 4C). Further, hemocytes showed broadly equivalent hydrolyzing activities with all three substrates (data not shown), indicating that MPLI did not stimulate the sole metaloprotease in hemocytes after injection of MPLI. However, there was no increase in the hydrolyzing activity with synthetic substrates for serine proteases such as Var- Pro-Arg-NH 2 -Mec, Ile-Glu-Gly-Arg- NH 2 -Mec, Phe-Ser- Arg- NH 2 -Mec or Gln-Arg-Pro- NH 2 -Mec (data not shown). Thus, as we speculated, injection of MPLI activated hemocyte metalloproteases, which contributed to the deg- radation of the neurilemmal matrix. MPLI-induced apoptosis of brain cells The final question is whether MPLI-induced elevation of brain dopamine levels results in changes in the brain cells. To examine this, brains dissected from the larvae 20 h after injection of MPLI were studied by transmission electron microscopy. Condensed chromatin was observed in the brain cells of the MPLI-injected larvae (Fig. 5A), suggesting that the brain cells were undergoing apoptosis as the result of the MPLI injection. This was substantiated by evidence that the number of TUNEL-positive cells was increased in the brains of the larvae 12 h after injection of MPLI. Prevention of the MPLI-induced dopamine elevation decreased the insecticidal effect of MPLI To confirm the contribution of dopamine to the mortality of insects, we tried to avoid elevating hemolymph dopamine concentrations after the MPLI injection and observed the lethality. Prior injection of 3-iodotyrosine, a competitive inhibitor of tyrosine hydroxylase, completely blocked the MPLI-induced increase in hemolymph dopamine (Fig. 6A). We analyzed the effects of this pretreatment with 3-iodotyrosine on the mortality of MPLI-injected insects. The survival rate of MPLI-injected larvae gradually decreased soon after the injection, and was only 20% 72 h after the injection. When the larvae were injected with 3-iodotyrosine before the MPLI injection, none of them had died 72 h after the injection of MPLI (Fig. 6B). Further, the number of TUNEL-positive cells in the brain of the 3-iodotyrosine-injected larvae was obviously decreased 12 h after injection of MPLI (Fig. 6C,D). However, the surviving 3-iodotyrosine-pretreated larvae did not meta- morphose normally to pupae and died before pupation (data not shown). Fig. 5. Transmission electron micrographs (A,B) and TUNEL-staining (C,D) of MPLI-injected larval brains. Electron microscopic observation of brains of armyworm larvae 20 h after injection of 17.4 pmol per larva of MPLI (A) or BSA (B). Note that MPLI treatment induced obvious condensation of chromatins (indicated with white arrows). TUNEL staining of brains of the armyworm larvae 12 h after injection of MPLI (C) or BSA (D). Note that MPLI treatment induced TUNEL-positive neural cells (indicated with black arrows). Ó FEBS 2003 Death from stress in insects (Eur. J. Biochem. 270) 3473 Discussion Dopamine plays a fundamental role as a neurotransmitter in the mammalian central and peripheral nervous systems. It is closely involved in a variety of important physiological and behavioral processes such as modulation of motor skills and higher-order cognitive function [20]. Insects have two separate pools of dopamine: nervous system and integu- ment [16,17,21,22]. Dopamine is the most abundant mono- amine in the nervous system and may serve as a neurotransmitter and neuromodulator [15,16]. Extremely high concentrations of dopamine are present in the integu- ment where it is used as an essential intermediate of cross- linking agents in cuticle formation throughout insect Fig. 6. Insecticidal effect of dopamine in hemolymph. (A) Hemolymph dopamine levels in armyworm larvae treated with MPLI and/or 3-iodotyrosine. Day-0 last-instar larvae of the armyworm were injected with 17.4 pmol per larva of MPLI (n ¼ 4) or BSA (n ¼ 4). 3-Iodotyrosine was administered previously to the test larvae as described in Materials and methods. Hemolymph was collected for dopamine measurement 6 h after injection with MPLI or BSA. Each column represents the mean ± SD for the number of determinations in parentheses. (B) Survival of larvae treated with MPLI and/or 3-iodotyrosine. Day-0 last-instar larvae injected with 17.4 pmol per larva of MPLI (n ¼ 10) (m). Larvae pretreated with 3-iodotyrosine before injection with 17.4 pmol per larva of MPLI (n ¼ 10) (h). Larvae injected with 17.4 pmol per larva of BSA (n ¼ 10) (d). This result was a typical case from four independent experiments, but the probabilities of significant survival difference between 3-iodotyrosine- treated and nontreated animals were 100%. (C, D) TUNEL-staining of brains of the armyworm larvae pretreated with BSA (C) or 3-iodotyrosine (D) 12 h after injection of MPLI. Note that 3-iodotyrosine pretreatment decreased the number of TUNEL-positive neural cells. Other explanations as in Fig. 5. 3474 H. Matsumoto et al.(Eur. J. Biochem. 270) Ó FEBS 2003 development [21,22]. Previous studies indicate that the dopamine concentration in the integument is about 50 times that found in the hemolymph. Further, we found that integument dopamine was secreted into incubation medium in vitro [19]. Therefore, it is reasonable to expect that integument dopamine is released into the hemolymph. If this is true, the large increase in hemolymph dopamine concentrationinMPLI-injectedlarvae(showninFig.1) would also be due to its release from the integument. Even though the dopamine concentration is significantly increased in the hemolymph, dopamine cannot normally penetrate the hemolymph/brain barrier because it is thought that the neural sheath cells comprising this barrier are selective to the exchange of metabolites and ions between the blood and the underlying brain in healthy insects [23–26]. However, once the neural sheath is damaged, as seen in the MPLI-treated larval brain (Fig. 3), dopamine can enter the brain through the neural sheath. Therefore, it is plausible that the increased dopamine in the brain of MPLI-injected larvae shown in Fig. 1 is due to influx from the hemolymph. The MPLI-induced increase in brain dopamine is smaller than the increase in hemolymph dopamine. However, this difference may be mostly due to the difference in the rate of dopamine metabolism in the two tissues: dopamine is metabolized more rapidly in the brain than in the hemolymph, therefore more dopamine may have passed into the brain than was measured. Many studies on dopamine-induced apoptosis of neural cells as well as culture cells have been published over the last few years [27–30]. To our knowledge this is the first to provide evidence of this phenomenon occurring in insects. The increased numbers of apoptotic neural cells after injection of MPLI suggests that apoptosis of brain cells may be the cause of MPLI-induced mortality. As mentioned above, insects have a vast dopamine pool in their integuments. These amounts, it would appear, are enough to actually kill the insects. 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