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Ismail A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1989 b04OO\q ABSTRACT ROLE OF BIOGENIC AMINES IN BIOCHEMICAL AND BEHAVIORAL CHANGES INDUCED IN INSECTS BY FORMAMIDINE PESTICIDES BY Saad M. M. Ismail In these studies, the possible biochemical mechanisms of anorexia caused by formamidine pesticides and biogenic amines in the American cockroach Periplaneta americana L. and the tobacco homworm larvae, Manduca sexta, were investigated. Chlordimeform (CDM) is known to cause intense anorexia in the American cockroach at doses as low as 1 to 5 ugfrnsect. My results indicate that such a phenomenon was accompanied by an increase in haemolymph glucose level with simultaneous decrease in the trehalose level. The combined glucose and trehalose level remained relatively constant during the test period of 5 hr. These changes in haemolymph sugars were found to be due to the activation of trehalase in the thoracic muscles by in vivo adminstration of CDM or in vitro incubation with N-demethylchlordimeform ( DCDM). These phenomena were similar to the ones produced by octopamine (OA) both in vivo and in vitro. Phentolamine (PA) antagonized such actions of 0A and DCDM in the thoracic muscles. Furthermore, both DCDM and 0A could elevate CAMP levels in vitro in muscle homogenate preparation. These results support the notion that CDM, after metabolic activation to DCDM, acts on 0A receptors causing activation of the CAMP mediated- response system. The results of this activation is an increase of trehalase activity in the thoracic muscles and probably other tissues, which leads to the conversion of trehalose to glucose. Such a rise in haemolymph glucose could be the biochemical cause for anorexia in the American cockroach. In the tobacco homworm larvae, the antifeeding activity of CDM, DCDM, and CA was accompanied by elevation of haemolymph glucose, trehalose and total sugar levels. PA antagonized the elevation of haemolymph sugars by CDM, DCDM, and 0A in viva. In addition, PA inhibited the activation of haemolymph trehalase induced 0A and DCDM in vitra. The effect of OA and DCDM was not additive in vitra and in viva. These results support the notion that CDM and or DCDM act on the 0A receptors as has been shown in other insects. Such a rise in haemolymph sugars may be the biochemical cause for the antifeeding activity in this species. The interaction of CDM, DCDM, and amitraze with 0A receptors and the resulting enhancement of cAMP production in vitra were investigated in homogenates of the two- spotted spider mite, Tetranychus urticae Koch . DCDM and amitraze stimulated the production of cAMP in mite homogenate. Among various biogenic amines tested OA and synephrine were the most active in elevating cAMP levels while dopamine and 5- hydroxytryptarnine showed only marginal potency in this regard. The results indicate that formamidines are likely to act on 0A receptors which result in the elevation of the intracellular cAMP level. PA and propranolol antagonized the effect of DCDM on cAMP levels. The action of formamidines was very potent in elevating CAMP level as compared to the nonformamidine pesticides tested. Both DCDM and 0A caused an increase in phosphorylation activities on proteins which were also phosphorylated by exogenously added CAMP-dependent protein kinase. The results of pharmacological characterization studies confirmed the overall theory that the agonistic effects of formamidines are expressed primarily through the OA-sensitive adenylate cyclase system ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. Fumio Matsumura for his unending guidance and scientific insights. I thank the other members of my guidance committee ( Dr. J. Miller, Dr. K. Klomparens, Dr. M. Zabik, Dr. R. Hollingworth, and Dr. D. Newson ) for their many helpful suggestions concerning this research and my entire Ph. D. program. Thanks also to Hugh Olsen for his assistance in the University of California Davis. ii TABLE OF CONTENTS Page LIST OF TABLES ..................................................................................... v LIST OF FIGURES ................................................................................ Vii GENERAL INTRODUCTION ..................................................................... 1 CHAPTER 1. Studies on the biochemicalmechanismsofanorexiacause by formamidine pesticides in the American cockroach, Periplanera americana L ................................................................................. 7 INTRODUCTION .......................................................................... 8 MATERIALS AND METHODS ...................................................... . 10 InseCts ............................................................................... 10 Chemicals ........................................................................... 10 Studies on the anorectic effect of CDM ...................................... 10 Determination of haemolymph sugars .......................................... 11 Studies on trehalase activity in the thoracic muscles .................... . 12 Preparation of tissue extract for enzyme assay. ....... 12 Assay of trehalase activity ................................................ 13 Studies on the presence of octopamine receptors in the thoracic muscles and nervous system ....................................... 13 A. 3H-oct0pamine binding assay ........................................ 13 B. Adeneylate cyclase and CAMP assay ............................ . 14 Studies on protein phosphorylation in the nervvous system .............. 15 RESULTS ......................................................................................... 16 DISCUSSION .................................................................................... 37 CHAPTER 2. Studies on the biochemical mechnisms of anorexia caused by formamidine pesticides in the tobacco homworm Manduca saxra 40 INTRODUCTION ............................................................................. 41 MATERIALS AND METHODS .......................................................... 43 Insects .................................................................................. 43 Chemicals ............................................................................. 43 Studies on the anorectic effecr of CDM ........................................ 43 Determination of haemolymph sugars ......................................... 44 Studies on trehalase activity in the haemolymph ........................... 45 Determination of haemolymph lipids .......................................... 45 RESULTS ...................................................................................... 46 DISCUSSION .................................................................................. 61 iii CHAPTER 3. Influence of pesticides and nemoactive amines on CAMP levels of two-spotted spider mite ( Acari :Tetranychidae ) ........................... 65 INTRODUCTION ............................................................................. 66 MATERIALS AND METHODS ........................................................ 67 Mites .................................................................................... 67 Chemicals .............................................................................. 68 Analysis of CAMP in the homogenate ........................... . .............. 68 Studies on protein phosphorylation ............................................ 69 RESULTS ....................................................................................... 70 DISCUSSION .................................................................................. 82 GENERAL DISCUSSION.AND CONCLUSIONS ................................ 84 APPENDD(.UItrastrucmral studies on formamidine— induced Changes of neurosecretion in the American cockroach, Periplaneta americana L .......... 88 LITERATURE CITED ..................................................................... 92 iv Table 10. 11. 12. 13 14. 15. LIST OF TABLES Page Anorectic effect of CDM. DCDM, and other neuroactive amines in the American cockroach ...................................................................... 17 Efi‘ect of CDM. DCDM, and OA on haemolymph sugarsof the American cockroach ................................................................................... 19 Effect of ATP and Mg2+ on trehalase activity of thoracic muscles ............. 23 Effect of OA agonistic and antagonists on trehalase activity in viva ............ 26 Effect of OA agonists and antagonists on trhalasc activity in vitra .............. 27 Effect of different compounds on the binding of 3H»OA in the nervous sysrem and thoracic muscles ............................................................ 31 Effect of piperoyl butoxide on 3H-OA binding in the nervous system and thoracic muscles ...................................................................... 32 Effect of OA, DCDM, and 8-Br-CAMP on endogenous phosphorylation of total proteins in homogenates of nervous system. The data were obtained by denistomen'ic scaning of radioautograms of SDS- polyacrylamide gel electrophoresis, and are expressed as relative intensitirs in % of the total lane intensity of the control (=100) .................. 36 Anorectic effect of CDM, DCDM, and 0A in the tobacco homworm larcae... 47 Effect of CDM, DCDM, and CA on body weight gain by tobacco homworm larvae ............................................................................. 48 Effect of CDM, DCDM, and CA on fecal production of tobacco homworm larvae ......................................................................................... 49 Effect of OA antagonists and agonistis on haemolymph trehalase activity of the tobacco homworm in vitra .......................................................... 58 Effect of OA agonists and antagonists on haemolymph sugars in the tobacco homworm larvae in viva ................................................................... 59 Effect of OA, Cdm, and DCDM on lipid levels in haemolymph of the tobacco homworm larvae ........................................................................... 60 Effect of octopamine and DCDM on CAMP levels in haemolymph of two— spotted spider mites ......................................................................... 74 16. 17. Efl'ect of various pesticides on CAMP levels in homogenates of two-spotted spider mites in virra ......................................................................... 78 Effect of octopamine, N-demethylchlordimeformmCDM) and 8-Br-CAMP on endogenous phosphorylation of total proteins in homogenates of two- sponed spider mites. The data were obtained by densitometric scanin g of radioautograms of SDS -polyacrylamide gel electrophoresis, and are extirgased as relativ intensities in % of the total lane intensity of the control 8 = ) .......................................................................................... 1 vi Figure 992v LIST OF FIGURES P826 Structure of biogenic amines ............................................................... 3 Structure of formamidine pesticides ...................................................... 4 Structure of nonformamidine pesticides ................................................. 5 Efl’ecronOug CDM/inseCton glucose(-)andtrehalose(o-) levels in the American cockroach at difl'erent times. Results are means 1 SD of 3 experiments each performed in triplicate. Means with the same letter for each sugar are not significantly different at the 5% level according to the LSD after ANOVA- - ....................... 21 Efi'ect CDM of (-o-), DCDM (-o-), and CA (+) on trehalase activity in thoracic muscles. Results are means 1; SD of 3 experiments each performed in triplicate. Trehalase activity of control = 1513.1 i 25.9 nmol glucose/mg protein/hr. Means with the same letter for each level of concentration are no: significantly different by Tukey's tesr at the 5% level .............................................. 22 Efi'ecr of 2 mM CDM ('0') and 2 mM CA (-I- ), 10 ul injeCtion/rnseCt. on trehalase aCtivity in thoracic muscles at different times. Results are means i SD of 3 experiments each performed in triplicate. Means with the same letter are not significantly different for each time level by Tukey's test at the 5% level after ANOVA. The control acdvity at 0 time was 1,528.5 1:1 .4 nmole glucose/mg protein/hr ............................. 24 Activation of trehalase activity in the thoracic muscles by OA ( o ) and DCDM ( . ) in virra. EC5o of OA and DCDM are 8 x 10-9M and 3 x 10’7 M respecrively. Trehalase activity of conu'ol = 1433 132.9 nmol glucose/mg prorein/hr. Results are means 3; SD of 4 experiments each performed in triplicate ............................................. 28 Inhibitory effect of phentolamine on trehalase aCtivity of the thoracic muscles caused by 10'9 M OA ( o ) and 10‘7 M DCDM ( . ) in vitro IC50 = 10-6 M and 3 x 1043 M phentolamine for CA and DCDM respecrively. All data have been expressed as the absolute values in glucose formation above that (1468 i 138 nmol glucose/mg protein/hr) observed in the absence of additions. Trehalase aCtivities caused by 10’9 M OA and 10‘7 M DCDM were 705 3; 44.4 and 749 i 55.6 nmol glucose/mg protein/hr above basal activity. Results are means :SD of 4 experiments each performed in triplicate ................................................................... 29 vii 9. Efi'ects of OA, DCDM, and 8-Br-CAMP on endogenous phosphorylation of specific proteins in preparations of the nervous system. Lanes shown are: connol (Lane 1), lmM OA (Lane 2), 10 mM OA (Lane 3), 1 mM DCDM (Lane 4), 10 mM DCDM (Lane 5), 1 mM 8-Br-CAMP (Lane 6), and 10 mM 8-Br—CAMP (Lane.7).The figure shows autoradiograph of SDS-polyacrylamide gel-electrophoresis. Note that the proteins were first phosphorylated (non-radioactive) using endogenous protein kinase, and second, the remaining unphosphorylated proteins were phosphorylated using gamma-32P-ATP and exogenously added PKA. Therefore, the increase in activity of endogenous protein kinases on a given protein expressedas dredecreaseintheintensity ofacorrespondingprotern bandon the electrophoretogram ....................................................... 33 10. Effect of CDM (~o-), DCDM (+), and OA 00-) on glucose levels in haemolymph of the tobacco homworm larvae after 1 h in viva. Results are means 1: SD of 3 experiments each performed in triplicate. Glucose level of conn'ol = 54 3; 4.4 glucose/ 100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injecrion) are nor significantly different by Tukey’s tesr at the 5% level after factorial AN VA 11. Efi'ecr of CDM (-0- ), DCDM (+), and CA (+) on glucose levels in haemolymph of the tobacco homworm larvae after 6 h in viva. Results are means 3; SD of 3 experiments each performed in triplicate. Glucose level of control = 57 i 3.9 mg glucose/100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injeCtion) are n0t significantly difi'erent by Tukey’s test at the 5% level after factorial ' ANOVA ...................................................................... . 52 12. Efl'ect of CDM (0-), DCDM be), and OA (qr) on trehalase levels in haemolymph of the tobacco homworm larvae after 1 h in viva. Results are means 1; SD of 3 experiments each performed in triplicate. Trehalose level of control = 467.3 1; 86.7 mg trehalose/ 100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injecrion) are n0t significantly different by Tukey's test at the 5% level after factorial AN OVA .......................................................... 53 13. Efl’ect of CDM {-0- ), DCDM (+), and CA (+) on trehalose levels in haemolymph of the tobacco homworm larvae after 6 h in viva. Results are means 1 SD of 3 experiments each performed in triplicate. Trehalose level of control = 503 1: 123 mg trehalose/lOO ml haemolymph. Means with the same letter for each level of concentration (10 ul injection) are not significantly different by Tukey's tesr at the 5% level after factorial ANOVA .......................................................... 54 14. EECCt of CDM (-o-), DCDM (+), and CA (-a-) on total sugar levels in haemolymph of the tobacco homworm larvae after 1 h in viva. Results are means 1: SD of 3 experiments each performed in triplicate. Total sugar level of control = 521.6 3 91 mg total sugar/ 100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injection) are not significantly different by Tukey's test at the 5% after factorial ANOVA....... .................................................... 55 viii 15. .16. 17. 18. 19. Efi'ect of CDM (-o-), DCDM (+), and CA (+) on t0tal sugars levels in haemolymph of the tobacco homworm larvae after 6 h in viva. Results are means 3 SD of 3 experiments each performed in triplicate. Total sugar level of control = 560.6 i 126 mg total sugar/ 100 ml haemolymph. Means with the same level of concentration (10 ul injection) are not significantly difi'erent by Tukey’s test at the 5% level after factorial AN OVA ....................................................... 57 Efi'ect of (+) octopamine; (~o-) synephrine; (s) norepinephrinm 0*") epinephrine; (43-) 5-hydroxyuytamine; and (at) dopamine on CAMP levels in homogenates of two-spotted spider mites in vitra. Data are means i SD as % of control (=100) for 3 experiments with 3 replicates for each concentration. The control was 0.59 3; 0.03 pmol CAMP/mg protein/min .................................................. 71 Effects of (-o-) chlordirneform; (.5) N-demethylchlordimeform; 00-) N- didemethylchlordimeform; and (-o-) amitraz on CAMP levels in homogenates of two-sponed spider mites in vitra. Results are means 1; SD as % of control (=100) for 3 experiments with 3 replicates for each concentration. The control was 0.59 i 0.03 pmol CAMP/mg protein/min ..... 72 Lineweaver-Burk plots of adenylate cyclase in homogenate of two-sported spider mites treated with (:1) octopamine and ( e) octopamine plus DCDM . DCDM concentration was 1 uM. Each point is the mean of three separate assays. IN = l/velocity (pmol CAMP/mg protein/min) and 1/8 = lloctopamine concentration ( M). Control velocity = 0.51 :1; 0.02 pmol CAMP/mg protein/min .................................................................. 75 Inhibition of DCDM stimulation by octopamine receptor antagonists. (+) phentolamine; and (O) propranol in homogenates of two-spotted spider mites. Data are means 1: SD as % of inhibition of DCDM for 3 experiments with 3 replicates for each treatment. DCDM concentration was 0.1 mM. 75 Effects of octopamine, DCDM, and 8-Br-CAMP on endogenous phosphorylation of specific proteins in homogenates of twoospOtted spider mite. Lanes shown are: lmM DCDM (Lane 1), 10mM DCDM (Lane 2), lmM OA (Lane 3), 10mM OA (Lane 4), control (Lanes 5 and 6), lmM 8-Br-CAMP (Lane 7), and 10mM 8-Br-CAMP (Lane 8). The figure shows autoradiograph of SDS-polyanylamide gel- electrophoresis. Note that the proteins were first phosphorylated (nonradioactive) using endogenous protein kinase, and second, the remaining unphosphorylated proteins were phosphorylated using gamma-32P-ATP and exogenously added PKA. Therefore, the increase in activity of endogenous protein kinases on a given protein expressed as the decrease in the intensity of a corresponding protein 79 band on the electrophoretogram ...................................................... GENERAL INTRODUCTION The action of the majority of pesticides is to disrupt the normal functioning of the insect nervous system (Corbett et al., 1984). Pesticides that mediate their actions via changes in the levels of second messengers could thus act at a variety of different sites such as the receptors themselves or any of the steps between the activation of the receptor and the actual response system (Evans and Davenport,l986). In addition to these direct effects of pesticides on second messenger systems, they may also have indirect effects induced by the release of endogenous neurohormones. Thus a variety of pesticides have been demonsn'ated to release a range of nem'ohormones, including diuretic hormone (Casida and Maddrell, 1971), adipokinetic hormone (Singh and Orchard, 1982) and octopamine (Davenport and Evans,1984). Biogenic amines are Chemical messengers in various tissues. In the nervous system, biogenic amines are located primarily in the neurosecretory system. Biogenic amines themselves may act as bioactive Chemicals regulating various physiological and behavioral functions. Furthermore, amines are known to function as either neurotransmitters, neuromodulators or neurohormones in invertebrate species (Barker et al., 1972; Robertson et al., 1976; Evans,1980 and 1985; Evans and Davenport,l986; Evans et al., 1988). The insect nervous system contains a large number of different biogenic amines (Figure 1) including octopamine, dopamine, serotonin (S-hydroxytryptamine), and noradrenaline (norepinephrine) (Evans,1980). Octopamine (CA) is known to be a major neurotransmitter (Orchad et al.,1983), neurohorrnone (Downer,1979a and 1979b) and neuromodulator in insects (Morton and Evans,1984). So far as is known, type-2 octopamine receptors are associated with the membrane-bound enzyme, adenylate cyclase. Binding of octopamine to type-2 oCtoparnine receptor activates this enzyme and thereby causes an increase in intracellular CAMP levels in the effector cells (N athanson,1985). In turn CAMP aCtivates various protein kinases which carry out the messages initiated by OA. Type-2 0A receptors have never been identified in vertebrate species (Bodnaryk,1982). Octoparrrine itself is present in only minute concentrations in vertebrates (Nathanson,1985). Such an observation raises the possibility that type-2 OA receptors are selectively localized in invertebrates. From the viewpoint of development of selective pesticides, the use of type-2 OA agonists (such as formamidine-based pesticides) may be desirable since they may be able to cause distruption of the hormonal and transmitter functions of OA in insects without noticeable ill effects on vertebrates (Nathanson, 1985 and 1987). The mechanism whereby formamidines (Figure 2) protect plants and animals from arthropod attack is complex, with dose-dependent lethal and sublethal effects being involved, particularly at critical points in the life cycle. The sublethal effects on behavior are associated with an increase in arousal and excitability of the insect with Changes in locomotory activity, reduction in feeding and disruption of reproductive behaviour (Beeman and Matsumura,1978; Hollingworth and Lund, 1982; Knowles, 1982; Matsumura and Beeman, 1982; Davenport and Wright,1985; Nathanson,1987). The work in this dissertaton is an attempt for understanding : (1) The possible mechanisms of appetite control in insects, such as the American cockroach Periplaneta americana and the tobacco homworm Manduca sexta , using Chlordimeforrn as a tool for probing the anorectic mechanisms in these species., and (2) The function of biogenic amines in the mite species Tetranychus urticae Koch in terms of the CAMP-second messenger system. This study was devised first to establish the methodology to study biogenic amine-sensitive systems in mites and second to find the dominant biogenic amines in T etranychus urticae. In addition, I have also examined similar effects of formamidine pesticides in as much as that they produce unique behavioral changes which have been postulated to be mediated through the alteration of the aminergic response systems. Furthermore, several nonformamidine pesticides (Figure 3) were tested to determine which pesticides induce alteration of the aminergic response systems. 3 Figure 1. Structures of biogenic amines. Octopamine: l-(p -hydroxypheny1)-2-aminoethanol. HOCHCHZ N32 0!! Synephrine: 1-(4-hydroxyphenyl)-2-methylaminoethanol. HOCHCHZNHC83 OH Serotonin: 5-hydroxytryptarnine. H Dopamine: 3,4-dihydroxyphenethylamine. Ho— / \ mzmzm" 6’ HO Figure 1 contnued: Norepinephrine: HOCHCHQNHQ 2—amino-1-(3,4-dihydroxyphenyl)ethanol. OH OH Epinephrine: KOCH l~(3,4-dihydoxyphenyl)—2-methylamino)ethanol. CHZNHCH‘J OH 0!! Figure 2. Structures of formamidine pesticides. Chlordimeforrn: N'-(4-chloro-a -tolyl)-N,N -dimethylformamidine. 933 I R) R=H. R'=CH3 / \ / C 1 —-x N - CIIN\ DCDM _ \ CH ( R') =H. R'=H CH 3 3 DDCDM Aminaz: 1,3-di(2,4—dimethy1phenylimino)-Z-methyl azapropane. C33 113C CH3 N —CH— N—CII- N ca C33 Figure 3. SnuCtures of nonformamidine pesticides. Chlorobenzilate: m14,4' ' zil . e y -drchloroben ate 0!! \ \ \ / C1 or / T __ cooczag DDT: dichlorodiphenyltrichloroethane. / \ 5““ F C 1 \ CH \ \>-—c 1 Dicofol: 1,1-bis(p- Chlorophenyl)-2,2,2-trichloroethanol OH ./ \ L__/ \ .31 — CCI.3 ‘—' HCH: 1,2,3,4,S,6-Hexachlorocyclohexane I ; \ ---c1 Aldicarb: CI-I3SC(CH3)CH=NOCONHCH3 2-methyl-2-(methylthio)propionaldehyde 0 -(rnethylcarbamoyl)oxime. Parathion: 0,0 -diethyl O-p -nitrophenyl phosphorothioate. C2H50\ S \ ll P—O no C 2 II 50 Fenitrothion: 0,0 -dimethy10- (3-methyl—4-nitrophenyl) phosphorothioate. Fenvalerate: 4—Chloro- 0(-1(methylethyl)benzeneacetic acid cyano(3-phenoxyphenyl)methyl CSICI' Deltamethrin: 3-(2,2-Dibromoethenyl)-2,2-dimethylcyclopropanecarboxylic acid cyanot 3- phenoxyphenyl)methyl ester. Br CHAPTER 1 Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the American cockroach Periplaneta americana L. INTRODUCTION Chlordimeform (CDM) is a unique pesticide which elicits multiple effects in various insect and acarina species. It is being used to suppress pest populations of ticks, mites, and insects (Beeman,1982). Many of the biochemical and physiological responses have been described in target and non-target species (Maitre et al., 1978; Lund et al., 1979; Baily et al., 1982; Beeman, 1982) indicating very unique action mechanisms of this type of pesticide. In brief, toxicological responses of insects to CDM appear to result form the action of CDM on membrane ion Channels (Lund et al., 1979; Beeman and Matsumura, 1982; Hollingworth and Lund, 1982) at high doses and the interaction of CDM or DCDM, with OA-sensitive receptors at low doses (Evans and Gee,1980: Hollingworth and Murdock, 1980; Nathanson and Hunnicutt, 1981; Gole et al., 1983). Octopamine (OA) is now firmly established to be a major neurotransmitter and neurohormone in insects (Nathanson, 1985). So far as known, 0A receptors are associated with a membrane-bound enzyme, adenylate cyclase which synthesizes CAMP (Rodbell,1984). Binding of CA to its receptor activates this enzyme and thereby causes an increase in the intracellular CAMP levels in the effector cells (N athanson, 1985). Elevation of CAMP levels resulting from the activation of 0A receptors is known to eventually cause the activation of various protein kinases which in turn, increase the levels of phosphorylation of various target proteins. It is this last step of reactions that bring about the diverse physiological responses of the cell to the applied biogenic amines (Nestler and Greengard,1984). Octopamine is an effector of several excitatory processes in insects (Downer, 1980). It appears that in lepidopterous insects and in locusts, OA acts as an excitatory neurotransmitter, particularly at the thoracic ganglia (Evans and Gee, 1980). It has also been reported that OA causes enhancement of trehalase activity in muscle and hemolymph of the American cockroach, Periplaneta americana L. (Jahagirdar et al., 1984). Furthermore, it has been reported that OA and CAMP stimulate protein phosphorylation in the central nervous system of Schistacerea gregaria (Rotondo et al., 1987). CDM and its analogs provide a good tool for the study of aminergic, particularly octopaminergic function in insects. The formamidines are particularly useful in that they appear to penetrate the central nervous system and its barrier systems while OA itself cannot penetrate through the nerve sheath and plasma membranes. It has been reported that CDM causes intense anorexia in the American cockroach Periplaneta americana L. at low doses such as 1 to 5 uglinsect (Beeman and Matsumura,1978). Beeman and Matsumura (1978) concluded that such an effect could not be the result of CDM's repellent action, since the same phenomenon could be reproduced by injecting CDM. Nor is it likely that physiological impairment per se causes the effect, since the roaches poisoned by fenitrothion, while showing significant visible toxicological effects, consumed as much food as the untreated roaches. Such a potent and rather specific anorectic effect of CDM might mean that aminergic, including octopaminergic, neurons are involved in controlling the sensation of hunger in some insects (Beeman and Matsumura, 1978). There is some supporting evidence for this hypothesis. In the above study, OA itself caused anorexia. The antifeeding activity of CDM in the tobacco homworm, Manduca sexta was greatly enhanced by the simultaneous application of a phosphodiesterase inhibitor (Nathanson, 1985). However, since the actions of formamidines are complex, one must conduct a careful study to establish a cause-effect relationship in proving or disproving such a hypothesis. Moreover, the mechanisms of appetite control in insects are poorly understood. Reported herein are the results of my investigative efforts on this subject using CDM as a tool for probing the anorectic mechanism in this species. 10 MATERIALS AND METHODS Insects Adult male American cockroaches, Periplaneta americana L. were used in all experiments. They have been maintained in our laboratory for several generations under standard conditions (Beeman and Matsumura, 1978). Chemicals D,L-octopamine hydrochloride (OA), L-norepinephrine (NE), isoproterenol, GTP, ATP, trehalose and the glucose assay kits were obtained from Sigma Chemical Company (St. Louis, MO). Phentolamine hydrochloride (PA) was obtained from CIBA-Geigy (Summit, NJ). Fenitrothion was from EPA (Research Triangle Park, NC). Chlordimeform (CDM), N -demethylchlordimeform (DCDM) as hydrochloride salts were synthesized and purified in our laboratory, 3H-D, L—OA hydrochloride (3,4-ring 3H- labeled, specific activity 12 Ci /mmol and the CAMP assay kits were obtained from Amersham Corporation (Arlington Heights, IL). Studies on the anorectic effect of CDM CDM, DCDM, and several neuroactive amines were assayed for anorectic activity in starved American cockroach, Periplaneta americana, using the method of Beeman and Matsumura (1978). Prior to the feeding experiments, the insects were held in battery jars without food (water only) for 10 to 14 days. At the end of this conditioning period and at the start of the experiment, the test Chemicals were injected abdominally with 5 ul of water, and the insects were held for 1 hr without food or water. The roaches were then isolated in 11 l-pint cardboard ice cream cartons covered with cheesecloth and each insect was offered a small, preweighed piece of Purina High Protein Dog Chow (50-100 mg). After a feeding period of 5 hr, food consumption was measured to the nearest 0.1 mg. In each experiment, simultaneous controls were run by using roaches taken from the same culture boxes treated by injection with the same volume of water and tested for food consumption as described previously (Beeman and Matsumm'a, 1978). Determination of hemolymph sugars Adult, male American coclo‘oaches were used (10 insects per test) for this purpose. The Chemicals (CDM and DCDM) at 1-50 uglinsect and CA at 50-400 ugfrnsect were injected abdominally with 5 ul of water. Control insects were treated in an identical manner with 5 ul of water, and always were run parallel to each test. The insects were held for a few minutes without food and water after the treatment, then individually isolated in l-pint cardboard ice cream cartons covered with cheesecloth and starved for additional 5 hr (water only). In some experiments, the roaches received 20 ug CDM, but did not receive any food or water during the entire test periods, and the hemolymph sugars were measured at different times. At the end of starvation period hemolymph was collected from the insects by the method of Stemburg and Conigan (1959) by cutting the tip of the antennae, placing upside down in a retainer inside a glass centrifuge tube and by brief centrifugation. The non-reducing disaccharide, trehalose is the major hemolymph sugar in most insect species and provides an important source of metabolic energy. Because trehalose yields glucose on acid hydrolysis, it may be estimated as reducing sugar. Determination of hemolymph sugars was carried out, using the method of Steele (1961) with some modifications. Two samples (20 ul each) of pooled hemolymph for each dose group were taken and placed in separate small ampules. Both samples received 20 ul each of 2N HCl but one sample was treated immediately with 2N NaOH to neutralize the 12 effect of HCl for the glucose assay. The other sample was used for trehalose assay by autoclaving at 15 lb./in.2 for 30 min, and then neutralized by 2N NaOH. Ten ul of each sample was taken for determination of hemolymph glucose and trehalose respectively, using the method developed by Sigma Chemical Company (Bulletin No. 15-UV) which depends on the following reactions: Glucose + ATP + Hexokinase -—> G-6-P + ADP G-6-P + NADP + G-6-PD -—) 6-PG + NADPH The level of NADPH was assessed spectrophotometrically at 340 nm. Studies on trehalase activity in the thoracic muscles The procedures used in these experiments are based on those used by Jahagirdar et al. (1984). Adult male insects were injected abdominally with 10 ul of cockroach Ringer’s solution (Hoffman and Downer, 1976) containing the desired concentration of test compounds at a range of 10‘2 - 10'5M using a Hamilton microsyringe. Preparation of tissue extract for enzyme assay Muscle tissues were dissected rapidly after 30 min from injection, rinsed in Ringer's solution and then frozen in liquid nitrogen and subsequently pulverized. The tissue powder was then sonicated for l min in 100 mM phosphate buffer pH 6.3 using Branson Sonifier Cell Disrupter at 200 W. Following centrifugation of the sonicated suspension at 500 x g for 10 min, the resulting supernatant was assayed for enzyme activity. The time interval between isolation of tissues and commencement of trehalase assay was approximately 15 min. In in vitra experiments, muscle tissues were dissected, rinsed in insect saline (Yamasaki and Narahashi, 1959) solution and incubated at room 13 temperature for 30 min in solutions of the test compounds at 10‘3M. Insect saline (Yamasaki and Narahashi, 1959) was used as a vehicle for the test Chemicals (10 ul injection) in the experiments for time-course study and the effects of octopamine agonists and antagonists on trehalase activity. Assay of trehalase activity Trehalase activity was measured by monitoring the release of glucose from trehalose without acid hydrolysis as described in the previous section. The reaction mixture for the muscle trehalase comprised of 28 mM trehalose, 100 mM phosphate buffer pH 6.3, and an aliquot (10 or 20 ul containing approximately 20-25 ug protein) of enzyme preparation in a total volume of 1.0 ml. The incubation was carried out at 40°C for 60 min. Under these conditions, the production of glucose due to the trehalase activity was linear over the incubation period for 60 min and gradually declined thereafter. Studies on the presence of octopamine receptors in the thoracic muscles and nervous system of the American cockroach A. 3H-octopamine binding assay The method adopted was similar to the one developed by Hashemzadeh et a1. (1985) for the octopamine receptor of the firefly light organ. The thoracic muscles and the nervous system (the brain and the ventral nerve cord) were dissected from 8 insects kept on an ice bath with care to remove traces of fat body and integument. Freshly dissected thoracic muscles and the nervous system were rinsed and homogenized separately in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4), containing 5 mM MgC12, using a glass/teflon homogenizer. The homogenates were centrifuged at 500 x g for 10 min to yield the crude nuclear fraction as a pellet. The supernatant was centrifuged at 40,000 x g 14 at 4°C for 1 hr. The second supernatant fraction was carefully discarded and the final pellet was resuspended in the same Tris-HCl buffer at 20-30 mg protein per ml using a small homogenizer. Tissue binding experiments with 3H-OA were conducted in 10 x 130 mm culture tubes at 27°C in a shaking water bath. Each tube received 20-30 ul of the washed membrane protein (190-220 ug protein/assay tube), 100 ul of unlabeled test Chemicals, 22- 25 ul of 3H-OA and 856-869 ul of 50 mM Tris-HCl buffer containing 5 mM MgC12 and 2 mM ascorbic acid, pH 7.4, to make the final incubation volume of 1 ml. Each treatment was done in triplicate. The 3H—OA and unlabeled drugs were dissolved in the same Tris/MgCIQ/ascobate buffer, pH 7.4 (Hashemzadeh et al., 1985). The reaction was started by the addition of 3H—OA, continued for 10 min and terminated by 10-fold dilution of the reaction mixture with ice-cold Tris buffer (pH 7.4) containing 5 mM MgC12, followed immediately by vacuum filtration through a pre-rinsed Whatman GF/C glass fiber filter. The filters were immediately rinsed 4 times, each time with 4 ml aliquot of the same Tris buffer, and placed in plastic vials for scintillation counting. Competition experiments were performed to compare the ability of octopaminergic congeners to inhibit 3H-OA binding. All Chemicals were tested at 10 uM except GTP and pepronyl butoxide which were tested at 100 uM and 1.0 uM respeCtively for their ability to compete with 10 nM 3H—OA for specific binding sites in the nervous system and thoracic muscles. B. Assay of octopamine receptor functions by using adenylate cyclase and CAMP levels in thoracic muscles The procedures used for measuring CAMP levels in the nervous system and thoracic muscles of the American cockroach were essentially identical to those developed by Nathanson and Greengard(l973), Nathanson and Hunnicutt (1981) and Nathanson (1985). Freshly dissected thoracic muscles as above, were rinsed and then homogenized (50 15 mg/ml) in 6 mM Tris-malate, pH 7.4 containing 2 mM EGTA (Nathanson, 1985) using a glass/teflon homogenizer. The CAMP levels were determined in test tubes, each with 0.2 ml of 80 mM Tris- malate (pH 7.4) buffer containing 10 mM theophylline, 8 mM MgC12, 0.1 mM GTP, 0.5 mM EGTA, 2 mM ATP, and 10 ul of muscles homogenate containing 0.31 to 0.33 mg protein. The test compounds (OA and DCDM) were added with 20 ul of reaction buffer. The enzyme reaction was initiated by the addition of ATP carried on for 5 min at 30°C, stopped by heating to 90°C for 2 min and centrifuged at 1000 x g for 10 min to remove insoluble materials. CAMP in the supernatant was measured by the protein-binding assay of Brown et al. (1971). In all cases, protein concentrations were estimated using the assay kit of Bio-Rad Laboratories, CA (No. 500-0001).Lyophilized preparations of bovine plasma globulin were used as the protein standard. Studies on protein phosphorylation in the nervous system The method adopted was essentially identical to the one developed by Costa et al.(1982) . For this purpose, the freshly dissected nervous system (brain and the ventral nerve cord) from 15 insects were homogenized (20 mg/ml) in 6 mM Tris-malate, pH 7.4 containing 2 mM EGTA (Nathanson, 1985) using a glass/teflon homogenizer. A 0.1 ml aliquot of nerve homogenate containing 310-335 ug protein was added to each tube. The test Chemicals were added with 100 it] of 80 mM Tris-malate (pH 7.4) buffer containing 10 mM theophylline, 8 mM MgC12, 0.1 mM GTP, 0.5 mM EGTA and 2 mM ATP. The reaction mixurre was incubated for 5 min at 30°C, and stopped using 20 ul of 1% sodium dodecyl sulfate (SDS) and quickly heating at 100°C for 2 min. On cooling in an ice water bath, 20 ul of 10% Triton-X 100 was added, the system thoroughly vortexed, centrifuged at 16,000 x g for 5 min at room temperature and the supernatant was collected. To each 16 tube, 1.78 ml of 50 mM histidine-HCI buffer (pH 6.5) was added, and after mixing, transferred to a Centricon 30 tube with a membrane filter (Aminco Inc.). The content was centrifuged at 3000 x g for 60 min to reduce the volume to about 40 ul. A 20 ul aliquot of this concentrated protein solution was transferred to a small test tube containing 10 ul (600 n g) of catalytic subunit of protein kinase. The 32P-phosphorylation reaction was initiated by the addition of 20 ul of 2 uCi of gamma-32P-ATP in distilled water. After 10 min the reaction was stopped with 40 ul of 2 X "treatment buffer" (4% SDS, 20% glycerol, 10% 2-mercaptoethanol in 0.125 M Tris- HCl pH 6.8) and heating to 100°C for 2 min. The entire volume of the reaction product in each tube was transferred to an electrophoresis well. The method of SDS-polyacrylamide gel-electrophoresis used was that of Takacs (1979) using a Bio-Rad protein 11 System at 30 mA (with 1.5 mm Spacer). All statistical analyses were carried out by analysis of variance followed by a multiple comparisons test protocol. The latter was used to determine significance of difference between means at the 5% level in each experiment (Steel and Torrie, 1980). RESULTS To study the possible mechanisms of anorexia, the effects of formamidine derivatives (CDM and DCDM) and several other neuroactive amines were assessed by the total food consumption test method (Beeman and Matsumura, 1978). None of the mean values of food consumption among the control groups were significantly different from each other at the 5% level. The overall mean of food consumption (38D) for all control groups was 16.7 :13 mg (range = 14.8;t-_19.3 mg). Table 1 shows the potent anorectic effect of CDM and DCDM. At 1 u g/msect food consumption was reduced by 76% and 79% for CDM and DCDM, respectively. These differences are highly significant from the control values in both cases. At a dose of 5 17 Table 1. Anorectic effect of CDM, DCDM, and other netnoactive amines in the American cockroach. Compounds Dose Number of Meari‘ of food (118) Insects tested consumption (mg/insects/S hr) Treated Control Treated Control "t" Chlordimeform 1 12 10 3.6 15.2 90.30“ 5 14 13 0.9 15.7 128.08" 10 12 12 0.1 14.8 120.16“ N-demethyl- Chlordimeform 1 10 10 3.4 16.3 96.15" 5 15 13 0.5 14.9 126.61 10 16 16 0.2 18.1 168.64" Octopamine 50 15 15 4.8 15.7 99.57" 100 17 15 3.4 18.3 139.87"I 200 20 23 0.3 16.2 173.34“ Isoproterenol 100 19 17 17.9 17.6 0.00 200 22 18 18.3 18.1 0.00 Fenitrothion 1 12 10 6.2 19.3 101.98“ 5 11 8 1.4 16.9 111.19“ Norepinephrine 100 22 19 6.7 15.9 98.45“ 200 15 16 3.2 17.1 128.91* a: Mean values of food consumption were compared using pooled variance and Student’s "t" mm for independent means. For each pair of means, the statistic was calculated. *Statistically different from respective control at P<0.05. 18 uglinsect the effect lasted at least 7 hr post-treatment. The insects however, recovered their appetites after 24 hr. Fenitrothion which often caused poisoning symptoms, showed somewhat less anorectic effect than CDM or DCDM at a dose of 1 uglinsect. Three adrenergiC agonists (D,L-octopamine, L-norepinephrine and D, L- isoproterenol) were tested for anorectic activity. Octopamine had potent anorectic activity at the dose of 50 ug, 100 ug, and 200 ug. The anorectic effect of norepinephrine was significant either at a dose of 100 ug or 200 ug. In contrast, isoproterenol (the N-isopropyl analog of norepinephrine) had no anorectic activity at a dose of 100 ug and 200 ug. In all cases, no toxic symptoms could be observed with any of the treatments except in the case of fenitrothion, where in few cases tremors and convulsions were observed. A l hr-post injection incubation was adopted to allow sufficient time for metabolic activation and transport of the test compounds to their action sites. A feeding period of 5 hr was considered to be long enough for the insects to eat their fill. The possibility that the anorectic effect of CDM, DCDM, and CA may be related to Changes in hemolymph sugars was investigated by measuring glucose, trehalose and total sugar levels in the hemolymph of the male American coclooaches. Hemolymph sugar levels in insects receiving no food for 5 hr after treatment are presented in Table 2. The data show that CDM, DCDM, and OA increased glucose levels but decreased trehalose levels especially with CDM and DCDM. The total hemolymph sugars, however, remained relatively constant. At a dose of 50 uglinsect, the glucose levels were 789.3 i 62.9, 893.0 1 12.3, and 254 i 2.0 mg glucose/ 100 ml hemolymph for CDM, DCDM, and CA respectively, whereas control glucose level was 173.6 1'. 4.1 mg glucose/ 100 ml hemolymph. These results appear to indicate that those agents causing anorectic effects have a common property to increase the level of glucose and to reduce the level of trehalose in the hemolymph at the same time. 19 Table 2. Effect of CDM, DCDM, and CA on haemolymph sugars of the American cockroach mg sugar/ 100 ml haemolymph Compound Dose (mean _-t_-SD) (ug) Glucose Trehalose Total Sugars CDM 0 177.0 1 26.6 1,018.3 ;t-_ 75.3 1,195.3 i 6.0 1 271.0 i 46.8 909.7 i 31.9 1,180.7 i 78.6 5 453.7 i 44.0 759.0 :1; 30.0 1,194.7 i 53.1 10 721.7 i 104.2 369.3 i 67.1 1,091.0 i 37.7 50 789.3 i 62.9 128.7 3; 18.3 918.0 1 54.6 DCDM 0 169.0 $15.7 1,030.7 3; 36.1 1,199.6 i 51.5 1 887.0 138.7 307.3 1; 20.0 1,194.3 _-l_- 52.0 5 564.3 $43.7 623.0 i 15.1 1,187.3 1; 54.6 10 819.0 i 25.7 274.0 i 32.0 1,093.0 i 53.0 50 893.0 :12.3 113.0 i 18.0 989.3 5; 35.9 OA 0 174.7 1; 6.8 1,007.8 1; 9.5 1,182.0 1; 10.5 50 254.0 i 2.0 1,331.0 3; 62.9 1,585.0 i 64.8 100 359.7 1; 10.2 1,269.7 gt; 56.8 1,629.3 :1; 54.0 200 265.3 :1: 4.0 1,026.3 :I; 89.6 1,291.7 3; 85.7 400 339.0 1; 9.64 483.3 :1; 16.2 822.3 :9; 20.8 Results are expressed as means iSD of 3 experiments each performed in triplicate. 20 The time course of such a biochemical Change was studied next and the results are shown in Figure 4. They indicate again that the glucose level was elevated significantly at different times especially at 0.5 hr post-treatment as compared to zero time control values, the maximum level of increase being 363% above zero time. At the same time, the trehalose level decreased after 0.5 hr and its level remained relatively constant until 5 hr. Since the maximum elevation of glucose level was after 0.5 hr, all subsequent determinations of hemolymph sugars were made after 0.5 hr from treatment. It was of interest to know whether the elevation of hemolymph glucose after injection with CDM, DCDM, and 0A resulted from the activation of trehalase in the thoracic muscles, since the activation of trehalase is expected to result in the hydrolysis of trehalose to glucose. The results shown in Figure 5 Clearly demonstrate that CDM, DCDM, and OA have the property to increase trehalase activity in viva even at the lowest concentration tested (10 ul of 10 uM injection). The maximum activation of trehalase was 3304.2 i 79.4 nmol glucose/mg protein/hr at 1000 uM DCDM (10 ul injection) as compared to the control enzyme activity of 1513.1 5; 25.8 nmol glucose/mg protein/hr. The possibility that the CDM-mediated enhancement of trehalase activity is due to the activation of a pre-existing enzyme by a protein phosphorylation mechanism was tested by incubating enzyme preparations from both control insects and CDM-treated insects with ATP and Mg2+. The addition of these phosphorylation promoting materials enhanced trehalase activity in both preparations from CDM-treated insects (1 mM CDM) and control insects (Table 3). A time course study on the activating effect of CDM and OA on the trehalase activity in thoracic muscles of the American cockroach was carried out (Figure 6). Both CDM and CA with 10 ul injection of 2 mM concentration Clearly stimulated trehalase. The maximum activation of trehalase was seen at 30 min for CDM (3032.2 1; 102.0 nmol glucose/mg protein/hr). However, the time course of the activation by CA was somewhat .: O. E _>_. ‘e’ 3 4° 1: e o 5” S e 03 3 (I) O: E l I l I l 2 3 4 5 6 Time ( hours) Figure 4. Effect of 20 ug CDM/inseCt on glucose (I- ) and trehalose (c- ) levels in the American cockroach at different times. Results are means 1; SD of 3 experiments each performed in triplicate. Means with the same letter for each sugar are not significantly different at the 5% level according to the LSD after ANOVA. 22 3500-1 3000-1 5 . E 2500- 9 Q 2000- c: E t 8 0 1500-1 0 a G 3 1000- o ‘ E c sea- 0 r """]f' """'r ‘ ' "mW’—**'*""r ' "'""1 10° 10‘ 102 103 104 105 Concentration uM (10 ul injection) Figure 5. Effect of CDM (-o-),DCDM (-o-), and 0A (1.) on trehalase activity in thoracic muscles. Results are means 1; SD of 3 experiments each performed in triplicate.Trehalase activity of control = 1513.1 i 25.9 nmol glucose] mg protein/hr. Means with the same letter for each level of concentration are not significantly different by Tukey’s test at the 5% level. 23 Table 3. Effect of ATP and Mg2+ on trehalase activity of thoracic muscles in vitra Treatment Trehalase activity nmol glucose/mg protein/h Control 1546.0 1; 50.7 a CDM 2309.5 r. 531.2 b Control + Mg2+ 2769.5 i 166.2 bc Control + ATP 2930.0 i 33.4 bd Control + Mg2+ + ATP 3388.7 i 153.3 be CDM + Mg2+ 3980.5 i 229.3 ef CDM + ATP 4445.2 i1365.1 fg CDM + Mg2++ ATP 5753.7 $11689 h Assay mixture was preincubated for 10 min before the addition of trehalose (28 mM). Buffer used was 0.1 M phosphate, pH 6.3. ATP and Mg2+ concentrations were used at 0.05 mM and 0.1 mM, respectively. The final assay mixture was 1.0 ml. Results are expressed as means iSD for 3 experiments each performed with 4 replicates. Means are iglngrezntly different, if do not share a common letter on the 5% level of the LSD after 24 4000 ‘1 3000' ' 2000 nmol glucose/mg protein/hr TIME (hours) Figure 6. Effect of 2mM CDM (43-) and 2mM OA (+),10 ul injection/insect on trehalase actrvrty in thoracic muscles at different times. Results are means $ SD of 3 experiments each performed 111 mphcate. Means with the same letter are nor significantly different for each time level by Tukey’s test at the 5% level after AN OVA. The control activity at 0 time was 1528.5 $1.4 nmol glucose/mg protein/hr. 25 different. The plateau was reached at 30 min (2469 $ 93.9 nmol glucose/mg protein/hr), but the activity remained almost constant until the end of the experiment. If the above hypothesis of activation through phosphorylation on trehalase (or its modulating proteins) is correct, the activation of trehalase by CA should be blocked by phentolamine, an alpha-adrenergic antagonist. Indeed, in the study shown in Table 4, phentolamine was found to be potent in antagonizing the stimulatory effect of octopamine. However, phentolamine itself decreased drastically trehalase activity. CDM and DCDM at 1 mM again showed the same tendency. Additional evidence that the actions of 0A are antagonized by phentolamine comes from the in vitra study on the effect of agonists and antagonists of CA on trehalase activity. The results (Table 5) show that 1 mM phentolamine antagonized 1 mM OA and was also very potent in antagonizing the action of 1 mM DCDM. On the other hand, when 1 mM OA and 1 mM DCDM were mixed and tested as a single treatment, the resulting enhancement of trehalase was the same as that of 1 mM DCDM. These results support the notion that all these agents act at the same octopamine receptor and therefore, their effects are not additive. In such a case, OA and DCDM must act as agonists and phentolamine as an antagonist. EC50 values of OA and DCDM which were required to cause 50% activation of trehalase activity were found to be about 8 x 10-9 M OA and 3 x 10-7 M DCDM (Figure 7). Furthermore, effects of various concentrations of phentolamine on trehalase activities in the thoracic muscles after treatment with 10-9 M OA and 10-7 M DCDM (Figure 8) were found to be antagonistic to the effects of both OA and DCDM especially at higher concentrations. IC50 was found to be in the neighborhood of 1 x 10'6 M and 3 x 10‘8 M phentolamine for the net increases of trehalase activity caused by 10"9 M OA and 10'7 M DCDM, respectively. 26 Table 4. Effect of OA agonists and antagonists on trehalase activity in viva Treatment Trehalase Activity nmol glucose/mg protein/h Control 1527.2 3; 22.2 d CDM 2580.0 $77.4 C DCDM 3304.2 $79.4 a CA 2902.0 $34.7 b OA+ PA 1641.0 $27.3 d PA 962.0 $30.3 e Results are means $SD of 3 experiments each performed in triplicate. All test compounds used at 1 mM (10 ul injection). Means are significantly different from each other, if do not share a common letter on the 5% level of the LSD. 27 Table 5. Effect of OA agonists and antagonists on trehalase activity in vitra Compound Trehalase activity nmol glucose/mg protein/h Control 1572.0 $ 81.2 Cd CDM 1799.0 $ 156.0 C DCDM 3210.5 $ 123.1 a 0A 2331.5 $ 102.6 b OA +DCDM 3360.5 $ 253.0 a CA + PA 1569.0 $184.0 Cd DCDM + PA 1248.2 $146.5 d Note: All compounds were used at 1 mM. Results are means $SD for 3 experiments each performed in triplicate. Means do not share a common letter are significantly different on the 0.05 level of the LSD. 28 120 100% 80" 60-1 Trehalase activity (% of control) Agonist concentration. log M Figure 7. Activation of trehalase activity in the thoracic muscles by CA ( o ) and DCDM ( e ) in virra.EC50 of OA and DCDM are 8 x 10‘9 M and 3 x 10‘7 M respectively. Trehalase activity of control = 1433 $ 32.9 nmol glucose/mg protein/hr. Results are means + SD f 4 ' perfo in triplicate. __ 0 experiments each 29 100-1 ’E .2 g 80-1 .2 .E o\° " 60-1 2‘ IE "3 40" Q m .‘E 1 (U z 2 20-1 p. 0 ' I ' t . , I ' -10 -8 -6 -4 -2 Phentolamine concentration. log M Figure 8. Inhibitory effect of hentolarnine on trehalase activity of the thoracic muscles caused by 10- M OA ( o ) and 10-7 M DCDM( . ) in vitra I C50 = 10'6 M and 3 x 10‘8 M phentolamine for DA and DCDM respectively. All data have been expressed as the absolute values in glucose formation above that (1468 $138 nmol glucose/mg protein/hr) observed in the absence of additions. Trehalase activities caused by 10'9 M OA and 10-7 M DCDM were 705 a 44.4 and 749 r. 55.6 nmol glucose/mg protein/hr above basal activity. Results are means $ SD of 4 experiments each performed in triplicate. 30 The possibility that octopamine receptors exist in the thoracic muscles of the American cockroach was investigated by two different methods. In the first method, I tested 3H—OA binding and was able to show the presence of a saturable, high-aff'urity binding site for 3H- OA in the thoracic muscles and the nervous system (Table 6). The results shown in Table 6 also indicate that the levels of the specific binding of 3H—OA are affected by various compounds. GTP, DCDM, NE, and PA reduced 3H-OA specific binding in muscles indicating the possibility that they act at the same binding site. However, piperonyl butoxide (PB) also reduced the binding (Table 7). The addition of 10 uM nonlabeled OA did not cause further reduction of 3H-OA binding as in the case of the nervous system. The results appear to indicate that the major 3H—OA binding component is the mixed function oxidase. Therefore, I took another approach to prove the existence of octopamine receptors in the thoracic muscles, i.e., correlating the action of OA and DCDM to adenylate cyclase. The results Clearly showed that both 10uM OA and 10 uM DCDM have the property to activate adenylate cyclase, resulting in the elevation of CAMP levels from a control value of 0 to 6.0 $ 0.56 and 9.8 $ 5.65 pmol CAMP/mg protein/min respectively. These results support the hypothesis that the DCDM-sensitive octopamine receptor also exist in the thoracic muscles. To ascertain that the formamidines-induced increase in CAMP levels actually evoke functional Changes in the cells of the nervous system, the changes in protein phosphorylation patterns were studied by incubating homogenates of the American cockroach nervous system with gamma-32P-ATP in the presence and the absence of DCDM, OA, and 8-Br-CAMP. After the reaction, the proteins were solubilized with SDS and polyacrylamide—SDS gel electrophoresis was developed. The resulting autoradiogram of labeled phosphoprOtein indicate that OA, DCDM, and 8-Br-CAMP Clearly stimulated the endogenous phosphorylation in the preparation (Figure 9). The molecular weights of the major protein bands were 22, 39, 79, and 158 KDa. To confirm the results of the visual observation, a densitometric assessment was made on the same autoradiogram and the 31 6:53:38“... n m Z deg—25.3 n 2 oem 2: a 2:22.26 358555 8: 2m 0322 =23 £55 2222 2:8 65 .23 2822 28:22 E eon—Leta :23 2585?» m we QmH 838 mm 332er Ba 233m ”202 8 S. H .wwe e 53H 8x m 37H 2:. e we H Ow? m2 2: S + - 97—» 66 —" 45* 29* 35 results shown in Table 8, verifying that OA, DCDM, and 8-Br-cAMP increased endogenous phosphorylation of total proteins in homogenates of the nervous system as indicated by a reduction in 32P-phosphorylation. Note that in this mode the increase in endogenous protein kinase activities is expected to result in a decrease in overall 32P-phosphorylation because of the reduction in sites available for subsequent phosphorylation with gamma-32P-ATP. 36 Table 8. Effect of OA, DCDM, and 8-Br-cAMP on endogenous phosphorylation of total proteins in homogenates of nervous system. The data were obtained by densitometric scanning of autoradiograms of SDS, polyacrylamide gel electrophoresis, and are expressed as relative intensities in % of the total lane intensity of the control (= 100). Treatment Levels of protein phosphorylation % of control 1 mM OA 98.2 ;I-_ 2.6 a 10 mM OA 67.6 $1.9 c lmM DCDM 57.8 11.3 c 10 mM DCDM 29.9 3; 3.2 b 1 mM 8-Br-cAMP 62.6 3; 6.4 c 10 mM 8-Br-cAMP 30.6 55.7 b Note: The results expressed as means :80 of 2 densitometric measurements of 2 independent experiments. Means are significantly different from each other if do not share a common letter on the 5% level according to Tukey's test. 37 DISCUSSION In this study I have confirmed the original finding by Beeman and Matsumura (197 8) that Chlordimeform elicits a potent anorectic action in the American cockroaches at a dose far below the minimum dose required to elicit overt symptoms of acute neurotoxicity. The observation that both D, L-octopamine-HCI and Chlordimeform caused the same anorectic effect appears to be in line with the generally accepted view that at low doses chlordimeform's action is mediated through its action on the octopamine receptors (Beeman and Matsumura, 1978; Evans and Gee, 1980; Hollingworth and Murdock, 1980; Nathanson and Hunnicutt, 1981; Hollingworth and Lund, 1982; Cole et al., 1983; Hashemzadeh et al., 1985; Nathanson, 1985). My original hypothesis was that Chlordimeform and/or its metabolic products in vivo act on the octopamine receptors in the central nervous system, particularly those in the corpus cardiacum which are well known to mediate the release of hyperglycemic peptide hormone to the hemolymph (Steele, 1961), to elevate the hemolymph trehalose level. However, my initial test results demonstrated that such a phenomenon is accompanied with an increase of hemolymph glucose level, but not trehalose level. The combined glucose and trehalose level remained relatively constant. An in vitro study has shown that the activity of trehalase in the thoracic muscles did increase as a result of in viva administration of CDM or in vitro incubation with DCDM. Similar results were obtained by administration of octopamine in vitro and in vivo. The most pertinent observation in this regard has been that octopamine itself causes elevation of trehalase in vivo and in vitro in several tissues of the American cockroach including in the thoracic muscles (15). Thus, I have revised my original hypothesis and formulated a new one proposing that the main site of action of Chlordimeform to cause anorexia in this species is the octopamine receptor, which is present in many tissues 38 (Jahagirdar et al.,1984) and is coupled to regulation of trehalase activity in converting hemolymph trehalose to glucose. To support this hypothesis, I have first established the presence of octopamine receptors, second, shown Chlordimeform or its metabolites interact with octopamine receptors, and third, documented the activation of trehalase by both octopamine and formamidines in a cell free, in vitro system. The presence of specific high-affinity octopamine receptors has been reported in this species (Nathanson and Greengard, 1973; Evans and Gee,1980; Nathanson and Hunnicutt, 1981; Gole etal., 1983; Downer, 1988) supporting the current study. However, by the 3H-octopamine binding method employed, it was not possible to clearly demonstrate the presence of these receptors in muscle membranes themselves, probably because of the presence of interfering substances in the crude membrane preparation. Nevertheless, it is likely that the octopamine receptors are present in muscles, since octopamine clearly caused a rise in trehalase activity in a cell free system (Jahagirdar et al. (1984), and since phentolamine, an octopamine receptor antagonist in insects, blocks the activation of adenylate cyclase by octopamine (Downer, 1980; Cole et al., 1983). These observations, together with the demonstration of octopamine receptors in muscles of other insect species such as locust (Evans et al., 1988) provide compelling evidence that octopamine receptors exist in the thoracic muscles of the American cockroach. The results of the present study suggest that CDM mediated enhancement of trehalase activity may be effected through activation of a pre-existing enzyme by a phosphorylation mechanism since trehalase activity increased with the addition of the protein phosphorylation promoting factors ATP and Mg2+ to the incubated enzyme preparations from both control and CDM-treated insects. These results were similar to the reported observation that octopamine-mediated enhancement of trehalase occurs through activation of a pre-existing enzyme by a phosphorylation mechanism since the addition of ATP and Mg2+ enhances trehalase activity in both control and OA-treated insects 39 (J ahagirdar et al., 1984). This conclusion is consistent with the data obtained from the protein phosphorylation studies. Both 0A and DCDM clearly stimulated protein phosphorylation by activating endogenous protein kinases in the nervous system. The pattern of change in phosphorylation is very similar to that caused by 8-Br—cAMP, which is known to penetrate through the plasma membrane and thereby directly activate CAMP- dependent protein kinases present inside the cell. Such phosphorylation occurs on sites identical to those of the exogenously added CAMP—dependent protein kinase and supports the view that the formamidine-induced rises in cAMP level directly result in the activation of an endogenous CAMP-dependent protein kinase. Anorexia is a poorly studied subject in insects, therefore, it is possible that there are more than one cause for making insect anorectic. What I have established in the current study is that CDM and its active metabolites in viva definitely cause a rise in the trehalase activity, resulting in the rise of the hemolymph glucose level. Increases in blood glucose levels have been shown to be causally related to anorexia in other animals (Hendley et al., 1987). Therefore, there is an excellent chance that the increase in the glucose level is the major cause for anorexia in this species. CHAPTER 2 Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the tobacco homworm Manduca sexta 40 INTRODUCTION Chlordimeform (CDM) belongs to a structurally novel group of pesticides, the formamidines, which have an unusual spectrum of biological activity, being specially effective against some insects (Lepidoptera and Hemiptera) and acari. They are particularly effective in controlling those already resistant to other classes of insecticides (Hollingworth, 1976; Beeman, 1982). The mechanism whereby formamidine pesticides protect plants and animals from arthropod attack is complex, resulting from a combination of lethal and sub-lethal effects; the latter in many cases occurring only at critical points in the life cycle of pests. Other sublethal effects are, for example, disrupting feeding and reproduction, continued flight, increased sensitivities to sex-pheromones, etc. (Hollingworth, 1976; Beeman, 1982). Gladney et al. (1974) and Stone et al. (1974) have shown that CDM causes detachment of ticks from the host. Mites or lepidopterous larvae feeding on treated leaves become hyperactive, resulting in movement away from the food source (Doane and Dunbar, 1973). Hirata and Sogawa (197 6) found that CDM causes a decrease in feeding of several species of hemiptera and suggested that the mortality observed was the result of starvation and not a direct toxic action of the insecticide. Beeman and Matsumura (1978) reported that CDM causes an intense anorexia in the American cockroach at doses as low as l to 5 uglinsect. Lund et a1. (1979) concluded that decreasing plant consumption by tobacco homworm larvae is afforded by a nonlethal mechanism which probably arises from m0tor stimulation through actions on central non-cholinergic systems. Ismail and Matsumura (unpublished data) recently found that such a phenomenon of anorexia in the American cockroach is accompanied by an increase in haemolymph glucose levels, but not trehalose levels. The combined glucose and trehalose level remained relatively constant. In addition, an in vitra study has shown that trehalase activity in the thoracic muscles has increased as a result of in viva administration of CDM, or in virra incubation with 41 42 DCDM. These phenomena are identical to the ones produced by administration of 0A in vitra and in viva. The results indicate that the primary action site of this group of formamidines in this species, is at the octopamine receptor in muscles and other nonneural tissues which regulate the levels of trehalase and not at the neural site such as corpus cardiacum which regulates the release of hyperglycemic hormone. When CDM and 0A were applied as a spray to leaves, Manduca sexta larvae elicited an antifeeding activity in the tobacco homworm which was greatly enhanced by the simultaneous application of a phosphodiesterase inhibitor ((Nathanson, 1985). Thus aminergic, most likely octopaminergic processes, mediated by cAMP, appear to be involved in the above antifeeding effect of CDM in this species. Octopamine is known to be a major neurotransmitter and neurohormone in many insects (Nathanson, 1985) and an effector of excitatory processes in several insect species (Downer, 1980). It has been reported that 0A mediated the enhancement of trehalase activity in muscle and haemolymph of the American cockroach (Jahagirdar, 1984). Since aminergic, particularly octopaminergic actions of formamidines are regarded as their main biochemical action mechanisms (Hollingworth, 1976; Matsumura and Beeman, 1976; Beeman, 1982), leading to the behavioral and toxicological effects of these agents in insects and acarina species, study of their effects on key behavioral functions mediated by biogenic amines in insects is likely to yield valuable data. The main purpose of this investigation was to study the biochemical mechanisms of the antifeeding activity or anorexia caused by non-lethal levels of formamidine pesticides in Manduca sexta larvae. 43 MATERIALS AND METHODS Insects Tobacco homworms, Manduca sexta (Johanson), were obtained from Carolina Biological Company and reared in individual containers on artificial diet (Bio-Serv, Inc., Frenchtown, NJ.) at 27 i 1°C under a long-day (LD, 17:7) photoperiod (Bell and Joachim, 1976). The fifth-instar larvae were used in all experiments. Chemicals D,L-octopamine hydrochloride (OA), and (trehalose and glucose) assay kits were obtained from Sigma Chemical Company (St. Louis, MO). Phentolamine (PA) hydrochloride was obtained from CIBA-Giegy (Summit, N.J.). Chlordimeform (CDM) and N-demethylchlordimeform (DCDM) as hydrochloride salts were synthesized and purified in our laboratory. Studies on the anorectic effect of CDM CDM, DCDM, and CA were assayed for anorectic activity in the fifth-instar larvae of the tobacco homworm Manduca sexta. The larvae (8-10 larva per test) were weighed before treatment and at the end of the experiment. They were injected abdominally with 10 ul of saline solution (Yamasaki and Narahashi, 1959) containing the test chemicals with concentrations of 0.1 to 100 mM using a Hamilton microsyringe. Control insects were treated in an identical manner with 10 ul injection of saline solution only. The larvae were held for 2 min after injection without food, and then isolated in plastic cups separately and 44 offered a small pre-weighed piece of the prepared artificial diet (5-10 gm). Food consumption, body weight gain, and production of fecal pellets over 6h were measured. Determination of haemolymph sugars Larvae on the first day of the fifth instar were injected (10 larvae per test) abdominally with 10 ul of saline solution containing test chemicals in the range of (102 to 105 uM). Control insects were injected with 10 ul saline solution only. They were held for 2 min after injection, and then isolated in plastic cups separately and offered a small piece (5-10 gm) of the prepared artificial diet. Haemolymph was collected 1 and 6h post- injection into a chilled test tube through a wound caused by severing the second abdominal segment (Shapiro and Law, 1983). The collected haemolymph from 10 larvae/test was pooled. The non-reducing disaccharide, trehalose is the major haemolymph sugar in most insect species and provides an important source of metabolic energy. Because trehalose yields glucose on acid hydrolysis it may be estimated as reducing sugar. Determination of haemolymph sugars was carried out using the method of Steele (1961) with some modifications. Two samples (20 ul each) of pooled haemolymph for each concentration (10 ul injection) were taken and placed in separate small ampules. Both samples received 20 ul of 2N HCl, but one sample was treated immediately with 2N Na OH to neutralize the effect of HCl for glucose assay. The other sample was used for trehalose assay by autoclaving at 15 lbfrn.2 for 30 min, and neutralized thereafter using 2N NaOH. Ten ul of each sample was taken for determination of haemolymph glucose and trehalose respectively, using a spectroscopic method (Sigma Chemical Company, Bulletin No. 15 UV) which depends on the following reactions: 45 Glucose + ATP + Hexokinase —> G-6-P + ADP G-6-P + NADP + G-6-PD —-> 6 - PG + NADPH The level of N ADPH was assessed spectrophotometrically at 340 nm. Studies on trehalase activity in the haemolymph The procedures used in these experiments are based on those used by Jahagirder et al. (1984). Trehalase activity was measured by monitoring the release of glucose from trehalose without acid hydrolysis as described previously. The reaction mixture for haemolymph trehalase comprised of 28 mM trehalose, 100 mM sodium acetate buffer, pH 5.5, an aliquot (IO-20 ul containing about 25-50 ug protein) of enzyme preparation and test chemicals were added after being dissolved in sodium acetate buffer pH 5.5 to give final concentration of 10‘4M. The reaction mixture was incubated at 30°C for 60 min. In all cases protein concentrations were estimated using the assay kit of Bio-Rad Laboratories, CA (No. SOD-0001). Lyophilized preparations of bovine plasma globulin were used as the protein standard. Determination of haemolymph lipids The larvae on the first day of the fifth instar were treated in the same manner as before for sugar determination. The lipid level was measured 1h after the treatment. The procedures used for lipid determinations were based on a modified vanillin method of Jutsum and Goldsworthy (1974) and Holwerda et al. (1977). A 5 ul aliquot of pooled haemolymph for each treatment was mixed with 500 ul mixture of chloroformzmethanol (1:2). A 100 111 aliquot was placed in a test tube and the solvent was evaporated. To each tube 1 ml of concentrated sulfuric acid was added. The tubes were then heated in boiling water bath for 10 min and cooled at once. 100 ul portions of the cooled samples were 46 mixed with 2.5 ml vanillin-phosphoric acid reagent, incubated for 30 min at room temperature, and the reaction products were then measured at 546 nm. All statistical analyses were carried out by "analysis of variance" followed by a multiple comparison test protocol. The latter was used to determine the significance of difference between means at the 5% level in each experiment (Steel and Torrie, 1980). RESULTS To study the possible mechanisms of anorexia, the effects of formamidine derivatives (CDM and DCDM) and octopamine were assessed by using the total food consumption test method (Beeman and Matsumura, 1978). None of the mean values of food consumption by control groups were significantly different from each other at the 5% level. The overall mean of food consumption (i SD.) for all control groups was 3.85 :1; 0.75 gm. Table 9 shows the potent anorectic effect of CDM, DCDM, and OA. Food consumption was reduced after 6h by 69.5%, 82%, and 86%, respectively, at 10 mM (10 ul injection). All test chemicals, especially CDM and DCDM at 1 mM concentration produced fine tremors in the head within 30 min accompanied by increased locomotion. The tremor became very strong when the concentration was increased. All larvae showing these symptoms recovered within 0.5 - 6 h at all tested levels. The decrease in food consumption was dose dependent. Table 10 provides the data on the body weight gain by the fifth-instar larvae after the experimental period (6h). The results show that there was a decrease in body weight gain by the larvae which was dose-dependent in all tests. The overall mean of body weight gain for all control groups was 1.34 1; 0.09 gm after 6h. The fifth-instar larvae injected with CDM, DCDM, and 0A produced fewer fiass pellets than the corresponding control (Table 11). Thus all the data, the decrease in food consumption, body weight gain and 47 Table 9. Anorectic effect of CDM, DCDM, and 0A in the tobacco homworm larvae. Concentration mM Food consumption (gm/6h) Compound (10 ul injection) (mean i SD) "t" Treated Control CDM 0.1 2.91 i 0.16 3.23 i 0.13 6.6* 1.0 2.53 :1; 0.2 3.15 i 0.09 12.5* 10 1.07 :1; 0.12 3.51 1; 0.17 38.7* 100 0.64 i 0.01 3.97 i 0.13 47.6* DCDM 0.1 2.29 :3; 0.12 3.27 :1; 0.15 21.3““ 1.0 1.89 i 0.09 3.53 i 0.19 35.8* 10 0.77 1; 0.04 4.33 i 0.17 67 .4* 100 0.52 i 0.06 3.8 i. 0.2 733* 0A 0.1 2.73 :1; 0.15 3.33 i 0.12 12.3* 1.0 2.16 i 0.12 3.89 i; 0.17 37.7* 10 0.64 i 0.09 4.66 i 0.11 87.3* 100 0.39 -_I; 0.02 4.9 i 0.2 1006* Results are means i SD. of 3 experiments each performed in triplicate with 8-10 larvae/replicate. Data analyzed by two tailed student's "t" test for independent menas. *Statistically different from corresponding control with P < 0.05. 48 il‘able 10. Effect of CDM, DCDM, and DA on body weight gain by tobacco homworm arvae. Concentration mM Body Weight Gain (gm/6h) Compound (10 ul injection) (mean 1; SD) "t" Treated Control CDM 0.1 1.05 :1; 0.08 1.19 :1; 0.05 3.6* 1.0 0.83 i 0.09 1.37 i 0.11 14.3“ 10 0.61 i 0.2 1.41 ;l-_ 0.07 21.4* 100 0.33 3; 0.07 1.38 1; 0.15 27.9"I DCDM 0.1 1.28 1; 0.01 1.98 :1; 0.12 20.4* 1.0 1.02 i 0.03 2.03 i 0.16 27.6* 10 0.62 i 0.07 4.96 _-I; 0.09 36.4* 100 0.28 i 0.03 2.17 3; 0.1 548* 0A 0.1 1.55 i 0.01 2.1 1; 0.09 14.6“ 1.0 1.08 3; 0.05 1.97 :1; 0.13 25.0* 10 0.72 1; 0.03 2.21 i 0.04 41.5* 100 0.05 :1; 0.01 1.98 i 0.07 51.8* Results are menas i SD. of 3 experiments each performed in triplicate with 8-10 larvae per test. Data analyzed by two tailed student's "t" test for independent menas. *Statistically different from respective control with P < 0.05. t 49 Table 11. Effect of CDM, DCDM, and GA on fecal production of tobacco homworm larvae. Concentration mM % decrease in number (10 ul injection) of pellet (mean i S.D) CDM DCDM 0A 0.1 39.4 i 5.7a 31.0 i 3.9a 32.3 :1; 4.7a 1.0 47.3 1; 3.3b 43.6 i 11.6b 53.6 1 10% 10 53.7 i 13. c 60.1 i 6.4cd 68.7 i 3.5d 100 73.6 i 18 d 75.2 i 9.2de 87.5 +_ 6.1e Results are means i SD of 3 experiments each performed in triplicate. Fecal production ofconu'ol = 26.67 :1; 1.32 pellets/6h. Means do not share a common letter within each concentration (10 ul injection) level are significantly different on the 5% level of Tukey's test after factorial ANOVA. 50 fecal production, as compared to control, indicate that these chemicals cause anorexia in the fifth-instar of the tobacco homworm larvae. The possibility that the anorectic effect of CDM, DCDM, and 0A may be related to changes in haemolymph sugars was investigated by measuring glucose, trehalose, and total sugar levels in the haemolymph of the fifth-instar larvae l and 6h post-injection with test chemicals. The results shown in Figure 10 indicate that all active agents increased glucose levels after 1h from treatment DCDM. and 0A caused maximum increases in glucose levels 177 i 7 and 118 i 7.4 mg glucose/100 ml haemolymph, respectively, at 103 uM (10 ul injection), but CDM caused maximum increase 110.8 i 9.1 mg glucose/ 100 ml haemolymph at 104 uM. Results of the haemolymph glucose levels after 6h (Figure 11) show that the glucose levels were higher at 100 uM for all test compounds. There was a decline in the glucose level with higher concentrations (10 ul injection) to approximate the value of control. The results in Figure 12 indicate that trehalose levels increased slightly for all test chemicals after 1 h. The levels were 534.6 1; 5.3, 558 _-1_-_ 12.1 and 638.8 i 19.9 mg glucose/100 ml haemolymph for OA, DCDM, and CDM respectively at 103 uM (10 ul injection), whereas control level of trehalose was 467.3 1; 86.7 mg trehalose/100 ml haemolymph. On the other hand, after 6h (Figure 13) the levels of trehalose were increased significantly for all test chemicals and the maximum increases of trehalose were 730.2 i 21.2, 821.2 i 10.9 and 758.8 i 18.3 mg trehalose/100ml haemolymph for CDM, DCDM, and 0A respectively at 103 uM (10 ul injection). These results indicate that these chemicals elevate both glucose and trehalose levels. The enhancement of glucose levels resulted from CDM, DCDM, and 0A were higher after 1h than after 6h. On the other hand, the trehalose level was significantly higher after 6h.than after 1h. This tendency was more noticeable for DCDM and 0A than for CDM. The total haemolymph sugar after 1h (Figure 14) were clearly elevated for all test chemicals and the maximum elevation of total sugars levels were 735 i 19.1 and 653 i 51 200 E g 160' E m «1 f 120d E o 2 B 80- (I) o o 1 2 a a 40-4 E O . tn...“ . in“... . ..nfl1—qu—v—I-rmfi' 10‘ 102 103 104 105 106 Concentration uM (10 ul injection) Figure 10. Effect of CDM (-o-), DCDM ( -o- ), and 0A (-a- ) on glucose levels in haemolymph of the tobacco homworm larvae after 1 h in viva . Result are means : SD of 3 experiments each performed in uiplicate. Glucose level of control = 54 i 4.4 mg glucose/100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injection) are net significantly different by Tukey’s test at the 5% level after factorial ANOVA. 52 140 1: 120- O. 2: O E 100- Q m 1 .C .5- 80-1 8 I: 60" Q U) 1 § 40- a i U, E 20" o v vvvvvvq v v vuvvvv' I v I I‘VII' I 1 vvv-vv' r V were: 101 102 103 104 105 106 Concentration uM (10 ul injection) Figure 11. Effect ofCDM (-o- ), DCDM (+), and 0A (-¢- ) on glucose levels in haemolymph of the tobacco homworm larvae after 6 h in vivo .Results are means i SD of 3 experiments each performed in triplicateGlucose level of control = 57 i 3.9 mg glucose/ 100 ml haemolymph.Means with the same letter for each level of concentration (10 ul injection) are not significantly different by Tukey’s test at the 5% level after factorial ANOVA. 53 1000 c 1 2' 3 800- 3 a 3 a a f 600- E C 53 B 400d In 2 (U 5 9 0-0 200‘ c: E o . ....... ........ . um... ......... ...... 101 102 103 104 105 106 Concentration uM (10 ul injection) Figm'e 12. Effect of CDM (-O-), DCDM (+), and 0A (41-) on trehalose levels in haemolymph of the tobcco homworm larvae after 1 h in vivo Results are means i SD of 3 experiments each performed in triplicate. Trehalose level of control = 467.3 i 86.7 mg trehalose / 100 ml haemolymph. Means with the letter for each level of concentration(10 ul injection) are not significantly different by Tukey’s test at the 5% level after factorial ANOVA. 54 1000 c 1 g 2 8004 O E q Q) at -= 600- E + O O t 400- C) m 2 (U .3 g 200-1 a: E 0 U i " 'I'"' I ‘ "U'U'I I U I‘D'I'I I ‘ 'V'UUU' V V "U" 101 102 103 104 105 10‘5 Concentration 0M (10 ul injection) Figure 13. Effect of CDM (-o-), DCDM (+ ), and 0A (+) on trehalose levels in haemolymph of the tobacco homworm larvae after 6 h in viva. Results are means i SD of 3 experiments each performed in triplicate. Trehalose level of control = 503 i 123 mg trehalose/ 100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injection) are not significantly different by Tukey’s test at the 5% level after factorial AN OVA 55 1000 J: O. E. 3 800 " E Q N C — d E 600 o O E 400 - (U or 3 4 (D 6 § 200 '4 U, E 0 Y t """I V ‘I 'IV‘I'I ' I 'fi'"' I—V YI'I'I' ' ' "Y" 101 102 103 104 105 106 Concentration uM (10 ul injection) Figure. 14. Effect of CDM (-O- ), DCDM (+ ), and 0A (.a. ) on total sugar levels it haemolymph of the tobacco homworm larvae after 1 h in vivo. Results are means i SD of 3 experiments each performed in triplicate. Total sugars level of control = 521.6 :1; 91 mg total squ 100 ml haemolymph. Means with the same letter for each level of concentration (10 ul injection) are not significantly different by Tukey,s test at the 5% level after factorial ANOVA. 56 12.7 (mg total sugar/100 ml haemolymph) for DCDM and OA, respectively with 10 til 103 uM, whereas the maximum increase in the case of CDM (791.6 i 27.1 mg total sugars/ 100 ml) was obtained at 104 uM. At 105 uM (10 ul injection), the total sugar was less than the control level for DCDM and CA but higher than control in the case of CDM. The results shown in Figure 15 indicate that the level of total haemolymph sugars was increased for all test compounds and the maximum increases were 794.8 i 23.7, 923.5 i 13.9 and 816.6 :25.3 mg total sugar/ 100 haemolymph for CDM, DCDM and 0A respectively at 103 uM (10 ul injection). The elevation of total haemolymph sugars was higher in the case of DCDM and 0A than CDM. These results appear to indicate that these agents causing anorectic effects have a common property to increase the levels of glucose, trehalose and total haemolymph sugar at the same time in the larvae of tobacco homworm. It was of interest to know whether the elevation of haemolymph glucose after injection of CDM, DCDM and 0A resulted from the activation of trehalase in the haemolymph since the activation of trehalase is expected to result in the hydroxlysis of trehalose to glucose. The results in Table 12 clearly demonstrate that all these agents have the property to increase trehalase activity in vitro. When DCDM and 0A were tested at the same concentration 10'4M, trehalase activity was almost the same. Phentolamine (PA) inhibited trehalase activity when mixed and tested with CA and DCDM. The inhibition of trehalase activity induced by DCDM was higher than the inhibition of trehalase activity induced by OA. Phentolamine alone, at the same concentration, inhibited trehalase activity. Table 13 shows that, when the larvae were injected with 10 ul (10'3M) of OA, CDM and DCDM, glucose, trehalose and total sugar levels were increased significantly over the controls. When DCDM and 0A were used as one treatment, the levels of glucose, trehalase and total sugars were the same as that of DCDM alone. Phentolamine antagonizedboth 0A and DCDM, when mixed with each as one treatment. In addition, phentolamine itself, when applied as one treatment, decreased the levels of glucose, 57 1000 a 4 E 5‘ 8001 E g 4 1.: a E 600-1 c 8 , 2 4004 (U c: 3 tn 2 zoo-i o 4 E 0 . ......, ”m", . ”m“, ........, 101 102 103 104 105 106 Concentration uM (10 ul injection) Figure 15. Effect of CDM(-o-), DCDM (-o- ), and 0A (+) on total sugars levels in haemolymph of the tobacco homworm larvae after 6 h in viva. Results are means i SD of 3 experiments each performed in triplicate.Total sugars level of control = 560.6 3 126 mg total sugars/100 ml haemolymph. Means with the same letter for level of concentration ( 10 ul injection) are not significantly diffsrent by Tukeyts test a the 5% level after factorial AN OVA. 58 Table 12. Effect of 0A antagonists and agonists on haemolymph trehalase activity of the tobacco homworm in vitra. Treatment nmol glucose/mg protein/h Control 325.0 1; 11.1 df CDM 381.3 $20.1 d DCDM 576.7 1; 16.0 b 0A 516.6 1; 18.5 b 0A +DCDM 523.0 1; 10.1 bc 0A + PA 335.0 -_i-_ 14.5 de DCDM + PA 158.7 a 69.9 g PA 68.0 g 9.9 a Results are means 1 SD of 3 experiments each performed in triplicate. All compounds were tested at 10‘4M. Means with the same letter are not significantly difl‘erent on the 5% level of the LSD after ANOVA. 59 Table 13. Effect of 0A agonists and antagonists on haemolymph sugars in the tobacco homworm larvae in viva Treatment mg sugar/ 100 ml haemolymph Glucose Trehalose Total sugars Control 73.2 :_i-_ 9.3 (1 475.0 5; 18.7 a 548.2 ;I-_ 20.7 a 0A 168.4 -_1-_ 6.4 b 626.0 i 8.7 b 777.7 1 33.1 b CDM 109.4 1 4.9 c 679.1 i 9.5 cd 788.5 1 11.4 d DCDM 252.1 i 7.0 a 660.0 _-+_- 18.3 ab 912.1 _-i_- 11.3 a 0A + DCDM 252.5 i 6.0 a 648.1 i 9.8 ab 900.7 _-i_- 16.8 a 0A + PA 75.6 i 5.6 (1 491.7 :2; 17.1 c 567.3 _-1_- 22.5 c DCDM + PA 61.2 i 3.1 d 467.2 i 7.1 c 549.4 i 41.9 c PA 38.4 i 4.5 e 317.0 -_i-_ 18.0 (1 355.4 1'. 19.1 d Results are means 1 SD of 3 experiments each performed in triplicate. Means with the same letters are not significantly different for each sugar on the 5% level of the LSD after ANOVA. All compounds used at 10’3M (10 ul injection) using saline solution as solvent and determinations were done after 1 h from injection. 60 Table 14. Effect of OA, CDM, and DCDM on lipid levels in haemolymph of the tobacco homworm larvae. Concentration mM (10 ul injection) ug lipids/ul haemolymph 0A CDM DCDM 0.1 5.71 i 0.57 a 5.03 i; 0.15 a 5.67 i 0.16 a 1.0 15.60 i 1.36 b 5.53 i 0.64 a 4.90 3; 0.40 a 10 19.07 i 1.20 c 4.97 i 0.05 a 5.28 i 0.89 a 100 10.36 i 5.97 d 4.47 1: 0.42 a 4.98 3; 0.24 a Results are means i SD of 3 experiments each performed in triplicate. Lipids level of control was 5.76 i 0.04 ug lipids/ul haemolymph. Means with the same :ctter are not significantly different within concentrations of each compound by Tukey's to st at the 5% level after AN OVA. 61 trehalose and total sugar in the haemolymph. These results indicate that OA, CDM and DCDM have the property to stimulate trehalase activity as well as that they may also activate another enzyme such as glycogen phosphorylase which elevates trehalose level of the haemolymph. It was of interest to study the effect of OA, CDM and DCDM on haemolpmyh lipid level, since lipids are considered to be a main source of metabolic energy in many insect species. The results in Table 14 show that only CA has the ability to elevate haemolymph lipid level. CDM, and DCDM have no effect on the lipid level. The maximum, elevation of haemolymph lipid was 19.07 i 1.2 ug lipid/ul haemolymph produced by injection with 10 til 10 mM octopamine. DISCUSSION It has been reported that Chlordimeform causes antifeeding activity or anorexia in different insect species such as American cockroach (Beeman and Matsumura, 1978) and lipidopterous larvae (Doane and Dunbar, 1973; Lund et al., 1979; Davenport and Wright, 1987). To date nothing has been reported on the possible biochemical mechanisms of anorexia in insects caused by formamidine pesticides.l have previously shown that the anorectic effect of CDM in the American cockroach is related to changes in haemolymph sugars levels. These changes include elevation of haemolymph glucose but not trehalose. The total sugars levels remained relatively constant. The elevation of glucose levels apparently resulted from trehalase activation by CDM, DCDM and CA on that species. In the case of Manduca sexta, however, I observed an increase in the level of both glucose and trehalose in the haemolymph as a result of injection of these anorectic agents. The increase in glucose levels can be explained by the ability of these anorectic agents to increase trehalase activity. The increase in trehalase levels, on the other hand, must be due 62 to an increase in glycogen breakdown, which could be triggered by the release of the hyperglycemic hormone from its storage site in the corpus cardiacum. Since such a release mechanism is known to be mediated by octopaminergic neurons (Orchard, 1984), such a possibility does exist. Nevertheless, one must be cautious in concluding such a cause- effect relationship, since neither CDM nor DCDM caused an increase in adipokinetic hormone, which is operated in a similar fashion via octopaminergic neurons in the corpus cardiacum (Orchard, 1984). Indeed, among the compounds tested, OA clearly caused an increase in lipid levels. Thus, a balanced view of the evidence indicates that the increase in haemolymph trehalose levels are not mediated by release of hyperglycemic hormone from the corpus cardiacum. Instead, in this insect, an agonistic action on universally distributed octopamine receptors, for example, on the fat body,(Downer, 1979a and 97%), results in an increase in trehalose levels. Alternatively, the increase in glucose levels may somehow be tightly coupled to glycogen breakdown. More work is needed to clarify these questions. As for the actual cause of formamidine evoked-anorexia in this insect species, two major candidates may be proposed; the first is the intense neurotoxic effect on the central nervous system caused by these pesticides as originally described by Lund et al. (1979). The second involves increased haemolymph glucose levels, as observed in the current study. In the case of the American cockroach, it is clear that increased haemolymph glucose levels are involved, since the neurotoxic symptoms in this species develop only at doses approximately 1000 fold higher than that which causes anorexia. However, in Manduca sexta, these doses are very close and therefore it is not easy to separate them. A close examination on Lund et al.'s work (1979) reveals that the median effective dose to elicit the convulsant effect (TD50) in the fifth-instar larva was 0.7 mg / larva. The median concentration of CDM to cause neurotoxic effects (ECso) on isolated nervous system was approximately 1 uM. The lowest dose I have used in the current study was 0.232 mg/ larvae or 0.028 ug/g (1 larvae weighs 8.26 i 1.42 g) which did not produce a fine tremor in the head region, while at 2.32 mg / larva such a symptom was clearly recognizable. In 63 their electrophysiological study about 1/3 of EC5o was the threshold concentration. Thus the lowest dose I adopted appears to be approximately the threshold dose for the neurotoxic effects. It must be noted that even at this dose, the level of haemolymph glucose rose to 350% (T able 13) of the control value within 1h from the time of injection of DCDM. Also, while all symptoms disappeared within 1 to 3h even at 2.32 ug/larva, the level of haemolymph glucose stayed high even after 6h. Despite these considerations in the final analysis, one must conclude that probably both factors are likely to play significant roles in reducing the food intake by M. sexta larvae. The extent of reduction in food consumption at 0.1 mM (0.232 ug/larvae) of CDM is not very great. The rise in glucose level as a result of DCDM administration was not as spectacular as in the case of the American cockroach. Furthermore, in the cae of CDM the rise in the glucose level at this or even at higher doses was significant, but not as impressive as DCDM, despite the fact that the extent of the reduction in the body weight gain (Table 10) caused by the former was comparable to that caused by the latter compound. Nevertheless, these data indicate that the rise in haemolymph sugar levels must cause at least some degree of anorexia in Manduca sexta larvae. For instance, even at the lowest dose tested where no visible neurotoxic effects were observed, the food consumption, the body weight gain and the fecal production were significantly reduced within the short time period (6h). Moreover, OA itself, which did not cause the neurotoxic symptoms at 0.1 or 1.0 mM levels, elicited the same level of anorexia and the increase in the level of haemolymph glucose. The results of this study support the hypothesis that formamidines act on the octopamine receptor as an artificial agonist since phentolamine, which is known to block the effect of octopamine at the receptor level, also inhibited the elevation of trehalase activity induced by both octopamine and DCDM in viva. Furthermore, the effect of 64 octopamine and DCDM on haemolymph sugars levels were not additive in viva with 10 ul 1 mM of both octopamine and DCDM. In conclusion, I have shown that CDM and its active metabolite DCDM cause an increase in the haemolymph sugar level, particularly that of glucose. Such biochemical changes, along with CDM-induced neurotoxic effects, are likely to contribute to the overall phenomenon of the CDM—induced decrease in food consumption in this species. CHAPTER 3 Influence of pesticides and neuroacuve amines on cAMP levels of two-spotted spider mite (Acari: Tetranychidae) 65 INTRODUCTION Considerable biochemical and physiological evidence indicates that biogenic amines function as neurotransmitters, neuromodulators and circulating neurohormones in invertebrate species (Barker et al., 1972; Walker et al., 1972; Robertson et al., 1976; Evans, 1980). A large number of different biogenic amines are known to be present in the insect nervous system (Evans, 1980). Among them octopamine plays the most dominant role in elevating the levels of cAMP (Nathanson and Greengard, 1973), and shows a widespread distribution in insect nervous tissue (Evans, 1978, and 1985). It is found to be associated specifically with the cells of the dorsal unpaired median (DUM) cell system (Hoyle, 1975; Heyle and Barker, 1975; Evans and O'Shea, 1977, 1978) which function as modulators of neuromuscular transmission and muscle contraction (Evans and O'Shea, 1977; O'Shea and Evans, 1979; Evans and Siegler, 1982; Morton and Evans, 1984) and also as excitatory neurons for the light organs of fireflies (Christensen and Carlson, 1981, 1982; Christensen et al. 1983). Octopamine also functions in some insects as a neurotransmitter controlling the release of adipokinetic hormone (Orchard and Loughton, 1981; Orchard et al., 1983) and as a neurohormone (Goosey and Candy, 1980; Davenport and Evans, 1984 a and 1984 b) that mobilizes energy reserves from stores of both carbohydrates (Matthews and Downer, 1974; Downer, 1979 a.and 1979 b; Jahagirdar et al., 1984) and lipids (Orchard et al., 1981, and 1982). Whether or not a particular cell will respond to a biogenic amine will depend upon whether it possesses active receptors for that amine on its surface membrane. Activation of these receptors will initiate a pre-programmed response from the cell either by changing the permeability of the membrane to specific ions or by the activation of specific second messenger systems within the cell. While our knowledge of the pharmacology and mode of action of biogenic amine receptors in insects is rapidly increasing,at present only a few pesticides are known to activate biogenic amine receptors or to directly interfere with the 66 67 second messenger systems (Evans and Davenport, 1986). In systems that use cAMP as a second messenger, membrane bound receptors on the outer surface of the cell membrane control the interaction of the regulatory and catalytic subunits of adenylate cyclase, the enzyme that synthesizes cAMP (Rodbell, 1984). The increased levels of cAMP, resulting from receptor activation, cause the activation of specific cAMP-dependent protein kinases which in turn, increase the levels of phosphorylation of various target proteins (Nestler and Greengard, 1984). It is the activation of the latter proteins that brings about the diverse physiological responses of the cells to the applied biogenic amine. Since no direct experimental evidence has been produced for such a function for biogenic amines in mite species, it seemed important to first establish the methodology to study biogenic amine-sensitive systems in a mite species and second to find which of them are dominant biogenic amines in that species. In the current study we have also examined similar effects of formamidine pesticides in as much as that they produce unique behavioral changes which have been postulated to be mediated through alteration of aminergic response systems. MATERIALS AND METHODS Mites A susceptible strain of two-spotted spider mites, Tetranychus urticae Koch were kindly donated by Dr. Beth Grafton-Cardwell of the Department of Entomology, University of California, Davis. These mites were reared in our laboratory on lima bean plants, Phasealus vulgaris (Henderson's Bush variety) in a growth chamber maintained at 27-28°C with a 16:8 (LzD) photoperiod. 68 Chemicals D,L-octopamine (OA), D,L—dopamine (DA), D,L-S-hydroxytryptamine (S-HT), D,L- synephrine (SN), L-norepinephrine (NE), L-epinephrine (EN), propranolol (PR), Catalytic subunit of protein kinase and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP) were obtained from Sigma Chemical Company, (St. Louis, MO). Chlordimeform (CDM), N-demethylchlordimeform (DCDM), and N-didemethylchlordimeform (all hydrochloride salts) were synthesized and purified in our laboratory. Phentolamine (PA) was obtained from CIBA-Giegy (Summit, NJ). Amitr'az was supplied by Nor-AM Chemical Company (Wilmington, DE). Deltamethrin, fenvalerate, DDT, dicofol, chlorobenzilate, BHC mixture, parathion and aldicarb were obtained from EPA (Research Triangle Park, NC). cAMP assay kits and gamma-32P-ATP (3000Ci/mmole at the beginning) were obtained from Amersham Corporation, (Arlington Heights, IL). Analysis of cAMP levels in the homogenate Our procedures using mite homogenates are based on those used by Nathanson and Greengard, 1973; Nathanson and Hunnicutt, 1981, and Nathanson (1985) with insects. Prior in vitra experiments using exogenous cAMP had shown that under the following conditions, cAMP levels are optimized. Approximately 50mg of two-spotted spider mites were homogenized in 1ml of 6mM Tris-malate, pH 7.4 containing 2mM EGTA (Nathanson, 1985), and 1 mM 2- mercaptoethanol using a glass-teflon homogenizer. The cAMP levels were determined in test tubes, each with 0.2ml of 80mM Tris- malate (pH 7.4) buffer containing 10mM theophylline, 8mM MgClz, 0.1mM GTP, 0.5mM EGTA, 2mM ATP, lmM 2-mercaptoethanol and 10ul of mite homogenate containing 0.4 - 0.45mg protein. The test compound was added with 20ul of reaction buffer (OA, DA, SN, 69 NE, EN, S-HT, CDM, DCDM, DDCDM, PA and PR) or 1 ul of ethanol (deltamethrin, fenvalerate, DDT, chlorobenzilate, dicofol, BHC, parathion, aldicarb, and amitraz). Appropriate solvent controls were run in parallel for all experiments. The enzyme reaction(5 min at 30°C) was initiated by the addition of ATP, stopped by heating to 90°C for 2 min, and then centrifuged at 1000 g for 10 min to remove insoluble material. Cyclic AMP in the supernatant was measured by the protein-binding assay of Brown et al. (1971). Protein concentrations were estimated using the method of Lowry et. al. (1951). Studies on protein phosphorylation The method adopted was similar to the one developed by Costa and Catterall (1982). For this purpose, approximately 0.5 g of two-spotted spider mites were homogenized in the same 6mM Tris-malate buffer. A 0.1 ml aliquot of mite homogenate was added to each tube with 100 ul of the same reaction buffer containing the appropriate final concentration of test substances. The reaction mixture was incubated for 5 min at 30° C and the reaction was stopped using 20 ul of 1% sodium dodecyl sulfate (SDS) and quickly heating at 100° C for 2 min. On cooling in an ice water bath, 20 ul of 10% Triton- X 100 was added, the system thoroughly vortexed, centrifuged at 16,000 x g for 5 min at room temperature and the supernatant was collected. To each tube 1.78 ml of 50 mM histidine-HCl buffer (pH 6.5) was added, and after mixing transferred to a centricon 30 tube with a membrane filter (Aminco Inc.). The content was centrifuged at 3000xg for 60 minutes to reduce the volume to about 40 ul. A 20 ul aliquot of this concentrated protein solution was transferred to a small test tube containing 10 ul (600 ng) of catalytic subunit of protein kinase. The 32P—phosphorylation reaction was initiated by the addition of 20 ul of 2uCi of gamma-32P-ATP in distilled water. After 10 min the reaction was stOpped with 40 ul of 2x "treatment buffer" (4% SDS, 20% glycerol, 10% 2-mercaptoethanol in 0.125 M Tris-HCl pH 6.8) and heating to 100° C for 2 rrrin. The entire volume of the reaction 70 product in each tube was transferred to an electrophoresis well. The method of SDS polyacrylamide slap gel-electrophoresis used was that of Takacs (1979) using a Bio-Rad protein 11 System at 30 mA (with 1.5 mm spacer). RESULTS Preliminary experiments were conducted to determine the levels of GTP required for optimal production of cAMP in mite homogenates and the rate of cAMP production. This was done by measuring octopamine-mediated cAMP production in mite homogenates in the presence of varying concenu'ations of GTP. The data indicated that optimal cAMP production occurred when 0.1mM GTP was included in the incubation reaction mixture. The results of a time course study on cAMP production indicated that there is a linear relationship, up to 5 min, in control incubation and in preparations to which biogenic amines and formamidines were added in order to stimulate the activity of adenylate cyclase. Therefore, all subsequent determinations were carried out for 5 min. The effects of octopamine, synephrine, norepinephrine, epinephrine, 5- hydroxytryptamine, and dopamine on cAMP levels in mite homogenates under these conditions were assessed and the results are illustrated in Figure 16. Octopamine caused the most pronounced increase in cAMP level with a 700% increase over the control value at an octopamine concentration of lmM. Synephrine, norepinephrine, epinephrine, 5- hydroxytryptamine, and dopamine at lmM elevated cAMP levels by 508%, 231%, 28%, 9% and 0% respectively. Incubation of mite homogenates in the presence of N-demethylchlordimeform, and amitraz also caused increased cAMP levels, whereas Chlordimeform, and N- didemethylchlordirneform caused little enhancement of cAMP levels (Figure 17). N- demethylchlordimeform and amitraz increased cAMP production approximately 7.5 times at 71 1000 800‘ 600' 400' 200q Cyclic AMP (percent 01 control) o . . n... 010° 101 102 103 104 1o5 Biogenic Amine Concentration (11M) Figure 16. Effect of ( +) octopamine; (~o-) synephrine; ( +) norepinephrine; («n.- ) epinephrine; ( a- ) 5-hydroxytrytamine; and ( -- ) dopamine on cAMP levels in homogenates of two-spotted spider mites in vitra. Data are means 1; SD as % of control (=100) for 3 experiments with 3 replicates for each concentration. The control was 0.59 i- 003 pmol cAMP/mg protein/min.Means with the same letter for each level of concentratiOn are not significantly different by Tukey’s test at the 5% level. 72 Cyclic AMP (percent of control) 10° 10‘ 102 103 104 105 Concentration of Formamidine (11M) Figure 17. Effects of (-o-) Chlordimeform; (+) N-demethylchlordimeform; (-o-) N-didemethylchlordimeform; and (-o-) amitraz on cAMP levels in homogenates of two-spotted spider mites in vitra. Results are means 1; SD as % of control (=100) for 3 experiments with 3 replicates for each concentration. The control was 0.59 i 0.03 pmol cAMP/mg protein/min.Means with the same letter for each level of concentration are not significantly different by Tukey’s test at the 5% level. 73 lmM, whereas Chlordimeform and N-didemethylchlordimeform caused a maximal increase of approximately 2.5 and 3.65 times in cAMP levels respectively at 100uM. The possibility that these formamidines may interact with the same binding site as octopamine (i.e. high affinity octopamine receptor) in mite homogenates was investigated by examining the additive effects of N-demethylchlordimeform and octopamine on the enhancement of cAMP production. The results shown in Table 15 indicate that the level of evoked cAMP production above basal activity due to a combination of lmM octopamine and lmM DCDM (500%) was not greater than the stimulation caused by lmM octopamine (718%) or lmM DCDM (512%) alone. These results provide some evidence that DCDM and octopamine affect the same receptor. Additional evidence that DCDM and octopamine indeed compete for the same site comes from experiments in which the adenylate cyclase aetivity was stimulated by low concentrations of octopamine in the presence or absence of a fixed, low concentration of DCDM. It was found that in the presence of luM DCDM, the enzyme activity increased 203% by concentration of luM octopamine whereas the increment was only 91% and 150% for luM octopamine and DCDM, respectively (Table 15). These results show that there were additive effects when octopamine and DCDM were used at low concentration. When the concentration of octopamine was approximately the same as that of lmM DCDM, it was the DCDM (because of its probable greater affinity) that primarily determined receptor response. The data in Figure 18 show the kinetic aspect of this additive effect of enzyme activity. The results indicate that at higher concentrations of octopamine (over 40uM) the effect of DCDM becomes unrecognizable. It was of interest to determine whether the activation of adenylate cyclase by DCDM could be blocked by antagonists known to block the octopamine receptor in other arthropod species (Nathanson and Green gard, 1973; Nathanson and Hunnicutt, 1981; and Hashemzadeh et.al., 1985). In the study shown in Figure 19 the effects of different concentrations of phentolamine, an alpha-adrenergic antagonist, and propranolol, a beta- adrenergic antagonist on cAMP production were studied in the presence of 0.1mM DCDM 74 Table 15. Effects of octopamine and DCDM on cAMP levels in homogenates of two- spotted spider mites. Treatment pmol cAMP/mg protein/min Control 0.54 i 0.06 a 1 uM octopamine 1.04 i 0.05 b 1 uM DCDM 1.36 :1; 0.02 c 1 uM octopamine + luM DCDM 1.65 i 0.21 d 1mM octopamine 4.45 i 0.03 e 1mM DCDM 3.33 i 0.02 f 1mM octopamine + 1mM DCDM 3.26 3; 0.19 f,g Data expressed as means i SD for 3 experiments each performed in triplicate. Means are significantly different (P _<_ 0.05) from each other if do not share a common letter by Student-Newman-Kuels test (Steel and Torrie, 1980). 75 1.0 0.8 ‘ 0.6 " 1N '- 0.4 " 0.2 ‘ 0.0 ' 1 ' I ' 0.0 0.1 0.2 0.3 1/8 Figure 18. Lineweaver-Burk plots of adenylate cyclase in homogenate of two- spotted spider mites treated with ( c1 ) octopamine and ( o ) octopamine plus DCDM. DCDM concentration was 1 uM. Each point is the mean of three separate assays. IN = 1/velocity (pmol cAMP/mg protein/min) and 1/8 = l/octopamine concentration ( M). Control velocity = 0.51 i 0.02 pmol cAMP/mg protein/min. 76 120 100 "‘ on O l 60 L 1 l l 40‘ °/o Inhibition of DCDM 20‘ o ..,..,..,..,..,.. 0 2 4 6 81012 Concentration of Antagonist (mM) Figure 19. Inhibition of DCDM stimulation by octopamine receptor antagonists, (+) phentolamine; and (+) propranol in homogenates of two-spotted spider mites. Data are means _-_+-_ SD as % of inhibition of DCDM for 3 experiments with 3 replicates for each treatment. DCDM concentration was 0.1 mM. 77 which was used as an agonist. Both antagonists caused a dose-dependent decrease on DCDM-induced adenylate cyclase activities. Phentolamine was more potent than propranolol in this regard. These data, together with the previous results support the notion that the stimulatory action of DCDM on mite homogenates adenylate cyclase occurs through activation of the octopamine-receptor. Although the predominant action of DCDM was stimulation, the compound at a high concentration (10mM) also exhibited inhibitory effects on octopamine-sensitive adenylate cyclase activity. The effects of various pesticides on cAMP levels in homogenates of two-spotted spider mites were investigated. The results (Table 16) illustrated that only DCDM and amitraz clearly stimulated the levels of cAMP. Deltamethrin, fenvalerate, DDT, BHC showed no such effect, whereas dicofol, chlorobenzilate, parathion, and aldicarb caused slight increases in cAMP production. To ascertain that the acaricide-induced increase in cAMP levels evoke functional changes in mite cells, the changes in protein phosphorylation patterns were studied by incubating the homogenate with gamma-32P-ATP in the presence and the absence of DCDM, octopamine and 8-Br-cAMP. After the reaction the proteins were solubilized with SDS and polyacrylamide-SDS gel electrophoresis was developed. The resulting autoradiogram of labeled phosphoproteins indicate that octopamine, desmethylchlordimeform, and 8-Br-cAMP clearly stimulated the endogenous phosphorylation in the homogenates (Figure 20). The molecular weights of the major protein bands were 29, 38 and 96 KD. Note that in this mode the increase in endogenous protein kinase activities is expected to result in a decrease in overall 32F - phosphorylation.To confirm the results of the visual observation, a densitometric assessment was made on the same autoradiogram and the results are shown in Table 17. 78 Table 16. Effects of various pesticides on cAMP levels in homogenates of two-spotted spider mites in virra. Treatment pmol cAMP/mg protein/min Control 0.45 ;I-_ 0.03 a Deltamethrin 0.43 i 0.02 a Fenvalerate 0.45 i 0.06 a DDT 0.42 -_l-_ 0.02 a Dicofol 1.09 :l_- 0.03 b Chlorobenzilate 0.88 1 0.02 c BHC 0.42 i 0.02 a Parathion 0.61 i: 0.06 d Aldicarb 0.94 i: 0.05 c Amitraz 1.73 5; 0.02 e DCDM 2.49 i 0.03 f Octopamine 3.18 i 0.19 g All chemicals tested at 10'5 M. Data expressed as means 1; SD for 3 experiments each with 3 replicates for each treatment. Means with different letters are significantly different from each other (P _<_ 0.05) by Student-Newman—Kuels test (Steel and Torrie, 1980). 79 Figure 20. Effects of octopamine, DCDM, and 8-Br-cAMP on endogenous phosphorylation of specific proteins in homogenates of two-spotted spider mite. Lanes shown are: 1mM DCDM (Lane 1), 10mM DCDM (Lane 2), 1mM OA (Lane 3), 10mM OA (Lane 4), control (Lanes 5 and 6), 1mM 8-Br-cAMP (Lane 7), and 10mM 8-Br-cAMP (Lane 8). The figure shows autoradiograph of SDS- polyanylamide gel-electrophoresis. Note that the proteins were first phosphorylated (nonradioactive) using endogenous protein kinase, and second, the remaining unphosphorylated proteins were phosphorylated using gamma-32P-ATP and exogenously added PKA. Therefore, the increase in activity of endogenous protein kinases on a given protein expressed as the decrease in the intensity of a corresponding protein band on the electrophoretogram. 80 Mero3 1 2 205 —> 1 1 6 —. 97 —> idogrzi; 66 _" iidtm 011:: 111671.?! of SDS inf-i 45 -> rcmiliii 111111 29 -> 115an 15111 013 81 Table 17. Effects of octopamine N—demethylchlordimeform (DCDM) and 8-Br-cAMP on endogenous phosphorylation of total proteins in homogenates of two-spotted spider mites. The data were obtained by densitometric scanning of autoradiograms of SDS- polyacrylamide gel electrophoresis, and are expressed as relative intensities in % of the total lane intensity of the conu'ol (=100). Treatment Levels of protein phosphorylation % of control 1 mM octopamine 49.4 _t 12.4 b 10 mM octopamine 16.4 i 1.7 a lmMDCDM 47.9_-i_-_1.l bc 10mM DCDM 17.8 :1; 3.5 a 1 mM 8-Br-cAMP 28.2 i 8.1 abc 10 mM 8-Br-cAMP 3.03 i 0.04 a The results of two densitometric measurements of two independent experiments: means :1; standard deviation. Means are significantly different (P 5 0.05) from each other if do not share a common letter by Student-Newman-Kuels test (Steel and Torrie, 1980). 82 DISCUSSION At low doses the formamidines cause marked behavioral abnormalities in susceptible arthropods, suggesting that some part of their pesticidal activity is through a neurotoxic effect (Beeman and Matsumura, 1982). In addition to direct killing action, active formamidines affect acarine feeding, reproduction, and locomotion (Knowles, 1982). In the case of Tetranychus urticae Koch, and the carmine spider mite, Tetranychus cinnabarinus Boisduval, formamidines induced two types of dispersal behavior, walk-off and spin-down (Gemrich et al., 1976 a, and 1979 b; Franklin and Knowles, 1984). The observation that formamidine derivatives (DCDM and amitraz) increase cAMP levels in mite homogenates agrees with the report that DCDM is an agonist of octopamine- sensitive adenylate cyclase in different arthropod species: e.g. the nerve cord of the American cockroach Periplaneta americana (Downer et al., 1985), or firefly light organ (Nathanson and Hunnicutt, 1981; Nathanson, 1985) or locust muscle (Davenport et al., 1985), and raise the possibility of similar actions of formamidine derivatives (DCDM and amitraz) and octopamine in different artluopod species including mites. These formamidines are highly acaricidal, yet, their mechanisms of action are not known. Furthermore, the nature of aminergic systems in acarina species is not well understood. Dopamine is the most frequently mentioned biogenic amine present in acarina species (Scott et al., 1985). Perhaps the most significant aspect of the current study results is that, for the first time, the predominance of octopamine in activating adenylate cyclase system has been demonstrated in an acarina species. Neither dopamine nor 5HT showed any appreciable activities in this regard. The results of the present study suggest the possibility that these formamidines were acting as agonists of octopamine-sensitive responses. Comparison of the formamidine-mediated enhancement of cAMP levels and those induced by octopamine, synephrine, norepinephrine and epinephrine, indicate that the response to formamidine is 83 more similar to the response to octopamine than to norepinephrine or epinephrine. The antagonists which are known to block the activation of adenylate cyclase by octopamine in other arthropod species (Nathanson and Greengard, 1973; Nathanson and Hunnicutt, 1981; Hashemzdeh et al., 1985) demonstrate a similar tendency to block the activation of the same enzyme by DCDM in this species. Thus my results support the theory that octopamine acts as a neurotransmitter in two-spotted spider mites and that the primary site of action of these formamidines in elevating cAMP levels in mite homogenates is through an agonism of octopamine-receptor. The described actions of acaricidal formamidines appear to be specific, as none of the nonacaricidal but highly insecticidal compounds tested, including deltarnethrin, fenvalerate, DDT, and BHC, showed such an effect. Interestingly, other acaricidal chemicals, dicofol, chlorobenzilate, parathion, and aldicarb showed slight increases in cAMP. The meaning of such results is not clear, but the data at least indicate that the action of formamidines is very potent as compared to any of the other pesticides tested, and that the significant rise is cAMP associated with a specific action of chemicals acting through the octopamine receptor. This conclusion is consistent with the data obtained from the protein phosphorylation studies. Both octopamine and DCDM clearly stimulated protein phosphorylation by activating endogenous protein kinases. The fact that the pattern of change in phosphorylation is very similar to that caused by 8-Br-cAMP and that such phosphorylation occurs on sites identical to those of the exogenously added cAMP-dependent protein kinase supports the view that the formamidine-induced rise in cAMP levels directly results in activation of an endogenous cAMP-dependent protein kinase. GENERAL DISCUSSION AND CONCLUSIONS It has been reported that octopamine-2 receptors appear to be associated with adenylate cyclase whereas octopamine-1 receptors do not (Evans, 1984). Octopamine- sensitive adenylate cyclase has not been demonstrated in vertebrates (Bodnaryk, 1982). In addition, octopamine may be a selective neurotransmitter, having a physiological role in invertebrates but not vertebrates (Nathanson, 1985). This selectivity should allow the development of potent octopamine analogs which have selective toxicity for insects but not for mammals and other vertebrates (Nathanson, 1987). Therefore, the use of type-2 OA agonists (such as formamidine-based pesticides) may be able to cause disruption of hormonal and transmitter functions of 0A in insects without noticeable ill effects on vertebrates (Nathanson, 1987). The mechanisms whereby formamidines protect plants and animals from arthropod attack is complex, with dose-dependent lethal and sublethal effects, particularly at critical points in the life cycle. The sublethal effects on behaviou r are associated with an increase in arousal and excitability of the insect with changes in locomotory activity, reduction in feeding and disruption of reproductive behavior (Beeman and Matsumura, 1978; Hollingworth and Lund, 1982; Knowles, 1982; Matsumura and Beeman, 1982; Nathanson, 1987). My results confirmed the original finding that CDM and OA cause anorectic effects in the American cockroach Periplaneta americana (Beeman and Matsumura, 1978) and the tobacco homworm Manduca sexta (Nathanson, 1985). These results indicate that these phenomena are accompanied by changes in haemolymph sugars in different insect species used in this work. In the American cockroach the anorectic effect caused by formamidines and OA was accompanied by increse of haemolymph glucose level, but not trehalose level. The combined glucose and trehalose level remained relatively constant. These phenomena were very similar to the ones produced by CA both in viva and in vitra .The results indicate 84 85 that the elevation of haemolymph glucose was due to the activation of muscle trehalase. These observations agree with the report that OA activates u'ehalase in the thoracic muscles and haemolymph of the American cockroach (Jahagirdar et al., 1984). Phentolamine antagonized such actions of OA and DCDM in the thoracic muscles. Furthermore, both DCDM and OA could elevate cAMP levels in virra in muscle homogenate preparations. These results support the notion that CDM, after metabolic activation to DCDM in viva, acts on the OA receptor of the American cockroach causing activation of the cAMP- mediated response system. These results agree with the demonstration of OA receptors in muscles of other insect species such as locust (Evans et al., 1988). In addition, the results of the activation of the cAMP-mediated response system is an increase of trehalase activity in the muscles and probably other tissues, resulting in increased conversion of trehalose to glucose. Since increases in blood glucose levels are related to anorexia in other animals Hendley et al., 1987) my results suggest that such a rise in haemolymph glucose is one of the most likely biochemical causes for anorexia in this species. On the other hand, I have not shown that the rise in haemolymph glucose level is the only cause for anorexia in this species. In addition, anorexia is a poorly studied subject in insects, and more studies would be needed to fully understand the underlying biochemical causes. In the case of Manduca sexta , hower, I have observed an increase in the level of both glucose and trehalose in the haemolymph as a result of injection of these anorectic agents. The increase in glucose levels can be explained by the ability of these anorectic agents to increase trehalase activity. The increase in trehalose levels, on the other hand, must be due to an increase in glycogen breakdown, which could be triggered by the release of the hyperglycemic hormone from its storage site in the corpus cardiacum. Since such a release mechanism is known to be mediated by octopaminergic neurons (Orchard,1984), such a possibility does exist.Nevertheles, one must be cautious in concluding such a cause- effect relationship, since neither CDM nor DCDM caused an increase in adipokinetic hormone (Orcard,1984). Indeed, among the compounds tested , OA clearly caused an 86 increase in lipid levels. Thus a balanced view of the evidence indicates that the increase in haemolymph trehalose levels are not mediated by the release of hyperglycemic hormone from the corpus cardiacum. Instead, in this species, an agonistic action on universally distributed octopamine receptors, for example on the fat body (Downer, 1979a and 1979b), results in an increase in trehalose levels. As for the actual cause of formamidine evoked-anorexia in this insect species, two major candidates may be proposed; the first is the intense neurotoxic effect on the central nervous system caused by these pesticides as originally descriped by Lund et al., 1979a. The second involves increased haemolymph glucose levels, as observed in the current study. In the case of the American cockroach, it is clear that increased haemolymph glucose levels are involved, since the neurotoxic symptoms in this species develop only at doses approximately 1000 fold higher than that which causes anorexia. However, in Manduca sexta , these doses are very close and therefore it is not easy to separate them. Nevertheless, the obtained data indicate that the rise in haemolymph sugar levels must cause at least some degree of anorexia in Manduca sexta larvae. For instance, even at the lowest dose tested where no visible neurotoxic effects were observed, the food consumption, the body weight gain and the fecal production were significantly reduced within the short time period (6h). Moreover, OA itself,which did not cause the neurotoxic symptoms at 0.1 or 1.0 mM levels, elicited the same level of anorexia and the increase in the level of haemolymph glucose. In conclusion, I have shown that CDM and its active metabolite DCDM cause an increase in the haemolymph sugar level, particularly that of glucose. Such biochemical changes, along with CDM-induced neurotoxic effects, are likely to contribute to the overall phenomenon of the CDM-induced decrease in food consumption in this species. The obtained results from the two-spotted spider mite Tetranychus urticae indicate for the first time the predominance of octopamine in activating the adenylate cyclase system in an acarina species. Nither dopamine nor 5HT showed any significant activities in this 87 regard. My results do not agree with the report that dopamine is the most frequently mentioned biogenic amine present in acarina species (Scott et al., 1985). The results of the present study suggest that formamidines act as agonists of octopamine-sensitive responses. The response to formamidines was more closely resembled the response to octopamine than the response to any other biogenic amine. Phentolamine was more active than propranolol in antagonizing the activation of the same enzyme by DCDM in Tetranychus urticae. Thus my results provide a support for the theory that octopamine acts as a neurotransmitter in two-spotted spider mites and that the primary site of action of these formamidines in elevating cAMP level in mite homogenates is through the activation of octopamine receptors. The action of formamidines was very potent as compared to any of the other pesticides tested. The significant rise in cAMP is associated with a specific action of chemicals acting through the OA receptor. This conclusion is consistent with the data obtained from the protein phosphorylation studies. Both OA and DCDM clearly stimulated protein phosphorylation by activating endogenous protein kinases. The fact that the pattern of change in phosphorylation was very similar to that caused by 8-Br-cAMP, and that such phosphorylation occurs on sites identical to those of the exogenously added cAMP-dependent protein kinase, supports the view that the formamidine-induced rise in cAMP levels directly results in the actiVation of an endogenous cAMP-dependent protein kinase. APPENDIX Ultrastructural studies on formamidine-induced changes of neurosecretion in the American cockroach, Periplaneta americana L INTRODUCTION It is well established that one of the major actions of insecticides is to cause multiple release of neurohormones (Maddrel, 1980). Detailed studies have shown that peptide hormones such as hyperglycaemic ,adipokinetic, hypertrehalosaemic and hypotrehalosaemic (insuline-like) are produced and stored in specialized tissues such as the corpus cardiacum ( Normann, 1980). Hormone release in such cases could be triggered in a variety of ways including administration of insecticides. Formamidines are a relatively new class of pesticidal chemicals. It is generally known that the major effects of these compounds are mediated through the aminergic receptors in insects (Beeman and Matsumura,1978; Evans and Gee, 1980, Murdock and Hollingworth, 1980; Singh et al., 1981; Osborne, 1986; Downer, 1988). The release of adipokinetic hormone from the neurosecretory cells of the glandqu lobe of the locust corpus cardiacum is under the synaptic control of axons in nervous corpus cardiacum II (NCC II) ( Orchard and Loughton, 1981a). Octopamine appears to be the prime candidate as the transmitter mediating the release of this hormone from glandular cells ( Orchard and Loughton, 198 1b). Actions of formamidines upon the octopaminergic site of nerve cells have been reported in the firefly lantern organ ( Murdock and Hollingworth, 1980) , neuromuscular transmission at the locust extensor-tibae muscle ( Evans and Gee, 1980), and neuroglandular cells of the locust corpus cardiacum (Singh et al., 1981). Singh et al., 88 89 (1981) found that the octopamine-mediated release of adipokinetic hormone from the locust CC was mimicked by the formamidine compounds chlordimeforrn (CDM) and N- demethylchlordimeform (DCDM). Furthermore, both CDM and DCDM were capable of potentiating the release induced by electerical stimulation of NCCII. These studies provide physiological evidence for the postsynaptic action of formamidines. Form an ultrastructural viewpoint, it has been shown that treatment of isolated locust corpora cardiaca with 5 uM DCDM causes depletion of neurosecretory materials from the intrinsic neurons, although it does not appear to reduce granule numbers in neurosecretory terminals of extrinsic cells. Such an ultrastructural effect could be prevented by 5 uM of the alpha-aminergic antagonist phentolamine, indicating that the effects brought about by actions of the formamidines upon aminergic receptors on the neuroglandular cells (Singh and Barker, 1983). The formamidines at low doses cause anorexia in the American cockroach. The increase in blood sugar has been regarded as one of the main causes for anorexia in higher animals. Therefore, I have hypothesized that CDM and or its metabolic products could interact with the aminergic, particularly the octopaminergic, neurons in the corpus cardiacum and thereby induce a release of a hyperglycaemic peptide hormone ( Steele, 1961). This hypothesis is also supported by the report of Singh and Barker (1983) indicating a similar action pattern in the locust. Reported herein are electron microscopic studies on CDM-induced changes of neurosecretion in the corpus cardiacum of the American cockroach, Periplaneta americana L. MATERIALS AND METHODS The chemical used in this study, Chlordimeform (CDM) hydrochloride, was synthesized and purified in this laboratory. The insects were adult male American cockroaches reared under standard conditions for several generations (Beeman and Matsumura,1978). 90 The insects were injected with 10 ul of saline solution (Y amasaki and Narahashi,1959) containing 20 ug CDM/insect. The solution was injected into the abdominal hemocoal between the fifth and sixth tergites using a Hamilton microsyringe. Control insects were injected withr 10 ul of saline solution. Both groups of insects were held for 2 hrs without food (only water). The corpora cardiaca were dissected from treated and control insects and fixed separately for 2 hrs on ice in 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The glands were then washed 3 times for 30 min each in the same buffer and post-fixed in 1% osmium tetraoxide in the phosphate buffer for 2 hrs at room temperature. Following post- fixation the tissue was washed 2 times for 15 min each in the phosphate buffer. Following dehydration in a graded series (25 min each step) of 25%, 50%, 75% and 3x 100% ethanol, the tissues were infiltrated and embedded in a mixture of Spurrs and Epon-Araldite epoxy resin (Klomparens et al., 1986). Silver (70-80 nm) sections were cut with a diamond knife and picked up on copper girds. The sections were post-stained with 2% aqueous uranyl acetate for 30 min followed by staining in Reynolds lead citrate for 3 min (Reynolds,1963). The sections were examined in a JEOL 100CX II transmission electron microscope operated at 100kv. 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