Changes in catecholamine levels in the gut and frass of the corn earworm, Helicoverpa zea induced by dietary l -azetidine-2-carboxylic acid

Vol. 27, No. 5, pp. 431-438, 1997 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0965.1748/97 $17.00 + 0.00

Insect Biochem. M&c.

PII: SO9651748(97)00015-S

Pergamon

Bid.

Changes in Catecholamine Levels in the Gut and Frass of the Corn Earworm, Helicoverpa zea Induced by Dietary L-Azetidine-2Carboxylic Acid B. MOSES Received

OKOT-KOTBER,?

22 October

OLUSOLA

A. ADEYEYE*t

1996; revised and accepted

6 February

1997

Several studies have shown that a plant-derived lower homologue of proline, L-azetidine-2carhoxylic acid (AZC), fed to larvae of the corn earworm, Helicoverpu zeu, induces toxicity leading to an array of developmental defects including incomplete pupal cuticular sclerotization or tanning. Destabilized cuticular tanning has recently been shown to be correlated with a deficiency in a hemolymph catecholamine, N-&tlanyldopamine (NBAD) (Okot-Kotber and Adeyeye, 1995). Analysis of gut and frass catecholamine contents has revealed that an AZCsupplemented diet induces a two-fold increase in the excretion of dopamine (DA) and palanine. Also, more 3,4-dihydroxyphenylalanine (DOPA) was excreted in this group than in the controls, although overall, less DOPA was detected than DA in both cases. Four catechols of unknown structure, designated FCl, FC2, FC3 and FC4 according to their relative chromatographic retention times, were found in frass extracts with relatively higher levels in the AZC-treated larvae than in the controls. Three of these, FCl, FC2 and FC3 were also found in the diet, but FC4 was detected only in frass. Gut tissue catecholamines were much higher (by approximately loo-fold) than found in the frass. Approximately two-fold higher DOPA and seven-fold higher DA concentrations were detected in the guts of pupae with incomplete tanning, that had developed from AZC-treated larvae, compared to the controls. It appears, therefore, that the deficiency in NBAD observed in the hemolymph of AZC-fed larvae is largely caused by a slower rate of acylation of DA into NBAD, which results in the accumulation of DA in the gut. The data suggests that there is a defective P-alanyldopamine synthetase in the AZC-treated insects rather than an increased catabolism of NBAD. 0 1997 Elsevier Science Ltd L-Azetidine-2-carboxylic acid Proline analogue Helicoverpa zea Corn earworm Larvae Pupae cle sclerotization Tanning Toxicity Dopamine N-P-Alanyldopamine Gut Frass Excretion

INTRODUCTION

(1989) showed that besides the inhibition of growth and development, dietary azetidine-2-carboxylic acid (AZC), a proline homologue, induced severe malformation of appendages and partial tanning of Helicoverpa zea pupae. These data suggested that AZC caused a curtailed cuticular sclerotization process. Tanning or sclerotization is vital for the normal formation and stabilization of insect cuticle (Andersen, 1979). The ill effects caused by some protective phytochemicals on the cuticle may be caused, in part, by their interference with that process. Apart from proteins and chitin, catecholamines play a fundamental role in stabilizing an insect cuticle during its formation. Notable in that role are the N-acyldopamine compounds which generate

Insect cuticle is important as an exoskeleton for muscle attachment, support, and as a barrier protecting the insect from adverse effects of the environment. Disturbance in this structure renders the insect vulnerable. Some plant allelochemicals destabilize insect cuticle. For example, severe pupal deformities have been reported in Munduca sextu that had been fed a diet containing canavanine (Rosenthal and Dahlman, 1975). Adeyeye and Blum

*Author for correspondence. TDepartment of Biological Sciences, PA 15282, U.S.A.

Duquesne

University,

Cuti-

Pittsburgh,

431

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quinone metabolites essential for cuticular tanning (Andersen, 1985, 1990; Kramer et al., 1988: Sugumaran, 1988; Hopkins and Kramer, 1992). Initial interest in Nacyldopamines was generated by Karlson and Sekeris (1962) who reported that N-acetyldopamine was the sclerotizing agent of the insect cuticle. The significance of these N-acylated dopamine (DA) metabolites became more evident when Andersen (1971) isolated phenolic compounds from sclerotized locust cuticle and reported that N-acetyldopamine was the primary diphenol involved in the cross-linking and stabilization of the locust cuticle. It was subsequently discovered that N-/3alanyldopamine (NBAD) is the major precursor of quinone metabolites crucial for cross-linking and stabilization of the cuticle of a lepidopteran insect, Manduca sexta (Hopkins et al., 1982, 1984; Morgan et al., 1987). Although the mechanism of cross-linking is still poorly understood, we have recently found evidence of covalent cross-linking between some cuticular proteins and an NBAD metabolite in M. sexta pharate pupae (OkotKotber et al., 1994, 1996), signifying the importance of the acylated DA derivatives in the stabilization of the cuticle. In an earlier attempt to determine the causes of cuticular lesions observed in corn earworm pupae after the feeding of larvae on a diet containing AZC, we found that corn earworms had lower levels of catecbolamines, especially NBAD (Okot-Kotber and Adeyeye, 1995). These data suggested that the observed lesions may have resulted from a destabilized cuticle caused by a catecbolamine deficiency. It was not clear, however, whether this deficiency was due to a failure in the synthesis of NBAD, its precursor, or was caused by its increased catabolism. We report here the results of a study designed to determine whether there is an increased excretion of NBAD or its metabolites in corn earworms reared on a diet containing AZC. We monitored catecholamine levels in the guts and the excreta during the final larval instar and in the guts of early pupal instars to determine the rate of excretion of these cross-linking agents compared with insects reared on a normal diet. MATERIALS AND METHODS

Sigma Chemical Co. 6-AminoquinolyI-N-hydroxysuccinimidyl carbamate (AQC) with AccQ-Fluor amino acid analysis reagent kit was supplied by Waters Corp. (Milford, MA, U.S.A.). Other chemicals and solvent~, unless otherwise noted, were purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). Insects

Corn earworm eggs were supplied by Insect Biology and Population Management Research Laboratory. USDA, Tifton, GA, U.S.A., and reared in our laboratory on a pinto bean artificial diet as modified by Perkins et al. (1973) at 24 + I°C, 14:10 h light:dark photoperiod. When the larvae developed into third instars, they were reared singly either on a diet containing 2 mM AZC (larvae that pupate late), 5 mM AZC (larvae that never pupate), or on a normal diet (control group). These individuals were observed daily, and larvae that molted into the fifth instar were isolated. One set of insects were used for gut material and a second set for frass samples. Three to five animals were used for the daily collection of materials. Extraction q f catechols f r o m whole guts

Guts from fiflh-instar larvae were dissected from day zero until pupation (day 8) and from 24 h-old pupae. Guts from larvae on a 5 mM AZC diet were also dissected on days 8 and 9. Dissected guts were meticulously cleaned of any adhering tissues (fat body, tracheoles, muscles and Malpighian tubules) in the dissection buffer containing antioxidants, protease and phenol oxidase inhibitors as described previously (Okot-Kotber et ell., 1994). The contents of each gut lumen were teased out and discarded. The guts were subsequently thoroughly rinsed with several changes in the dissection buffer before weighing and extracting by homogenization in Eppendorf tubes using a hand-held, battery-powered pellet pestle motor (Kontes) on ice. The extraction solution consisted of 1 M HCI (final concentration) containing an antioxidant, 5 mM ascorbic acid, and 1.86 p,M DHBA as an internal standard. Each extraction was followed by centrifugation at 13,000g lbr 15 min at 4°C and the supernatants of individual samples were pooled appropriately.

Chemicals

Catecholamine standards: dopamine (DA); 3,4-dihydroxybenzylamine (DHBA); 3,4-dihydroxyphenylalanine (DOPA); 3,4-dihydroxyphenylacetic acid (DOPAC); 3,4dihydroxyphenylglycol (DOPEG); epinephrine (EPI); N-acetyldopamine (NADA); N-acetylnorepinephrine (NANE); and norepinephrine (NE) were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). 3,4-Dihydroxyphenylethanol (DOPET) and 3,4-dihydroxyphenylketoethanol (DOPKET); N-/~-alanyldopamine (NBAD); and N-~-alanylnorepinephrine (NBANE) were donated by Dr Karl J. Kramer (ARS, USDA, Manhattan, KS, U.S.A.). L-Azetidine-2-carboxylic acid (AZC) and amino acid standards including 13-alanine were obtained from

Frass collection, catechol and amino acid extraction

The larvae isolated for frass collection were transferred daily to fresh diet so as to minimize contamination with the diet. The collection of frass started on day zero (the day of molt into the fifth instar) until day 3 in control larvae and day 5 in those on the AZC-treated diet. No further frass was produced after this interval as the insect prepares to pupate. Freshly produced frass pellets were carefully collected, avoiding contamination, once every 24 h. Each pellet was divided in half; one was used for catechol and the other for/3-alanine determinations. Each half was weighed immediately. The frass aliquots were subsequently extracted several times with a known vol-

CATECHOLAMINES IN THE CORN EARWORM ume of extraction solution as described in the gut extraction section above for catechols and with 1 M acetic acid for /3-alanine. These extracts were centrifuged at 13,000g with refrigeration and the supernatants used for analyses.

Catechol analysis Catechoi extracts were subjected to alumina recovery as previously described (Morgan et al., 1987; OkotKotber et al., 1994) and the eluates were loaded onto a C18 reversed-phase column (Biophase ODS 5 ~m, 4.6 × 250 ram; Bioanalytical Systems, West Lafayette, IN, U.S.A.) employing Beckman System Gold Autosampler 502, and were chromatographed on a high-performance liquid chromatography (HPLC) system fitted with an amperometric dual electrode electrochemical detector (Bioanalytical Systems). The catechols were monitored with the downstream electrode set at - 100 mV after oxidation at + 700 mV with an upstream electrode and a full-scale response set at 10nA. Dual-series electrochemical detection was used to improve selectivity and detection limits, thereby facilitating the identification of the diphenols. Catecholamine peaks were identified by retention times, using two isocratic mobile phases consisting of either 26% acetonitrile containing sodium lauryl sulfate (SLS) as an ion-pairing agent (SLS mobile phase) or 13% methanol containing sodium octyl sulfate (SOS) as the ion-pairing agent (SOS mobile phase), as previously described (Morgan et al., 1987; Okot-Kotber et al., 1994). The flow rate was 1 ml/min and the column temperature maintained at 300C with a jacketed CERA Column Heater 250 (Cera Corp., San Diego, CA, U.S.A.). Concentrations were estimated by comparing peak areas with those of known standards of corresponding compounds using Beckman System Gold software for data analysis through a Beckman Analog Interface Module 406 connected to the BAS detector system, and using DHBA as an internal standard.

HPLC quantitation of [3-alanine A highly sensitive and specific reagent for primary and secondary amino acids, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, was used for derivatization as described by Cohen and Michaud (1993), and adopted in the procedure accompanying the AccQ-Fluor amino acid analysis reagent kit supplied by Waters Corp. Frass amino acid extracts as well as amino acid standards, including /3-alanine, were similarly treated, derivatized and analysed by HPLC. Precolumn derivatized frass amino acid extracts and the standards were subjected to HPLC analysis on a C18 reversed-phase Nova-Pak column 4 t~m, 3.9 × 150 mm (Waters) using a Beckman chromatograph controlled by System Gold software. The samples were loaded either manually or using a Beckman Autosampler 502. Amino acids were separated employing a modified mobile phase gradient system described by Cohen and Michaud (1993). Mobile phase A consisted of 140raM sodium

433

acetate with 17 mM triethylamine and pH adjusted to 5.02 with phosphoric acid. Mobile phase B was 60% acetonitrile in water (v/v). The gradient modification consisted of linear segments set as initial 0% B, 6, 10, 37, and 100% B at 19, 30, 45, and 47 rain respectively, then held at 100% B for 3 min, and subsequently equilibrated at 100% A for 10 min before the next injection. The flow rate was 1 ml/min and the column was maintained at a constant temperature of 38°C using a jacketed Cera column heater. The eluent was monitored at 254 nm (Hong, 1994) using a uv/vis Beckman Programmable Detector Module 166, and data were collected and analysed by the System Gold software. RESULTS

Catecholamines in larval and pupal gut extracts Acid extracts of larval and day-zero pupal gut tissue from normal and AZC-treated corn earworms were analysed by HPLC-EC and revealed that 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylethylamine (dopamine or DA) were the predominant catecholamines. However, in the later stages of larval development and in the pupae, N-/3-alanylnorepinephrine (NBANE) and norepinephrine (NE) were also detected (Fig. 1). The concentration of DOPA was highest on day zero of AZC-fed fifth instars, averaging approximately 2.3/~mol/g fresh weight. DOPA concentration was also highest at this age in larvae fed a normal diet, however it was more than three-fold lower than in the AZC-fed individuals (Fig. 2). Thereafter, the DOPA concentration fluctuated between 0 and 350 nmol/g fresh weight in the normal, and between 180 and 360 nmol/g fresh weight in the AZC-fed individuals, following essentially a similar pattern. Changes in DA concentrations in the guts of larvae fed on a control diet and those on the AZC-containing diet were more drastic. Whereas no DA was detected from day 0 to day 2 in the guts of larvae that fed on a normal diet, a sudden rise occurred, peaking on day 4 reaching approximately 180 nmol/g fresh weight (Fig. 3). This peak was followed by a drop to 30--40 nmol DA/g fresh gut between days 5 and 6 and a second rise to approximately 150nmol/g fresh weight just before pupation on day 8. The pattern in the individuals that were fed on the AZC-supplemented diet was similar to that described above. However, moderate levels were present right from day zero and the peak level of 230 nmol DA/g fresh weight was achieved a day later than in the untreated insects (day 5). This was followed by an abrupt drop on day 6 and a subsequent sudden rise, a pattern similar to that in the controls, culminating in a sharp peak (approximately 450 nmol DA/g fresh weight) on day 8 of the instar, and finally a sharp drop to approximately 150 nmol/g fresh weight on day 9. The analysis of catecholamines in the guts of tanning pupae (24 h-old) that developed from larvae reared on a

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Larval Age (Days) FIGURE 2. Levels of 3,4-dihydroxyphenylalanine (DOPA) concentrations in the gut tissue during the development of fifth instar larvae of H. zea fed on normal (control) and AZC-supplemented diet (treated) insects. Data points represent mean concentrations and vertical bars are _+ SE of the means (n = 4). Where vertical bars are not apparent, the SE is equal to or less than the size of the data point symbol.

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FIGURE 1. HPLC of a standard mixture of catecholamines (a) and 1 M HCI extract of gut tissue from H. zea zero-day-old pupa (b). The catecholamines were resolved on a C18 reversed-phase column using an SLS-containing isocratic mobile phase as described in Materials and Methods. Solid line represents oxidation and dotted line reduction. Abbreviations for the catechols are as in the text.

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normal diet and those fed on an AZC-fortified diet revealed that nearly twice as much DOPA (430 nmol/g fresh weight) was found in the guts of treated pupae (Fig. 4) compared with that found in normal pupae (approximately 250nmol/g fresh weight). Likewise, higher DA levels were detected in the guts of treated pupae (approximately 1300 nmol DA/g fresh gut) than in the guts of normal pupae (approximately 200 nmol DA/g fresh gut), which constitutes a six- to seven-fold difference (Fig. 4).

Catecholamines in larval frass HPLC-EC analysis of larval frass extracts revealed that frass contained relatively low quantities of DOPA, DA and occasional trace amounts of NE and N B A N E (Fig. 5). Four peaks that did not correspond in mobility with any of the standard catecholamines commonly found in insect hemolymph or cuticle were also detected (Fig. 5). Their chromatographic mobilities were compared with those of other catechols occasionally detected

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Larval Age (Days) FIGURE 3. Levels of 3,4-dihydroxyphenyldopamine (DA) concentrations in the gut tissue during the development of fifth instar larvae of H. zea fed on normal (control) and AZC-supplemented diet (treated) insects. Data points represent mean concentrations and vertical bars are _+ SE of the means (n = 4), Where vertical bars are not apparent. the SE is equal to or less than the size of the data point symbol.

in insects, namely: DOPAC, DOPKET, DOPEG, DOPET, and even with epinephrine, and none of them matched with these standards either. They were, nonetheless, presumed to be catechols, on the basis of their electrochemical activity at the dual electrode under the voltage conditions characteristic of catechols described above. The four compounds of unknown nature, hence-

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forth referred to as FC1, FC2, FC3 and FC4 (designated according to their relative mobility in the SLS mobile phase during chromatography on a C 18 RP column, with FC 1 being the fastest and FC4 the slowest; FC standing for "frass catechol"). Analysis of freshly prepared diet revealed that three of the four unknown catechols: FC1, FC2 and FC3 were also present in the diet in similar proportions as in the frass. Quantities of DOPA in the frass obtained from larvae on the control and AZC-containing diet remained relatively low (approximately 300-600 pmol/g fresh frass) throughout the first 3-4 days (Fig. 6). However, there was more than a three-fold increase in excreted DOPA in the frass of AZC-treated larvae in the next day that they continued producing frass. Changes in DA concentrations in the frass are also shown in Fig. 7. On day 0, twice as much DA (5.3 nmol/g fresh frass) was detected in the frass of AZCtreated individuals than that of larvae fed a normal diet (2.5 nmol/g fresh frass). However, in the subsequent 2 days, both control and treated larvae excreted approximately the same quantities of DA in their frass. Whereas a three-fold increase in DA excretion was noted in the frass from controls, DA excretion was depressed in the AZC-treated larvae to very low levels (less than 0.5 nmol/g fresh weight) in the last 2 days that they were still producing frass.

FIGURE 5. A representative HPLC-EC chromatogramshowing peaks of catecholamines extracted from larval frass of H. zea compared with standards. Shown are: (a) Chromatogram of standard catecholamines and (b) Chromatogram of catecholamines extracted from frass. Solid line represents oxidation and dotted line reduction. The peaks were resolved on a C18 reversed-phase column using an SLS-containing mobile phase and the abbreviations for catecholamines are as described in Materials and Methods. [3-Alanine in f r a s s

The levels of/3-alanine were low in both treated, and control animals, and were consistently slightly higher in the frass of treated larvae than in that of untreated larvae (Fig. 8). A downward trend was observed in both cases from day 0 (40 and 45 nmol/g fresh frass in the normal and treated, respectively) to a minimum level on day 3 (15 and 20 nmol/g fresh frass, respectively). The AZCtreated larvae continued evacuating their guts for another 2 days. During this period they experienced another 1.5fold increase in /3-alanine excretion (up to 30 nmol/g fresh gut) by the end of the experiment. DISCUSSION

It has become more evident that tanning or sclerotization in lepidopterans primarily requires N-/3-alanyldopamine (NBAD) to generate the metabolites necessary for

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cross-linking and stabilization of the cuticle. The role of N B A D in this process has been inferred mainly from numerous indirect experiments, as a result of the complexity and relative inaccessibility o f the structures concerned. Furthermore, the intermediary molecules formed during the conversion of N B A D into cross-linking agents, are short-lived and highly reactive, and therefore difficult to investigate. Despite this drawback, there is some evidence suggesting that /3-hydroxylation of N B A D by the participation of diphenoloxidases occurs on the side chain, producing N B A N E (Morgan e t al., 1987). The enzymatic conversion o f N B A D facilitates the formation of covalent bonds with nucleophilic groups

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FIGURE 8. Concentrations of /3-alanine in the frass of H. zea tilth instar larvae fed on normal (control) and AZC-supplemented diet (treated). Data points represent mean concentrations and vertical bars are _+SE of the means (n = 4). (especially from proteins) through the ring or side-chain /3-carbon (Schaefer et al., 1987; Sugumaran, 1988; Andersen, 1989). We have, indeed, demonstrated the occurrence of protein-catecholamine adducts purified from M a n d u c a s e x t a cuticular proteins (Okot-Kotber et al., 1994, 1996), notably an adduct of N B A D which released N B A N E under mild acid hydrolysis. It appears, therefore, that correlations between elevated N B A D titers and cuticular sclerotization (Hopkins et al., 1982, 1984; Krueger e t al., 1989) were not simple coincidences. Investigations towards understanding the mechanism underlying the abnormal sclerotization of pupae that develop from larvae fed a diet containing A Z C established a correlation in the levels of N B A D in the hemolymph of H. z e a and cuticular tanning (Okot-Kotber and Adeyeye, 1995). Later and lower levels of N B A D and its precursor, DA, were observed in the treated individuals that failed to pupate than in the controls, suggesting an altered metabolism of N B A D or its quinone derivatives utilized in cuticular cross-linking. Little is known about the site of N B A D catabolism, let alone the pathway involved in the removal of excess or unutilized N B A D from the hemolymph. However, it appears that/3-alanyldopamine hydrolase may be involved, as described at least for D r o s o p h i l a (Wright, 1987), where this enzyme hydrolyses N B A D into its constituent components, DA and /3-alanine. It is not surprising therefore that we detected both DA and/3-alanine in the frass in the present study. However, the scenario involving N B A D metabolism appears to be more complex, particularly in situations where larvae ingested AZC. First, we determined that D O P A is the predominant catecholamine in the guts of treated and control larvae, although significant amounts of DA were also found. It is likely that D O P A originated via tyrosine hydroxylation. The enzyme and site of synthesis are not known in insects, but it has been

CATECHOLAMINES IN THE CORN EARWORM

suggested that integumentary tyrosinase (Aso et al., 1984; Kramer and Hopkins, 1987) could be important. It is conceivable that in H. zea, the gut epithelium may be one of the major sites for the synthesis of DOPA and its subsequent conversion into DA required for acylation. This notion is supported by the elevated levels of DOPA and DA detected in the gut compared to those determined earlier in the cuticle or hemolymph (Okot-Kotber and Adeyeye, 1995). Changes in the levels of DOPA are comparable in both treated and control animals throughout the fifth instar, with peaks of greater than 350 nmol/g fresh tissue. This level is more than 20-fold higher than the levels reported in the cuticle of H. zea (Okot-Kotber and Adeyeye, 1995). In that study, no appreciable levels of DOPA in the hemolymph was found. Furthermore, in control pupae, where the process of sclerotization is more intense, we observed approximately the same concentrations of DOPA and DA as in the larval stage. This indicates that there is a balance between the synthesis of DOPA, its conversion into DA, and subsequent utilization. However, in the AZC-treated group, where there was poor sclerotization or tanning, an almost two-fold increase in DOPA concentration was found in the gut over larval levels, whereas DA concentration increased seven-fold. The data indicate that the rate of DA synthesis significantly surpasses that of its utilization, resulting in its accumulation and to a lesser extent that of DOPA. It is likely, therefore, that the abnormal cuticular sclerotization in AZC-treated insects is, in part, caused by the under-utilization of DA for the synthesis of NBAD. An alternative hypothesis that may account for the accumulation of DA in the gut of pupae that developed from AZC-fed larvae is that insects fail to utilize synthesized NBAD, leading to its increased catabolism, and therefore accumulation of DA in the gut. However, it is not clear why much lower levels of DOPA and DA were excreted in the frass (approximately 100-fold less than found in the gut) in both cases. It is probable that the insect conserves these important catecholamines in the gut for reutilization when required. On a molar-ratio basis, almost 10-fold more/3-alanine was excreted than DA, indicating either that DA is more easily resorbed than/3-alanine for reutilization or that more/3-alanine is excreted from other sources of catabolism such as the degradation of uracil and aspartic acid (Ross and Monroe, 1972). Although some traces of NE and NBANE were detected late in the frass from AZC-treated individuals, no detectable quantities of NBAD were found. The origin of NE and NBANE traces could be the foregut cuticular lining because these catecholamines were previously detected in the abdominal cuticle (Okot-Kotber and Adeyeye, 1995), and Krueger et al. (1989) found appreciable quantities of NBAD in the foregut of M. sexta. Several unknown electrochemically active compounds were found in the frass from both AZC-treated and control larvae. All of the four compounds were more abun-

437

dant in the AZC-treated individuals than in the controls. Although their chemical nature was not determined, from their chromatographic and electrochemical behavior, we inferred that three of them are more hydrophilic than DOPA, and presumably are charged catechols. This notion is based on the observation that their mobility was considerably influenced by the pH of the mobile phase, and that they were both oxidizable and reducible in the dual-redox electrode under the characteristic potentials used in this study for catechol detection. Attempts were not made to determine their concentrations because their exact nature is not known, more so because three of them were also found in freshly prepared diet, indicating that they originated from the diet. It appears that losses of free catechols through excretion might not account for the lowered levels of NBAD and DA that had previously been reported in the hemolymph (Okot-Kotber and Adeyeye, 1995). It is probable, therefore, that the lowering of NBAD levels in the AZC-treated H. zea is a result of the impeded utilization of DA for NBAD synthesis, possibly through interference with the normal functioning of/3-alanyldopamine synthetase. Conversely, utilization of synthesized NBAD might be impeded by a hormonal imbalance induced by AZC, leading to catabolism of under-utilized NBAD and accumulation of DA. We are currently investigating the tissue distribution and activity of/3-a!anyldopamine synthetase in normal pharate pupae vs AZC-treated instars to determine the influence of AZC on the functioning of this enzyme during metamorphosis. We are also investigating the effects of an AZC diet on the metabolism of juvenile hormone and ecdysone which are known to control metamorphic processes.

REFERENCES Adeyeye O. A. and Blum M. S. (1989) Inhibition of growth and development of Heliothis zea (Lepidoptera: Noctuidae) by a non-protein imino acid, L-azetidine-carboxylic acid. Environ. EntomoL 18, 608~511. Andersen S. O. (1971) Phenolic compounds isolated from insect hard cuticle and their relationship to sclerotization process. Insect Biochem. 1, 157-170. Andersen S. O. (1979) Biochemistry of insect cuticle. Annu. Rev. Entomol. 24, 29~51. Andersen S. O. (1985) Sclerotization and tanning of the cuticle. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 3 (edited by Kerkut G. A. and Gilbert L. 1.), pp. 5974, Pergamon Press, Oxford. Andersen S. O. (1989) Oxidation of N-/3-alanyldopamine by insect cuticles and its role in cuticular sclerotization. Insect Biochem. 19, 581-586. Andersen S. O. (1990) Sclerotization of insect cuticle. In Molting and Metamorphosis (edited by Ohnishi E. and Ishizaki H.), pp. 133155. Japan Science Society Press, Tokyo/Springer-Verlag, Berlin. Aso Y., Kramer K. J., Hopkins T. L. and Whetzel S. F. (1984) Properties of tyrosinase and dopa quinone imine conversion factor from pharate pupal cuticle of Manduca sexta L. Insect Biochem. 14, 463-472. Cohen S. A. and Michaud D. P. (1993) Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino

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acids via high-pertormance liquid chromatography. Analyl. Biochem. 211, 279 287. Hong J. L. (1994) Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography with ultraviolet detection..L Chromamgr. A. 670, 59 66. Hopkins T. L., Morgan T. D., Aso Y. and Kramer K. J. (1982) N-/3Alanyldopamine: major role in insect cuticle tanning. Science 217, 364-366. Hopkins T. L., Morgan T. D. and Kramer J. K. (1984) Catecholamines in hemolymph and cuticle during larval, pupal and adult development of Manduca sexta (L.). Insect Biochem. 14, 533-540. Hopkins T. L. and Kramer K. J. (1992) Insect cuticle sclerotization. Annu. Rev. Entomol. 37, 273-302. Karlson P. and Sekeris C. E. (1962) N-Acetyldopamine as sclerotizing agent of insect cuticle. Nature (London) 195, 183-184. Kramer K. J. and Hopkins T. L. (1987)Tyrosine metabolism for insect cuticle tanning. Archs. Insect Biochem. Physiol. 6, 279-301. Kramer K. J., Hopkins T. L. and Schaefer J. (1988) Insect cuticle structure and metabolism. In Biotechnology fi~r Crop Protection (edited by Hedin P. A., Menn J. J. and Hollingworth R. M.), pp. 160-185. American Chemical Society, Washington, DC. Krueger R. A., Kramer K. J., Hopkins T. L. and Spiers R. D. (1989) N-/3-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation. Insect Biochem. 19, 169-175. Morgan T. D., Hopkins T. L., Kramer K. J., Roseland C. R., Czapla T. H., Tomer K. B. and Crow F. W. (1987) N-/3-alanylnorepinephrine: biosynthesis in insect cuticle and possible role in sclerotization. h~sect Biochem. 17, 255-263. Okot-Kotber B. M. and Adeyeye O. A. (1995) The influence of dietary L-azetidine-2-carboxylic acid on catecholamine levels in the hemolymph and cuticle of the corn earworm, Helicoverpa zea. Insect Biochem. Molec. Biol. 25, 959-966. Okot-Kotber B. M., Morgan T. D., Hopkins T. L. and Kramer K. J. (1994) Characterization of two high molecular weight catechol-

containing glycoproteins from pharate pupal cuticlc ol thc tobacco hornworm, Manduca .~exta. Insect Bioehem. Molee. Biol. 24, 787 802. Okot-Kotber B. M., Morgan T. D.. Hopkins T. L. and Kramcr K..l. (1996) Catecholamine-containing proteins from the pharate pupal cuticle of the tobacco hornworm, Manduca sexta. Ins'cot Biochem. Moh,c. Biol. 26, 475~-t84. Perkins W. D., Jones R. C., Sparks A. N., Wiseman B. R., Snow J. W. and McMillan W. W. (1973) Artilicial diets lor mass rearing of the corn earworm (Heliothis zea). In USDA ARS Produc~ Research Report, pp. 154, Washington, DC. Rosenthal G. A. and Dahlman D. L. (1975) Non-protein amino acidinsect interactions. 1I. Effects of canaline urea cycle amino acids on growth and development of the tobacco hornworm, Manduca sexta (L.)(Sphingidae). Comp. Biochem. Physiol. 52A, 105-108. Ross R. H. and Monroe R. E. (1972) /3-Alanine metabolism in the housefly, Musca domestica: concentrations in the larvae, pupae. and adults. J. lnseet Physiol. 18, 791-796. Schaefer J., Kramer K. J., Garbow J. R., Jacob G. S., Stejskal E. O.. Hopkins T. L. and Speirs R. D. (1987) Aromatic cross-links in insect cuticle: detection by solid state J~C and ~SN NMR. Science 235, 1200 1204. Sugumaran M. (1988) Molecular mechanisms for cuticular sclerotization. Adv. Insect Physiol. 21, 179-231. Wright T. R. F. (1987) The genetics of biogenic amine metabolism. sclerotization, and melanization in Drosophila melanogaster. Adv. Genet. 24, 127-222.

A c k n o w l e d g e m e n t s - - W e wish to acknowledge gratefully comments from Drs T. L. Hopkins, K. J. Kramer and Mr T. D. Morgan on an earlier draft. We also acknowledge B. Giacomazzo, I. Grakalic and D. Steele for their laboratory assistance. This research was supported in part by National Science Foundation grant no. DEB 92207642 to Dr Olusola A. Adeyeye.