In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.)

Peptides 23 (2002) 1759–1763

In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.) S. Harshini a , R.J. Nachman b , S. Sreekumar a,∗ b

a Department of Zoology, University College, Trivandrum 695 034, Kerala, India Food Animal Protection Research Laboratory, Agriculture Research Service, US Department of Agriculture, 2881 F & B Road, College Station, TX 77845, USA

Received 17 December 2001; accepted 20 May 2002

Abstract The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO3 H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[␾1]WP-I, 542␾1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 ◦ C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Amylase; Coconut pest; Digestive enzyme release; FMRF amide; Leucomyosupressin; Leucosulfakinins; Neuropeptide; Opisina arenosella; Perisulfakinin; Protease

1. Introduction Neurosecretions emanating from the brain are known to regulate the secretion of digestive enzymes in insects. Immunological studies have revealed the presence of several vertebrate like neuropeptides in insects which include insulin-like peptide in hymenopteran insects [11] and glucagon-like peptide in Manduca sexta [30]. Recently a large number of insect-specific neuropeptides have been identified, isolated, and sequenced by various research groups [20]. These peptides have been assayed for their effect on myocontractile activity. It is reported that several neuropeptides exhibit almost similar activity either by stimulating or inhibiting muscle contraction. This finding suggests that the actual biological functions of these peptides can be ascertained only after evaluating their effect on different ∗

Corresponding author. Tel.: +91-471-332-934. E-mail address: sree [email protected] (S. Sreekumar).

physiological systems. The role of individual neuropeptides in digestion and related processes is not fully understood since most of the studies have been done with the crude extracts of the brain or employing indirect methods [3]. In this paper, we report the activity of certain FMRF amide related insect neuropeptides and analogues having amino acid sequence homology with vertebrate gastrin/cholecystokinin, in digestive enzyme release from the midgut of the lepidopteran larva of Opisina arenosella employing an in vitro method.

2. Materials and methods Three- to four-day-old final (eighth) instar larvae of the coconut pest O. arenosella were used. Insects were reared in the laboratory, following the method of Santhosh-Babu and Prabhu [26] in a glass jar provided with fresh coconut leaves.

0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 1 5 2 - 3

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2.1. Preparation of midgut for bioassay The larvae were beheaded and cut posteriorly at about the eighth segment. The alimentary canal was pulled out from the posterior end using a pair of fine forceps. It is washed in insect saline and adhering tissues such as fat bodies and tracheal tubes were removed. The midgut (9–11 mm long) was then separated and the contents were flushed out by injecting insect saline into the lumen of the gut with a syringe. The two ends of the open midgut tube were ligated with human hair. The ligated midguts were used in the bioassay. 2.2. Preparation of neuropeptide solution The neuropeptides FMRF amide, leucomyosupressin (LMS), and neuropeptide analogues leucosulfakinin I (FLSK), leucosulfakinin II (LSKII Ser (SO3 H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[␾1]WP-I, 542␾1, and 378A[5b]WP-I were used for studying their effect on secretion of amylase and protease in midgut preparations (Table 1). Neuropeptide solutions (10−6 M) were prepared by dissolving the neuropeptide samples in required volume of insect saline (120 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2 , 0.56 mM dextrose). For studying the dose effect, midgut preparations were incubated with varying concentrations of neuropeptides solutions and the digestive enzyme levels were measured subsequently. 2.3. Bioassay The neuropeptide solution (2 ml) was taken in the bioassay apparatus and the midgut preparation was incubated in it for 30 min at 37 ◦ C, bubbling a small stream of oxygen into the solution. The bioassay apparatus is a glass cylinder, (5 cm×1 cm diameter), open above, with a slanting side tube near the bottom [29]. The side tube was fitted with a rubber stopper. A hypodermic needle inserted into the chamber of

the apparatus through the side tube served for the delivery of oxygen. A glass rod was placed at the open end of the bioassay apparatus to suspend the midgut preparation with a thread. The bioassay apparatus was kept in a water bath at 37 ◦ C. After incubation, the midgut preparation was taken out and washed in insect saline. It was opened and the contents were collected in 0.5 ml distilled water for estimating amylase and protease levels. The method of Neolting and Bernfeld [21] was employed with some modifications for estimating amylase activity. The reaction mixture for amylase consisted of 0.2 ml lumen contents, 0.4 ml 1% starch and 0.2 ml glycine–NaOH buffer (pH 8.8). It was incubated for 30 min at 37 ◦ C. The reaction was terminated by adding 1.2 ml of dinitrosalicylic acid reagent and heating at 100 ◦ C in a water bath for 5 min. The absorbency of the solution was read at 550 nm and quantified as micrograms maltose equivalents liberated by using a maltose (0.1–1%) standard curve. Protease activity was assayed by incubating 0.2 ml lumen contents with 0.4 ml of 1% casein (vitamin-free) solution and 0.2 ml of glycine–NaOH buffer (pH 9.8), according to the method of Birk et al. [1]. After 10 min of incubation at 37 ◦ C, the reaction was stopped by adding 1.2 ml 1% trichloroacetic acid. The incubation mixture was centrifuged at 10,000 × g at 4 ◦ C for 10 min and the supernatant was read at 280 nm with a UV-Spectrophotometer. A standard curve prepared by using varying concentrations of tyrosine, ranging from 0.005 to 0.1% was used to calculate the amount of tyrosine liberated in the reaction in micrograms. In control experiments, ligated midgut preparations were incubated in insect saline without neuropeptides. 2.4. Statistical analysis Two way analysis of variance followed by multiple comparison test based on least significant difference (LSD) at 0.05 level of significance were carried out to determine the difference between the means.

3. Results Table 1 Amino acid sequence of neuropeptides used for bioassay Neuropeptide

Amino acid sequence

FMRF amide FLSK

Phe–Met–Arg–Phe-NH2 Glu–Gln–Phe–Glu–Asp–Tyr (SO3 H)–Gly–Phe–Nleu–Arg–Phe-NH2 Glu–Asp–Tyr–Gly–Nleu–Pro–Phe-NH2 pGlu–Asp–Val–Asp–His–Val–Phe–Leu– Arg–Phe-NH2 His–Val–Phe–Cpa–Arg–Phe-NH2 pGlu–Ser(SO3 H)–Asp–Asp–Tyr (SO3 H)–Gly–His–Met–Arg–Phe-NH2 Glu–Asp–Tyr (SO3 H)–Gly–His–Met–Pro–Phe-NH2 Glu–Gln–Phe–Asp–Asp–Tyr (SO3 H)–Gly–His–Met–Arg–Phe-NH2 Glu–Gln–Phe–Glu–Asp–Tyr–Gly–His–Met– Arg–Phe-NH2

542␾1 LMS 378A[5b]WP-I LSK II Ser (SO3 H) PLSK PSK 14A[␾1]WP-I

The contents of midgut preparations showed an increase in amylase level after incubation with FMRF amide and FLSK, as revealed by statistical evaluation by two-way ANOVA followed by LSD and excluding peptides grouped along with the control. FLSK was effective in increasing amylase level at all the three doses and it elicited maximum response (83.53 ± 6.98 amylase units) at 10−6 M concentration. A decrease in amylase level was observed when midgut preparations were incubated with LSK II Ser (SO3 H), PSK, and 14A[␾1]WP-I. This effect was maximum with PSK at all doses (Table 2). FLSK, PSK, 14A[␾1]WP-I, and 542␾1 caused an increase in level of protease when these peptides were used to incubate the midgut preparations. Maximum protease activity of 444.44 ± 67.58 was observed with 14A[␾1]WP-I

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Table 2 Effect of FMRF amide and analogues on secretion of amylase in midgut preparations of larvae of Opisina arenosella Neuropeptides

Amylase units (M) 10−6

FMRF amide FLSK LSK II Ser (SO3 H) PLSK PSK 14A[␾1]WP-1 542␾1 LMS 378A[5b]WP-I Control

23.91 83.53 8.70 12.37 4.22 10.56 15.22 13.40 22.95 18.12

10−7 ± ± ± ± ± ± ± ± ± ±

1.94 6.98 2.07 0.98 0.61 0.73 1.74 1.47 5.78 1.06

(5) (6) (5) (6) (6) (6) (9) (5) (6) (8)

jk m abcd cdefgh ab bcdef defgh defgh jk ghij

39.78 29.31 17.81 12.66 2.21 11.18 12.99 13.04 14.49 18.12

10−8 ± ± ± ± ± ± ± ± ± ±

3.80 (8) l 1.58 (6) k 6.67 (6) ghij 0.87 (6) defgh 0.89 (9) a 0.56(6) bcdefg 1.18 (6) defgh 2.53 (5) defgh 5.89 (6) defgh 1.06 (8) ghij

5.43 39.75 19.33 17.21 9.66 18.12 12.08 11.96 16.61 18.12

± ± ± ± ± ± ± ± ± ±

2.06 (5) abc 2.44 (6) l 4.53 (6) hij 1.02 (6) fghij 2.02 (6) bcde 3.5 (6) ghij 1.91 (6) cdefg 3.15 (5) cdefg 0.98 (6) efghi 1.06 (8) ghij

Values are mean ± S.E.M. of number of observations indicated between parenthesis. The units indicate the amount of enzyme required to liberate 1 ␮g of maltose equivalents from starch per minute. The significant difference between the group was analyzed by two-way analysis of variance. Mean values with different letters significantly differ (P < 0.05) as determined by multiple comparison test (LSD).

Table 3 Effect of FMRF amide and analogues on secretion of protease in midgut preparations of larvae of Opisina arenosella Neuropeptides

Protease units (M) 10−6

FMRF amide FLSK LSK II Ser (SO3 H) PLSK PSK 14A[␾1]WP-1 542␾1 LMS 378A[5b]WP-I Control

288.88 388.80 133.83 222.22 351.85 444.44 412.22 148.14 198.14 212.96

10−7 ± ± ± ± ± ± ± ± ± ±

13.89(5) efghijkl 18.14 (6) lmno 48.52 (5) abc 44.14 (6) bcdef 42.48 (6) jklmno 67.58 (6) o 16.10 (8) no 67.79 (5) abcd 43.71 (6) bcdef 24.50 (8) bcdefg

134.44 339.62 339.63 314.80 222.22 240.74 148.15 133.33 105.20 212.96

10−8 ± ± ± ± ± ± ± ± ± ±

46.85 (8) abc 69.54 (6) hijklmno 22.2 (6) hijklmno 61.83 (6) ghijklmn 30.25 (9) cdefghi 51.25 (6) cdefghi 34.93 (9) abcd 54.56 (5) abc 26.10 (6) ab 24.50 (8) bcdefg

66.67 358.15 265.56 370.37 178.89 296.30 345.56 192.59 216.73 212.96

± ± ± ± ± ± ± ± ± ±

47.95 (5) a 39.30 (6) jklmno 37.41 (6) defghijk 64.85 (6) klmno 17.69 (6) abcde 64.85(6) ghijklm 22.82 (6) ijklmno 69.44 (5) bcdef 35.20 (6) bcdefgh 24.50 (8) bcdefg

Values are mean ± S.E.M. of number of observations indicated between parenthesis. The units indicate the amount of enzyme required to liberate 1 ␮g of tyrosine from casein per minute. The significant difference between the group was analyzed by two-way analysis of variance. Mean values with different letters significantly differ (P < 0.05) as determined by multiple comparison test (LSD).

at 10−6 M concentration. FLSK showed significant and sustained effect at all three concentrations, viz. 10−6 , 10−7 , and 10−8 M. FMRF amide caused a decrease in protease level at 10−8 M concentrations (Table 3). Comparison of dose effect of peptides on amylase and protease shows that at higher concentration of 10−6 M is required for eliciting maximum response, as may be seen from Tables 2 and 3.

4. Discussion In the present study some of the neuropeptides and their analogues sharing a tetrapeptide C-terminal identity have been used to evaluate their effect on digestive enzyme secretion in the midgut of larvae of O. arenosella. The neuropeptides FMRF amide, FLSK, LMS, and 378A[5b]WP-I possess an FXRFamide sequence at the C-terminal where ‘X’ can be either leucine or methionine. Meanwhile, the C-terminal of the neuropeptides LSK II Ser (SO3 H), PLSK,

PSK, and 14A[␾1]WP-I has an HMRF amide sequence. The results of the present study shows that incubation of midgut preparations with some of the neuropeptides and neuropeptide analogues caused either an increase or decrease in the levels of digestive enzymes in lumen contents. This effect is, evidently, due to stimulation or inhibition of digestive enzyme release from the midgut tissue into the gut lumen. Leucosulfakinin (LSK), is a sulfated neuropeptide isolated from the head extracts of Leucophaea maderae exhibiting myotropic activity [15,16]. FLSK, LSK II Ser (SO3 H), PSK, and PLSK used in the present study are analogues of LSK. These peptides contain a tyrosine sulfate residue and exhibit a remarkable octapeptide C-terminal sequence homology with vertebrate gastrin/CCK hormone family, [17,18] the octapeptide sequence of gastrin being Glu–Ala–Tyr(SO3 H)–Gly–Try–Met–Arg–Phe-NH2 . In 14A[␾1]WP-I and 542␾1 tyrosine residue is not sulfated, but share a C-terminal identity with LSK and its analogues. The stimulatory effects of FLSK on protease and amylase release and LSK II Ser (SO3 H), PLSK, PSK, 14A[␾1]WP-I,

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and 542␾1 on protease release from the midgut of O. arenosella, revealed in the present study, may be due to their structural similarity with gastrin/CCK, particularly the presence of nonbasic tyrosine residue in the neuropeptide sequences. Leucosulfakinins have a similar action in Rhynchophorus ferrugineus [19]. An additional effect of sulfakinins in insects could be inhibition of food intake as reported in Schistocerca gregaria [31,28]. The present study shows that LSK II Ser (SO3 H) inhibited amylase release along with PSK and 14A[␾1]WP-I in O. arenosella despite sharing striking amino acid sequence similarities with gastrin/CCK and possessing tyrosine sulfate or tyrosine residue. The C-terminal of LSK II Ser (SO3 H), PSK, and 14A[␾1]WP-I has an HMRF amide sequence which may be inhibiting amylase release. The inhibitory effect of LSK II Ser (SO3 H) can as well be due to the N-terminal pyroglutamate blocking. It is reported that removal of pyroglutamate at the N-terminal increases the activity of some of the peptides [14]. FMRF amide is a cardioexcitary peptide with only the C-terminal tetrapeptide sequence found in other neuropeptides. It is first identified from the mollusc Macrocallista nimbosa [24]. The leucine derivative of FMRF amide is known as FLRF amide. This peptide is also found in molluscs [9] and has been shown to inhibit midgut muscle contraction in Sphingid moth [6] and locust [12]. In this study FMRF amide caused a decrease in protease and amylase levels at a lower concentration of 10−8 M. However, a higher concentration (10−7 M) of FMRF amide increased the amylase release. It is already reported that the dissociated cells from the digestive tract of the scallop Pecten maximus release amylase on treatment with FMRF amide and FLRF amide [5]. Cells displaying FLRF amide like immunoreactivity have been observed in the midgut of Locusta migratoria [12]. In this insect FMRF amide content in midgut epithelial cells varies with feeding-related events [13]. Similarly, a decrease in FMRF amide immunoreactivity has been observed in the midgut endocrine cells of Aedes aegypti following ingestion of blood [2]. Genes encoding FMRF amide related peptides are identified in the central nervous system of Drosophila melanogaster [27]. Also, several FMRF amide related immunoreactive materials have been purified in D. melanogaster [22]. Such observations suggest that FMRF amide may be functioning as a midgut hormone in insects. LMS is known to inhibit hindgut contractile activity in L. maderae [10] and midgut muscle contraction in Diploptera punctata [7]. LMS extracted from the central nervous system of Periplaneta americana is effective in inhibiting visceral muscle activity [23]. In D. punctata it stimulates amylase and invertase activity in the midgut lumen contents [8]. 378A[5b]WP-I is an LMS analogue. LMS and analogue revealed no significant activity in the present study. A comparison of dose-effects of neuropeptides shows that in most cases a higher concentration, as much as 10−6 M, is required for causing maximum activity in the release of protease and amylase in vitro. Nachman et al. [16] have

observed that an analogue of LSK, synthetic gastrin I and gastrin II remained inactive in stimulating hindgut muscle contractions up to a concentration of 10−6 M. FMRF amide and HMRF amide elicited no response on cockroach hindgut preparations even up to 10−5 M [17]. Similarly in the Scallop, Pecten maximus, FMRF amide showed maximum activity in stimulating amylase secretion from isolated midgut cell at a concentration of 10−6 M [5]. However, it is known that vertebrate hormones work at concentrations close to 10−9 to 10−8 M. The discrepancy observed in these studies could be due to the reason that in the in vitro system the neuropeptides behave as paracrine effectors, than as genuine hormone [5]. It is observed in this study that FMRF amide stimulates amylase release at higher doses, and inhibits the release of digestive enzymes at a lower dose of 10−8 M. Many vertebrate hormones exhibit differential action in relation to dose. Pancreatic polypeptide, for example, elicits different effects at different concentrations. It stimulates secretion of gastric juice and inhibits gastric mobility at a lower concentration while producing the opposite effects at higher concentrations. Some of the peptides used in this study exhibited varying degrees of responses with different doses. As the midgut epithelium of insects contain endocrine cells, it is possible that some of the effects obtained in this study could be indirect, through releasing gut hormones. The neuropeptides have multiple target tissues and actions. Besides regulating enzyme secretion from midgut cells, the neuropeptides FMRF amide, PSK, 14A[␾1]WP-I and 542␾1 that share sequence similarity with gastrin/CCK, are capable of stimulating release of secretions concerned with buffering in R. ferrugineus, in a dose-dependent manner [25]. Changes in chemical nature of gut contents and therefore pH, caused by the peptides can possibly interfere with the release of digestive enzymes from the midgut tissue as well as enzyme activity. It is generally held that the C-terminal sequence of a peptide is crucial for its physiological activity and immunoreactivity while the N-terminal part of the molecule modulates the biological potency of action [4]. The results show that a peptide as short as FMRF amide can influence release of digestive enzymes from the midgut tissue of larvae of O. arenosella. The C-terminal tetrapeptide sequence of neuropeptides, therefore, may represent the active core. The results of the present study indicate that in larvae of O. arenosella amylase release can be stimulated by peptides with FXRF amide at the C-terminal (‘X’: leucine or methionine). Leucosulfakinin peptides with C-terminal HMRF amide sequence are found to inhibit the release of amylase. Meanwhile, protease release is effected by peptides having C-terminal sequence with both FXRF and HMRF amides. It is probable that in O. arenosella protease and amylase are secreted by specific cells. Information on the active core of neuropeptides sharing common aminoacid sequence may have significance as it provides a template for the rational design of pseudopeptides and nonpeptide mimetics for pest management. It can be further used to trim the length of the

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DNA that is required to alter the physiological functions through recombinant DNA technology.

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