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Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae)
Comparative Biochemistry and Physiology Part B 132 (2002) 353–358
Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae) S. Harshinia, R.J. Nachmanb, S. Sreekumara,* 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 and B Road, College Station, TX 77845, USA
b
Received 12 September 2001; received in revised form 31 January 2002; accepted 2 February 2002
Abstract Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 8C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 Aw1x WP-2, LPK and 434 wf2x WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (XsAsn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the Cterminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Amylase; Coconut pest; Digestive enzyme release; Leucokinin; Neuropeptide; Opisina arenosella; Pyrokinin; Protease
1. Introduction Neuropeptides are diverse chemical messengers known in the nervous system of metazoans includ¨ ing insects (Nassel, 1993). The large number of neuropeptides structurally identified from different insect groups highlights the complexity of the neurosecretory system in regulating various physiological processes (Predel and Eckert, 2000). In Leucophaea maderae alone, more than 12 neuropeptides are isolated of which, all, except leucomyosuppressin, exert a myotropic action (Holman et al., 1990). This observation suggests that myotrop*Corresponding author. Tel.: q91-471-332-934. E-mail address: sree [email protected] (S. Sreekumar).
ic action may be one of the many biological activities of these peptides. To date, it is not possible to draw any conclusion regarding the major functional role of these peptides as it will require the elucidation of all their diverse activities in different physiological and biochemical systems. The probable role of neuropeptides in digestion and feeding needs to be investigated, for the stomatogastric nervous system, gut endocrine cells and the nerves innervating the gut contain peptiˇ dergic principles (Sehnal and Zitnan, 1990). In the present study, we demonstrate the effect of leucokinins, a family of eight peptides isolated from L. maderae, their related peptide analogues and leucopyrokinin on in vitro release of amylase and protease from the midgut epithelial tissue of
1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 2 . 0 0 0 4 7 - 7
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S. Harshini et al. / Comparative Biochemistry and Physiology Part B 132 (2002) 353–358
Table 1 Amino acid sequence of neuropeptides used for bioassay Neuropeptide
Amino acid sequence
LPK LK-I LK-II LK-III LK-IV LK-V LK-VI LK-VII LK-VIII FFSWG amide 122Aw1xWP-2 434wf2xWP-1
pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2 Asp-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 Asp-Pro-Gly-Phe-Ser-Ser-Trp-Gly-NH2 Asp-Gln-Gly-Phe-Asn-Ser-Trp-Gly-NH2 Asp-Ala-Ser-Phe-His-Ser-Trp-Gly-NH2 Gly-Ser-Gly-Phe-Ser-Ser-Trp-Gly-NH2 pGlu-Ser-Ser-Phe-His-Ser-Trp-Gly-NH2 Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 Gly-Ala-Ser-Phe-Tyr-Ser-Trp-Gly-NH2 Phe-Phe-Ser-Trp-Gly-NH2 Phe-Phe-Aib-Trp-Gly-NH2 pGlu-Leu-Phe-Phe-Aib-Trp-Gly-NH2
LPK: Leucopyrokinin; LK: Leucokinin. FFSWG amide, 122A (1) WP-2 and 434 (f2) WP-1 are synthetic leucokinin analogues.
the larvae of the lepidopteran moth, Opisina arenosella. 2. Materials and methods Three- to four-day-old final (eighth) instar larvae of the coconut pest Opisina arenosella were used. Insects were reared in the laboratory, following the method of Santhosh-Babu and Prabhu (1987) in a glass jar provided with fresh coconut leaves. 2.1. Preparation of midgut for bioassay The larvae were beheaded and cut posteriorly at approximately 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 Leucokinins (LK I–VIII), the Leucokinin analogues FFSWG amide, 122A w1x WP-2, 434 wf2x WP-1 and Leucopyrokinin (LPK) were used for studying the effect on secretion of amylase and protease in midgut preparations (Table 1). 10y6 M solutions of the
neuropeptides were prepared by dissolving the neuropeptide samples in insect saline (120 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 0.56 mM glucose). For studying the dose responses, midgut preparations were incubated with varying concentrations of neuropeptide (LK III, LK VIII and LPK) 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 8C, bubbling a small stream of oxygen into the solution. The bioassay apparatus is a glass cylinder, (5=1 cm diameter), open above, with a slanting side tube near the bottom (Sunitha et al., 1999). 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 8C. 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 of distilled water for estimating amylase and protease levels. The method of Noelting and Bernfeld (1948) 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 8C. The reaction was terminated by adding 1.2 ml of dinitrosalicylic acid reagent and heating at 100 8C in a water bath for 5 min. The absorbence of the solution was read at 550 nm and quantified as microgram 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. (1962). After 10 min of incubation at 37 8C, the reaction was stopped by adding 1.2-ml 1% trichloroacetic acid. The incubation mixture was centrifuged at 10 000=g at 4 8C for 10 min and the supernatant was read at 280 nm. A standard curve prepared by using varying concentrations of tyrosine, ranging from 0.005 to 0.1%, was used to calculate the
S. Harshini et al. / Comparative Biochemistry and Physiology Part B 132 (2002) 353–358 Table 2 Effect of LPK, leucokinins and LK analogues on secretion of protease and amylase in midgut preparations of larvae of Opisina arenosella Neuropeptides
Protease unitsa
Amylase unitsb
LPK LK I LK II LK III LK IV LK V LK VI LK VII LK VIII FFSWG amide 122A w1x WP-2 434 wf2x WP-1 Control
111.1"16.63 (6)* 155.60"67.2 (5) 96.29"51.85 (5)* 44.40"20.37 (5)** 118.49"27.41 (5)* 251.90"30.37 (5) 207.40"28.15 (5) 170.41"55.22 (12) 453.70"46.27 (5)* 118.50"47.94 (5)* 160.37"22.78 (6) 117.78"95.17 (6)* 212.96"24.50 (8)
2.54"0.72 (6)*** 10.86"4.97 (5) 12.67"4.66 (5) 4.98"4.31 (8)** 18.46"5.45(5) 19.91"5.83 (5) 23.53"3.44 (5) 19.45"3.77 (8) 15.58"3.54 (5) 0 (5) 11.78"3.39 (6)** 14.50"4.13 (6) 18.12"1.06 (8)
Values are mean"S.E.M. of number of observations indicated between parentheses. The significance was analyzed by Student’s t-test. * P-0.05. ** P-0.01. *** P-0.001. a 1 Unitsamount of enzyme required to liberate 1 mg of tyrosine from caseinymin. b 1 Unitsamount of enzyme required to liberate 1 mg of maltose equivalents from starchymin.
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amount of tyrosine liberated in the reaction in micrograms. In control experiments, ligated midgut preparations were incubated in insect saline without neuropeptides. 3. Results 3.1. Digestive enzyme levels in midgut preparations incubated with neuropeptides The midgut preparations incubated with neuropeptides LK III, FFSWG amide, 122Aw1xWP-2, LPK, revealed a decrease in amylase secretion when compared with controls. The protease levels decreased following incubation with FFSWG amide, LK II, LK III, LK IV, LPK and 434 wf2xWP-1. LK VIII caused an increase in the level of protease (Table 2). 3.2. Dose response studies With LK III and LPK digestive enzyme levels in midgut preparations decreased with increasing concentration of neuropeptides. However, a 10y6 M solution of LK VIII stimulated amylase secretion. Figs. 1 and 2 shows the dose response of
Fig. 1. Dose-response of LK III, LK VIII and LPK in protease secretion in midgut preparations of larvae of Opisina arenosella. Results are expressed as mean"S.E.M. (ns6). The significance of data was analyzed by Student’s t-test (*P-0.05, **P-0.01).
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Fig. 2. Effect of varying concentration of LK III, LK VIII and LPK on amylase secretion in midgut preparations of larvae of Opisina arenosella. Results are expressed as mean"S.E.M. (ns6). The significance of data was analyzed by Student’s t-test (**P-0.01, ***P-0.001).
these neuropeptides on protease and amylase secretion. 4. Discussion A large number of neuropeptides specific to insects have been discovered and sequenced. Leucokinins are a group of eight myotropic peptides isolated from the head extracts of L. maderae assayed with a sensitive hind gut preparation (Holman et al., 1986a,b, 1987a,b). In addition to the established myotropic action, these peptides stimulate lipid mobilization and inhibit protein synthesis in L. maderae (Goldsworthy et al., 1992). Leucokinins and their homologues may also function in the control of water and ion balance in numerous insects (Schoofs et al., 1993). In Drosophila melanogaster, leucokinins are reported to increase the intracellular calcium in the stellate cells (O’Donnell et al., 1998). The leucokinins are designated as LK I–VIII, and comprise a group of structurally related peptides with sequence identity at positions 4, 6, 7 and 8. They are also the first to be recognized as a natural
analogue series of isopeptides in one species. In this study, leucokinins, LK analogues and leucopyrokinin have been bioassayed for evaluating their effect on digestive enzyme secretion from the larval midgut of O. arenosella. Of the eight LK peptides investigated, LK III revealed maximum activity as it causes significant decrease in the levels of protease and amylase in the midgut preparations. LK II and LK IV were effective only in inhibiting the release of protease. LK I, LK V, LK VI and LK VII showed no activity. Meanwhile, LK VIII exerted a stimulatory effect by increasing the level of protease. Regarding the structure activity relationships of the leucokinins, our study indicates that the leucokinin structure is recognized by the midgut receptors of O. arenosella. Leucokinins have an identical C-terminal pentapeptide sequence identified as FXSWG amide (Phe-X-Ser-Try-Gly-NH2 ) where ‘X’ is a variable being Asn, His, Ser, or Tyr. This common sequence, represents the active core of the molecule (Nachman and Holman, 1991). LK II, LK III and LK IV, which inhibited the release of digestive enzymes to varying
S. Harshini et al. / Comparative Biochemistry and Physiology Part B 132 (2002) 353–358
degrees, possess at the fifth position the amino acids Ser, Asp and His, respectively. These amino acids can be encountered at the same positions in LK I, LK V, LK VI and LK VII which have no effect on enzyme secretion. These observations suggest that substitution of amino acids at the fifth position does not influence the activity of the peptides. LK II shares with LK-III a similar amino acid sequence except at position 2 where it has proline instead of glutamine. LK II and LK VII have similar amino acid sequence except at the third position where LK II has glycine, and in LK VII it is alanine. This single substitution of alanine in place of glycine in LK VII may be responsible for its loss of inhibitory activity. This is further supported by the fact that the same substitution is present in LK I which reveals no activity in the bioassay. LK IV which has inhibited the release of protease shares with LK II and LK III aspartic acid at position 1 and has serine at position 3 as in leucopyrokinin, another inhibitory peptide. Unlike other peptides in the series, LK VIII stimulated the release of protease. This peptide is unique among leucokinins in having a tyrosine residue in the fifth position and perhaps this is responsible for its stimulatory action in O. arenosella as in L. maderae where it causes increased myotropic activity (Holman et al., 1987b). In the present study, the neuropeptides LK V and LK VI had no activity, on the release of digestive enzymes. These peptides have different sequences of amino acids at the variable part, the N-terminal, which is supposed to modulate the physiological actions (Duve et al., 1994). The synthetic peptide FFSWG amide, with only the common core, is highly effective as it inhibits the release of amylase totally and to a significant extent the release of protease as well. 122Aw1x WP-2 is an analogue of leucokinins, resistant to an endopeptidase known as angiotensin converting enzyme found in vertebrates and insect tissues due to the presence of aminoisobutyric acid (Aib). Aib-containing analogues of the insect kinin neuropeptide family can disrupt the water andyor ion balance in insects (Nachman et al., 1997b). 122Aw1x WP-2 differs from FFSWG amide in lacking serine, a common residue present in the core segment of leucokinins. This peptide is less effective as it inhibits the secretion of amylase only. 434 wf2x WP-1 is another enzyme resistant analogue that mimics the activity of leucokinins. Compared to 122Aw1xWP-2, 434 wf2x WP-1 pos-
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sesses an additional sequence of pGlu-Leu at the N-terminus. It is found to be effective in inhibiting the release of protease. The pyrokinins are a large family of myotropic peptides (Predel et al., 1999) known to regulate diverse processes like pheromone biosynthesis, egg diapause and pupation in different insects (Imai et al., 1991; Raina, 1993; Zdarek et al., 1998). In the present bioassay, leucopyrokinin (LPK) showed an inhibitory effect on the release of digestive enzymes from the midgut tissue of lepidopteran larvae. LPK possesses an N-terminal blocking pyroglutamate and proline at position 6. N-terminal pyroglutamate blocking can influence the activity of peptides. For instance, removal of N-terminal pyroglutamate from LPK leads to an increase in myotropic activity over the natural product in L. maderae (Nachman and Holman, 1991). The existence of proline residue in pyrokinins may also determine the activity, as it may turn the C-terminal part of the molecule (Nachman and Holman, 1991; Nachman et al., 1993). Dose response studies with LK III and LPK showed an increase in inhibitory effect with increasing concentration of the neuropeptides. Among LK peptides, LK III has shown maximum activity followed by LK II and LK IV. However LK III stimulated enzyme secretion at a concentration of 10y8 M, when compared with control, thus indicating differential effects at higher and lower concentrations. It is known that in vertebrates, pancreatic polypeptide 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. Information on active core sequence of neuropeptides can be of great value in developing new strategies in pest management, particularly those involving recombinant DNA technology (Holman et al., 1990). The present study reveals that the minimum requirement for achieving inhibition of digestive enzyme secretion in larvae of O. arenosella may be the C-terminal penta peptide sequence FXSWG amide as exemplified by FFSWG amide. It is recently reported that some of the neuropeptides belonging to the leucosulfakinin family are effective in stimulating digestive enzyme release in the larvae of the red palm weevil Rhynchophorus ferrugineus (Nachman et al., 1997a). This is the first report on insect neuropeptides demonstrat-
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ing inhibitory activity in the release of digestive enzymes from midgut tissue. References Birk, Y., Harpez, I., Ishaaya, I., Bondi, A., 1962. Studies on the proteolytic activity of beetles Tenebrio and Tribolium. J. Insect Physiol. 8, 417–429. Duve, H., Rehfel, J.F., East, P., Thorpe, A., 1994. Localisation of sulphakinin neuronal pathways in the blow fly Calliphora vomitoria. Cell Tissue Res. 275, 177–186. Goldsworthy, G.J., Coast, G., Wheeler, C., Cusinato, O., Kay, I., Khambay, B., 1992. The structure and functional activity of neuropeptides. In: Crampton, J.M., Eggleston, P. (Eds.), Insect Molecular Science. Academic Press, London, pp. 205–225. Holman, G.M., Cook, B.J., Nachman, R.J., 1986a. Isolation, primary structure and synthesis of two neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins. Comp. Biochem. Physiol. 84C, 205–211. Holman, G.M., Cook, B.J., Nachman, R.J., 1986b. Primary structure, and synthesis of two additional neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins. Comp. Biochem. Physiol. 84C, 271–276. Holman, G.M., Cook, B.J., Nachman, R.J., 1987a. Isolation, primary structure and synthesis of leucokinins V and VI: myotropic peptides of Leucophaea maderae. Comp. Biochem. Physiol. 88C, 27–30. Holman, G.M., Cook, B.J., Nachman, R.J., 1987b. Isolation, primary structure and synthesis of leucokinins VII and VIII: the final members of this new family of cephalomyotrophic peptides isolated from head extracts of Leucophaea maderae. Comp. Biochem. Physiol. 88C, 31–34. Holman, G.M., Nachman, R.J., Wright, M.S., 1990. Insect peptides. Ann. Rev. Entomol. 35, 201–217. Imai, K., Konno, T., Nagasawa, Y., et al., 1991. Isolation and diapause hormone of the silkworm Bombyx mori. Proc. Jpn. Acad. 67, 98–101. Nachman, R.J., Holman, G.M., 1991. Insect Neuropeptides: Chemistry, Biology and Action, Vol. 453. American Chemical Society, Washington DC, pp. 194–214. Nachman, R.J., Holman, G.M., Haddon, W.F., 1993. Leads for insect neuropeptide mimetic development. Arch. Insect Biochem. Physiol. 22, 181–197.
Nachman, R.J., Giard, W., Favrel, P., Suresh, T., Sreekumar, S., Holman, G.M., 1997a. Insect myosuppressins and sulfakinins stimulate release of the digestive enzyme a— amylase in two invertebrates: the scallop Pecten maximus and insect Rhynchophorus ferrugineus. Ann. N.Y. Acad. Sci. 814, 335–338. Nachman, R.J., Isaac, R.E., Coast, G.M., Holman, G.M., 1997b. Aib-containing analogue of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensin converting enzyme and potent diuretic activity. Peptides 18, 53–57. ¨ Nassel, D.R., 1993. Neuropeptides in the insect brain: a review. Cell Tissue Res. 273, 1–29. Noelting, G., Bernfeld, P., 1948. Surles enzymes amylolytiques-III. La b-amylase: Dosage d’ active et controle de l’ absence d’alpha-amylase. Helv. Chim. Acta 31, 286–290. O’Donnell, M.J., Rheault, M.R., Davies, S.A., et al., 1998. Hormonally-controlled chloride movement across Drosophila tubules is via ion channels in stellate cells. Am. J. Physiol. 43, R1039–R1049. Predel, R., Kellner, R., Nachman, R.J., Holman, G.M., Rapus, ¨ J., Gade, G., 1999. Differential distribution of pyrokininisoforms in cerebral and abdominal neurohaemal organs of the American cockroach. Insect Biochem. Mol. Biol. 29, 139–144. Predel, R., Eckert, M., 2000. Neurosecretion: peptidergic systems in insects. Naturwissenschaften 87, 343–350. Raina, A.K., 1993. Neuroendocrine control of sex pheromone biosynthesis in Lepidoptera. Ann. Rev. Ent. 38, 329–349. Santhosh-Babu, P.S., Prabhu, V.K.K., 1987. How many larval instars are there in Opisina arenosella? Entomon. 12, 39–42. Schoofs, L., Vanden Broeck, J., De Loof, A., 1993. The myotropic peptides of Locusta migratoria: Structure, distribution, functions and receptors. Insect Biochem. Mol. Biol. 23, 859–881. ˇ Sehnal, F., Zitnan, D., 1990. Endocrines of insect gut. In: Epple, A., Scanes, C.G., Stetson, M.H. (Eds.), Progress in Comparative Endocrinology. Wiley–Liss, New York, pp. 510–515. Sunitha, V.B., Reena, T., Harshini, S., Sreekumar, S., 1999. Is gut pH regulated by midgut endocrine system in the larvae of Rhynchophorus ferrugineus (Fab.)? Ind. J. Exp. Biol. 37, 423–426. Zdarek, J., Nachman, R.J., Hayes, T.K., 1998. Selective action and structure–activity relationships of the insect neuropeptides of the pyrokininyPBAN family on pupation in flesh fly (Neobelleria bullata) larvae. Eur. J. Entomol. 95, 9–16.
Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae) S. Harshinia, R.J. Nachmanb, S. Sreekumara,* 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 and B Road, College Station, TX 77845, USA
b
Received 12 September 2001; received in revised form 31 January 2002; accepted 2 February 2002
Abstract Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 8C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 Aw1x WP-2, LPK and 434 wf2x WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (XsAsn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the Cterminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Amylase; Coconut pest; Digestive enzyme release; Leucokinin; Neuropeptide; Opisina arenosella; Pyrokinin; Protease
1. Introduction Neuropeptides are diverse chemical messengers known in the nervous system of metazoans includ¨ ing insects (Nassel, 1993). The large number of neuropeptides structurally identified from different insect groups highlights the complexity of the neurosecretory system in regulating various physiological processes (Predel and Eckert, 2000). In Leucophaea maderae alone, more than 12 neuropeptides are isolated of which, all, except leucomyosuppressin, exert a myotropic action (Holman et al., 1990). This observation suggests that myotrop*Corresponding author. Tel.: q91-471-332-934. E-mail address: sree [email protected] (S. Sreekumar).
ic action may be one of the many biological activities of these peptides. To date, it is not possible to draw any conclusion regarding the major functional role of these peptides as it will require the elucidation of all their diverse activities in different physiological and biochemical systems. The probable role of neuropeptides in digestion and feeding needs to be investigated, for the stomatogastric nervous system, gut endocrine cells and the nerves innervating the gut contain peptiˇ dergic principles (Sehnal and Zitnan, 1990). In the present study, we demonstrate the effect of leucokinins, a family of eight peptides isolated from L. maderae, their related peptide analogues and leucopyrokinin on in vitro release of amylase and protease from the midgut epithelial tissue of
1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 2 . 0 0 0 4 7 - 7
354
S. Harshini et al. / Comparative Biochemistry and Physiology Part B 132 (2002) 353–358
Table 1 Amino acid sequence of neuropeptides used for bioassay Neuropeptide
Amino acid sequence
LPK LK-I LK-II LK-III LK-IV LK-V LK-VI LK-VII LK-VIII FFSWG amide 122Aw1xWP-2 434wf2xWP-1
pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2 Asp-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 Asp-Pro-Gly-Phe-Ser-Ser-Trp-Gly-NH2 Asp-Gln-Gly-Phe-Asn-Ser-Trp-Gly-NH2 Asp-Ala-Ser-Phe-His-Ser-Trp-Gly-NH2 Gly-Ser-Gly-Phe-Ser-Ser-Trp-Gly-NH2 pGlu-Ser-Ser-Phe-His-Ser-Trp-Gly-NH2 Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 Gly-Ala-Ser-Phe-Tyr-Ser-Trp-Gly-NH2 Phe-Phe-Ser-Trp-Gly-NH2 Phe-Phe-Aib-Trp-Gly-NH2 pGlu-Leu-Phe-Phe-Aib-Trp-Gly-NH2
LPK: Leucopyrokinin; LK: Leucokinin. FFSWG amide, 122A (1) WP-2 and 434 (f2) WP-1 are synthetic leucokinin analogues.
the larvae of the lepidopteran moth, Opisina arenosella. 2. Materials and methods Three- to four-day-old final (eighth) instar larvae of the coconut pest Opisina arenosella were used. Insects were reared in the laboratory, following the method of Santhosh-Babu and Prabhu (1987) in a glass jar provided with fresh coconut leaves. 2.1. Preparation of midgut for bioassay The larvae were beheaded and cut posteriorly at approximately 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 Leucokinins (LK I–VIII), the Leucokinin analogues FFSWG amide, 122A w1x WP-2, 434 wf2x WP-1 and Leucopyrokinin (LPK) were used for studying the effect on secretion of amylase and protease in midgut preparations (Table 1). 10y6 M solutions of the
neuropeptides were prepared by dissolving the neuropeptide samples in insect saline (120 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 0.56 mM glucose). For studying the dose responses, midgut preparations were incubated with varying concentrations of neuropeptide (LK III, LK VIII and LPK) 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 8C, bubbling a small stream of oxygen into the solution. The bioassay apparatus is a glass cylinder, (5=1 cm diameter), open above, with a slanting side tube near the bottom (Sunitha et al., 1999). 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 8C. 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 of distilled water for estimating amylase and protease levels. The method of Noelting and Bernfeld (1948) 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 8C. The reaction was terminated by adding 1.2 ml of dinitrosalicylic acid reagent and heating at 100 8C in a water bath for 5 min. The absorbence of the solution was read at 550 nm and quantified as microgram 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. (1962). After 10 min of incubation at 37 8C, the reaction was stopped by adding 1.2-ml 1% trichloroacetic acid. The incubation mixture was centrifuged at 10 000=g at 4 8C for 10 min and the supernatant was read at 280 nm. A standard curve prepared by using varying concentrations of tyrosine, ranging from 0.005 to 0.1%, was used to calculate the
S. Harshini et al. / Comparative Biochemistry and Physiology Part B 132 (2002) 353–358 Table 2 Effect of LPK, leucokinins and LK analogues on secretion of protease and amylase in midgut preparations of larvae of Opisina arenosella Neuropeptides
Protease unitsa
Amylase unitsb
LPK LK I LK II LK III LK IV LK V LK VI LK VII LK VIII FFSWG amide 122A w1x WP-2 434 wf2x WP-1 Control
111.1"16.63 (6)* 155.60"67.2 (5) 96.29"51.85 (5)* 44.40"20.37 (5)** 118.49"27.41 (5)* 251.90"30.37 (5) 207.40"28.15 (5) 170.41"55.22 (12) 453.70"46.27 (5)* 118.50"47.94 (5)* 160.37"22.78 (6) 117.78"95.17 (6)* 212.96"24.50 (8)
2.54"0.72 (6)*** 10.86"4.97 (5) 12.67"4.66 (5) 4.98"4.31 (8)** 18.46"5.45(5) 19.91"5.83 (5) 23.53"3.44 (5) 19.45"3.77 (8) 15.58"3.54 (5) 0 (5) 11.78"3.39 (6)** 14.50"4.13 (6) 18.12"1.06 (8)
Values are mean"S.E.M. of number of observations indicated between parentheses. The significance was analyzed by Student’s t-test. * P-0.05. ** P-0.01. *** P-0.001. a 1 Unitsamount of enzyme required to liberate 1 mg of tyrosine from caseinymin. b 1 Unitsamount of enzyme required to liberate 1 mg of maltose equivalents from starchymin.
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amount of tyrosine liberated in the reaction in micrograms. In control experiments, ligated midgut preparations were incubated in insect saline without neuropeptides. 3. Results 3.1. Digestive enzyme levels in midgut preparations incubated with neuropeptides The midgut preparations incubated with neuropeptides LK III, FFSWG amide, 122Aw1xWP-2, LPK, revealed a decrease in amylase secretion when compared with controls. The protease levels decreased following incubation with FFSWG amide, LK II, LK III, LK IV, LPK and 434 wf2xWP-1. LK VIII caused an increase in the level of protease (Table 2). 3.2. Dose response studies With LK III and LPK digestive enzyme levels in midgut preparations decreased with increasing concentration of neuropeptides. However, a 10y6 M solution of LK VIII stimulated amylase secretion. Figs. 1 and 2 shows the dose response of
Fig. 1. Dose-response of LK III, LK VIII and LPK in protease secretion in midgut preparations of larvae of Opisina arenosella. Results are expressed as mean"S.E.M. (ns6). The significance of data was analyzed by Student’s t-test (*P-0.05, **P-0.01).
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Fig. 2. Effect of varying concentration of LK III, LK VIII and LPK on amylase secretion in midgut preparations of larvae of Opisina arenosella. Results are expressed as mean"S.E.M. (ns6). The significance of data was analyzed by Student’s t-test (**P-0.01, ***P-0.001).
these neuropeptides on protease and amylase secretion. 4. Discussion A large number of neuropeptides specific to insects have been discovered and sequenced. Leucokinins are a group of eight myotropic peptides isolated from the head extracts of L. maderae assayed with a sensitive hind gut preparation (Holman et al., 1986a,b, 1987a,b). In addition to the established myotropic action, these peptides stimulate lipid mobilization and inhibit protein synthesis in L. maderae (Goldsworthy et al., 1992). Leucokinins and their homologues may also function in the control of water and ion balance in numerous insects (Schoofs et al., 1993). In Drosophila melanogaster, leucokinins are reported to increase the intracellular calcium in the stellate cells (O’Donnell et al., 1998). The leucokinins are designated as LK I–VIII, and comprise a group of structurally related peptides with sequence identity at positions 4, 6, 7 and 8. They are also the first to be recognized as a natural
analogue series of isopeptides in one species. In this study, leucokinins, LK analogues and leucopyrokinin have been bioassayed for evaluating their effect on digestive enzyme secretion from the larval midgut of O. arenosella. Of the eight LK peptides investigated, LK III revealed maximum activity as it causes significant decrease in the levels of protease and amylase in the midgut preparations. LK II and LK IV were effective only in inhibiting the release of protease. LK I, LK V, LK VI and LK VII showed no activity. Meanwhile, LK VIII exerted a stimulatory effect by increasing the level of protease. Regarding the structure activity relationships of the leucokinins, our study indicates that the leucokinin structure is recognized by the midgut receptors of O. arenosella. Leucokinins have an identical C-terminal pentapeptide sequence identified as FXSWG amide (Phe-X-Ser-Try-Gly-NH2 ) where ‘X’ is a variable being Asn, His, Ser, or Tyr. This common sequence, represents the active core of the molecule (Nachman and Holman, 1991). LK II, LK III and LK IV, which inhibited the release of digestive enzymes to varying
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degrees, possess at the fifth position the amino acids Ser, Asp and His, respectively. These amino acids can be encountered at the same positions in LK I, LK V, LK VI and LK VII which have no effect on enzyme secretion. These observations suggest that substitution of amino acids at the fifth position does not influence the activity of the peptides. LK II shares with LK-III a similar amino acid sequence except at position 2 where it has proline instead of glutamine. LK II and LK VII have similar amino acid sequence except at the third position where LK II has glycine, and in LK VII it is alanine. This single substitution of alanine in place of glycine in LK VII may be responsible for its loss of inhibitory activity. This is further supported by the fact that the same substitution is present in LK I which reveals no activity in the bioassay. LK IV which has inhibited the release of protease shares with LK II and LK III aspartic acid at position 1 and has serine at position 3 as in leucopyrokinin, another inhibitory peptide. Unlike other peptides in the series, LK VIII stimulated the release of protease. This peptide is unique among leucokinins in having a tyrosine residue in the fifth position and perhaps this is responsible for its stimulatory action in O. arenosella as in L. maderae where it causes increased myotropic activity (Holman et al., 1987b). In the present study, the neuropeptides LK V and LK VI had no activity, on the release of digestive enzymes. These peptides have different sequences of amino acids at the variable part, the N-terminal, which is supposed to modulate the physiological actions (Duve et al., 1994). The synthetic peptide FFSWG amide, with only the common core, is highly effective as it inhibits the release of amylase totally and to a significant extent the release of protease as well. 122Aw1x WP-2 is an analogue of leucokinins, resistant to an endopeptidase known as angiotensin converting enzyme found in vertebrates and insect tissues due to the presence of aminoisobutyric acid (Aib). Aib-containing analogues of the insect kinin neuropeptide family can disrupt the water andyor ion balance in insects (Nachman et al., 1997b). 122Aw1x WP-2 differs from FFSWG amide in lacking serine, a common residue present in the core segment of leucokinins. This peptide is less effective as it inhibits the secretion of amylase only. 434 wf2x WP-1 is another enzyme resistant analogue that mimics the activity of leucokinins. Compared to 122Aw1xWP-2, 434 wf2x WP-1 pos-
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sesses an additional sequence of pGlu-Leu at the N-terminus. It is found to be effective in inhibiting the release of protease. The pyrokinins are a large family of myotropic peptides (Predel et al., 1999) known to regulate diverse processes like pheromone biosynthesis, egg diapause and pupation in different insects (Imai et al., 1991; Raina, 1993; Zdarek et al., 1998). In the present bioassay, leucopyrokinin (LPK) showed an inhibitory effect on the release of digestive enzymes from the midgut tissue of lepidopteran larvae. LPK possesses an N-terminal blocking pyroglutamate and proline at position 6. N-terminal pyroglutamate blocking can influence the activity of peptides. For instance, removal of N-terminal pyroglutamate from LPK leads to an increase in myotropic activity over the natural product in L. maderae (Nachman and Holman, 1991). The existence of proline residue in pyrokinins may also determine the activity, as it may turn the C-terminal part of the molecule (Nachman and Holman, 1991; Nachman et al., 1993). Dose response studies with LK III and LPK showed an increase in inhibitory effect with increasing concentration of the neuropeptides. Among LK peptides, LK III has shown maximum activity followed by LK II and LK IV. However LK III stimulated enzyme secretion at a concentration of 10y8 M, when compared with control, thus indicating differential effects at higher and lower concentrations. It is known that in vertebrates, pancreatic polypeptide 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. Information on active core sequence of neuropeptides can be of great value in developing new strategies in pest management, particularly those involving recombinant DNA technology (Holman et al., 1990). The present study reveals that the minimum requirement for achieving inhibition of digestive enzyme secretion in larvae of O. arenosella may be the C-terminal penta peptide sequence FXSWG amide as exemplified by FFSWG amide. It is recently reported that some of the neuropeptides belonging to the leucosulfakinin family are effective in stimulating digestive enzyme release in the larvae of the red palm weevil Rhynchophorus ferrugineus (Nachman et al., 1997a). This is the first report on insect neuropeptides demonstrat-
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