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A brain peptide stimulates release of amylase from the midgut tissue of larvae of Opisina arenosella Walk. (Lepidoptera: Cryptophasidae)
Neuropeptides Neuropeptides 37 (2003) 133–139 www.elsevier.com/locate/npep
A brain peptide stimulates release of amylase from the midgut tissue of larvae of Opisina arenosella Walk. (Lepidoptera: Cryptophasidae) S. Harshini 1, V. Reshmi, S. Sreekumar
*
Department of Zoology, University College, Trivandrum, Kerala 695 034, India Received 2 January 2003; accepted 25 March 2003
Abstract Brain extracts from 3 to 4 day old final (eighth) instar larvae of Opisina arenosella (Lepidoptera) stimulate amylase release from midgut preparations maintained in vitro. This effect of the brain extract was both time and dose dependent. The brain factor stimulating enzyme release may be a peptide as it is heat stable and susceptible to treatment with proteolytic enzymes. For purification of the brain factor, a head extract prepared in 2% NaCl was first precipitated in 80% aqueous acetone and then fractionated by DEAE cellulose ion exchange chromatography. The fraction OCF2 ; from ion exchange chromatography was further purified on a Sephadex G25 column. The fraction designated as OCF2:3 obtained by gel filtration showed maximum activity and it was selected for HPLC analysis. HPLC elution profiles of OCF2:3 showed two major peaks separated by a time interval of 0.107 min. The two overlapping peaks of OCF2:3 may represent either different forms of a peptide or different peptides of a family. The molecular weight OCF2:3 was estimated to be 1070 Da. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Opisina arenosella; Coconut pest; Neuropeptide; Digestive enzyme release; Amylase; Insect midgut
1. Introduction Immunological studies have revealed the presence of vertebrate gut peptides and hormones in insect nervous system, e.g., gastrin/cholecystokinin-like material in the brain, suboesophageal ganglion, thoracic ganglion and corpus cardiacum (Duve and Thorpe, 1984) and brain of Calliphora erythrocephala (Duve and Thorpe, 1981), insulin, gastrin and pancreatic polypeptide-like immunoreactivity in the brain of Bombyx mori (Yui et al., 1980) and substance P-like peptides in Periplaneta americana brain, retrocerebral complex and subesophageal ganglia (Verhaert and De Loof, 1985). The recent advancement in techniques for purification and sequencing of neuropeptides as well as the introduction of molecular cloning has contributed information relating *
Corresponding author: Tel.: +91-471-2332934. E-mail address: [email protected] (S. Sreekumar). 1 Present address: Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui-230 026, P R China.
to the existence of the neuropeptide isoforms within a given species and the existence of extensive families of related peptides. Some of the insect specific neuropeptides such as leucosulfakinins and leucomyosuppressin that exhibit amino acid sequence homology with vertebrate gastrin/CCK are demonstrated to have effects on digestive system by stimulating either the release of digestive enzymes or secretions for pH buffering (Nachman et al., 1997; Harshini et al., 2002b, Sajjaya et al., 2001). This report details the characterization, isolation and partial purification of a brain peptide causing the release of amylase in the larvae of Opisina arenosella.
2. Materials and methods Three to four day old final (eighth) instar larvae of O. arenosella were used for the present study. The insects were reared in the laboratory following the method of Santhosh-Babu and Prabhu (1987) in a glass jar provided with fresh coconut leaves every day.
0143-4179/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0143-4179(03)00025-8
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2.1. Preparation of brain extract The head capsule of final instar larvae was pinned to a wax bottomed Petri dish and was cut along the middorsal line. The two halves thus separated were pulled apart to expose the brain. The exposed area was washed in insect saline (Harshini et al., 2002a). The brain was carefully pulled out using a pair of fine forceps and transferred to saline. Insect saline containing tissues was boiled for 10 min, to denature the hydrolytic enzymes present in them. It was cooled and homogenized in a glass homogenizer by hand. The homogenate was centrifuged at 10,000g for 10 min at 4 °C. The supernatant obtained was used as incubation solution in the bioassay. The concentration of the extract was adjusted to 2 brains/10 ml saline. 2.2. 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 with 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 and washed in several changes of saline. The two ends of the open midgut tube were ligated with human hair. The ligated midguts were used in the bioassay.
activity was assayed by incubating 0.2 ml lumen contents with 0.4 ml 1% starch and 0.2 ml glycine–NaOH buffer (pH 8.8). After 30 min of incubation at 37 °C, the reaction was stopped by adding 1.2 ml of dinitrosalicylic acid reagent and heated at 100 °C in a water bath for 5 min. The absorbency of the solution was read at 550 nm and quantified as lg maltose equivalents liberated, using a maltose (0.1–1%) standard curve. Amylase activity was represented in units, one unit is the amount of enzyme required to liberate 1 lg of maltose equivalents from starch/min. Differences between control and experimental means were tested for statistical significance by applying StudentÕs ÔtÕ test. 2.5. Dose–response of amylase release by brain extract Extracts of brain with concentrations equivalent to 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 brains/10 ml saline were used for incubating the midgut preparations of O. arenosella larva. After incubation for 30 min the contents of midgut preparations were analyzed for estimating amylase activity. 2.6. Time course of amylase release by brain extract To study the time course effect, the midgut preparations were incubated with brain extract (2 brains/10 ml saline) for 10, 30, and 60 min. Amylase activity of the contents was estimated subsequently. 2.7. Treatment of brain extract with proteolytic enzymes
2.3. Bioassay The brain extract (2 ml) was taken in the bioassay apparatus and the midgut preparation was incubated in it for 30 min bubbling a small stream of oxygen into the solution. The bioassay apparatus is a glass cylinder (51 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 °C. After incubation, the midgut preparation was taken out and washed in insect saline. It was opened and the contents were collected in 0.2 ml distilled water for estimating amylase levels. In control experiments, ligated midgut preparations were incubated in insect saline. 2.4. Estimation of amylase activity For estimating amylase activity, the method of Neolting and Bernfeld (1948) was followed with modifications as employed by Harshini et al. (2002a). Amylase
Pepsin, trypsin and a-chymotrypsin were used for the study. Fifty mg of the enzyme was dissolved in 50 ml distilled water to which was added five drops of 0.1 N HCl. The brain extract was prepared by boiling insect saline containing tissues for 10 min and after homogenization, it was centrifuged at 10,000g for 10 min to obtain a clear solution. The initial concentration of the extract was 2 brains/2.5 ml saline. The brain extract was treated separately with pepsin, trypsin and a-chymotrypsin by adding 0.5 ml of enzyme solution to 2.5 ml of the extract and incubated at 37 °C for 4 h. After incubation, the brain extract was made up to 10 ml with insect saline. The resulting solution was studied for its biological activity. Enzyme solution heated at 100 °C for 10 min was used in control experiments. 2.8. Differential precipitation in aqueous acetone The heads from 200 larvae were dissected and cleaned. They were pooled in 5 ml 2% ice cold NaCl solution and the extract was prepared as described earlier. The crude extract was cooled to 4 °C and ice cold acetone was added to it to obtain 70% aqueous acetone. It was allowed to stand for 90 min at 4 °C. This solution
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was then centrifuged at 10,000g for 20 min at 4 °C. The supernatant was made up to 80% aqueous acetone and after standing at 4 °C for 90 min, the precipitate formed was centrifuged down at 10,000g for 20 min at 4 °C. The precipitate was collected in 2 ml saline and bioassayed for activity in stimulating digestive enzyme secretion.
The Kav values were plotted against their molecular mass to obtain a straight line. The Kav of the active fraction was similarly calculated and its molecular weight was read from the graph.
2.9. Ion exchange chromatography
The active fractions from gel filtration were screened for their activity and the fraction OCF2:3 was subjected to HPLC analysis employing the method of Holman et al. (1987) with certain modifications. The HPLC column and operating conditions are as follows: Shimpack CLC-ODS (M) C18, 4.6 mm 25 cm (Shimadzu C-R7A e plus). Conditions: Solvent A ¼ 0:01 M ammonium acetate buffer (pH 6.1). Solvent B ¼ 25% acetonitrile in water, made to 0.01 M ammonium acetate. Initial conditions: 100% solvent A for 8 min then linear programme to 100% solvent B over 30 min. Flow rate: 1.5 ml/min. Temperature: ambient. Detector range: 2.0 aufs at 214 nm.
The protein precipitate from the head was applied to a 0.3 g DEAE-Cellulose column (10 30 cm i.d) equilibrated with 0.1 M phosphate buffer, pH 7. The proteins were eluted with a total of 200 ml NaCl, increasing molarity step-wise by successive washings with 25 ml each of 0.1, 0.2, 0.4, 0.6, and 0.8 M NaCl, 40 ml 1 M NaCl, and 50 ml of 1.5 M NaCl. Two ml fractions were collected and the absorbency of the solution was measured at 280 nm. The chromatography was carried out at 4 °C. The fractions coming under a peak were pooled. 0.01 ml of this solution was withdrawn and made up to 10 ml. The activity of the solution was evaluated in the bioassay.
2.12. HPLC fractionation
3. Results 2.10. Gel filtration chromatography A Sephadex G-25 column (1.842 cm), equilibrated with 0.01 M ammonium acetate buffer (pH 6.1) containing 0.2 % sodium azide as antibacterial agent was employed to further purify the fractions from the ion exchange columns. The void volume was determined with blue dextran. Following sample application directly on to the surface of the gel column, the first 40 ml of the elutant was discarded and then 2 ml fractions were collected (flow rate 1 ml/2 min) and the elution profile was monitored at 280 nm. 0.01 ml of the sample from the peak fractions was bioassayed for activity. The fractions were designated as Opisina cephalic factors (OCF) and each one was given two numbers denoting the serial number of peaks from ion exchange and gel filtration, respectively. For estimation of proteins in the fractions the method of Lowry et al. (1951) was employed. 0.1% Bovine serum albumin was used as standard.
Incubation of midgut preparations with the extracts of brain (2 brain/10 ml saline) caused an increase in amylase level (29.589.57 units) in the lumen contents
Fig. 1. Dose–response of brain extract on amylase levels in midgut preparations of larvae of O. arenosella. *, significant at 0.05 level; **, significant at 0.01 level.
2.11. Molecular weight determination The molecular weight of amylase secretion releasing factor was determined on Sephadex G-25 column after caliberating it with DOPA, FMRF amide (Harshini et al., 2002b), Leucokinin VII (LK VII) (Harshini et al., 2002a), Leucomyosupressin (LMS) and Leucosulfakinin II (LSK II Ser (SO3 H)) (Harshini et al., 2002b), having molecular weights 195, 670, 810, 1250 and 1550, respectively. The Kav value for each molecular marker was calculated applying the equation Kav ¼ Ve Vo =Vt Vo where ÔVe Õ is the elution volume of the molecular weight marker, ÔVo Õ the void volume and ÔVt Õ the total volume.
Fig. 2. Time course of activity of brain extract on amylase levels in midgut preparations of larvae of O. arenosella. *, significant at 0.05 level; **, significant at 0.01 level.
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Table 1 Digestive enzyme levels in midgut preparations of larvae of O. arenosella incubated with proteolytic enzyme-treated brain extracts Enzyme used for treatment of brain extract
Amylase level Control unitsa
Pepsin Trypsin a-Chymotrypsin
Test unitsa
21.72 5.04 (8) 31.07 11.27(8) 8.56 3.64 (8)
% Reduction
11.16 3.36(6) 11.41 3.01(8) 6.52 3.08(7)
48.39 63.64 25
Values are mean SEM of number of observations indicated between parentheses. a 1 unit ¼ amount of enzyme required to liberate 1 lg of maltose equivalents from starch/min. * Significant at 0.01 level.
when compared with the controls (17.972.52 units) The amylase levels increased with increasing concentrations of the brain extracts, i.e., from 0.5 to 3 brains/10 ml saline (Fig. 1). With the increase in incubation period from 10 min to 60 min, a corresponding increase in activity of amylase was observed (Fig. 2). Treatment of brain extract with proteolytic enzymes caused significant reductions in amylase levels in the bioassay. With pepsin treated brain extract the amylase level was reduced to 48.39%, while the reduction with trypsin treated brain extract was 63.64%. The reduction in amylase level in midgut preparations incubated with a-chymotrypsin treated brain extract was 25% (Table 1). Results of differential precipitation of brain extract showed that precipitates obtained in 80% aqueous acetone had maximum activity (48.91 units), in stimulating
amylase secretion, followed by 70% aqueous acetone precipitates (32.61 units). DEAE cellulose chromatography with head extract yielded four peaks exhibiting digestive enzyme secretion stimulating activity. The active fractions were designated as OCF1 , OCF2 , OCF3 and OCF4 (Fig. 3). Of these, OCF2 showed high activity in the bioassay. Gel filtration of OCF2 yield five peaks having activity (Fig. 4). They are referred to as OCF2:1 , OCF2:2 , OCF2:3 , OCF2:4 , and OCF2:5 . The head fraction OCF2:3 revealed a 70-fold increase in specific activity over the ion exchange fraction. The total activity and specific activity of the fractions at different steps of purification are given in Table 2. The HPLC elution profile of OCF2:3 showed two major peaks, eluting at 1.97 and 2.077 min (Fig. 5). The molecular weight of OCF2:3 was found from the graph plotting Kav of molecular weight markers against molecular mass (Fig. 6). The Kav of OCF2:3 was found to be 0.4118 and hence its molecular weight may be read as 1070.
4. Discussion
Fig. 3. DEAE cellulose chromatography: elution profile of precipitate obtained by treating head extract in 80% aqueous acetone.
Fig. 4. Gel filtration profile of the fraction, OCF2 , fractionated from DEAE cellulose column.
Neurosecretions from brain are held to be involved in the regulation of digestive enzyme secretion in insects (Prabhu and Sreekumar, 1994). A decline in digestive enzyme levels is often observed if the midgut tissue is not accessible to brain hormone following treatments such as decapitation, ligation of head, cauterization or extirpation of neurosecretory cells. These methods however have provided only inconclusive results on neurohormonal regulation of digestion in insects, as the effects can be indirect. For example, Muraleedharan and Prabhu (1979) have reported that extirpation of neurosecretory cells leads to a decline in secretion of protease and amylase in Dysdercus cingulatus. However, they have attributed this effect to reduced intake of food. The present in vitro study rules out such indirect effects and involvement of neuronal and endocrine influences from other centres. It has been shown in bioassay that incubation of empty midgut tubes of larvae of O. arenosella with brain extracts causes an increase in activity of amylase in lumen contents. This effect is, presumably,
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Table 2 Summary of results of purification of head factor stimulating amylase release in the larvae of O. arenosella Step
Acetone precipitation Ion-exchange chromatography Gel filtration chromatography
Fraction
80% OCF2 OCF2:3
Peak fraction number
21 38
Amylase activity in bioassay Total activitya
Specific activityb
48.91 5.435 16.30
4810 4756 345,561
Each value is mean of two observations. One unit ¼ Amount of enzyme required to liberate 1 lg of maltose equivalents from starch/min. b Total activity/mg protein. a
Fig. 5. HPLC elution profile of the fraction OCF2:3: .
due to the release of amylase from the midgut tissue into the lumen. The stimulatory effect of brain extract on midgut tissue is found to be both dose and time dependent. An earlier study shows that in the larvae of Oryctes rhinoceros, a midgut peptide, probably hormone, has a similar action (Sreekumar and Prabhu, 1988).
Fig. 6. Kav of molecular weight markers plotted against molecular mass. LK VII: Leucokinin VII, LMS: Leucomyosuppressin, LSKIISer(SO3 H): Leucosulfakinin II.
The present study reveals that the treatment of brain extracts of larvae of O. arenosella with pepsin and trypsin reduces the amylase release stimulating activity by about 48% and 63%, respectively. Instability to treatment with proteolytic enzymes is a characteristic of proteinaceous substance (Frontali and Gainer, 1977). The prothoracicotrophic hormone from B. mori is inactivated on treatment with pepsin, trypsin and a-chymotrypsin, while exopeptidases like aminopeptidases and carboxypeptidases are found to be without effect (Ishizaki and Suzuki, 1980). The diapause hormone in B. mori (Isobe and Goto, 1980) and the diuretic hormone in Rhodnius prolixus (Aston and Hughes, 1980) could similarly be inactivated by trypsin and a-chymotrypsin, but not by exopeptidases. These differences in inactivation properties of various enzymes can be attributed to their specificity towards bonds constituted by specific amino acids in the substrate. It is also accepted that peptides are stable to boiling at neutral pH (Leeman et al., 1977). In the present study, the head extracts of larvae of O. arenosella have been prepared in boiling 2% NaCl solution for the purification of active factors. This method is followed to cause inactivation of proteolytic enzymes present in the epithelial extract and precipitation of structural proteins of tissues. The same method has been followed by Ishizaki and Suzuki (1980)
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for the extraction of prothoracicotropic hormone from B. mori. In this study, acetone is subsequently used to precipitate the amylase releasing factor from the crude head extract. It is found that the fraction obtained in 80% aqueous acetone retains maximum activity when compared with other fractions precipitated at 70% and 90% aqueous acetone. Some of the vertebrate-like gut peptides isolated and purified from nervous tissues include insulin-like material in Calliphora vomitoria (Duve et al., 1979) and gastrin-like peptide in Manduca sexta (Kramer et al., 1977) and C. vomitoria (Dockray et al., 1981). In the present study the amylase release stimulating factor from head tissue of larvae of O. arenosella has been partially purified employing DEAE cellulose ion exchange chromatography, get filtration on Sephadex G-25 column and HPLC. Ion exchange chromatography has yielded four fractions from head extract. Further purification of the fraction called OCF2 from ion exchange chromatography, on Sephadex G-25 column has revealed five active fractions. Of these the fraction OCF2:3 has shown considerable activity in the bioassay by stimulating amylase release. Hence, the fraction OCF2:3 was subjected to HPLC analysis. The HPLC elution profile of OCF2:3 shows two major, PK No. 3 and PK No.4 separated by a time interval of 0.107 min. In another study it is observed that HPLC analysis of two midgut epithelial fractions called OMF1:1 and OMF3:1 which stimulate enzyme release in this insect, have similar elution profiles with two peaks separated by a time interval of 0.103–0.108 min (Harshini and Sreekumar, 2001). It is known that neuropeptides are produced in an inactive form and they are subsequently modified post-transcriptionally to the active form. Some of the post-transcriptional modifications include amidation at C-terminal and sulfation of tyrosine as in vertebrate gastrin/CCK peptides and insect sulfakinins (Nachman et al., 1997). It appears that the two major peaks of OCF2:3 represent the different forms of a same peptide. Alternatively, they may be two different peptides, possibly of the same family, that overlap under the chromatographic conditions employed. For example, eight octaptides designated as leucokinins (LK I–VIII) have been isolated from the head of Leucophaea maderae. They are recognized to be a natural analogue series of isopeptides (Holman et al., 1987). The molecular weight of OCF2:3 has been estimated from the Kav graph by running molecular weight markers on Sephadex G-25 column. It is found that this head factor has a molecular weight of 1070. It is note worthy that the digestive enzyme release stimulating peptide OMF3:1 isolated from the midgut tissue (Harshini and Sreekumar, 2001) has striking similarity with OCF2:3 with respect to gel filtration (Sephadex G-25) and HPLC elution profile and Kav value indicating them to be the same peptide. The present study thus indicates that the
factor stimulating amylase release may have a dual distribution in both the brain and the midgut. It is generally accepted that in vertebrates many of the biologically active peptides are common to the nerve cells in the brain and endocrine cells in the gut (Dockray, 1984). This appears to be the case in insects as well. This view is supported by the observations in P. americana which contains dual distribution of vertebrate like gut hormones and neuropeptides in the central nervous system and midgut tissue (Schols et al., 1987). It is worth mentioning in this context that several peptides may exhibit the same function. In some instance the same peptide may have different sites of action, an example being procolin which can stimulate contractions of both hindgut musculature and heart (Mordue and Morgan, 1985). These observations suggest that multiple sites and actions are of common occurrence with most of the gutneuropeptides in both vertebrates and invertebrates. Any conclusion regarding the major functional role of these peptides is possible only after elucidation of all their diverse activities in different physiological and biological systems as suggested by Holman et al. (1987).
Acknowledgements We are grateful to Dr. R.J. Nachman, USDA, USA for providing neuropeptide samples and Dr. M. Anpu, Scientist, Regional Research Laboratory, Thiruvananthapuram, for his help in HPLC.
References Aston, R.J., Hughes, L., 1980. Diuretic hormone – extraction and chemical properties. In: Miller, T.A. (Ed.), Neurohormonal Techniques in Insects. Springer, New York, Heidelberg, Berlin, pp. 91–115. Dockray, G.J., 1984. The chemistry of neuropeptides. Frontiers of Hormonal Research 12, 8–15. Dockray, G.J., Duve, H., Thorpe, A., 1981. Immunocytochemical characterization of gastrin/cholecystokinin-like peptides in the brain of the blow fly Calliphora vomitoria. General and Comparative Endocrinology 45, 491–496. Duve, H., Thorpe, A., 1981. Gastrin/cholecystokinin-like immunoreactivity neurons in the brain of the blow fly Calliphora erythrocephala (Diptera). General and Comparative Endocrinology 43, 381–391. Duve, H., Thorpe, A., 1984. Immunocytochemical mapping of gastrin/ cholecystokinin-like peptides in the neuroendocrine system of the blow fly Calliphora erythrocephala (Diptera). Cell Tissue Research 237, 309–320. Duve, H., Thorpe, A., Lazarus, N.R., 1979. Isolation of material displaying insulin-like immunological and biological activity from the brain of the blow fly, Calliphora vomitoria. Biochemical Journal 184, 221–227. Frontali, N., Gainer, H., 1977. Peptides in invertebrate nervous system. In: Gainer, H. (Ed.), Peptides in Neurobiology. Plenum Press, New York, London, pp. 259–293.
S. Harshini et al. / Neuropeptides 37 (2003) 133–139 Harshini, S., Nachman, R.J., Sreekumar, S., 2002a. Inhibition of digestive enzyme release by neuropeptides in the larvae of Opisina arenosella (Lepidoptera: Cryptophasidae). Comparative Biochemistry Physiology B 132/2, 353–358. Harshini, S., Nachman, R.J., Sreekumar, S., 2002b. In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.). Peptides 23/10, 1759–1763. Harshini, S., Sreekumar, S., 2001. Isolation and partial purification of the digestive enzyme-release peptide hormone from the midgut of larvae of Opisina arenosella Walk.(Lepidoptera: Cryptophasidae. In: Muraleedharan, K. et al. (Eds.), Advances in Entomology. Association for Advancement of Entomology, Trivandrum, India, pp. 191–197. Holman, G.M., Cook, B.J., Nachman, R.J., 1987. 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. Comparative Biochemistry Physiology C 88, 31–34. Ishizaki, H., Suzuki, A., 1980. Prothoracicotrophic hormone. In: Miller, T.A. (Ed.), Neurohormonal Techniques in Insects. Springer, New York, Heidelberg, Berlin, pp. 24–76. Isobe, M., Goto, T., 1980. Diapause hormone. In: Miller, T.A. (Ed.), Neurohormonal Techniques in Insects. Springer, New York, Heidelberg, Berlin, pp. 216–243. Kramer, K.J., Speirs, R.D., Childs, C.N., 1977. Immunological evidence for a gastrin-like peptide in insect neuroendocrine system. General and Comparative Endocrinology 32, 423–426. Leeman, S.E., Mroz, E.A., Carrawaj, R.E., 1977. Substance P and neurotensin in peptides. In: Gainer, H. (Ed.), Neurobiology. Plenum Press, New York, London, pp. 99–144. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with folin–phenol reagent. Journal of Biological Chemistry 193, 265–275. Mordue, W., Morgan, P.J., 1985. Chemistry of peptides. In: Kerkurt, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 4. Pergamon Press, Oxford, New York, Toronto, Sydney, Paris, Frankfurt, pp. 153– 183. Muraleedharan, D., Prabhu, V.K.K., 1979. Role of the median neurosecretory cells in secretion of protease and invertase in the red
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A brain peptide stimulates release of amylase from the midgut tissue of larvae of Opisina arenosella Walk. (Lepidoptera: Cryptophasidae) S. Harshini 1, V. Reshmi, S. Sreekumar
*
Department of Zoology, University College, Trivandrum, Kerala 695 034, India Received 2 January 2003; accepted 25 March 2003
Abstract Brain extracts from 3 to 4 day old final (eighth) instar larvae of Opisina arenosella (Lepidoptera) stimulate amylase release from midgut preparations maintained in vitro. This effect of the brain extract was both time and dose dependent. The brain factor stimulating enzyme release may be a peptide as it is heat stable and susceptible to treatment with proteolytic enzymes. For purification of the brain factor, a head extract prepared in 2% NaCl was first precipitated in 80% aqueous acetone and then fractionated by DEAE cellulose ion exchange chromatography. The fraction OCF2 ; from ion exchange chromatography was further purified on a Sephadex G25 column. The fraction designated as OCF2:3 obtained by gel filtration showed maximum activity and it was selected for HPLC analysis. HPLC elution profiles of OCF2:3 showed two major peaks separated by a time interval of 0.107 min. The two overlapping peaks of OCF2:3 may represent either different forms of a peptide or different peptides of a family. The molecular weight OCF2:3 was estimated to be 1070 Da. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Opisina arenosella; Coconut pest; Neuropeptide; Digestive enzyme release; Amylase; Insect midgut
1. Introduction Immunological studies have revealed the presence of vertebrate gut peptides and hormones in insect nervous system, e.g., gastrin/cholecystokinin-like material in the brain, suboesophageal ganglion, thoracic ganglion and corpus cardiacum (Duve and Thorpe, 1984) and brain of Calliphora erythrocephala (Duve and Thorpe, 1981), insulin, gastrin and pancreatic polypeptide-like immunoreactivity in the brain of Bombyx mori (Yui et al., 1980) and substance P-like peptides in Periplaneta americana brain, retrocerebral complex and subesophageal ganglia (Verhaert and De Loof, 1985). The recent advancement in techniques for purification and sequencing of neuropeptides as well as the introduction of molecular cloning has contributed information relating *
Corresponding author: Tel.: +91-471-2332934. E-mail address: [email protected] (S. Sreekumar). 1 Present address: Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui-230 026, P R China.
to the existence of the neuropeptide isoforms within a given species and the existence of extensive families of related peptides. Some of the insect specific neuropeptides such as leucosulfakinins and leucomyosuppressin that exhibit amino acid sequence homology with vertebrate gastrin/CCK are demonstrated to have effects on digestive system by stimulating either the release of digestive enzymes or secretions for pH buffering (Nachman et al., 1997; Harshini et al., 2002b, Sajjaya et al., 2001). This report details the characterization, isolation and partial purification of a brain peptide causing the release of amylase in the larvae of Opisina arenosella.
2. Materials and methods Three to four day old final (eighth) instar larvae of O. arenosella were used for the present study. The insects were reared in the laboratory following the method of Santhosh-Babu and Prabhu (1987) in a glass jar provided with fresh coconut leaves every day.
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2.1. Preparation of brain extract The head capsule of final instar larvae was pinned to a wax bottomed Petri dish and was cut along the middorsal line. The two halves thus separated were pulled apart to expose the brain. The exposed area was washed in insect saline (Harshini et al., 2002a). The brain was carefully pulled out using a pair of fine forceps and transferred to saline. Insect saline containing tissues was boiled for 10 min, to denature the hydrolytic enzymes present in them. It was cooled and homogenized in a glass homogenizer by hand. The homogenate was centrifuged at 10,000g for 10 min at 4 °C. The supernatant obtained was used as incubation solution in the bioassay. The concentration of the extract was adjusted to 2 brains/10 ml saline. 2.2. 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 with 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 and washed in several changes of saline. The two ends of the open midgut tube were ligated with human hair. The ligated midguts were used in the bioassay.
activity was assayed by incubating 0.2 ml lumen contents with 0.4 ml 1% starch and 0.2 ml glycine–NaOH buffer (pH 8.8). After 30 min of incubation at 37 °C, the reaction was stopped by adding 1.2 ml of dinitrosalicylic acid reagent and heated at 100 °C in a water bath for 5 min. The absorbency of the solution was read at 550 nm and quantified as lg maltose equivalents liberated, using a maltose (0.1–1%) standard curve. Amylase activity was represented in units, one unit is the amount of enzyme required to liberate 1 lg of maltose equivalents from starch/min. Differences between control and experimental means were tested for statistical significance by applying StudentÕs ÔtÕ test. 2.5. Dose–response of amylase release by brain extract Extracts of brain with concentrations equivalent to 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 brains/10 ml saline were used for incubating the midgut preparations of O. arenosella larva. After incubation for 30 min the contents of midgut preparations were analyzed for estimating amylase activity. 2.6. Time course of amylase release by brain extract To study the time course effect, the midgut preparations were incubated with brain extract (2 brains/10 ml saline) for 10, 30, and 60 min. Amylase activity of the contents was estimated subsequently. 2.7. Treatment of brain extract with proteolytic enzymes
2.3. Bioassay The brain extract (2 ml) was taken in the bioassay apparatus and the midgut preparation was incubated in it for 30 min bubbling a small stream of oxygen into the solution. The bioassay apparatus is a glass cylinder (51 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 °C. After incubation, the midgut preparation was taken out and washed in insect saline. It was opened and the contents were collected in 0.2 ml distilled water for estimating amylase levels. In control experiments, ligated midgut preparations were incubated in insect saline. 2.4. Estimation of amylase activity For estimating amylase activity, the method of Neolting and Bernfeld (1948) was followed with modifications as employed by Harshini et al. (2002a). Amylase
Pepsin, trypsin and a-chymotrypsin were used for the study. Fifty mg of the enzyme was dissolved in 50 ml distilled water to which was added five drops of 0.1 N HCl. The brain extract was prepared by boiling insect saline containing tissues for 10 min and after homogenization, it was centrifuged at 10,000g for 10 min to obtain a clear solution. The initial concentration of the extract was 2 brains/2.5 ml saline. The brain extract was treated separately with pepsin, trypsin and a-chymotrypsin by adding 0.5 ml of enzyme solution to 2.5 ml of the extract and incubated at 37 °C for 4 h. After incubation, the brain extract was made up to 10 ml with insect saline. The resulting solution was studied for its biological activity. Enzyme solution heated at 100 °C for 10 min was used in control experiments. 2.8. Differential precipitation in aqueous acetone The heads from 200 larvae were dissected and cleaned. They were pooled in 5 ml 2% ice cold NaCl solution and the extract was prepared as described earlier. The crude extract was cooled to 4 °C and ice cold acetone was added to it to obtain 70% aqueous acetone. It was allowed to stand for 90 min at 4 °C. This solution
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was then centrifuged at 10,000g for 20 min at 4 °C. The supernatant was made up to 80% aqueous acetone and after standing at 4 °C for 90 min, the precipitate formed was centrifuged down at 10,000g for 20 min at 4 °C. The precipitate was collected in 2 ml saline and bioassayed for activity in stimulating digestive enzyme secretion.
The Kav values were plotted against their molecular mass to obtain a straight line. The Kav of the active fraction was similarly calculated and its molecular weight was read from the graph.
2.9. Ion exchange chromatography
The active fractions from gel filtration were screened for their activity and the fraction OCF2:3 was subjected to HPLC analysis employing the method of Holman et al. (1987) with certain modifications. The HPLC column and operating conditions are as follows: Shimpack CLC-ODS (M) C18, 4.6 mm 25 cm (Shimadzu C-R7A e plus). Conditions: Solvent A ¼ 0:01 M ammonium acetate buffer (pH 6.1). Solvent B ¼ 25% acetonitrile in water, made to 0.01 M ammonium acetate. Initial conditions: 100% solvent A for 8 min then linear programme to 100% solvent B over 30 min. Flow rate: 1.5 ml/min. Temperature: ambient. Detector range: 2.0 aufs at 214 nm.
The protein precipitate from the head was applied to a 0.3 g DEAE-Cellulose column (10 30 cm i.d) equilibrated with 0.1 M phosphate buffer, pH 7. The proteins were eluted with a total of 200 ml NaCl, increasing molarity step-wise by successive washings with 25 ml each of 0.1, 0.2, 0.4, 0.6, and 0.8 M NaCl, 40 ml 1 M NaCl, and 50 ml of 1.5 M NaCl. Two ml fractions were collected and the absorbency of the solution was measured at 280 nm. The chromatography was carried out at 4 °C. The fractions coming under a peak were pooled. 0.01 ml of this solution was withdrawn and made up to 10 ml. The activity of the solution was evaluated in the bioassay.
2.12. HPLC fractionation
3. Results 2.10. Gel filtration chromatography A Sephadex G-25 column (1.842 cm), equilibrated with 0.01 M ammonium acetate buffer (pH 6.1) containing 0.2 % sodium azide as antibacterial agent was employed to further purify the fractions from the ion exchange columns. The void volume was determined with blue dextran. Following sample application directly on to the surface of the gel column, the first 40 ml of the elutant was discarded and then 2 ml fractions were collected (flow rate 1 ml/2 min) and the elution profile was monitored at 280 nm. 0.01 ml of the sample from the peak fractions was bioassayed for activity. The fractions were designated as Opisina cephalic factors (OCF) and each one was given two numbers denoting the serial number of peaks from ion exchange and gel filtration, respectively. For estimation of proteins in the fractions the method of Lowry et al. (1951) was employed. 0.1% Bovine serum albumin was used as standard.
Incubation of midgut preparations with the extracts of brain (2 brain/10 ml saline) caused an increase in amylase level (29.589.57 units) in the lumen contents
Fig. 1. Dose–response of brain extract on amylase levels in midgut preparations of larvae of O. arenosella. *, significant at 0.05 level; **, significant at 0.01 level.
2.11. Molecular weight determination The molecular weight of amylase secretion releasing factor was determined on Sephadex G-25 column after caliberating it with DOPA, FMRF amide (Harshini et al., 2002b), Leucokinin VII (LK VII) (Harshini et al., 2002a), Leucomyosupressin (LMS) and Leucosulfakinin II (LSK II Ser (SO3 H)) (Harshini et al., 2002b), having molecular weights 195, 670, 810, 1250 and 1550, respectively. The Kav value for each molecular marker was calculated applying the equation Kav ¼ Ve Vo =Vt Vo where ÔVe Õ is the elution volume of the molecular weight marker, ÔVo Õ the void volume and ÔVt Õ the total volume.
Fig. 2. Time course of activity of brain extract on amylase levels in midgut preparations of larvae of O. arenosella. *, significant at 0.05 level; **, significant at 0.01 level.
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Table 1 Digestive enzyme levels in midgut preparations of larvae of O. arenosella incubated with proteolytic enzyme-treated brain extracts Enzyme used for treatment of brain extract
Amylase level Control unitsa
Pepsin Trypsin a-Chymotrypsin
Test unitsa
21.72 5.04 (8) 31.07 11.27(8) 8.56 3.64 (8)
% Reduction
11.16 3.36(6) 11.41 3.01(8) 6.52 3.08(7)
48.39 63.64 25
Values are mean SEM of number of observations indicated between parentheses. a 1 unit ¼ amount of enzyme required to liberate 1 lg of maltose equivalents from starch/min. * Significant at 0.01 level.
when compared with the controls (17.972.52 units) The amylase levels increased with increasing concentrations of the brain extracts, i.e., from 0.5 to 3 brains/10 ml saline (Fig. 1). With the increase in incubation period from 10 min to 60 min, a corresponding increase in activity of amylase was observed (Fig. 2). Treatment of brain extract with proteolytic enzymes caused significant reductions in amylase levels in the bioassay. With pepsin treated brain extract the amylase level was reduced to 48.39%, while the reduction with trypsin treated brain extract was 63.64%. The reduction in amylase level in midgut preparations incubated with a-chymotrypsin treated brain extract was 25% (Table 1). Results of differential precipitation of brain extract showed that precipitates obtained in 80% aqueous acetone had maximum activity (48.91 units), in stimulating
amylase secretion, followed by 70% aqueous acetone precipitates (32.61 units). DEAE cellulose chromatography with head extract yielded four peaks exhibiting digestive enzyme secretion stimulating activity. The active fractions were designated as OCF1 , OCF2 , OCF3 and OCF4 (Fig. 3). Of these, OCF2 showed high activity in the bioassay. Gel filtration of OCF2 yield five peaks having activity (Fig. 4). They are referred to as OCF2:1 , OCF2:2 , OCF2:3 , OCF2:4 , and OCF2:5 . The head fraction OCF2:3 revealed a 70-fold increase in specific activity over the ion exchange fraction. The total activity and specific activity of the fractions at different steps of purification are given in Table 2. The HPLC elution profile of OCF2:3 showed two major peaks, eluting at 1.97 and 2.077 min (Fig. 5). The molecular weight of OCF2:3 was found from the graph plotting Kav of molecular weight markers against molecular mass (Fig. 6). The Kav of OCF2:3 was found to be 0.4118 and hence its molecular weight may be read as 1070.
4. Discussion
Fig. 3. DEAE cellulose chromatography: elution profile of precipitate obtained by treating head extract in 80% aqueous acetone.
Fig. 4. Gel filtration profile of the fraction, OCF2 , fractionated from DEAE cellulose column.
Neurosecretions from brain are held to be involved in the regulation of digestive enzyme secretion in insects (Prabhu and Sreekumar, 1994). A decline in digestive enzyme levels is often observed if the midgut tissue is not accessible to brain hormone following treatments such as decapitation, ligation of head, cauterization or extirpation of neurosecretory cells. These methods however have provided only inconclusive results on neurohormonal regulation of digestion in insects, as the effects can be indirect. For example, Muraleedharan and Prabhu (1979) have reported that extirpation of neurosecretory cells leads to a decline in secretion of protease and amylase in Dysdercus cingulatus. However, they have attributed this effect to reduced intake of food. The present in vitro study rules out such indirect effects and involvement of neuronal and endocrine influences from other centres. It has been shown in bioassay that incubation of empty midgut tubes of larvae of O. arenosella with brain extracts causes an increase in activity of amylase in lumen contents. This effect is, presumably,
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Table 2 Summary of results of purification of head factor stimulating amylase release in the larvae of O. arenosella Step
Acetone precipitation Ion-exchange chromatography Gel filtration chromatography
Fraction
80% OCF2 OCF2:3
Peak fraction number
21 38
Amylase activity in bioassay Total activitya
Specific activityb
48.91 5.435 16.30
4810 4756 345,561
Each value is mean of two observations. One unit ¼ Amount of enzyme required to liberate 1 lg of maltose equivalents from starch/min. b Total activity/mg protein. a
Fig. 5. HPLC elution profile of the fraction OCF2:3: .
due to the release of amylase from the midgut tissue into the lumen. The stimulatory effect of brain extract on midgut tissue is found to be both dose and time dependent. An earlier study shows that in the larvae of Oryctes rhinoceros, a midgut peptide, probably hormone, has a similar action (Sreekumar and Prabhu, 1988).
Fig. 6. Kav of molecular weight markers plotted against molecular mass. LK VII: Leucokinin VII, LMS: Leucomyosuppressin, LSKIISer(SO3 H): Leucosulfakinin II.
The present study reveals that the treatment of brain extracts of larvae of O. arenosella with pepsin and trypsin reduces the amylase release stimulating activity by about 48% and 63%, respectively. Instability to treatment with proteolytic enzymes is a characteristic of proteinaceous substance (Frontali and Gainer, 1977). The prothoracicotrophic hormone from B. mori is inactivated on treatment with pepsin, trypsin and a-chymotrypsin, while exopeptidases like aminopeptidases and carboxypeptidases are found to be without effect (Ishizaki and Suzuki, 1980). The diapause hormone in B. mori (Isobe and Goto, 1980) and the diuretic hormone in Rhodnius prolixus (Aston and Hughes, 1980) could similarly be inactivated by trypsin and a-chymotrypsin, but not by exopeptidases. These differences in inactivation properties of various enzymes can be attributed to their specificity towards bonds constituted by specific amino acids in the substrate. It is also accepted that peptides are stable to boiling at neutral pH (Leeman et al., 1977). In the present study, the head extracts of larvae of O. arenosella have been prepared in boiling 2% NaCl solution for the purification of active factors. This method is followed to cause inactivation of proteolytic enzymes present in the epithelial extract and precipitation of structural proteins of tissues. The same method has been followed by Ishizaki and Suzuki (1980)
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for the extraction of prothoracicotropic hormone from B. mori. In this study, acetone is subsequently used to precipitate the amylase releasing factor from the crude head extract. It is found that the fraction obtained in 80% aqueous acetone retains maximum activity when compared with other fractions precipitated at 70% and 90% aqueous acetone. Some of the vertebrate-like gut peptides isolated and purified from nervous tissues include insulin-like material in Calliphora vomitoria (Duve et al., 1979) and gastrin-like peptide in Manduca sexta (Kramer et al., 1977) and C. vomitoria (Dockray et al., 1981). In the present study the amylase release stimulating factor from head tissue of larvae of O. arenosella has been partially purified employing DEAE cellulose ion exchange chromatography, get filtration on Sephadex G-25 column and HPLC. Ion exchange chromatography has yielded four fractions from head extract. Further purification of the fraction called OCF2 from ion exchange chromatography, on Sephadex G-25 column has revealed five active fractions. Of these the fraction OCF2:3 has shown considerable activity in the bioassay by stimulating amylase release. Hence, the fraction OCF2:3 was subjected to HPLC analysis. The HPLC elution profile of OCF2:3 shows two major, PK No. 3 and PK No.4 separated by a time interval of 0.107 min. In another study it is observed that HPLC analysis of two midgut epithelial fractions called OMF1:1 and OMF3:1 which stimulate enzyme release in this insect, have similar elution profiles with two peaks separated by a time interval of 0.103–0.108 min (Harshini and Sreekumar, 2001). It is known that neuropeptides are produced in an inactive form and they are subsequently modified post-transcriptionally to the active form. Some of the post-transcriptional modifications include amidation at C-terminal and sulfation of tyrosine as in vertebrate gastrin/CCK peptides and insect sulfakinins (Nachman et al., 1997). It appears that the two major peaks of OCF2:3 represent the different forms of a same peptide. Alternatively, they may be two different peptides, possibly of the same family, that overlap under the chromatographic conditions employed. For example, eight octaptides designated as leucokinins (LK I–VIII) have been isolated from the head of Leucophaea maderae. They are recognized to be a natural analogue series of isopeptides (Holman et al., 1987). The molecular weight of OCF2:3 has been estimated from the Kav graph by running molecular weight markers on Sephadex G-25 column. It is found that this head factor has a molecular weight of 1070. It is note worthy that the digestive enzyme release stimulating peptide OMF3:1 isolated from the midgut tissue (Harshini and Sreekumar, 2001) has striking similarity with OCF2:3 with respect to gel filtration (Sephadex G-25) and HPLC elution profile and Kav value indicating them to be the same peptide. The present study thus indicates that the
factor stimulating amylase release may have a dual distribution in both the brain and the midgut. It is generally accepted that in vertebrates many of the biologically active peptides are common to the nerve cells in the brain and endocrine cells in the gut (Dockray, 1984). This appears to be the case in insects as well. This view is supported by the observations in P. americana which contains dual distribution of vertebrate like gut hormones and neuropeptides in the central nervous system and midgut tissue (Schols et al., 1987). It is worth mentioning in this context that several peptides may exhibit the same function. In some instance the same peptide may have different sites of action, an example being procolin which can stimulate contractions of both hindgut musculature and heart (Mordue and Morgan, 1985). These observations suggest that multiple sites and actions are of common occurrence with most of the gutneuropeptides in both vertebrates and invertebrates. Any conclusion regarding the major functional role of these peptides is possible only after elucidation of all their diverse activities in different physiological and biological systems as suggested by Holman et al. (1987).
Acknowledgements We are grateful to Dr. R.J. Nachman, USDA, USA for providing neuropeptide samples and Dr. M. Anpu, Scientist, Regional Research Laboratory, Thiruvananthapuram, for his help in HPLC.
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