Isolation and immunochemical characterization of ...

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetle Leptinotarsa decemlineata

D MURALEEDHARAN* and D SHEELA DEVI Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, India

*Corresponding author (Fax, 91-471-447158; Email, dmur@ md3. vsnl.net.in ).

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC- 18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug, Dysdercus cingulatus and the castor semilooper, Achaea janata as well.

1. Introduction

Neuropeptides typically signify small signalling molecules of neuronal origin several of which have been localized even at extraneural sites in many insect species in recent years. During the last decade both general and specific roles of neuropeptides in the neural control of physiology, behaviour and development of insects at the molecular level have been elucidated in detail. Evident features of co-synthesis and co-release of several neuropeptides suggest their shared functions and many different neuropeptides are often known to be derived from the same precursor (Sossin et al 1989; O'Brien and Tagert 1994). FMRF-amide, the tetrapeptide (Phe-Met-Arg-Phe-amide) having sequence homology to vertebrate opioid and corticotropin releasing factors have been immunochemically localized in the midgut endocrine cells of several representative insect Orders by Zitnan etal (1993). Scattered information on the distribution of neuropeptides in insect brain-enterogastric system have been reviewed recently (Zitnan etal 1993; Nassel etal 1994; Muraleedharan 1995). Insect midgut has been implicated to be the source of several myotropic neuropeptides including FMRF-amide and AKH some of which have been isolated and characterized (Jenkins et al 1989; Yi etal 1992, 1995; Schoofs etal 1993;

Muraleedharan et al 1994). Midgut endocrine cells show immunoreactivity to the neuropeptide allatostatin in cockroach midgut (Reichwald etal 1994) and these allatostatins are implicated to control midgut enzyme activity (Veenstra et al 1997).

We report here our findings relating to the immunohistochemical localization of MAC-18 and MAC-3 (monoclonals to cephalic neurosecretory cell antigens of Colorado beetles) positive midgut endocrine cells. We also tried to isolate, and partially characterize the midgut neuroendocrine factors. Functional role of the identified peptides was also assessed, since Luffy and Dorn (1992) suggested that gut endocrine cells deliver hormonal input in addition to nervous input from the stomatogastric nervous system.

2. Materials and methods

2.1 Animals

Colorado potato beetle Leptinotarsa decemlineata Say were mass reared in the laboratory under long day conditions on fresh potato leaves and from this 20 day-old reproductive adult beetles were used for the present investigations. The other two species of insects used for testing immunoreactivity of isolated peptides were 3 day- old adult red cotton bug, Dysdercus cingulatus Fabr. and

Keywords. Leptinotarsa decemlineata; midgut; Dysdercus cingulatus; AKH; FMRF-amide; MAC; impedance converter; Achaeajanata

J. Biol., 24, No. 2, June 1999, pp 185-191. © Indian Academy of Sciences 185

186 D Muraleedharan and D Sheela Devi

1 day-old final instar castor semilooper, Achaea janata Linn, both reared under controlled conditions in the laboratory on soaked cotton seeds and fresh castor (Ricinus communis) leaves respectively.

2.2 lmmunocytochemistry

Gravid beetles were injected with colchicine (1.5 lal/ insect) and after two days they were dissected under cold Ringer solution; midguts were collected, fixed in Bouin- Hollande sublime, 4% paraformol, gluteraldehyde-picric acid-acetic acid mixture and Susa. Paraplast embedded sections were immunochemically stained with indirect peroxidase-antiperoxidase (PAP) method (Sternberger 1977). Monoclonals to anti-Colorado potato beetle antigens (MACs) developed by Schooneveld etal (1989), AKH- 241 antiserum (gift from Prof. H H Boer, Amsterdam) and FMRF-amide antiserum (gift from Dr H Schooneveld, Netherlands) were used as primary antibodies. Endo- genous peroxidase activity in sections was inhibited by treating it with 0.01% H202. Rabbit anti-mouse peroxidase complexes were used as secondary antibodies while DAB as the peroxidase substarte. Optimal dilutions for MACS were 1 : 1000 while that for AKH-241 and FMRF-amide were 1 : 500. Omission of primary antibodies treatment to check unspecific binding of second antibody and compounds applied later in the procedure served as negative controls.

2.3 Peptide extraction

Midgut from 280 numbers of fully fed beetles were dissected out under cold insect Ringer and transferred to homogenizing medium (methanol/water/acetic acid mixture in 90 : 1 : 29) at room temperature. After homo- genization and subsequent ultrasonic disruption, the supernatant was methanol-evaporated and the lipids were removed with hexane-water mixture treatment. The crude remainder sample was partially purified using C18 Sep- Pak cartridges (Waters associates) for removing high molecular weight proteins. The sample was finally eluted and collected in 0.1% TFA in methanol/water mixture (70 : 30). It was evaporated to remove acetonitrile content under nitrogen gas until a 1 ml prepurified sample was obtained. Samples of 50 [.tl at a time were fractionated with RP-HPLC (Shimadzu) and C4 column by means :~f a linear gradient of 0-60% acetonitrile over ! h. Two ml fractions (26 nos) were collected and lyophilized. Lyophilized fractions were subsequently redissolved either in water for electrophoretic separation or in casein enriched saline for hindgut bioassay.

2.4 Bioassay

Midguts/hindguts of well-fed healthy adult beetles were dissected out. Midguts were placed in an embryodish with

2 ml saline with 1% BSA and changes on the peristaltic contraction pattern as a consequence of peptide administration to the bath were visually recorded. Hindguts were mounted in a 500 lal incubation chamber using minute insect pins. Peptide fractions were applied first removing the saline BSA medium and then adding the sample dissolved in the same saline medium. Changes in contraction of both amplitude and frequency were monitored by an impedance recorder (Biocom, Gould brush). Only those guts which were fully relaxed and practically immobile could be used for assaying the stimulatory effect of isolated peptide fractions. A positive response is defined as a significant increase in contraction frequency and amplitude within 1.5 min that lasts for at least 1 min while rinsing with the Ringer usually abolishes the response.

2.5 SDS-PAGE and immunoblotting

Lyophilized HPLC fractions (H24, H25 and H26 ) were s.eperated by SDS-PAGE along with biotinylated range of low molecular weight marker proteins (Bio-rad) on 30% polyacrylamide using Bio-Rad Miniprotean II electro- phoresis apparatus. Peptides were electroblotted for 3 h (Bio-Rad Transblot Cell) on to immobilon (Millipore) sheets and fixed on the membrane with gluteraldehyde vapour (60°C for 1 h). Blots were then immunostained with antibodies and AB complex HRP (Dakopatts) for markers. Unstained active peptides were also eluted for subsequent bioassay (MAC-18 and MAC-3 positive fractions).

2.6 Localization of MAC-positive cells in brain

Brains from 3 day-old adult Dysdercus/final instar larvae of Achctea were dissected out and fixed in Bouin's; embedded in paraplast and sections were immunostained with primary antisera of both MAC-18 and MAC-3. Control sections of brain were processed by omitting the primary antisera to check non-specific binding. Optimal dilutions of both MAC-18 and MAC-3 were 1 :' 1000. The secondary antibodies used were peroxidase conjugated rabbit immunoglobulins to mouse antiserum and developed with 0.05% DAB.

3. Results

Several of the MACs (monoclonals) tested for immunochemical localization in the midgut of the Colorado potato beetle gave positive immunoreactivity to MAC-3, MAC-8, MAC-9 and MAC-18. Of these MAC- 18 and MAC-3 recognized endocrine cells abundantly and intensely in the epithelial layer of the midgut (figure IA, B). Immunostained cells are interspersed throughout the

Midgut neuropeptides in Colorado potato beetle 187

Figure 1. lmmunopositive endocrine cells in the midgut of adult L. decemlineata. Open type MAC-18, MAC-3 (A and B) and AKH241, and FMRF-amide (C, D, E) positive endocrine cells (arrows). Due to their slender long shape, portions of cells often visualized. Bm, basement membrane; E, epithelial cells: L, lumen of midgut (x 400).


entire length of the midgut but appear to be more concentrated at the anterior region. Immunopositive endocrine cells to polyclonals of AKH-241 and FMRF- amide have also been frequently noticed through the entire length of the midgut (figure IC, D, E) and their frequency being more at the anterior part. Immunostained cells have a basal region adjacent to the basal lamina and often exhibit apical extensions. All these putative endocrine cells are of the 'open' type which are very slender, standing on the basement membrane reaching for the gut lumen. Generally these endocrine cells seemed

intensely localized at the anterior gut while fewer numbers are located in the narrower central and posterior parts. In total several hundred endocrine cells could be estimated to be present, but their contents could only be made visible under conditions where secretions accumulate.

HPLC-elution profile of methanol soluble midgut extract is as shown in figure 2. The fractions collected after a retention time of 42 min (fractions 24, 25 and 26) contained most of the immunoreactive substances (figure 3a, b). The elution diagram shows 3 different peaks in this

I 24 I 25 I 26 I

Figure 2. HPLC elution profile of midgut extract. Acetonitrile gradient ran from 0-60% for 60 min. Of the 2 ml collected fractions, 24th, 25th and 26th selected for further immunochemical characterization and myotropic assay.

Figure 3. Immunoblots of peptides from HPLC fractions (46, 48 and 50 min) stained with MAC-18 (a) and M'AC-3 (b). Same fraction shows additional feeble positive staining also. (c) Immunoblots of eluted single peptide each showing positive reactions either to MAC-18 or to MAC-3.


Three to four numbers of immunoreactive peptidergic neurosecretory cells showing positive reaction to both MAC-18 and MAC-3 could be localized in position of the lateral group of brain neurosecretory cells in A. janata (figure 5a, b). In D. cingulatus 1-7 numbers of neurosecretory cells on both sides of the pars intercerebralis region showed immunopositive reaction to both MAC-18 and MAC-3 (figure 5c, d).

Figure 4. Imepedence converter recordings of hindgut bioassay depicting myotropic activity of both 22 kDa (Mac-18) and 26 kDa (MAC-3) peptides isolated. Samples applied at times indicated by arrow heads. Rinsing commenced on points of lower arrows. (A), MAC-18 positive (22 kDa) peptide; (B), MAC-3 positive (26 kDa) peptide. Note contractions initiated within seconds after supply.

area. When each of these fractions were subjected to electrophoretic separation followed by blotting and immunostaining either with MAC-18 or MAC-3 few of the immunopositive bands were noticed (figure 3a, b). All the 3 fractions contained intense bands having a molecular mass of 22 kDa (MAC-18 positive) and 28 kDa (MAC-3 positive). Separate elution of each of the prominent MAC-18 positive and MAC-3 gave single 22 kDa and 26 kDa peptide bands respectively (figure 3c).

Application of all the 3 active peptide fractions (F24, F25 and F26) to in vitro gut preparations have an immediate stimulatory influence causing contraction of gut circular muscles. Since midguts are known to be innervated through the stomatogastric nervous network, direct myogenic effect of these isolated fractions on whole midgut preparations seemed unrealistic as has been doubted by Luffey and Dorn (1992). Hence a well- organized in vitro hindgut preparation for recording the myogenic effect of the recovered active peptide samples in the impedance converter set up was resorted to. Addition of all the 3 fractions stimulated the contraction of both the amplitude and frequency of the hindgut after a variable lagtime of seconds. The effect was also found to be dose-dependent and the lowest dosage to give a positive response was less than 0-07 midgut equivalents. Slight individual variations on the sensitivity of hindguts could also be noticed. Testing with the final eluted single peptide fractions (22 kDa and 26 kDa) separately also showed similar significant stimulation both in the amplitude and the frequency of hindgut contraction in a dose-dependent manner (figure 4).

Immunostaining with monoclonals (MAC-18 and MAC-3) of the sections of brain tissue of both cotton bug and castor semilooper localized immunopositive neurosecretory cells in the brain of both these species (figure 5).

4. Discussion

Endocrine cells secreting peptidergic factors in the midgut of the Colorado beetles could be localized immunocytochemically using MAC-18 and MAC-3 monoclonals. These monoclonals are antisera developed against lateral and medial brain neurosecretory cell groups of the beetle and it is interesting to localize the same secretory peptides in the midgut. The role of such endocrine cells in gut physiology has not been elucidated so far even though midgut endocrine cells have been localized in several other insect species (Endo and Nishiitsutsuji-Uwo 1981; Brown et al 1986; Jenkins et al 1989; Zitnan et al 1993). Increased number of endocrine cells have been localized towards the anterior region of the midgut even though less numbers have always been scattered throughout the midgut region. The same type of distribution of endocrine cells have been found in dipterans studied (Duve and Thorpe 1982; Brown et al 1986). However in Heliothis a more scattered type of distribution has been noticed (Jenkins e ta l 1989). We extracted immunoreactive products from midgut homogenates and tested them for myogenic activity using midgut and hindgut. The two isolated active midgut peptides showed immunoreactivity to both MAC-18 and MAC-3 indicating that both peptides are present in the gut extract having molecular masses of 22 kDa and 26 kDa, respectively. All the three HPLC fractions showed stimulatory influence on the gut contractility. The final 22 kDa and 26 kDa peptides characterized after elution of the bands, both had a distinct stimulatory effect on gut contractlity establishing that these peptides have regulatory role on gut contraction in Colorado beetle.

Recently Yi e ta l (1995) isolated and purified a myotropic peptide (Mas-Mg-Mt-I) from the midgut of Manduca sexta the molecular mass of which has not been estimated. Allatostatin genes have been shown to be expressed in the midgut endocrine cells of cockroach and in the mosquito Aedes aegypti (Reichwald e ta l 1994; Veenstra et al 1997). Using polyclonals against FMRF- amide and AKH-241 neuropeptides almost a similar pattern of distribution of midgut endocrine cells immunopositive to these two peptides could be localized in the midgut of the beetle signifying that the two peptides isolated and characterized are analogous to FMRF-amide and AKH-241. Since MAC-18 and MAC-3 positive cells


Figure 5. Immuno-sensitive MAC-positive neuroscecretory cells in the brain of 5th instar larve ofA. janata (a and b) and adult D. cingulatus (c and d). Arrows denote immunopositive neurosecretory cells. PAP method-DAB stain (x 400).

could also be demonstrated in two different species of insects like Achaea and Dysdercus in analogous groups of neurosecretory cells (as lateral and median), it becomes clear that these two peptides isolated are not species specific. It is not surprising that neuropeptides are localized at different extraneuronal sites in several insect species as has been observed by (Nassel et al 1994) and the evident features of cosynthesis and co-release of several neuropeptides suggest that they may have shared functions (O'Brien and Tagert 1994). Neuropeptide-F-like

molecules localized within the paraneurot~s of mesenteric epithilium suggested a signalling function for NPF(S) within the gut (Verhaert etal 1993, 1994). In Colo rado beetle NPF type peptides have been demonstrated both in the nervous system and in the 'open' paraneurons of midgut epithelium (Spittaels et al 1996). After further identification of structure of our isolated substances, we will hopefully be able to obtain the peptides in pure chemical form and determine their precise structure.


Acknowledgements

Part of this work was carried out in the Wageningen Agricultural Universi ty, Netherlands with a NUFFIC Fel lowship awarded to the senior author. The authors gratefully acknowledge the helps rendered by Dr H Schooneveld and H M Smid of Wageningen Agricultural University, Netherlands. Finanacial assistance rendered by the Council of Scientific and Industrial Research, New Delhi, also is thankfully acknowledged.

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MS received 8 October 1998; accepted 24 February 1999

Corresponding editor: VIDYANAND NANJUNDIAH

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