Neuropeptides associated with the frontal ganglion of larval Lepidoptera

Peptides 26 (2005) 11–21

Neuropeptides associated with the frontal ganglion of larval Lepidoptera Neil Audsley∗ , June Matthews, Robert J. Weaver Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK

Abstract The occurrence of neuropeptides in the frontal ganglia of larvae of the tobacco hawkmoth, Manduca sexta, the tomato moth, Lacanobia oleracea and the cotton leafworm, Spodoptera littoralis was investigated using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and enzyme-linked immunosorbent assay (ELISA). Only three types of peptides could be identified or assigned from frontal ganglion extracts; M. sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT), and F/YXFGL-NH2 allatostatins. The peptide profiles of frontal ganglion of L. oleracea and S. littoralis were similar, with ten identical [M + H]+ ions, seven of which could be assigned to known lepidopteran peptides (Manse-AT, cydiastatin 2, 3, 4 and helicostatin 1, 5, 9). In addition, mass ions corresponding to helicostatin 7 (which was confirmed by MALDI-post source decay analysis) and Manse-AS were present in frontal ganglia of L. oleracea and helicostatin 6 in frontal ganglia of S. littoralis. Only four mass ions from M. sexta frontal ganglia corresponded to known peptides, cydiastatin 3 and 4, helicostatin 1, and Manse-AT. The only difference between the profiles of frontal ganglia from different stages of L. oleracea were mass ions which could not be assigned, and no differences were observed in the allatoregulatory peptides present. In HPLC fractions of M. sexta frontal ganglia, F/YXFGL-NH2 allatostatin-like immunoreactivity was widespread suggesting that more allatostatins were present than were identified. Crown Copyright © 2004 Published by Elsevier Inc. All rights reserved. Keywords: Peptidomics; Insect; Lepidoptera; Allatostatin; Allatotropin

1. Introduction The stomatogastric nervous system that innervates the gut has been reported to be involved in a variety of functions including feeding, crop emptying, and defecation [34]. Central to this system is the frontal ganglion, which is situated on the anterior wall of the esophagus and is linked to the brain by a pair of frontal connectives. This ganglion innervates parts of the stomodaeum, anteriorly via the frontal nerve, and posteriorly through the recurrent nerve. In larvae of the tobacco hawkmoth, Manduca sexta, the frontal ganglion has been implicated in the regulation of feeding. Using a combination of electrophysiological recordings and severance of the recurrent nerve, Miles and Booker [33] showed that foregut contractions were controlled by the frontal ganglion. In adult Periplaneta americana [41], nymphal and adult Schistocerca gregaria [26], and in adult ∗

Corresponding author. Tel.: +44 1904 462628; fax: +44 1904 462111. E-mail address: [email protected] (N. Audsley).

Heliothis zea [7], removal of the frontal ganglion results in the accumulation of food in the foregut. A similar effect was observed by cutting the recurrent nerve in larval M. sexta [38]. Using immunocytochemical techniques, the allatoregulatory peptides M. sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT) and allatostatins of the Y/FXFGL-NH2 family have been localized in large neurosecretory cells of the frontal ganglia of various lepidopteran larvae [16,18,19,21]. These peptides are also present in the recurrent nerve, and branches of this nerve supplying the muscles of the crop and the region of the stomodeal valve [19,21]. Manduca sexta allatotropin has also been detected in the frontal ganglia of larval M. sexta using both immunohistochemistry and in situ hybridization [6]. This tissue localization implies that these peptides are myoactive on the foregut. Spontaneous contractions of the foregut of larval Cydia pomonella, Helicoverpa armigera and L. oleracea are inhibited by Y/FXFGLNH2 allatostatins [16,18,19]. Manduca sexta-AS also inhibits foregut peristalsis in larval L. oleracea [19]; whereas, Manse-

0196-9781/$ – see front matter Crown Copyright © 2004 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.10.011

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AT has been shown to have a stimulatory effect on the foregut of larval H. armigera and L. oleracea [18,19]. Other peptides have also been detected in the frontal ganglion and/or stomatogastric nervous system of lepidopterans using immunochemical techniques. Zitnan et al. [47] reported FMRF-NH2 -like immunoreactivity in the ganglia (including frontal ganglion) and nerves of the stomatogastric system of the waxmoth, Galleria mellonella, and Golubeva et al. [24] reported pheromone biosynthesis activating neuropeptide (PBAN)-like immunoreactivity in the esophageal nerve and anterior midgut of the Gypsy moth, Lymantria dispar. Immunoreactivity to adipokinetic hormone and eclosion hormone have been detected in the recurrent nerve of pharate adult M. sexta [27] and pheromonotropic melanizing peptide (PMP)-like immunoreactivity has been detected in the frontal ganglion and esophageal nerve of larval Helicoverpa zea [37]. In Lepidoptera, there are around fifty characterized neuropeptides with molecular masses of less than 2500 Da. Of these, 16 are F/YXFGL-NH2 allatostatins and approximately, 20 others are peptides that are also myoactive on the gut, including myokinins, myotropins, and myoinhibitory peptides [23]. Some of these peptides may be active on the foregut and may also be present in the frontal ganglion. The aim of this study was to profile and compare the neuropeptides in the frontal ganglion of three lepidopteran species, L. oleracea, M. sexta, and Spodoptera littoralis using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS).

2. Materials and methods 2.1. Experimental animals Lacanobia oleracea and Spodoptera littoralis were reared at 20 ◦ C and 65% relative humidity, under a 16-h light: 8-h dark photoperiod as described by Corbitt et al. [8]. Larvae were fed on a maize-flour based noctuid artificial diet (BioServ, Frenchtown, NJ, USA). Manduca sexta were reared from eggs supplied by Prof. S. Reynolds, University of Bath, using methods described by Yamamoto [45]. 2.2. Tissue extraction and liquid chromatography For single tissue extraction, frontal ganglia were dissected from last day fourth instar larval M. sexta or last day fifth instar larval L. oleracea and S. littoralis (same physiological age) and placed into Eppendorf tubes containing 10 ␮l of 0.1% (v/v) trifluoroacetic acid (TFA). For separation by reversed-phase high performance liquid chromatography (RP-HPLC), 170 frontal ganglia from fifth stadium larval M. sexta or 230 frontal ganglia from sixth stadium larval L. oleracea were dissected into 500 ␮l of 0.1% TFA. The tubes were agitated at room temperature for 15 min, and then centrifuged at 10,000 × g for 5 min. For single tissue extracts 0.5 ␮l of

supernatant was applied directly to the MALDI sample plate (as described below); whereas, the supernatant for HPLC separation was diluted with 0.1% TFA and loaded, via a Rheo˚ analytical dyne loop injector, onto a Jupiter C18 10 ␮m 300 A column (250 mm × 2.1 mm i.d.; Phenomenex, Macclesfield, UK) fitted with a guard column (30 mm × 2.1 mm i.d.) of similar packing material. The column was eluted with a linear gradient of 5–60% acetonitrile/0.1% TFA, over 55 min at a flow rate of 0.2 ml/min, and elution monitored at 214 nm, using a Beckman 32 Karat chromatographic system (Beckman Coulter Ltd., UK), comprising a dual pump programmable solvent module 126 and a UV detector module 166. Fractions (1 min, 0.2 ml) were collected and concentrated to c.10 ␮l by centrifugal evaporation for mass analysis. The elution positions of Manse-AS, Manse-AT, and cydiastatin 4 were determined using the same HPLC conditions. 2.3. Mass analysis Mass spectra were acquired on a Voyager DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Warrington, UK). The matrix, ␣-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) was prepared at a concentration of 10 mg/ml in 50% acetonitrile/0.05% (v/v) TFA. Samples (0.5 ␮l) were added to the MALDI sample plate followed by 0.5 ␮l of matrix, and dried at room temperature. Standards (bradykinin, angiotensin, somatostatin, and adrenocorticotropic hormone; Sigma-Aldrich, UK) were added adjacent to samples. Spectra represent the resolved monoisotopic [M + H]+ masses in reflector mode within the mass range m/z, 500–5000. Spectra are the accumulation of 5 × 50 shots. The measured monoisotopic masses [M + H]+ were compared to the calculated monoisotopic masses [M + H]+ of known peptides. Monoisotopic masses were calculated using protein prospector (University of California, San Francisco). Analyses by MALDI-PSD were performed on the same instrument and samples, using angiotensin as the standard. A PSD spectrum was produced from 7–8 spectral segments and stitched together using the Voyager software. 2.4. ELISA Indirect ELISAs for Manse-AS, Manse-AT, and cydiastatin 5 were used to measure immunoreactivity in HPLC fractions, using methods reported by Audsley et al. [1]. Briefly, HPLC fractions and synthetic peptides were dried onto multiwell plates (Sigma-Aldrich, UK; Cat. No. M4034) at 37 ◦ C then incubated overnight at 4 ◦ C with 100 ␮l of 0.1 M bicarbonate (coating) buffer (pH 9.6). Plates were washed three times with 150 ␮l of 10 mmol/l phosphate buffer/0.1% TWEEN-20 (PBS), blocking solution (150 ␮l; 2% non-fat milk in PBS) was added, and the plates incubated for 90 min at 37 ◦ C. After a further PBS wash, 100 ␮l of primary antiserum (dilutions: 1:3000, Manse-AS; 1:5000, Manse-AT; 1:5000, cydiastatin 5) were added to each well and the plates incubated for another 90 min at 37 ◦ C. One hundred micro-

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liters of goat anti-rabbit antiserum conjugated to horseradish peroxidase (1:3000 dilution in PBS) were added as secondary antibody after washing three times with PBS. Plates were then incubated for 40 min at 37 ◦ C. After final PBS washing (3×), 100 ␮l of substrate solution (10 mg O-phenylenediamine, 10 ␮l H2 O2 in 25 ml citrate buffer, pH 5.0) were added to each well and incubated for 40 min at 37 ◦ C. The reaction was stopped by addition of 50 ␮l 1.0N H2 SO4 to each well and optical density read at 492 nm on a Labsystems Multiskan MCC/340. 2.5. Foregut contractions Sixth instar L. oleracea larvae were starved overnight, anaesthesized with CO2 and cut open along their dorsal surface to one side of the heart from the head to the third abdominal segment. The cuticle was pinned back to expose the foregut and anterior midgut, which were rinsed several times in physiological saline. The composition of saline was Na+ 154 mM, K+ 2.7 mM, Ca2+ 1.8 mM, Cl− 160 mM, glucose 22 mM and hydroxyethylpiperazine ethanesulphonic acid 12 mM, at pH 7.2 [9]. After rinsing, the gut was bathed in 200 ␮l of saline at 22 ± 2 ◦ C and the preparation viewed under a dissecting microscope to observe peristaltic contractions of the foregut. A baseline frequency of contractions was established over 2 × 1 min periods. Control saline was then replaced by saline containing either cydiastatin 4 or Manse-AT at various doses. Frequency of contractions was again counted over 2 × 1 min periods, after which the gut was washed several times to remove peptides and return the foregut to baseline peristatlsis. Once this was achieved, another solution of saline + peptide could be added and foregut peristalsis observed again. 2.6. Synthetic peptides and antisera Manduca sexta allatostatin and cydiastatin 4 were custom synthesized at the Advanced Biotechnology Centre, Imperial College School of Medicine, Charing Cross Hospital, London. Manduca sexta allatotropin and proctolin were purchased from Sigma-Aldrich (UK). Polyclonal antisera to Manse-AS were custom prepared by Genosys Biotechnologies (Cambridge, UK), using ManseAS conjugated to keyhole limpet haemocyanin. Polyclonal antisera to Manse-AT and cydiastatin 5 were gifts from Prof. A. Thorpe, Queen Mary and Westfield College, London, UK. 3. Results 3.1. Comparison between single frontal ganglion extracts from M. sexta, L. oleracea, and S. littoralis The MALDI spectra of single frontal ganglia, extracted in 0.1% TFA, from the last day of the penultimate instar of larval M. sexta, L. oleracea, and S. littoralis, are compared in Fig. 1, and the measured monoisotopic masses ([M + H]+ )

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Table 1 A comparison between the measured monoisotopic mass ions [M + H]+ from M. sexta, L. oleracea, and S. littoralis frontal ganglia showing similarities between species at the same developmental stage Peptide

Cydiastatin 2 Cydiastatin 3 Cydiastatin 4 Helicostatin 1 Helicostatin 5 Helicostatin 6 Helicostatin 7 Helicostatin 9 Manse-AT

Monoisotopic masses [M + H]+ Calculated

Measured M. sexta

L. oleracea

S. littoralis

2168.1 925.5 909.5 934.4 911.5 953.5 926.5 1393.7 1486.7

– 925.5 909.5 – – – – – –

2168.1 925.5 909.5 – 911.5 – 926.5 1393.7 1486.7

2168.2 925.5 909.6 934.5 911.5 953.5 – 1393.7 1486.8

Masses were calculated using protein prospector (University of California, San Francisco).

compared in Table 1. Although, the signal intensities were low, a number of significant ion signals were observed for all three samples. Analysis of single frontal ganglion extracts gave reproducible spectra. Only two masses ([M + H]+ ) corresponding to known lepidopteran peptides were measured in frontal ganglion extract from M. sexta; 909.6 and 925.6, which correspond to cydiastatin 4 (909.5; ARPYSFGL-NH2 ) and cydiastatin 3 (925.5; SRPYSFGL-NH2 ), respectively (Fig. 1A, Table 1). These two were the only [M + H]+ ions in M. sexta that were also present in L. oleracea and S. littoralis frontal ganglion extracts (Table 1). The most prominent signal in frontal ganglion extract from M. sexta was an unknown, with a monoisotopic mass of 2194.2. Masses in agreement with the monoisotopic masses of cydiastatin 3, 4, and helicostatin 5 (911.5; ARAYDFGL-NH2 ) and helicostatin 7 (926.5; LPMYNFGL-NH2 ) had the highest signal intensities in single FG extracts from L. oleracea (Fig. 1B, Table 1). In addition, monoisotopic masses corresponding to helicostatin 9 (1393.7; ERDMHRFSFGL-NH2 ), Manse-AT (1486.7; GFKNVEMMTARGF-NH2 ), and cydiastatin 2 (2168.1; AYSYVSEYKRLPVYNFGL-NH2 ) were also measured. From the frontal ganglion of S. littoralis, signals corresponding to allatostatins were also the most intense (Fig. 1C, Table 1). The monoisotopic masses 909.6, 925.5, 911.5, and 953.5 correspond to cydiastatin 4, 3, and helicostatin 5, 6 (953.5; LPMYNFGL-NH2 ), respectively. Other monoisotopic masses were in agreement with helicostatin 1 (934.4; SPHYDFGL-NH2 ), helicostatin 9 (1393.7), ManseAT (1486.8), and helicostatin 2 (2168.2). Ion signals 1277.8 and 2223.2 are unknowns, but were present in both S. littoralis and in L. oleracea FG extracts. 3.2. Comparison between single frontal ganglion from larval L. oleracea at different developmental stages The spectra of single frontal ganglion extract from day 5 VIth instar (feeding) larval L. oleracea is shown in Fig. 2.

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Fig. 1. Mass spectra of single frontal ganglion extracts from last day penultimate instar larval M. sexta (A), L. oleracea (B) and S. littoralis (C) showing monoisotopic masses [M + H]+ .

The monoisotopic masses are compared to a last day Vth instar (non-feeding) larval L. oleracea (Fig. 1B). At both stages of development, masses (909.5, 911.5, 925.5, 926.5, 1393.7, 2168.1) corresponding to the same F/YXFGL-NH2 peptides (cydiastatin 2, 3, 4 and helicostatin 5, 7, 9) and Manse-AT (1486.7) were present. The notable differences in the measured masses were 1570.9 (only measured in FG extracts of last day Vth instar larvae) and 1605.1 (only measured in day 5 VIth instar larvae). These ion signals do not correspond to any known lepidopteran peptides.

3.3. MALDI-TOF MS of frontal ganglia extracts from L. oleracea after HPLC separation. Analysis of HPLC fractions from an extract of 230 frontal ganglia by MALDI-TOF MS gave reproducible spectra. Numerous signals were observed in HPLC fractions, those corresponding to known lepidopteran peptides are listed in Table 2. As with single ganglion extracts, only [M + H]+ masses corresponding to allatoregulatory peptides could be identified in HPLC fractions from larval L. oleracea. The mass

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Fig. 2. Mass spectra of single frontal ganglion extracts from day 5, VIth instar larval L. oleracea showing monoisotopic masses [M + H]+ .

spectrum from fraction 32 is shown in Fig. 3A. The measured monoisotopic masses are identical to the calculated monoisotopic masses of cydiastatin 4 (909.5), helicostatin 5 (911.5), cydiastatin 3 (925.5), and helicostatin 1 (934.4), the latter of which was not detected in single ganglion extracts from L. oleracea (Table 1). Fig. 3B is a composite of fractions 34 (i) and 45 (ii). Two masses were in very close agreement (0.1 Da) with known lepidopteran peptides, cydiastatin 9 (1393.7), and Manse-AT (1486.7), and the measured mass (1889.1) in fraction 45 was close (0.2 Da) to the calculated monoisotopic mass of Manse-AS (1888.9; pEVRFRQCYFNPISCF-OH). The [M + H]+ mass 1905.8, corresponding to Manse-AS with a Q instead of pE at position 1 (1905.9; QVRFRQCYFNPISCF-OH) was also measured. Other measured masses in agreement with known lepidopteran allatostatins were 926.5 (helicostatin 7) and 2168.1 (cydiastatin 2), both of which eluted in other fractions. Fragmentation by MALDI-PSD of the precursor ion 926.5 produced a number of C-terminal, N-terminal and internal ions consistent with helicostatin 7 (Fig. 4). Fragmentation of preTable 2 Calculated and measured monoisotopic masses [M + H]+ of peptides from L. oleracea and M. sexta frontal ganglia identified or assigned by MALDI-TOF MS Peptide

Monoisotopic masses [M + H]+ Calculated

Cydiastatin 2 Cydiastatin 3 Cydiastatin 4 Helicostatin 1 Helicostatin 5 Helicostatin 7 Helicostatin 9 Manse-AT Manse-AS Q1 Manse-AS

2168.1 925.5 909.5 934.4 911.5 926.5 1393.7 1486.7 1888.9 1905.9

cursor ions 909.5, 911.5 and 925.5 by MALDI-PSD were also in agreement with cydiastatin 4, helicostatin 5, and cydiastatin 3, respectively (results not shown). Mass ions corresponding to cydiastatin 4, Manse-AT, and Manse-AS all eluted in the same positions as their respective synthetic peptides. All other measured monoisotopic masses did not correspond to any known lepidopteran peptide. The low abundance of other ions precluded analysis by MALDI-PSD. 3.4. MALDI-TOF MS of frontal ganglia extracts from M. sexta after HPLC separation Analysis of HPLC fractions of an extract of 170 M. sexta frontal ganglia by MALDI-TOF MS identified only four monoisotopic masses in agreement with known lepidopteran peptides (Fig. 5, Table 2). These were 909.6 (cydiastatin 4), and 925.6 (cydiastatin 3), both of which were present in single ganglion extracts, plus 934.6 (helicostatin 1) and 1486.7 (Manse-AT), which were not detected in single ganglion extracts. Other ion signals were measured, but none corresponded to any known lepidopteran peptide. The identities of cydiastatin 3 and 4 were confirmed by fragmentation by MALDI-PSD (results not shown). Analysis of other ions by MALDI-PSD was precluded by low signal intensities. 3.5. ELISAs of M. sexta FG HPLC fractions

Measured L. oleracea

M. sexta

2168.1 925.5 909.5 934.4 911.5 926.5 1393.7 1486.7 1889.1 1905.8

– 925.6 909.6 934.6 – – – 1486.7 – –

Masses were calculated using protein prospector (University of California, San Francisco).

Fig. 6 shows immunoreactivity in HPLC fractions of larval M. sexta FG to Y/FXFGL-NH2 allatostatins (Fig. 6A), Manse-AT (Fig. 6B) and Manse-AS (Fig. 6C). A number of fractions were immunoreactive to antisera against cydiastatin 5, and those fractions (32 and 33) containing peptides with masses corresponding to known Y/FXFGL-NH2 allatostatins are labeled. Immunoreactivity to Manse-AT was detected in the same HPLC fraction (34) as the corresponding ion (1486.7) and synthetic Manse-AT (Fig. 6B). Even though an ion in agreement with the monoisotopic mass of ManseAS was not detected by MALDI-TOF MS, Manse-AS-like immunoreactivity was detected in HPLC fractions of M. sexta

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Fig. 3. (A) Mass spectra of fraction 32 from HPLC separation of sixth stadium larval L. oleracea frontal ganglia extract, showing monoisotopic masses [M + H]+ . Masses in agreement with cydiastatins 3, 4 and helicostatins 1, 5 are labeled, other masses are unknowns, and (B) composite mass spectra of fraction 34 (i) and 45 (ii) from HPLC separation of sixth stadium larval L. oleracea frontal ganglia extract, showing monoisotopic masses [M + H]+ . Masses in agreement with helicostatin 9, Manse-AT, and Manse-AS are labeled. Other masses are unknowns.

frontal ganglia (Fig. 6C), eluting in the same position as synthetic Manse-AS (fraction 45). 3.6. Effects of peptides on larval L. oleracea foregut peristalsis The effects of cydiastatin 4 and Manse-AT on spontaneous foregut contractions of sixth stadium L. oleracea are shown in Fig. 7. Control rates of foregut contractions were 6.2 ± 0.4 per

minute which could be stimulated on average up to two-fold by Manse-AT (0.1–100 fmol/l), in a dose-dependent manner (Fig. 7A), although individual rates were often highly variable (n = 16–20). Cydiastatin 4 (Fig. 7B) caused a dosedependent inhibition of foregut contractions over the range 10–100 nmol/l. The effects of both peptides were reversible. The effect of proctolin on the frequency of foregut muscle contractions in sixth stadium L. oleracea was also investigated. There was no significant difference between

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Fig. 4. Post-source decay analysis of precursor ion 926.5 from HPLC fraction 31 of L. oleracea frontal ganglia. Ion signals labeled are C-terminal, N-terminal and internal fragments.

Fig. 5. Mass spectra of fraction 32 from HPLC separation of fifth stadium M. sexta frontal ganglia extract, showing monoisotopic masses [M + H]+ . Masses in agreement with those for cydiastatins 3, 4, and helicostatin 1 are labeled.

control rate of muscle contractions (9.6 ± 1.1 per minute) and treatment with 1 ␮M proctolin (10.1 ± 1.3 per minute; n = 15). Lower doses of 100 nM and 10 nM were also inactive (n = 14–20).

4. Discussion The peptides present in the frontal ganglion of three species of Lepidoptera (L. oleracea, S. littoralis, and M. sexta) that extract into 0.1% TFA, have been investigated

using RP-HPLC, MALDI-TOF MS, and ELISA. By comparing mass ions [M + H]+ of spectra obtained from frontal ganglion extracts with calculated monoisotopic masses [M + H]+ of known lepidopteran peptides, measured monoisotopic masses could either be assigned, or identified as known peptides, depending on whether a particular peptide had been characterized from that species before. Some of the known peptides corresponding to measured monoisotopic masses were confirmed by MALDI-PSD analysis. Many peptides of insects that are related (within an order) are often identical (e.g. allatostatins from cockroaches [5,43] and allatostatins

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Fig. 6. ELISA of HPLC fractions of separation of fifth stadium M. sexta frontal ganglia extract showing immunoreactivity to cydiastatin 5 (A), Manse-AT (B) and Manse-AS (C).

and AKH from moths [15,23]. It is therefore likely that measured masses from the frontal ganglia of the three species investigated in this study correspond to known lepidopteran peptides with matching masses. Only three types of peptides were identified in extracts of single or HPLC separated frontal ganglia. These were ManseAS, Manse-AT, and allatostatins of the F/YXFGL-NH2 family. Manduca sexta allatostatin and Manse-AT were first isolated from heads of pharate adults of M. sexta [29,30]. They have since been shown, by immunocytochemistry and in situ hybridization, to be localized in neurosecretory cells in the frontal ganglia of M. sexta, L. oleracea, H. virescens, and S. frugiperda [6,19,21]. In the present study, a mass ion corresponding to Manse-AT was measured in M. sexta, S. littoralis and L. oleracea; whereas, Manse-AS was only detected in L. oleracea. This could be because Manse-AS is present in such low amounts in the frontal ganglion that its signal is masked by other peptides/proteins present. In support of this, ManseAS was not detected in single ganglion extracts, but only after HPLC separation, and even then its signal intensity was low. In HPLC fractions of M. sexta frontal ganglion, ManseAS-like immunoreactivity was present, and co-eluted with synthetic peptide, even though a corresponding mass ion was not detected by MS. In a previous study, a mass ion corresponding to Manse-AS, but not Manse-AT, was detected in

Fig. 7. Frequency of foregut muscle contractions of sixth stadium larval L. oleracea: stimulation by Manse-AT (A) and inhibition by cydiastatin 4 (B). Means ± S.E., n = 16–20.

methanol extracts of brains, after HPLC separation, of both M. sexta and L. oleracea [3]. Using immunochemical techniques, Manse-AT has also been detected in the brain of both these insects [2,46]. A mass corresponding to Manse-AT was measured in frontal ganglion of L. oleracea extracted in 0.1% TFA, but not methanol (result not shown). These results suggest that Manse-AT does not extract as well into methanol as it does into 0.1% TFA. Allatostatins of the Y/FXFGL-NH2 family, originally identified in cockroaches [35,44] have also been identified in lepidopterans. A total of eight YXFGL-NH2 allatostatins (cydiastatins) were characterized from C. pomonella and ten (helicostatins) from H. armigera, four of which are identical between the two species [15]. Some of these allatostatins have been identified in L. oleracea and M. sexta using MALDITOF MS and MALDI-PSD analysis [3,4]. Allatostatins of the F/YXFGL-NH2 family have also been shown to be localized in the frontal ganglion of larval L. oleracea, M. sexta, and S. frugiperda using immuncytochemical techniques [19,21]. However, because there are several Y/FXFGL-NH2 allatostatins in a single species (up to 14 in cockroaches and 9 in moths), all with similar C-terminal sequences, antisera could be cross-reacting with a number of these peptides. Hence, immunochemical techniques, using polyclonal antisera, do not

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identify the specific peptide present. By comparing measured masses with the masses of known allatostatins, MALDI-TOF MS helps to identify which Y/FXFGL-NH2 allatostatins are present and augments immunocytochemical data. Cydiastatin 3, 4 and helicostatin 1 are the only F/YXFGL-NH2 allatostatins detected in the frontal ganglia of larval M. sexta, and all had previously been detected in the brain of this insect using the same techniques [3]. Fragmentation by MALDI-PSD of precursor ions corresponding to cydiastatin 3 and 4 confirmed their identities in the brain of larval M. sexta, and in this study, in the frontal ganglion. All three of the aforementioned Y/FXFGL-NH2 allatostatins are common to the frontal ganglion of all three species studied, although helicostatin 1 was only detected in frontal ganglion of M. sexta and L. oleracea after HPLC separation. Using an ELISA to cydiastatin 5, the F/YXFGL-NH2 -like immunoreactivity in HPLC fractions of M. sexta frontal ganglion was widespread. This would suggest that there are more F/YXFGL-NH2 allatostatins in the frontal ganglion of this insect than currently identified by MALDI-TOF MS, although the spread of immunoreactivity could also be due to cross-reactivity of antisera with unrelated compounds. Some of the unknown mass ions measured in HPLC fractions may be novel F/YXFGL-NH2 allatostatins, especially as the three F/YXFGL-NH2 allatostatins assigned or identified from M. sexta frontal ganglia are restricted to only two of the immunoreactive HPLC fractions. Seven mass ions from larval L. oleracea and S. littoralis frontal ganglion correspond to F/YXFGL-NH2 allatostatins, six of which are identical between the two species. As with M. sexta, all seven mass ions from larval L. oleracea frontal ganglion that corresponded to F/YXFGL-NH2 allatostatins were also measured in brain extracts from this insect, of which three, cydiastatin 3, 4 and helicostatin 5 were positively identified by MALDIPSD [3]. Fragmentation of precursor ions corresponding to cydiastatin 3, 4 and helicostatin 5 also confirmed identity of these peptides in the frontal ganglion of L. oleracea. In addition, MALDI-PSD analysis of precursor ion 926.5 produced ion fragments that are consistent with helicostatin 7. Other Y/FXFGL-NH2 allatostatins present in M. sexta and L. oleracea, and all peptides in S. littoralis, have been assigned to known peptides with matching masses, although these have not been confirmed by MALDI-PSD. Cydiastatins 3 and 4 are common to all lepidopterans studied to date, having been structurally characterized, or assigned by matching molecular masses, in C. pomonella, H. armigera, L. oleracea, M. sexta, and S. littoralis [3,4,15,16]. Cydiastatin 4 is more widespread; a gene encoding an identical peptide, along with other Y/FXFGL-NH2 allatostatins, has been identified in six species of cockroaches [5,12,13], the locust, S. gregaria [39] and the cricket, Gryllus bimaculatus [32]. Cydiastatin 4, however, was not one of the Y/FXFGL-NH2 allatostatins characterized from four species of Diptera [14,22,31,40], the shore crab, Carcinus maenus [17] or tiger prawn, Penaeus monodon [20], despite the relatively large number of Y/FXFGL-NH2 allatostatins identified from these species. Allatoregulatory peptides appear to be

19

the most abundant mass ions present in the frontal ganglia of the lepidopteran species investigated in this study, because no other mass ions in agreement with the calculated mass of any other known lepidopteran peptide were detected. Interestingly, there was no evidence for FMRF-NH2 -like peptides, AKH, PMP, PBAN or eclosion hormone, even though immunoreactivity to these peptides has been reported in the frontal ganglion and/or other regions of the stomatogastric nervous system [24,27,37,47]. There was also no mass ions in agreement with the monoisotopic masses of other myoactive active peptides characterized from Lepidoptera [23]. Most of these peptides have been shown to be active on the hindgut or heart [23]. It is possible that these peptides have no myoactivity on the foregut, or they are not localized in the frontal ganglion. Alternatively, these peptides may be present, but of very low abundance, or not readily detectable by MALDITOF MS. Due to the relationship between the frontal ganglion and foregut [19,21,33,34], the peptides identified or assigned in the frontal ganglion in this study are implicated in the regulation of foregut movements. The way in which these peptides interact, and why there are two types of inhibitory peptides (Manse-AS and Y/FXFGL-NH2 allatostatins) and only one stimulatory peptide (Manse-AT) to regulate foregut movement is not understood. The Y/FXFGL-NH2 allatostatins have been shown to inhibit spontaneous foregut contractions in C. pomonella, H. armigera [16,18], and L. oleracea (using cydiastatin 5 and helicostatin 8) [19]. As shown here, the most common allatostatin, cydiastatin 4, has a similar inhibitory effect on foregut peristalsis in larval L. oleracea. Likewise, M. sexta AT stimulates foregut muscle contractions in larval L. oleracea over a dose range (10−16 –10−12 mol/l) similar to that reported for H. armigera [18]. Although Manse-AT had previously been shown to act on L. oleracea foregut, no dose–response curve had been reported [19]. Duve and Thorpe [21] have shown that different combinations of Manse-AS, Manse-AT, and Y/FXFGL-NH2 allatostatins are localized in the nerves supplying the muscles of the crop in larval M. sexta, L. oleracea and S. frugiperda. To add further to the complexity, MALDI-TOF MS has shown that there are at least seven Y/FXFGL-NH2 allatostatins in the frontal ganglion of Lepidoptera. Proctolin, the first insect peptide to be structurally characterized, has myotropic activity on various tissues in a number of different insects [23]. Using mass spectrometric techniques, proctolin has been detected in the stomatogastric nervous system of P. americana [36]. Immunoreactivity to proctolin has been detected in the brain and retrocerebral complex [46] and mandibular and maxillary cells of the subesophageal ganglia [11] of M. sexta and in the nervous system of L. dispar [10], but there are no reports of proctolin-like immunoreactivity in the stomatogastric nervous system in Lepidoptera. There was no evidence of a mass ion corresponding to proctolin in frontal ganglion extracts from M. sexta, L. oleracea or S. littoralis. Even though proctolin stimulates foregut contractions in S. gregaria [25], this peptide had no effect on

20

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the foregut of larval L. oleracea, suggesting that in this insect stimulation of foregut peristalsis may be predominantly due to allatotropin. Proctolin also had no effect on the isolated gut of S. frugiperda [28], but has been shown to evoke contractions of the hindgut in larval Pieris rapae [42]. In conclusion, a number of lepidopteran peptides have been identified or assigned from frontal ganglion extracts of larval M. sexta, L. oleracea, and S. littoralis. These peptides are of three types: Manse-AS, Manse-AT, and Y/FXFGLNH2 allatostatins, no other type of peptide was identified. It would appear that these peptides are the major peptides associated with the frontal ganglion, and hence are primary regulators of foregut muscle activity.

[12]

[13]

[14]

[15]

[16]

Acknowledgement The authors acknowledge support from the Pesticide Safety Directorate, DEFRA.

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