Identification of neuropeptides in the midgut of parasitized insects: FLRFamides as candidate paracrines

Molecular and Cellular Endocrinology 133 (1997) 19 – 32

Identification of neuropeptides in the midgut of parasitized insects: FLRFamides as candidate paracrines Timothy G. Kingan a,b,*, Dusan Zitnan c, Howard Jaffe d, N.E. Beckage b a

USDA ARS Insect Neurobiology and Hormone Laboratory, BARC-East Bldg. 306 Rm. 322, Belts6ille, MD 20705, USA b Department of Entomology, 5419 Boyce Hall, Uni6ersity of California, Ri6erside, CA 92521, USA c Institute of Zoology, Slo6ak Academy of Sciences, Dubra6ska cesta 9, 84206 Bratisla6a, Slo6akia d NIH NINDS LNC, Bldg. 36 Rm. 4D20, Bethesda, MD 20892, USA Received 31 March 1997; accepted 15 July 1997

Abstract Parasitism of Manduca sexta (Lepidoptera: Sphingidae) larvae by the braconid wasp Cotesia congregata (Hymenoptera: Braconidae) leads to accumulation of peptides in host neurons and neurosecretory cells of the central nervous system (CNS) and neurons and endocrine/paracrine cells of the midgut. This accumulation has now facilitated the characterization of two new members of the FLRFamide family from midguts of parasitized larvae. The peptides, given the names F24 and F39, are 24 and 39 amino acids in length with the sequences VRDYPQLLDSGMKRQDVVHSFLRFamide and YAEAAGEQVPEYQALVRDYPQLLDSGMKRQDVVHSFLRFamide. The sequence of F24 is identical to the C-terminal 24 amino acids of F39. The C-terminal 10-mer of each is identical to a previously characterized decapeptide neurohormone (F10). This sequence is preceded by a potential processing site. In nonparasitized insects F39 was present at several-fold the amount of F24. In parasitized insects F24 and F39 accumulate in the middle and posterior regions of the midgut, which are enriched in endocrine/paracrine cells reacting with FLRFamide antisera. In the combined brain and subesophageal ganglion F39 was not detected and the amount of F24 never exceeded 2 fmol per Br/SEG. Of the three peptides, only F10 was found in the hemolymph. Thus, F24 and F39 may be intermediates in the biosynthesis of F10 and may themselves be released locally from endocrine/paracrine cells in the midgut epithelium. © 1997 Elsevier Science Ireland Ltd. Keywords: Tobacco hornworm; Manduca sexta; Gut endocrine cells; FLRFamide; Wasp; Cotesia congregata; Enzyme immunoassay

1. Introduction The midgut is the primary digestive organ of insects, secreting digestive enzymes and mediating absorption of nutrients. It is innervated throughout by elements of the enteric nervous system (ENS). The ENS comprises the neurons of the enteric plexus, which may regulate muscular activity and neurons of the CNS which form neurohemal endings, primarily at the boundary between the midgut and hindgut. In addition to this * Corresponding author. Tel.: +1 909 7874369; fax: + 1 909 7873087; e-mail: [email protected]

innervation, the midgut harbors a number of endocrine and/or paracrine cells scattered amongst the cells of the epithelium. All three groups of cells exhibit a peptidergic phenotype in diverse orders of insects (Sehnal and Zitnan, 1996; Zitnan et al., 1993), but the full complement of chemical messengers has not been characterized for any of the groups. Amongst those neuropeptides found in the midgut of insects by immunocytochemistry are the FMRFamides. Members of this structural family of neuropeptides, containing Phe-X-Arg-Phe-NH2 (where X is Met, Leu, or Ile) at the C-terminus, are found in the CNS of most, perhaps all, orders of insects. In lepidopterous

0303-7207/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 0 3 - 7 2 0 7 ( 9 7 ) 0 0 1 4 0 - 8

20

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

insects FMRFamide-like peptides (FLP) are known to occur in all three groups of neural or endocrine elements in the midgut (Zitnan et al., 1993; Copenhaver and Taghert, 1989; Zitnan et al., 1995a). In the tobacco hornworm Manduca sexta a morphological subset of intrinsic neurons in the anterior midgut is reactive with FMRFamide antisera (Copenhaver and Taghert, 1989), while the proportion of endocrine/paracrine cells that are reactive with antisera has not been determined (Zitnan et al., 1995a). Is the complement of endocrine cells and neurons capable of producing and releasing neuropeptides fully reflected by immunocytochemical results from normal insects? In M. sexta, parasitism by braconid wasps leads to a dramatic increase in the number of endocrine cells in the midgut (gut endocrine cells; GEC) that are revealed with FMRFamide antisera (Zitnan et al., 1995a). Together with findings from parallel studies on CNS structures (Zitnan et al., 1995b), these results showed that parasitism affects accumulation of neuropeptides in both peripheral and central elements of the host, possibly reflecting a widespread failure in secretory function. Parasitism of lepidopterous insects by braconid wasps often leads to cessation of feeding and developmental arrest late in the host’s larval life. While developmental arrest is permissive for completion of the parasite’s development, we do not have a complete understanding of the physiological changes associated with arrest. In several species, juvenile hormone (JH) titers are elevated and ecdysteroid titers are suppressed in parasitized last instar larvae (Lawrence and Lanzrein, 1993; Beckage and Riddiford, 1982). Because decline in the titer of JH in the final instar is apparently required for the release of prothoracicotropic hormones (PTTHs) (Nijhout and Williams, 1974) and the steroidogenic competence of the prothoracic glands (Watson and Bollenbacher, 1988), it is possible that the failure to clear JH partially accounts for developmental arrest following parasitization. Failure to release PTTHs would be manifested by their accumulation in the CNS, as, indeed, was found in the CNS of parasitized Manduca sexta larvae (Zitnan et al., 1995b). The latter finding is not specific for the PTTHs, but instead reflects a general accumulation of multiple neuropeptides in the CNS (Zitnan et al., 1995b). While it is known that members of the FMRFamide family from the CNS of several insect species (Walker, 1992), including M. sexta (Kingan et al., 1990, 1996) are myotropic or myosuppressive on skeletal and visceral muscle (Kingan et al., 1990, 1996; Holman et al., 1986; Fonagy et al., 1992; Lange et al., 1994; Robb et al., 1989), the function of peptide chemical messengers from the gut of insects remains enigmatic. Thus, it seemed possible that a model comprising the parasitized insect might be useful in determining functions for

midgut peptides by revealing a link between accumulation of FMRFamides and a deficit in some specific aspect of gut function. As a first step in determining whether these peptides could act as endocrines or paracrines and ultimately what their role might be in the function of the alimentary canal, we sought to characterize the immunoreactive peptides.

2. Materials and methods

2.1. Animals and dissections Larvae were reared and parasitized as either first or fourth instars by the braconid wasp Cotesia congregata as described earlier (Zitnan et al., 1995a; Beckage et al., 1994). Midguts were removed from the following groups of insects: (1) nonparasitized feeding larvae on the first (N1) and third day (N3) after ecdysis to the 5th instar, weights 2.5–3 g and 6–8 g, respectively; (2) N1 insects, at 2.5–3 g, starved for 7 days and then used for purification of F24 from whole midguts (S7); (3) N1 insects, at 3–3.5 g, starved for 7 days for use in fractionation of extracts from midgut fragments and CNS structures (S7); (4) second gate wandering larvae on day 5, weights 8–10 g (W); (5) parasitized insects, weights 2.5–5 g, 7 days after emergence of wasp larvae P(Em7). Midguts were removed and cut longitudinally to remove the peritrophic membrane and any remaining gut contents. After rinsing in M. sexta saline (Riddiford et al., 1979), guts were blotted on tissue paper, frozen together on dry ice, and stored at − 80°C. For analysis of peptides in the CNS, the combined supraesophageal (brain), frontal and subesophageal ganglia (Br/SEG) were removed from groups of :50 nonparasitized, starved and parasitized insects. Tissues were rinsed, pooled for each group and stored frozen at − 80°C.

2.2. Immunohistochemistry The procedure for staining FLP in the midguts was similar to that described earlier (Zitnan et al., 1993, 1995a). Midguts from N1 and P(Em7) larvae were dissected and cleaned as described above, fixed in Bouin’s fixative and washed in 70% ethanol followed by phosphate-buffered saline with 0.5% Tween-20 (PBST). After pre-incubation with 5% normal goat serum, tissue was incubated with rabbit antiserum to FMRFamide (‘c671M’, generously provided by Dr Eve Marder, Brandeis University) and diluted 1:1000 in PBST for 2 days at 4°C. The rabbit IgG was detected by incubation with biotinylated goat-anti rabbit IgG followed by streptavidin-biotinylated horseradish peroxidase complex (Jackson Immunoresearch, West Grove, PA). Finally, peroxidase was stained with 3,3%-

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

diaminobenzidine (Sigma Chemical, St. Louis, MO) and the stained tissue was mounted in glycerol.

2.3. Immunoassays FLRFamides were assayed during the purification process by ELISA as previously described (Kingan et al., 1990; Kingan, 1989) with the use of F10 (Kingan et al., 1990) as a calibration standard. The ELISA uses an adsorbed FLRFamide – BSA conjugate in competition with sample peptide for binding to FMRFamide antibodies. The rabbit antiserum was the same as that used for immunohistochemistry, except that it was diluted 1:30 000. It is equally reactive toward FMRFamide and FLRFamide, but its reactivity toward nonamidated FMRF is at least 104-fold lower (Marder et al., 1987). For quantitation of individual FLRFamides in extracts of midgut and central ganglia a new enzymelinked immunoassay (EIA) was developed to optimize sensitivity. This assay uses a peptide-enzyme conjugate in competition with sample peptide for binding to antibody. The preparation of the conjugate was by a two-step glutaraldehyde procedure (Harlow and Lane, 1988). Briefly, 200 ml 10% glutaraldehyde in water was added to 80 nmol F7G (MasFLRFamide II (Kingan et al., 1996)) in 200 ml PBS and the mixture was allowed to stand at room temperature for 1 h. Under these conditions of excess glutaraldehyde, the single primary amine in the peptide was acylated, but the peptide did not cross-link with itself. After adsorption of the glutarylated peptide in a C8 Sep-Pac Lite cartridge (Millipore, Milford, MA), the unreacted glutaraldehyde was removed by rinsing with 2 mM sodium phosphate, pH 6.0. The derivatized peptide was then eluted with 0.6 ml 2 mM sodium phosphate:acetonitrile, 1:1. This volume was reduced 50% by vacuum centrifugation. Then :10 nmol (0.45 mg) horseradish peroxidase (Sigma) was added in 300 ml 40 mM sodium phosphate/0.3 M NaCl, pH 7.4. The reaction mixture was allowed to stand overnight at 4°C. Uncoupled peptide was removed from the F7G-HRP conjugate by size exclusion chromatography (G3000 SWXL, 7.8 mm ×300 mm; TOSO HAAS, Montgomery, PA) in 0.1 M Na + /K + phosphate, pH 7.0. Fractions were diluted and assayed for peroxidase activity with tetramethylbenzidine (TMB)/ H2O2 as in ELISA. Active fractions were pooled, diluted with one volume of glycerol and stored at − 20°C. Standard solutions of peptides were prepared for use in EIA after quantitation by amino acid analysis. Assays were carried out as follows: Firstly, 0.5 mg affinitypurified goat anti-rabbit IgG or Fc (Jackson Immunoresearch) was adsorbed in 70 ml 0.01 M sodium phosphate/0.15 M NaCl, pH 7.4 (PBS), to the wells of an ELISA plate (Corning Easy Wash ELISA plate, Corning, NY) for 15 – 18 h at room temperature. The

21

contents were discarded and without rinsing 315 ml 0.02 M sodium phosphate/0.15 M NaCl/1 mM Na2EDTA/ 0.1% BSA, pH 7.4 (EIA buffer) with 0.002% sodium azide was added to each well. After 1 h the plate was rinsed with PBS containing 0.05% Tween-20. The rinse was discarded and 75 ml rabbit anti-FMRFamide (Marder et al., 1987), diluted 1:30 000 in EIA buffer, was added to each well. Samples and standards were then added in 25 ml aliquots, followed by 25 ml of 1:125 diluted F7G-peroxidase conjugate. The plate contents were then mixed for 5 min on a plate shaker (Mini-Orbital Shaker, Bellco Biotechnology, Vineland, NJ) and then incubated overnight, without mixing, at 4°C. The plate was then rinsed, developed with TMB/H2O2 for 10–20 min, quenched with 1 M H3PO4 and read at 450 nm as described previously (Kingan, 1989). This assay is similar to that described for atriopeptin (McLaughlin et al., 1987), except that the latter assay used acetylcholinesterase (from electric eel) coupled to peptide with a heterobifunctional reagent. The reactivities in EIA of F10 and the FLRFamides described in this report are equal (Fig. 1).

2.4. Purification of FLRFamides from midguts Peptides were purified from two pooled samples, each of : 60 midguts. Midguts were placed frozen in a ground glass homogenizer with 10-fold excess (volume:weight) 90% methanol/9% water/1% acetic acid (90:9:1). The tissue was then homogenized by hand with a ground glass pestle until fully disrupted. The homogenate was centrifuged at 12 000× g for 15 min at 4°C. The supernatant was removed and the pellet was

Fig. 1. Dose response curve in EIA for F10, F24, and F39. This assay is based on the competition of free peptide with peptide covalently coupled to HRP (see Section 2). The fitted curve for F10 values was calculated by desktop computer with a four-parameter logistic equation. Note that the three peptides are equipotent, showing that the N-terminal extensions of the longer peptides do not participate in their reactivities. Thus, determinations from an assay calibrated with F10 reflect actual levels of all three peptides. Very low coefficients of variation (typically 54%) made quantitation at 2 fmol/well routine.

22

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

resuspended with a glass stirring rod in 1/2 the original volume of fresh 90:9:1. This mixture was centrifuged as above, the supernatants were combined and reduced in volume by :90% in a rotary evaporator. The contents were removed and the flask was rinsed with several milliliters of 0.1% trifluoroacetic acid (TFA). The combined extract and rinse were desalted on three C18 Sep-Pac cartridges attached in tandem with plastic pipette tips. After adsorption of the extract the cartridges were rinsed with 0.1% TFA and eluted with 60% acetonitrile/0.1% TFA. This material was diluted with four volumes of 0.1% TFA and loaded into a 1 cm C8 HPLC column (Dynamax, Rainin Instruments) by multiple injections via a 9 ml sample loop. The chromatogram was developed with a gradient of acetonitrile in 0.05 M sodium phosphate, pH 6.0 at a flow rate of 1.0 ml/min for the first batch of midguts and 1.3 ml/min for the second batch. One minute fractions were collected and 1 ml from each fraction was removed and placed directly in wells of a pre-coated ELISA plate (see Section 2.3). Immunoreactive fractions were pooled and chromatographed using a 4.6 mm C4 column (Vydac, The Separations Group, Hesperia, CA) with a gradient of 2-propanol in 0.1% TFA and a flow rate of 0.7 ml/min. Finally, immunoreactive fractions were pooled and chromatographed in a 2.1 mm C4 column (Vydac) with a gradient of acetonitrile in 0.1% TFA and a flow rate of 0.2 ml/min. To compare the complement of FLP found in parasitized and nonparasitized insects, we separately purified, by procedures identical to those described above, FLRFamides from the midguts of N4, W, and S7 insects.

2.5. Sequence determination and peptide synthesis In each case a small portion of the purified peptides was used for determination of its molecular weight by electrospray mass spectrometry (ESMS) in a triple quadrupole mass spectrometer (model TSQ-700, Finnigan-MAT, San Jose, CA). For the midguts of parasitized insects, an aliquot comprising 5% of the purified material was set aside and the remainder was subjected to automated Edman degradation (pulsed liquid protein sequencer, model 473A, Applied Biosystems, Culver City, CA). Peptides were synthesized commercially (Biotechnology Facility, Texas A and M University). The crude material was found by HPLC and ESMS to be :50% oxidized in the single methionine of each peptide. Therefore, methionines were first reduced with N-methylmercaptoacetamide (5% reagent in 1% acetic acid for 15 h at 30°C; see Houghten and Li, 1979). Then peptides were purified by HPLC in the 1 cm C8 column (see Section 2.4) using a gradient of acetonitrile in 0.05 M sodium phosphate, pH 6.0, followed by a second run in the same column with a gradient of acetonitrile in 0.1% TFA.

2.6. Quantitation of FLRFamides in tissues Individual FLRFamides were quantified in fragments of midgut, in batches of Br/SEGs and in hemolymph. Single midguts from nonparasitized or parasitized insects (N1, S7, P(Em7)) were dissected, freed of Malpighian tubules, cut longitudinally, cleaned and cut transversely into three pieces of approximately equal sizes. Batches of Br/SEGs from : 50 insects of different groups were collected and stored at − 80°C. Tissues were homogenized in 90:9:1 and the insoluble material was removed by centrifugation. The pellet was rinsed once with fresh 90:9:1. The combined supernatants were diluted 10-fold with 0.1% TFA and desalted on Sep-Pac Lite cartridges as in Section 2.5, peptides were eluted with 50% acetonitrile in 0.1% TFA. This material was either partially dried by vacuum centrifugation (midgut fragments) or diluted 5-fold with 0.1% TFA (Br/SEGs) prior to loading onto the 2.1 mm C4 column and HPLC. The flow rate was 0.2 ml/min and 0.5, 1 or 2 min fractions were collected in different regions of the chromatogram, depending on the resolution desired. Aliquots of 20 mg BSA in water were added to each fraction, which were then frozen and dried by vacuum centrifugation. The dried fractions were reconstituted in EIA buffer for quantitation by EIA as described above. For hemolymph, the dorsal horn was cut and hemolymph was collected into a test tube held on ice. Whole hemolymph was heated to 90°C for 3 min and centrifuged at 12 000× g for 3 min. The supernatant was removed and the pellet was washed with an equal volume of 0.1% TFA. After centrifuging the supernatants were pooled and passed through 0.2 mm syringe filters. An amount corresponding to 150 ml hemolymph was chromatographed. BSA was added to each fraction as above and the samples were frozen and dried by vacuum centrifugation. They dried samples were resuspended in 150 ml EIA buffer, dissolved by sonication in a water bath for 2 min, frozen at − 70°C and then thawed for assay. Recovery with this protocol was determined to be : 50% for each peptide from hemolymph samples spiked with F10, F24 and F39 prior to heating.

3. Results

3.1. Characterization of new FLRFamides from the midguts of parasitized lar6ae The CNS of M. sexta larvae harbors a number of FLRFamides, three of which have been purified and sequenced (Kingan et al., 1990, 1996). Thus, it seemed possible that similar or identical peptides would be in the midgut. After finding a dramatic increase by immunoreactive FLP in midguts from parasitized relative

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

23

Fig. 2. Purification of an immunoreactive FLRFamide from midguts of parasitized M. sexta. Desalted extract was first fractionated (see Section 2) in (A) a 1 cm C8 column with a gradient of acetonitrile in 0.05 M sodium phosphate, pH 6.0; flow rate was 1.3 ml/min. Fractions 9 –68 were then chromatographed in (B) a 4.6 mm C4 column with a gradient of 2-propanol in 0.1% TFA. Fraction 34 was then run in (C) a 2.1 mm C4 column with a gradient of acetonitrile in 0.1% TFA. Fraction 49 from (C) was subjected to ESMS and sequence analysis by automated Edman degradation.

24

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

to nonparasitized larvae (Zitnan et al., 1995a), we wanted to identify the reacting peptides. This accumulation in parasitized larvae facilitated the purification of peptides from the extracts of two batches of midguts. In one purification, the majority of FLP was found in a single fraction, eluting very late in the chromatogram (Fig. 2A). Fractions c 68 – 69 from this chromatogram were pooled and fractionated with a different solvent system and column (see Section 2); again FLP was largely confined to a single fraction (Fig. 2B). Fraction c 34 from this chromatogram was then run in a narrow bore column (Fig. 2C), yielding immunoreactive peptide in fraction c49. A portion of fraction c49 was analyzed by ESMS, and the main component in this fraction had a mass of 4527.3 Da. The remainder of the fraction was subjected to automated Edman degradation, revealing a peptide with 39 amino acids of the following sequence: YAEAAGEQVPEYQALVRDYPQLLDSGMKRQDVVH*SFLR*F* (determinations in starred cycles were with less than the usual degree of certainty). These assignments are, however, consistent with the mass determination. In addition, the reactivity of the peptide with the FMRFamide antiserum indicated the C-terminal Phe is present as the amide rather than the free acid (see Section 2); amidation is also consistent with the mass determination. A second peptide was then purified from a separate pooled sample of midguts (see Section 2). The purified material was found by ESMS to have a mass of 2906.5 Da. Edman degradation unambiguously revealed a 24 amino acid peptide of the sequence: VRDYPQLLDSGMKRQDVVHSFLRF. This sequence is identical to the presumptive C-terminal 24 amino acids of the 4527.3 Da peptide. A portion of this purified material was also digested with endoproteinase Lys-C. Upon fractionation of the digest by HPLC two purified peptides were obtained and the sequences were unequivocally determined to be: VRDYPQLLDSGMK and RQDVVHSFLRF. These sequences are predicted from that of the intact peptide and their determination relieves the uncertainty in the assignment during Edman degradation of the longer peptide. The new FLRFamides, designated F39 and F24, were chemically synthesized with C-terminal amides and purified. When the synthetic peptides were added to their presumptive tissue peptides and chromatographed (under conditions identical to those in Fig. 2C), single peaks with the expected retention times and integrations were obtained (not shown). The sequences of the new peptides are shown with the other members of this family from M. sexta in Fig. 3. Notably, the C-terminal ten amino acids of F24 and F39 are identical to the previously characterized sequence of a peptide neurohormone from the Br/SEG (Kingan et al., 1990). The ‘Q’ (Gln) in the longer

Fig. 3. Amino acid sequences of the midgut peptides and the previously characterized FLRFamides from M. sexta.

peptides, if exposed as an N-terminal amino acid, would be enzymatically or spontaneously cyclized to give rise to the ‘pE’ (pyroglutamyl) residue. Since the new peptides are named according to their length, the decapeptide hormone is renamed F10. The previously characterized heptapeptides from M. sexta (Kingan et al., 1996) are renamed F7G and F7D to conform with the new, simpler terminology.

3.2. Distribution of FLRFamides in the midgut: relationship to feeding In earlier studies an increased number of GEC stained with FMRFamide antisera and quantity of extractable FLP was found in the midguts of parasitized insects (Zitnan et al., 1995a). To address the possibility that the newly identified FLRFamides could account for changes after parasitization, we took two approaches. Firstly, the identities of F24 and F39 in tissues from nonparasitized insects were confirmed by purification and ESMS. In addition, F24 was identified in tissue from starved insects by its reactivity in ELISA and its chromatographic properties in three HPLC systems. The yields of these peptides after purification are shown in Table 1. Significantly, the yield of F24 from midguts of feeding insects is much lower than from midguts of parasitized, starved and wandering insects. While the latter groups have the cessation of feeding in common, we cannot conclude that this condition alone (or in part) is responsible for the high levels seen in parasitized insects (see Section 4), which had ceased their food consumption 9 days earlier. In the second approach, we quantified F24 and F39, as well as other identified FLRFamides, in anterior, middle and posterior fragments of midgut. In addition to learning about the effects of parasitism, we wanted to correlate these determinations with the distribution of cellular elements in the ENS (Copenhaver and Taghert, 1989; Zitnan et al., 1995a). The fractionation of identified M. sexta FLRFamides and extracts of midgut fragments from N3 larvae and P(Em7) larvae parasitized in the fourth instar are shown in Fig. 4. Peaks with chromatographic properties identical with the five identified FLRFamides were found in nonpara-

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

25

Fig. 4. FLRFamides in fractionated extracts of midguts. (A) Fractionation of identified M. sexta FLRFamides with the same conditions used for tissue extract. Retention times are: F7G, 28.4 min; F7D, 31.0 min; F10, 36.5 min; F24, 38.5 min; F39, 43.8 min. (B) Nonparasitized 5th instar larvae (N1) and (C) parasitized 5th instar larvae 7 days after emergence of parasitoids (P(Em7)). The bars represent the total amount of immunoreactive peptide in the midgut and the proportions of that total in each fragment.

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

26

sitized insects (Fig. 4B). Additional preparations, similarly fractionated, (not shown) also revealed peaks of FLI at 33 and 54 – 56 min (Fig. 4B) and in some parasitized insects, 46 min (not shown). These peptides have not yet been identified. The findings with respect to F39, F24 and F10 are based on analyses of three N3 and three P(Em7) insects (three fractionations/assays per insect) and can be summarized as follows: 1. In nonparasitized insects the amount of F39 is several-fold greater than F24 (Fig. 4B). 2. F39 is 52-fold greater in parasitized than in nonparasitized insects. The range of F39 in parasitized insects was between 500 fmol and 5.5 pmol per midgut (Fig. 4C). 3. F24 was several-fold greater in parasitized insects than in nonparasitized feeding insects (compare Fig. 4C and B). The amount of F24 in midgut fragments from nonparasitized insects was always 5 400 fmol, while in parasitized insects it was between 3 (Fig. 4C) and 12 pmol (not shown) per midgut. 4. F24 and F39 were relatively depleted anteriorly and in some individuals, they could be almost exclusively confined to the middle portion of the midgut (Fig. 4C). This distribution resembled that for FLP at 54–56 min in the chromatogram from nonparasitized insects (Fig. 4B). Greater variability existed in the quantities of F39 and F24 in parasitized insects relative to nonparasitized insects. 5. F10 was always relatively low in the middle region of the midgut and was sometimes highly enriched in the anterior region (Fig. 4B) or in the posterior region (not shown). It was shown earlier that parasitization evokes a dramatic increase in the number of GEC staining with Table 1 Yields of FLRFamides from midguts of M. sexta Group

Peptide isolated

Yield (pmol/g)

N3

F24 (2907.1 Da), oxid’d F24 (2922.2 Da)

W

F39 (4540.7 Da) F24

22 7

S7 P(Em7) P(Em7)

F24 F39 (4527.3 Da) F24 (2906.5 Da)

33 130 109

0.8

Yields of identified FLRFamides from midguts of different experimental groups. Not all peptides were identified in each pool of midguts. Values are in pmol peptide per g wet weight of tissue. The molecular weights of peptides were obtained by ESMS. In some cases the single methionine of each peptide was oxidized, adding 16 Da to the expected mass. Abbreviations (see also Section 2): N3, nonparasitized, day 3 of 5th instar; W, wandering, B24 hr after initiation of the behavior; S7, 5th instar larvae, starved for 7 days; P(Em7), parasitized, 7 days after emergence of the parasitoids.

a FMRFamide antiserum (Zitnan et al., 1995a). Now, the enrichment of F24 and F39 in the middle and posterior midgut prompted us to reexamine the distribution of FLP-containing cellular elements in the anterior–posterior axis. As noted earlier, the somata of the intrinsic neurons (Fig. 5A and D) are found in the anterior midgut (Copenhaver and Taghert, 1989; Zitnan et al., 1995a). On the other hand, a great many more stained GEC are found in the mid- and posterior regions than in the anterior region (Fig. 5B and C). Moreover, while parasitization leads to the appearance of some stained GEC in the anterior midgut (Fig. 5D), the response is especially dramatic in the middle and posterior regions (e.g. compare Fig. 5B and E). As observed earlier (Zitnan et al., 1995a), small GEC are often of the open-type, containing apical processes (Fig. 5C). In some preparations from parasitized insects affording an oblique view of the epithelium, nearly all GEC were found to have apical processes (data not shown). A prominent feature of GEC from parasitized insects was the appearance of basal processes (Fig. 5G and H).

3.3. FLRFamides in the central ner6ous system Thus, we established that F10, F24 and F39 are candidate chemical messengers in the midgut. Since F10 has been determined to be a CNS peptide (Kingan et al., 1990, 1996), it was of interest to determine if the longer peptides are also present in the CNS. In addition, because earlier immunocytochemical studies showed that FLPs accumulate in neurosecretory cells and other neurons of the CNS (Zitnan et al., 1995b), we wanted to know if parasitization affects the Br/SEG content of members of this family. Extracts of tissue from N4, S7 and P(Em7) insects were fractionated and compared in their profiles of immunoreactive peptides. With two exceptions, all three extracts contained the same set of peaks, and a similar amount of each immunoreactive peak (51.5fold difference) was found in the three extracts. The first exception was from S7 insects; here an immunoreactive peak containing :15 fmol per Br/SEG was present at 40–41 min (not shown). The second exception was from P(Em7) insects in which 20 fmol per Br/SEG was found at 56 min (Fig. 6). This amount was 5-fold greater than that found in nonparasitized insects and :3-fold greater than that found in starved insects (not shown). The latter peptide is probably the same as that found enriched in the middle midgut (Fig. 4B); its identity has not yet been determined. The most abundant peptides were F7G at 28.5–29 min and F10 at 36.5–37 min (Fig. 6). These peptides were identified previously in similarly fractionated ex-

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

27

Fig. 5. FLI in the midgut of nonparasitized N1 (A–C) larvae and parasitized P(Em7) (D – F) and P(Em14) (G, H) larvae. In the anterior region (A), staining was detected in neurons (arrowheads) and axons of the ENS and a few small GEC, while middle and posterior regions (B, C, respectively) showed reaction in several types of GEC and axons of the ENS. Note that large GEC (arrowheads) were only detected in the middle region under the longitudinal muscle bands that were innervated by axons of the ENS. The number of neurons exhibiting FLI in the ENS is similar in parasitized and nonparasitized larvae, but the number of GEC was considerably higher in parasitized insects, particularly in the middle and posterior regions. Most GEC of P(Em14) larvae contained strongly reacting basal processes (G, H, middle and posterior regions, respectively), indicating accumulation of FLPs. Scale bar: 100 mm in A—F; 500 mm in G and H.

tracts (Kingan et al., 1996). Additional peaks that could be reliably quantified were found at 22 – 23, 33 and 56 min. Small peaks were found in all three extracts at 38 – 39, 46–47 min and in starved insects, 40 – 41 min. The small peak at 38 – 39 min corresponds in retention time with F24. The immunoreactivity in these fractions consistently rose above that in the adjacent fractions at 37.5 and 40 min. The amount of material quantified was very low (see Section 4), :1 – 2 fmol in each fraction per Br/SEG. We did not detect FLP in 43–44 min, which would contain F39, that rose above that in the adjacent fractions. The limit of sensitivity under the conditions of our assay is : 0.4 fmol per Br/SEG.

3.4. FLRFamides in the hemolymph The identification of F24 and F39 from the midgut and their presumptive localization in GEC raises the possibility that they function as hormones. In fact, our use of the term ‘endocrine’ here is based on immunocytochemical and morphological criteria and is not yet documented by biochemical studies of release. Thus, as a first step in addressing the possibility that F24 and/or F39 function as hormones, we fractionated hemolymph by HPLC after a preliminary heat precipitation. In all nonparasitized feeding insects tested, the most abundant circulating FLRFamide was F10 (Fig. 7A; 36

28

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

Fig. 6. Immunoreactive FLRFamides in the Br/SEG of parasitized 5th instar larvae. Note that 0.5, 1, and 2 min fraction sizes were collected in order to increase resolution in specific regions of the chromatogram. Peptides previously identified include those at 28.5 – 29 min (F7G) and at 36.5–37 min (F10). Additional peaks of immunoreactivity are at 23 – 24, 33, and 56 min. Smaller peaks are consistently found at 38 – 39, 46–47, and, in starved insects, 40–41 min.

min). An additional peak of immunoreactivity, which has not been identified, was found at 40 – 41 min; this corresponds in retention time with the material that accumulates in the Br/SEG of starved insects (see Section 3.3). However, the distribution of immunoreactivity does not support a finding of circulating F24 or F39: the reactivity of those fractions which would contain F24 (38 min) or F39 (43 min), was never higher than in their adjacent fractions. Here, the limit of detection was 0.14 pmol/ml hemolymph. The profile of heat-stable and UV absorbing material in hemolymph from parasitized insects was profoundly altered from that in nonparasitized insects (Fig. 7B). F10-like material and the peak at 40 – 41 min were each several-fold lower than in the hemolymph of nonparasitized insects. However, a new peak of immunoreactivity, at 47–48 min appeared, with amounts in each fraction corresponding to \ 10 nM ir-F10 in hemolymph. This material is not specific to parasitized insects, in that it also appears in starved insects (Fig. 7C). However, its concentration in hemolymph from parasitized insects was typically 10-fold higher than in starved insects, whether parasitized insects were 7 days (Fig. 7B) or 4 days (not shown) post-emergence. The results of the CNS and hemolymph determinations are summarized in semi-quantitative form in Table 2.

4. Discussion We have isolated and characterized two neuropeptides in the FLRFamide family from larval midguts of parasitized M. sexta. The sequence of F24 is identical to the C-terminal 24-mer of F39. While immunocyto-

chemical studies have documented the presence of neuropeptides in the midgut for a number of orders of insects (Zitnan et al., 1993; Brown et al., 1986; Jenkins et al., 1989; Montuenga et al., 1989; Reichwald et al., 1994), only three other peptides from this tissue have been characterized: an otherwise unrelated RFamidecontaining peptide from Periplaneta americana (Veenstra and Lambrou, 1995), an allatostatin from the cockroach Diploptera punctata (Reichwald et al., 1994) and a myotropic pentapeptide from M. sexta (Yi et al., 1995). Excluding the C-terminal RFamide, F24 and F39 have no homology with the pancreatic polypeptides or peptide YYs or the recently characterized neuropeptide F (Spittaels et al., 1996). The new peptides are striking in that the sequence of a previously characterized decapeptide from M. sexta, F10 (Kingan et al., 1990), is conserved at their C-termini. The pyroglutamyl residue of F10 would arise in biosynthesis by cyclization of an N-terminal glutamine. The glutamine in F24 and F39 is preceded by a pair of basic amino acids, Lys-Arg. Together, these features indicate that F24 and F39 could serve as precursors for F10, since the domains of biologically active peptides in precursors are usually flanked by pairs of basic amino acids (Douglass et al., 1984). A pair of basic amino acids is also found in the D. punctata allatostatin (Reichwald et al., 1994), which is apparently not processed in the midgut. F24 and F39 have some demonstrated and predicted structural features of processing intermediates in a biosynthetic pathway for F10 in the midgut and CNS. Indeed, F10 is found along with the longer peptides in fractionated extracts of midguts. However, it is possible that the major portion of F24 and F39 is not processed

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

29

Fig. 7. Immunoreactive FLRFamides in hemolymph from 5th instar caterpillars. FLPs in fractions 20 – 59 were quantified. Those in which the concentration exceeded 0.14 pmol/ml hemolymph are represented by a bar in the histogram. (A) Feeding, non-parasitized larvae, N1, 3.0 g weight. The peak in 36 min has previously been identified as F10. (B) Parasitized larva, 7 days after emergence of wasp larvae (P(Em7)). Note the difference in the y-axis scale of A and B. The appearance of the UV profile is significantly altered from that of the N1 and S5 (below) larvae. (C) Insect starved for 5 days (S5), beginning as a 3.0 g larva on the second day of the fifth instar.

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

30

Table 2 Br-SEG/hemolymph ratios for FLRFamides in larvae of M. sexta Group

36 (F10)

38.5–39 (F24)

40 – 41

47 – 48

56 – 57

N1 P(Em7) S7

+++/++ ++++/+ ++++/−

+/− +/− +/−

−/++ −/− ++/+

+/− +/++++ +/++

+/− +++/− ++/−

FLRFamides as a Br-SEG/hemolymph ratio for five FLPs. FLPs are identified by their retention times in HPLC (see Figs. 6 and 7). The range of the Br/SEG entries are, per structure (fmol): −, B0.4; +, 1 – 5; ++, 6 – 10; +++, 11 – 20; ++++, 21 – 60. The range of hemolymph entries are, per ml (pmol): −, B0.14; +, 0.14–0.3; ++, 1–3; ++++, 25. Abbreviations as in Table 1.

to F10, but is stored in neurons or GEC separate from those containing F10. We have attempted to address this possibility by determining the regional distribution of the three peptides. The cell bodies of intrinsic neurons are found anteriorly in the midgut along bands of longitudinal muscles; some neurites extend posteriorly into the mid- and posterior regions of the midgut (Copenhaver and Taghert, 1989; Zitnan et al., 1995a). The neurohemal endings of the proctodeal nerve are largely confined to the posterior regions, although some processes extend anteriorly (Zitnan et al., 1995a). We show here that GEC are highly enriched in the midand posterior regions (Fig. 5). The finding, then, that F24 and F39 are almost completely confined in some insects to the middle portion of the midgut suggests that they are produced in the GEC. If so, this would not exclude the possibility that some F10 is produced in these cells; however, the relatively small amount of F10 in the middle portion of the midgut suggests that it could be attributable to endings of the intrinsic neurons and/or neurohemal endings from the proctodeal nerve. Conclusive experiments in determining cellular distributions of F10, F24 and F39 must await the availability of specific probes. The amount of F10 in the Br/SEG is relatively high and the amount of F24 is very low (Table 2). It will be important to confirm the finding of small amounts of F24 in the CNS by independent measurements, for instance, by pulse-chase studies and immunoprecipitation. Nevertheless, the tissue distributions of FLRFamides raise the possibility that a larger precursor or the prohormone itself gives rise to different sets of peptides in the CNS and gut, as has been shown to occur in mammalian studies (Dickerson and Noel, 1991). For instance, the primary product of prosomatostatin produced by cerebral cortical cells in culture (Robbins and Reichlin, 1983) and rat pancreas and pyloric antrum (Patel and O’Neil, 1988) is somatostatin-14, while in the rat jejunal mucosa the primary product is somatostatin-28 (Patel and O’Neil, 1988). The relationships of F24/F10 and F39/F10 pairs of peptides are suggestive of processing events mediated by subtilisin-like convertases (Steiner et al., 1992). In addition, the F39/F24 pair suggests a possible chymotrypsin-like processing event. Cleavages of this spe-

cificity are relatively rare in the processing of precursors for peptide hormones and little is known of the enzymatic process (Cretien et al., 1989). The existence in the posterior pituitary of, for instance, the Ala1-Leu17 fragment of the propressophysin C-terminal glycopeptide (Holwerda, 1972) reveals a possible Leu-Leu processing event. However, at this time both types of processing in M. sexta are speculative and will require experimental documentation. Parasitization by the braconid wasp C. congregata causes an increase in the number of GEC expressing detectable FLI and an enhancement in accumulation in individual cells (Zitnan et al., 1995a and this report). A biochemical basis for these observations is provided with the finding that accumulation of identified FLRFamides may exceed by many-fold that found in the midguts of feeding insects. This increase far exceeds that found in the Br/SEG for any of the FLRFamides, showing that parasitization differentially affects neurosecretory and endocrine cells. Since parasitized insects had not been fed for 7 days prior to removal of their midguts, the accumulation of F24 in midguts of starved insects (Table 1), raises the possibility that some of the influence of parasitism and starvation on peptide accumulation is attributable to similar physiological events. The increase in content of F24 in post-feeding (wandering) nonparasitized insects, when compared to feeding insects of similar size and age, further suggests an inverse relationship between feeding and peptide content in the midgut. F24 may increase nearly 10-fold upon cessation of feeding and onset of wandering (Table 1). Thus, the peptide content of the midgut increases during three different states of non-feeding. The mechanisms by which parasitization or starvation lead to accumulation of FLRFamides is not yet known. The data from midgut and Br-SEG raise the possibilities that either biosynthesis is increased or release is decreased. The depletion of F10 and the FLP at 40–41 min from hemolymph in parasitized insects (Fig. 6) favors an explanation incorporating a block in normal release. However, a simple and global block in peptide release cannot explain all the results, in that the FLP at 47–48 min increases in hemolymph with starvation and parasitism (Table 2).

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

It is possible of course that endocrine events in the host associated with developmental arrest, namely the suppression of ecdysteroids and the failure to clear juvenile hormone (Beckage and Riddiford, 1982), could be linked with accumulation of FLRFamides. An interesting parallel in M. sexta is the recent observation that FLRFamides are normally depleted from motoneurons of the segmental ganglia under the influence of the pupation peak of ecdysteroids and that peptides accumulate if cultured in the ecdysteroid-free environment of the diapausing pupa (Witten and Truman, 1996). Thus, if intrinsic neurons and endocrine/paracrine cells of the midgut are similarly regulated, the accumulation of FLRFamides could occur because the appearance of ecdysteroids associated with pupation is prevented. However, the increase in midgut F24 after the commitment peak of ecdysteroids and the onset of wandering (Table 1) argues against the suppression of ecdysteroids as being the sole determiner in accumulation of FLRFamides. Additional insight into regulatory events might be gained when the effect of the pupation peak of ecdysteroids on midgut FLRFamides is determined. The role played by peptides from intrinsic neurons and endocrine/paracrine cells in the function of the midgut is not known. The movement of foodstuff through the foregut into the midgut occurs by bulk flow and by the action of myogenic contractions of the foregut. Neurites from neurons of the enteric plexus in the anterior midgut invest the circular muscles between the longitudinal bands (Copenhaver and Taghert, 1989) and could respond to stretch via reflex arcs. In another sphingid moth, F10 has been shown to be myoinhibitory in the adult midgut (Fujisawa et al., 1993). Thus, it is possible that some FLRFamides serve to relax visceral muscle to facilitate movement of food into the midgut with foregut contractions. A similar principle operates in the mammalian small intestine in which relaxation of circular muscles mediated by descending inhibitory neurons allows progression of contents through the gut that occurs with peristalsis (Sundler et al., 1989; Furness et al., 1992). GEC, which are found primarily in the mid- and posterior midgut, rest on the basal lamina amongst absorptive and secretory cells of the epithelium. Those GEC of the ‘open-type’ have apical processes with microvilli extending to the gut lumen (Endo and Nishiitsutsuji-Uwo, 1981) and might be expected to respond to the changing milieu of the gut lumen. Indeed, in some preparations of the midgut, most of the small GEC could be seen to have apical processes. Cells of the closed type, without apical processes, might respond to changing mechanical properties of the gut during feeding. If as suggested above, F24 and F39 are produced and released by GEC, the observation that they cannot be detected in the hemolymph argues against an endocrine function. The possibility that peptides are

31

released as paracrines is supported by findings in parasitized insects, in which many cells are revealed to have basal processes (Fig. 5G and H). Furthermore, their accumulation during states of non-feeding suggests that release is regulated by feeding. Thus, it will be important to test experimentally the possibilities that peptides found in the midgut regulate some critical function of the digestive process. These functions could include the production or release of digestive and antibiotic enzymes, as well as the regulation of the trans-epithelial electrical potential thought to be essential for absorptive events.

Acknowledgements This work was supported by the US Department of Agriculture National Research Initiative Competitive Grants 93-37302-8968 (T.G.K.) and 92-37302-7470 (N.E.B.). We thank Dawn Harrison for carrying out the mass determinations, Dr Chuck Woods for the peptide quantitations by amino acid analysis and Frances Tan for overseeing insect rearing. We are grateful to Dr A.K. Raina for his support of this work.

References Beckage, N.E., Tan, F.F., Schleifer, K.W., Lane, R.D., Cherubin, L.L., 1994. Characterization and biological effects of Cotesia congregata polydnavirus on host larvae of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem. Physiol. 26, 165 –195. Beckage, N.E., Riddiford, L.M., 1982. Effects of parasitism by Apanteles congregatus on the endocrine physiology of the tobacco hornworm, Manduca sexta. Gen. Comp. Endocrinol. 47, 308– 322. Brown, M.R., Crim, J.W., Lea, A.O., 1986. FMRFamide- and pancreatic polypeptide-like immunoreactivity in midgut endocrine cells of a mosquito. Tissue Cell 18, 419 – 428. Copenhaver, P.F., Taghert, P.H., 1989. Development of the enteric nervous system in the moth I. Diversity of cell types and the embryonic expression of FMRFamide-related neuropeptides. Dev. Biol. 131, 70 – 84. Cretien, M., Sikstrom, R., Lazure, C., Mbikay, M., Benjannet, S., Marcinkiewicz, M., Seidah, N.G., De Seve, J.A., 1989. Expression of the diversity of neural and hormonal peptides via the cleavage of precursor molecules. In: Martinez, J. (Ed.), Peptide Hormones as Prohormones: Processing, Biological Activity, Pharmacology. Ellis Horwood, Chicester, pp. 1 – 25. Dickerson, I.M., Noel, G., 1991. Tissue-specific peptide processing. In: Fricker, L.D. (Ed.), Peptide Biosynthesis and Processing. CRC Press, Boca Raton, FL, pp. 71 – 109. Douglass, J., Civelli, O., Herbert, E., 1984. Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu. Rev. Biochem. 53, 665 – 715. Endo, Y., Nishiitsutsuji-Uwo, J., 1981. Gut endocrine cells in insects: the ultrastructure of the gut endocrine cells of the lepidopterous species. Biomed. Res. 2, 270 – 280. Fonagy, A., Schoofs, L., Proost, P., Van Damme, J., Bueds, H., De Loof, A., 1992. Isolation, primary structure and synthesis of neomyosuppressin, a myoinhibiting neuropeptide from the gray

T.G. Kingan et al. / Molecular and Cellular Endocrinology 133 (1997) 19–32

32

fleshfly, Neobellieria bullata. Comp. Biochem. Physiol. 102C, 239 – 245. Fujisawa, Y., Shimoda, M., Kiguchi, K., Ichikawa, T., Fujita, N., 1993. The inhibitory effect of a neuropeptide, ManducaFLRFamide, on the midgut activity of the sphingid moth, Agrius con6ol6uli. Zool. Sci. 10, 773–777. Furness, J.B., Bornstein, J.C., Murphy, R., Pompolo, S., 1992. Roles of peptides in transmission in the enteric nervous system. Trends Neurosci. 15, 66 – 71. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. Holman, G.M., Cook, B.J., Nachman, R.J., 1986. Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut. Comp. Biochem. Physiol. 85C, 329–333. Holwerda, D.A., 1972. A glycopeptide from the posterior lobe of pig pituitaries. Eur. J. Biochem. 28, 340–346. Houghten, R.A., Li, C.H., 1979. Reduction of sulfoxides in peptides and proteins. Anal. Biochem. 98, 36–46. Jenkins, A.C., Brown, M.R., Crim, J.W., 1989. FMRF-amide immunoreactivity and the midgut of the corn earworm (Heliothis zea). J. Exp. Zool. 252, 71–78. Kingan, T.G., 1989. A competitive enzyme-linked immunosorbent assay: Applications in the assay of peptides, steroids, and cyclic nucleotides. Anal. Biochem. 183, 283–289. Kingan, T.G., Shabanowitz, J., Hunt, D.F., Witten, J.L., 1996. Characterization of two myotropic neuropeptides in the FMRFamide family from segmental ganglia of the moth, Manduca sexta: candidate neurohormones and neuromodulators. J. Exp. Biol. 199, 1095 – 1104. Kingan, T.G., Teplow, D.B., Phillips, J.M., Riehm, J.P., Rao, K.R., Hildebrand, J.G., Homberg, U., Kammer, A.E., Jardine, I., Griffin, P.R., et al., 1990. A new peptide in the FMRFamide family isolated from the CNS of the hawkmoth, Manduca sexta. Peptides 11, 849 – 856. Lawrence, P.O., Lanzrein, B., 1993. Hormonal interactions between insect endoparasites and their host insects. In: Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds.), Parasites and Pathogens of Insects. Academic Press, New York, pp. 59–86. Lange, A.B., Peeff, N.M., Orchard, I., 1994. Isolation, sequence and bioactivity of FMRFamide-related peptides from the locust ventral nerve cord. Peptides 15, 1089–1094. Marder, E., Calabrese, R.L., Nusbaum, M.P., Trimmer, B., 1987. Distribution and partial characterization of FMRFamide-like peptides in the stomatogastric nervous systems of the rock crab, Cancer borealis and the spiny lobster, Panulirus interruptus. J. Comp. Neurol. 259, 150–163. McLaughlin, L.L., Wei, Y., Stockmann, P.T., Leahy, K.M., Needleman, P., Grassi, J., Pradelles, P., 1987. Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin. Biochem. Biophys. Res. Comm. 144, 469–476. Montuenga, L., Barrenechea, M.A., Sesma, P., Lopez, J., Vazquez, J.J., 1989. Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust, Schistocerca gregaria (Forskal). Cell Tissue Res. 258, 577–583. Nijhout, H.F., Williams, C.M., 1974. Control of moulting and metamorphosis in the tobacco hornworm, Manduca sexta (L.): cessation of juvenile hormone secretion as a trigger for pupation. J. Exp. Biol. 61, 493 – 501.

.

Patel, Y.C., O’Neil, W., 1988. Peptides derived from cleavage of prosomatostatin at carboxyl- and amino-terminal segments. J. Biol. Chem. 263, 745 – 749. Reichwald, K., Unnithan, G.C., Davis, N.T., Agricola, H., Feyereisen, R., 1994. Expression of the allatostatin gene in endocrine cells of the cockroach midgut. Proc. Natl. Acad. Sci. USA 91, 11894 – 11898. Riddiford, L.M., Curtis, A.T., Kiguchi, K., 1979. Culture of the epidermis of the tobacco hornworm, Manduca sexta. J. Tissue Culture Methods 5, 277 – 290. Robb, S., Packman, L.C., Evans, P.D., 1989. Isolation, primary structure and bioactivity of SchistoFLRF-amide, a FMRF-amidelike neuropeptide from the locust, Schistocerca gregaria. Biochem. Biophys. Res. Comm. 160, 850 – 856. Robbins, R.J., Reichlin, S., 1983. Somatostatin biosynthesis by cerebral cortical cells in monolayer culture. Endocrinology 113, 574– 581. Sehnal, F., Zitnan, D., 1996. Midgut endocrine cells. In: Lehane, M.J., Billingsley, P.F. (Eds.), The Biology of the Insect Midgut. Chapman and Hall, London, pp. 56 – 85. Spittaels, K., Verhaert, P., Shaw, C., Johnston, R.N., Devreese, B., Van Beeumen, J., De Loof, A., 1996. Insect neuropeptide F (NPF)-related peptides: isolation from Colorado potato beetle (Leptinotarsa decemlineata) brains. Insect Biochem. Mol. Biol. 26, 375 – 382. Steiner, D.F., Smeekens, S.P., Ohagi, S., Chan, S.J., 1992. The new enzymology of precursor processing endoproteases. J. Biol. Chem. 267, 23435 – 23438. Sundler, F., Bottcher, G., Ekblad, E., Ha˚kanson, R., 1989. The neuroendocrine system of the gut. Acta Oncol. 28, 303 –314. Veenstra, J.A., Lambrou, G., 1995. Isolation of a novel RFamide peptide from the midgut of the American cockroach, Periplaneta americana. Biochem. Biophys. Res. Comm. 213, 519 – 524. Walker, R.J., 1992. Neuroactive peptides with an RFamide or Famide carboxyl terminal. Comp. Biochem. Physiol. 102C, 213– 222. Watson, R.D., Bollenbacher, W.E., 1988. Juvenile hormone regulates the steroidogenic competence of Manduca sexta. Mol. Cell. Endocrinol. 57, 251 – 259. Witten, J.L., Truman, J.W., 1996. Developmental plasticity of neuropeptide expression in motoneurons of the moth Manduca sexta: steroid hormone regulation. J. Neurobiol. 29, 99 – 114. Yi, S.-X., Tirry, L., Bai, C., Devreese, B., Van Beeumen, J., Degheele, D., 1995. Isolation, identification, and synthesis of Mas-MG-MT I, a novel peptide from the larval midgut of Manduca sexta (Lepidoptera: Sphingidae). Arch. Insect Biochem. Physiol. 28, 159 – 171. Zitnan, D., Kingan, T.G., Beckage, N.E., 1995a. Parasitism-induced accumulation of FMRFamide-like peptides in the gut innervation and endocrine cells of Manduca sexta. Insect Biochem. Mol. Biol. 25, 669 – 678. Zitnan, D., Kingan, T.G., Kramer, S.J., Beckage, N.E., 1995b. Accumulation of neuropeptides in the cerebral neurosecretory system of Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata. J. Comp. Neurol. 356, 83 – 100. Zitnan, D., Sauman, I., Sehnal, F., 1993. Peptidergic innervation and endocrine cells of insect midgut. Arch. Insect Biochem. Physiol. 22, 113 – 132.