Methods and materials Directory UMM :Journals:Journal of Insect Physiology:Vol47.Issue2.Feb2001:

1424 A. Seinsche et al. Journal of Insect Physiology 46 2000 1423–1431 ised as myotropic factors on the basis of the bioassay employed. Later, their ability to stimulate fluid secretion andor to depolarise the transepithelial potential of Mal- pighian tubules of Aedes aegypti and A. domesticus was shown Hayes et al., 1989; Coast et al., 1990. They also seem to be involved in the regulation of lipid concen- tration in the haemolymph and protein synthesis in the fat body Goldsworthy et al., 1992. So far, kinins have been isolated from A. domesticus Holman et al., 1990, L. migratoria Schoofs et al., 1992, Culex salinarius Hayes et al., 1994, A. aegypti Veenstra, 1994, Helico- verpa zea Blackburn et al., 1995, P. americana Predel et al., 1997, M. domestica Holman et al., 1998 and Drosophila melanogaster Terhzaz et al., 1999. It is the C-terminal pentapeptide sequence common to all insect kinins that seems to be necessary to elicit both the diuretic and the myotropic response in vitro. In the cricket A. domestica the active core sequence FYPWG- amide is equipotent with the parent peptide in both the hindgut and Malpighian tubule assays Holman et al., 1987a,b; Coast et al., 1990. Within this sequence the conformation of the aromatic residues Phe residue 1 and Trp residue 4 seems to play a key role in both activities. By contrast, position 2 tolerates wide vari- ations in the composition of the side-chain, although compounds containing aromatic residues were the most potent in bioassays Nachman et al. 1991, 1994; Nach- man et al., 1993a. Several members of the kinin family Culex depolariz- ing factor II, Lem Kinin I and II and Lom kinin were shown to be inactivated by an angiotensin-converting enzyme ACE from M. domestica through removal of a C-terminal dipeptide amide fragment Lamango et al., 1997. In mammals, the enzyme, a Zn 2 + -metallopeptid- ase, is responsible for the activation of the vasoconstric- tor angiotensin II and the inactivation of the vasodilator bradykinin. It also inactivates several amidated peptides by the removal of di- or tripeptide amides, e.g. Leu- Met-enkephalinamide and substance P. The M. dom- estica ACE-related enzyme is inhibited by several human diuretics such as captopril, enalapril-maleate and lisinopril. The precise role of ACE in insects is not known, but its ability to degrade a variety of different neuropeptides and its high titre in the haemolymph sug- gest that this endopeptidase plays a role in the degra- dation of regulatory peptides in insects. The effects of human ACE inhibitors on larval development of M. sexta Lamango, 1994 and the lethal phenotype of two AnCE mutants of Drosophila melanogaster Tatei et al., 1995, however, indicate that the enzyme is of vital importance for normal development. For our study, we chose the helicokinins as represen- tatives of the kinin family occurring in an important pest species. The primary aim of the current study was to determine whether synthetic helicokinins based on those isolated from H. zea could be shown to affect water bal- ance in the closely related species Heliothis virescens. A further aim was to investigate the role of ACE in the regulation of these neuropeptides in an economically important species of noctuid.

2. Methods and materials

2.1. Insects H. virescens were reared on artificial diet and main- tained under natural light conditions and RT. The Mal- pighian tubules MTs for the in vitro bioassay were obtained from 2- to 4-day-old fifth-instar larvae. 2.2. Test substances All peptide sequences tested were synthesized on an automatic peptide synthesizer Applied Biosystems 431A using Polystyrene AM RAM amide resin RAPP- Polymere, Tu¨bingen, Germany following standard FmoctBu protocols. The resin-cleavage and deprotec- tion steps were performed with trifluoroacetic acid TFAwaterthioanisol1,2-ethanedithiol 10:0.5:0.5:0.25. The crude peptides were precipitated with ether and lyophilized from water. The peptides were further pur- ified by RP–HPLC acetonitrilewater, 0.1 TFA using isocratic conditions. The peptides were characterized by ESI–MS. HezK I: Tyr-Phe-Ser-Pro-Trp-Gly-amide; C 39 H 46 N 8 O 8 ; mz = 755 [M + 1] + HezK II: Val-Arg-Phe-Ser-Pro-Trp-Gly-amide; C 41 H 58 N 12 O 8 ; mz = 847 [M + 1] + HezK III: Lys-Val-Lys-Phe-Ser-Ala-Pro-Trp-Gly- amide; C 45 H 68 N 12 O 9 ; mz = 921 [M + 1] + Phe-Ser-Pro-Trp-Gly-amide; C 30 H 37 N 7 O 6 ; mz = 592 [M + 1] + Ser-Pro-Trp-Gly-amide; C 21 H 28 N 6 O 5 ; mz = 445 [M + 1] + Pro-Trp-Gly-amide; C 18 H 23 N 5 O 3 ; mz = 358 [M + 1] + The ACE inhibitors were obtained from Sigma, Ger- many. 2.3. Bioassays 2.3.1. In vitro Diuretic activity was measured using a modification of the Ramsay assay Ramsay, 1954. The MTs were dissected from the larvae and were placed individually in 30 µ l droplets of Pringle’s saline 154 mM NaCl, 0.27 mM KCl, 0.18 mM CaCl 2 , 22 mM glucose; Pringle, 1938 under water-saturated paraffin oil. The open ends were secured outside the droplets, allowing the secreted fluid to accumulate in a separate droplet. After an equili- 1425 A. Seinsche et al. Journal of Insect Physiology 46 2000 1423–1431 bration period the MTs were allowed to secrete without any stimulation for 30 min control rate, and the diam- eters of secreted fluid droplets were measured using an ocular micrometer. The droplets were then discarded. Fresh saline 30 µ l containing the test substances was added to the saline. After 30 min the diameters of the secreted droplets were measured again. 2.3.2. In vivo In vivo effects were assessed during the feeding-phase of fifth-instar larvae of H. virescens 2–3 days after last moult, 250–300 mg of weight. Larvae were temporarily immobilised by cooling on ice before being placed in isolated Petri dishes. Injections were carried out manu- ally using a 10 µ l glass syringe with 5 µ l of the test compound being applied by inserting a 26-gauge needle through the cuticle at the base of the last abdominal limb. Controls were treated with saline. The larvae were weighed and placed individually on food. They were reweighed at least every 24 h and further development until the pupal stage 12–15 days duration was recorded. The faeces were collected every day until pupation and weighed. Water contents of the faeces were calculated by subtracting faecal weight after drying 110 ° C, 24 h. Differences between control and treatment means were tested for statistical significance using a two-tailed, paired Student’s t-test.

3. Results