Interactions between allatostatins and allatotropin on spontaneous contractions of the foregut of larval Lacanobia oleracea

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Journal of Insect Physiology 53 (2007) 75–83 www.elsevier.com/locate/jinsphys

Interactions between allatostatins and allatotropin on spontaneous contractions of the foregut of larval Lacanobia oleracea H.J. Matthews, N. Audsley, R.J. Weaver Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK Received 16 August 2006; received in revised form 17 October 2006; accepted 19 October 2006

Abstract The interactions between the activity of three neuropeptides, Manduca sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT) and cydiastatin 4, on the spontaneous foregut contractions of the tomato moth, Lacanobia oleracea, were investigated. Bioassays revealed that application of Manse-AS to the foregut at high concentrations (107 M) stopped contractions completely, and this inhibition could not be reversed by Manse-AT. Conversely, Manse-AS could inhibit a Manse-AT stimulated tissue. In contrast, Manse-AT reversed the inhibition of foregut peristalsis by cydiastatin 4 (107 M), and cydiastatin 4 counteracted the stimulation by Manse-AT. These results imply that the Manse-AS inhibitory effect is dominant over the stimulatory action of Manse-AT. However, when two peptides with opposing actions were added together, the overall effect on foregut peristalsis was determined by the relative concentrations of each peptide, suggesting that in these experiments, no peptide was dominant over the other. When Manse-AS and cydiastatin 4 were applied to foregut tissues simultaneously the overall effect was not significantly different to the individual peptides, i.e. there was no additive effect. This suggests that the individual activities of Manse-AS and cydiastatin 4 are suppressed by an undetermined mechanism in the presence of the other peptide. These results question the need for two structurally different allatostatins that have the same physiological effect on foregut peristalsis in L. oleracea larvae. Crown Copyright r 2006 Published by Elsevier Ltd. All rights reserved. Keywords: Insect; Lepidoptera; Myoactivity; Neuropeptide

1. Introduction In insects, the stomatogastric nervous system that innervates the gut has been reported to be involved in crop motility and feeding (Penzlin, 1985). An integral part of this system is the frontal ganglion, and in Lepidoptera its physiological importance in the control of foregut motility was demonstrated in larvae of the tobacco hawkmoth, Manduca sexta (Miles and Booker, 1994, 1998) and in adults of the corn earworm, Heliothis zea (Bushman and Nelson, 1990). Several peptides have been detected and/or identified in the stomatogastric nervous system in larval Lepidoptera using immunocytochemical and mass spectrometric techniques. M. sexta allatostatin Corresponding author. Tel.: +44 1904 462629; fax: +44 1904 462111.

E-mail address: [email protected] (H.J. Matthews).

(Manse-AS), M. sexta allatotropin (Manse-AT) and allatostatins of the Y/FXFGL-NH2 family (cydiastatins, helicostatins) are all localised in the frontal ganglion and detected in the axons of the recurrent nerve that innervate the muscles of the foregut of larval Lacanobia oleracea and M. sexta (Duve et al., 2000; Duve and Thorpe, 2003; Audsley et al., 2005; Duve et al., 2005). As implied by their localisation, these allatoregulatory peptides have also been shown to have myoactivity on the foregut of larval Lepidoptera, the allatostatins inhibit whereas allatotropin stimulates foregut peristalsis (Duve et al., 1997, 1999, 2000; Audsley et al., 2005). How these peptides interact to regulate the muscles of the foregut, and hence movement of food through this region of the gut is poorly understood. When assayed on isolated foregut of larval Helicoverpa armigera the stimulatory effect of allatotropin was shown to be dominant

0022-1910/$ - see front matter Crown Copyright r 2006 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.10.007

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over the inhibitory effect of the helicostatins tested (Duve et al., 1999). The authors suggest that the stimulatory allatotropin effect may be more important than the allatostatin inhibition because contraction of the gut is necessary to move food along the alimentary canal. In L. oleracea spontaneous contractions of the foregut have previously been shown to be inhibited by Manse-AS and Y/FXFGL-NH2 type allatostatins (helicostatin 8, cydiastatins 4 and 5), and are stimulated by allatotropin (Duve et al., 2000; Audsley et al., 2005). The aim of this study was to investigate the interactions and possible synergism between the myoinhibitory effects of two representative allatostatins from L. oleracea (Manse-AS (pEVRFRQCYFNPISCF-OH) and cydiastatin 4 (ARPYSFGL-NH2) and the myostimulatory role of allatotropin (GFKNVEMMTARGF-NH2) on foregut peristalsis of L. oleracea larvae.

M. sexta allatostatin and cydiastatin 4 were custom synthesised at the Advanced Biotechnology Centre, Charing Cross and Westminster Hospital Medical School, London, UK. M. sexta allatotropin was purchased from Sigma-Aldrich (UK).

Insects were discarded if there was no gut movement (approx. 20%). One of three types of experiment were then carried out. In experiments to study the sequential application of peptides, control saline was replaced with the same volume of solution containing one peptide at a specific concentration and contractions monitored as above. Previous work and current work presented here had shown that the concentrations of peptides chosen would either initiate a maximum response (Manse-AS 107 M, Manse-AT 108 M, cydiastatin 4107 M) or a ca. 50% response (Manse-AS 1010 M) (Audsley et al., 2005; Matthews et al., 2006). The solution was removed and then either the same volume of saline was added containing a different peptide, or the foregut preparation was washed three times in 200 ml physiological saline and then the same volume of saline added containing a different peptide. Contractions were monitored as above. To determine the effects of simultaneous addition of peptides, control saline was replaced with the same volume of saline containing a combination of peptides (either (Manse-AS+Manse-AT) or (cydiastatin 4+Manse-AT)) at specific concentrations. Contractions were monitored over two 2-min periods. The interactions between Manse-AS and cydiastatin 4 were studied by replacing control saline with the same volume of saline containing the peptides either individually or together. Peptides were tested at concentrations that either gave o25% response (Manse-AS (1013 M), cydiastatin 4 (1010 M)) or gave 425 but o50% response (Manse-AS (1010 M, cydiastatin 4 (109 M)). Ten different larvae were used for each concentration of peptide(s) tested. The frequency of contractions in the presence of peptide(s) was then compared to the frequency of control (baseline) levels.

2.3. Physiology

2.4. Statistical analysis

Sixth instar L. oleracea larvae that had been starved overnight were anaesthetised with CO2 and dissected in a longitudinal well made in the wax base in a dissecting dish. The dorsal surface was opened by cutting from the level of the second proleg to the back of the head capsule, allowing exposure of the foregut and anterior midgut. The cuticular flaps were pinned down and the foregut washed several times with 200 ml of physiological saline of the following composition (mM): Na+ 154, K+ 2.7, Ca2+ 1.8, Cl 160, hydroxyethylpiperazine ethanesulphonic acid (HEPES) 12, and glucose 22, pH 7.2 (Cook and Holman, 1978). The foregut preparation was then immersed in 200 ml saline. Foregut contractions were measured using two electrodes positioned either side of the foregut linked to an impedance converter (model 2991; UFI, Morro Bay, CA, USA) and changes in impedance were monitored on a chart recorder (Pharmacia). The number of peristaltic contractions were monitored over two 2-min periods to establish baseline contractions.

Differences in percentage inhibition/stimulation were analysed using either a paired t-test or the Student’s t-test.

2. Materials and method 2.1. Insects L. oleracea were reared as described by Corbitt et al. (1996) on an artificial diet and kept at 20 C, 65% relative humidity, in a 16 h light: 8 h dark photocycle. Larvae were fed on a maize-based noctuid artificial diet (Bio-Serv, Frenchtown, NJ, USA). 2.2. Peptides

3. Results 3.1. Effects of Manse-AS, cydiastatin 4 and Manse-AT on control rates of foregut peristalsis The frequency of spontaneous contractions of the foregut of L. oleracea under control (saline only) conditions was variable, between 4 and 32 contractions per minute, but could be inhibited by both Manse-AS and cydiastatin 4, or stimulated by Manse-AT (Figs. 1a–c). Using high doses (107 M) of Manse-AS, spontaneous contractions of the foregut were inhibited completely, and this inhibition could not be reversed despite repeated washes with physiological saline, containing no peptide (Fig. 1a). By contrast, although 107 M cydiastatin 4 also completely inhibited foregut muscle contractions, its effects

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wash

a

Manse-AS 10-7M

Saline

Impedance (Volts)

Saline

100

(A)

(B)

150 320

370 450

(C)

500

Time (Seconds) wash

b

Cydiastatin 4 10-7M

Saline

Impedance (Volts)

Saline

140

(A)

240 290

(B)

390 470

(C)

570

Time (Seconds) wash

c

Manse-AT 10-8M

Saline

Impedance (Volts)

Saline

120

(A)

170 240

(B)

290 390

(C)

440

Time (Seconds) Fig. 1. (a) Impedance converter trace showing (A) control foregut peristalsis of sixth instar L. oleracea and (B) inhibition of peristalsis by Manse-AS 107 M. After saline washes contractions were not resumed (C). (b). Impedance converter trace showing (A) control foregut peristalsis of sixth instar L. oleracea and (B) inhibition of peristalsis by cydiastatin 4107 M. After saline washes contractions were restored back to control levels (C). (c). Impedance converter trace showing (A) control foregut peristalsis of sixth instar L. oleracea and (B) stimulation of peristalsis by Manse-AT 108 M. After saline washes contractions were restored back to control levels (C).

could be reversed by repeated washing of the tissues with saline to remove peptide (Fig. 1b). Both the frequency and amplitude of contractions were increased by the addition of

108 M Manse-AT to the L. oleracea larval foregut, and subsequent return to baseline levels was achieved after saline washing (Fig. 1c).

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3.2. Interactions between Manse-AS and Manse-AT 3.2.1. Sequential effects The inhibitory effects of 107 M Manse-AS on the foregut could not be reversed by replacing the bathing saline with saline containing high concentrations (108 M) of Manse-AT (Fig. 2a). Even after the Manse-AS inhibited foregut had been washed several times with physiological saline alone, adding Manse-AT had very little effect on the rate of foregut muscle contractions (Fig. 2b). Although some tissues were stimulated to contract, the effects were highly variable and not significantly different (t9 ¼ 1.50, P40.05) from Manse-AS inhibited guts (zero contractions). In contrast, Manse-AT stimulated guts could be completely inhibited by replacing the bathing saline with saline containing 107 M Manse-AS, even without tissue washing (Fig. 2c). The inhibitory effects of lower doses of Manse-AS (1010 M), which inhibit foregut peristalsis by

a

only 58.777.8% were also not significantly affected by 108 M Manse-AT (t8 ¼ 0.21, P40.05) (Fig. 3a). However, contractions returned to control levels after saline washing, after which adding 108 M Manse-AT to the bathing saline stimulated contractions to maximal levels (195.2765.7%; Fig. 3b). 3.2.2. Simultaneous addition of peptides The ability of Manse-AS to inhibit the stimulatory effect of 108 M Manse-AT on foregut peristalsis, when both peptides were added together, was dose dependant (Fig. 4). In this set of experiments 108 M Manse-AT increased foregut contractions by ca. 50%. In the presence of 108 M Manse-AT, a dose of 106 Manse-AS was required to completely inhibit foregut contractions, and the inhibitory effects of Manse-AS diminished with decreasing concentrations. When both peptides were present at equal amounts (108 M) there was no significant difference in

b

c Manse-AT 10-8M

200

% change from control

150 100 50

Manse-AS Manse-AT 10-8M 10-7M

0

12

Manse-AS 10-7M 345

saline wash

Manse-AS 10-7M

Manse-AT 10-8M 67

-50 -100 -150

Fig. 2. Sequential effects of Manse-AS (107 M) and Manse-AT (108 M) on frequency of foregut peristalsis of sixth instar L. oleracea. The first peptide was added to the foregut, removed, and then a different peptide added either with no wash in between (a and c) or after washing three times with saline (b). Means7SE, n ¼ 10.

a

b

Manse-AT 10-8M

% change from control

300

200

100 Manse-AS 10-10M

Manse-AT 10-8M

Manse-AS 10-10M

saline wash

0

-100 Fig. 3. Sequential effects of Manse-AS (1010 M) and Manse-AT (108 M) on frequency of foregut peristalsis of sixth instar L. oleracea. The first peptide was added to the foregut, removed, and then a different peptide added either with no wash in between (a) or after washing three times with saline (b). Means7SE, n ¼ 10.

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100

% change from control

50

0

-50

-100

-150

AT (8)

AT/AS (8/6)

AT/AS (8/7)

AT/AS (8/8)

AT/AS (8/9)

AT/AS (8/10)

Negative log peptide conc. (M) Fig. 4. Dose response for the inhibition of the effects of Manse-AT (AT) by Manse-AS (AS) on frequency of foregut peristalsis of sixth instar L. oleracea. Means7SE, n ¼ 10. Negative log peptide concentrations (M) are given in parentheses.

of 108 M Manse-AS, a dose of 107 M Manse-AT was required to fully stimulate foregut muscle contractions.

100

% change from control

50

0

3.3. Interactions between cydiastatin 4 and Manse-AT

1

5

6

AS/AT (8/10)

AS/AT (8/11)

-50

-100

-150

AS (8)

AS/AT (8/7)

AS/AT (8/8)

AS/AT (8/9)

Negative log peptide conc. (M) Fig. 5. Dose response for the inhibition of the effects of Manse-AS (AS) by Manse-AT (AT) on frequency of foregut peristalsis of sixth instar L. oleracea. Means7SE, n ¼ 10. Negative log peptide concentrations (M) are given in parentheses.

frequency of contractions from the control (t17 ¼ 0.29, P40.05), and at 1010 M, Manse-AS had no significant effect on the frequency of foregut contractions compared to Manse-AT alone (t18 ¼ 0.35, P40.05). The effects of Manse-AT on the inhibition of foregut peristalsis by 108 M Manse-AS were variable, but dose dependant (Fig. 5). A dose of 1010 M Manse-AT had no significant effect on the inhibition of foregut peristalsis compared to Manse-AS alone (t13 ¼ 1.72, P40.05). As the concentration of Manse-AT increased, the inhibitory effects of Manse-AS diminished, and the frequencies of contractions were significantly increased by 109 M (t12 ¼ 9.46, Po0.001), 108 M (t11 ¼ 5.45, Po0.01) and 107 M (t11 ¼ 6.98, Po0.001) Manse-AT. In the presence

3.3.1. Sequential effects Cydiastatin 4 (107 M) inhibited the frequency of foregut contractions by 97.3%. Replacement of the bathing saline with saline containing 108 Manse-AT resulted in an increase in frequency of foregut contractions, greater than control levels and not significantly different to Manse-AT alone (t18 ¼ 0.68, P40.05; Fig. 6a). In a separate experiment the rate of foregut contractions was doubled by the addition of 108 M Manse-AT to the bathing saline compared to control levels. Replacement of this saline with saline containing 107 M cydiastatin 4 resulted in a complete inhibition of foregut contractions, similar to that produced by cydiastatin 4 alone (Fig. 6b). 3.3.2. Simultaneous addition of peptides When added together in the gut bathing saline the ability of cydiastatin 4 to inhibit the stimulatory effect of 108 M Manse-AT on foregut peristalsis was dose dependant (Fig. 7). At low doses of cydiastatin 4 (1010 M) there was no significant effect on the rate of contractions compared to 108 M Manse-AT alone (t18 ¼ 0.64, P40.05). At equimolar concentrations of Manse-AT and cydiastatin 4 contraction rates were not significantly different to control rates (t19 ¼ 0.18, P40.05). As the amount of cydiastatin 4 increased from 108 M, gut contractions were reduced to less than control rates until complete inhibition occurred at a dose of 105 M cydiastatin 4 (in the presence of 108 M Manse-AT). The stimulatory effect of Manse-AT on the inhibition of foregut peristalsis by cydiastatin 4 was also dose dependant, but the effects of Manse-AT were variable (Fig. 8). A dose of 107 M Manse-AT in the presence of 108 M

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a

b

200

Manse-AT 10-8M

% change from control

150 Manse-AT 10-8M

100 50

Cydiastatin 4 10-7M

Cydiastatin 4 10-7M

0 -50 -100

Fig. 6. Sequential effects of cydiastatin 4 (107 M) and Manse-AT (108 M) on frequency of foregut peristalsis of sixth instar L. oleracea. The first peptide was added to the foregut, removed, and then a different peptide added (a, b) Means7SE, n ¼ 10.

% change from control

100

50

0

-50

-100

AT (8)

AT/C4 (8/5)

AT/C4 (8/6)

AT/C4 (8/7)

AT/C4 (8/8)

AT/C4 (8/9)

AT/C4 (8/10)

Negative log peptide conc. (M) Fig. 7. Dose response for the inhibition of the effects of Manse-AT (AT) by cydiastatin 4 (C4) on frequency of foregut peristalsis of sixth instar L. oleracea. Means7SE, n ¼ 10. Negative log peptide concentrations (M) are given in parentheses.

cydiastatin 4 resulted in stimulation of foregut peristalsis in a manner similar to Manse-AT alone (t14 ¼ 1.69, P40.05). This stimulation gradually diminished as the concentration of Manse-AT was reduced until Manse-AT had no significant effect at concentrations p1013 M (P40.05).

3.4. Interactions between Manse-AS and cydiastatin 4 Fig. 9 shows the inhibition of foregut peristalsis achieved when Manse-AS and cydiastatin 4 were applied either individually or in combination to the L. oleracea foregut bathing saline at different concentrations. There were no significant differences in the % inhibition when Manse-AS and cydiastatin 4 were applied in combination compared to

when each was applied individually (P40.005), i.e. the effects were less than additive.

4. Discussion In larval L. oleracea, A-type allatostatins (cydiastatins, helicostatins), the C-type allatostatin (Manse-AS) and M. sexta allatotropin (Manse-AT) are co-localised in a pair of anterior neurones in the frontal ganglion. Axons from these neurones innervate the muscles of the crop and stomodeal valve via the recurrent nerve (Duve et al., 2000, 2005; Duve and Thorpe, 2003). Consistent with their tissue localisation, these peptides are myo-active on the foregut of L. oleracea larvae.

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150

% change from control

100

50

0

1

3

4

5

C4/AT (8/8)

C4/AT (8/9)

C4/AT (8/10)

6

7

8

9

C4/AT (8/12)

C4/AT (8/13)

C4/AT (8/14)

-50

-100 C4 (8)

C4/AT (8/7)

C4/AT (8/11)

Negative log peptide conc. (M) Fig. 8. Dose response for the inhibition of the effects of cydiastatin 4 by Manse-AT (AT) on foregut peristalsis of sixth instar L. oleracea. Means7SE, n ¼ 10. Negative log peptide concentrations (M) are given in parentheses.

60

% inhibition

50 40 30 20 10 0 AS (13)

C4 (10)

AS(13) +C4(10)

AS (10)

C4 (9)

AS(10) +C4(9)

Fig. 9. Percentage inhibition of foregut peristalsis of sixth instar L. oleracea by either Manse-AS (1013 M), cydiastatin 4 (1010 M) and in combination, or by Manse-AS (1010 M), cydiastatin 4 (109 M) and in combination. Negative log peptide concentrations (M) are given in parentheses.

Foregut peristalsis is stimulated by Manse-AT whereas both types of allatostatins are myo-inhibitory (Duve et al., 2000; Audsley et al., 2005; Matthews et al., 2006). The functional significance of these peptides, which have opposing effects on foregut, being localised in the same neurone is unclear. This situation is further complicated by the identification of at least seven A-type allatostatins plus Manse-AS, in the frontal ganglion of L. oleracea larvae, which all appear to have the same physiological role on the foregut (Audsley et al., 2005). Duve et al. (1999) report that various members of the A-type allatostatin family in H. armigera are similar in their inhibitory effects on the crop, with 107–106 M causing complete cessation of muscle activity and the frequency and amplitude of contractions being significantly reduced by doses of 1010–109 M allatostatin.

High concentrations (107 M) of Manse-AS completely inhibit contractions of the foregut of larval L. oleracea, and neither the application of Manse-AT or saline washes could reverse this inhibition, although the length of time that this inhibitory effect lasted was not determined. The biological activity of a peptide (e.g. allatostatin) results from receptor occupancy coupled with the ability to transform the receptor into an active state supporting signal transduction (Tobe et al., 2000). As long as the signalling molecule (peptide) remains bound, the receptor is activated. These results would suggest that Manse-AS has a higher affinity than Manse-AT has for their corresponding receptors. At high concentrations it appears that Manse-AS occupies its receptors for prolonged periods and does not dissociate from them readily, even after rinsing tissues several times with saline. These results may

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also help to explain why a single high dose of Manse-AS results in a reduction in feeding, retarded growth and increased mortality when injected into L. oleracea larvae (Audsley et al., 2001), presumably due to a persistent inhibition of foregut peristalsis by Manse-AS. However, Manse-AT can suppress the inhibitory effects of lower doses of Manse-AS (1010 M), but only after tissue rinsing. In contrast, a foregut stimulated with high concentrations of Manse-AT can be inhibited with Manse-AS, even without tissue rinsing. Hence, Manse-AS readily suppresses the stimulatory effects of Manse-AT showing that the Manse-AS inhibitory actions are dominant in this set of experiments. By comparison, a Manse-AT stimulated gut can be inhibited by replacement of the bathing saline with saline containing cydiastatin 4, and a gut inhibited by cydiastatin 4 can be stimulated by Manse-AT. Compared to Manse-AS, the inhibitory effects of cydiastatin 4 do not appear to be as persistent and results suggest that cydiastatin 4 has a lower affinity for, and dissociates more readily from, its receptor(s). In H. armigera larvae, a foregut inhibited by a helicostatin, can be stimulated by allatotropin, but in the reverse experiment, helicostatin cannot inhibit an allatotropin stimulated gut. It was suggested that the allatotropin effect was not hindered by allatostatin receptor occupancy and the allatotropin effect in stimulating gut contractions is dominant over the inhibitory effects of A-type allatostatins (Duve et al., 1999). When Manse-AS and Manse-AT or cydiastatin 4 and Manse-AT were applied to the foregut simultaneously the overall response of the foregut was determined by the relative peptide concentrations, and the effects were dose dependant. When present in equimolar amounts, the opposing effects of the allatostatins and allatotropin were counteractive, with no significant or very little change from control rates of muscle contraction, implying neither peptide has a dominant effect over the other. If the allatostatin concentration was greater than the allatotropin concentration then the overall effect was inhibitory and vice versa. However, very high doses (105 M) of cydiastatin 4, in the presence of 108 M Manse-AT were required to fully inhibit foregut peristalsis and very low amounts (1013 M) of Manse-AT in the presence of 108 M cydiastatin 4 could still suppress the full inhibition of gut motility. This suggests that Manse-AT stimulation may be more dominant than cydiastatin 4 inhibition, and/or Manse-AT is a more potent peptide. Allatotropin has been shown to be active at doses as low as 1016 M on the foreguts of larval H. armigera (Duve et al., 1999) and larval L. oleracea (Audsley et al., 2005). When assayed on juvenile hormone (JH) synthesis by the CA of adult female S. frugiperda, Manse-AS was shown to have no effect on basal rates of JH production, but reduces or abolishes the stimulatory effects of Manse-AT when the two peptides are added to the incubation medium together (Oeh et al., 2000). Oeh et al. (2003) suggest that contractions of the gut muscles of Heliothis virescens are stimulated by myotropic peptides (helicokinins and Manse-AT) binding to G

protein coupled receptors resulting in an influx of Ca2+ into the cells mediated by a protein kinase C (PKC) pathway. Hinton et al. (1998) also postulates that the stimulation of foregut contractions by proctolin involves Ca2+ and PKC. The same may be true of Manse-AT stimulation of foregut contractions in larval L. oleracea. The combined effects of the two different types of allatostatins (Manse-AS and cydiastatin 4) showed that their effects were not significantly different to the effects of the peptides alone, i.e. there was no additive effect of the two peptides at the range of doses tested. Coast (1995) reported that low concentrations of Locusta-diuretic peptide and locustakinin acted co-operatively to stimulate fluid secretion by isolated Malpighian tubules of Locusta migratoria greater than the individual peptides alone. This synergism is believed to be due to the peptides acting via different second messengers (cyclic AMP and Ca2+) to cooperatively stimulate KCl and NaCl transport and hence fluid secretion. Cydiastatin 4 and Manse-AS are associated with different, but related, G protein coupled receptors, and two homologues of each receptor have been identified in Drosophila melanogaster (reviewed by Claeys et al., 2005). Second messengers have not been measured in gut tissues treated with allatostatins, but no evidence of the involvement of cyclic nucleotides was apparent in corpora allata inhibited with A-type allatostatins of the cockroach Diploptera punctata (Cusson et al, 1992). It is therefore unclear which second messengers are involved and whether cydiastatin 4 and Manse-AS are activating the same or different G protein subunit types to induce a change in foregut peristalsis in larval L. oleracea. By comparison the activation of G-protein-coupled receptors in membranes of human leukaemia (HL60) cells by chemotatic factors (N-formylmethionyl-leucyl-phenylalanine and complement component 5a) was also non-additive. It was suggested that these receptors share the same G protein pool in the cell membranes and that activation of these G proteins by one of the two receptors decreases the availability of G proteins for the other receptor (Wieland et al., 1992). Whatever the mechanisms of action, these two types of allatostatin do not co-operate in their physiological actions, even though they both inhibit muscle contraction in the foregut when applied alone. One can only speculate as to whether this interference, which precludes an additive effect of the two types of allatostatin, occurs at receptor binding or signal transduction level. Additive effects of different A-type allatostatins on juvenile hormone synthesis by the corpora allata from the cockroach, D. punctata were reported by Tobe et al. (2000). It was suggested that the differing biological activities of the allatostatins tested were partly due to differences in receptor affinity, receptor antagonism and receptor activation, and that the allatostatins regulate JH biosynthesis through differing affinity interactions with receptors in the corpora allata. The interaction between different A-type allatostatins on foregut peristalsis was not investigated.

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The lack of an additive response of cydiastatin 4 and Manse-AS raises the question of the need for two allatostatin types, co-localised in the same cells in the frontal ganglion, having the same physiological effect on the same tissue in the same insect. In the blow fly, Calliphora vomitoria different A-type allatostatins (Leucallatostatins) affected motility of the gut in different, but adjacent, regions (Duve et al., 1996). Two functional types of muscle, gut compressors and gut dilators, were identified in larvae of M. sexta which work antagonistically on the foregut to produce foregut movements that serve to either move food toward the midgut (waves of peristalsis) or retain it in the foregut (synchronous constrictions) (Miles and Booker, 1994). It is therefore possible that different Atype allatostatins, Manse-AS and Manse-AT exert their effects on different regions of the foregut in larval Lepidoptera, but was not observed in this study. In conclusion, the present study has provided some insight into the interactions between two types of allatostatins and allatotropin in their effects on the L. oleracea foregut. The interaction between peptides are likely to be due to different affinities for their respective receptors, differential breakdown of peptides, differences in receptor trafficking and/or different receptor juxtaposition. Given that a total of eight A-type allatostatins and ManseAS have been identified in this species (Audsley and Weaver, 2003), there is likely to be considerable complexity in how the differences in affinities and activities of these peptides integrate to produce the desired biological effects. Acknowledgements The authors are very grateful to Hannah Bradish for the supply of insects used in this study. This work was funded by the Pesticides Safety Directorate (DEFRA). References Audsley, N., Matthews, J., Weaver, R.J., 2005. Neuropeptides associated with the frontal ganglion of larval Lepidoptera. Peptides 26, 11–21. Audsley, N., Weaver, R.J., Edwards, J.P., 2001. In vivo effects of Manduca sexta allatostatin and allatotropin on larvae of the tomato moth, Lacanobia oleracea. Physiological Entomology 26, 181–188. Audsley, N., Weaver, R.J., 2003. Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDITOF mass spectrometry and post-source decay. Peptides 24, 1465–1474. Bushman, D.W., Nelson, J.O., 1990. The role of the frontal ganglion and corpora cardiaca corpora allata complex in post-feeding weight loss in adult Heliothis zea. Physiological Entomology 15 (3), 269–274. Claeys, I., Poels, J., Simonet, G., Franssens, V., Van Loy, T., Van Hiel, M.B., Breugelmans, B., Vanden Broeck, J., 2005. Insect neuropeptide and peptide hormone receptors: current knowledge and future directions. Vitamins and Hormones 73, 217–282. Coast, G.M., 1995. Synergism between diuretic peptides controlling ion and fluid transport in insect Malpighian tubules. Regulatory Peptides 57, 283–296. Cook, B.J., Holman, G.M., 1978. Comparative pharmacological properties of muscle function in the foregut and hindgut of the cockroach

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