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
3.1. Ramsay assay The effect of the three synthetic helicokinins HezK
I–III on secretory activity of isolated Malpighian tubules from fifth-instar H. virescens is shown in Fig. 1.
All three helicokinins stimulated fluid secretion in a dose-dependent manner over a range from 10
212
to 10
27
M with EC
50
concentrations of 2.9 ×
10
211
M HezK I, 2
× 10
211
M HezK II and 3.6 ×
10
210
M HezK III. At 10
29
M the kinins increased fluid secretion by 410 HezK I, 354 HezK II and 207 HezK III. The
highest rates of secretion were induced with concen- trations of 10
28
M with increases of 6.1 times for HezK I, 5.3 times for HezK II and 4.2 times for HezK III.
3.2. In vivo microinjections The injection of the helicokinins led to a reduction in
weight gain in all cases Fig. 2. Four hours after the treatment, the control group gained approximately 2 in
weight, while the treated animals lost weight 2.8 HezK I; 7.07 HezK II and 2.6 HezK III. After 24
h the control animals injected with saline had generally gained about 29 in weight while the HezK III treated
Fig. 1. Responses of isolated Malpighian tubules from fifth-instar H.
virescens to helicokinin I–III. Each point represents the mean
± SD
of 10–15 observations. Significant differences between control and test means p0.005.
Fig. 2. Effects of helicokinin I–III on weight gain of fifth-instar lar-
vae of H. virescens 4 h and 24 h after the treatment. Larvae were injected with 50 pmol of the helicokinin in 5
µ l saline. The animals
were permitted to feed before and after the treatment. The control group was treated with saline. Mean values
± SD are shown for groups
of 10–15 larvae. Significant difference p0.005.
larvae increased their weight only by 1. The HezK I and II treated animals even lost weight: the HezK I
group about 7.3 and the HezK II ones about 2.6. By contrast, the amount of diet consumed during 4 h and
the amount of faeces produced during the observation period did not differ significantly between control and
1426 A. Seinsche et al. Journal of Insect Physiology 46 2000 1423–1431
Table 1 Rates of mortality in percent after combined injections with helicokinins 50 pmol and ACE inhibitors 1
µ mol 5 days after the treatment.
Each set of experiments comprised 15–20 larval H. virescens. The controls were injected with saline. Significant differences are marked with an asterisk p,0.01
Injection of saline
captopril enalapril-maleate
lisinopril +
saline 18.8
± 3.6
7.3 ±
5.9 4.5
± 3.6
8.5 ±
2.8 +
HezK I 43.8
± 6.7
83.3 ±
12.3 55.5
± 6.9
33.3 ±
5.6 reduced pupal weight
+ HezK II
11.1 ±
5.3 22.2
± 5.7
50 ±
8.3 45.4
± 7.2
reduced pupal weight +
HezK III 16.7
± 3.7
22.2 ±
4.2 25
± 9.8
33.3 ±
4.3
treated groups Table 2. The faeces from the treated groups, however, contained more water 80–85 than
in the controls 65–75 Table 2. Of the HezK I treated animals, 43 died within 6 days after the treat-
ment, the levels of mortality in HezK II and III treated larvae did not differ significantly from the controls
Table 1.
3.3. Determination of the active core sequence of HezK I
To determine the active core sequence of HezK I YFSPWG-amide, truncated sequences were tested in
vitro Fig. 3 over the same concentration range as the original peptide 10
212
–10
27
M. Only the pentapeptide FSPWG-amide was able to induce a dose-dependent
increase in secretion similar to that found for HezK I EC
50
= 1.8
× 10
210
M. Neither the tetra- SPWG-amide
Fig. 3. Responses of isolated Malpighian tubules from fifth-instar H.
virescens to helicokinin I and truncated analogues. Each point rep-
resents the mean ±
SD of 10–15 observations. Significant differ- ences p0.005.
nor the tripeptide PWG amide increased the fluid secretion significantly above the control levels.
Although the C-terminal pentapeptide sequence of HezK I was sufficient to elicit a complete in vitro
response, in vivo it was not able to mimic the activity of the parent peptide data not shown. The treated larvae
showed no significant loss of weight or disruption in development.
To evaluate the significance of the amino acid side- chains, an alanine scan was carried out. Analogues were
synthesised in which one of the six amino acids of the original peptide was substituted by an alanine residue.
All peptides were tested in the Ramsay assay over the same concentration range as HezK I. The results are
shown in Fig. 4. The exchange of Tyr residue 1, Ser residue 3 and Pro residue 4 had no significant effect
on the dose-dependent increase in secretion compared with the results for HezK I. Substitution of Phe residue
2, Trp residue 5 and Gly residue 6, however, led to a complete loss of diuretic activity of the analogues.
Fig. 4. Responses of isolated Malpighian tubules from fifth-instar H.
virescens to helicokinin I and alanine scan analogues. Bars show mean
rates of urine production ±
SD, N =
10–15. Significant differences p0.005.
1427 A. Seinsche et al. Journal of Insect Physiology 46 2000 1423–1431
Fig. 5. Effects of ACE inhibitors on weight gain of fifth-instar H.
virescens 24 h after the injection. The animals were treated with 1
µ mol of the ACE inhibitor in 5
µ l saline. The control group was treated
with saline. The animals were allowed to feed before and after the treatment. Mean values of control and test animals N
= 10–12 did not
differ significantly p.0.005.
3.4. ACE inhibitors The Ramsay assay and in vivo microinjections were
used to investigate whether the ACE inhibitors captopril, enalapril-maleate and lisinopril could influence in vitro
fluid secretion or larval development. In the Ramsay assay the substances were tested over a concentration
range from 10
27
to 10
24
M. None of the three ACE inhibitors affected the rate of fluid secretion in vitro data
not shown. Microinjection of 1 µ
mol of these com- pounds had no effect on larval development Fig. 5. The
amount and water content of faeces produced by the treated animals did not differ significantly from the con-
trols Table 2.
In a second set of experiments the larvae were injected with a combination of 50 pmol HezK and 1
µ mol ACE
inhibitor in 5 µ
l of saline. The results Table 1 show that 83.3 of the larvae treated with a combination of
HezK I and captopril died within 5 days after the injec-
Table 2 Comparison of the amounts of faeces until pupationdiet consumption within 24 h and water content of faeces between saline-injected larval
H. virescens control group and larvae treated with helicokinins 50 pmol and ACE inhibitors 1
µ mol. Each set of experiments comprised 15–
20 larval H. virescens. Significant differences are marked with an asterisk p,0.01 Faeces mg
Water content Diet comsumption mg
Control group 943.3
± 240.2
68.9 ±
2.3 581.6
± 168.5
Hez K I treated 1160.2
± 212.8
82.6 ±
3.7 574.3
± 121.4
Hez K II treated 916.1
± 283.5
79.8 ±
5.6 611.3
± 187
HezK III treated 1027.0
± 140
73.4 ±
5.9 555.6
± 241
Captopril treated 1111.6
± 337.6
68.7 ±
3.9 Enalpril treated
957.4 ±
252 64.5
± 8.9
Lisinopril treated 1024.1
± 124.5
71.2 ±
5.4
tion, compared with 43.3 for HezK I alone see above. The surviving animals developed to pupae weighing
14.2 less than the controls. A combination of HezK I and enalapril-maleate increased mortality to a level of
55.5. HezK II and III, which when injected alone did not increase mortality, caused 30–50 mortality in com-
bination with the ACE inhibitors. Enalapril-maleate combined with HezK II led to a mortality rate of 50
and a weight reduction of 13 for the surviving pupae. The combination of HezK with lisinopril led to a sig-
nificant increase in mortality: 33.3 for HezK III and 45.5 for HezK II.
4. Discussion
