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Inhibition of flight in Periplaneta americana (Linn.) by a trehalase inhibitor, validoxylamine A
Pergamon
0022-1910(94)EOOO8-4
J. Insecr Physiol. Vol. 40. No. 6, pp. 455461, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910194 $7.00 + 0.00
Inhibition of Flight in Periplaneta americana (Linn.) by a Trehalase Inhibitor, Validoxylamine A YOSHIAKI KONO,*,t MASAKAZU TAKAHASHI,* YUKIHIKO KAMEDA,§ EITARO HORIf
KAZUHIRO
MATSUSHITAJ
MASAMI NISHINAJ
Received 16 September 1993; revised 8 December 1993
Male adults of Peripluneta americana were graded into three groups according to their flight activity; very active (continuous flight longer than 5 min), active (l-5 min) and inactive (shorter than 1 min). ‘H-NMR analysis showed that approx. 30 mM trehalose was present in the haemolymph of each of the above categories of untreated cockroaches. After exhaustive flight, the haemolympb trehalose concentration in very active individuals decreased to < 10 mM. When validoxylamine A, which is reported to be a potent and specific inhibitor of trehalase in various organisms, was injected into the cockroaches, they were unable to continue flying for >2.5 min. The trehalase activity of the flight muscle was inhibited about 70% and the haemolymph trehalose concentration increased to about 3 times the normal level without change in the other haemolymph components. Trehalose also accumulated in the tissues of the cockroaches injected with validoxylamine A. These observations suggest that validoxylamine A inhibits the muscle trehalase and prevents the use of trehalose as a source of energy for flight. Trehalase inhibitor
Validoxylamine A
Flight Periplaneta americana NMR-analysis
INTRODUCTION
lecterial gland as a result of trehalase inhibition (Takeda et al., 1990; Yao et al., 1991). VAA injection markedly elevated haemolymph trehalose levels in B. mori (Kono et al., 1993) and in most other species examined including, the cabbage armyworm, Mamestra brassicae, the housefly, Musca domestica, the blowfly, Calliphora nigribarbis and the locust, Locust migratoria (Kono et al.,
Validoxylamine A (VAA, Fig. l), an amino sugar whose glucoside, validamycin A, has been used for a long time as a fungicide against the rice sheath blight, is a potent and specific inhibitor of trehalases from various organisms (Kameda et al., 1987). VAA inhibits trehalases competitively because its chemical structure resembles that of trehalose [Fig. 1; Asano et al. (1990)]. Time dependent, slow-binding reversible inhibition has been shown against porcine kidney trehalase (Salleh and Honek, 1990). Administration of VAA evoked various physiological changes as well as lethal effects in insects (Asano et al., 1990; Kono et al., 1993, 1994). Injection of VAA into the pupa of Bombyx mori induced a change in egg determination in the adults from diapause egg producers to non-diapause egg producers (Takeda et al., 1988) and prevented production of glutinous material in the col-
unpublished data). Relaxation of the segmental muscles and the decrease of pulsation of the dorsal vessel were also observed in VAA treated Mamestra larvae (Kono et al., unpublished data). These effects of VAA seem to be related to the prevention of trehalose utilization. In the present paper, the effect of VAA on flight activity is observed in Periplaneta americana, a species which utilizes mainly glucose for flight fuel (Chino et al., 1992). The effect of the drug on the haemolymph components, especially trehalose, was analyzed with ‘Hand 13C-NMR spectrometry. MATERIALS
*Department of Medical Entomology, The National Institute of Health, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan. tTo whom all correspondence should be addressed. IDepartment of Medical Zoology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 305-04, Japan. &School of Pharmacy, Hokuriku University, Kanagawa-machi, Kanazawa 920- 11, Japan.
AND METHODS
Insects
The stock culture of Periplaneta americana was maintained with rabbit food and water under long-day conditions (25”C, LD 16: 8). Male adults 2&50 days post emergence were used for the experiments. 455
YOSHIAKI KONO et al.
456
CH,OH
CH,OH
dH
dH
Validoxylamine FIGURE
A
a,ol-Trehalose
1. Chemical structures of validoxylamine A and cc,a-trehalose.
Chemicals
VAA (Fig. 1) was purified as previously described (Kameda et al., 1986). [‘3C]1-Glucose (99 atom %)
Trehalose peak I TSP peak FIGURE 2. Calibration curve for quantification of trehalose concentration. A linear relationship, Y = 3.83X, was obtained. Y, trehalose concentration (mg/ml), X, signal height of middle peak of trehalose C-6 methyl moiety at 3.46 ppm/signal height of TSP at 0 ppm.
and D,O were purchased from Cambridge Isotope Laboratories (England) and CEN Sacley (France), respectively. Other chemicals used were of analytical grade.
TSP
FIGURE 3. ‘H-NMR spectrum of haemolymph of the American cockroach. 1, leucine; 2, valine; 3, lactate; 4, alanine; 5, acetate; 6, methionine; 7, acetone; 8, choline; 9, phosphoryl choline; 10, trehalose; 11, glycine. TSP, 1 mM TSP as an inner standard.
INHIBITION TABLE
OF FLIGHT
1. Effect of VAA injection on the haemolymph trehalose concentration
29.5 63.1 77.1 70.4 94.9
Control 0.4 2.0 10.0 50.0 *Average
Measurement of Jlight activity A small piece of paper folded into a T shape was attached to the pronotum of the cockroach with an instant binding agent. The cockroach was hung horizontally with a small vascular clamp to hold the paper on the pronotum. Air current (24 m/s) was blown over the cockroach from the front by a blower, and the duration of sustained flight was measured. Flight durations varied from < 1 min to > 20 min. Cockroaches were divided into three groups according to the duration of flight; inactive: < 1 min, active: l-5 min, very active: > 5 min.
Trehalose cont. (mM)* mean + SD
Dose @g/insect)
of 3 males
451
+ 9.5 k 15.8 & 28.3 + 36.5 + 21.0
1 day after injection.
Znjection of VAA NMR analysis
Five microliters aqueous solution of the described concentration of VAA was injected into the dorsal body cavity of the cockroach with a microsyringe. The same amount of tiater was injected into the control cockroaches.
Ten to 40 ~1 haemolymph was collected from the cut hind leg coxa and centrifuged with a small amount of phenylthiourea at 2000g for 10 min at 4°C. The supernatant was diluted to 600 ~1 with heavy water (D,O) and
TSP
I
I
,,
,
4
I
I,
I
I
I
,,
,
,
,
,
,
,
,
3
,
(
,
2
,
,
,
,
,
,
,
,
,
,,
,
,,,
,
,
,
1
mm
FIGURE enhanced
4. Effect of VAA on haemolymph components the trehalose concentration (10) two-fold within
of I’. americana. Injection of 5Opg VAA/insect (bottom trace) 24 h. Phosphoryl choline (9) decreased, compared with the control (top trace).
,
,
0
458
YOSHIAKI TABLE
2. Haemolymph trehalose trations after sustained flight
No.
Time of flight (min)
Without flight 1 2 Immediately 3 4 5 6 7
Trehalose
KONO
Measurement
concen-
(mM)
29.8 20.5
21.6 18.7 7.6 7.4 9.9
I day after flight 8 11 9 13 10 17
21.6 29.8 18.9
filtered through a molecule sieve (MolcutTM) to exclude molecules > 10 kDa. The eluate was stored at -20°C until the NMR analysis. Haemolymph samples (450~1) to which was added 50~1 of 10 mM 3trimethylsilyl 2,2,3,3_tetradeuteropropionate (TSP) or 100 mM dioxane in D,O as an internal standards were (400 MHz) or 13C-NMR subjected to ‘H-NMR (100 MHz) analysis using a JEOL JNM-EX400 FTNMR spectrometer. Assignment of the signals on the NMR chart was made by adding synthetic standards to the sample and by checking the overlapping of the signals. To observe the effect of VAA on sugar metabolism, 200 pg [‘3C]1-glucose in 5 ~1 water per insect was injected into the cockroach 2 h after an injection of 20 pg VAA/insect or an equivalent volume of water. For the determination of haemolymph trehalose concentration, ‘H-NMR was used. A calibration curve of trehalose concentration (Y) against the ratio (X) of signal strength of trehalose (methyl signal at C-6) at 3.46 ppm to signal strength of TSP at 0 ppm was drawn (Fig. 2). The concentrations could be calculated based on the linear relationship between Y and X: Y = 3.83X (mglml). Whole body homogenates (5 adults/3.5 ml D,O) of the [‘3C]1-glucose injected cockroaches whose haemolymph had been sampled, were analyzed using the same procedure as for haemolymph.
TABLE
3. Effect
of VAA
Category of flight activity Inactive Active Very active Without VAA injection Active Very active
of trehalase activity
Dissected flight muscle was homogenized with 10 times (w/v) phosphate buffer solution (pH 7.2) and centrifuged at ZOOOg,for 30 min. One tenth ml of the homogenate supernatant mixed with 0.2 ml of 50 mM trehalose (substrate) was incubated at 30°C for 1Omin and bathed in boiling water for 5 min to stop the trehalase reaction. The reaction mixture was diluted with 0.5 ml distilled water and again centrifuged at 2000g for 10 min. A 0.2 ml sample of the supernatant was incubated with 1 ml of glucose oxidase assay mixture including glucose oxidase, peroxidase, 4-aminoantipyrine and phenol (Glucose B-test Wako, Wako Chem. Indust.) at 30°C for 20 min to measure glucose. The glucose concentration in the mixture was determined by the increase in absorbance at 505 nm.
cont.
(very active)
after flight 3 5 13 15 18
et al.
RESULTS
Haemolymph components detected by ‘H-NMR
analysis
Figure 3 shows a ‘H-NMR analysis of the haemolymph of the male adult. The haemolymph used was collected from 5 males (60 ~1) and diluted lo-fold. The indication of leucine (l), valine (2), lactate (3), alanine (4), acetate (5), methionine (6), acetone (7), choline (8), phosphoryl choline (9), trehalose (10) and glycine (11) was confirmed by the addition of each substance to the sample. Phosphoryl choline, choline, methionine and glycine were found in high concentration. Based on the relative signal strength of the trehalose C-6 methyl signal and TSP signal (Fig. 3), the average trehalose concentration of ten individuals was determined to be 31.3 &-8.8 mM (f SD). The mean trehalose concentration (f SD) of untreated males in the inactive, active and very active flight groups was 35.9 + 7.6 mM (n = 3), 33.3 If: 13.7 mM (n = 2) and 25.1 &-6.7 mM (n = 2), respectively. The trehalose concentration of the very active group was lower than that of the inactive group, but the difference was not statistically significant. Efect
of VAA on haemolymph trehalose concentration
The concentration of haemolymph trehalose 1 day after injection of VAA (0.4-50 pgg/insect) is shown in Table 1 (in this experiment, cockroaches were treated
injection on flight concentration
activity
and
haemolymph
trehalose
No. of insect used
Time of flight (min) 1 day after injection mean k SD
Trehalose cont. (mM) immediately after flight mean If: SD
I 4 3
105.7 94.1 f 15.5 77.2 & 37.3
2 3
4.0 + 1.4 15.3 + 2.5
20.2 + 2.1 8.3 + 1.4
INHIBITION OF FLIGHT
459
-9
TSP
I
FIGURE 5. Effect of VAA on the components of body homogenate supernatant. ‘H-NMR charts of control (top) and treated (bottom) cockroaches. Nos I-10: same as Fig. 3, 12: glucose.
regardless of flight activity). The lowest dose of VAA (0.4 pg/insect) enhanced the trehalose level about 2-fold, and the highest dose (50pg/insect), about 3-fold, At intermediate doses, the increase in trehalose concentration was more variable. At 50 ,ug VAA/insect, the trehalose levels of active and very active groups increased to 80.9 + 15.2 mM (n = 2) and 75.6 rt 11.7 mM (n = 3), respectively. Figure 4 shows the ‘H-NMR analysis of haemolymph taken from cockroaches injected with 50 pg VAA/insect. This treatment caused an obvious increase in trehalose concentration (10). No change, however, occurred in the other haemolymph components, except for a decrease in phosphoryl choline (9).
Table 2. It can be seen that trehalose levels dropped only slightly in cockroaches Nos 3 and 4, which stopped flying after a few minutes, but was below 10 mM immediately after flight to exhaustion in very active cockroaches (Nos 5-7). The level of trehalose was restored to near normal levels within 24 h (Nos 8-10). As shown in Table 3, when the cockroach was injected with 50,ug VAA/insect just after flight and kept under normal conditions for 1 day, it lost its ability to fly. All cockroaches treated with VAA 24 h previously, stopped flying within l-2.5 min (< 1.5 min on average). Their haemolymph trehalose was maintained at the elevated level even after flight. Inhibition qfflight muscle trehalase activity by VAA
night activity and trehalose concenfration Trehalose concentrations were measured in cockroaches immediately after and 1 day after flight to exhaustion. The relationship between flight duration and haemolymph trehalose concentration is shown in
Trehalase activity of flight muscle dissected from cockroaches 1 day after injection of 5 and 50 pg VAA/insect was determined. About 2 I.rmol glucose was produced by the muscle equivalent of one untreated male adult (200 mg fresh weight) in 10 min. VAA injection of
460
YOSHIAKI
KONO
et al.
DISCUSSION
5 and 50 pg/insect inhibited trehalase activity 74.0 f 8.2% (mean *SD, n = 3) and 68.2f6.4% (n = 3), respectively. A dose dependent inhibition was not observed at these dose levels.
VAA strongly inhibits trehalase activity in various organisms: fungi, yeast, insects and mammals (Kameda et al., 1987). For instance, in vitro IC-50s against trehaE#ect of VAA on sugar metabolism lases of Spodoptera litura larvae, baker’s yeast, and Haemolymph and supernatants of whole body porcine kidney were 4.8 x lo-‘M, 3.0 x 10m9M and homogenates were analysed with ‘H-NMR and 13C- 4.5 x 10m9M, respectively (Kameda et al., 1987; Salleh NMR spectrometry 1 day after injection of [‘3C]1-glu- and Honek, 1990). In vivo injection of VAA also strongly case. A comparison of signal strength for each inhibited trehalase activity in ovaries (loo%, Takeda chemical in ‘H-NMR analysis shows that VAA en- et al., 1988) and in arborescent regions of colleterial hanced the haemolymph trehalose level 3.2 times glands (78%, Yao et al., 1991) of Bombyx mori, as well (treated: 69.0 mM, control: 21.6 mM), while phosphoryl as in flight muscle of Periplaneta americana in the choline level decreased to 60% of the control present experiments (ca 70%). sample in one day. In the supernatant of the body In contrast to the potent inhibition of trehalases, VAA homogenate (Fig. 5) the trehalose and lactate concenshowed no significant inhibition of other kinds of sugar trations increased to 2.8 and 1.8 times that of the hydrolases; cellulase, pectinase, chitinase, c1-amylase, control, respectively, while the glucose level decreased to a-glucosidase and /?-glucosidase (Asano et al., 1987). about 38% of the control. These changes in sugar VAA, therefore, appears to be specific for trehalases. level due to VAA treatment were confirmed by 13C- The complete conversion of labeled glucose to trehalose NMR analysis (Fig. 6). In haemolymph, all of the indicates that trehalose synthesis is not affected by VAA treatment. The increase in trehalose and decrease in injected [13C]l-glucose disappeared and was incorporated into trehalose. In whole body supernatant, levels of glucose concentration in tissue homogenates of VAA treated cockroaches is considered to be the result of a labeled trehalose and glucose were very low. Glycogen (signal at 101 ppm in 13C-NMR) was not detected in decrease in the rate of trehalose conversion into glucose. Accumulation of trehalose in the haemolymph may also whole body supernatants of either treated or control have resulted from a disturbance of trehalose utilization. cockroaches.
I
(b)
I”
105
100
95
90
85
80
75
70
65
60
55
PPm
PPm
Cd)
105 100
9.5
90
85
80 PP*
75
70
65
60
55
105
100
95
90
85
80
75
70
65
60
55
PPm
FIGURE 6. Effect of VAA on trehalose metabolism. ‘)C-NMR charts of body homogenate supernatant, control (a) and 50 ,ug VAA/insect injection (b); and of haemolymph, control (c) and 50 pg VAA/insect injection (d). 200 pg/insect [“C]l-glucose was injected into all cockroaches 2 h after VAA injection.
INHIBITION
Periplaneta americana mainly utilizes carbohydrates in the form of trehalose as its flight fuel because of a deficiency in the lipid transporting system and the low content of fat body diglycerides (Chino et al., 1992). Furthermore, 13C-NMR analysis showed that only low levels of glycogen was stored in the tissues (fat body and muscle). Elevation of haemolymph trehalose in response to flight (King et al., 1986) was not observed in the present experiment. In fact, rapid decrease of haemolymph trehalose during flights of 10-20 min supports the idea of active utilization of trehalose for flight energy when the provision of trehalose from carbohydrates reserves is insufficient. The drastic effect of VAA on flight activity in the cockroach seems to depend on the specific mechanism that provides energy for flight. In another species, Locusta migratoria, which uses both carbohydrates and lipids as flight fuels (Goldsworthy, 1983), VAA showed no marked inhibitory effect on flight. It does, however, enhance the haemolymph trehalose level, indicating the inhibition of tissue trehalase (Kono et al., unpublished data).
REFERENCES Asano N., Yamaguchi T.. Kameda Y. and Matsui K. (1987) Effect of validamycins on glycohydrolases of Rhi:octonia solani. J. Antibiotics 40, 526-532. Asano N., Takeuchi M., Kameda Y.. Matsui K. and Kono Y. (1990) Trehalase inhibitors, validoxylamine A and related compounds as insecticides. J. Antibiotics 43, 722-726.
OF FLIGHT
461
Chino H., Lum P. Y., Nagao E. and Hiraoka T. (1992) The molecular and metabolic essentials for long-distance flight in insects. J. camp. Physiol. B 162, 101-106. Goldsworthy G. J. (1983) The endocrine control of flight metabolism in locusts. Adv. Insect Physiol. 17, 149-240. Kameda Y., Asano N., Yamaguchi T.. Matsui K., Horii S. and Fukase H. (1986) Validamycin G and validoxylamine G, new members of the validamycins. J. Antibiotics 39, 1491-1494. Kameda Y., Asano N., Yamaguchi T. and Matsui K. (1987) Validoxylamines as trehalase inhibitors. J. Antibiotics 40, 563-565. King L. E., Steele J. E. and Bajura S. W. (1986) The effect of flight on the composition of haemolymph in the cockroach. Periplaneta americana. J. Insect Physiol. 32, 649455. Kono Y., Takeda S., Kameda Y., Takahashi M., Matsushita K., Nishina M. and Hori E. (1993) Lethal activity of a trehalase inhibitor, validoxylamine A, and its influence on the blood sugar level in Bombyx mori. Appl. Entomol. Zool. 28, 379-386. Kono Y., Takeda S. and Kameda Y. (1994) Lethal activity of a trehalase inhibitor, validoxylamine A, against Mamestra brassicae and Spodoptera litura. J. Pesticide Sci. 19, 3942. Salleh H. M. and Honek J. F. (1990) Time-dependent inhibition of porcine kidney trehalase by aminosugars. FEBS Letf. 262, 3599362. Takeda S., Kono Y. and Kameda Y. (1988) Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor. Entomol. exp. appl. 46, 291-294. Takeda S., Kono Y. and Kameda Y. (1990) Non-glutinous egg production by using a trehalase inhibitor. validoxylamine A, in Bombyx mori. J. Seric. Sci. Japan. 59, 360-365. Yao X., Fugo S. and Takeda S. (1991) Effect of validoxylamine A on the trehalase activity in the colleterial glands of the silkworm, Bombyx mori. J. Serie. Sci. Japan. 60, 296--301.
Acknowledgement-The study was partly supported by a Grant-in Aid for General Scientific Research (No. 03454057) from the Ministry of Education, Science and Culture.
0022-1910(94)EOOO8-4
J. Insecr Physiol. Vol. 40. No. 6, pp. 455461, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910194 $7.00 + 0.00
Inhibition of Flight in Periplaneta americana (Linn.) by a Trehalase Inhibitor, Validoxylamine A YOSHIAKI KONO,*,t MASAKAZU TAKAHASHI,* YUKIHIKO KAMEDA,§ EITARO HORIf
KAZUHIRO
MATSUSHITAJ
MASAMI NISHINAJ
Received 16 September 1993; revised 8 December 1993
Male adults of Peripluneta americana were graded into three groups according to their flight activity; very active (continuous flight longer than 5 min), active (l-5 min) and inactive (shorter than 1 min). ‘H-NMR analysis showed that approx. 30 mM trehalose was present in the haemolymph of each of the above categories of untreated cockroaches. After exhaustive flight, the haemolympb trehalose concentration in very active individuals decreased to < 10 mM. When validoxylamine A, which is reported to be a potent and specific inhibitor of trehalase in various organisms, was injected into the cockroaches, they were unable to continue flying for >2.5 min. The trehalase activity of the flight muscle was inhibited about 70% and the haemolymph trehalose concentration increased to about 3 times the normal level without change in the other haemolymph components. Trehalose also accumulated in the tissues of the cockroaches injected with validoxylamine A. These observations suggest that validoxylamine A inhibits the muscle trehalase and prevents the use of trehalose as a source of energy for flight. Trehalase inhibitor
Validoxylamine A
Flight Periplaneta americana NMR-analysis
INTRODUCTION
lecterial gland as a result of trehalase inhibition (Takeda et al., 1990; Yao et al., 1991). VAA injection markedly elevated haemolymph trehalose levels in B. mori (Kono et al., 1993) and in most other species examined including, the cabbage armyworm, Mamestra brassicae, the housefly, Musca domestica, the blowfly, Calliphora nigribarbis and the locust, Locust migratoria (Kono et al.,
Validoxylamine A (VAA, Fig. l), an amino sugar whose glucoside, validamycin A, has been used for a long time as a fungicide against the rice sheath blight, is a potent and specific inhibitor of trehalases from various organisms (Kameda et al., 1987). VAA inhibits trehalases competitively because its chemical structure resembles that of trehalose [Fig. 1; Asano et al. (1990)]. Time dependent, slow-binding reversible inhibition has been shown against porcine kidney trehalase (Salleh and Honek, 1990). Administration of VAA evoked various physiological changes as well as lethal effects in insects (Asano et al., 1990; Kono et al., 1993, 1994). Injection of VAA into the pupa of Bombyx mori induced a change in egg determination in the adults from diapause egg producers to non-diapause egg producers (Takeda et al., 1988) and prevented production of glutinous material in the col-
unpublished data). Relaxation of the segmental muscles and the decrease of pulsation of the dorsal vessel were also observed in VAA treated Mamestra larvae (Kono et al., unpublished data). These effects of VAA seem to be related to the prevention of trehalose utilization. In the present paper, the effect of VAA on flight activity is observed in Periplaneta americana, a species which utilizes mainly glucose for flight fuel (Chino et al., 1992). The effect of the drug on the haemolymph components, especially trehalose, was analyzed with ‘Hand 13C-NMR spectrometry. MATERIALS
*Department of Medical Entomology, The National Institute of Health, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan. tTo whom all correspondence should be addressed. IDepartment of Medical Zoology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 305-04, Japan. &School of Pharmacy, Hokuriku University, Kanagawa-machi, Kanazawa 920- 11, Japan.
AND METHODS
Insects
The stock culture of Periplaneta americana was maintained with rabbit food and water under long-day conditions (25”C, LD 16: 8). Male adults 2&50 days post emergence were used for the experiments. 455
YOSHIAKI KONO et al.
456
CH,OH
CH,OH
dH
dH
Validoxylamine FIGURE
A
a,ol-Trehalose
1. Chemical structures of validoxylamine A and cc,a-trehalose.
Chemicals
VAA (Fig. 1) was purified as previously described (Kameda et al., 1986). [‘3C]1-Glucose (99 atom %)
Trehalose peak I TSP peak FIGURE 2. Calibration curve for quantification of trehalose concentration. A linear relationship, Y = 3.83X, was obtained. Y, trehalose concentration (mg/ml), X, signal height of middle peak of trehalose C-6 methyl moiety at 3.46 ppm/signal height of TSP at 0 ppm.
and D,O were purchased from Cambridge Isotope Laboratories (England) and CEN Sacley (France), respectively. Other chemicals used were of analytical grade.
TSP
FIGURE 3. ‘H-NMR spectrum of haemolymph of the American cockroach. 1, leucine; 2, valine; 3, lactate; 4, alanine; 5, acetate; 6, methionine; 7, acetone; 8, choline; 9, phosphoryl choline; 10, trehalose; 11, glycine. TSP, 1 mM TSP as an inner standard.
INHIBITION TABLE
OF FLIGHT
1. Effect of VAA injection on the haemolymph trehalose concentration
29.5 63.1 77.1 70.4 94.9
Control 0.4 2.0 10.0 50.0 *Average
Measurement of Jlight activity A small piece of paper folded into a T shape was attached to the pronotum of the cockroach with an instant binding agent. The cockroach was hung horizontally with a small vascular clamp to hold the paper on the pronotum. Air current (24 m/s) was blown over the cockroach from the front by a blower, and the duration of sustained flight was measured. Flight durations varied from < 1 min to > 20 min. Cockroaches were divided into three groups according to the duration of flight; inactive: < 1 min, active: l-5 min, very active: > 5 min.
Trehalose cont. (mM)* mean + SD
Dose @g/insect)
of 3 males
451
+ 9.5 k 15.8 & 28.3 + 36.5 + 21.0
1 day after injection.
Znjection of VAA NMR analysis
Five microliters aqueous solution of the described concentration of VAA was injected into the dorsal body cavity of the cockroach with a microsyringe. The same amount of tiater was injected into the control cockroaches.
Ten to 40 ~1 haemolymph was collected from the cut hind leg coxa and centrifuged with a small amount of phenylthiourea at 2000g for 10 min at 4°C. The supernatant was diluted to 600 ~1 with heavy water (D,O) and
TSP
I
I
,,
,
4
I
I,
I
I
I
,,
,
,
,
,
,
,
,
3
,
(
,
2
,
,
,
,
,
,
,
,
,
,,
,
,,,
,
,
,
1
mm
FIGURE enhanced
4. Effect of VAA on haemolymph components the trehalose concentration (10) two-fold within
of I’. americana. Injection of 5Opg VAA/insect (bottom trace) 24 h. Phosphoryl choline (9) decreased, compared with the control (top trace).
,
,
0
458
YOSHIAKI TABLE
2. Haemolymph trehalose trations after sustained flight
No.
Time of flight (min)
Without flight 1 2 Immediately 3 4 5 6 7
Trehalose
KONO
Measurement
concen-
(mM)
29.8 20.5
21.6 18.7 7.6 7.4 9.9
I day after flight 8 11 9 13 10 17
21.6 29.8 18.9
filtered through a molecule sieve (MolcutTM) to exclude molecules > 10 kDa. The eluate was stored at -20°C until the NMR analysis. Haemolymph samples (450~1) to which was added 50~1 of 10 mM 3trimethylsilyl 2,2,3,3_tetradeuteropropionate (TSP) or 100 mM dioxane in D,O as an internal standards were (400 MHz) or 13C-NMR subjected to ‘H-NMR (100 MHz) analysis using a JEOL JNM-EX400 FTNMR spectrometer. Assignment of the signals on the NMR chart was made by adding synthetic standards to the sample and by checking the overlapping of the signals. To observe the effect of VAA on sugar metabolism, 200 pg [‘3C]1-glucose in 5 ~1 water per insect was injected into the cockroach 2 h after an injection of 20 pg VAA/insect or an equivalent volume of water. For the determination of haemolymph trehalose concentration, ‘H-NMR was used. A calibration curve of trehalose concentration (Y) against the ratio (X) of signal strength of trehalose (methyl signal at C-6) at 3.46 ppm to signal strength of TSP at 0 ppm was drawn (Fig. 2). The concentrations could be calculated based on the linear relationship between Y and X: Y = 3.83X (mglml). Whole body homogenates (5 adults/3.5 ml D,O) of the [‘3C]1-glucose injected cockroaches whose haemolymph had been sampled, were analyzed using the same procedure as for haemolymph.
TABLE
3. Effect
of VAA
Category of flight activity Inactive Active Very active Without VAA injection Active Very active
of trehalase activity
Dissected flight muscle was homogenized with 10 times (w/v) phosphate buffer solution (pH 7.2) and centrifuged at ZOOOg,for 30 min. One tenth ml of the homogenate supernatant mixed with 0.2 ml of 50 mM trehalose (substrate) was incubated at 30°C for 1Omin and bathed in boiling water for 5 min to stop the trehalase reaction. The reaction mixture was diluted with 0.5 ml distilled water and again centrifuged at 2000g for 10 min. A 0.2 ml sample of the supernatant was incubated with 1 ml of glucose oxidase assay mixture including glucose oxidase, peroxidase, 4-aminoantipyrine and phenol (Glucose B-test Wako, Wako Chem. Indust.) at 30°C for 20 min to measure glucose. The glucose concentration in the mixture was determined by the increase in absorbance at 505 nm.
cont.
(very active)
after flight 3 5 13 15 18
et al.
RESULTS
Haemolymph components detected by ‘H-NMR
analysis
Figure 3 shows a ‘H-NMR analysis of the haemolymph of the male adult. The haemolymph used was collected from 5 males (60 ~1) and diluted lo-fold. The indication of leucine (l), valine (2), lactate (3), alanine (4), acetate (5), methionine (6), acetone (7), choline (8), phosphoryl choline (9), trehalose (10) and glycine (11) was confirmed by the addition of each substance to the sample. Phosphoryl choline, choline, methionine and glycine were found in high concentration. Based on the relative signal strength of the trehalose C-6 methyl signal and TSP signal (Fig. 3), the average trehalose concentration of ten individuals was determined to be 31.3 &-8.8 mM (f SD). The mean trehalose concentration (f SD) of untreated males in the inactive, active and very active flight groups was 35.9 + 7.6 mM (n = 3), 33.3 If: 13.7 mM (n = 2) and 25.1 &-6.7 mM (n = 2), respectively. The trehalose concentration of the very active group was lower than that of the inactive group, but the difference was not statistically significant. Efect
of VAA on haemolymph trehalose concentration
The concentration of haemolymph trehalose 1 day after injection of VAA (0.4-50 pgg/insect) is shown in Table 1 (in this experiment, cockroaches were treated
injection on flight concentration
activity
and
haemolymph
trehalose
No. of insect used
Time of flight (min) 1 day after injection mean k SD
Trehalose cont. (mM) immediately after flight mean If: SD
I 4 3
105.7 94.1 f 15.5 77.2 & 37.3
2 3
4.0 + 1.4 15.3 + 2.5
20.2 + 2.1 8.3 + 1.4
INHIBITION OF FLIGHT
459
-9
TSP
I
FIGURE 5. Effect of VAA on the components of body homogenate supernatant. ‘H-NMR charts of control (top) and treated (bottom) cockroaches. Nos I-10: same as Fig. 3, 12: glucose.
regardless of flight activity). The lowest dose of VAA (0.4 pg/insect) enhanced the trehalose level about 2-fold, and the highest dose (50pg/insect), about 3-fold, At intermediate doses, the increase in trehalose concentration was more variable. At 50 ,ug VAA/insect, the trehalose levels of active and very active groups increased to 80.9 + 15.2 mM (n = 2) and 75.6 rt 11.7 mM (n = 3), respectively. Figure 4 shows the ‘H-NMR analysis of haemolymph taken from cockroaches injected with 50 pg VAA/insect. This treatment caused an obvious increase in trehalose concentration (10). No change, however, occurred in the other haemolymph components, except for a decrease in phosphoryl choline (9).
Table 2. It can be seen that trehalose levels dropped only slightly in cockroaches Nos 3 and 4, which stopped flying after a few minutes, but was below 10 mM immediately after flight to exhaustion in very active cockroaches (Nos 5-7). The level of trehalose was restored to near normal levels within 24 h (Nos 8-10). As shown in Table 3, when the cockroach was injected with 50,ug VAA/insect just after flight and kept under normal conditions for 1 day, it lost its ability to fly. All cockroaches treated with VAA 24 h previously, stopped flying within l-2.5 min (< 1.5 min on average). Their haemolymph trehalose was maintained at the elevated level even after flight. Inhibition qfflight muscle trehalase activity by VAA
night activity and trehalose concenfration Trehalose concentrations were measured in cockroaches immediately after and 1 day after flight to exhaustion. The relationship between flight duration and haemolymph trehalose concentration is shown in
Trehalase activity of flight muscle dissected from cockroaches 1 day after injection of 5 and 50 pg VAA/insect was determined. About 2 I.rmol glucose was produced by the muscle equivalent of one untreated male adult (200 mg fresh weight) in 10 min. VAA injection of
460
YOSHIAKI
KONO
et al.
DISCUSSION
5 and 50 pg/insect inhibited trehalase activity 74.0 f 8.2% (mean *SD, n = 3) and 68.2f6.4% (n = 3), respectively. A dose dependent inhibition was not observed at these dose levels.
VAA strongly inhibits trehalase activity in various organisms: fungi, yeast, insects and mammals (Kameda et al., 1987). For instance, in vitro IC-50s against trehaE#ect of VAA on sugar metabolism lases of Spodoptera litura larvae, baker’s yeast, and Haemolymph and supernatants of whole body porcine kidney were 4.8 x lo-‘M, 3.0 x 10m9M and homogenates were analysed with ‘H-NMR and 13C- 4.5 x 10m9M, respectively (Kameda et al., 1987; Salleh NMR spectrometry 1 day after injection of [‘3C]1-glu- and Honek, 1990). In vivo injection of VAA also strongly case. A comparison of signal strength for each inhibited trehalase activity in ovaries (loo%, Takeda chemical in ‘H-NMR analysis shows that VAA en- et al., 1988) and in arborescent regions of colleterial hanced the haemolymph trehalose level 3.2 times glands (78%, Yao et al., 1991) of Bombyx mori, as well (treated: 69.0 mM, control: 21.6 mM), while phosphoryl as in flight muscle of Periplaneta americana in the choline level decreased to 60% of the control present experiments (ca 70%). sample in one day. In the supernatant of the body In contrast to the potent inhibition of trehalases, VAA homogenate (Fig. 5) the trehalose and lactate concenshowed no significant inhibition of other kinds of sugar trations increased to 2.8 and 1.8 times that of the hydrolases; cellulase, pectinase, chitinase, c1-amylase, control, respectively, while the glucose level decreased to a-glucosidase and /?-glucosidase (Asano et al., 1987). about 38% of the control. These changes in sugar VAA, therefore, appears to be specific for trehalases. level due to VAA treatment were confirmed by 13C- The complete conversion of labeled glucose to trehalose NMR analysis (Fig. 6). In haemolymph, all of the indicates that trehalose synthesis is not affected by VAA treatment. The increase in trehalose and decrease in injected [13C]l-glucose disappeared and was incorporated into trehalose. In whole body supernatant, levels of glucose concentration in tissue homogenates of VAA treated cockroaches is considered to be the result of a labeled trehalose and glucose were very low. Glycogen (signal at 101 ppm in 13C-NMR) was not detected in decrease in the rate of trehalose conversion into glucose. Accumulation of trehalose in the haemolymph may also whole body supernatants of either treated or control have resulted from a disturbance of trehalose utilization. cockroaches.
I
(b)
I”
105
100
95
90
85
80
75
70
65
60
55
PPm
PPm
Cd)
105 100
9.5
90
85
80 PP*
75
70
65
60
55
105
100
95
90
85
80
75
70
65
60
55
PPm
FIGURE 6. Effect of VAA on trehalose metabolism. ‘)C-NMR charts of body homogenate supernatant, control (a) and 50 ,ug VAA/insect injection (b); and of haemolymph, control (c) and 50 pg VAA/insect injection (d). 200 pg/insect [“C]l-glucose was injected into all cockroaches 2 h after VAA injection.
INHIBITION
Periplaneta americana mainly utilizes carbohydrates in the form of trehalose as its flight fuel because of a deficiency in the lipid transporting system and the low content of fat body diglycerides (Chino et al., 1992). Furthermore, 13C-NMR analysis showed that only low levels of glycogen was stored in the tissues (fat body and muscle). Elevation of haemolymph trehalose in response to flight (King et al., 1986) was not observed in the present experiment. In fact, rapid decrease of haemolymph trehalose during flights of 10-20 min supports the idea of active utilization of trehalose for flight energy when the provision of trehalose from carbohydrates reserves is insufficient. The drastic effect of VAA on flight activity in the cockroach seems to depend on the specific mechanism that provides energy for flight. In another species, Locusta migratoria, which uses both carbohydrates and lipids as flight fuels (Goldsworthy, 1983), VAA showed no marked inhibitory effect on flight. It does, however, enhance the haemolymph trehalose level, indicating the inhibition of tissue trehalase (Kono et al., unpublished data).
REFERENCES Asano N., Yamaguchi T.. Kameda Y. and Matsui K. (1987) Effect of validamycins on glycohydrolases of Rhi:octonia solani. J. Antibiotics 40, 526-532. Asano N., Takeuchi M., Kameda Y.. Matsui K. and Kono Y. (1990) Trehalase inhibitors, validoxylamine A and related compounds as insecticides. J. Antibiotics 43, 722-726.
OF FLIGHT
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Chino H., Lum P. Y., Nagao E. and Hiraoka T. (1992) The molecular and metabolic essentials for long-distance flight in insects. J. camp. Physiol. B 162, 101-106. Goldsworthy G. J. (1983) The endocrine control of flight metabolism in locusts. Adv. Insect Physiol. 17, 149-240. Kameda Y., Asano N., Yamaguchi T.. Matsui K., Horii S. and Fukase H. (1986) Validamycin G and validoxylamine G, new members of the validamycins. J. Antibiotics 39, 1491-1494. Kameda Y., Asano N., Yamaguchi T. and Matsui K. (1987) Validoxylamines as trehalase inhibitors. J. Antibiotics 40, 563-565. King L. E., Steele J. E. and Bajura S. W. (1986) The effect of flight on the composition of haemolymph in the cockroach. Periplaneta americana. J. Insect Physiol. 32, 649455. Kono Y., Takeda S., Kameda Y., Takahashi M., Matsushita K., Nishina M. and Hori E. (1993) Lethal activity of a trehalase inhibitor, validoxylamine A, and its influence on the blood sugar level in Bombyx mori. Appl. Entomol. Zool. 28, 379-386. Kono Y., Takeda S. and Kameda Y. (1994) Lethal activity of a trehalase inhibitor, validoxylamine A, against Mamestra brassicae and Spodoptera litura. J. Pesticide Sci. 19, 3942. Salleh H. M. and Honek J. F. (1990) Time-dependent inhibition of porcine kidney trehalase by aminosugars. FEBS Letf. 262, 3599362. Takeda S., Kono Y. and Kameda Y. (1988) Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor. Entomol. exp. appl. 46, 291-294. Takeda S., Kono Y. and Kameda Y. (1990) Non-glutinous egg production by using a trehalase inhibitor. validoxylamine A, in Bombyx mori. J. Seric. Sci. Japan. 59, 360-365. Yao X., Fugo S. and Takeda S. (1991) Effect of validoxylamine A on the trehalase activity in the colleterial glands of the silkworm, Bombyx mori. J. Serie. Sci. Japan. 60, 296--301.
Acknowledgement-The study was partly supported by a Grant-in Aid for General Scientific Research (No. 03454057) from the Ministry of Education, Science and Culture.