Camp. Eiochem. Physiol. Vol. 106C. No. 2, pp. 351-357, 1993 0742-8413/93 $6.00 + 0.00 Printed in Great Britain 0 1993 Pergamon Press Ltd

DELTAMETHRIN-INDUCED DEREGULATION OF THE WATER BALANCE IN THE MIGRATORY LOCUST,

LOCUSTA MIGRATORIA

JACQUES PROUX,* ABDELILAH ALAOUI,* BRIGITTE MORETEAU~ and AMINA BASKALI*

‘Laboratoire de Neuroendocrinologie des Insectes, URA CNRS 1138, Universitd de Bordeaux 1, Avenue des Facultts, 33405 Talence Cedex, France; and tLaboratoire de Biologie et Gbnetique Evolutives, UPR

CNRS 2411, Avenue de la terrasse, 91198 Gif-sur-Yvette Cedex, France

(Received 24 May 1993; accepted for publication 25 June 1993)

Abstract-l. Several insecticides were tested for their ability to induce a water deregulation in the larval migratory locust. All of them provoked an accelerated dehydration (when compared to sham-operated insects). Deltamethrin and baygon were the most potent.

2. This enhanced dehydration due to deltamethrin in adult locust resulted from an increase in the water loss through the feces. This increase was not due to a direct effect of deltamethrin on urine production by the Malpighian tubules but to a hormonal deregulation.

3. Intoxicated insects produced large amounts of the vasopressin-like insect diuretic hormone. This higher synthesis activity occurs within the hours following the insecticide injection and is accompanied by an increase in water loss.

4. These hormonal and metabolic modifications are transient. Hormonal level and diuresis rate both return to the basal levels 7 hr after the insecticide injection.

INTRODUCTION

One of the most obvious effects of the majority of the commercially available insecticides is a massive stimulation of the nervous system leading to hyperac- tivity, uncoordinated movements, tremor, convul- sions then knockdown. This nervous stimulation has been well documented for the pyrethroid deltamethrin, one of the most wide-spread insecti- cides currently used. Occurring in vertebrates as well as in invertebrates, this stimulation is mostly a conse- quence of changes in nerve membrane permeability resulting from a prolongation of the sodium current during membrane excitation (see Vijverberg and Van Den Bercken, 1990 for review). In insects, one of the consequences of this long lasting depolarization is a prolonged repolarization phase with an increase in neurotransmitter release (Salgado et al., 1983) and a depletion of synaptic vesicles (Schouest et al., 1986) in the peripheral nervous system. Evidence available from rat brain studies (Aldridge et al., 1978; Doherty et nl., 1987) suggests similar effects on the central nervous system. However, these primary manifes- tations of toxicity are rarely themselves lethal and it is still appropriate to ask why neurointoxicated in- sects die (Lund, 1986).

In one of the most recent and comprehensive reviews on insecticides, this author concluded that there was relatively little information available on our knowledge on the physiological consequences of neu- rointoxication in insects. Even though the relation- ship between insecticide intoxication and nervous system disfunction is obvious, the ultimate causes of

death are still unknown. The data mentioned above, suggesting that neurohormonal pyrethroid-induced releases could occur in the central nervous system of insects, are in agreement with the hypothesis of Samaranayaka-Ramasamy (1978) of a disturbance of several hormone-controlled metabolisms due to a non-selective release or neurohormones. Thus, intoxi- cation followed by insect death might result from the addition of these imbalances in some physiological processes crucial for the insect life.

This hypothesis was then summarized in Lund’s explanatory scheme (1986). The nervous system dis- ruption due to the insecticide treatment would have two major consequences: 1. a release of multiple neurohormones; and 2. correlated behavorial and physiological changes (including the inability to feed) leading to a general breakdown in homeostatic mech- anisms, to an alteration in blood composition, incon- sistent with cellular integrity, and ultimately to the death.

Although attractive, this statement has remained up to now an hypothesis. Samaranayaka (1974) tried to demonstrate a release of the hypoglycaemic and adipokinetic factors from the corpora cardiaca of poisoned gregarian locust (Schistocerca gregaria) but failed, since no difference in hormonal activity was observed neither in the tissue nor in the haemolymph extracts. Therefore, no one ever demonstrated a change in neurohormone metabolism as a direct consequence of the nervous system breakdown ob- served in insecticide-treated insects.

An almost constant physiological consequence of insecticide intoxication reported to-date is a severe

351

352 J. Paoux et al.

water loss, but little is known about this disruption in homeostasis. We took advantage of our knowledge of the hormonal control of diuresis in the migratory locust to try to correlate the lethal effect of several insecticides on this insect with changes in diuresis regulation. We measured first the influence of differ- ent insecticides on live weight and water content in larvae and then we focused our interest in the most potent and used insecticide: deltamethrin. We investi- gated its influence on adult diuresis, from behavioral studies to the dosing of one of the locust diuretic hormones: the vasopressin-like Insect Diuretic Hor- mone (AVP-like IDH). This hormone is an 18-residue antiparallel dimer made from two molecules of a nonapeptide bound by disulfide bridges (Proux et al., 1987). The AVP-like IDH and its monomer (devoid of any known biological activity) are synthesized in the suboesophageal ganglion (SOG) of the migratory locust and are transported together in all the central nervous system to be released into the haemolymph. The AVP-like IDH acts on the Malpighian tubules in a diuretic manner by increasing the primary urine excretion (Proux et al., 1988).

MATERIALSANDMETHODS

Insects

Fifth instar male and female larvae and adult 20-day old male migratory locusts (Locusta migra- toria) were reared under crowded conditions at 30°C 70% humidity and 12L/12D. They were fed fresh grass and dry food (mostly bran). Experiments were conducted at the same hour each day. Experimental animals were starved overnight before experimen- tation. Only still-living animals were considered. Those dying during the experiments were discarded. Each experimental group was kept in a small cubic cage (20 cm each side) or in a little plastic box where urine was colleted on filter papers.

Chemicals

Deltamethrin [(S) - tl - cyano - m - phenoxybenzyl (lR,sR) - 3 - (2,2 - dibromovinyl) - 2,2 - dimethyl -cycle - propane carboxylate)] was a gift from Roussel- Uclaf (France), lindane (hexachlorocyclohexane y stereoisomer) fenthion [O,O-dimethyl, 0-(methyl-3- methylthio+phenyl) thiophosphate] and baygon (0 - isopropoxyphenyl - N - methylcarbamate) were purchased from Pepro (France) and Bayer (Nether- lands), respectively. Trifluoroacetic acid (TFA) was from Merck (Germany) and propanol (2-PrOH) was from Prolabo (France.).

Insecticide application

Larvae. Each insecticide was dissolved in acetone and applied at the neck level (on the permeable membrane laying between the head and the thorax). Doses producing a lethal effect of 50% (LD& after 18 hr of intoxication were previously determined. They correspond to 5, 3, 0.3 and 0.125 pg/g of locust

live weight for fenthion, lindane, baygon and deltamethrin, respectively. Acetone dilutions for each insecticide were adjusted to deliver the insecticide in a 4~1 aliquot. Sham-operates received 4 ~1 of acetone.

Adults. Deltamethrin was freshly prepared to avoid an isomerization leading to inactivation (Perschke and Hussain, 1992). It was first dissolved in pure ethanol (stock solution) and used at two doses: 0.20 and 0.23 pg/g of locust live weight (corresponding to LD~~ and LD,~ after 48 hr of intoxication, respectively). Each dose was prepared in 1 pl of alcohol adjusted to 10~1 with saline and injected into the abdomen haemocoel. Sham-operates received 10 ~1 of the same mixture without deltamethrin.

Weight measurements

Live weight (LW) was measured twice: immedi- ately after insecticide application (larvae) or injection (adults) then at the end of the experience. Dry weight (DW) was obtained after a 12 hr-80°C drying. Final water percent was the ratio (LW - DW)/LW. Pre- liminary measurements demonstrated that DW was not modified during the experience. This allowed us to determine the initial water percent of each exper- imental insect and to calculate the change in water content.

Experiments lasted 18 hr for larvae, and up to 48 hr for adults.

Fluid excretion appraising

Excreted feces from six locusts were allowed to fall on the filter-paper collectors (area 84cm2) which absorbed the spotting fecal fluid. The total surface of the spots was measured using a video-camera, a computer and a surface analysis program (Biocam 200 Image Anaiyser) when the experiment was over. Comparison of surfaces between insecticide-treated and sham-operated insects leads to a fair appreciation of their respective fluid excretions.

Fluid excretion measurement

In vivo. Urine excretion was monitored using a test based on Malpighian tubules rapid excretion of a dye, the amaranth (Mordue, 1969). Insecticide-treated or sham-operated male adults were injected with 20 p 1 of 0.50% amaranth in 0.9% NaCl (to avoid an osmotic stress) 4 or 24 hr after the treatment. Half an hour after injection, 40 ~1 of dyed haemolymph was removed and diluted in 1 ml methanol. The samples were centrifuged for 20 min at 35,000 g to pellet large molecules and to avoid turbidity. Supernatants were then spectrophotometrically measured at 523 nm. Dye concentrations were calculated using a reference curve. An increase in urine excretion leads to a faster amaranth release (i.e. to a lower optical density).

In vitro. Diuretic activity of isolated Malpighian tubules was measured according to Proux et al. (1988). Briefly, sets of Malpighian tubules were iso- lated, cannulated and bathed in oxygenated saline

Deltamethrin and water balance in a locust 353

(Hanrahan and Phillips, 1982) complemented or not with deltamethrin. The insecticide (previously pre- pared in alcohol as a stock solution) will dissolve in the saline as demonstrated by previous results from Chalmers et al. (1987), Lombet et al. (1988) and Amar et al. (1992). Urine flows into the cannula and reaches paraffin oil where it forms circular droplets measured with an ocular equipped with reticle. Urine excretion rate was monitored per 15-min period during 2 hr: H 1 (equilibration) and H2 (deltamethrin or alcohol addition). The excretion of each quarter of H2 was compared to that of the last quarter of Hl and the difference expressed as a percentage.

Extraction and pur$cation of the A VP-like peptides

Individual or pooled SOG were homogenated in methanol at 4°C for 30 set using an ultrasonic probe (Ultrasons 150 SF, Annemasse S.A., France). Hom- ogenates were centrifuged for 30min at 4°C and 35,000 g, supernatants were dried using a Speed-Vat concentrator (Savant Instruments, Farmingdale, New York, U.S.A.) and saved at -25°C for enzyme immunoassay or subsequent purification.

As stated above, the AVP-like IDH and its monomer are present together in the SOG. We used a classical reversed-phase liquid chromatographic (RPLC) procedure (Proux and Beydon, 1992) to separate them from the dried SOG supernatants.

A

1 2 3 4 5

treatment

Briefly, supernatants from groups of 10 SOG were first prepurified using disposable reversed-phase car- tridges (Sep-Pak Cl8, Waters Associates, Milford, MA, U.S.A.). AVP-like peptides were eluted in a 30% 2-PrOH/O.l% TFA fraction which was then concentrated, dissolved in 0.1% TFA and loaded with a 1 -ml loop on a 25 x 0.46 cm analytical RPLC column of 7pm C4 packing (Zorbax). Elution was performed at a rate of 0.5 ml/min and 1 ml-fractions were collected. A 30-min linear gradient of 5-30% 2-PrOH in 0.1% TFA was applied using a Beckman system Gold liquid chromatograph equipped with a binary solvent delivery system and a model 168 detector. Chromatographic fractions were dried then saved at -25°C for subsequent screening using an enzyme immunoassay.

Enzyme immunoassay of the A VP-like peptides

AVP-like peptides contained in individual SOG extracts or in RPLC fractions were dosed using an enzyme immunoassay recently developed (Proux et’& 1993) and based on the use of insect AVP-like IDH antibody and AVP-like IDH acetylcholinester- ase conjugate as enzymatic tracer. Sensitivity is high for AVP-like IDH (0.3 nmol/l) and meagre for its monomer (14 nmol/l).

Amounts of AVP-like IDH or of its monomer contained in the RPLC fractions were directly ex-

B

._ 1 2 3 4 5

Treatment

Fig. I. Change in live weight (A) and water content (B) in fifth instar male (D) and female (a) larvae after a 18 hr period with several insecticides. 1: control, 2, 3, 4 and 5: larvae receiving 5, 3, 0.3 and 0.125 rg/g of locust live weight of fenthion, lindane, baygon and deltamethrin (LD~), respectively.

M + SEM; n = 15;* and **: P i 0.05 and 0.01, respectively. _

3. PROUX ef al.

OH 6H 12ti 24H 46H

Time (hour*)

on 621 ?2 H 24 H 46 H

Time (hours) Timr (hours)

2 5

0'

z E - 5’

.F i

:! = -10- S u

$ 6 -16

-2o-

-26J ’ OH 6H 12 H 24 H 46 H

Time (hourr)

2

Fig, 2. Change in live weight (A) and water content (B) in deltamethrin-treated or sham-operated adult male locusts during a 48 hr period. 1,2: treatment with 0.20 and 0.23 pg of dei~rnet~~u~g of locust live weight (LB% and LD,&, rpspecti~ely, I): sham-operated group; 0: de~~rnet~~~-tr~t~ group. M + SEM;

n f 8 to 10-F P < 0.01. 7

Deltamethrin and water balance in a locust 355

pressed in pg of these molecules and amounts of the mixture of the two AVP-like peptides contained in the SOG extracts were expressed in pg of AVP-like IDH equivalent.

Statistical treatment of data

Standard errors are shown in graphs, Comparisons between the experimental groups were performed by means of variance analysis. In larval insects compari- sons were done within each experimental group (data at t = 0 versus data at t = 18); in adults, comparisons were made between the experimental groups (insecti- cide-treated versus sham-operated).

RESULTS

Influence of insecticides on live weight and water content

Larvae. All insecticide-treated insects exhibited sig- nificant live weight loss and decreased water content after an 18-hr intoxication period, when compared to sham-operated locusts (Fig. 1) in both males and females. Maximal live weight loss and dehydration were observed when using deltamethrin or baygon.

Adults. Comparison between insecticide-treated group and sham-operated group shows that deltamethrin causes a significant decrease in water content and live weight (Fig. 2A and 2B). The difference between the two deltamethrin doses (LD,,

and LD,J is not significant, likely because data deal only with still-alive animals. This difference in weight loss (and consequently in water content loss) is mostly noticeable during the first hours following injection as demonstrated in Fig. 3. Then it becomes weaker, and decreasing rates are almost similar.

This consequence of intoxication is soon noticeable as demonstrated when monitoring live weight every hour (Fig. 3). Intoxicated locusts start to lose live weight three hours after the injection of deltamethrin, i.e. earlier than sham-operated insects.

Behaviour of insecticide-treated insects in relation to water loss

The origin of water loss was studied first since it can occur through different processes including evap- oration, degurgitation or diuresis. Locusts (two groups of eight) were injected with alcohol (sham-op- erated) or deltamethrin (insecticide-treated) and their behaviour was observed during 4 hr. Sham-operated insects had coordinated movements and excreted feces at a normal rate. Intoxicated animals exhibited, first of all, a period of hyperactivity with uncoordi- nated movements followed by a knockdown period. An abdominal peristaltism was observed with pro- duction of big and damp feces. On the other hand, no degurgitation was observed. These first results are in agreement with a diuretic origin for the observed water loss. They were confirmed when measuring the urine spots on the sheets of filter-paper. Areas of the urine spots were measured after 6 and 24 hr stays

a-

-1 -

a-

-3-

‘0 2 4 6 I

8

Time (hours)

Fig. 3. Change in live weight in deltamethrin-treated (0.23 pg of deltamethrin/g of locust live weight, LDT5) or sham-operated adult male locusts during a 7 hr period. 0: sham-operated group; 0: deltamethrin-treated group.

M + SEM, n = lo;**: P < 0.01. -

above filter papers of sham-operated or insecticide- treated locusts. These latter have a diuresis signifi- cantly higher than that of sham-operated locusts since spot areas were 4.44 or 27.95 cm* (6 hr) and 6.02 or 37.96 cm’ (24 hr) for sham-operated or intoxicated groups, respectively.

Measurement of primary urine excretion

In vitro. Primary urine excretion was measured on isolated Malpighian tubules bathed on standard or deltamethrin-supplemented saline (1 p g/ml, i.e. an insecticide concentration equivalent to that injected in a standard locust when an LD,~ is required). No difference was observed (result not shown), ruling out a direct action of deltamethrin on Malpighian tub&s.

In vivo. Dye excretion was measured 4 and 24 hr after the injection of deltamethrin (0.23 pgg/g of live weight, i.e. a LD,J_ At f = 4, insecticide-treated lo- custs exhibited an excretion rate significantly higher than that calculated for sham-operated locusts. This difference was no longer observed at t = 24 (Table 1).

Table I, Dye amounts in 40 p I haemolymph samples collected from deltamethnn-treated (D) or sham-operated (SO) locusts submitted to the in virro bioassay 4 hr or 24 hr after the injection. M + SEM;*:

P < 0.05; N.S. non-significant difference

Haemolymph amaranth amount (pg/40 ~1)

MkSEM n P

SOf=4 23.6 * 2.2 I7 Dt=4 17.3 * I.5 I7 < 0.05 SO t = 24 22.47 f 1.2 I5 Dr=24 20.11 * 1 7 I2 N.S.

356 J. PROUX et al.

Measurement of A VP-like peptides in the sub- oesophageal ganglion

Total amount (A VP-like IDH + its monomer). The

amounts of AVP-like peptides were measured over time from locusts treated with deltamethrin and compared to those of sham-operated animals (Fig. 4). Intoxication provokes a large increase in AVP-like peptide content at t = 1 followed by a return towards the basal level at t = 7. Sham-operated animals ex- hibit an increase in AVP-like peptide content, but to a much lower extent. High standard errors demon- strate the importance of the individual responses to intoxication (due, at least partly, to the use of a LD,J. Additional measurements were carried out at t = 16 and t = 24, as observed at t = 7; AVP-like peptide amounts were similar in the treated and sham-oper- ated locusts (result not shown).

Specific amounts (AVP-like IDH or its monomer). The previous results left unsolved one problem in- herent to the assay itself. Due to the cross-reaction of the monomer when dosing AVP-like IDH in the SOG extracts, we did not know to what extent this monomer accounted for the observed AVP-like pep- tides increase. To answer this question, SOG were dissected out either immediately (t = 0) or 1 hr (t = 1) after alcohol (SO: sham-operated group) or deltamethrin (D: intoxicated group) injection. Then, AVP-like IDH and its monomer were separated by RPLC and quantified using the enzyme immuno- assay. Results are summarized in Table 2.

0 1 3 7

lime (hours)

Fig. 4. Content of AVP-like peptides in SOG of sham-oper- ated (II or deltamethrin-iniected W locusts (0.23 ug of

.-I

deltamethrm/g of locust hv; weigh;, ID,,) at t = 0, ; = 1, t = 3 or t = 7 hr after the injection. M f SEM, n = 8.

Table 2. Amounts of Mm and AVP-like IDH (pg) and Mm/AVP- like IDH ratios in IO-SOG pools from sham-operated (SO) and deltamethnn-InJected (D) locusts (0.23 pg of deltamethrin/g of locust hve weght, LD,~). Measurements were done immediately (I = 0) or

I hr (I = I) after the injectlon

Mm (PS) Dm (PP) MmjDm

sot=0 216 88 5 sot=1 948 423 4 Dt=O 263 149 3.6 Dt=l 1552 845 3.6

At the beginning of the experiment (t = 0), the respective amounts of monomer and AVP-like IDH as well as the monomer/AVP-like IDH ratio are comparable in the two experimental groups. One hour later (t = l), all of these amounts were in- creased, but these increases were more important in the insecticide-treated group than in the sham-oper- ated group, as already shown (Fig. 4). On the other hand, the monomer/AVP-like IDH ratio remained unchanged.

DISCUSSION

All the tested insecticides decrease water content and live weight in larval locusts. Similar results were obtained in adults when using deltamethrin. They are due to an increase in water loss through the Malpighian tubules. These data are in agreement with those of Casida and Maddrell (1971) which demon- strated in Rhodnius fifth instar larvae that several insecticides increased Malpighian tubules excretory activity. On the other hand, they do not fit with those of Gerolt (1976, 1983) which proposed that locust lethal water loss could result from a degeneration of the tegument, being no longer watertight.

In contrast with Casida and Maddrell (1971) who used an in vivo assay, the use of an in vitro test allowed us to demonstrate that deltamethrin does not act directly on Malpighian tubules but through other regulatory level(s).

One of them seems to be the metabolism of the AVP-like peptides. The comparison between the dosed amounts of AVP-like IDH and of its monomer in SOG of different experimental groups clearly demonstrates that synthesis of the two AVP-like peptides was disturbed. This is partly due to the prick and/or the injection of alcohol since the AVP-like peptides amounts were increased in the sham-oper- ated group but also, to a higher extent, to deltamethrin itself as demonstrated by the changes in the insecticide-treated group. This increase could be due to a deregulation in the AVP-like peptide syn- thesis itself and/or to an increase in some brain neuromodulators strongly suspected to regulate the AVP-like peptides balance in the migratory locust (Proux and Girardie, 1982).

Our results are in agreement with the Sama- ranayaka-Ramasamy’s (1978) hypothesis. Although other hormone mechanisms might be taken into

Deitarn~th~n and water balance in a locust 357

consideration in the insect death, it is likely that the disturbing effect of deltamethrin on the AVP-like IDH metabolisms can be involved.

This ins~ticide-indu~d hormonal deregulation and its consequences on Malpighian tubule fluid excretion seem to be very transient. They mostly occur at the beginning of the intoxication (t = 3) and do not last very long since AVP-like peptide contents and rates of diuresis from Malpighian tubules be- come very close again in the different experimental groups, 7 and 24 hr after the injection, respectively. This transitory effect is also noticeable when consid- ering the evolution in live weight during the first 7 hr following treatment since insecticide-treated insects lost weight earlier than sham-operates.

The lethal outcome of the insecticide-treated in- sects cannot be totally attributed to the observed disruption in water metabolism. It is probably also due to other behavioral and metabolic disruptions, but might be facilitated by the transient increase in dehydration which made the insects weaker.

To conclude, our results are the first direct demon- stration of an insecticide-induced hormonal pertur- bation leading (at least partly) to a disruption in homeostasis. It is likely that the initial insect dehy- dration is a direct consequence of the release then the use by the Malpighian tubules of this hormonal overload. Although this transient disorganization cannot, by itself, cause the insect death, it has likely had some influence on it.

Investigations are in progress to understand more precisely how, and to what extent, deltamethrin acts on AVP-like IDH metabolism, and to determine how this insecticide could act on the other neurohormones involved in the locust water regulation.

~C~no~Iedgements-We wish to thank C. Belloc and F. Gautron for their technical assistance.

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