Insecticide poisoning: Disruption of a possible autonomic function in pupae of Tenebrio molitor

PESTICIDE

BIOCHEMISTRY

insecticide

AND

PHYSIOLOGY

(1987)

Poisoning: Disruption of a Possible Autonomic in Pupae of Tenebrio molitor K.

*Institute Czechoslovakia,

29, 25-34

of Organic Chemistry and tDepartment

SLAMA*

T. A.

AND

and Biochemistry of Entomology,

Function

MILLERt

(OOCHB), U Salamounky 41, 15800 Prague University of California, Riverside, California

5, 92521

Received February 10, 1987; accepted May 14, 1987 Poisoning of pupae of Tenebrio molitor by pyrethroid, carbamate, or organophosphate insecticides was monitored by recording pressure in the hemocoel. The pressure was controlled automatically at around a negative 0.5 kPa value and predictable sharp peak increases in pressure of OS-l.0 kPa occurred in bursts. The pressure pulses in Tenebrio pupae were thought to represent the activity of an important autonomic function in insects. Insecticide poisoning increased the frequency of the pressure pulses and their amplitudes as high as 6 kPa in a dose-dependent manner. The pattern in pyrethroid-poisoned pupae was distinctly different from that following carbamate or organophosphorus poisoning. At 12-18 hr after treatment, the pattern of pressure bursts in pupae recovering from poisoning began to resemble the pattern before treatment, with peaks of pressure. In pupae not recovering from poisoning, the pattern was distinctly different, with only single peak pulses of pressure. Thus, it was possible to determine the fate of poisoned pupae after 12 hr. 0 1987 Academic Press. Inc.

tonomic processes in greater detail. A short series of rapid hemocoel pressure increases and decreases that occurred periodically as discrete bursts of pulses was discovered by chance in 1971 (3). The pulses were later found to be accompanied by (and presumably caused by) contractions of the intersegmental muscles in the abdomens of pupae (4). The pattern of pressure pulse bursts was altered by 1°C changes in ambient temperature, by hemolymph pressure changes of less than 1 Pa, and by changes in osmotic pressure caused by adding a few nanoliters of distilled water to the hemolymph (5). The pulse bursts were also seen to be highly coordinated with the timing of the opening of spiracles and the pressure thus created caused air to move into and out of the tracheal system (6). Thus, the pressure pulses were thought to be involved directly or to reflect indirectly the function of parts of the autonomic system of insects. In pupae of Tenebrio molitor the pressure pulses were shown to be created by synchronous contraction of intersegmental muscles in the abdomen. The contractions

INTRODUCTION

The final stages or endpoints of insecticide poisoning in insects have been arbitrarily determined and remain poorly defined (1, 2). For practical purposes, most laboratory determinations of toxicity are made a day or two after treatment; however, insects sometimes recover from chemical poisoning after being paralyzed for weeks. Part of the reason why the cause of death from insecticide poisoning is so poorly understood is because the physiology of most autonomic processes is not understood well enough. The autonomic processes include those that continue to maintain respiration, homeostasis, and water balance, for example, in paralyzed insects. It has not been clear what role the insect nervous system plays in autonomic functions. Obviously the insect nervous system can be severely poisoned with a loss of all locomotion, and yet autonomic functions continue . The discovery of hemocoel pressure pulses may provide a means of studying au25

0048-3575187

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Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

26

SLAMA

AND

and pulses stopped when the mesothoracic ganglion was lesioned or separated from the lower ventral nerve cord. They were not disturbed by any lesion or extirpation of nerve centers or connectives higher than the mesothoracic ganglion (6). Some information on the action of neuroactive drugs on the pressure pulses was reported previously (7, 8). We report here the difference between the actions of anticholinesterase (including previously reported results (7)) and pyrethroid insecticides on Tenebrio pupae as monitored by changes in the patterns of hemocoel pressure pulses. MATERIALS

AND

METHODS

Pupae of the mealworm, T. molitor L., were reared and stored at 27°C but measurements were done at room temperature (21 to 24°C). For measurements of hemolymph pressure, the pupae were installed on the needle of a hydraulic transducer as previously described (3). Pressures were recorded on a M-1000 multichannel tensometric apparatus manufactured by Mikrotechna Co. (Prague, Czechoslovakia), as described by Slama (9). The measurements of extracardiac pulsations were initiated a few hours before treatments. The values of hemolymph pressure were expressed in Pascal units relative to the local barometric (zero) pressure. The methods of decerebrating, ligaturing, or sectioning the nerve cord have been previously described (6). Pyrethroids used were: allethrin; bioresmethrin; pyrethrum (2% pyrethrins); cypermethrin and isomers S-3206, and S-2703; deltamethrin (Decis); fenvalerate analogs S-5602, S-5602A, S-5602Ad, S-5602B, and S-5439; permethrin; phenothrin. The S-numbered compounds were obtained from the Sumitomo Chem. Co. (Osaka, Japan). Nine organophosphorus compounds were used: malathion, thiometon (Ekatin), pirimiphos-methyl (Actellic). acephate (Orthene), methamidophos (Tamaron), mevinphos (Phosdrin), dichlorvos (Hogos), omethoate (Folimat), and dimefox (Terra-

MILLER

Systam). Five carbamates were tested: carbofuran, pirimicarb, propoxur, thiofanox, and methomyl. DDT and y-HCH (lindane) were also tested. Upon arrival, compounds were refrigerated. They were dissolved in acetone to make final concentrations for topical applications and the dilutions were discarded after 3 days. Each pyrethroid treatment was replicated at least six times independently. The figures used here were taken as representative sample records from 2 years of tests using the 2 insecticides listed above. RESULTS

Lethality was difficult to determine. Treated pupae were kept for several days and hemocoel pressure was monitored along with development to the imaginal molt. Since we were interested in the physiology of the poisoned insects, and in particular, in the functioning of autonomic processes, we were mostly concerned about how these processes responded to poisoning. Attempts were made to measure the LD,, value of compounds on 4-day-old pupae of T. molitor. All insecticides were applied topically in acetone in a range of concentrations from 0.1 to 10 p.g. None of the pyrethroids tested in this way caused outright mortality, even after 3 days, despite profound changes in the autonomic functions. However, pupae treated with 10 kg doses all completed development successfully to the imaginal molt, but failed to eclose properly. Thus, we considered this a “toxic” dose. Higher doses (ca. 10 kg) of organophosphorus compounds caused mortality in a day or two. All development ceased, the pupae desiccated, the hemocoel pressure increased to zero from normally negative values, and pupae gradually blackened from bacterial infection. These lethal actions represented the greatest difference between pyrethroid and organophosphorus poisoning.

INSECTICIDE

POISONING

AND AUTONOMIC

According to all observations, the pyrethroids exhibited tremendously rapid action on the system regulating hemolymph pressure in pupae. Concentrations of around 10 pg or more caused symptoms of intoxication after 1 1 - 13 min (Table I), whereas organophosphorus and/or carbamate insecticides usually acted much later (>30 min to several hr). Our 4-day-old pupae were in the middle of the interecdysial period. The baseline hemocoel pressure was usually -0.1 to -0.5 kPa, corresponding to - 10 to - 50 mm of H,O hydrostatic pressure. Pupae of this age normally showed a stable pattern of repeated pressure pulses (Fig. 1). The pressure peaks occurred in the form of several, usually 8 to 10, sometimes more, pulsations with 25- to 40-min rest periods. The amplitude and frequency of the pulsations in one such series is shown in more detail in Fig. 1B. At room temperature the frequency of individual pulsations was normally 0.6-0.8 Hz, with an amplitude of 0.3 to 0.5 kPa. After topical treatment of 10 pg of permethrin, the pupa continued showing normal extracardiac pulsations but at 13- 14 min the regular pulses were replaced by irregular oscillations in hemolymph pressure, which progressively increased until a typical intoxication pattern of constant contractions occurred (Fig. IC after 15 min). Records taken over a longer time course at low sensitivity and extremely slow chart speed lacked details but were most instructive. Hemolymph pressure changes moniTABLE Lutenq

Period

between

Application

und First

FUNCTION

27

tored a few hours before and several hours after intoxication showed reversible intoxication to a sublethal dose of pyrethrum (1 pg, Fig. 2A). A return to the normal pattern of the pressure pulsations occurred after several hours (Fig. 3A and 3C). Some of the normal pauses between bouts can be seen between the 6th and 7th hr on this trace (Fig. 3C). The amplitude of the pulsations following this sublethal dose decreased after about 10 hr (Fig. 2A), but appeared relatively normal after the 18th hr (Fig. 3C). Figure 2B shows responses from application of a larger dose of pyrethrum (10 kg). Intensive contractions reaching 2.5 kPa or more of hemolymph pressure were recorded about an hour after treatment. The high amplitude and high frequency contractions continued for a very long time (Figs. 3B and 3D). The amplitude of contractions declined gradually after 14 hr (Fig. 3B) At this time the record showed an absence of any coordinated pulsations (Fig. 3D), and there were only single peaks related to sudden contractions of intersegmental muscles. This pattern continued until the pupae failed to eclose properly. Pyrethroid insecticides are generally more toxic at colder temperatures. Since the pattern of pressure pulses recorded during pyrethroid poisoning was distinctive, we were interested in demonstrating the reversibility of poisoning by altering temperature. The syringe needle was inserted into pupae as usual for recording hemocoel pressure. Pupae were topically treated with 1 Symptoms

of Irritation

on Pressure

Records

Pyrethroid

Dose 64

n

Range (min)

Average time

Permethrin Deck Pyrethrum

10 10 10

12 8 8

9.5-14.5 9.0-15.0 8.9- 14.5

11 min, 40 set 10 min, 50 set 12 min, 10 set

Note. Incomplete data of the average time (less than five measurements) for bioresmethrin allethrin 11 min; phenothrin 30 min; fenvalerate 20-45 min; and cypermethrin 40 min.

10 min, 50 set;

SLAMA

-o*sc

, 0

AND

1 30

MILLER

60

FIG. 1. (A) Pulsations of hemocoel pressure in a 4-day-old normal pupa 23°C. (3) One of the bouts of activity recorded at higher sensitivify and faster tive pressure of the baseline at about -300 Pa. (C) Pressure pulses recorded following topical application of IO kg permethrin (arrow), 23°C.

20 ng of Ambush (formulated permethrin) and kept in the room (24°C). After symptoms were observed and recorded (about 1Y2 hr after treatment), pupae mounted on the syringe needle were placed into a small chamber where the temperature was kept at 37°C. A thermometer mounted next to the pupae showed the ambient temperature. We did not record the internal temperature of the pupae. When pupae were placed at the warmer temperature (Fig. 4A), the constant pressure pulses decreased within a minute. When pupae were returned to room temperature, the pressure pulses characteristic of poisoning returned in about 1 minute (Fig. 4B). If the pupae were again placed in the warmer temperature, the symptoms of poisoning again decreased to zero (Fig. 4C)

90

120

of Tenebrio molitor at speed. Note the negafrom a 6-day-old pupa

in about 1 minute. The pupae could be held poisoned at 24°C or not poisoned at 37°C for several hours with each state being readily reversed upon changing the temperature . After the period of acute intoxication was over, there remained small residual contractions which lasted for many hours until death. In most cases doses of 10 kg of pyrethroids (many times the lethal dose on adults) did not kill the pupae outright. Those that survived had a decreased baseline pressure due to water loss (decrease of internal volume). Those that developed to adult emergence (7th day after pupal ecdysis) never formed morphologically perfect adults. Physostigmine poisoning of intact T. molitor pupae (7) produced responses some-

INSECTICIDE

0

I

0II

2-

I"

l-

2 x

o-

POISONING

4

2

AND

AUTONOMIC

29

FUNCTION

6

a

12

10

-1 -

-1.

'

1111

FIG. 2. (A) Hemocoel pressure in c14-duy-old prtprr of Tenebrio molitor at 23°C. Conrinuous record during application (arrow) of a lethal dose of pyrethrum extract (I pg, 2% pyrethrins). (B) Hemocoel pressure in II I-duy-old pupa of Tenebrio molitor at 23°C. Continuous recording during application (arronz) of a lethal dose of pyrethrum extruct (IO wg. 2% pyrethrins).

what similar to those shown here. Pulsations in hemolymph pressure were completely abolished in pupae with the nerve cord transsected between the first and second abdominal segments (See (6)).

Pupae deprived of the brain showed a normal regimen of extracardiac pulsations, as evidenced by initial phase of the record in Fig. 5A. Subsequent topical treatment by 10 kg of deltamethrin caused symptoms ost

1

m

2

-0.5

0 -1 -1

IdAA

-2 I 8

12

I

1

I

14

1

1

16 HRS.

FIG. 3. (A) and (C) Same pupa as in Fig. ZA, recorded dose. (B) and (D) Same pupa as in Fig. ZB, recorded ufter

11

0

11

2

11

’

4

6

1”

a

MIN.

after 18 hr of poisoning with 18 hr of poisoning by a lethal

a sublethal dose.

1

10

30

SLAMA

12 I

c

13I

AND

MILLER

b 14I

15I

16

17

I 21

1 22

I 23

I

I

24

1.6-

0-a -

O-

18

I 19

I 20 TIME

(Mln

)

FIG. 4. The effect of two different ternperutures (24 und 3PC) on the pvretllroid-indLtced responses in hernolyrnph pressure (topicul treutment by 20 ng c?fAmbrrsh 92 min before the time indicated here us zero) in I-duy-old pupae of Tenebrio molitor. The du.rhed line indicntes on& M’hen pupae wsere pluced info u 3PC chumber (A, inset) or removed to umbient room temperature ut 24°C (B. inset): it does not represent the actual internul temperutrrre of the pupae.

and patterns in hemolymph pressure similar to those from treatment of intact pupae by pyrethroids, but the contractions were weaker, i.e., changes in hemolymph pres-

sure reached only about 1 or 1.5 kPa, whereas normally deltamethrin treatment produced pressure peaks reaching 5 to 6 kPa. However, the time course of changes

Y

i

t

c

A

1

I

I o-5

0.5I

I

I

1

I

I 1

HOURS

I

I

I

1.51

1.5

7

I

I

2I

2

I

I

FIG. 5. Hernocoel pre~swc rec,orded from \‘crriolri j-day-old puptre of‘ Tetlebrio molitor. thc~t /ltrd (A) beet1 decerebrtrted 5 hr earlier; (B) connecti\~es le.vioned betnleen wboesophaaed and prothoracic garglirr 24 hr enrlier; and (C’) connerti~w bemren the second rend third abdorninol ganglicr sectioned 5 hr earlier. All pupae were treated (cwows) with (7 lerl7al dose of deltctrnethri~~ (IO pg of Deck). Note the clecrrly srnctller- pressure pulses drtring poisotling in the latter case (C). All pupae \tlere at 23°C. Note rrlso the o\~erall itlcwase in burr1 Iletnocoel pre,s.cuw occcrsioncrlly lusting 30 nljn or lorl~pr in euch record.

0

I

I 0 II-

0

1

2

I

2.5L

2.5

--=-CA

w c

2

2

3

3

9

32

SLtiMA

AND

MILLER

abdominal segments. Then there were unas shown in Fig. SA remained basically coordinated contractions mixed with the preserved. Similar results were obtained with pupae pulses and later very frequent contractions that had the nerve cord sectioned between mixed with the puises and still later very without apparent the suboesophageal ganglion (SC) and the frequent contractions first thoracic ganglion (Fig. 5B). Figure .5B spasms. The peaks in hemolymph pressure in orshows that the presence of the nervous or carbamate poisoning system located in the head (i.e., brain and ganophosphorus SG) is not essential for appearance of the were not associated with spectacular toxicological symptoms of pyrethroid ac- spasms; the movements were mostly inconspicuous. Sometimes we observed protions on hemolymph pressure, although longation of the abdomen with no apparent they affected the intensity of contractions and so the size of the peaks. spasms. The differences between pyreThe record in Fig. 5C shows responses throids and anticholinesterase agents were following IO p,g topical treatment with del- evident also in the type of the residual pressure pattern tamethrin on a pupa with the nerve cord changes in hemolymph Figure 6A previously sectioned anterior to the second several hours after intoxication. abdominal ganglion. Here very small re- shows the most common type of residual sponses were obtained in hemolymph pres- pattern in hemolymph pressure which was sure. Since the first abdominal ganglion is maintained for prolonged periods, somefused with the metathoracic ganglion and times several days, after treatment of natcannot be surgically removed, pressures ural and many synthetic pyrethrins. were exclusively due to contractions of the A slightly different pattern was observed with phenothrin. as shown in Fig. 6B. but intersegmental muscles between the first and second abdominal segment which are still there were contractions of large and innervated from the first abdominal gan- small amplitude occurring in succession. glion. All other intersegmental muscles did The typical residual pattern after intoxicanot contract. tion with the carbamate methomyl (Lannate) (Fig. 6C) and the organophosphorus These, and additional ligation expericompound methamidophos (Tamaron) (Fig. ments, strongly suggested that pyrethroids acted in this system via the thoracic ganglia 6D) shows for comparison the steady of the nerve cord, i.e., through the system spasms and contractions with regular presgoverning the regulation of hemolymph sure peaks resulting from the inhibition of cholinesterase. pressure. By contrast to eserine, and other anticholinergic drugs and insecticides, pyDISCUSSION rethroid actions were expressed as convulsions in pressure only if the nervous conRhythmic pulsations in hemocoel (or henections between the central pattern gener- molymph) pressure occurring periodically ator in the mesothoracic ganglia and the are a feature of every pterygote insect exeffector muscles were intact. amined so far. Although these pulses were In organophosphorus poisoning of Tene- first discovered in quiescent adult Pyrdzocoris optrrrrs bugs, the pulses are largely brie pupae (7) intoxication was manifested by large spasms seen in the flexible abdomoverwhelmed by larger pressure changes inal segments, which were repeated at created during ordinary locomotion in adult insects. Therefore, pupae provided the more or less regular 30-set intervals. With pyrethroids, no such consistent spasms oc- more obvious choice of insect stage to curred. The initial phases of intoxication by study pressure pulses in more detail. organophosphates showed tremors of the The pressure pulses appeared to be im-

INSECTICIDE

POISONING

AND

portant for proper mixing of hemolymph since lesioning of the mesothoracic ganglion (the neural origin of the pulses) did not impair cardiac function or development, but led to necrotic degeneration of extremities in pupae of T . molitor. Injection of distilled water into the hemocoel produced an increase in hemocoel pressure with increased pulsation intensity accompanied by increased spiracular water loss. When the original water balance was reattained, the pressure pulses returned to their pretreatment pattern. Injection of oil or isotonic saline in a similar manner caused an initial increase in pressure, but no change in the pattern or character of pulsations and no increased water loss. The details of how these autonomic responses

AUTONOMIC

FUNCTION

are controlled are not known, but they can be monitored by simultaneously recording hemocoel pressure and respiratory gas exchanges. Insecticide poisoning usually is never followed to a conclusion physiologically for practical reasons. The point at which insects expire is difficult to determine and usually does not occur until a long time after treatment, even weeks or months later. There has been an interpretation in recent years that death from insecticide poisoning is caused by a disruption in those parts of the nervous system that control automatic (or autonomic) functions. The control of water balance has been recognized as absolutely critical to the survival of in-

34

SLAMA

AND MILLER

sects, and the loss of water by poisoned insects is a much studied subject (IO). Although it has been shown that large releases of different types of neurosecretory materials are a general phenomenon accompanying insecticide poisoning, these studies have never made the clear connection between a specific neuroendocrine function and a fatal lesion (cf. I I). Perhaps a better description of the nervous control of autonomic functions in insects would be helpful. We view the discovery of rhythmic pressure pulsations and, in particular, the neural origin of these rhythms in the mesothoracic ganglion of T. molitor. as an example of the role of the nervous system in autonomic function, and therefore a real opportunity to define the vague final stages of insecticide poisoning. ACKNOWLEDGMENTS The Eastern European Exchange Program of the National Academy of Sciences awarded an exchange fellowship to T.M. and made this collaborative study possible. We thank Dr. Hirosuke Yoshioka. Sumitomo Chemical Co. Ltd.. Takarazuka, Japan. for pyrethroid insecticides. REFERENCES I. J. R. Bloomquist and T. A. Miller. Neural correlates of flight activation and escape behavior in house flies recovering from insecticide poisoning, Arch. Insect Bioclwm. Physiol. 3, 55 I (1986).

2. C. Collins, J. M. Kennedy, and T. A. Miller. Sublethal poisoning: A comparison of behavior and histological changes in house fly CNS. Prstic,. Biochem. Pilysiol. 11, I35 (1979). 3. K. Slama. Insect haemolymph pressure and its determination, Actu Entomol. Bohernoslo~~. 74, 362 (1976). 4. K. Slama. Recording of haemolymph pressure pulsations from the insect body surface, J. Camp. Physiol. 154B, 635 (1984). 5. A. Provansal-Baudez and K. Slama. Effect of perisympathetic organs on extracardiac pulsation in Tmebrio molirctr (Coleoptera). Actrt Etr/0/~70/. Bohernosh~. 82. I61 ( 1985). 6. K. Slama, N. Baudry-Partiaoglou, and A. Provansal-Baudez, Control of extracardiac haemolymph pressure pulses in Ter7cbuio editor., J. Insect PI1ysiol. 25, 825 ( 1979). 7. K. Slama. Cholinergic control of extracardiac pulsations in insects, E.uperirntitr 42. 54 (1986). 8. J. Zdarek and G. Fraenkel. Pupariation in flies: A tool for monitoring effects of drugs. venoms. and other neurotoxic compounds, Adc. Iturc’t Bioc~hc~rn. Physiol., in press. 9. K. Slama, Microrespiratory in small tissues and organs. in “Measurement of Ion Transport and Metabolic Rate in Insects” tT. J. Bradley and T. A. Miller. Eds.). pp. 101-129. SpringerVerlag. New York. 1984. 10. J. B. Buck and M. L. Keister, Respiration and water loss in the adult blowfly, Phot~m’u reginu. and their relation to the physiological action of DDT. Biol. B/r//. 97. 64 (1949). I I, G. J. P. Singh and 1. Orchard. Is insecticide-induced release of insect neurohormones a secondary effect of hyperactivity of the central nervous system’? Pestic,. Biochrtn. Physiol. 17, 232 (1982)