Central vs. Peripheral Action of Pyrethroids on the Housefly Nervous

9 Central vs. Peripheral Action of Pyrethroids on the

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Housefly Nervous System T. A. MILLER and M . E . ADAMS Department of Entomology, University of California, Riverside, Calif. 92502

Despite years of research, the site and mode of action of pyrethroids have defied description, even the simplest c l a s s i f i c a tion as peripheral or centrally acting has not been possible to date. On one hand, pyrethroids act in a manner resembling DDT, which is known to be a peripheral neurotoxin in insects (1). The activities of both DDT (1) and pyrethrum (2) exhibit a negative dependence on temperature, being more toxic at lower temperature. Both DDT and pyrethroids produce negative after potentials, and repetitive discharge to single stimuli in axons (3). And both DDT and pyrethrum are extremely sensitive in causing trains of sensory nervous impulses when perfused on leg preparations of insects (4, 5, 6). The actions of DDT and a l l e t h r i n are also similar on the l a t e r a l - l i n e organ of the clawed toad, Xenopus laevis (7). Despite the impressive actions of pyrethroids on sensory nerve structures and the similarity between the actions of DDT and pyrethroids on isolated preparations on the nervous system, there is evidence of actions by pyrethroids on the central nervous system. Burt and Goodchild (8) found that speed of knockdown was proportional to the distance between the site of topical application and the central nervous system. They considered this to suggest strongly that knockdown, even the rapid knockdown reported by Page and Blackith (9), is due to an action on the central nervous system. Burt and Goodchild (10) found that the isolated and perfused central nervous system of Periplaneta amerieana was sensitive to extremely low concentrations of pyrethrin I (below 5 X 10 M ) . In contrast, DDT was without effect on the thoracic ganglia of Peviptaneta even when applied in emulsions of 4.5 X 10 M concentration (6). This latter observation is the best demonstration of a difference between the actions of DDT and pyrethroids. The ultimate actions of pyrethroids, then, could involve central and peripheral nervous structures—which of these might be involved during poisoning has been d i f f i c u l t to show u n t i l in vivo recording methods were developed to record the activity 8

3

98

Elliott; Synthetic Pyrethroids ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9.

M I L L E R

of

flight

(11, a

A N D

motor

12,

13,

central

the it

whole was

poisons


central was

house

that

ize

f l y during

the pattern

was u n i q u e 12).

patterns

of

poisons,

it

acting

results

were

of

from

since

motor

neurons

Using

recorded that

poisoning

the monitoring

of

this

activity

similar

method,

d u r i n g DDT

centrally

the pyrethroid

was c o n c l u d e d

of

from

acting

trans-

t o DDT a n d u n l i k e

that

trans-Barthrin

(12). challenged

extensive

the actions

flight

activity

and d i s t i n c t

showed

f l y during

allows

poisoning.

of

Furthermore,

nervous

a more

between

99

System

house

preparation

intact

peripherally that

the intact

This

coordination

These so

of

15).

neural

(11,

Barthrin

units

14,

found

poisoning

Action in the Nervous

A D A M S

informally

examination

by Paul

was u n d e r t a k e n

Burt

to

(16),

character-

pyrethrins.

Methods Three

pyrethroids

Tetramethrin

hydrophthalimidomethyl fast a

knockdown

sample

were

from

Berkhamsted.

toxicity

Richard Hart, sample

(+)eis

was s a i d

on insects

Wellcome

Research

was r e p o r t e d l y

to have

(17).

We

very obtained

Laboratories,

a +25/75

0

was chosen

(NRDC

examination. 2,3,4,5-tetra=

eis/trans

mixture. cis-

perties

for extensive phthalthrin,

chrysanthemate)

b u t poor This

chrysanthemate

Cis-methrin

chosen

(synonyms=neopynamin,

119).

tetramethrin

for intermediate

Cis-methrin

is

k i l l

and knockdown

pro-

5-Benzyl-3-furylmethyl

chrysanthemate:

eismethrin The

third

compound

examined

Procida

Chemical

Company

Othrin,

Bioethanomethrin)

through

was RU11679, Wellcome

provided

Research

5-Benzyl-3-furylmethyl

by the

(synonyms=k-

(+)trans

ethanochrysanthemate.


100

SYNTHETIC

For

purposes

o f comparison,

carbamate

insecticide

furanyl-7

W-methyl

and

dissected Nervous


using

bathed

in

dissolved The to

a grease

then

acetone,

removal

impulses

were

l e g o f t h e house l e g was stapled

tungsten

wire

cuticle was

droplets

flight

elsewhere muscles

(12,

in (20).

large

portion

line box area

bristles

shown

on thedorsal

located

quite

through

the cuticle

place.

Only

silver

sufficiently

large

o r Brush

these

cells

simple.

Compounds

For the

in

T h ef r o n t

surface of

ganglion

electrodes

down.

t h emesothoracic Compounds once

IV-dissecting

were

in

flies,

of legs

ganglion

was l e f t

perfused

onto

saline

and also

this

study. with

diameter

ym

to monitoring Since

intact

in

acetone.

of pyrethroids on were

then

removed

implanted

mounted

with

upside

andthe ventral The furcasternite

as

its

removal

caused

ganglion.

t h e exposed described

used

just

thepotentials a r e

was exposed.

to the thoracic

to be

amplification.

muscles, were

major

muscles

o f 25

directly

females

Calliphora

in

flight

wires

without

The

of

a n d waxed i r t

used

o f house

segment

medium,

were

a

of the thoracic

t o t h e t i p o f abdomen

the appropriate

disturbance

of interest

steel

occupy

of the mid

a r eplaced

toxicity

two p a i r s

cells

side

flight

f o r muscoid

insertions

wires

innervated,

applied

detail

of the thorax.

of the inherent

of the thoracic

excessive usually

were

measurements

thoracic

wire

a r esingly

pattern

muscle

muscle

muscles

recorder

in

The position

a s t o be connected 220

electrode

t o p i c a l l y in Ο . ΐ λ

on the rear area

joint, etched

through the

described

the f i b r i l l a r

or stainless

oscilloscope muscle

allows

from

metathor-

to wet the tarsus.

on either

F i g . 1.

dorsolongitudinal

non-insulated

f i b r i l l a r

originate in

applied

the general

t h emuscles

recorded

was i n j e c t e d

of dorsolongitudinal

and electrode

over

saline.

the opening

a t t h e coxa-femur

enough

cavity

thorax

accurately

Potentials are

of giant

Themuscles is

into

was punched

were

on the anterio-dorsal

of insertion

were

times

the isolated

h a s been

follows

of the thoracic

( F i g . 1).

Compounds 20

An indifferent

The arrangement

S i xpairs

were

Anelectrolytically

diameter

preparation

13).

and insert

from

Compounds

Musea domestiaa

flies

in

which was j u s t

motor

pro

tissues

solution

removal

t h e femur.

t h e femur.

o f acetone

The

After

urn)

near

of the cockroach,

through

Enough

were

6).

the l e g .

t o wax a s above.

1 m i l (25

in

the tibia

recorded

f l y .

of the tibia

placed

in

(19).

the

intact

to ordinary

Exposed

a t least

of the tarsus.

t h e hemolymph

(5,

nerve

according

a t p H 6.9

into

f l y l e g ,

l e g preparations

(18).

diluted

was i n j e c t e d

Nervous the

electrode saline

after

asthe

f l y in b o t h

andWeiant

the crural

recorded

a carbonate

displace

acic

were

in

solution

left

by Roeder

ascending

amerieana,

t h e house

o f t h e house

Cockroach

developed

impulses

included

motor

preparations.

those

Pevvplaneta cedures

preparations

l e g , theflight

essentially

as well

(2,2-dimethyl-2,3-dihydrobenzo=

carbamate).

Physiological cockroach

DDT w a s u s e d

carbofuran

PYRETHROIDS

thoracic

by Berridge

b y Thomson

(22)

ganglion (21)

as

f o r Phormia.


M I L L E R

A N D A D A M S

Action in the Nervous System

101


9.

Figure 1. Dorsal view of thoracic musculature of Musca domestica. Two dorsolongitudinal flight muscles (DLM) run longitudinally in the dorsal thorax, each comprised of six fibers. The six fibers of each DLM are stacked unihterally and are designated 1-6 from ventral to dorsal Recordings were made by inserting fine wires just below the cuticle into the appropriate motor unit. The desired unit was located in relation to the major bristles depicted in the map with circles.


102

SYNTHETIC

The

saline

pH was a d j u s t e d

compounds. study

Theprocedure

There


f i b r i l l a r

are 2 dorsolongitudinal each

a r e single

have

a single

dorsal-most

tion

separate

true

The

by a

of

c e l l

In unit

This

it

in

(23); cides,

is

concerning The

(23). that

to

activate

remain

of

under almost

the 6

in

muscle

and each

a D L M is

nerve

impulse

of a neighboring

thought

to be a

that

strong

neurons. o f anymotor

A similar

the left

on the units

condition

of

DLM.

lateral

of

likely

the right

Thenature

unit

of the

the

left

occurs

of

these

connec-

at

present.

of neuro-active

only

in

innervation

o f c i r c u i t r y a r e n o t known

to emphasize

4 of

central

DLM a r e w e a k l y

inhibitory

the action

in

(23).

lateral

has similar

Musca

influence

unit

and the other

axons

the units

sufficient

a few basic

i n s e c t i -

properties

t h e DLM u n i t s .

when

individual control used

t h e wing

DLM f l i g h t

in

motor

of firing

a l l normal

with

of

presumed

lateral

7

Hz a t 20°C

(12).

temperature

reflexly

of

similar,

t h e wing

on loss

of

lowrate

Thebasal

rate

of

or

units

downstroke.

t h e DLMf l i g h t

rather

continues (12). tarsal

are reinforced

motor

of

f i r e

and below.

decapitation

on, the units

units

motor

Thef l y can increase

each

inhibition.

t h e motor

the flight

t h e DLM f l i g h t

t h e power

at a

room

start

Once

t h e DLM m u s c l e s f o r the generation

At rest,

or without

feedback.

of a l l of

over

flight

stupor

units

conditions,

f i r e

around

cold

flight.

or decrease

during

downstroke.

spontaneously

always

only

spontaneously prostrate

on by sensory

rate

two

innerva-

one neuron

neurons

any one neuron

of analyzing

to initiate

increase

is

motor

the details

unison

The

The

of

flight

to involve

to cause

fact,

each

bifurcates

ascending

no r e s e t t i n g

force

near

axon

pattern

an antidromic

rhythm

is

between

a r e presumably

In

unit

t h e 5 motor that

assumed

impulses

which

contact

is

f l y has lost

in

them.

DLMi n n e r v a t i n g

A motor

between

It

purposes

it

of

such

connection

to units

however,

in

since

4

5 R a n d 6 R in F i g . 1 )

n e u r o n whose

the right

the firing

connection

thought

For

in

units.

each

Calliphova

is

motor

t h e 12

through

innervating

DLM ( l a b e l l e d

of

fibers

innervates.

and implies

tions

1

the l e f t DLM.

connections

connected

Fibers

cells

neurons

Antidromic

Musoa

giant

the muscle

connected

DLM have

neuron

called

T h e same

DLM ( 2 3 , 2 4 ) .

right

motor

cells.

the right

5 motor

functional

nervous

the

previous

Musca

Each

fibers.

canreset

inhibitory

a r e sometimes

single

Calliphora,

functionally

DLM

addition of

a

( D L M ) in

( F i g . 1).

the muscle

5 motor

comprise

muscle

in

muscles

6 fibers

multinucleate

reaching

is

cells

fibers

fibers

innervated

before

containing

muscle

they

the

in

and Discussion

domesticcLy

one

after

briefly

(14).

Results

are

to 6.9 and rechecked was d e s c r i b e d

PYRETHROIDS

to

decrease to However,

motor

units

as a result of firing


is

of


9.

M I L L E R

A N D

A D A M S


System

103

P r e l i m i n a r y s t u d i e s suggest t h a t a group of nerve c e l l b o d i e s a r e l o c a t e d n e a r t h e l e f t l a t e r a l edge o f t h e g a n g l i o n and in b e t w e e n t h e p r o t h o r a c i c and m e s o t h o r a c i c n e u r o m e r e s . T h i s group of c e l l s i n c l u d e s t h e 5 motor neurons i n n e r v a t i n g the l e f t DLM. A l t h o u g h no e v i d e n c e e x i s t s , one may assume a s a w o r k i n g h y p o t h e s i s t h a t l a t e r a l i n h i b i t o r y c o n n e c t i o n s between t h e s e 5 neurons occur w i t h i n t h e i r immediate l o c a l i t y . The m o t o r n e u r o n s s e n d axons d o r s a l l y out the main d o r s a l n e r v e which l e a v e s the gang l i o n a t a p o i n t above the c e l l b o d i e s . The m o n o p o l a r n e u r o n s a l s o send b r a n c h e s d o r s a l l y t h e n a r c h i n g toward t h e mid l i n e o f the g a n g l i o n to presumably connect w i t h symmetrical branches of t h e r i g h t DLM m o t o r n e u r o n s . The f i n e d e t a i l s o f t h e s e l a t t e r c o n n e c t i o n s a r e unknown, b u t t h e y a r e p r e s u m e d t o s u p p o r t t h e c o u p l i n g b e t w e e n r i g h t DLM and l e f t DLM u n i t s . I n s e c t Leg B i o a s s a y s . P l o t s of times r e q u i r e d to produce t r a i n s of ascending sensory impulses from t o p i c a l treatment to the house f l y l e g ( F i g . 2) o r p e r f u s i o n of p y r e t h r o i d s through the c o c k r o a c h l e g ( F i g . 3) were f a i r l y s i m i l a r . Compounds p r o d u c i n g f a s t knockdown w e r e more effective in producing t r a i n s of s e n s o r y p u l s e s . T e t r a m e t h r i n was s l i g h t l y more p o t e n t t h a n c i s - m e t h r i n on t h e h o u s e f l y l e g ( F i g . 2 ) , b u t b o t h t e t r a m e t h r i n and c i s - m e t h r i n w e r e f a r b e t t e r t h a n k - O t h r i n in p r o d u c i n g ascending t r a i n s of neuron impulses. The p o t e n c y o f k - O t h r i n was more s i m i l a r t o t h a t o f DDT t h a n t h e two p y r e t h r o i d s e x h i b i t i n g knockdown p r o p e r t i e s . F l i g h t Motor B i o a s s a y s . F l i g h t motor p a t t e r n of a normal f l y d u r i n g t e t h e r e d f l i g h t shows t h a t m o t o r u n i t s a r e a c t i v a t e d a t t h e same r a t e w i t h s l i g h t d i f f e r e n c e s in e x a c t t i m i n g ( F i g . 4 ) . T o p i c a l t r e a t m e n t o f the house f l y w i t h 1 yg o f c a r b o f u r a n c a u s e s h y p e r a c t i v i t y in a few m i n u t e s , t h e n c o n v u l s i o n s in a b o u t 5 minutes. By 1 0 m i n u t e s f o l l o w i n g t r e a t m e n t , t h e f l i g h t m u s c l e p o t e n t i a l s show u n c o u p l i n g b e t w e e n t h e l e f t and r i g h t DLM u n i t s ( F i g . 5, t r a c e s m a r k e d : 6R & 6 L ) . C o m p a r i s o n b e t w e e n 6 L and 5 L shows t h a t t h e m u s c l e p o t e n t i a l s o v e r l a p e x a c t l y r e f l e c t i n g t h e i r common i n n e r v a t i o n by t h e same m o t o r n e u r o n . T h i s " u n c o u p l i n g " between i n d i v i d u a l u n i t s of the f l i g h t m o t o r n e u r o n s i m p l i e s t h a t c a r b o f u r a n is a c t i n g on t h e c e n t r a l n e r v o u s s y s t e m w i t h o u t an a c t i o n on the p e r i p h e r a l n e r v o u s system. No c o n c l u s i o n s c a n be drawn c o n c e r n i n g t h e s i t e o r mode o f a c t i o n of c a r b o f u r a n in c a u s i n g t h i s a b n o r m a l u n c o u p l i n g r e s p o n s e because other c e n t r a l l y a c t i n g n e u r o t o x i n s a l s o cause u n c o u p l i n g : p i c r o t o x i n , l i n d a n e , d i e l d r i n and o r g a n o p h o s p h a t e s . DDT e x e r t s l i t t l e o r no d i r e c t a c t i o n o n t h e c e n t r a l n e r v o u s s y s t e m , b u t c a n be r e a d i l y c h a r a c t e r i z e d by m o n i t o r i n g f l i g h t motor p o t e n t i a l s ( 1 2 ) . L e t h a l d o s e s o f DDT c a u s e a g r a d u a l i n c r e a s e in f l i g h t m o t o r activities and s p l i t t i n g o f f l i g h t p o t e n t i a l s i n t o 2 and s o m e t i m e s m u l t i p l e s p i k e s ( F i g . 6 , a r r o w s ) . This i n c r e a s e in a c t i v a t i o n e v e n t u a l l y l e a d s t o a s t a t e o f c o n s t a n t


104

SYNTHETIC


Latency (min.)

PYRETHROIDS

FLY LEG T3 tetramethrin

6

5

4 3 - L o g Concentration (ug/.1 ul)

2

Ο

1

Figure 2. Dose-response relationships for insecticides applied to the isolated metathoracic leg of Musca. Dose is expressed on the abscissa as (-)log concentration vs. latency to sensory trains in the crural nerve. Latency is short for tetramethrin and cis-methrin, but significantly longer for k-Othrin, which lacks activity at lower concentrations. Barthrin and DDT analogs have effects in the range of k-Othrin.


9.

M I L L E R

A N D A D A M S


Latency(min.)

105

System

Ρ a m e n c a n a - T 3 Perfusion


20 h

.001

.01

.1

1

10

Concentration (ppm) Figure 3. Flot of dose vs. latency to sensory trainsinthe isolated metathoracic leg of the cockroach, Periplaneta american. The same trends apply here as in Musca, although cis-methrin assumes a more intermediate position between tetramethrin and k-Othrin.

4R-*-4—X—~L-«-*X- >,.L—^-L-—X

eRn^T-f^f

1

f

L-

f "t "'f—

Figure 4. Flight motor pattern of a normal housefly during tethered flight. Each trace represents a discrete motor unit. A common firing frequency is maintained by all motor units, but a loose phase relationship prevents units from firinginunison. The pohrity of 6R and 6L is reversed for comparison. Calibration: 100 msec.


SYNTHETIC PYRETHROIDS

106 motor

output

even

in

several

deep

examination also

pheral


the

of

showed Thus,

the

a

uncoupling

seen

system,

in

by

the

time

DDT a n a l o g s

in

in

other By

carbofuran, separated

by

in

motor

flight

rates

with

is

pyrethroids and

it

further

throids

were

isolated ent

peri-

flight

motor

recorded

following

a

treatment

increase

cursory

at

in

a c t i -

examination,

the

and

build

rate very

high

The

period of

centrally

the one

of

to

such

of

would

gradually

frequency of

as

varying

bursts

to

higher

Immediately

preceding

active

length start

inactivity

the

was

burst.

insecticides

characterization

during

nervous

Often

strength

between to

insecticides, bursts

distinguish

of

the

and

action

between

of

central

poisoning.

central on

the

system

or

leg

to

peripheral action, preparations

obtain

pyre-

and

on

the

some m e a s u r e

of

inher-

potency.

with

quickly. single

One m i n u t e began

nervous

coupled

both

between

the

and

4L).

recorded between

This

not

yet

fly

was

exhibiting

obtained

from

records

this

dose,

sidered

from

at are

units.

tetramethrin

of

occurred

this

1

few and

and

is

flies

down",

but

at

minutes

1/2

in

discharge

hyperactivity

house

"knocked

the

a l l

hours

lost

under this of

abdomen

flight (Fig. (Fig. 6R

a l l

same

arrows)

8, and

units

times

and

pauses

of

the

the

nervous

(Fig. system

investigation. and

poisoning

the

was

Within

locomotory

ability

of

this

sublethal

dose

of

tetramethrin

there

was

house

similar

minutes

and

period

The record

very

4

6L

occa-

were

at con-

several

hours. At

and

the

an

a

4R)

4R w i t h

units

over

still

6R,

treatment,

point

from

was

one in

0.1

very

of

later.

recovered

at

muscle

8,

however,

cf.

where

common

discharge

first

8

8,

approximately

determined

the

the

pattern,

(Fig.

F i g .

to

the

hyperactivity

same m u s c l e

seen

obtained the

the

be

origin

been

of

following

has

overall

muscles

active

The

units

on

discharges

left

exaggerated

arrow).

treatment,

and

can

2 minutes

The

treated developed

multiple

impulses.

right

When flies

following

between

are

house

showing

potentials

At sional

Poisoning.

tetramethrin,

potentials

to

a

remains

important

the

steady from

acting

of

the

examined

Tetramethrin

9,

from

evidence

central

low

enables

also

DDT a s

activity

convulsive

inactivity.

crucial

for

a

bursts

peripheral actions As

yg

at

difference

analogs

a

and,

inactivity.

units

(12).

of

in

brief

preparation

DDT

hours

that A

well.

produce

relative

predictable

This DDT

to

complete

The by

units

this

Another

overall

centrally

convulsive

preceding roughly

as

the

tended

the

Note

insecticides

activity

poisoning.

motor

units

contrast,

of

characterized

flight

motor

poisons. of

to

action

central

recordings

after

were

the

similar

between

7).

coupled.

on

trans-Barthrin,

acting

nature

(Fig.

remains

centrally

by

from

motor

activity

and

caused

difference, considerable

of

treatment

flight

main difference

poison

was

after

the

pyrethroid,

pattern

the

nerve

vation

hours

poisoning,

some


M I L L E R

A N D A D A M S

Action in the Nervous System

6R


6L ULUillliUJ llllllillllllllMllllllllllUllllli I 51 ΓΤΓΤΠΓΤΓΎΙΒΙΙΒΓ 111 mm uni iniimi 11II III 11111II ι II11 Figure 5. Disruption of coordination between motor units 6L and 6R caused by a lethal dose (1 μβ) of carbofuran. This effect on central coordination is characteristic of cholinesterase poisons. The firing of 5L mirrors that of 6L, indicating common input from a single motor neuron. Calibration: 1 sec.

4L 4R

Jt-JU-JU-i-JU-JU—-Jul—L-JuJt-—I

X^XJ^XUJ^^XXXiXik^

6R Figure

6. Flight motor pattern during tethered flight 1 hr after treatment with 1 pg DDT. Splitting of spikes is evident (arrows). Coupling between individual motor units is maintained despite symptoms of hyperactivity and locomotory in coordination at this stage of poisoning. Calibration: 100 msec.

4L.IUU \ HHUU44U-4U-14RÎ111 6L uni

1 I H H J U j l - U L 1 ίίίΐ i ί lima nu u ι • u ιι mi \

6RJIUJ—ll

ι ill,

j,ιιι

ii ι . ί I ll

ji

ι

ι

Figure 7. Condition of the flight motor almost 4 hr after treatment with 1 g DDT. The insect is in tetany and flight motor activation is almost continuous, yet the coupling between units is intact. Calibration: 0.5 sec.


108

SYNTHETIC

evidence

of

abdomen ment

uncoupling.

produced more

( i . e . ,

doses

applied

(%s-methrin the

abdomen

burst


more

in

than

accompanied One

after

by

after

near

still

period

ing

lost

LD50

(cf.

X

trace

a l l

traces

cis-methrin There

11,

dots)

to

occur

of

the

the treat-

house

highly

The

f l y

on

exaggerated

discharges

or

A)

B).

could

This

for

Topical

greater high

in

single

occur

activity

of

hours

o

t

n

n

in

was

of

qualitatively

house first

evidence

fly

was

several of

uncoupling

short

burst

units. house

flies

other the

before

k-Othrin

an

flies

still

to

by

period)

0.1

the

at

exaggerated

appearance

hastened

similar

by

than

term

the

house

abdomen

e

the the

frequency

symptoms

treatment

LD50)

which were

was

no

for

in

while

d e s c r i p t i o n of

lasted

the

treatment

a b i l i t y

Treatment

produced

15

which

(25

10).

top

on

thoracic

ganglion).

treatment

(Fig.

10,

locomotory

Poisoning.

symptoms.

symptoms

10,

(Fig.

the

quiescence Othrin

(Fig.

treatment.

k-Othrin

did

thoracic

Topical

(Fig.

continued

doses

tetramethrin as

hyperactivity.

hour

discharges

the

2 minutes

following

having

one

of

c i s - m e t h r i n produced

units

unit

hour

prostrate, minutes

within

single

one

nearer

\xg o f

0.1

doses

uncoupling

Poisoning.

with

discharges

occurred

Higher

obvious

PYRETHROIDS

of yg

or

poisonof

k-

appearance

responses

to

of

lower

doses. 14 ated

minutes

burst

uncoupling. occurred muscle than

20

(Fig.

(Fig.

minutes 13).

13,

with

in

the

than

its

The

same m u s c l e

phenomenon w i l l CNS

Assays.

nervous

time

from

treatment

by

pyrethroids

case)

appearance over

a

of

range

The

high

system

potency

parison

k-Othrin.

to

that

the

tral

site

and

in

view was

fact,

of

of in

pyrethroids of

CNS

of

flight

same

13,

compare

motor

units

to

uncoupling

the

uncoupling

to

further

study

elsewhere.

of

pyrethroids

fly

was

(thoracic motor

on

the

determined.

concentrations

the

the

i n i t i a l l y

(Fig. that

subjected

house

in

obvious

susceptible

some

uncoupling

units

units

detail

exposed

in

of

potency

the

2

exagger-

plus

ganglion

in

saline

units

was

cenThe

in to

this the

plotted

concentrations.

in

potency

of

k-Othrin

arrows),

more

details

being

various

uncoupling of

are

of

suggested

more

greater

the

in

pairs This

relative

of

yg 12,

pronounced

was

however,

in

(CNS)

surprising their

6R).

perhaps

on

The

tral

6R)

opposing

muscles;

reported

treatment

4R w i t h

during poisoning

be

0.1

(Fig.

u n c o u p l i n g between

unit

were

between

by

recorded

after

compare

opposite

units

treatment

were

u n c o u p l i n g between

6L

and

following

discharges

action

t e t r a m e t h r i n on

its

poorer

In

fact,

the in

comparing

same

range

of

similar

were the

thoracic

tetramethrin possessed

Unfortunately,

our

sample

the

toxicity

of

(Fig.

CNS p r e p a r a t i o n house

f l y

the

three

pyrethroids,

14).

This

in

in

f i t

at

ganglion

of

the

house

best

com-

suggested

potency the

was

to

the

cenfly

f i t .

t e t r a m e t h r i n was

a

mixture


of

M I L L E R

A N D A D A M S


6L


System

en

U—Jk- WUULU-A- -I |T I I! 'I'M! « !

4L

iT^t|îfî(^Mf~~1

4R ••^^-•ffr rV,V ~1 , |

Υ.

Figure 8. Flight motor output 1 min after a topical dose of 0.1 / A g tetramethrin. Symptoms of hyperactivity were obvious and accompanied by slight splitting of flight motor potentials (arrows). Calibration: 100 msec.

6R - 4 J U J L — U J J J U 4L

—|f^--ipT|^——Υ^γ^Υ^γνγ

Figure 9. An exaggerated burst (arrow) in 6L occurs 2 min after treatment with 0.1 μg tetrameth rin. Slight uncoupling is evident between 6L and 6R and 4R (hollow arrow). Calibration: 100 msec.

4L

U4JM#.

—HHUJ

ο 6

ΜΙεμ-

i\Mm

1

— f f M * i —

—

^

—

Figure 10. Topical treatment with 0.1 μ-g cis-methrin elicited exagger ated burst discharging in motor units separately (a.) or in unison (b). Calibration: 100 msec.



110

SYNTHETIC

PYRETHROIDS

Figure 11. Flight potential pattern 1 hr after treatment with 0.1 μ-g cis-methrin. Flight motor is uncoupled and hursts of high frequency discharge occur separately in different motor units. Calibration: 1 sec.

4R

-—~—*^$mmi

6L 6R

f j

^

^

-

^0Hh ^

^

f —

'

— —

Figure 12. Onset of high frequency discharge (arrows) and uncoupling of flight motor coordination 14 min after treatment with 0.1 pg of k-Othrin (25 X LD ). Calibration: 100 msec. 50

4RÎ

,1

1 • • 1 • ι η •! 4 •..

6L frftTtiTftirrnrnninrwrnfriHrTrHT ι ί éRi'Hririi-rri'fitiiriTHiTiii ffr t i n Figure 13. Pronounced uncoupling between flight motor neurons 20 min after treatment with 0.1 μg k-Othrin. Calibration: 1 sec.


.—

9.

M I L L E R

and

cis CNS

The

compound

of

CNS the

properties trains of

can

long

time,

less

of

It

of

activity

nervous

toxicity

a

are

determine

the

in

3.

CNS

a

i . e . ,

for

a

lethal

misleading

may

be

examined

and

Barthrin

without

axons.

i f

a

sufficiently dose

Structure-activity

be

1.

a b i l i t y

sensory

process,

in

(leg

with

peripheral

detoxication

the

activity

conclusions:

correlated

temporal

or

at

properties. would

to

peripheral

preliminary

impulses

metabolism

data

and

for

is

accumulate

knockdown

111

instructive

pyrethroids

pyrethroids

can

be

System

isomers.

basis

in

resist

it

would

resolved

provide

Toxicity

using

the

analysis

produce

2.


of

does

Knockdown to

isomers.

trans

activity

assays)


A N D A D A M S

regard-

studies

accounting

for

metabolism. These throids

hypotheses

examined

COMPOUND

here

by

as

comparing

shown

in

PERIPHERAL

CENTRAL

2

Leg

Exposed-

on

Treated

Trains

Paper

1.0

KD

Min.

CNS

ng

3

10

M

yg/fly

Uncoupling

X

Alone

Min.

Min.

pyre-

I. TOXICITY

KNOCKDOWN Min

the

Table

PB

SR

TETRAMETHRIN

5.75

0.5

18.7

+

7

.29

.07

CIS-METHRIN

7.97

0.8

24.9

+

7

.08

.03

2.8

1.2

20.7

+

5

.005

.003

1.7

2.3

>60

.66

.2

3.3

k-OTHRIN

31.2

BARTHRIN

TABLE central

I.

Comparative

nervous

action

knockdown,

and

toxicity

k-Othrin

at

1.7

has

onyl

butoxide

of

the

compounds

with

a

synergistic

k-Othrin higher other

synergistic

Barthrin action

are

Barthrin

are

reported

for

which than

As

a

a

2)

pyrethroids 2

Although

as of

there

I.

at

are

3.3 more

with

piper-

Tetramethrin higher

than

readily.

tetramethrin

is

in a

trains

the

by

Dr.

the

Even

with

sensory

nervous nervous between nerve

actions on

can

E l l i o t t

for

its

centrally

that

the be

of

DDT

toxic

the

owe

active

house

their

syn-

potency

In

effect,

responses

mask

system. knockdown

impulses

for

and

fly

by

modern

system.

peripheral

central

correlation of

bioassay but

account

of

pyrethroids

central and

could that

peripheral in

(Barthrin),

the

action

effects

produce

that

pioneered of

the

similar

suggest

action

sites

important

to

very

DDT-like action

improved are

DDT m o r e

coincidence, are

factors

action,

26). of

(Fig.

ratio

Table

detoxified

CNS p o t e n c y

These

ability

and

that

thetic

more

4

in

poor

of

an

ratios

(25,

virtue

there

of

latter

sensory

pyrethroids.

synergistic

compared

has

Barthrin

to

the

lowest

4

resembling

pyrethroids. leg.

ratio

suggesting

synergists

the

peripheral

for

4

and the


the 4

the

112

SYNTHETIC

PYRETHROIDS


" J

1

10 MINUTES

100

(log scale)

Figure 14. Dose-response curves for pyrethroids applied directly on the exposed CNS in saline. Dose is plotted on ordinate as (—) log concentration in mol/h against mean time to uncoupling on the abscissa. Tetramethrin shows slightly higher potency than cis-methrin or k-Othrin, but all fall in approximately the same range.



9.

MILLER

AND

ADAMS


System

113

p y r e t h r o i d s examined, t h i s does not prove that knockdown is caused e n t i r e l y by p e r i p h e r a l a c t i o n s . Uncoupling i n d i c a t e s chemical poisoning in the c e n t r a l nervous system. However, we do not know yet i f c e n t r a l nervous poisoning can occur without uncoupling or before uncoupling. T h e r e f o r e , the f r i e n d l y disagreement between Paul Burt and ourselves concerning c e n t r a l versus p e r i p h e r a l poisoning w i l l not be r e s o l v e d u n t i l more is known about the c e n t r a l s i t e and mode of a c t i o n of p y r e t h r o i d s . There is l i t t l e doubt, however, that some p y r e t h r o i d s are a c t i n g both on p e r i p h e r a l nerves and on the CNS. At p r e s e n t , it is not e n t i r e l y p o s s i b l e to assess the con t r i b u t i o n from p e r i p h e r a l a c t i o n and that from c e n t r a l a c t i o n to symptoms of p o i s o n i n g . Acknowledgement The authors wish to thank D r s . Richard J. Hart and Charles P o t t e r of Wellcome Research L a b o r a t o r i e s , Berkhamsted, England f o r p r o v i d i n g compounds and f o r t h e i r very u s e f u l d i s c u s s i o n s . Some of the r e s u l t s included here are from work by V . Salgado and J. Kennedy to be published in greater d e t a i l elsewhere. The r e s e a r c h was supported in p a r t by Environmental P r o t e c t i o n Agency Grant No. R-804345-01. The contents do not necessar i l y r e f l e c t the views and p o l i c i e s of the Environmental P r o t e c t i o n Agency, nor does mention of trade names or commercial products c o n s t i t u t e endorsement or recommendation f o r use. Literature 1.

2.

3. 4.

5. 6.

7.

Cited

Vinson, Ε. B. and C. W. Kearns. (1952). Temperature and the action of DDT on the American roach. J. Econ. Ent. 45: 484. Blum, M. S. and C. W. Kearns. (1956). Temperature and the Action of Pyrethrum in the American Cockroach. J. Econ. Ent. 49: 862. Narahashi, T. (1971). Effects of insecticides on excitable tissues. Adv. Insect Physiol. 8: 1. Singh, K. M., S. Pradhan and C. Dakshinamurti. (1972). Differential susceptibility of insect neuron to i n s e c t i cides. Indian J. Ent. 34(4): 263-271. Roeder, K. D. and E . A. Weiant. (1946). The s i t e of action of DDT in the cockroach. Science N.Y. 103: 304-306. Roeder, K. D. and E . A. Weiant. (1948). The effect of DDT on sensory and motor structure of the cockroach leg. J. Cell Comp. Physiol. 32: 175-186. van den Bercken, J., L . M. A. Akkermans and J. M. van der Zalm. (1973). DDT-like action of a l l e t h r i n in the sensory nervous system of Xenopus laevis. Europ. J. Pharmacol. 21: 95-106.


114

8.

9.


10.

11.

12. 13.

14.

15.

16. 17. 18.

19.

20. 21. 22. 23. 24.

SYNTHETIC PYRETHROIDS

Burt, P. E. and R. E. Goodchild. (1974). Knockdown by pyrethroids: its role in the intoxication process. Pestic. Sci. 5: 625-633. Page, A. B. P. and R. E . Blackith. (1949). The mode of action of pyrethrum synergists. Ann. appl. Biol. 36: 244249. Burt, P. E. and R. E. Goodchild. (1971). The site of action of pyrethrin I in the nervous system of the cockroach, Periplaneta americana. Ent. exp. & Appl. 14: 179-189. M i l l e r , T., L . J. Bruner and T. R. Fukuto. (1971). The effect of l i g h t , temperature, and DDT poisoning on housef l y locomotion and flight muscle activity. Pestic. Biochem. Physiol. 1: 483. M i l l e r , T. and J. M. Kennedy. (1972). Flight motor activity of house f l i e s as affected by temperature and insecticides. Pestic. Biochem. Physiol. 2: 206. M i l l e r , T. and J. M. Kennedy. (1973). In vivo measurement of house f l y temperature, flight muscle potentials, heartbeat and locomotion during insecticide poisoning. Pestic. Biochem. Physiol. 3: 370. M i l l e r , T., J. M. Kennedy, C. Collins, and T. R. Fukuto. (1973). An examination of temporal differences in the action of carbamate and organophosphorus insecticides on house f l i e s . Pestic. Biochem. Physiol. 3: 447. M i l l e r , T. A. (1976). Distinguishing between carbamate and organophosphate insecticide poisoning in house f l i e s by symptomology. Pestic. Biochem. Physiol. 6: 307-319. Burt, P. E. (1974). Personal communication. Hart, R. J. (1975). Personal communication. Wilkens, L . A. and G. E . Wolfe. (1974). A new electrode design for en passant recording, stimulation, and i n t r a cellular dye infusion. Comp. Biochem. Physiol. 48A: 217-220. M i l l e r , T. and J. James. (1976). Chemical sensitivity of the hyperneural nerve-muscle preparation of the American cockroach. J. Insect Physiol. 22: 981-988. Nachtigall, W. and D. M. Wilson. (1967). Neuromuscular control of dipteran f l i g h t . J. Exp. Biol. 47: 77. Berridge, M. J. (1966). Metabolic pathways of isolated Malphigian tubules of the blowfly functioning in an artificial medium. J. Insect Physiol. 12: 1523-1538. Thomson, A. J. (1975). Regulation of crop contraction in the blowfly, Phormia regina Meigen. Can. J. Zool. 53: 451-455. Mulloney, B. (1970b). Organization of f l i g h t motor neurons in Diptera. J. Neurophysiol. 33: 86-95. Wilson, D. M. (1968). The nervous control of insect flight and related behavior. Adv. Insect Physiol. 5: 289.


9.

25.

Action

in the Nervous

System

115

Jao, L . T. and J. E . Casida. (1974). Esterase inhibitors as synergists for (+)-trans-Chrysanthemate insecticide chemicals. Pestic. Biochem. Physiol. 4: 456-464. Miyamoto, J. and T. Suzuki. (1973). Metabolism of tetramethrin in houseflies in vivo. Pestic. Biochem. Physiol. 3: 30.


26.

M I L L E R AND ADAMS


Central vs. Peripheral Action of Pyrethroids on the Housefly Nervous

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