[ 9 7 ]

TRANSPIRATION THROUGH THE CUTICLE OF INSECTSBY V. B. WIGGLESWORTH, Agricultural Research Council Unit of Insect

Physiology, London School of Hygiene and Tropical Medicine

(Received 14 November 1944)

(With Plates 1 and 2 and six Text-figures)

The cuticle of most insects is an effective barrier tothe evaporation of water. Since the work of Kiihnelt(1928,1939) it has been known that this impermeabilityis a property of the outermost layer. It is usuallyascribed to the epicuticle—defined as the layer, ofthe order of 1 ^ in thickness, that is not penetrated bythe pore canals, contains no chitin, and resists solu-tion in cold concentrated hydrochloric or sulphuricacids. From chemical tests Kuhnelt concluded thatthe epicuticle contained fatty acids and cholesterol-like bodies. He therefore refers to it as a 'lipoidcuticula'.

The presence of lipoid material or of waxes in theouter parts of the insect cuticle has been indicatedfurther by Wigglesworth (1933), Bergmann (1938),Pryor (1940), Hurst (1940) and others. But it hasnever been clear whether such materials were to beregarded as impregnating the lipoid-free substanceof the epicuticle, as being chemically condensed orpolymerized to compose the epicuticle or as forminga discrete layer upon its outer surface.

It was shown by Ramsay (1935) that the cockroachowes its impermeability to water to a thin and ap-parently mobile layer of lipoid on its surface. At acritical temperature of about 30° C. this lipoid seemsto undergo a change of phase, and it then allowswater to pass freely through it. This interestingobservation has never been confirmed on other in-sects. It forms the starting-point of the presentstudy.

PART I. THE EFFECT OF TEMPERATUREON TRANSPIRATIONMATERIAL AND METHODS

Representative insects of diverse groups from dif-ferent habitats have been exposed for short periodsin dry air and their loss of weight by evaporationmeasured at different temperatures. In most ex-periments the insects have been first killed withhydrogen cyanide and the spiracles occluded withcellulose paint. They have been placed in a smallbasket of metal gauze, usually three or four together,and suspended in a conical flask over phosphoruspentoxide. The flask is immersed in a water bath andthe temperature recorded on a thermometer with thebulb close beside the suspended basket. The small

opening to the flask, combined with the large surfaceof the desiccating agent and the relatively small airspace, ensure the maintenance of an almost dryatmosphere around the insect.

When the same insects are used the same rates ofevaporation are obtained whether they are exposedin this apparatus for 15, 30 or 60 min. Equilibriumis probably established very rapidly. In most experi-ments the short exposures have been employed inorder that the entire series of readings may be takenwithout depleting the water content of the insect un-duly. Provided the spiracles are sealed, there is nodifference in the rate of evaporation from dead orliving insects.

The rate of loss has been expressed in mg. per sq.cm. of surface per hour. The estimation of theeffective 'surface' of the insect is a difficult matter.Where the rate of passage of water is very slow everylittle convolution and irregularity in the surfaceought probably to be taken into account. Where thepermeability of the cuticle is greater, the concentra-tion of water vapour in the finer irregularities willmake the gross surface a truer measure of the ef-fective evaporating area. For the present purposethe fine foldings of the surface have been disregarded.The cuticle of an insect of known weight has beencut up. and its surface area measured on squaredpaper. From this figure the value of k in the formulaS = kx W$ has been found and so the surface area insq. mm. (S) of any other member of the same speciescalculated from the weight in mg. (W). The followingare examples of the values of k that have been em-ployed: Agriotes larva, n-o; Tenebrio larva, 8-4;Calliphora l a rva , 6 - 8 ; Nematus l a rva , 8 5 ; Pierislarva, 98 ; pupa, 8-4; Rhodnius nymph (recently fed),8-i. This procedure gives only approximate values;but the specific differences in evaporation are sogreat that an error of 50 % in the value of k will notaffect the conclusions drawn.

The rate of evaporation from a free-water surfacewas obtained by exposing a shallow capsule brimfulof water in the same apparatus.

RESULTS

Text-fig. 1 shows the results obtained on a smallseries of insects representative of different habitats.Each point represents a mean value from four or

V. B. WlGGLESWORTH

eight individuals. The curve for water loss in Blat-tella germanica agrees fairly well with that obtainedby Ramsay (1935) on Periplaneta; it shows a ' criticaltemperature', with an abrupt increase in evaporationa little above 300 C. The curve of the leaf-eatinglarvae of the sawfly Nematus ribesii and the cater-pillar Pieris brassicae do not differ greatly from that ofBlattella. But the insects from dry environments,which can survive long periods without feeding(larvae of Tenebrio molitor and nymphs of Rhodniusprolixus) show a critical temperature 20-30° C.

oleracea and Hepialus lupulinus, the rate of evation at low temperatures is less and there is evilof a ra ther ill-defined critical tempera ture .

Differences in the rate of transpiration th rough thecuticle and in the critical tempera ture may be wellshown in the different stages of a given insect. T h i sis i l lustrated in Text-fig. 2. In Tenebrio there is littledifference in the t ranspira t ion curves of larva, pupaor adult . Bu t there is a great increase in the water-proofing of Bibio on pupa t ion ; and in the pupa ofPieris, which has to wi ths tand exposure in the open

0

Text-fig.

10 20 30 40 50 60 70

1. The rate of evaporation of water from dead insects at different temperatures.

higher . Below the critical tempera ture the t ranspira-t ion th rough the cuticle is very small in all these in-sects, though it is noteworthy that at say 20° C. therate is generally higher in the insects with, the lowercritical tempera ture .

O n the other hand, the group of insects from thesoil, which normally exist in an a tmosphere saturatedwi th mois ture , all show a high rate of t ranspirat ioneven at the lowest temperatures . In the larvae ofBibio marci the rate approximates to that of a free-water surface, wi th no visible break in the curve. Inthe larvae of wireworms Agriotes spp. , of Tipula

for m a n y m o n t h s , the critical t empera tu re exceedseven that of Tenebrio and Rhodnius. I n these experi-ments the spiracles of the imagines of Bibio, Tenebrioand Pieris were no t blocked. T h e t rue curve ofevaporat ion for the adults should therefore probablylie slightly to the r ight .

These and other results are summarized in Table 1,in which the standard of comparison used is thetemperature at which the loss of weight by evapora-tion amounts to 5 mg. per sq. cm. of surface perhour. This figure has been read off the evaporationcurves; it gives a value perhaps 8 or 10° C. above the

Transpiration through the cuticle of insects 99

70 80

-fig. 2. The rate of evaporation of water from dead insects in larval, pupal andadult stages at different temperatures.

Table i. Temperature in ° C. at which evaporation into dry air equals 5 mg. per sq. cm. per hr.

Insect

Bibio marci (Dipt. Bibionidae)Pterostichus madidus (Col. Carabidae)Agriotes sp. (Col. Elateridae)Aphodius fimitarius (Col. Scarabaeidae)Agrotis segetum (Lep. Noctuidae)Tipula oleracea (Dipt. Tipulidae)Phyllopertha horticola (Col. Scarabaeidae)Hepialus lupulinus (Lep. Hepialidae)Blattella germanica (Orth. Blattidae)Calliphora erythrocephala (Dipt. Muscidae)

» (prepupa)„ (puparium)

Nematus ribesii (Hym. Tenthredinidae)Pieris brassicae (Lep. Pieridae)P. rapae (Lep. Pieridae)Ephestia knehniella (Lep. Pyralidae)Achroia grisella (Lep. Pyralidae)Tenebrio molitor (Col. Tenebrionidae)IUmdnius prolixus (Hem. Triatomidae)

Lqrva

< 5< I O< I O

15-51 9 02 3 0

25-52 8 0—

41 04 1 0

41-0 (65-o)t43-S46-548-057-559558-S64-5

Pupa

39-S*40-5*44'S*—

63-56 2 0

34-°42-0—

57-o——

6656 5 0

61-5S7-o6 3 0—

Imago

40-5*400*46 0*———

45-0*—

42-S43-5*—

46-0*—

47-0*—

54-0*—

Spiracles not blocked. t Three days old.

IOO V. B. WlGGLESWORTH

critical temperature, which is itself difficult to definewithout taking measurements at a large number oftemperatures.

Evaporation from Rhodnius prolixusThe foregoing comparative results have been

based on measurements of evaporation at a few tem-peratures only, usually at intervals of io° C. Moredetailed studies were made on nymphs of Rhodniusprolixus. 4th stage and 5th stage nymphs 1 or 2 daysafter feeding have been used; they have identicalcurves of evaporation. As shown in Text-fig. 1,measurements were made at intervals of 2-5° C.

On the other hand, this constancy is not gmaintained if the insect is exposed alternately to jand low temperatures. After exposure to tempera-tures above the critical level there is some slightpermanent increase in transpiration on return to alower temperature; and this increase is greater thehigher the temperature has been. Thus in successiveexposures for 15 min. the loss of weight in mg. perhour in one group of 5th-stage nymphs was asfollows: 50° C. 0-9; 650 C. 124; 50° C. 2-2; 80° C.568; 500 C. 3-8. In another group the rates of losswere as follows: 50° C. 14; 80° C. 580; 50° C. 38;50° C. (after keeping 24 hr.) 3-2; 50° C. (after keep-

Exposed pupa

Puparium(3 days)

-fig. 3. The rate of evaporation of water from Calliphora in larval andpupal stages at different temperatures.

around the critical temperature, each point beingbased on the averages of from four to twelve insects.It can be seen that there is an abrupt change in therate of transpiration at about 57-5° C.; but even at70° C. the evaporation is not more than one-sixth ofthat from a free-water surface at that temperature.. The rate of transpiration at a given temperature

seems to be a definite figure which remains constanton repetition. Thus in a group of three 5th-stagenymphs with spiracles blocked the loss of weight inmg. per hour at 6o° C. in four successive exposuresof 15 min. was: 2-4, 2-8, 2-0, 2-8. The same insectsexposed at 700 C. gave losses per hour in three suc-cessive exposures of 18-6, 17-6 and 176 mg.

ing a further 3 days) 3-8. The exposure to high tem-peratures has clearly done some slight permanentinjury to the water-proofing layer.

Evaporation from larvae and pupae ofCalliphora erythrocephala

The results obtained with larvae and pupae of Calli-phora are set out in Text-fig. 3. The insects have beenkilled with cyanide and the spiracles blocked. Feed-ing larvae removed from the meat, larvae (so-called' prepupae') which have left the meat and evacuatedthe gut, and fully darkened puparia 24 hr. old, allshow curves for evaporation that are substantiallyalike.

Transpiration through the cuticle of insects 101

the other hand, there is a very striking dis-pl3Wment of the curve, with a great reduction inevaporation at high temperatures, in puparia 3 daysold in which pupation has occurred. This increasedwater-proofing is due, not to any change in thepuparium, but to the delicate cuticle of the truepupa; for when the puparial shell is peeled off andthe young pupa exposed, there is only a relativelyslight increase in permeability at high temperatures—such as would result from the removal of a com-paratively permeable outer sheath.

The pupal cuticle in Calliphora is excessivelyfragile. These observations therefore afford furtherevidence of the importance of the thinnest layers inthe prevention of evaporation.

Evaporation from larvae of AgriotesEach point on the curve for evaporation from

Agriotes larvae as given in Text-fig. 1 was derivedfrom a separate group of larvae. For it was foundthat in these larvae the rate of evaporation at a giventemperature diminished at each successive exposure.Thus a larva weighing 37-6 mg. was exposed for sixsuccessive periods of 15 min. at 30° C. The rates ofloss expressed in mg. per sq.cm. per hour were asfollows: 17-2, 11-6, 9'6, 7-6, 4-4, 2-8. The low finalvalue may have been influenced by the depletion ofwater in the body; but the rapid initial fall must bedue to a progressive reduction in the permeabilityof the cuticle*. A similar effect was seen in some ofthe Aphodius larvae; but the phenomenon has notbeen more closely studied.

The significance of the great and varied permea-bility of the insects living in the soil will be discussedfurther when the action of abrasive dusts has beenconsidered. Further results on evaporation from thewireworm will then be given (p. 106 and Text-fig. 6).

PART II. THE EFFECT OF ABRASIVEDUSTS ON TRANSPIRATION!

It has been known for many years that insects in dryenvironments may be killed by exposure to certainchemically inert dusts, and it has been generallyagreed that the cause of death is desiccation. Thesubject has been fully reviewed recently by Alex-ander, Kitchener & Briscoe (1944). These authors(and cf. Parkin, 1944) have made an extensive com-parative study of the action of such dusts and haveproved conclusively that they act by increasing therate of transpiration through the cuticle. They sug-gest that this may be due to the 'crystalline forces'at the surface of .the particles of dust exerting aspecific attraction on the fatty film of the epicuticle

* Dr D. L. Gunn has pointed out to me that a similardecrease in permeability to water following desiccationis seen in non-living membranes (cf. King, 1944).

f Certain of these results have been published in apreliminary note (Wigglesworth, 1944a), Confirmatoryobservations were reported by Kalmus. (1944).

and thus interrupting the continuity of the water-proofing layer.

In the present experiments the chief dust used hasbeen alumina, which Alexander et al. (1944) haveshown to be one of the most active, but some com-parative observations have been made with powderedquartz and slate dusts.

Effect of dusts on evaporation in RhodniusAlexander et al. (1944) found that the dusts had

no action on dead insects. This has been readily con-firmed in Rhodnius. 5th stage nymphs of Rhodnius1 day after feeding, with an average weight of 132 mg.,were killed with cyanide and the spiracles sealed withcellulose paint. They were then kept at 300 C. overphosphorus pentoxide for 24 hr., half being thicklydusted with alumina and half serving as controls.The controls lost an average of 2-0 mg. (1-5 % of theweight), the insects covered with alumina lost 1-7mg.(i-3%). ;

Similar insects, with the anus blocked with paraffin,allowed to run on filter paper lightly dusted withalumina under the same conditions were completelyshrunken and dead in 24hr., having lost 46-5% of theirweight. Insects running on clean filter paper lost only2-2%.

But the living state of the insect does not appearto be the determining factor. Living 5th stagenymphs with normal spiracles were suspended inmid-air by sealing the thorax to the head of a bentpin with paraffin and sealing the point of the pin tothe wall of a small capsule (Text-fig. 4A). Controlinsects suspended in dry air at 300 C. lost i-8% oftheir weight in 24 hr. Insects heavily dusted withalumina all over the body, including the spiracles,lost 1-9%.

The insects suspended in this manner remainmotionless during the experiment. It seemed likelythat it is the movement of the insect which is neededfor the action of the dust. It was observed that as theengorged Rhodnius nymph crawls on a surface theposterior region of the ventral abdomen touches theground and is continually rubbed (Text-fig. 4B). Ifa small amount of paraffin wax is placed on thisbearing surface so that it is held away from theground (Text-fig. 4C) and the insect is then allowedto run on the dusted filter paper, it suffers com-paratively little desiccation and instead of dyingwithin 24 hr. in dry air it will survive for days. Andthis is so in spite of the fact that the whole of thebody soon becomes covered with a film of dust.

Experiments along these lines, using alumina,finely powdered quartz (with particles of o-s-io/x)*and fine slate dustf are summarized in Table 2. Asseen in the same table, a considerable increase inevaporation occurs if the insect runs on fine emerycloth.

* Kindly supplied by Dr G. Nagelschmidt.t Kindly supplied by Dr I. Thomas.

I O 2 V. B. WlGGLESWORTH

These experiments suggest that the dusts act assimple abrasives and that they increase the permea-bility of the cuticle only if this is rubbed upon thedusted surface. As seen in Table 2, there is someincrease in evaporation even when the bearing sur-face is protected by wax. As will be shown later

Table 2. Average percentage loss of weight in 24 hr.in dry air at 300 C. from recently fed 5th stagenymphs of Rhodnius

Running on clean filter paper 21Running on emery cloth no. o 78Running on filter paper dusted with slate dust 84Running on filter paper dusted with slate dust 43

with mound of wax on the bearing surfaceRunning on filter paper dusted with fine quartz 178

dustRunning on filter paper dusted with fine quartz 5'2

dust with mound of wax on the bearingsurface

Running on filter paper dusted with alumina 46-5Running on filter paper dusted with alumina 76

with mound of wax on the bearing surface

demonstrated on the dead insect. Dead 4nymphs, recently fed, and with the spiracles blo^BPd,in dry air at 30° C. for 5 hr. lost o-8 % of their initialweight. Similar insects pressed firmly down into athick layer of alumina lost 0-9%. Insects pressedlightly against dusted filter paper and at the sametime drawn along, lost 30-0%. In this last experi-ment the dust was removed from the cuticle withinless than a minute of application by brushing witha camel's-hair brush under a stream 6i water.

Effect of dusts on evaporation in some other insectsIt is clear that the impermeable film on the surface

of Rhodnius is interrupted only by the mechanicalaction of the dusts. Dust in stationary contact withthe cuticle will not adsorb lipoid material and sobreak the film. But the cuticular wax of Rhodnius is ahard material with a high melting-point (Beament,1945) and a high critical temperature. It is possiblethat adsorption alone may serve to remove the water-proofing layer in insects with waxes of lower melting-point.

This has been tested in larvae of the sawfly(Nematus) and in young puparia of Calliphora. Thesawfly larvae were killed with cyanide and exposed at220 C. over phosphorus pentoxide in the presence ofcyanide vapour. The Calliphora puparia were ex-posed at 300 C. In each experiment untreated con-

B DText-fig. 4. A, method of suspending 5th stage nymph of Rliodnius. B, 5th stage nymph of Rlmdnius, fed; showinghow the abdomen rubs against the ground. C, the same; abdomen held away from the ground by a mound ofparaffin. D, 5th stage nymph, unfed; abdomen out of contact with the ground.

(p. 103), this is due to the chance abrasion of otherparts and to the dust getting between the moving sur-faces of the limb joints and causing their abrasion.A similar difference can be seen when fed and unfednymphs are allowed to run on dusted filter paper.In the unfed insect the abdomen is held above thesurface and only rubs against it occasionally (Text-fig. 4D). In one such experiment, during 24 hr. indry air at 300 C, the loss of weight was as follows:

Fed insects on clean filter paper 6-5 mg.Fed insects on dusted filter paper (alumina") 47 7 mg.Unfed insects on clean filter paper O'4 mg.Unfed insects on dusted filter paper (alumina) 7-0 mg.

As can be seen in Table 2, the quartz dust is lesseffective than the alumina, and the slate dust lesseffective still. A difference can be readily appreciatedby the tip of the finger drawn across the dustedpaper. That treated with alumina feels like exceed-ingly fine sandpaper; the quartz dust feels lessabrasive; the slate dust smoother still.

The necessity for abrasion by the dust can be

Table 3

Insect and treatment

Nematus larvaA. ControlB. Rubbed with aluminaC. Sprinkled with alumina

Calliphora puparium 24A. ControlB. Rubbed with aluminaC. Sprinkled with alumina

Calliphora puparium 3—4A. ControlB. Rubbed with aluminaC. Puparial shell largely removed

Loss of weight% in 24 hr.

8 2665

8 3

hr. old1 4 651-015-0

days old4'54-069

trols were compared with insects rubbed on filterpaper dusted with alumina and with insects sprinkledwith the dust. The results (Table 3) show that in

Transpiration through the cuticle of insects

insects, as in Rhodnius, rubbing with the dustd greatly the rate of evaporation but that

sprinkling with the dust had no action. In thepuparium of Calliphora 3-4 days old, in whichpupation has taken place, rubbing with the dustnaturally has no effect.

In the cockroach, as was shown by Ramsay (1935),the waterproofing lipoid is far more mobile; forexample, it will spread over the surface of waterdroplets sprayed on to the cuticle and protect themfrom evaporation. Table 4 shows the results of anexperiment with the cockroach (female Blatta),killed with cyanide and exposed to dry air in thepresence of cyanide. Control insects (A) were com-pared with insects (B) in which the dorsal and ventralsurfaces of the abdomen were rubbed on filter paperdusted with alumina and the dust then wiped off,and with others (C) sprinkled all over with alumina.It can be seen that in the cockroach simple dustingwithout rubbing causes rapid desiccation. This isapparent within the first 4 hr. after dusting. Duringthis period the rate of water loss was greater in theinsects which had been rubbed with the dust; butduring the next 20 hr., whereas the rate of loss fromthe rubbed insects had diminished, the loss frorn theinsects sprinkled with the dust had increased. Duringthe ensuing 24 hr. the rubbed insects showed afurther diminution; the sprinkled group wouldprobably have lost water at a still greater rate but forthe fact that they had become completely dried up.

Table 4. Evaporation from dead cockroach (Blatta) at20° C. in dry air expressed as mg. per hr. during suc-cessive periods of exposure. Average initial weight:880 mg.

Treatment

A. Control insectsB. Tergites and ster-

nites of abdomenrubbed with alu-mina

C. Sprinkled withalumina

First4 hr.

1 997

6-6

Next20 hr.

I S5-2

9 9

Next24 hr.

1'43'4

(7-2)*

Totalloss %

8-43 2 0

46-8

* Almost fully desiccated.

Visible effects of dust on the cuticleThe cuticle of Rhodnius, where it has been rubbed

with alumina by allowing the 5th stage nymph tocrawl on dusted filter paper or by pressing the insectagainst the filter paper and drawing it along, has beenexamined microscopically in surface view and insections. No injury to the epicuticle or to the bristlescan be detected. The layer which prevents evapora-tion probably lies, therefore, on the outer surface ofthe epicuticle.

Now it has been shown by Schmallfuss and others(i933) that the outer layers of the insect cuticle cpm-monly contain dihydroxyphenyl-alanine, dihydroxy-

IO3

phenyl-acetic acid or other polyphenols which pro-vide the chromogens used in the production ofmelanin; and Pryor (1940) has found evidence toshow that the quinones produced by oxidation of thepolyphenols, by 'tanning' the cuticular proteins, areresponsible for the hardening of the cuticle aftermoulting. The polyphenols are readily demonstratedby exposure to ammoniacal silver hydroxide, whichthey reduce to give a deep brown stain (Lison, 1936).PI. 1, fig. 3, shows a section of a fresh Rhodniuscuticle, cut with the freezing microtome and im-mersed for 1 hr. in ammoniacal silver hydroxide.The epicuticle is everywhere stained dark buown.

If the living Rhodnius nymph, however, is im-mersed in freshly prepared 5 % ammoniacal silverhydroxide, washed in distilled water, and the cuticlethen dissected off, fixed in Carnoy's fixative andmounted flat, there is no sign of staining. The poly-phenols are clearly separated from the solution by aprotective layer (PI. 1, fig. 2). But if the insect hasbeen rubbed with alumina, or allowed to crawl onfilter paper dusted with alumina or on fine emerycloth, there is very obvious brown staining over therubbed areas. This staining is confined to the promi-nent points of the cuticle: the domes of the raisedplaques from which the bristles arise and the crestsof the stellate folds into which the epicuticle isthrown (PI. 1, figs. 1, 4). The protective layer hasclearly been rubbed off these points.

When the insect has been crawling on dusted filterpaper there are brown staining patches also wherethe tibia and tarsus have been drawn across the sur-face, and between the articulations of the limb jointswhere these have rubbed together. The insects thathave crawled on emery cloth often suffer a morebrutal abrasion and obvious scratches can be seen(PI. i, ng. s).

In section the brown staining of the prominentcrests is seen to be confined to the epicuticle (PI. 1,fig. 4). Nymphs rubbed with the dust have beenimmersed in the silver solution for periods rangingfrom 5 min. to 2 hr. The prolonged exposure causesonly a slight increase in the extent of the brownstaining areas. These spots therefore probably repre-sent the true limits .of the areas from which the pro-tective layer has been abraded.

Similar results have been obtained with otherinsects. Calliphora puparia show no staining on im-mersion in the silver solution; but after rubbinggently on the dust, brown staining spots appear on allthe prominent ridges (PI. 1, fig. 6). Larvae of Tene-brio kept for a few hours on dusted filter paper showbrown staining areas where the coxae rub against thethorax (PI. 1, fig. 7), between the limb joints, and atall those points on the ventral surface of the abdomenwhere this touches the ground as the larva creepsalong. Larvae of Ephestia on dusted filter paper arevery rapidly affected and become desiccated withina few hours. Treatment with silver shows that notonly has the protective layer been abraded from the

104 V. B. WlGGLESWORTH

prolegs and wherever the cuticle has touched theground, but the dust has also got into the surfacefolds everywhere so that staining occurs whereverone point on the surface rubs against another. Ingeneral, the rate of water loss from an insect treatedwith the dust runs parallel with the extent of theabraded areas as shown by treatment with silver.

Recovery of impermeability after treatment with dust

5th stage nymphs of Rhodnius 24 hr. after feedingwere treated with alumina by laying them on theirbacks on dusted filter paper and drawing them overthe surface for a standard distance while pressingthem gently down with the finger. In this way arounded area of fairly constant extent on the dorsalsurface is abraded. The dust was removed at oncewith a camel's-hair brush under a stream of water.The rate of evaporation in dry air at 30° C. wasmeasured immediately after abrasion and after theinsect had been kept at 25° C. in saturated air forseveral days. The results are shown in Table 5.

Table 5. Loss of weight of 5th stage nymphs ofRhodnius, with mean initial weight of 120 mg.,during 24 hr. in dry air at 30° C.

A. Immediately after rubbing with alumina 311 mg.B. After keeping 1 day in moist air at 25° C. 5-2C. After keeping a days in moist air at 25° C. 3-4D. After keeping 3 days in moist air at 25° C. 24E. Normal controls 15

Similar recovery has been demonstrated in larvaeof Tenebrio and in pupae of Pieris brassicae afterrubbing with the dust (Table 6).

Table 6. Loss of weight during 24 hr.in dry air at 30° C.

1. Pupae of Pieris brassicae (mean initial weight 370 mg.):A. Immediately after rubbing with alumina 421 mg.B. After keeping 2 days in moist air at 200 C. 8-6C. After keeping 6 days in moist air at 20° C. 43D. After keeping 2 weeks in moist air at 32

2O°C.E. Normal control o#7

2. Larvae of Tenebrio molitor (mean initial weight oomg.):A. Immediately after rubbing with alumina 465 mg.B. After keeping 4 days in moist air 55C. Normal control 10

It is clear that to a large extent the impermeabilityof the cuticle can be restored, although recoverynever seems to be complete. This recovery is de-pendent on the activity of the epidermal cells andtakes place only if they are living. Thus 4th stagenymphs of Rhodnius were dusted by rubbing as de-scribed above and the dust removed immediately.Some of these were exposed to dry air at once; theremainder were kept in saturated air for 24 hr., halfof them living and half of them after exposure tohydrogen cyanide. At the end of 24 hr. those ex-posed to cyanide appeared dead, but the gut was still

Table 7. Loss of weight of \th stageRhodnius, with mean initial weight of 95 -^ng-,during 24 hr. in dry air at 30° C.

A. Alive: immediately after rubbing with 36'1 mg.alumina

B. Killed with HCN: immediately after rub- 373bing with alumina

C. Alive: after keeping in moist air for 1 day 92D. Killed with HCN: after keeping in moist 366

air for 1 day

showing peristaltic movements. Table 7 shows theevaporation from these three groups of insects.

The importance of the living cells is shown also byexperiments in which the cuticle has been abradedby dusting in the later stages of moulting, at a timewhen the old cuticle has become detached from thecells and the new cuticle is being laid down but hasnot yet become impermeable. The 5th stage nymphmoults at about one month after feeding. Table 8shows the absence of recovery in insects dusted atthree weeks.

Table 8. Percentage loss of weight of 5 th stage nymphsrubbed with alumina and the dust removed at once,kept for 1 day at 25° C. in moist, air and then ex-posed for 24 hr. in dry air at 30° C.

A. 3 days after feeding 38B. 3 weeks after feeding 319

We have seen that in Rhodnius and most otherinsects adsorption by the dust will not by itself breakdown the protective film. But once this film has beeninterrupted by abrasion the presence of the dust mayseriously interfere with the recovery process. Thismay be shown by comparing the efficiency of re-covery in insects in which the dust has been removedimmediately after dusting, with others in which thedust has been left on (Table 9). When the dust is lefton the cuticle, recovery is very incomplete even aftermany days.

Table 9. Percentage loss of weight of $th stage nymphsrubbed with alumina, exposed for 24 hr. in dry airat 300 C.

A. Immediately after rubbing; dust left on 482B. Immediately after rubbing; dust removed 475C. After keeping in moist air for 2 days; dust 168

left onD. After keeping in moist air for 2 days; dust 31

removed

In the cockroach, on the other hand, as shown inTable 4, adsorption by the dust quickly leads to in-creased evaporation. The high degree of mobility inthe waterproofing grease of the cockroach not onlyleads to its removal by adsorption, but, as Table 4also shows, it allows a considerable amount of re-covery in the dead insect—by the spreading of theoil over the abraded areas.

Transpiration through the cuticle of insects

Visible changes during recovery

If the dust has been removed from the Rhodniusnymph as completely as possible immediately afterapplication, and the insect then kept in moist air fora few days, the rubbed cuticle develops a milkybloom. The effect is more striking if the dust is lefton; what is at first an almost imperceptible film isthen converted within 2 or 3 days into a conspicuouschalky white patch, more or less firmly adherent tothe surface of the cuticle. In both cases this waxysecretion is more evident if the insect after recoveryis kept in a dry atmosphere, just as certain plants(which likewise are able to regenerate their waxycoating) produce less wax if they are grown in adamp atmosphere (Haberlandt, 1914).

As the new wax is secreted the cuticle becomes lessreadily wetted by water. At first droplets of waterwill adhere to the surface; after a few days the drop-lets fall from the cuticle without wetting it. Measure-ments of the angle of contact between water and thecuticle (kindly made for me by Mr Beament) showthat this increases from 102° to nearly 1200 duringthe first 48 hr. as the wax is being secreted. It is ofinterest to note that the angle increases also, thoughmore slowly, on the normal cuticle as moulting pro-ceeds; by 21 days after feeding it has increasedgradually from 1020 to 117°. An extra quantity ofwax thus appears to be secreted on to the surface ofthe old cuticle before the epidermal cells separatefrom it and so lose the power of making good anyabrasion that may occur.

The presence of the wax may be demonstratedalso with fat stains. If the cuticle of the insect whichhas recovered from dusting is heated with BlackSudan B at 50° C, the waxy deposits, chiefly on thesides of the epicuticular folds, are deeply stained.This staining is very much more conspicuous if thedust has not been removed (PI. 1, fig. 10).

As the wax is secreted, and the impermeability ofthe cuticle is restored, the patches that stain withsilver become smaller; and after recovery has pro-ceeded for a few days no reduction takes place onimmersion in the silver solution. PI. 1, figs. 8 and 9show two stages in this process. From the way inwhich the individual star-shaped areas on the crestsof the folds are broken up it is evident that thesecretion which covers them is coming through thesubstance of the cuticle everywhere. It clearly doesnot spread out from the openings of the ducts of thedermal glands; it must be the product of the generalepidermal cells.

During the process of recovery after abrasion thereare striking changes also in the underlying epidermalcells. In spite of the fact that there is no visible in-jury to the epicuticle the underlying cells behave asthough they had been wounded (Wigglesworth,1937). They migrate towards those regions that havebeen most severely rubbed, leaving areas with sparsecells between. This response is not the result of in-

jury to the cells by desiccation, for it occurs equallyin insects that have been kept in air saturated withmoisture. Indeed, the cells behave as though theirliving substance extended not only by way of thepore canals up to the epicuticle, but through theepicuticle up to the protective film on its surface.

During the next day or so small droplets whichstain with fat stains and reduce osmic acid appear inthe epidermal cells most affected. This is probably afurther reaction to injury; the droplets do not seemto be related with the secretion of wax that is occur-ring at this time. Another change is the failure of theepidermal cells under the rubbed area to develop thelarge amounts of red pigment or the white granules(believed to be uric acid) which they normally con-tain.* At no stage is there any sign of renewedactivity in the dermal glands.

Natural abrasion of the cuticle in soil insects

It is evident from Text-fig. 1 that the larvae ofinsects from the soil lose water very readily at quitelow temperatures. The question naturally ariseswhether this is the result of abrasion by particles ofearth. Immersion of the larvae in ammoniacal silverhydroxide reveajs in all of them extensive areas overwhich the layer of polyphenols has been exposed,and in most of them these arena are made up ofobvious scratches.

In Hepialus, after immersion in the silver solution,there are dark brown areas over all the projectingfolds of the body, particularly towards the anteriorend (Text-fig. 5A). On microscopic examinationthese areaB are seen to be made up of scratches criss-crossing in all directions. Scratches are very evidentalso on the outer surface of the true legs (PI. 1,fig. 11). In Agrotis the results are the same. It isnoteworthy that in both these caterpillars the cuticleis much more resistant to scratching over the tuberclesand over the head where there is a hard exocuticle.In Aphodius and Phyllopertha similar results are ob-tained : reduction of the silver appears in the form ofscratches on the prominent lateral folds, on the analextremity and the legs, but particularly over the sur-face of the rounded back (Text-fig. 5 B; PI. 1, fig. 13).The scratching of the back is less evident in Phyllo-pertha, in which the protective spicules are morestrongly developed.

In the wireworm Agriotes the body is covered

* There is also an interesting effect t>n growth. Themoulting of the insect is in no way delayed by extensiveabrasion of the epicuticle, provided desiccation is pre-vented. But the process which leads to the determinationof new plaques is upset. The number of plaques on one-half of an abdominal tergite when the 4th instar nymphmoults to a 5th instar increases from about 200 to 250(Wigglesworth, 1940). In a nymph which had beenrubbed on one side of the abdomen only there were 206plaques on the rubbed side, 258 on the normal. Injuryto the cells caused by rubbing the layers outside the epi-cuticle has prevented the determination of new plaques.

i o 6 V. B. WlGGLESWORTH

almost everywhere with hardened cuticle which re-sists extensive abrasion. But there are ventro-laterallines of soft cuticle and some areas of soft cuticle atthe base of the limbs which always show extensivebrowning in the silver (PL i, fig. 12). And whenexamined with a higher magnification the hardenedcuticle everywhere shows innumerable fine scratches.The degree of this visible scratching runs parallelwith the rate of evaporation. Thus in one series ofwireworms exposed at 40° C. the most permeablelost 39-2% of its weight in 15 min.; another lostonly 17-2%. PI. 2, figs. 1 and 2, show correspondingareas of cuticle from these two insects. The morepermeable is much more severely scratched.

In the larva of Pterostichus the hard cuticle is re-stricted to plates or sclerites set like islands in thesoft body surface (PI. 2, fig. 3). Here the abrasionof the soft areas is so extensive that in many placesthey show a uniform brown staining with the silver,and individual scratches cannot be seen. But where

at the lowest temperatures, the curve turns st|upwards as soon as the critical temperature is reacnca.

It is concluded therefore that the permeability ofthe cuticle in soil insects is the result of abrasion. Ifthis is so, the insect which has moulted and beenkept out of contact with the soil ought not to showany notable permeability to water until the criticaltemperature is reached. This has been tested on thewireworm. Larvae of Agriotes were placed singly inspecimen tubes with a pledget of moist cotton-wooland a small piece of carrot. The date of moulting wasnoted and the larva was then kept for 2 weeks. Text-fig. 6 shows the temperature-evaporation curve forsuch a larva. It falls into line with that of insectsfrom other environments: there is little evaporationuntil the critical temperature is reached. In an ex-periment done less than 24 hr. after moulting therew*as considerable evaporation at low temperaturesand exposure to silver showed irregular diffuse areasof brown staining.

Text-fig. 5. Larva of Hepialus (A) and Aphodius (B) from the soil, after immersion in ammoniacal silver,showing the distribution of the chief areas where the wax layer has been abraded.

there are depressed folds, as between the body seg-ments, abrasion is incomplete and in some placeslinear scratches appear. These scratches are some-times continuous with the scratches on the sclerites.On the sclerites, as on the hard parts of the otherinsects, abrasion is limited to fine scratches, runningmostly in the long axis of the body (PI. 2, fig. 4).

The larva of Bibio is everywhere covered with littleplaques bearing rows of blunt spines. The plaquesshow some abrasion, and in the soft cuticle betweenthem there are innumerable small areas which stainwith the silver (PI. 2, fig. 6). These areas do not takethe form of scratches, but in view of the observationson the soft cuticle of other insects they are probablythe result of abrasion. Likewise in the larva ofTipula, there are small scattered points over thecuticle which stain with the silver; in many placesthese lie in rows and are almost certainly due toabrasion (PI. 2, fig. 5).

In all these insects the rate of evaporation of waterat low temperatures runs parallel with the extent ofthe silver staining areas (Text-fig. 1). In Bibio andPterostichus evaporation is so great that there is nosign of a critical temperature. In Agriotes, Tipula,Hepialus, etc., in spite of the high rate of water loss

Abrasion of the cuticle and the entry of insecticides

Not only is the loss of water by evaporation in-creased after abrasion by rubbing with the dusts, butalso the uptake of liquid water. For example, 5thstage nymphs of Rhodnius killed with cyanide 24 hr.after feeding and then immersed in distilled waterfor 24 hr. showed an average increase in weight (onan initial weight of about 120 mg.) of 0-4 mg. in thenormal insect, and 4-1 mg. after rubbing gently onfilter paper dusted with alumina. This observationfalls into line with the osmotic uptake of water bynormal (and therefore abraded) wireworm larvaeliving in the soil (Evans, 1943).

It was to be expected that this increased permea-bility of the cuticle would be reflected in an increasedsusceptibility to insecticides. This has been testedwith nicotine and with rotenone. 5th stage nymphsof Rhodnius with uniformly thick cuticles were fed,and glass capsules of the type previously described(Wigglesworth, 1942) sealed to the dorsum of theabdomen with cellulose paint. A solution of 2%nicotine, in distilled water, was introduced into thecapsule and the top sealed with a cover-glass. Thenormal insects were slightly affected in 6 hr.; they

Transpiration through the cuticle of insects 107

affected but had not collapsed in 24 hr.in which the back had first been lightly

rubbed with alumina dust collapsed and were unableto walk within 20 min.

The experiment was repeated using dry powderedrotenone (Stafford Allen and Sons go % rotenone) asthe insecticide. The normal insects were unaffectedat the end of 3 weeks; those rubbed with the dustshowed weakness in 8 hr. and were dead in less than24 hr.

PART III. THE EFFECT OF SOLVENTSAND DETERGENTS ON TRANSPIRATIONTHROUGH THE CUTICLE

Since oils and waxes are responsible for maintainingthe impermeability of the insect cuticle to water,lipoid solvents and detergents should break downthe protective layer.

Experiments along these lines have been madechiefly on Rhodnius. Chloroform was selected as a

40°C; 29-2

em

a

Ir-

0 50 60"0 10 20 30

Text-fig. 6. Evaporation from the wireworm larva, pupa and adult at different temperatures.

T h e s e observations suggest that the activity ofinsecticidal powders may be enhanced if an abrasivedus t is incorporated wi th the filler. Special studieswill be needed to prove whether this happens inpractice, bu t it is well known that the efficiency of agiven insecticide is greatly influenced by the natureof the filler (Turner , 1943). Hockenyos (1939) notedthat hydrated lime increased the rate of entry ofsodium fluoride through the cuticle of the cockroach,and at t r ibuted this to an effect on the oil film.

good general solvent for oils and waxes. Tab le 10

Tab le 10. Loss of water during 1 hr. in dry air at30° C. from 5 th stage nymphs of Rhodnius 1 dayafter feeding. Average initial weight 130 mg.

A. Dead, normal controls O'2 mg.B. After extraction with chloroform for 15 2'7

min. at 200 C.C. After extraction with chloroform for 15 34'6

min. at 500 C.

io8 V. B. WlGGLESWORTH

shows that there is a considerable increase inevaporation after extraction with chloroform atroom temperature, a very great increase after extrac-tion at 50° C. Insects from these three groups weresubsequently immersed in ammoniacal silver hy-droxide and the cuticle of the abdomen examined.The controls as usual showed no staining. Thoseextracted for 15 min. at room temperature showedstaining over the muscle insertions (PI. 2, fig. 7) butvery little elsewhere. Those extracted for 15 min. at500 C. showed well-marked staining over the muscleinsertions and widespread staining elsewhere, par-ticularly in the hollows of the epicuticular folds(PI. 2, fig. 8).

The wax extracted with hot chloroform from thecast cuticle of Rhodnius is readily soluble in chloro-form in the cold (Beament, 1945). The foregoingexperiments show that extraction from the surface ofthe cuticle is very much more difficult, and that theease of extraction varies in different regions. Thestructural basis of these observations will form thesubject of a separate paper (Wigglesworth, 1944 c);but the general conclusion may here be stated: thatthe wax layer overlying the epicuticle is further pro-tected by a layer of cement which varies in hardnessand resistance to removal in different insects andin different parts of the same insect.

The hardness of the wax in Rhodnius is such thatthe permeability of the insect is not' very greatlyaffected if it is killed with chloroform vapour. Butin the soft-skinned larva of the sawfly Nentatus, andin the cockroach Blatta, exposure to the. vapour ofchloroform at room temperature for \-1 hr. is suf-ficient to increase enormously the permeability of thecuticle to water (Table 11).

Table 11. Loss of weight in 4 hr. at 220 C. of Nematuslarvae with average initial zveight of 80 mg., andBlatta adults with average initial weight of 850 mg.,and in 24 hr. at 300 C. of Rhodnius nymphs, withaverage initial weight of no mg.

Nematus larvae killed with HCN I O mg.Nematus larvae exposed to chloroform vapour 393

for 1 hr.Blatta adult females, killed with HCN 76Blatta adult females, exposed to chloroform 49-7

vapour for 1 hr.Rhodnius nymphs killed with HCN 1 -4Rhodnius nymphs exposed to chloroform 3-8

vapour for 1 hr.

We have seen (p. 105) that after abrasion with alu-mina dust the insect kept in a moist atmosphere will,to a great extent, recover its impermeability to water,and the points where the layer containing phenolswas exposed once more become covered by wax.This recovered area does not resist extraction withchloroform as does the normal cuticle. Thus 5thstage nymphs of RJiodnius were rubbed with aluminaon one-half of the abdomen only and then allowedto recover for 5 days in moist air, On treatment with

ammoniacal silver after this period they shovu^fcnostaining on either side. But after immersi^^inchloroform for £ nr- at room temperature theyshowed on the normal side some staining over themuscle insertions and in the form, of scattered pointsin the depressions between the cuticular folds. Onthe side that had been rubbed there was in additionextensive staining over the crests of the folds—though this staining was not quite so widespread asin the newly rubbed insect.

The same effect is apparent if the rate of evapora-tion after extraction with chloroform is observed(Table 12).

Table 12. Loss of weight in ing. in 4 hr. at 30° C. indry air from $th stage nymphs of Rhodnius 4 daysofter feeding (average weight 120 mg.)

A. Control 07 mg.B. The same extracted with chloroform for 69

15 min. at 200 C.C. 3 days after rubbing with alumina 14D. The same extracted with chloroform for 171

15 min. at 200 C.

From these results it appears that the wax in thelayer which overlies the epicuticle of Rhodnius iscovered with or associated with materials which pro-tect it from extraction. If the protective layer is tobe broken down and the insect rendered permeableto water (and perhaps to insecticides) an agent mustbe used that will attack both the wax and the cementsubstance and at the same time be sufficientlyhydrophil for water to pass readily through it.

With this end in view a long series of wettingagents and detergents and other chemicals have beentested. These have been applied as a thin smear overthe dorsal surface of the abdomen of living 4th stagenymphs of Rhodnius 24 hr. after feeding. Groups ofthree or four nymphs have been used for each com-pound and with many of the compounds the experi-ments were repeated several times. The anus of thenymphs was occluded with paraffin and they werethen exposed for 4 and 24 hr. at 300 C. in dry air,and the percentage loss of weight noted. A selectionfrom the results is shown in Table 13.

The substances used show a very wide range ofactivity in increasing the rate of loss of water, butcertain general principles can be discerned. Simplewater-soluble wetting agents such as Turkey-red oil,'Agral', 'Perminal', 'Aerosol OT', have very littleeffect. Where such water-soluble substances havedefinite detergent action, as with 'Lissapol' orsodium cetyl sulphate, their effect is somewhatgreater. Refined petroleum oils (P31) have verylittle effect, and so have olive oil and lanoline. Butwhen oils with polar groups such as ricinoleic acid,linoleic acid, oleic acid, or the naphthenic acids areapplied, the effect is much increased.

In general it is the oil-soluble substances withhydrophilic properties that are most effective. This

Table 13. Loss of weight in $th stage nymphs of Rhodnius, 24 hr. after feeding, exposed at 300 C.in dry air after smearing the dorsal surface of the abdomen with various chemicals

Chemical substance

ControlSodium salt of a sulphonated naphthalene derivativeSodium salt of a sulphonated aromatic hydrocarbonRefined medium petroleumAmmonium salt of ricinoleic sulphuric acid esterCarboxylic acids from petroleum. Acid value: 230 mg.

KOH/g. .B-naphthyl ether of polyethylene glycol, /?(OC2H4)5OH

(approx.)B-naphthyl ether of polyethylene glycol, R (OCaH^OH

(approx.)' Lecithin'

Sodium secondary alkyl sulphates (chain length

A phthalic glycerol alkyd resinCondensation compound of naphthalene sulphonic

acid and formaldehydePolyglycerol ester of coconut oil fatty acids (glycerol;

fatty acid = 20 : 4)Ricinoleic acidPolyethylene glycol monoiso-octyl tolyl etherA straight chain sodium alkyl sulphateDiethylaminoethyloleylamide acetateComplex alkylated benzene ether of polyethylene gly-

col, fl(OCaH4)8OH (approx.)An alkyl naphthalene sulphonic acidTriethanolamine soapSorbitol laurateSorbitol oleateLinoleic acidTwitchell's baseOleic acidPolyglycerol ester of coconut oil fatty acids (glycerol:

fatty acid 20 : 2)Sodium cetyl sulphateSodium salt of sulphonated ethylmethyloleylamideCoconut oil fatty acids mono-diglyceride (88 : 12)Sulphonated fatty estersDiglycol oleateSorbitan monoleatePetroleum oil containing sodium naphthasulphonate

or similar compoundsComplex sulphonateComplex sulphonatePolyglycerol ester of coconut oil fatty acids (glycerol:

fatty acid = 20 : 6)Polyglycerol ester of coconut oil fatty acids (glycerol:

fatty acid = 20 : 10)Carboxylic acids from petroleum. Acid value: I7omg.

KOH/g.Ammonium laurate

Sodium naphthasulphonate 50% in P31Diglycol lauratePolyglycerol ester 0 coconut oil fatty acids (glycerol:

fatty acid = 20 : 20)Propylene glycol laurateSodium alkyl sulphate (C17 with slightly branched

cnainjTriethanolamine esterGlyceryl monolaurate.OvolecithinCetyl ether of polyethylene glycol,

CX6H33(OC2H1)1,OH (approx.)Cetyl ether of polyethylene glycol,

^,iQtlsa{\J\*s2rii)i\jri ^approx.jptyl ether of polyethylene glycol,C16H88(OC2Hd)8OH (approx.)

Trade name or codenumber and maker

Perminal BX (I.C.I.)Agral 2 (I.C.I.)P31 (white oil) (Shell Co.)Turkey-red oilNaphthenic acid NA9 (Technical

Products Ltd.)R1660 (I.C.I.)

R1661 (I.C.I.)

Alco Soya Lecithin (AmericanLecithin Co.)

Teepol X (Technical Products Ltd.)

Emulsifier B1956 (Rohm and Haas)Belloid T.D. (Geigy Colour Co.)

Lever Bros.

Triton NE (Rohm and Haas)Lissapol C (I.C.I.)Sapamine A (Ciba Co. Inc.)R2206 (I.C.I.)

Belloid NW (Geigy Colour Co.)Belloid F (Geigy Colour Co.)Glyco Products Co. Inc.Glyco Products Co. Inc.

—Technical Products Ltd.

—Lever Bros.

I.C.I.Igepon T (General Dyestuff Corp.)Lever Bros.Calsolene oil HS (I.C.I.)Glyco Products Co. Inc.Span 80 (Atlas Powder Co.)Soluble 'cutting' oils (Shell Co.)

Foamacrex (Rex Campbell Co.)Emulsifier SZ (Rex Campbell Co.)Lever Bros.

Lever Bros.

Naphthenic acid NA17 (TechnicalProducts)

Ammonium laurate S (Glyco Co.Inc.)

Technical Products Ltd.Glyco Products Co. Inc.Lever Bros.

Prolaurin (Glyco Co. Inc.)Technical Products Ltd.

Belloid F.R. (Geigy Colour Co.)Glyceryl laurate S (Glyco Co. Inc.)British Drug HousesR2204 (I.C.I.)

R2211 (I.C.I.)

C09993 (I.C.I.)

Loss of weight% in 24 hr.

1-5-2-52 - 42 9

3 33 43 6

3-75

3 9

3'9

4 2

4'3

4 5

4-64-64-94'9

5-86 26 26 36-46-66-76-7

6 96 97-27-2

7-57-68-7

9-1

9 69 6

1 0 5

1 0 6

I I - I

1 2 31 2 4

1 3 3

1 4 1

15-2

1 5 71 6 3

1 6 72 4 7

3 9 5

48-0

JEB.21, 3 , 4

no V. B. WlGGLESWORTH

is well seen in the series of polyglycerol esters of thecoconut oil fatty acids, in which the percentage lossof water rises as the ratio of glycerol to fatty acid fallsand the oil solubility increases. Thus with a glycerol/fatty acid ratio of 20 : 2 the loss of weight is 6-7 %;20 : 4, 4-5 % ; 20 : 6, 96 % ; 20 : 10, 10-5 % ; 20 : 20,

13-3 %•But these properties do not provide a full ex-

planation of the differences observed. Materialssuch as Twitchell's base or sodium naphthasul-phonate are readily soluble in oil and lead to itsspontaneous emulsification in contact with water,and yet they are only moderately effective in in-creasing the loss of water through the cuticle. Thepolyethylene glycol ethers of ring compounds(R1660, R1661 and Triton NE), all of which arewater-soluble, have very little action. The cetyl etherof polyethylene glycol (C 09993) is the most effective

Table 14. Percentage loss of weight in \th stage nymphsof Rhodnius 1 day after feeding, exposed at 30° C.in dry air for 24 hr. after smearing the dorsal surfaceof the abdomen with cetyl ether of polyethylene glycol(C 09993) ot different concentrations in water

100%

5°%10%

1%

Control i-8

45-323-51194'S

It appears therefore that some otherinvolved besides an affinity for both oil and v^»cr.It may be that the long cetyl chain of the compoundsC09993, R2211, etc., is particularly effective inassociating with the long chains of the wax and thusleading to its dispersion.

That there is a relation between the quantity ofmaterial applied and the effect produced has beenshown by applying C 09993, dispersed in distilledwater in varying concentrations, as a thin film overthe dorsum of the abdomen (Table 14).

It may be that the wide differences between theaction of the different materials in Table 13 is de-termined as much by the cement which covers thewax layer in Rhodnius as by the wax itself. If this isso one might expect to find large differences in therelative effectiveness of the materials when appliedto different insects—in which differences in the pro-perties of both wax and cement probably occur.A small number of materials showing large dif-ferences in their action when applied to Rhodnius,have been applied to some other insects and theirrelative effects compared (Table 15). In all cases thecetyl ethers of polyethylene glycol (R22H andC09993) were the most efficient substances, but innone was their relative effect so outstanding as inRhodnius. In all these other insects the refinedparaffin (P31) was relatively more effective than inRhodnius.

Table 15. Percentage loss of weight from various insects smeared with different materials (see Table 13)and exposed for 24 hr. in dry air. Rhodnius nymphs smeared on the dorsum of the abdomen; the otherinsects smeared all over; Rhodnius, Pieris and Tenebrio exposed at 30° C, Nematus at 20° C.

Material

ControlP 3 iDiglycol oleateGlyceryl monolaurateR22IIC09993

Rhodnius nymph

2 0

3 37-S

1 6 339-54 8 0

Pieris pupa

o - i

4-68-68-6

n-6n-8

Nematus larva

7 91 7 76 1 2

54-876673-8

Tenebrio larva

2-58-9

16-42 2 9

29-62 7 7

Table 16. Percentage loss of weight from Calliphora puparium and pupa during 4 hr. at 300 C. in dry air

Object and treatment

Normal puparium 24-48 hr. oldNormal puparium before pupation, thinly smeared with C 09993Normal puparium after pupation,* thinly smeared with C 09993Pupa exposed by stripping puparium from posterior halfPupa exposed by stripping puparium from posterior half, thinly

smeared with C09993

Loss of weight/o

1-9467

i'72-3

17-2

* As determined by the extrusion of the pupal horns.

of all the compounds (see PI. 2, fig. 9) and it com-bines oil solubility with dispersibility in water. Butthe similar compound (R2204) with the longer poly-ethylene glycol chain is also a very effective substancealthough it dissolves readily in water and shows littlesolubility in oil.

In the puparium of Calliphora the effect ofC 09993, like the effect of abrasive dusts, depends onwhether pupation has occurred (Table 16). It ispossible, when pupation has taken place, to stripaway the puparial shell and smear the exposed pupawith the detergent.

Transpiration through the cuticle of insects i n

Effect of detergents on the entry of insecticidesIt has been shown by Fulton & Howard (1938

1942), Hurst (1940, 1943), Wigglesworth (1942) andothers that the solvent used for an insecticide in-fluences greatly its rate of passage through thecuticle. It seems likely that the differences betweendifferent emulsifying substances in their ability tobreak down the waterproof layer of the cuticle will bereflected in differences in facilitating the entry ofinsecticides. This is a large subject which will requirecareful study. A few experiments only have beenmade.

Capsules were secured to the backs of 5 th stagenymphs of Rhodnius with uniformly thick cuticles,by means of cellulose paint, 1 day after feeding, andleft 48 hr. to harden (Wigglesworth, 1942). A sus-pension of rotenone (Stafford Allen 90%: o#2 g. in2 ml. of oil) was then introduced and the capsulesclosed with cover-glasses sealed down with paraffin.Rotenone in refined paraffin (medicinal paraffin) hadproduced no effect in 7 days. Rotenone in the cetylether of polyethylene glycol (R2211) caused thecomplete collapse of all the nymphs in 24 hr. Nico-tine, 2% in refined paraffin (P31), similarly appliedhad almost no effect in 2 days; in the cetyl ether ofpolyethylene glycol (R2211) all were dead in 24 hr.

DISCUSSION

The results described confirm the belief that the layerof the cuticle which is impermeable to water is verysuperficial and very thin. The effective layer lies infact over the surface of the epicuticle. The pupariumof Calliphora forms an apparent exception, for here,as was shown by Hurst (1941), the permeability is notincreased if the lipoid layer on the surface is brokendown. Hurst attributed this property to the con-densed chitin-protein complex of the puparial shell;but it has been shown in the present paper that in theprepupa and young puparium of Calliphora the im-pervious layer is wholly superficial as in other insectsand that the hardening of the puparium does notincrease its impermeability to water. The greaterresistance of the three-day puparium to loss of wateris due in fact to the very fragile cuticle of the truepupa. So long as this is unbroken it is far moreeffective in restricting water loss than the hardpuparium that covers it. These observations suggestcomparison with other multilayered systems, suchas the insect egg (cf. Slifer, 1937).

The waterproof layer is of a waxy nature. Itseffectiveness is presumably dependent on theorientation and close packing of the long wax mole-cules. At a critical temperature, which varies Widelyin different insects, the packing of the molecules isloosened and water can pass through more readily.As was proved on the cockroach by Ramsay (1935),this will account for the fact that the loss of waterby evaporation from insects per unit of saturation

deficiency rises with rising temperature—as wasfound by Koidsumi (1934) in the pupa of Milioniaand by Evans (1934) in the egg of Lucilia. Theevidence for these ideas is given in the paper byBeament (1945) on the properties of the isolatedwaxes.

As shown by Beament (1945) the waxes of thedifferent insects have graded properties; they rangefrom the soft grease with polar characters in thecockroach Blatta, to the very hard apolar wax ofRhodnius or the pupa of Pieris. In the living insect,however, there are further complexities. In thecockroach'the greasy layer seems to be freely exposedand to be so mobile that it is largely removed by ad-sorption on to dusts, and is able to spread slowlyback over areas which have been abraded. In otherinsects the wax is covered and protected to a variableextent by a layer of cement. It will be shown else-where (Wigglesworth, 1944 c) that (at least inRhodnius) this layer is the product of the dermalglands and is poured out over the surface of theorientated wax which is itself the product of theepidermal cells.

The deposition of these layers has not been fullyconsidered in this paper; but the secretion of thelong wax molecules by the epidermal cells throughthe substance of the epicuticle presents an inte-resting problem. Histologically there appears to bea condensed epicuticular layer of say 1 /x in thicknessbeyond the limits of the pore canals. According tothe estimate of Richards & Anderson (1942) it isunlikely that this is perforated by spaces larger than0-002/A in diameter. But the wax molecules areprobably something like 0-004^ in length. The epi-cuticular pores will not therefore admit the passageof the wax in emulsified form. The wax moleculesmust either be synthesized in situ on the surface ofthe epicuticle or, associated perhaps with somesolubilizing substance, they must pass through theepicuticle singly or in small alined groups. It is con-ceivable that their discharge in this way may favourthe close packing and orientation of the wax mole-cules needed to form an impermeable film.

This view that impermeability to water is due to acontinuous layer of closely packed and orientatedwax molecules contrasts with the view of Hurst(1943) who regards the surface of the cuticle (ofblowfly larvae) as being not homogeneous but com-posite and made up of heterogeneous protein/lipoidassociations in patches. It is admitted, however, thatother materials, often perhaps protein, may bepresent in the form of a covering layer for the wax.Such materials may even be chemically linked withthe wax. In any case it is quite evident that there aregreat differences in different insects. The permeationof lipoid through the cuticle framework, if it occursat all, seems to be of no importance so far as the pre-vention of evaporation is concerned.

The observations described emphasize the livingnature of the insect cuticle. The retention of water

8-3

112 V. B. WlGGLESWORTH

seems to be a passive process dependent on the formof the wax layer; but the slightest injury to this layeris reflected in the behaviour of the living cells below.They react as though they had been wounded(Wigglesworth, 1937); they immediately cluster to-wards the injured site (although the epicuticle hasnot been visibly damaged) and they then proceed tosecrete fresh wax to repair the damage.

The abrasion of this delicate surface layer must bea common occurrence in the life of the insect, andwere the insect unable to make good slight injuriesto its surface layer it would soon cease to be im-permeable to water. In the insect larvae'in the soilthe abrasion is usually so severe that they have losttheir power to retain water. The wireworm, forexample, is scratched all over. In spite of its hydro-phobe cuticle it loses water by evaporation veryrapidly and liquid water passes in and out in accor-dance with the osmotic equilibrium (Evans, 1943).Thus, although they retain the waterproof cuticlecharacteristic of their class, the soil insects mustremain in moist surroundings.

In discussing the mode of action of fine dusts incausing insects to dry up, Germar (1936) does notaltogether rule out the possibility of their sharpedges wounding the cuticle, but he is inclined todoubt this explanation because he could see no in-juries under the microscope. The use of ammoniacalsilver provides a ready method of demonstrating suchinjuries to the waxy layer. Mechanical abrasion thusprovides a simple explanation of the effect of inertdusts—in the sense that the cuticular surface mustbe rubbed against the dust if this is to have anyaction. But, as Alexander et al. (1944) and Parkin(1944) have shown, there are very striking dif-ferences between different dusts and different in-sects. Before the true mechanism of their action isunderstood we need to know more about the physicalnature of 'abrasion'. It may be that the attractiveforces at the surface of crystalline particles, suggestedby Alexander et al. as important in the working ofthe inert dusts, play a part in abrasive action. Parkin(1944) and Alexander et al. (1944) have suggestedthat adsorption of the cuticular lipoid by the dustmay be important in interrupting the waterproofingfilm. This is certainly so in the cockroach in whichthe 'lipoid' is a soft grease. But in most insects,where the impervious layer is an orientated wax, ad-sorption is effective only during recovery fromabrasion, when new wax is being secreted to repairthe interruptions.

The complex nature of the surface layer of thecuticle goes some way to explain the difficulty withwhich poisons enter by this route. If an insecticideis to pass through the cuticle it must be in a mediumwhich will disrupt both the wax film and its pro-tective layer of cement. As Hurst (1943) says, dis-persant action must involve both lipoid and proteincomponents. The possible action of abrasive dusts infacilitating the entry of insecticides will require

careful study. It is perhaps the abrasionworm cuticle which renders it so permeable to arsenic(Woodworth, 1938)—though the normal cuticle ofthe cockroach (O'Kane & Glover, 1935) and of thetick Ixodes (Burtt, 1944) also allow arsenic to passthrough.

The importance of different emulsifying agentsand detergents in breaking down the protective layersalso needs studying from this point of view. Lennox(1940) has pointed out the toxicity of lipoid solvents,which he attributes to the solution of the epicuticularlipoids; and O'Kane, Glover, Blickle & Parker (1940)have noted that wax solvents pass most readilythrough the cuticle of the cockroach. Klinger (1936)and Umbach (1934) describe the increased permea-bility of the cuticle after extraction with chloroformor ether (cf. Bredenkamp, 1942 and Eder, 1940) andHurst (1943) notes that prolonged immersion ofblowfly larvae in fat solvents results in an irre-versible increase in cuticle permeability to water,alcohol, etc. Lipoid solvents are efficient carriers ofinsecticides into the insect because of their dis-persive action on the packed lipoids (Hurst, 1943;Wigglesworth, 1942). Hurst (1941) has also de-scribed more subtle effects: the desiccation of hard-bodied insects when smeared with lecithin; the ex-cessive evaporation from Tenebrio larvae when incontact with larvae of Calliphora, which is attributedto the more hydrophilic character of the free lipoidin the cuticle of Calliphora. Many further examplesof this sort of action have been described in thepresent paper, and the principles involved are dealtwith in the paper by Beament (1945) on the isolatedwaxes.

SUMMARY

Transpiration through the cuticle of insects is re-stricted by a thin layer of orientated wax on the outersurface of the epicuticle. In some insects at least thiswax layer is covered by a thin layer of cement.

When heated to a certain temperature the waxlayer shows an abrupt increase in permeability towater. This 'critical temperature' varies widely indifferent species and in different stages of the samespecies. It is highest in those insects which are mostresistant to desiccation.

In the newly formed puparium of Calliphora theimpermeable film of wax is wholly superficial as inother insects. But after pupation the main imperme-able layer is on the surface of the true pupa. Thecritical temperature of the pupa is much higher thanthat of the puparium.

Abrasion of the wax layer results in a great increasein transpiration through the cuticle. Inert dustscause the desiccation of insects by getting betweenthe moving surfaces of the cuticle and abrading thewax layer. Such dusts in stationary contact with thecuticle will not remove the wax by adsorption; hencethey are without action on dead or motionless insects.

Transpiration through the cuticle of insects

i cockroach is an exception to this. Here therproof layer is a soft grease, freely exposed on

the surface; it is largely removed by adsorption onto the dusts.)

The places where the wax has been abraded canbe demonstrated by immersing the insect in am-moniacal silver solution. The phenol-containingepicuticle stains deep brown only where the pro-tective layer of wax has been removed.

Although the epicuticle shows no visible injury asthe result of abrasion, the underlying cells react asthough they had been wounded, and growth pro-cesses in the epidermis are affected.

Insect larvae from the soil show great but variableevaporation of water. This is the result of abrasionof the cuticle by soil particles. If the wirewormAgriotes is allowed to moult out of contact with thesoil it has an impermeable cuticle like other insects.

After abrasion the living insect is able to secretemore wax through the substance of the cuticle andso to restore its impermeability. Adsorption of thewax by dusts while it is being secreted interfereswith this process of recovery.

Lipoid solvents remove the wax layer from thesurface and so increase transpiration. A long seriesof wetting agents and detergents has been tested.They show widely different effects on permeabilityof the cuticle to water.

Removal of the wax layer by means of abrasivedusts or tSuitable detergents increases the rate ofentry of insecticides through the cuticle.

I have received valued help in the supply of insectmaterial from Mr S. G. Jary, Dr C. Potter, Mr A.Roebuck, Dr I. Thomas and Dr I. H. H. Yarrow.I am indebted to the Geigy Colour Co., ImperialChemical Industries Limited, Messrs Lever Bros.,Messrs Rex Campbell and Company, and TechnicalProducts Limited for the supply of chemicals andinformation. Samples of chemicals of Americanorigin were obtained through the good offices of theBritish Central Scientific Office in, Washington.Finally it is a pleasure to acknowledge the largeamount of careful technical assistance given by MissW. Wall.

REFERENCES

ALEXANDER, P., KITCHENER, J. A. & BRISCOE, H. .V. A.(1944). Ann. Appl. Biol. 31, 143-59.

BEAMENT, J. W. L. (1945). J. exp. Biol. 21, 115-31.BERGMANN, W. (1938). Ann. Ent. Soc. Amer. 31, 315-

21.BREDENKAMP, J. (1942). Z. angew. Ent. 28, 519-49.BURTT, E. T. (1944). Unpublished work.EDER, R. (1940). Zool.Jb., Physiol., 60, 203-40. (Noted

from Biol. Abstr. 15 (1941), 20358.)EVANS, A. C. (1934). Parasitology, 26, 366-77.EVANS, A. C. (1943). Nature, Lond., 152, 21-2.FULTON, R. A. & HOWARD, N. F. (1938). J. Econ. Ent.

31, 405-10.FULTON, R. A. & HOWARD, N. F. (1942). J. Econ. Ent.

35. 867-70.GERMAR, B. (1936). Z. angew. Ent. 22, 603-30.HABERLANDT, G. (1914). Physiological Plant Anatomy.

London.HOCKENYOS, G. L. (1939). J. Econ. Ent. 32, 843-8.HURST, H. (1940). Nature, Lond., 145, 462.HURST, H. (1941). Nature, Lond., 147, 388.HURST, H. (1943). Trans. Faraday Soc. 39, 390-411.KALMUS, H. (1944). Nature, Lond., 153, 714-15.KING, G. (1944). Nature, Lond., 154, 575-6.KLINGER, H. (1936). Arb. Phys. angew. Ent., Berl., 3,

49-69, 115-51.KOIDSUMI, K. (1934). Mem. Fac. Sci. Agric. Taihoku,

12, 1-380.KOHNELT, W. (1928). Zool. Jb., Anat., 50, 219-78.KUHNELT, W. (1939). VII. Internat. Kongr. Entom.

Berlin, 1938, 2, 797-807.

LENNOX, F. G. (1940). Pamphl. Coun. Sci. Industr. Res.Aust. no. 101, pp. 69-131.

LISON, L. (1936). Histochimie Animate. Paris: Gauthier-Villars.

O'KANE, W. C. & GLOVER, L. C. (1935). Tech. Bull.N.H. Agric. Exp. Sta. no. 63, pp. 1-8.

O'KANE, W. C, GLOVER, L. C , BLICKLE, R. L. &PARKER, B. M. (1940). Tech. Bull. N.H. Agric. Exp.Sta. no. 74, pp. 1-16.

PARKIN, E. A. (1944). Ann. Appl. Biol. 31, 84-8.PRYOR, M. G. M. (1940). Proc. Roy. Soc. B, 128, 393-

407.RAMSAY, J. A. (1935). J. Exp. Biol. 12, 373-83.RICHARDS, A. G. & ANDERSON, T. F. (1942). J. Morph.

71, 135-83-SCHMALLFUSS, H . , HEIDER, A . & WlNKELMANN, K .

(1933). Biochem. J. 257, 188-93.SLIFER, E. H. (1937). Quart. J. Micr. Sci. 79, 493-506.TURNER, N. (1943). J. Econ. Ent. 36, 266-72.UMBACH.W. (1934). Mitt. Forstioirt. Forstwiss. 5, 216-18.WiGGLESWORTH, V. B. (1933). Quart. J. Micr. Sci. 76,

2707318.WiGGLESWORTH, V. B. (1937). J. Exp. Biol. 14, 364-81.WiGGLESWORTH, V. B. (1940). J. Exp. Biol. 17, 180-2OO.WIGGLESWORTH, V. B. (1942). Bull. Ent. Res. 33, 205-18.WiGGLESWORTH, V. B. (1944a). Nature, Lond., 153,

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V. B. WlGGLESWORTH

EXPLANATION OF PLATESPLATE I

Fig. i. Surface view of cuticle of Rhodnius 5th stagenymph treated with ammoniacal silver. Above: rubbedwith alumina; below: normal.Fig. 2. Section of cuticle of normal Rhodnius 5th stagenymph after immersion of the whole insect in ammoniacalsilver. No staining.Fig. 3. Section of cuticle of normal Rliodnius 5 th stagenymph cut from the fresh insect with the freezing micro-tome and then immersed in ammoniacal silver. All theepicuticle is stained.Fig. 4. Section of cuticle of Rhodnius 5 th stage nymphafter rubbing with alumina and immersion of the wholeinsect in ammoniacal silver. The epicuticle is stained overthe crests of the folds.Fig. 5. Ventral abdominal cuticle of Rhodnius 5 th stagenymph where it touches the ground. Whole insect im-mersed in ammoniacal silver after running on fine emerycloth.Fig. 6. Puparium of Calliphora, immersed in ammoniacalsilver after rubbing gently on filter paper dusted withalumina.Fig. 7. Larva of Tenebrio, immersed in ammoniacal silverafter crawling for some hours on filter paper dusted withalumina. The legs are drawn forwards to show the stainedareas where the coxae have rubbed against the thorax.There are abraded and stained areas at some other points,also indicated by arrows.Fig. 8. Cuticle of 5th stage nymph of Rhodnius immersedin ammoniacal silver soon after rubbing with alumina.Fig. 9. The same, after keeping the insect for 2 days inmoist air.Fig. 10. Cuticle of 5th stage nymph of Rhodnius 5 daysafter rubbing with alumina (the dust being left on),stained with Black Sudan B. The dusted area, which isstained, is above.

Fig. 11. Leg of Hepialus larva treated with ammoniacalsilver, showing the scratches on the exposed surface.Fig. 12. Ventral surface of Agriotes larva treated withammoniacal silver, showing abrasion of the ventro-laterallines of soft cuticle. The scratches in the hard generalcuticle are scarcely visible at this magnification (seePI. 2, figs. 1, 2).Fig. 13. Side of prothorax of larva of Aphodius treatedwith ammoniacal silver, showing scratches chiefly on theprominent folds.

PLATE 2

Fig. 1. Part of ventral surface of third abdominal seg-ment of wireworm treated with ammoniacal silver. Thiswas a less permeable larva.Fig. 2. The same, from a highly permeable larva.Fig. 3. Part of ventral surface of Pterostichus larva, un-treated, showing sclerites in the soft cuticle.Fig. 4. The same treated with ammoniacal silver, show-ing scratches in the sclerites and very extensive abrasionof the soft cuticle.Fig. 5- Cuticle of Tipula larva treated with ammoniacalsilver.Fig. 6. Cuticle of Bibio larva treated with ammoniacalsilver.Fig. 7 Cuticle of Rhodnius 4th stage nymph treated withammoniacal silver after extraction with chloroform for15 min. at 200 C. Staining over the muscle insertion andat small scattered points elsewhere (scarcely visible in thephotograph).Fig. 8. The same, after extraction for 15 min. at 50° C.Staining almost universal except over the plaques andhere and there over the crests of the folds.Fig. 9. The same, 24 hr. after smearing with C 09993(see Table 13). Staining chiefly in the depressions be-tween the folds.

JOURNAL OF EXPERIMENTAL BIOLOGY, 21, 3, 4 PLATE I

WIGGLESWORTH—TRANSPIRATION THROUGH THE CUTICLE OF INSECTSorti'ie r