-
Further Studies of the Third Instar Larval Cuticle ofCalliphora
erythrocephala
By L. S. WOLFE(From the Department of Zoology, Cambridge; now at
the Science Service Laboratory,
Department of Agriculture, London, Ontario)
With one plate (fig. i)
SUMMARY
The penetration and reduction of ammoniacal silver nitrate
solution in the epi-cuticle of the larva of Calliphora was studied.
The epicuticle of the third instar larvais more permeable over the
muscle insertions and cuticular sense organs. This findingis
related to their development at the previous moult.
A surface layer of orientated wax is not present. Proteinaceous
and fatty materialsfrom the feeding medium modify the properties of
the cuticle surface. Chloroform-methanol extracts a soft light
brown acidic lipide from the protein of the epicuticleafter
contaminants from the medium are removed.
The water loss from larvae and puparia of different ages and
after various treatmentswas studied. Young puparia recover from
abrasion but larvae do not. An hypothesisthat waxy substances are
liberated on to the surface of the puparium during hardeningand
darkening of the cuticle is presented and discussed.
The pore canals penetrate the endocuticle until they are cut off
from the epidermisby the development of the prepupal cuticle just
after the puparial contraction. Aninner endocuticle in which pore
canals were absent was not found. The structure ofthe pore canals
as shown by phase contrast examination is discussed. The pore
canalsare three times more concentrated in the lateral regions than
in the dorsal or ventralregions.
The oenocytes go through a secretory cycle during puparium
formation similar tothat occurring before moulting of the
larva.
INTRODUCTION
IN the course of a study of the deposition of the third instar
larval cuticleof Calliphora erythrocephala Meigen (Wolfe, 1954)
some new observationswere made on the structure and properties of
the cuticle. This paper reportsthese findings.
MATERIALS AND METHODS
The rearing of the larvae and the histological methods were the
same asdescribed previously (Wolfe, 1954). Ammoniacal silver
nitrate solutions werefreshly prepared before use. Water loss
through the cuticle was studied usingthe methods of Wigglesworth
(1945). The phase contrast microscope wasused for studying the
structure of the pore canals and the oenocytes. Specialtechniques
are described at the appropriate places in the text.
The occurrence and distribution of reducing substances in the
cuticle
Wigglesworth (1945, 1948) used ammoniacal silver nitrate
solution todemonstrate the presence of reducing polyphenols in the
insect cuticle and[ Q u a r t e r l y J o u r n a l of M i c r o s
c o p i c a l S c i e n c e , Vol . 9 6 , p a r t 2 , p p . 1 8 1 -
1 9 1 , 1955.1
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182 Wolfe—Further Studies of the
also to show the extent of damage to the epicuticle after
abrasion. Thismethod was used in this study not only to demonstrate
the presence ofreducing substances in the larval epicuticle of
Calliphora, but also as a quali-tative indication of regions in the
cuticle more readily penetrated by aqueoussolutions.
Third instar larvae of different age groups were immersed in a 5
per cent,ammoniacal silver nitrate solution at room temperature for
6 hours andthoroughly washed with distilled water. When examined
under a binocularmicroscope, a series of well-marked deposits of
silver were observed in theouter layers of the cuticle. These
deposits were localized in feeding larvae inregions of the muscle
insertions, cuticular sense organs, and functional andvestigial
spiracles. In mature larvae irregularly distributed deposits
werefound particularly in the spinous regions and were not
associated with senseorgans or muscle insertions. Sections of the
cuticle through these regionsrevealed that they were produced by
small lesions in the outer epicuticle.These lesions were probably
produced by the spines tearing and scratchingthe outer epicuticle
during the muscular contortions of the larvae whilefeeding. Though
probably present in young larvae they were not revealedby reduction
of silver solutions. Dennell (1947) showed that reducing
sub-stances appeared in the inner epicuticle only in mature
larvae.
Short periods of immersion in ammoniacal silver solution (2-3
hours)resulted in clusters of small deposits of silver at the
muscle attachments(fig. 1, A). These deposits were restricted to
the outer epicuticle. After longerimmersion periods (12 hours) the
solution penetrated through the endocuticleand was reduced at the
base of the tonofibrillae and the surrounding epi-
FIG. 1 (plate). A, reduction of ammoniacal silver nitrate
solution by the tips of the tono-fibrillae of the muscle
attachments in the epicuticle of a mature third instar larva.
Immersiontime, 2 hours.
B, oblique section through a cuticular sense organ of a mature
larva after immersion inammoniacal silver solution for 4 hours.
C, surface view of reduction at the cuticular sense organ to
show the intensity and extentof the reduction around the sensory
peg.
D and E, the intense reduction of ammoniacal silver nitrate
solution after abrasion of theouter epicuticle of a larva 12 hours
before puparium formation. D, abrasion with fine needle;E, abrasion
with powdered glass.
F, sagittal section through the cuticle of the 'white' puparium
to show the pore canalsextending into the endocuticle from the
epidermis and the absence of an inner endocuticle.The oenocytes are
shown immediately beneath the epidermis. Osmic acid / Orcein
stained;frozen section.
G, the pore canals in the cuticle of a mature third instar
larva, 'crop full' stage. The basaland distal portions of the pore
canal are easily distinguished. Phase contrast, oil
immersion,frozen section.
H, reduction of ammoniacal silver nitrate solution by the tips
of the pore canals in theinner epicuticle of a mature larva.
Immersion time, 24 hours.
i-N, phase contrast microphotographs of oenocytes at different
stages of their secretorycycle before puparium formation. Mounted
in Drosophila ringer. I, 'crop full' stage; j , 30hours before
puparium formation; K, 20 hours before puparium formation; L,
quiescentperiod just before puparial contraction; M, 'white'
puparium stage; N, 5-hour puparium justbefore the separation of the
oenocytes from the epidermal cells.
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FIG. I
L. S. WOLFE
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Third Instar Larval Cuticle of Calliphora erythrocephala 183
dermal cells. No reduction occurred in the endocuticle. In
feeding larvaereduction was localized to the sensory pegs of the
sense organs, but in themature larvae the cuticular depression also
showed deposits of silver (fig. 1,B and c). Sections of the cuticle
through the sense organs after 12-hourimmersion periods showed that
the silver solution had penetrated throughthe epicuticle of the
sensory pegs and down the distal processes to the sensecells within
the epidermis.
The localized reduction of ammoniacal silver solutions by the
sense organsand muscle insertions is interpreted as indicating a
greater permeability ofthe epicuticle over these regions. Further
confirmation for this conclusionwas obtained by immersing larvae
for 30 minutes in a saturated solution ofcobalt chloride, rinsing
in distilled water, and placing them in hydrogensulphide water.
Larvae thus treated showed black patches of cobalt sulphideover the
muscle insertions, cuticular sense organs, and spiracles. Addition
ofdetergents (Triton X, Co 9993, Teepol) to the cobalt solution did
not increasethe size of the sulphide deposits except for a greater
depth of penetrationdown the spiracles.
The modification of the epicuticle over the muscle insertions
and cuticularsense organs arises during development at the previous
moulting cycle (Wolfe,1954). The tonofibrillae and distal nerve
fibres are attached across the exuvialspace to the old cuticle
until just before ecdysis and the old epicuticle at thistime is
penetrated by fine fibres of the tonofibrillae at the muscle
attachmentsand the distal nerve fibres at the sense organs. When
the cuticle is shed abreak occurs at the region where the
tonofibrillae of the old cuticle penetratethe newly formed
epicuticle. Similarly, a break occurs at a level just belowthe
sense rods where the distal sensory nerve fibre penetrates the
epicuticleof the new sensory peg. It is the tips of the
cuticularized fibres of the tono-fibrillae and the sensory nerve
fibres that are thought to give the quick surfacereduction of
ammoniacal silver solution.
Kuhnelt (1949) reported the presence of reducing spots within
the cuticleof insects from widely different groups. He also found
deposits at the muscleinsertions, dermal sense organs, cuticle
lesions, and cuticular pores. Noopenings of ducts or pores were
found within the larval cuticle of Calliphoraexcept around the
spiracles.
The reducing ability of the muscle attachments and cuticular
sense organsis not attributed to phenolic substances, The reduction
at the cuticle lesions,however, is very probably due to exposure of
polyphenolic substances in theinner epicuticle. Feeding larvae
whose cuticles had been abraded either byrubbing in finely powdered
glass or scratched with a fine needle showed onlyslight browning at
the abraded areas after immersion in ammoniacal silversolution.
However, larvae similarly treated at the 'crop full' stage showed
anintense reduction (fig. 1, B and E). Powerful reducing substances
are addedto the inner epicuticle at this stage. Pryor (1940) and
Dennell (1947) haveconclusively shown that this strong reduction is
due to polyphenolic com-pounds, probably an o-dihydroxyphenol.
Mature larvae rubbed in alumina
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184 Wolfe—Further Studies of the
dust showed no increase in reduction within the cuticle when
immersed inammoniacal silver solution. The outer epicuticle is very
resistant and mustbe deeply abraded to expose the inner
epicuticle.
The epicuticle
The staining reactions of the epicuticle of Calliphora larvae
and the changesin these reactions at puparium formation differed
little from the closelyrelated species Sarcophaga falculata
(Dennell, 1946), and Rhagoletis cerasis(Wiesmann, 1938). However,
the outer epicuticle stained red in Mallory'sstain and black in
Heidenhain's haematoxylin. In young larvae the innerepicuticle
stained pink in Mallory's stain but in mature larvae became adeep
blue and also gave stronger Millon's and ninhydrin reactions.
Theappearance of phenolic substances and oxidase in the inner
epicuticle wasfound by Malek (1952) to coincide with the presence
of more protein.
The protein-lipide association in the inner epicuticle before
pupariumformation contains all the requisite materials for the
formation of the sclerotinof the exocuticle (Pryor, 1940, 1947).
Sclerotin formation commences in theinner epicuticle at puparium
formation and spreads to the outer endocuticularlayers. The term
'exocuticle' should be applied only to sclerotinized cuticlewhether
it is of epicuticular or endocuticular origin or both. The
exocuticleof the puparium consequently includes both endocuticle
and epicuticle whichbecome indistinguishable. The outer epicuticle
remains distinct, but its paleamber colour suggests that it also
contains sclerotin.
The surface of the larval epicuticle is hydrophil. An orientated
superficialwax layer on the epicuticle of the type described by
Beament (1945) andWigglesworth (1945) is absent from the Calliphora
larva. However, lipidesare incorporated in the epicuticle. The
epicuticle breaks down into oily drop-lets when treated with
concentrated nitric acid and potassium chlorate (cuti-culin
reaction). Chloroform extracts small quantities of a soft, almost
liquid,waxy material of indefinite melting-point from the
epicuticle. Beament (1945)'calculated a wax thickness of 0-27/0. on
the washed puparium and i-i/x onthe unwashed larval cuticle. He
attributed this difference of wax thickness-to the presence of
contaminants from the larval environment on the unwashedcuticle.
Experiments were performed to investigate the nature of the
hydro-phil cuticle and the extent the larval environment affects
cuticle wettability.
Two hundred unwashed mature Calliphora larvae were rolled in
aluminadust for 15 minutes, removed from the dust, and washed
quickly with a jetof alcohol. The alumina dust was then extracted
with 2:1 chloroform-methanol, a solvent mixture that extracts
little non-lipide material. This pro-cedure yielded 17-5 mg. of a
strongly smelling acidic grease with indefinitemelting-point. The
yield, after repeating the above procedure with larvaepreviously
washed in distilled water, was only 0-7 mg. An approximate
cal-culation gives a wax thickness of 1 JJ, on the unwashed and
0-004 /"• o n t n e
washed larva] cuticle. The latter value is so small that the
presence of a super-ficial wax layer on the epicuticle appears
unlikely. The waxy materials.
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Third Instar Larval Cuticle of Calliphora erythrocephala 185
extracted from the unwashed larvae originate as suggested by
Beament (1945)from substances in the feeding medium adsorbed on to
the epicuticle.
Three separate batches of fifty washed, isolated, and dried
cuticles ofmature Calliphora larvae were weighed and extracted for
1 hour with chloro-form-methanol. After removal of solvent, 3
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186 Wolfe—Further Studies of the
with epicuticle thickness. In the first and second instars the
epicuticle thick-ness is less than iju. whereas in the third instar
it varies from 3 to 7^, in
TABLE I
Percentage loss of weight of Calliphora erythrocephala larvae
and puparia aftervarious treatments and exposure to dry air over
P205for 4 hotirs at 25
0 C.
Object of treatment
A. Second instar larva untreatedThird instar larva feeding
,,Mature larva „White puparium ,,Puparium 4 hours „Puparium 40
hours ,,
B. Larva 'crop full', control„ immersed cold CHC13 3 minutes„
immersed hot CHC13 3 minutes,, rubbed with alumina dust; dust left
on,, heated first to 6o° C.„ smeared with Co 9993
C. Puparium 2 hours, control„ immersed in CHCL3 for 3 minutes„
immersed in benzol for 3 minutes„ immersed in ale.-ether (3 :1)„
immersed in acetone for 3 minutes„ immersed in abs. ale. for 3
minutes
D. Puparium 2 hours, control,, surface scraped to damage
epicuticle„ surface scraped; left 12 hours„ surface scraped; left
12 hours; smeared Co9993„ surface smeared Co 9993,, rubbed with
alumina dust,, rubbed with alumina dust; rinsed dist. water;
left 12 hoursE. Puparium 10 hours, control
,, surface scraped„ puparium removed in section to level of
pre-
pupal cuticleF. Puparium 35 hours, control
,, puparium removed post half„ puparium removed post half; pupal
cuticle
smeared with Co 9993„ ditto; pupal cuticle immersed in CHC13 for
3
minutes,, immersed with puparium intact in CHC13 for
3 minutes
Per cent,loss ofweight
6 42 9
i - 8I-S1 6I - I
2 1
2I-O44'O
3 - 2
9 52-8
1 "3S2'44 3 1
23-S2 2 9
5"1
1 "454'3
3-81 9 4
22"41 2 8
2 61-2
46 O
3O-4i - o2-7
1 0 5
2 1 0
1-2
thickness. Also an inner epicuticle cannot be seen in the
epicuticle of firstand second instar larvae. It may be that the
presence of an inner epicuticle isessential for the control of
water loss as well as water penetration. Richards,Clausen, and
Smith (1953) have recently shown that the inner epicuticle
ofSarcophaga bullata is essential for the ph-enomenon of
asymmetrical penetra-
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Third Instar Larval Cuticle of Calliphora erythrocephala 187
tion to occur. The suggestion by Bonnemaison and Cayrol (1951)
that theendocuticle thickness is a factor in resistance to
penetration of insecticidesseems less likely.
Treatment of the larva and early puparium with organic solvents
greatlyincreased the water loss through the cuticle (table 1, B and
C). This is almostcertainly a result of extraction of waxy
substances and disorganization ofthe epicuticular protein-lipide
complex. Larvae rubbed in alumina dust orsmeared with the powerful
detergent Co 9993 (cetyl ether of polyethyleneglycol) showed no
increase in water loss. This is further evidence for theabsence of
an orientated surface wax layer controlling water loss on the
larvalcuticle of Calliphora.
A curious difference was found between the mature larva and the
earlypuparium. Rubbing the early puparium with alumina dust led to
a significantwater loss, but if the puparium was left for 12 hours
impermeability wascompletely restored (table 1, D). Recovery also
occurred after light scrapingof the epicuticle of the puparium. The
reasons for this recovery reaction arenot clear. A possible
explanation is that wax is continuously secreted duringthe
darkening and tanning of the puparium. The puparium
progressivelydarkens and hardens during the first 20-25 hours, and
this is precisely theperiod before pupation when recovery from
abrasion was observed. However,wax could not have been secreted
continuously by the epidermis or fromgland cells during this period
because they are separated from the puparialcuticle 2 hours after
the puparial contraction by the formation of a very thinprepupal
cuticle.
Recovery of the larval cuticle from abrasion was not observed.
The pro-teinaceous and waxy materials on the surface of the larval
epicuticle derivedfrom the feeding medium are also present as a
solidified and oxidized layeron the surface of the puparium. The
puparium is not wetted as readily as thelarva and also shows a
higher resistance to water loss. Rubbing the pupariumin alumina
dust led to a much greater increase in water loss than in the
larva(table i, D). This indicates that the surface waxy materials
on the pupariumdo control water loss and suggests that besides the
contaminants carried overto the puparium from the feeding medium
there may be a wax layer formedon the puparium. Pryor (1940)
concluded that sclerotin formation made thecuticle 'lipophil'. He
regarded the epicuticle as a simple protein later tannedand
impregnated with lipides. It is suggested that the formation of
sclerotinwithin the protein-lipide epicuticle of the larva of
Calliphora during pupariumformation leads to the exclusion of
lipide on to the lipophil surface forminga distinct waxy layer.
Abrasion of this layer by alumina dust or its disruptionby
detergents might be expected to result in an increase in water
loss. Thisprocess of exclusion of waxy substances from within the
epicuticle on to itssurface would continue as long as the process
of hardening and darkeningoccurs. The puparium is not fully
hardened until pupation.
The prepupal cuticle does not control water loss in the early
puparium(table 1, E). At pupation, occurring 25 hours after
puparium formation, water
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188 Wolfe—Further Studies of the
loss is efficiently controlled by the waxy layer of the delicate
pupal cuticle(table i, F). This has been extensively studied by
Beament (1945).
The pore canals
The pore canals in newly moulted third instar Calliphora larvae
appear ascytoplasmic filaments extending as far as the inner
epicuticle. The depositionof endocuticle during the third instar
results in the retraction of the cyto-plasmic part of the filament;
the outer non-cytoplasmic portion then becomesextremely difficult
to distinguish from the surrounding endocuticle by usualstaining
procedures. Sections of cuticle, however, treated with 2 per
cent,osmic acid show the pore canals very clearly. This observation
suggests thatduring the retraction of the cytoplasmic filaments
from the inner epicuticlea little lipidal material is left in the
pore canals. Sudanophil material is alsopresent particularly in the
branching filaments just beneath the inner epi-cuticle. Pore canals
branching fan-like within the inner epicuticle have beenobserved by
Plotnikow (1904) in Bombyx, Dennell (1946) in Sarcophaga, andWay
(1950) in Diataraxia.
Fresh sections of mature larval cuticle when examined either
with trans-mitted light or under phase contrast show the pore
canals clearly differentiatedfrom the endocuticle. Phase contrast
examination has revealed several inter-esting points about their
structure in the mature cuticle (fig. 1, G). TWOdistinct regions of
the canal are shown. The basal portion contains epidermalcytoplasm
extending approximately one-third of the way through the
endo-cuticle (25-30 fi). The distal portion shows what appear to be
numerous finegranules within the laminae of the endocuticle and
ends in the inner epicuticle.The distal portion of the canals does
not show any lining and certainly doesnot look like a duct. The
canal is not helicoidal but runs an almost straightcourse through
the endocuticle. However, in young growing larvae, the porecanals
are very irregular in their course through the endocuticle and
appearas irregular wavy lines crossing the laminae of the
endocuticle. A spiral orhelicoidal course of the pore canals
through the endocuticle sufficientlyregular to ascribe a pitch to
the helix as recorded by Dennell (1946) forSarcophaga and Richards
and Anderson (1942) for Periplaneta has not beenobserved.
One of the functions of the pore canals is the secretion of the
inner epi-cuticle (Wolfe, 1954). Dennell (1946) reported the
presence in Sarcophagafalculata of an endocuticular layer, the
inner endocuticle, which containedno pore canals and was secreted
in the mature larva just before pupariumformation. This inner
endocuticle was not found in Calliphora. Pore canalswere found in
osmic acid Orcein stained preparations connected to the epi-dermis
right up to the commencement of browning of the puparium (fig. 1,
F).
Larvae at the 'crop full' stage when immersed in ammoniacal
silver nitratesolution for long periods (25-30 hours) showed series
of distinct spots ofsilver within the epicuticle (fig. 1, H). These
deposits corresponded to thepore canals in the inner epicuticle.
They are not shown in larvae immersed
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Third Instar Larval Cuticle of Calliphora erythrocephala 189
for short periods. The solution must penetrate through the outer
epicuticleto the tips of the pore canals before being reduced. The
outer epicuticle isslightly permeable to aqueous solutions in the
mature larva. The van Wisse-lingh chitin test was performed on
isolated pieces of cuticle which weremounted and examined in
surface view. The pore canals showed up as darkpurple dots on a
paler purple background, and were found to be moreconcentrated in
the lateral than the dorsal or ventral regions;
approximately17,400/sq. mm. in the lateral regions and 5,600/sq.
mm. in the mid-dorsaland ventral regions. Fresh, unstained,
transverse sections of the cuticleshowed the endocuticle laminae
crossed by many more lines in the lateralregions than
elsewhere.
The endocuticle increases in thickness during the growth of the
third instarlarva, reaching a maximum of 80-85 /n. The laminae of
the endocuticle are atall stages of growth penetrated by
cytoplasmic extensions of the epidermiswhich continue distally as
chitinized filaments into the inner epicuticle(fig. 1, G). An
examination of the pore canals of fresh sections of the
maturecuticle under phase contrast did not show any space between
pore canalcontents and surrounding endocuticle that might suggest
plugs or cords ofchitin within the pore canal lumen. Chitinous
filaments were not found pro-jecting from teased laminae of the
outer region of the endoculicle. However,in young larvae, filaments
were found projecting from endocuticular laminaein certain sections
that had become teased apart during section cutting. Thesefilaments
were pieces of the cytoplasmic part of the pore canals which in
thenewly moulted larva extend up to the inner epicuticle (Wolfe,
1954). Asthe larva grows and the endocuticle thickness rapidly
increases these cyto-plasmic filaments are retracted. A marked
differentiation still remains in thenewly secreted endocuticle
connecting the cytoplasmic portion of the porecanals to the inner
epicuticle. It is this distal portion of the pore canals thatgives
a strong chitin reaction as shown by Dennell (1946). The
cytoplasmicportion did not give a chitin reaction. This may be the
reason why Dennellwas unable to find pore canals in the inner
region of the endocuticle in themature larva.
The observations made above indicate that the pore canals remain
con-nected to the epidermis throughout the third stadium. Way
(1950) has shownin Diataraxia that in the soft cuticle the pore
canals function only duringthe early stages of development and are
then cut off from the epidermisby the development of an inner
endocuticle. In areas of hard cuticle therewas a thick heavily
tanned exocuticle that continued to develop throughoutthe stadium
and required the maintenance of the pore canal system from
theepidermis. In Calliphora, however, the darkening and hardening
processesoccur at the end of the third stadium when the cuticle has
reached its maxi-mum thickness. During the period immediately
before puparium formationpolyphenols, basic protein, and enzymes
accumulate in the inner epicuticle.It appears necessary that the
pore canal conducting system be maintainedbetween epidermis and
inner epicuticle until puparium formation.
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190 Wolfe—Further Studies of the
The pore canals of Calliphora, it is suggested, should not he
regarded asdistinct ducts or canals in the cuticle. Rather, they
are thought to be differ-entiated regions through the laminae of
the endocuticle possessing a greaterporosity and able to transport
materials necessary for the formation of thepuparium quickly and
selectively to the inner epicuticle.
The oenocytes at puparium formation
The oencytes exhibit a secretory cycle at moulting and at
puparium forma-tion. During the period when the larvae are
migrating away from the feedingmedium, the amount of secretory
granules within the oenocyte cytoplasmincreases rapidly and reaches
a maximum at the time of contraction of thecuticle to form the
white puparium and then decreases during the first 5 hoursof the
puparium. The secretory cycle was followed by examining, under
phasecontrast, fresh oenocyte groups dissected from the larvae and
puparium atdifferent times during the third stadium (fig. 1, I-N).
The staining propertiesof the oenocyte secretion elaborated before
moulting and puparium formationare those of an acidic, unsaturated
lipide in association with protein. Thelarval oenocytes are
completely histolysed by the 25th hour after the whitepuparium
stage.
The role of the oenocytes in puparium formation is very obscure.
Noneof the changes leading to the formation of the puparial cuticle
seem to becorrelated with oenocyte activity. It might be suggested
that the oenocytesare associated with the production of a component
necessary for the darkeningand hardening of the puparium. But the
phenolic precursors for this processare present in the inner
epicuticle before secretion appears in the oenocytecytoplasm.
Moreover, histochemical tests on isolated oenocytes for
phenols,oxidases, and dehydrogenases were negative. However, a
suggestive correla-tion exists between the secretory activity of
the oenocytes and the secretionof the prepupal cuticle during the
first 6 hours after the white puparium stage.At moulting oenocyte
secretive activity follows strikingly the deposition ofthe
protein-lipide epicuticle (Wigglesworth, 1948; Wolfe, 1954).
The secretion granules within the oenocyte cytoplasm as well as
the pre-pupal cuticle are stained by Sudan black. The peak in the
secretive activity ofthe oenocytes occurs just before the prepupal
cuticle is formed. Immediatelyafter the secretion of the prepupal
cuticle histolysis commences in the epi-dermal cells and oenocytes,
which separate from the cuticle and becomereplaced by bands of
actively dividing imaginal epidermal cells spreadingover the larval
epidermal cells displacing them into the interior of
thepuparium.
I wish to thank Professor V. B. Wigglesworth for his excellent
supervisionand criticism, and Dr. J. W. L. Beament and Dr. M. G. M.
Pryor for theirvaluable comments throughout the course of this
work. This research wascarried out during the tenure of an 1851
Exhibition Scholarship at theUniversity of Cambridge.
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Third Instar Larval Cuticle of Calliphora erythrocephala 191
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