AN ABSTRACT OF THE THESIS OF
I'RANK JOSEPH I-ONGO for the M. S. in
-(Nalrr-ei1o"g-r.")
z_ogtg.sJ
(Major)
Date thesis is presented _ _ SggS*_?q
-126!
Title CYTOPHOTOMETRIC DETERMINATIONS OF NUCLEAR DNA
CONTENT IN TISSUES OF THE WAX MOTH I-ARVA,
GALLERIA MELLONEL],A LINNAEUS.
Redacted for Privacy
Abstract approved
Tlrr":"";;G;;r)
The present study was undertaken to deterrnine, by measurernent of nuclear DNA, the degrees and frequencies of ploidy in the
last larval instar of the rnoth Ca]]g!a rnellonella Linnaeus. While
such data have been arrrassed
for a wide variety of vertebrate tissues,
few studies have been concerned with invertebrates and especially
insects where somatic polyploidy is developed to an unusually high
degree.
Relative arnounts of DNA were deterrnined cytophotornetrically
on individual Feulgen-stained nuclei of hypoderrnis, ventriculus, f.at
cells, blood cells, anterior silk gland, and proventriculus. Graphs
of the deterrnined DNA values indicated the presence of nuclear
classes falling into polyploid ratios,
The rnajority of blood ce11 and fat cel1 nuclei had class I
values (= diploid), while class II and III (= tetraploid and octoploid)
DNA values represented about I7 to 20 percent of the total nuclear
population. The rnajority of DNA values of the ventriculus nuclei fe11
into the second class while 6. 8 percent were considered as class I.
The hypodermal nuclei were mostly of class I, while class II values
represented 5. 5 percent of the total nurnber of determinations.
Nuclei of the proventriculus and the anterior silk gland showed
a broad spectrurn of DNA values ranging from classes IV to VIII
including rnany interrnediate value s.
The method of measurement was analyzed for sources of
error. Various interpretations were offered for the variations in DNA
values including the role of endornitosis, definitive polyploidy, aneu-
ploidy, differential gene replication, etc. In the case of the anterior
silk gland and the proventriculus a possible relationship between DNA
arnounts and ceI1 activity
is indicated.
CYTOPHOTOMETRIC DETERMINATIONS OT' NUCLEAR DNA
CONTENT IN TISSUES OT' THE WAX MOTH I,ARVA,
GALLERIA ME L]-ONE L]-A LINNAEUS
by
FRANK JOSEPH I.ONGO
A THESIS
submitted to
OREGON STATE UNIVERSITY
in partial fulfillment of
the requirernents for the
degree of
MASTER OF SCIENCE
June 1955
APPROVED:
Redacted for Privacy
Professor and Chairman of Departrnent of. ZooLogy
In Charge of Major
Redacted for Privacy
Dean of Graduate School
Date thesis is presented
Typed by Nancy Kerley
August 20, 1964
ACKNOWLEDGMENTS
Heartfelt thanks are expressed to Dr. Ernst J. Dornfeld for
recomrnending this study, for his critical advice and encouragernent,
and
for his help in the preparation of this thesis,
Sincere gratitude is acknowledged to Dr. A, Owczarzak for
his technical instruction and assistance and his critical guidance.
Appreciation is also expressed to Dr. G. C. Wittig for
providing the specirnens; to John Belton for his direction in the preparation of the rnany photornicrographs necessary to this study; and
to rny wife, Kitty, for her assistance in the nurnerous calculations
and her constant encouragement.
TABLE OF CONTENTS
Page
INTRODUCTION
I
MATERIAIS AND METHODS
Fixation and Staining
Photornetric Apparatus
Method of Measurernent
RESULTS
4
5
6
9
Hypoderrnal Nuc1ei
Ventriculus NucIei
Fat CelI Nuclei
Blood Cell Nuclei
Proventriculus NucIei
Anterior Silk Gland Nuclei
DISCUSSION AND CONC LUSIONS
Specificity of the Feulgen Stain
Sources of Error in Cytospectrophotornetry
Analysis of Results
9
t0
10
1I
LZ
IZ
I4
L4
15
L7
SUMMARY
ZZ
BIB LIOGRAPHY
z5
APPENDIX
30
Derivation of the Photornetric Forrnula
30
Plate
3Z
s
CYTOPHOTOMETRIC DETERMINATIONS OT' NUCLEAR DNA
CONTENT IN TISSUES OF THE WAX MOTH I,ARVA,
GALLERIA ME LI,ONE LI,A LII.trNAEUS
INTRODUCTION
It is a well established fact that desoxyribose nucleic acid
(DNA)
constitutes a rnajor cornponent of the hereditary nuclear material and
can be taken as an index of the chrornatin content within a
ee1l. Quan-
titative deterrninations of DNA are therefore useful in establishing
phases of chrornosorne replication and degrees of ploidy or polyteny
within
ce11
nuclei. For such studies Lessler (1953) considers
the
Feulgen nucleal reaction to be the rnost reliable. This reaction was
first described by Feulgen and Rossenback in I9Z4
ar.d since that time
it has been the rnost widely used quantitative cytochernical test for
DNA,
Early quantitative studies on DNA were based on biochemical
analysis of rnass nuclear suspensions, The rnethod was first used by
Bovin, Vendrely, and Vendrely (1948) on rnarrlrnalian tissues and
subsequently ernployed by Mirsky and Ris ( 19491 on investigations of
fish, arnphibian, reptile, and bird tissues. Results frorn such work
indicated that the nuclear DNA content is specific for any one species
and that
it rnay be viewed as a possible genetic cornponent, However,
the nuclear DNA content deterrrnined with sirnilar rnethods by Mirsky
and Ris (1949)
for different beef tissues dernonstrated results
Z
inconsistent with this theory. It was suggested that since the nuclear
suspension rnethod yields dete rrninations repre senting ave ra,ge value
s
for a great nurnber of nuclei no insight could be gained as to the DNA
cornposition of specific cells and therefore variations between in-
dividual nuclei were lost in the rnass exarninations.
Photornetric rneasurernents of individual nuclei present a rnore
cornplete picture of the variations in the DNA content between nuclei
of the salrre and different tissue types. The first cytospectrophoto-
rnetric rnethod, introduced by Caspersson (1935), dernonstrated that
the purines and pyrirnidines of nucleic acids have rnaxirnurn absorbencies when exarnined with ultraviolet light at 250 rnpr. The rnethod
of rneasuring the absorption of visible light by Feulgen stained nuclei
was later developed by Pollister and Ris (1947) and by Pollister and
Moses (1949). Although the rnethod is sirnple in theory, in practice
it is lirnited by heterogeneous chrornatin distribution and by variation
in nuclear size and shape. In I95Z Patau (l95Zl and Ornstein (L952)
independently devised a rnethod to circurnvent the above difficulties.
Later, in
1958 and 1961, Mendelsohn (1958b; 1961) rnodified the
newer rnethod by reducing the nurnber of operational steps and pro-
viding tables of standard calculations. Essentially the rnethod devised by Patau and Ornstein ernp1oys two wavelengths of light and is
not dependent on chrornatin distribution and nuclear geornetry.
Through the use of the photornetric rnethod considerable
3
attention has been given to the DNA variation in the rnitotic and
rneiotic cycles, the degree and frequency of ploidy, and DNA variations accornpanying physiological changes, Photornetric rneasurernents of ploidy in interphasic nuclei are particularly valuable, since
chrornosorne counts cannot be carried out and estirnates based on
nuclear size are unreliable. Increase in nuclear volurne does not
necessarily indicate an increase in chrornosome nurnber or in DNA
content. Polyploid classes and their f requencies have been deterrnined for a host of anirnal tissues, rnost of which have been verte-
brate types. With regard to studies on invertebrate tissues rather
Iittle has been done. In the latter group rnay be rnentioned the work
of Merriarn and Ris ( I954) on honey-bee tissues; Leuchtenberger and
Schrader \l95zl on the salivary glands of Helixi Lison and Pasteels
(1951) on blastorneres of the sea urchin; Swift and Kleinfeld (i953)
on grasshopper sperrnatogenesis, ocigenesis, and cleavage; Kurnick
and Herskowitz (1952) on salivary gland nuclei of Drosophila; and
Stich and Naylor (1958) on polytene chrornosornes of chironomids.
The present study was undertaken to extend this rather
lirnited body of inforrnation by producing data on the nuclear distribution of DNA in various 1arva1 tissues of the wax rnoth, Galleria
rnellonella Linnaeus.
4
MATERIAI,S AND METHODS
Fixation ar{Sta:!g!rrg.
Last instar larvae of Galleria rnellonella were obtained from
the Forest Science Laboratory of Oregon State University. They
average about 3.0 cm in length and 0.5 crn in diarneter, are relatively
unornarrlented, gray in co1or, and with few bristles. In a prelirninary
histological exarnination a nuclear size inventory was rnade on the
larval tissues. The rnaterial for investigation was prepared in
the
following rnanner: The larvae were injected with Carnoyrs fluid (three
parts of absolute ethanol to one part glacial acetic acid). The head
and part of the posterior end were rernoved and the anirnals were
bisected into anterior and posterior halves. The bisected portions
were placed into vials containing Carnoyrs fluid and fixed under
vacuurrr
for five hours, The f.luid u'as changed three tirnes to
cornpensate
for the change in concentration of the fixa.tive while under
a decreasing partial pressure. Following fixation the larvae were
dehydrated and ernbedded in paraffin in the usual histological manner.
Cross sections were rnade of the bisected halves according to the
results of the nuclear size inventory, specifically, at thicknesses
egual to one and one ha.1f tirnes the size of the nuclear type under
study. Sections were cut at 25 St f.or anterior silk gland and
proventriculus; l0 p for hypodermis, blood ceIls, and ventriculus;
and 1Z 1t f.or fat ce11s, Staining with the Feulgen reagent followed
a
modified method described by Stowell (19451. Tissue sections were
hydrolyzed in tN HCl at 60" c for ten minutes followed by staining
in sulphurous fuchsin reagent for one hour, Unhydrolyzed sections
were also stained for controls. Any one slide contained sections of
only one thickness. Hypodermal ce11s were found in all sections and
were used as a standard of cornparison. A11 the slides were stained
with the sarne stock Feulgen reagent kept colorless by storage in
a
tightly closed container at 5o C,
Photometric Apparatus
The photornetric apparatus (Plate VII) consisted essentially
of a rnicroscope and a Farrand photornultiplier unit. The light source
was a 115-vo1t tungsten coiled filament rnounted in a Spencer illurni-
nator connected through a Raytheon voltage stabilizer to a ll0-voIt
line source. One coil of the larnp was used. as a point source of
light. It was focused on the entrance slit of a Bausch and Lornb
grating rnonochrornator set at 0. 7 rnrn. The rnagnified irnage of the
rnicroscope field was projected through the bellows of a Leitz
Aristophot camera and viewed with a telescope carried on a rnodified
plate carrier sirnilar to the photohead assembly designed by Birge
(1959). The nuclear sizes and field areas could be rneasured in
6
rnicrons with an ocular rnicrometer in the telescope. By
sliding the telescope out of position the irnage was directed to an RCA
IPZI phototube. An iris diaphragm placed directly under the phototube
lirnited the projection on the photosensitive area to the desired field
area.
absorption measurernents were taken at
A11
49O
rnp and 514
rnp isolated frorn the light source by the Bausch and Lornb rnono-
chrornator. Bausch and Lornb
tives,
N.
4 rnrn and 28 rnrn achrornatic objec-
A. 0.55 and 0.08, respectively, were used together with
8X, l0X, and l5X Leitz periplan oculars depending on the magnification required. The objectives were rnounted on a centerable nose
piece, A Leitz aplanic, achrornatic centerable condenser,
N. A.
1.40, cornpleted the optical set-up. Photocurrents were read on an
RCA ultra-sensitive DC rnicroarnrneter of 0 to 1000 parnp range and
50 rnegohrns maxirnurn input resistance.
Method of Measurernent
The following data were recorded (see sarnple data sheet,
Plate VIII):
l)
Code nurnber
of specific nuclei
(#)
Z) Area of the rnicroscope field in rnicrons (B) containing
whole nucleus and a border of cytoplasrn
the
3) Microarnmeter deflection when light
of. 490 mp passed
through the nucleus (Ir r)
4l Microarnmeter deflection when light
of. 49O rnp passed
through the cytoplasm near the nucleus (IrO)
5) Transmission (T1) of light (490 rnp), given Uy It
t/ItO
5) Reciprocal of Tl (LI), given by 1-T,
7l Microarnrneter deflection when light of 514 mF, passed
through the nucleus (Irr)
8) Microarnmeter deflection when light of 514 rrrl, passed
through the cytoplasm near the nucleus (Ia6)
9) Transrnission (Tr) of tight (514 rnp), given by I22lI2g
I0) Reciprocal of T Z (LZl, given by 1-Ta
I
l) Factor (Q) expressing the product of LZ/LI
lZ) Correction factor (C) which is a function of Q. This
factor is given in chart form by Patau ll95z\ and can be cornputed
frorn the following equation:
.v = 11
frrr6f
l3) Relative
arnount of the absorbing substance
(y), or DNA
in the whole field area (B). This is calculated frorn the forrnula used
by Patau lI95Zl, viz.,
Y = KBLIC
where y is the arnount of DNA in arbitrary units, K is a constant
8
I
equal to * 1n t0 (where k,r is the extinction coefficient) and rnay be
*I
neglected when relative arnounts of y are desired, B is the area of the
microscope field containing the nucleus, L, is the reciprocal of I-T,
and C
is the correction factor given by Patau. Derivation of this
forrnula is given in the appendix.
14) Factor (M) representing the average relative arnount of
DNA
for a series of two readings.
Care was taken to measure only entire nuclei as deterrnined by
critically focusing beyond their upper and lower surfaces. Nuclei
which were cornpletely out of focus when other objects rernained
sharply in focus were assurned to be whole. Errors involved in the
rneasurernents of incomplete nuclei were thus avoided.
Magnification at the level of the phototube was adjusted in
such a way that the diarneter of the rneasured nucleus would not
exceed the range of two rnm to ten
mrn. Two rnillirneters was the
lower lirnit of uniform sensitivity of the phototube while ten rnrn was
the upper lirnit. Field areas were rneasured to the nearest tenths of
all magnifications. Microarnmeter deflection was estirnated to the
nearest thousandth.
RESULTS
Five last instar larvae were used for the present study. For
each larva about 15 nuclei of each cell type were measured
for DNA
content. Analysis of the DNA content per ce1l type was rnade on an
individual larva as well as a collective basis. Examination of the
graphs on Plates X, XII, XIV, XVI, XVIII, andXX dernonstrates that
no real significant differences exist alnong the five larvae. There-
fore the results will be discussed collectively by the tissue type.
Microscopic appearances of nuclei of the ventriculus, pro-
ventriculus, blood ceIls, fat ce1ls, hypoderrnis, and anterior region
of the silk gland are shown in P1ates II through VI.
Hypodermal Nuclei
Fifteen nuclei of the hypodermis were rneasured in each of the
five larvae. Distributions of the relative amounts of DNA are graphically recorded in Plates D( and X. The majority of DNA values,
ranging from 1.0 to 3.0, form a rather sharply delirnited and fairly
syrnrnetricalunimodalcurve. A few DNA values, 6.6 percent of the
total number of deterrninations, range from 3.5 to 4.0. DNA values
of hypodermal nuclei were thus grouped into two classes. The peak
value for class I nuclei (diploid) is 2.0 while the rnean and standard
deviation are I .94 arrd 0.L4, respectively. CIass II nuclei (tetraploid),
10
which include DNA values double the class I peak, show a mean and
standard deviation of 3. 8 and O.26.
Ventriculus Nuclei
The nurnber of ventriculus nuclei exarnined was 73. The re-
sults are graphed on Plates XI and XII. The majority of DNA values,
ranging frorn 3.0 to 6.5, form a fairly symmetrical unimodal curve
with a peak value of.4,0, These DNA values faIl into class II and
have a mean and standard deviation of 4. 25 and 0. 89, respectively.
A few DNA values, 6.8 percent of the total nurnber of determinations,
ranging from l.5 to 2,5, fall into class I; mean and standard deviation
are 2.0 and 0. 36.
Fat Ce1l Nuclei
The distribution of DNA values for 76 f.at cell nuclei are shown
on Plates
XIII
and
XIV. The results obtained are essentially similar
to those found in the hypoderrnal nuclei. Within the range of 0.50 to
3.0 the DNA values forrn a high unimodal curve with a peak value of
1,5 (class I nuclei). Sorne DNA values, l2 percent of the total, are
found in the range of.3.5 to 5,5 (class II nuclei), while a few DNA
values, 5.2 percent of the total nurnber of deterrninations, are found
in the range of 6,0 to 7,5 (cIass III, or octoploid nuclei). The rnean
and standard deviation of the class I values are 1.47 and 0.60,
ll
respectively. The slightly lower mean values of the class I nuclei
can be accounted
for by the degeneration of the fat ce1ls as the larvae
approach metamorphosis. Mention of this phenomenon will be made
in the discussion. For class II nuclei, the rnean and standard
deviation are 4.61 and 0.80, respectively; for class III they ate 7.0
and 0.55.
Blood CeII Nuclei
Distribution of DNA values for 47 blood ceIl nuclei are given
on Plates XV and
XVI. Due to the difficulty in finding these cells in
tissue sections 75 measurements (15 measurements per larva)
could not be made. Random rrreasurernents were made of various
blood cell nuclei without distinction of possible types, which proved
insurrnountably difficult. DNA values of class I nuclei forrn a birnodal curve ranging frorn 1.0 to 3.0 with peaks at 1.5 and 2.5. The
mean and standard deviation are I.82 and 0.57, respectively, for the
birnodal range. Lack of a syrnrnetrical unirnodal curve rnay be due
to an insufficient nurnber of readings and/or the inability of classifying the various blood
ce11
types. Fourteen percent of the total nurrr-
ber of deterrninations range frorn 3.5 to 5.0 and are considered to
be class II nuclei. Mean and standard deviation of the class II nuclei
are 4.ZL and 0.88, respectively. Class III nuclei, rnaking up 6.4 percent of the total deterrninations, range frorn 6,5 to 8.5. Mean and
IZ
standard deviation for the class III nuclei are 8. 0 and 0.85.
Proventriculus NucIei
Due to the large size and overlapping disposition of the pro-
ventricular nuclei difficulty was encountered in atternpting to measure
large numbers of single, entire nuclei. Distribution of t},e 52 DNA
values (graphs on Plates XVII and XVIII) form a rather wide spectrum
frorn 23 to
143
with no evidence of syrnrnetrical unimodal curves.
The bulk of the measurernents (62.5 percent) tend to fall into the
range of.44 to 82. Placement of the DNA values into nuclear classes
is at best difficult. A possible grouping would include in class IV
the
values LZ to 24, in class V 32 to 49, class VI 50 to 95, and class VII
98
to I43.
Such a breakdown would
yield successive doubling of rnean
values (tV = 23.0; \[ :: {Q.r); YI = 64.3; and V]I = III.6).
.
Further consid-
eration of this broad range of DNA values is deferred to the discussion.
Anterior Silk Gland Nuclei
The nurnber of anterior silk gland nuclei rneasured was 54
(graphs on Plates XIX and XX). As in the proventriculus, the silk
gland nuclei reach gigantic size and tend to overlap
in sections;
hence the standard quota of rneasurable nuclei could not be reached,
Distribution of the DNA values forrn a broad spectrurn, frorn t3 to
13
20L,5, rerniniscent of the values found in the proventriculus. The
bulk of the rneasurernents (59.2 percent) fal-I in the range
of. 29
to
76.
There exists no clear evidence of unirnodal curves or sequential
doubling of DNA values. Again placernent of the DNA values into
nuclear classes is at best difficult. Possible groupings would include
in class IV
97
13
to 28, class y 29 to 49, class VI 50 to 95, class VII
to I50, and class VIII
169
to Z0I. Such a breakdown would
roughly yield successive doubling of rnean values (IV = I3,0; Y = 37,6;
VI = 70;0; VII = 113.0;
arrd
VIII = 20L.5). Further consideration of
this situation will be deferre<1 to the discussion.
t4
DISCUSSION AND CONCLUSIONS
Specificity of the Feulgen Stain
The exact mechanism of the Feulgen nucleal reaction for DNA
has not been fully established. However, it is generally agreed that
a positive Feulgen reaction after acid hydrolysis under controlled
conditions coupled with a negative reaction without hydrolysis is
a
specific test for the presence of DNA-protein.
Ris and Mirsky (19491 have demonstrated the quantitative
nature of the Feulgen reaction by comparing results obtained on
cytological preparations with DNA values determined biochemically.
Work by Tamrn, Hodes, and Chargaff (1952), Overend (1950), and
overend and stacey (L9491 give evidence that controlled rnild acid
hydrolysis removes purines from DNA without significant loss of
polymerization of the remaining moIecule, thus providing specific
loci of attachment for leucofuchsin. Swift (1950) and Hoover
and.
Thornas (1951) have demonstrated that the color intensity of Feulgen
preparations varies directly with the thickness of the absorbing layer,
obeying the requirernents of the Beer-l,ambert 1aw. The constancy
in DNA values of Feulgen-stained nuclei, as well as the l:Z:4 ratios
reported in nurnerous studies of polyploid series offers good indica-
tion of the quantitative nature of this staining reaction. Further
15
studies on the Feulgen reaction are reviewed by Lessler (1953) and
Novikoff (1955) who have concluded the reaction to be highly re1iable.
Conditions such as temperature of the hydrolytic solution,
hydrolysis tirne, pH, and duration of staining in the Feulgen reagent
must be controlled if reproducible results are to be obtained, In the
course of this study standardized conditions were maintained.
Sources of Error in Cytospectrophotornetry
The various sources of error found in the two-wavelength
rnethod have been discussed and estirnated by Patau (l95zl,
Ornstein 11952), Pollister and Ornstein (1955), Mendelsohn (1958a;
1958b; 1951), and Garcia (L962ai I96Zbl.
Essentially, there exist in photometry two rnajor sources of
optical error which rnay be considered as the rnost irnportant. The
first is the rrdistributional error" which is caused by non-uniforrnity
of the dye distribution in the object and the second is the rrstray 1ight
errorrrwhich is due to extraneous light waves entering the photoreceive r.
In principle the 'rdistributional errorrr can be elirninated by
using a wavelength of light at which the stained object is so highly
transparent that all dye I'particlesrrare exposed to the sarne flux of
photons and there is no effective shadowing. Thus the arnount of light
absorbed out of a given light flux will be proportional to the nurnber
r5
of dye rrparticles" present. In effect the two-wavelength rnethod
utilizes such a principle, correcting for the "distributional error, "
Patau ll95?l has dernonstrated that this error (6)
v,
is
19
9Yg4r
equal
to
vv II
.,
Y'
where y is an approxirnation of the true amount of y, dye. W'ith increasing transparency (brought about by changes of the wavelengths)
6 will converge towards
zero, computation of this formula also
demonstrates that the field area (B) has no influence on y, Analytical
treatrnent of the rtdistributional errorrr (6) appears to be a hopeless
undertaking in view of the infinite nurnber of possible dye distribu-
tions. However, through the use of nuclear models that depict
selected and varied chrornatin distributions it can be demonstrated
that the two-wavelength method may have a rtdistributional error, of
less than two percent.
Stray light or the scattering of light into the rneasured bearn
by the surrounding tissues [referred to as the Schwarzschild-Villiger
effect and discussed by Naora ( 195 t ; l95Z; t 955)] was reduced
by closing down the vertical slits of the rnonochrornator to 0.? rnfil so
that the illurninated field was a few diarneters larger than the nucleus
to be rneasured. By ernploying wavelengths sornewhat off the Feulgen
absorption peak errors arising frorn high extinction values were
further rninirnized.
Other errors that rnay occur in photornetry rnay take the
forrn of non-specific light loss, rneasurernent of incornplete nuclei,
T7
and non-linearity of phototube response. Non-specific light loss rnay
occur due to the scattering of light out of the measured beam by
colloidal particles and by large bodies of different refractive indices
frorn the surrounding rnedium, The degree of this light loss may be
deterrnined by rneasuring undyed tissue otherwise identical to the
test object. Swift (1950) has reported that such rneasurernents rnay
yield negative absorption. In this case correction for such nonspecific light was found unnecessary.
Measurernent of incornplete nuclei was avoided by the rnethod
previously explained. It is unlikely that any incornplete nuclei were
rneasured.
Linearity of phototube response was checked before experirnental rneasurernents were rnade. As discussed earlier, linearity
was obtained at focal ptrane (leveI of the phototube) diameters of two
to ten rnillirneters. Magnifications were adjusted for rneasurernents
within these lirnits.
Analysis of Results
In the data presented, the rneasurernents forrn curves essentially sirnilar to those usually obtained in Feulgen photornetric
studies of nuclei, NucLear DNA classes indicated by the curves suggest a successive doubling of chrornatin rnaterial. Class II nuclei
rnay be considered as interphasic doubling of DNA prior to chrornatid
t8
separation were it not for the fact that rnitotic figures do not occur
in the exarnined tissue sections. Therefore it is likely that these
nuclei are definitively tetraploid or in an equivalent polytene condition.
Variation within classes may be accounted for by the overall
error involved in the cytophotometric method. The exact amount of
this error can not be assessed with certainty. Patau (l95zl calculates
that under the proper conditions this error rnay be kept below three
percent of the true dye content. Sorne of the variations rnay be
accounted
for by incomplete synthesis of DNA during endomitosis or
be attributed to aneuploidy. W'ithout actual chromosome counts
aneuploidy can not be estabLished with confidence, but it has been
chrornosornally dernonstrated in vertebrate tissues by Hsu and
Pornerat (1953), 'Walker and Boothroyd (I954), and others.
Low polyploid frequencies are found in the blood ceIIs,
hypoderrnis, ventriculus, and fat
ce11s
in contrast to the anterior
silk gland and proventriculus where these freguencies are high.
The
low frequencies, however, rnay be largely spurious and due to rnitotic
cycling,
The developrnent of f.at cells and their histolysis during insect
growth and rnetarno rpho si s has been discussed by Nakahara (1917),
Bishop (1922; L9Z3l, and Perez (1910); a general discussion is given
by Wigglesworth ( I953). As an insect approache s rnetarnorphosis fat
t9
celI nuclei becorne intensely active. The nuclear surface rnay be increased by arnitosis (Nakahara, 1917) or by the elongation or
branching of the nucleus (Bishop, tgZZ; 1g}3l. These phurro*"na may
be accompanied by the breakdown of the nuclear mernbrane and the
discharge of basophilic Feulgen-negative granules frorn the nucleus
(WiggLesworth, 1953), Exarnination of Plate IV will show the fat
cell nucleus undergoing several of these changes prior to pupation.
Lower DNA values tend to indicate the destruction or rernoval of
chrornatin rnaterial as cited by Bishop (l9ZZ; I9Z3l and Paillot and
Noel ll937l. However, to establish this change of nuclear content
unequivocally a photometric study would be necessary which would
follow the course of developrnent and histolysis in the fat
ce11s.
The proventriculus and anterior silk gland nuclei present
rather interesting cases. In view of the great number of nuclei with
apparent intermediate DNA values, it rnay be that DNA replication
or chrornatin duplication is not who11y euploid. Similar cases
have
been observed by Truong and Dornfeld ( 1955) and Leuchtenberger and
Schrader (1952). The possibility also exists that the excessive nurn-
ber of interrnediate DNA values reflect a high rate of endornitotic
activity thus resulting in a picture sirnilar to that obtained when
exarnining tissues with high rnitotic rates.
'Work by Breuer and Pavan (1955), Beerrnann
and Bahr lL954l,
and Mechelke (l95zl has dernonstrated that at certain stages of
z0
dipteran 1arva1 development specific bands of polytene chromosomes
show enlargernents called chrornosornal puff s
or Balbiani rings.
Investigations with the use of tritiated thlrrnidine by Ficq and Pavan
(1957 ) have shown
that there is a build-up of DNA followed by the
synthesis of RNA at these chrornosornal sites. The possibility of the
same
or a sirnilar phenornenon being responsible for the nurnber of
interrnediate DNA values in the larvae of Galleria mellonella can not
be
ignored. It is conceivable that during developrnent
sorrre genes
or groups of genes have enhanced physiological activities and are
engaged
in excessive replication, Thus different DNA values rnay be
obtained frorn ceIIs of the same tissue type depending on variables in
the tirnes and degrees of their activity.
Nuclear and cytoplasrnic changes accornpanying the various
phases
of secretory activity in the silk glands of the larvae of
Galleria rnel1onella have been described by'ffu (1930). It was suggested, although without certitude, that the anterior portion has 1itt1e
or no role in secretory activity. Intense activity, however, was
dernonstrated for the rniddle and posterior portions of the gland. The
high DNA values found in this study to occur in the anterior portion
of the silk gland nevertheless suggest sorne kind of synthetic activity.
The highly branched and attenuated nuclei of the rniddle and posterior
regions were, unfortunateJ.y, not amena.bl.e to DNA rneasurernent, but
it is quite obvious that they represent degrees of ploidy far in excess
Z1
of those in the anterior region (cf. Plate VI). It rnay be pertinent
to cornrnent that sirnilar nuclei frorn salivary glands of the heteropteran water- strider, Gerris lateralis, were found, by *-chrornosome
count, to be around Z}49-ploid (Geitler, 1938).
2Z
SUMMARY
1) Relative DNA amounts were lrreasured photometrically
individual Feulgen-stained nuclei of hypoderrnis, ventriculus,
ce11s, blood ce11s,
on
f.at
anterior silk g1and, and proventriculus of the last
instar larvae of Galleria mellonella Linnaeus. Graphs of the deterrnined DNA values indicated the presence of nuclear classes falling
into, and in sorne cases between, polyploid ratios,
Z) Seventy-five hypodermis nuclei were rrreasured in five
larvae. The rnajority of DNA values
fe11
into the first (or diploid)
class while a few values, 6.6 percent of the total nurnber of deter-
rninations, constituted class II (tetraploid);
3) The nurnber of ventriculus nuclei examined was ?3.
The
majority of the DNA values fell into class II while a few (6.8 percent)
were considered as class I nuclei.
4l The nurnber of DNA values deterrnined for the fat cell
nuclei was 76. The bulk of the DNA values feI1 into class I. The
rnean value of the class
I nucLei here was slightly lower than that ex-
perienced in the ventricuLus and hypoderrnis (I.47 as corrrpared to
2,0 and L.94, respectively). This lower value was attributed to the
presence of histolysis prior to rnetarnorphosis. CIass II and class
III values were present aLso, rnaking up tZ percent and 5. Z percent,
re spectively.
z3
5) Only 47 blood cell nuclei were rneasured,
due to the
difficulty in finding these cells in tissue sections. Randorn rneasurements were made without distinction of possible cell types. The
majority of the DNA values fell into the first class while l4 percent
and 6.4 percent of the total determinations were considered to be
classes II and III, respectively.
5) Due to their large size and over-lapping disposition only
52 proventriculus nuclei could be
rneasured. A broad spectrurn of
DNA values was found ranging frorn 23 to L43, with a great nurnber of
intermediate values and no evidence of unirnodal curves. This spec-
trum encompassed classes rv through vlr. various explanations
were proposed to account for this phenomenon.
7l The nurnber of anterior silk gland nuclei rneasured was 54.
Again a broad spectrurn of DNA values was found ranging frorn 13 to
201.5, with a great nurnber of interrnediate values and no evidence
of unirnodal curves. The highly branched, attenuated, and overlapping nuclei of the rniddle region of the silk gland were unrneas-
urable, but their DNA values very likely extend to class XI (2048ploid).
8) The sources of error in the
rrlea surements we
re
analy zed.
Various interpretations were given for variations in DNA values
which included endornitosis, definitive polyploidy, aneuploidy, dif-
ferential gene replication, etc. In the case of the silk gland and the
z4
proventriculus a strong relationship between DNA content and cell
activity appears to exist. The nature of this relationship needs
separate study.
z5
BIB LIOGRAPHY
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1959,
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Garcia, A. M, Studies on DNA in leucocytes and related cells of
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z6
Geitler, L, Ubur den Bau des Ruhekerns rnit besonderer
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Lison, L. and J. Pasteels. 6tudes histophotometrique sur 1a teneur
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Mechelke, F. Die Entstehung der polyploiden Zellkerne des
Antherentapetums bei Antirrhinurn rnajus L. Chrornosorna
246-295.
5:
1952.
Mendelsohn, M. L. The two-wavelength rnethod of rnicrospectrophotornetry- I. A microspectrophotometer and tests on rnodel
systerns. Journal of Biophysical and Biochernical CytoLogy 4:
407
-4r4.
rg58a.
Mendelsohn, M. L. The two-wavelength rnethod of microspectrophotornetry. II. A set of tables to facilitate the calculations.
Journal of Biophysical and Biochernical Cytology 4:4I5-424.
I
958b.
z7
Mendelsohn, M. L. The two-wavelength rnethod of rnicrospectrophotornetry. III. An extension based on photographic color
transparencies. Journal of Biophysical and Biochernical
Cytology 4:425-45I. 1958c.
Mendelsohn, M. L. The two-wavelength rnethod of rnicrospectrophotornetry. IV. A new solution. Journal of Biophysical and
Biochernical Cytology 1t:509-513. 1961.
Merriam, R. W. and H. Ris, Size and DNA content of nuclei in
various tissues of rna1e, fernale, and worker honey bees.
Chrornosorna 6:522-538. 1954.
Mirsky, A. E. and H. Ris. Variable and constant components of
chrornosomes. Nature 163:666-667.
1949.
Nakshara, W. Studies of arnitosis: Its physiological relations in
the adipose cells of insects, and its probable significance.
Journal of Morphology 30:483 -525. 19L7.
Naora, H. Microspectrophotornetry and cytochernical analysis of
nucleic acids. Science lL4zZ79. 1951 .
Naora, H. Schwarzschild-Villiger effect in rnicrospectrophotornetry.
Science lI5zZ48-249. 1952.
Naora, H. Microspectrophotornetry of cell nucleus stained by
Feulgen reaction. I. Microspectrophotornetric apparatus
without Schwarzschild-Villiger effect. Experirnental Cell
Research 82259-278, 1955.
Novikoff, A. B. Histochernical and cytochemical staining rnethods.
In: Analytical Cytology, ed. R. C. Mellors. McGraw-HiII
Book Co., Inc. , L955. p. L-63.
Ornstein, L. The distributional error in rnicrospectrophotornetry
of ce1ls. I-aboratory Investigation lzZ50-262. 1952.
Overend, W. G. Desoxy-sugars. Part XIII. Sorne observations of
the Feulgen nucleal reaction. Journal of the Chernical Society,
1950, p. 2769-2774.
Overend, W. G. and M. Stacey. Mechanisrn of the Feulgen nucleal
reaction. Nature 163:538 -540. 1949.
28
Paillot, A. and R. Noel. Nouvelles recherches sur I'histophysiologie
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Acad6rnie des Sciences (Paris) 205: t 09S- 1095. lgll
.
Patau, K. Absorption rnicrophotornetry of irregular-shaped objects.
Chromosoma 52341-362, 1952.
P€rez, C. Recherches histologiques sur le rndtarnorphose des
Muscides: Calliphora erythrocephala Mg. Archives de Zoologie
Exp6rimentale et Gdn6rale 44zl-274. 1910.
Pollister, A. W. and M. J. Moses. A sirnplified apparatus for
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Physiolo gy 32:567 -57 7 . 1949 .
Pollister, A. ltr. and L. Ornstein. Cytophotornetric analysis in the
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McGraw-HiIl Book Co. , Inc. , I955, p. 1-71.
Pollister, A. W. and H. Ris. Nucleoprotein deterrnination in
cytological preparations. Cold Spring Harbor Syrnposia on
Quantitative Biolo gy 12:147 -157 . 1947 .
Ris, H. and A. E. Mirsky. Quantitative cytochemical determinations
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Stich, H, F. and J. M. Naylor. Variation of desoxyribonucleic acid
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Stowell, R. E. Feulgen reaction for thyrnonucleic acid. Stain
Technolo gy
20:45-58.
1945.
Swift, H. H. The desoxyribose nucleic acid content of anirnal nuclei.
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29
Tamm, C, , M. E. Hodes, and E. Chargaff. The forrnation of
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534. r930_,-
APPENDIX
30
DERIVATION OF THE PHOTOMETRIC I'ORMULA y = KBLTC'i,
, have been selected so that the test object yields
extinctions E(\,) = 2E(r ,). If these measurernents are absolutely
}.
,
and tr
accurate, if there is not stray 1ight, and if the test object is a uni-
forrnly stained layer, then k, = Zki (where k is the extinction coefficient). Let, instead, the true ratio of extinction coefficients
kr:ka = I:Z(1+A).
parts: A= Ol *
OZ
be
srnall unknown error (a) is composed of three
* 43. A, is the sarnpling er.ror of the galvano-
rneter readings and can be rnade very srnall by taking a sufficient
nurnber of repeated readings. A, is approximately the difference of
the relative stray light errors cornrnitted in rneasurernent of E(\
,)
and E(\ r). These are the stray iight errors of the conventional
method. A, is caused by a non-uniform dye distribution within
the
test object. Distributional errors are caused by the existence of
different light paths along which photons have different probabilities
of being absorbed.
It is assurned that an object to be deterrnined is uniforrnly
absorbing and that the illurninating bearn consists of para1le1 light
with norrnal incidence then the following rnay be dernonstrated. Let
"r.
b. the unknown transrnission of the object at \r(v = l,Z). Let
'i'This derivation is condensed frorn Patau (1952).
31
F
S B be the unknown
total area of the object. The flux through F
in the absence of the object is F/B
T-T
^vl
B
-r'
B
I.rO; that
with the object is
'v0
Hence:
1r. = 1
B
- F Lr,
(1)
\ rr, the extinci;ion E.,, = - 1og 'Tr.. Because of k, = 2(1 + A)kl it is E, = Z(1 + A)E, anci 'r - ',2(l+A).
r,
The object has, at
This in conjunctionwithequation (I) leads to
r-1_1I+2a
e =LZ - I +'1.
LlI
I -',rl
Introducing
q(a)
=
I
I - 'fl
- 'i
(zl
rl+za
there is obtain"d 'it _ (o _ l)o (a). Hence:
Et - log I
O,
Equation (1) yields F = B",
*;
F=BL,
Because of Y =
I
\
- log q (A)
and.
(4)
I -(O- 1)n (a)
FEt, Equations (3) and (4) cornbine
y = KBLI
1-(O- 1)n (a)
(3)
to
lh#-hq(a)l
For A - 0, that is q (A) = l, equation (5) becornes
Y=
KBLIC
(5)
PI,ATE I
Dorsal aspect of dissected last instar larva of Galleria
rne11one1la L. (x7), Fat bodies have been
""-offi?greater
rno rpholo gi ca1 detail.
ABBREVIATIONS
ASG.
Anterior region of the silk gland
C
Crop
Co.
Colon
I
Ileurn
M.
Malphigian tubule
MSG
. Mid-region of the silk gland
PSG.
. Posterior region of the silk gland
Pv.
Proventriculus
R
Rectum
Sal G
Salivary gland
T
Tracheae
V
Ventriculus
33
iI
a
,
I
,,
t
I
,
a
I
t
PLATE II
Cross section of last instar larva of Galleria mellonella L.
through the preventriculus. (x30)
ABBREVIATIONS
ASG.
Anterior region of the silk gland
v
Cuticle
FB
Fat body
H
Hypodermis
MB
Musc1e band
Pv
Proventriculus
Sal G
Salivary gland
T
Trachea
NC
Nerve cord
JJ
t.\
i-..
\.i....
'j.t'|..
"'{'$'i
"{t\
'fill
'Tilil
lr.li
t:ii
*l:i
,fiil
'/iit
,HI
ili.'
tr!t
H/
i,:.r;
PI,ATE III
Cross section of last instar larva of Galleria rne11onella L.
through the ventriculus. (x30)
ABBREVIATIONS
C.
Cuticle
FB
Fat body
H
Hypoderrnis
MB
Muscle band
MSG
Mid-region of the silk gland
T
. Trachea
V
Ventriculus
NC
Nerve cord
37
I
I
i
PI,ATE IV
Figure l. Blood smear stained with Wrightts stain.
Magnification 1000x.
Figure 2. Cuticle (C) and hypodermis nuclei (H). f'eulgen
stain, Section thickness 10 p. Magnification
I 000x.
Figure 3, Fat celI nuclei stained with Feulgen reagent.
' Section thickness l2 p. Magnification I000x.
39
rr .?ir
,i
n:
.e,
f*-
{*
i-.
..
P]-ATE V
Figure 4. Ventriculus nuclei stained with Feulgen reagent.
Section thickness l0 p. Magnification 1000x.
Figure 5. Nuclei of the. proventriculus stained with Feulgen
reagent. Section thickness 25 p. Magnification
200x.
4l
PI,ATE VI
Figure 6. Squash preparation of mid-region of the silk gland
nuclei stained with aceto-orcein. Magnification
2
00x.
Figure 7. Nuclei of the anterior region of the silk gland
(left) and salivary gland (right) stained with
Feulgen reagent. Section thickness 25 p,
Magnification 1000x.
43
PI-ATE VII
Cytospectrophotornetric apparatus used in this study. Description is given in the text.
ABBREVIATIONS
L.
Light source (enclosed in black box)
M.
Monochrornator
P.
Phototube
PS
Power supply
R.
Microarnmeter
T.
Telescope
45
L
6/14/64
#
B
3.8
Irr
Iro
.505
.563
.505
. s63
1.010
6l
=49omp
T1 =
Fat Cell
L.
PI,ATE VIII
L =514mp
rtt/rto
L1 =
1-Tt
t 2-
lzz
lzo
.473
.8970
. 1030
t?6
.#8
s70
.568
.941
1.138
Tzz/Tzo
Lz=
1-T
2
Q=
LzlLt
c
Y =KBL
1
.
,8?59
L73t
1. 681
1.205
4.363
.82t3
1787
L.7LO
1.181
4.3?9
M:
4. 351
I
(O
t
Ol
FA
35. 1(
.503
.500
.560
.560
5.9:
1. OO3
t.t20
3.8
. s30
.565
. 533
.565
1. 063
t. t28
.530
.530
.565
.563
Ol
5.93 1. 060
.465
.
.46s
.568
. 933
1. 136
s68
.578
513
.578
1.o23
1. 156
s08
s08
. s75
t,L?8
1.016
1. 150
.523
.56s
5(,()
.5 7E
.523
.565
s03
.
r.o#
1.130
.520
.563
.523
.565
5.93 1.O43
L.t?3
I
I
1045
.510
ar,
(O
.895s
15 t6
pa
.9424
,0576
.
.
.9397
.0602
.
.575
.8849
.1151
1. 998
1.005
1.977
.883s
1165
1. 935
1. 031
2.
M:
3.8
$
.9257
.0743
.
1. @3
s78
r82
2.O79
.8676
1324
1,782
L.129
3,263
.8699
1 301
t.7?s
1.166
3.091
1.156
I
(O
I
Or
ra
35. 16
.9246
.0754
.503
.578
.5@
.575
1. OO3
1.153
M:
SAMPLE DATA SHEET
3. t77
'f
6
47
P
I.AT E IX
z4
3Z
30
z8
z6
?4
o
o
t
z
l+{
o
t{
o
zz
2A
l8
,o
) l6
z
E
L4
tz
l0
8
6
Relative Arnount of DNA
Distribution of DNA in the hypodermis nuclei of five larvae.
48
PI-ATE X
La.rva A
o
o6
z
t4
t{
O)
"oL
(
l,arVa
C
li
)
z
6
4
z
Irarva D
6
4
z
Relative Arnount of DNA
Distribution of DNA in the hypodermis nuclei of each larva.
49
P
]-AT E XI
34
32
30
z8
z6
z4
o
U
)
z
q{
??
o ?0
Ii
o
,o
l8
E
t
z l6
t4
LZ
10
8
6
4
z
Relative Arnount of DNA
Distribution of DNA in the ventriculus nuclei of five larvae.
50
PI-ATE
6
4
La.rva
z
6
4
Larva B
z
.il
o
o
Ftb
7
z(+{
o
4
h
.82
Larva
C
Larva
D
E
I
z
4
z
6
4
z
345
6
Relative Arnount of DNA
Distribution of DNA in the ventriculus nuclei of each larva,
5I
PI.ATE XIII
34
3Z
30
Z8
2,6
2,4
.il
0)
ZZ
U
d
z z0
rH
o
H
ts
z
1B
l6
t4
tz
IO
8
6
4
z
t234567
Relative Arnount of DNA
Distribution of DNA in the fat cell nuclei of five larvae.
5Z
PI-ATE XIV
5
4
Larva A
z
6
4
z
6
o
i4
z,+l
o
2
k
po
E
EE
a
4
La.rva D
z
6
4
Larva E
z
t2345678
Relative Amount of DNA
Distribution of DNA in the fat
ce11
nuclei of each larva.
53
PI,ATE XV
34
32
30
z8
z6
z4
'6zz
U
5
zz0
t+a
o
ro 18
p
E 15
a
Z
t4
tz
IO
I
6
4
?
345578
Relative Amount of DNA
Distribution of DNA in the blood cell nuclei of four larvae.
54
PI-ATE
6
4
z
6
4
Z
0)
i6
t
Larva C
z
,*4
o
t{
a)
-oE
J
Z
6
4
z
6
Larva E
4
z
No data available
345
b'78
Relative Arnount of DNA
Distribution of DNA in the blood celI nlrclei. of each larva.
PI-ATE
xvII
q)
o
t
zq{
o
h
q)
p
E
a
zr
30
40
50
60
70
80
g0
100 tto
tz1
130 143
Relative Arnount of DNA
Distribution of DNA in the proventriculus nuclei of five larvae.
(,I
Ltl
PI.ATE XVIII
La.rva A
I-a.rva B
l,arVa
o
F{
o
,
z
(+{
o
C
I
t{
o
La.rva D
.o 3
E
)
z
Larva E
40
50
60
70
80
g0
100
I
IO
LZO
130 I4O
Relative Arnount of DNA
Distribution of DNA in the proventriculus nuclei of each larva,
(rl
o.
PI.ATE XIX
.d
Q)
o
)
Z
r+{
o
t{
0)
.o
E
)zt
Relative Arnount of DNA
Distribution of DNA in the a nterior silk gland nuclei of five larvae.
(rl
-t
PLATE XX
I-a"rva A
,
ll
3
2
I
.d
o
ll lFt-l
-IX tHH
La.rva B
,
fl
I lll
il
llli |
F-lHl
o
r3
z
l,arva
,xz
o
,rl
pc)
rlltl
tl ll
r3
z
z
I
C
tl IHH
l,arva D
ntl tlfl
3
il t, r il
tHH
Larva E
Z
I
30 40 50 60
rlr+1
t43 r6e zol
LZ,
"o
*'ir",r'" .i,1"*', irol*o
Distribution of DNA in the anterior silk gland nuclei of each 1arva.
(rl
o