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Short Views on Insect

Molecular
Biology,
Vol.(1),Biology
2009
Short Views
on Insect
Molecular

Research
ResearchArticle
Article

Vol. (1), 159 173, 2009

Chapter 9

Autophagic programmed cell death in the peripheral fat body

tissues of the silkworm, Bombyx mori L.
Prosothaman Sumithra#, Raman Chandrasekar # and Muthukalingan Krishnan*#
* Biomedical Medical Diagnostic Laboratory, Department of Environmental
Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, India.
#
Insect Molecular Biology Laboratory, Department of Environmental Biotechnology,
School of Environmental Sciences, Bharathidasan University, Tiruchirappalli- 620 024,
Tamilnadu, India.
Abstract
Programmed cell death (PCD) generally occurs within a developmental context in response to physiological stimuli such
as hormones and is dependent on de novo gene expression. Despite the ubiquity of this phenomenon, little is known
about what tells a cell to die, and less still about the physiological and molecular mechanisms that bring about death. One
system that has proven to be very amenable for the study of PCD is the larval peripheral fat body (PF) tissues of the
silkworm, Bombyx mori. Here we assess the two major pathways of PCD via apoptosis and autophagic cell death both by
morphological and molecular methods in the disintegrated larval PF tissues during the larval pupal transformation.
Larval PF tissue began to disintegrate by the process of PCD on day of spinning (S0) and its structural integrity was
completely lost by day one of pupa (P1). PCD in PF was well characterized by chromatin condensation by acridine orange
staining, cytoplasmic budding, giant autophagic vacuoles, the disappearance of the smooth endoplasmic reticulum and
intercellular channels, and the fragmentation of the cytoplasm into membrane-bound bodies by morphological
examination and DNA fragmentation by agarose gel electrophoresis. Western blot analysis of proteasomal subunits
proved that the involvement of proteolytic activity during the PCD of peripheral tissues. Lysosomal participation during
the PCD of PF tissues was investigated by elevated level of marker enzyme acid phosphatase distinctly on the day 1 of
pupal period. Further to investigate the effective role of 20-hydroxyecdysone (20E) on these events, we cultured the larval
PF tissues with or without 20E and analyzed by chromatin condensation via acridine orange staining and DNA laddering
studies.
Key Words : Bombyx mori; peripheral fat body; PCD; 20E; 26S Proteasome; Acid phosphatase
* For correspondence (email: krish14156@yahoo.com)

Overview
1.
2.

Introduction
Materials and Methods
(a) Experimental animals
(b) Detection of chromatin condensation
(c) Tissue preparation for Electron Microscopy
(d) DNA extraction and Electrophoresis
(e) Gel Electrophoresis and Immunoblotting
(f) ACP assay
(g) In vitro culture of fat body tissues

3.

4.
5.
6.

Result
(a) Chormatin Condensation
(b) DNA laddering studies
(c) Lysosomal Acid Phosphatase a marker enzyme
(d) 26S & 20S Proteasome
(e) Morphological Examination
(f) Role of 20E on PCD
Discussion
Acknowledgement
Reference

The article has been scientifically edited by Chandrasekar R.

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1. Introduction
In contrast to vertebrate cells where times arrow tends to lead irreversibly to differentiation, death
and whole-tissue regeneration, insect cells follow a path of make do and mend, with cells regenerating
and changing their function through life. Some cells are irreplaceable, some cells complete their
functions and are then sacrificed, and some cells live a finite lifetime to be replaced by yet another
generation by the process of PCD. Genetic dissection of cell death programs has identified as conserved
elements of core cell-death pathway, as well as an understanding of how these proteins bring about the
destruction of a cell (Buszczak and Segraves 2000). Cells use different pathways for active selfdestruction as reflected by different morphology: while in apoptosis (or type I) nuclear fragmentation
associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic
degradation of cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of
PCD (Bursch, 2004). The most extensively studied category, apoptosis (type I) is characterized by cell
rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation and fragmentation,
nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-bound apoptotic
bodies that are rapidly digested by macrophages or neighboring cells (Kerr et al., 1972; Wyllie et al.,
1980; Clarke 1990; Takayama et al., 2003; Boyce et al., 2004; Danial and Korsmeyer 2004; Festjens et
al., 2004; Lorenzo and Susin 2004; Chandrasekar et al., 2008a & 2008b). Rather, cells may use different
pathways to commit suicide, including autophagic PCD or type II cell death. Its main features are the
appearance of abundant autophagic vacuoles in the cytoplasm, which is accompanied by mitochondrial
dilation and enlargement of the ER and the Golgi apparatus. However, apoptosis and autophagic PCD
are not mutually exclusive phenomena. PCD generally takes place in four steps: the decision to die,
death, engulfment and degeneration (Steller 1995). Indeed, Zakeri et al. (1995) detailed non-apoptotic
types of PCD with diverging molecular symptoms such as a lack of endonuclease activation and changes
in sub-cellular hydrolase traffic, and Bowen et al. (1998) described a number of examples of nonapoptotic vacuolar PCD in a range of invertebrates including insects. More recently, a number of
reviews covering morphological and functional features of autophagic PCD throughout the living world
were published (Clarke 1990; Zakeri et al., 1995; Bursch 2001; Bursch et al., 2004; Chandrasekar et al.,
2008a). The genetic and molecular background of the type II (autophagic) mechanism of PCD is not
well known, although, the morphological and biochemical features of autophagocytosis are characterized
in detail. Cells undergoing autophagic PCD the first changes visible at the electron microscope level
comprise formation of autophagic vacuoles (AV), which gradually degrade cytoplasmic structures.
These observations with dying cells meet well with the current concept of macroautophagy (herein
referred to as autophagy), namely that it ensues through a sequence of events highly conserved from
yeast to humans including : the sequestration of cytoplasmic constituents or certain organelles in a
random manner by so called isolatory membranes and the autophagosomes thus formed, subsequently
fuse with lysosomes forming autophagic vacuoles (AV), inside which the macromolecules of the
engulfed organelles are digested at acidic pH by the lysosomal enzymes (Klionsky and Emr 2000;
Reggiori and Klionskz 2002; Bursch 2004; Chandrasekar 2006; Chandrasekar et al., 2008a).
Ecdysteroids play a central role in the cell death of numerous in insect tissues (Chinzei 1975;
Truman and Schwartz 1984, Dai and Gilbert 1999; Terashima et al., 2000; Tsuzuki et al., 2001; Halaby
et al., 2003). During metamorphosis, ecdysteroids display different effects on cell death, i.e., either an
increasing or decreasing ecdysteroid titer can elicit PCD, depending on the developmental stage (Truman
and Schwartz 1984; Kimura and Truman 1990; Truman et al., 1992). Evidence is now mounting that
intracellular proteolysis is involved in PCD (Martin and Green, 1995). One of the major difficulties in
examining the biochemical pathways that regulate this process is that in most cases, condemned cells are
interspersed among healthy ones, making the observed biochemical changes difficult to interpret.
Ubiquitin mediated proteolysis is required not only for protein turnover during homeostasis and stress,
but also during the PCD of some cells (Muller and Schwartz 1995). In the muscle cells of Manduca sexta
committed to die an increase in the expression of polyubiquitin was described which has been shown as
an essential mediator of proteolysis (Myer and Schwartz 1996). Ubiquitin functions as a posttranslational adduct to proteins, where its best characterized role is to target proteins for proteolysis via
an ATP/ubiquitin-dependent proteinase, which contains the multicatalytic proteinase (MCP; proteasome)
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as an integral component. This complex degrades the target substrate and returns intact ubiquitin protein
to the cytoplasm (Rechsteiner et al., 1993).
The autophagic degradation of larval organs during metamorphosis can be regarded as a form of
PCD. The developmental process of different larval tissues during metamorphic transformation showed
that tissues such as the silk gland and gut are completely histolyzed (Shiba et al., 2001; Lee et al., 2002;
Uhlirova et al., 2003), while other tissues such as fat body undergo reorganization with histolysis (Rizki
1978; Rabossi et al., 2004) and predetermined imaginal tissues differentiate and grow into organs and
external structures (Fristrom and Chihara 1978; Uhlirova et al., 2003). Typically, PCD occurs in a
temporal window of opportunity following a molt. The intersegmental muscles that facilitate adult
eclosion degenerate in the first few days of adult life (Lockshin and Williams 1965; Schwartz and
Truman 1982; Schwartz, 1992). The motoneurons innervating these muscles undergo PCD concurrently
(Truman 1983). Similarly, the prothoracic gland degenerates following production of the ecdysteroids
that will stimulate adult differentiation (Ozeki 1966; Dai and Gilbert 1997).
Fat body in insects is one of the eukaryotic systems which epitomizes the complexity of gene
control as displays an orchestral expression of genes in response to intra and extra molecular cues. Our
previous studies on B. mori (Vanishree 1998) and Amsacta albistriga (Chandrasekar 2006;
Chandrasekar, et al., 2008a, 2008b) reported that PF tissues located near to the cuticle region reaches
high abundance during mid-larval period and begins to be disintegrated during the larval pupal
transformation while the ecdysone titer is high. This protrudes the amenable way at first time to
investigate the molecular studies on PCD in the larval PF tissues of the silkworm, B. mori, which has
spawned and provided an opportunity to address a number of intriguing questions surrounding apoptosis
in insects.
2. Materials and Methods
a. Experimental animals
Eggs of a hybrid of a multivoltine strain of B. mori (local Tamil Nadu White) and a bivoltine
strain, NB2D4, were purchased from the local Government Grainage, Tiruchirappalli, India, and
maintained at a temperature of 272C and 755% relative humidity. Larvae that hatched out were fed
with freshly chopped, tender leaves of mulberry, MR2 variety, until the third instar and were given
coarse leaves until the end of the fifth stadium. After the fourth molt, larvae were sexed and those
weighing 0.8g were selected for the experiments as described previsouly by Chandrasekar et al. (2007).
The life span of the silkworms under these conditions is 30-32 days, with the final fifth stadium, lasting
for 6 days. Fifth stadium larvae cease feeding to begin cocoon spinning on day 7 and the larval pupal
ecdysis occurs on day 8. For convenience, the day of spinning was termed by S0 and the unsclerotized
soft pupal stage day P0. The pre-pupal period lasts for 3 days (S0-S2) and the pupal period for 7 days (P1P7).
b. Detection of chromatin condensation
Acridine orange (Sigma-Aldrich) stain that is commonly used to reveal the condensed chromatin
that is characteristic of apoptosis (Hopwood et al., 2001). Using this method of detection, when viewed
under appropriate filters, apoptotic cells are highly fluorescent making them easily identifiable from nonapoptotic cells. The larvae were chilled on ice for 2-3 min before dissection in insect ringer solution.
The larval PF tissues were collected throughout the V instar, spinning stage and the pupal stage, placed
on a glass slide and tapped gently with a brass rod to disperse the tissue. Acridine orange (50M in
insect ringer solution) stain was added and the tissue was viewed immediately under a fluorescence
microscope.

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c. Tissue preparation for Electron Microscopy

According to Muller et al. (2004) insects were inflated with 4% paraformaldehyde, 0.5%
glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for 5 min at room temperature. Larval PF lobes (S2,
P0, P1) were dissected fixed under the fixative solutions for 1 h at 4C. After rinsing with 0.1M
cacodylate buffer, the fat body pieces were post-fixed for 1 h in 0.5% osmium tetroxide. After
dehydration in a graded series of ethanol (50% ethanol, 15 min; 70% ethanol, 25 min; 90% ethanol, 30
min; 100% ethanol two times each for 30 min) and then in propylene dioxide (10 min) at room
temperature, the tissue was embedded in Araldite (Araldite: Propyele oxide, 1:1, overnight). The
specimens were thermally cured at 50C for two days. Thin sections of 60nm-90nm were cut with a
diamond knife. Sections were picked in copper grids and stained with uranyl acetate for 8 min, lead
citrate for 2 min and then blotted dry. Preparations were analyzed with the Philips Electron Microscope
(Philips 410 LS, Austria) at an acceleration voltage of 75 kV.
d. DNA extraction and Electrophoresis
Genomic DNA was extracted from larval PF tissues throughout the V instar larval, spinning and
pupal stages by adopting the Sambrook et al. (1989) methodology with slight modification. PF tissues of
B. mori were collected and homogenized with 400 - 800l of TEN 9 buffer (50mM TrisCl, 100mM
EDTA, 200mM NaCl) and the homogenate was centrifuged at 12000 rpm at 4C for 10-15 min. Then,
equal volume of 20% SDS was added and the tubes were inverted few times and incubated at room
temperature for 30 min. The lysate was treated with 5l proteinase K (10mg/ml) for 4 h at 60C. At end
of the incubation, phenol and chloroform in the ratio of 1:1 was added and the tubes were inverted
several times and refrigerated for 15 -30 min. Then, the tubes were centrifuged at 15,000 rpm at 4C for
10-15 min and the aqueous phase was collected. To this added equal volume of 0.3M sodium acetate
and 3 volume of 70% ice cold ethanol. The tubes were inverted gently and kept at -20C over night.
The tubes were centrifuges at 15,000 rpm for 15 min at 4C and the pellet was washed with 70% ice cold
ethanol. Dried pellets were dissolved in 200l of TE buffer (10mM Tris HCl, 1mM EDTA). To this 5l
of DNase-free RNase (2mg/ml) was added to the samples and digestion proceeded for 30 min at 37C.
After washing with 70% ethanol the pellet were dissolved in 50l of TE buffer and stored at -85C.
DNA was subjected to electrophoresis in 1.2% agarose gel and was visualized by ethidium bromide
staining.
e. Gel Electrophoresis and Immunoblotting
PF tissues were homogenized in ice cold 20mM Tris HCl, 50mM NaCl, pH 7.5 and centrifuged at
12,000 rpm at 4C for 15 min. The supernatant was mixed with loading buffer and boiled for 5 min.
20g of total protein was subjected to electrophoresis on 12% sodium dodecyl sulphate-polyacrylamide
gels. Proteins were transferred to nitrocellulose paper for 30 min at 30 V according to the procedure of
Towbin et al. (1979). Following transfer, membranes were blocked in transfer buffer saline (20mM TrisHCl, 0.5M NaCl, pH 7.5) containing 3% non-fat dried milk for 1 h, at 20C. The blocked membranes
were then incubated in the presence of rabbit anti- ATPase subunit (47kDa) and 20S regulatory subunit
(29kDA) primary antibodies in TTBS (20mM Tris-HCl, 0.5M NaCl, 0.05% Tween 20, pH 7.5)
containing 2% non fat dried milk overnight at 4C. Antibody binding was visualized by 4-chloro-1naphthol/ hydrogen peroxide liquid chromogenic substrate using anti-rabbit secondary antibodies labeled
with hydrogen peroxidase .
f. ACP assay
The enzyme assay was carried out according to the method of Henrickson and Clever (1972). The
reaction mixture contained 150mM sodium acetate buffer (pH 5.0) and 20g of tissue homogenate
proteins. It was incubated at 37C for 10 min to exclude glucose-6-phosphatase activity (Beaufay et al.,
1954). The reaction was initiated by the addition of 5mol of substrate, p-nitrophenyl bisodium
phosphate (Sigma) to the assay mixture followed by incubation for 1 h at 37C. The reaction was
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terminated by the addition of 0.5ml of 0.1N NaOH, and the color developed was measured at 420nm
against substrate blank. The p-nitrophenol was used for the preparation of standard curve. The activity
of the enzyme was expressed as nmol of p-nitrophenol released /h/g of fat body protein.
g. In vitro culture of fat body tissues
To study the effect of 20-hydroxyecdysone on PCD of the PF tissues, (at S2 stage); different
concentration (1, 2, 3, 4 and 5g) of 20E was added to 1ml of Graces insect cell culture medium and
incubated for desired timing at 25C. Ethanol equivalents were also added to the control medium. The
effects of 20E on the PCD of the cultured tissues were determined by Acridine orange staining and DNA
laddering studies between 6 h intervals.
3. Results
Traditionally, peripheral and perivisceral fat body tissues were considered as one multifunctional
organ and frequently compared to mammalian liver and adipose tissue (Keeley 1985; Locke 2003). Our
previous studies on B. mori (Vanishree et al., 2005) and A. albistriga (Chandrasekar et al., 2008b)
clearly indicate the existence of functional differences between peripheral and perivisceral fat body
tissues, while PF tissue is predominantly responsible of biosynthetic activity, whereas the perivisceral fat
body is specialized as a storage organ. After the synthesis and sequestration of storage protein,
respective fat body cells undergo PCD (Chandrasekar et al., 2008a).

a.
b.
c.
d.
e.

S0 :
S1 :
S2 :
P0 :
P1:

day zero of spinning stage

day one of spinning stage
day two of spinning stage
day zero of pupal stage
day one of pupal stage

Fig.1. Fluorescent microscopic images of larval peripheral tissues stained with acridine orange showing the
onset of programmed cell death by the pattern of chromatin condensation in dying tissues. The live
tissues emit bright green fluorescence whereas the dying tissues emit bright orange fluorescence showing
the chromatin condensation.

(a). Chromatin Condensation

Acridine orange is one of the several nuclei acid stains routinely used to reveal the condensed
chromatin characteristic of apoptotic cells (Mc Gahon et al., 1995; Longthorne and Williams 1997;
Hopwood et al., 200l). Using this method of detection, and when viewed under blue filters, apoptotic
cells are highly fluorescent making them easily identifiable from non-apoptotic cells. The live cells emit
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an intense green fluorescence whereas the dead cells display an intense dark orange fluorescence. The
larval PF from day one larval (V1) to day one pupal (P1) tissues were freshly collected, stained with
acridine orange and viewed under fluorescence microscope using appropriate filter. An important
outcome of this study is that the larval peripheral tissues underwent PCD during the day one of pupal
period by emitting dark red fluorescence showing the presence of chromatin condensation (Fig. 1f) while
larval pupal transformation stages (V6, S0, S1, S2, P0) (Fig. 1a-e) emitted a bright green fluorescence
indicating the presence of active live cells.
Fig.2. Electrophoretic detection of DNA fragmentation in the total
genomic DNA isolated from larval peripheral fat body tissues of
the last larval instar. Strong DNA fragmentation with appearance
of low molecular weight DNA fragments can be observed during
the larval pupal transformation prominently on day one of
pupal period.
V2 V6 :
S0 S 2 :
P0 P 1 :

day two to day six of last larval instar

day zero to day two of spinning stage
day zero to day one of pupal stage

(b). DNA laddering studies

To examine the DNA fragmentation, genomic DNA was extracted and electrophoresed on 2%
agarose and examined under UV light. Clear DNA laddering pattern of low-molecular weight fragments
<1kb were observed in the disintegrated larval PF tissues during larval pupal transformation
significantly on day one of the pupal stage (Fig. 2). It is important to note that the larval peripheral
tissues lost its synthetic activity and underwent cell death in a programmed pattern.

Lysosomal activation is an integral part

of apoptosis in some systems, including insect
tissues during metamorphosis and degenerative
mammalian tissues (Halaby et al., 2003).
Lysosomal acid phosphatase is the ideal
enzyme for PCD and was estimated by
adopting the Hendrickson and Clever (1972)
methodology in peripheral tissues (V1 to P1) as
reflection of the lysosomal activity which in
turn indicates the status of the cell. The
present work lays the ground work for the
involvement of acid phosphatase during
autophagic degradation of disintegrated
peripheral tissues. Our current observation
demonstrates that the PF lysosomal acid
phosphatase activity increased gradually and
reached maximum on day one of the pupal
stage (Figure 3 and 4). On the other hand, the
enzyme activity was also analyzed in
polyacrylamide gels, which showed a
significant elevation of enzyme activity exactly
on the day one of pupal stage (P1) (Fig. 4)
where the complete disintegration of larval
peripheral fat tissues takes place.
164

Enzyme activities (nmoles of PNP

released /h/mg of fat body protein)

(c). Lysosomal Acid Phosphatase a marker enzyme

0.06
0.05
0.04
0.03
0.02
0.01
0
V1

V2

V3

V4

V5

V6

S0

S1

S2

P0

P1

Stages

Fig. 3 Graphical representation of lysosomal acid

phosphatase activity in the larval peripheral fat body
tissues in the B. mori showing the elevated level of
enzyme activity in the day one of pupal stage. The
enzyme activity is expressed in nmole of
pnitrophenol phosphate released per hour per mg of
fat body protein. The standard deviation and
standard error are from 5 experimental replicates.
V1 V6 :
day one to day six of last larval instar
S0 S2 : day zero to day 2 of spinning stage
P0 P1 : day zero to day one of pupal stage

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Fig. 4 Polyacrylamide Gel Electrophoretic pattern of

lysosomal acid phosphatase activity in the larval
peripheral fat body tissues of B. mori showing the
elevated level of enzyme activity on day one of pupal
stage. After electrophoresis the gel was incubated
with substrate solution containing acetate buffer (pH
5.0), sodium chloride, 10% Magnesium chloride,
sodium napthyl phosphate, fast blue RR salt stain
for half an hour in dark Purple color bands were
visualized by adopting the method of Vallejos, (1983).
V3 V6 : day three to day six of last larval instar
S0 S2 : day zero to day 2 of spinning stage
P0 P1 : day zero to day one of pupal stage

(d). 26S & 20S Proteasome

Total protein homogenates prepared from the PF of the larvae of B. mori were collected from each
day of the 5th larval stages and subjected to 12% SDS- PAGE (Fig. 5 a). The presence of the 26S
proteasome in the larval peripheral fat tissues throughout the V instar stages was analyzed by Western
blot technique using MS73 subunit of 26S proteasome antibody which recognizes an ATPase subunit of
the 26S proteasome regulatory cap. One distinct band with an apparent molecular mass of 47kDa was
identified in the Western blot (Fig. 5b) analysis. This protein was not detected at the last larval instar
(V4-V6) but increasing amounts of MS73 was started appearing on day of the spinning stage (S0) and
reached maximum on day one of pupal stage, indicating involvement of this proteolytic activity during
the PCD of larval PF tissues which occurs on day one of pupal stage (Fig.5a)

Fig.5. Protein profile (a) and immunodetection of proteasome subunits in the larval peripheral fat body
tissues of B. mori subjected to 12% SDS PAGE showing the cross reactivity of 47kDa (S6/MS73) - ATPase
subunit of the 26S proteasome (b) and 28kDa regulatory subunit of 20S proteasome (c).
V4 V6
S0 S2
P0 P1

:
:
:

day four to day six of last larval instar

day zero to day 2 of spinning stage
day zero to day one of pupal stage

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The presence of 20S proteasome activity in the larval fat tissues was analyzed by immunoblotting
using the anti MCP antibody (Thanks to Prof. Martin Rechsteiner) which recognizes the regulatory
complex (19S) of 20S proteasome complex. Three distinct bands with apparent molecular mass of
28kDa region were labeled in Western blot. Western blot profile of the samples demonstrated the lack of
regulatory complex of 20S proteasome throughout the feeding period, but the appearance of 19S protein
was revealed in cells of the PF during commencement of the 1st day of pupal stage (Fig. 5c).
(e). Morphological Examination
Morphology of the larval PF tissues undergoing PCD was examined by electron microscopic
techniques. Apoptotic morphology (eg. shrinkage of cells and nuclei, cytoplasmic blebbing, ruptured
mitochondria, or detached cells which lost their intercellular connections) was not observed by electron
microscopic analysis and the followings were the prominent changes observed during the disintegration
of larval peripheral fat tissues; The first step in the
formation of the autophagic vacuoles is the appearance of
the so-called autophagosomes (Ag) or segregosomes. On
day two of spinning larva great number of mitochondria
were seen which indicates the active lively stage whereas
on the same day it structured for its disintegration by the
formation of autophagosomes (Ag) (fig. 6a).
The
appearance of autophagosomes is in great numbers by the
end of the feeding period (S2) indicating the
commencement of the PCD process in these cells (Fig.
6a). In the day zero of pupa the formed autophagosomes

Fig.6 Electron micrographs of larval

peripheral fat body tissues undergoing
programmed cell death during larval
pupal transformation in the silkworm,
B. mori. Formation of autophagosome
on day two of spinning larva (a);
aggregation of autophagosome on day
zero of pupa (b, c); after 3 h on day one
of pupa the autophagosomes fuses with
lysosomes to form autophagic vacuoles
(d); after 10 hrs on day one pupa lytic
enzyme in the autophagic vacuoles
digested the aggregated materials and
forms undigested one as apoptotic
bodies (e).
(Scale bar = 1m).
AB Apoptotic Bodies;
Ag Autophagosomes;
Av Autophagic Vacuoles;
L Lipid;
M Mitochondria;
N - Nucleus

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aggregates together (Fig.6b, c) but after 6h the number of mitochondria are less (Fig. 6b) which indicates
that the larval peripheral fat body get ready for the disintegration. On the same day but after 12h, the PF
tissues prearranged themselves by complete disappearance of mitochondria (Fig. 6c). Later on the
aggregated autophagosomes fuse with lysosomes in which the digestion of the macromolecules begins
inside these organelles known as autophagic vacuoles after 3 hrs of day one of pupal stage in PF cells
(Fig. 6d). Autophagic vacuoles fuse very often with each other where degradation of the sequestered
organelles and materials began. Finally most of the cytoplasmic organelles were wrapped by autophagic
vacuoles (AV) and the undigested residues turned into electron-dense material after 10hrs in day one of
the PF cells. These undigested residues appear on the electron micrographs as dark, electron-dense spots
as apoptotic bodies (AB) or smears inside them (Fig. 6e). At the end of the larval (P1) stage, the
structure of the segregated organelles is totally destroyed. Only the undigested, homogenously electrondense material fills the inside of the large autophagic vacuoles (Fig. 6e).
(f). Role of 20E on PCD
Investigation of cell death during insect metamorphosis has provided new insights into the
mechanism by which a hormone, in which the steroid hormone 20-hydroxyecdysone, induces death in
some tissues while simultaneously promoting the further development of others. Changes in
responsiveness of the PF tissues to 20E, an effective inducer of PCD were examined by culturing the fat
body tissues in insect Graces medium supplemented with different concentrations of 20E and were
determined by chromatin condensation and DNA laddering studies.
Fig.7. In vitro studies on the effect of 20E on
chromatin condensation on the day two of spinning
stage peripheral fat body tissues of the silkworm,
B. mori after 12 h incubation at 25C. (Live cell
emits green fluorescence; dead cell emits dark
brown fluorescence)
Control treated with ethanol (a); 1g/ml of 20E
(b); 2g/ml of 20E (c); 3g/ml of 20E (d); 4g/ml of
20E (e); 5g/ml of 20E (f); 3g/ml of 20E (24h
incubation at 25C) (g); 4g/ml of 20E (24h
incubation at 25C) (h)

Fig. 8. In vitro studies on the

effect of 20E on DNA
fragmentation by 1.2% agarose
gel electrophoresis in the day
two of spinning stage peripheral
fat body tissues treated with 4
and 5g/ml of 20E after 12 h
incubation at 25C.

To test this hypothesis stating that ecdysteroids induce PF tissues PCD directly; S2 (spinning day
2) PF tissues were incubated in supplemented Insect Graces medium with or with out 20E (1 to 5
g/ml) for up to 24 h. Chromatin condensation appeared to be 20E dose and time dependent and was
examined regularly during 6 hours intervals. Early signs of chromatin condensation occurred by
increasingly after 12 h incubation with maximum concentration (5g/ml) of 20E (Fig. 7). After 24h,
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almost every cell contained a highly condensed and fragmented nucleus. Among the five doses of 20E
tested, 5g/ml was the most effective inducer of 100% chromatin condensation after 12 h incubation
(Fig. 7f). A dose of 3 and 4g/ml causes only partial nuclear condensation (Fig. 7g, h), whereas 1 and 2
had no effect even after 24 h incubation (data not shown). This approach led to the unexpected finding
that 20E (5g/ml) had direct effect on the PCD of larval PF tissues showing that the induction crop up
before 48h than the naturally occurring PCD of these tissues. induces PCD of peripheral tissues Control
experiments i.e. culture of PF tissues in 20E free Insect Graces medium were also examined for
chromatin condensation in order to study the effect of 20E as an effective inducer of PCD. In control
experiments there was no sign of chromatin condensation were observed by acridine orange staining
even after 24 h incubation.
Total genomic DNA was extracted from the PF tissues cultured in Insect Graces medium treated
with 3, 4 and 5g/ml after 12h incubation and subjected to agarose gel electrophoresis. Clear nuclear
fragmentation was noted in 5g/ml, which strongly support the hypothesis that 20E an effective
inducer of PCD in the PF tissue (Fig. 8).
4. Discussion
In the last decade a tremendous progress has been achieved in understanding the control of
apoptosis by survival and death factors as well as the molecular mechanisms of preparation and
execution of the cells suicide. Autophagic PCD appears to be a phylogenetically old phenomenon, it may
occur in physiological or disease status (Bursch 2001). However, it is an open question whether these
pathways exist and /or coexist during the physiological, developmental processes of insects. To approach
this problem, we studied the events of PCD in the larval fat body cells (PF tissues) during the last stage
of larval development of silkworm, B. mori. This organ can be regarded as a practically homogenous
tissue, consisting of a single cell type, the trophocytes, which die via autophagocytosis (Locke and
Collins 1966; Sass and Kovacs 1975; 1977; Chandrasekar et al., 2008a). Hence we made an attempt to
study the hallmarks of autophagic PCD in the larval PF tissues.
There are two major morphological categories of PCD: type I, apoptotic cell death, and type II,
autophagic cell death (Pilar and Landmesser 1976; Chu-Wang and Oppenheim 1978; Clarke 1990;
Lockshin and Zakeri 1996). Each type displays different specific morphological and biochemical
features. Based on our fluorescent studies, ultrastructural analysis, and DNA laddering studies, cell death
of larval PF tissues appears to display characteristics of both types, i.e. condensed chromatin under the
nuclear membrane, DNA fragmentation, apoptotic body formation (type I), as well as numerous
autophagic vacuoles, and intense membrane endocytosis (type II). Disintegrated, fragmented or
shrinkage cells of PF tissues were not registered through electron microscopic analysis and did not reveal
the characteristic nuclear morphology, or the blebbing. On other hand, extensive autophagy indicated
by the extremely quick and extensive increase in the lysosomal compartment of the cells showing the
elevated level of lysosomal marker enzyme, acid phosphatase (stage P1) which was observed in the PF
tissues from the beginning of the spinning period confirming the results of earlier studies made on other
lepidopteron insects (Locke and Collins 1966; Sass and Kovas 1975; 1977; Muller et al., 2004;
Chandrasekar et al., 2008a). These events definitely pave the way for DNA fragmentation exactly on the
day one of pupal stage in the PF tissues. These combined features have also been observed in other
cases, such as the dying cells in the posterior necrotic zone of the forelimbs of chick embryos that
display all the characteristics of type I cell death but contain numerous autophagic vacuoles as (Mottet
and Hammer 1972; Hurle and Hinchliffe 1978; Clarke, 1990). Our observations are in good agreement
with earlier descriptions (Schwartz 1992; Zakeri et al., 1993; Muller et al., 2004; Chandrasekar et al.,
2008a,b) showing that in the dying intersegmental muscles during the postembryonic development of
insects and the larval fat body cells of M. sexta, the classical apoptotic morphology can be recognized.
However, some other data suggest that the autophagic and apoptotic pathways may coexist in certain
degrading larval organs (salivary and prothoracic glands) of insects (Dai and Gilbert 1997).

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DNA fragmentation is an early hallmark for apoptosis and therefore an important criterion for
identifying the type of PCD. One of the aims of the present work was to see whether DNA fragmentation
occurs in dying insects PF cells showing a typical morphologic signs of autophagocytotic cell death.
Although this distinction appears very clear, the technique used to identify DNA fragmentation, such as
the DNA laddering technique, is quite critical. Using this technique, laddering was detected during the
final stage of the disintegration of PF tissues of B. mori, strongly indicating that the process is
autophagic phenotype. The DNA fragmentation, as an integral part of the PCD in natural developmental
processes of insects was described in those organs where the apoptotic and autophagic features were
simultaneously observed but the performance of the classic DNA ladder failed (Zakeri et al., 1993) with
the only exception of a highly synchronized in vitro cell culture triggered to death (Dai and Gilbert
1999). According to our best knowledge, it is the second report on the occurrence of DNA fragmentation
in larval PF tissues of B. mori with morphological pure autophagic phenotype where as the first report
was observed in larval fat body cells of M. sexta (Muller et al., 2004).
Lysosomes are normally associated with cytoplasmic turnover, whereas, in some cells undergoing
cell death, autophagic vacuoles that are derived from lysosomes play a significant role in cellular
breakdown (Beaulaton and Lockshin 1977; 1982; Clarke, 1990). Zahrebelski et al. (1995) studies
suggested that the release of hydrolytic enzymes from lysosomes might be the final event causing lysis
of the membrane and irreversible loss of viability. Acid phosphatase has been used as the marker
enzyme for lysosomes and a marker for apoptosis. It plays a critical role in the degradation of insect
tissues (salivary gland, intersegmental muscle, etc.) and mammalian tissues (mammary gland, prostate
gland, etc.) (Halaby et al., 2003). In silkworm, B. mori, the elevated level of lysosomal acid phosphatase
during the histolysis of the PF tissues may play a vital role in formation of autophagic vacuoles, which
may cleanse the system by engulfing apoptotic bodies or cell debris during the late stages of cell death
as reported for prothoracic gland cell death of Drosophila and M. sexta (Dai and Gilbert 1991). Based
on studies of professional phagocytes by Vieira et al. (2002) stated that in Caenorhabditis elegans
phagosomes likely fuse with endosomes /lysosomes, resulting in the formation of degradative
phagolysosomes. The trigger for cell death and the dramatic increase in polyubiquitin expression in the
degenerating intersegmental muscles (ISM) of M. sexta is the decline of the level of circulating molting
hormone. It is well known that ubiquitin may serve as a macromolecular tag attached to certain proteins
to mark them for degradation by the proteasome (Pickart 2001). The ubiquitin-protein conjugates may
also be decomposed in the lysosomal compartment of the cells (as it was described in various cell types)
(Laszlo et al., 1990; Mayer et al., 1991). Therefore, the regulatory ATPase subunit of the 26S
proteasome (47kDa- MS73) and the 28kDa - regulatory subunit of 20S proteasome may have a role in
the PCD of the larval PF tissues during metamorphosis. In M. sexta MS73 is found at high level only in
body wall muscles that are undergoing or are destined to undergo PCD (Low et al., 1997). MS73
immunoreactivity has now been found to be present in two other muscle types that undergo
developmentally PCD, the intersegmental muscles ISM1-2 and ISM7-8, which die just after pupal
ecdysis, and the proleg retractor muscle (PRM), which dies just before pupal ecdysis. In all three muscle
types, the level of MS73 more than doubles just before death occurs (Low et al., 1997). Our immunoblot
analysis was in good agreement with that of Low et al. (1997) showing in the dying PF cells that the
expression of 47 kDa ATPase subunit of 26S and 28 kDa regulatory subunit of 20S proteasome were
elevated just before the death occurs. The nuclear location and accumulation of ubiquitin and
proteasomes which coincide with the start of autophagy suggest that this highly conserved system may
be involved in the regulation of differential gene activation necessary to accomplish the process of
autophagocytosis in the larval fat body cells of M. sexta (Muller et al., 2004).
During an insects metamorphosis from larval to adult form, different tissue types respond to a
series of transient increases in the titer of a steroid hormone, 20E. The present data confirmed that 20E
act directly on larval PF tissue to trigger PCD. The effective dose 5g/ml trigger the PCD of peripheral
tissues 2 days before, that exactly on day 2 of spinning stage after 12h incubation, whereas in the
developmental PCD of these tissues occurs prominently on day 1 of pupal period. In previous studies,
the involvement of 20E in the onset of PCD of insect tissues was demonstrated in tissues such as
intersegmental muscles and motoneurons by in vivo observations (Lockshin and Williams 1965;
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Schwartz and Truman 1982) or by in vitro culture with 20E (Streichert et al., 1997). Although these
observations and in vitro manipulations provide strong evidence for the direct action of 20E on tissues,
the tissues in these studies consisted of several types of cells. This left open the possibility of
intercellular signaling that triggered PCD. Anterior silk glands are stimulated to full PCD by the rise of
hemolymph ecdysteroids in the photophase of day 2. The titer of approximately 1.5g/ml in the late day
2 (Kiguchi 1983) may trigger PCD. This indicates that the degeneration of tissues requires stronger
steroid stimulation than that needed for tissue differentiation (Terashima et al., 2000). The present study
provides an in vitro system for further investigations of the molecular and cellular mechanisms of PCD
in the larval PF tissues.
The pace in cell death research is frantic and, sadly, this has resulted in considerable confusion
caused by a lot of contradictory publications. In conclusion, the present study indicates that the PCD
seen in PF tissues during larval pupal transformation of B. mori lies between classic apoptosis and
autophagy, since its exhibits some characteristics of both phenomena. We have provided an overview of
state of the physiological PCD field in PF tissues of B. mori to highlight some of the unanswered
questions, as well as the genetic screening tools available to further this work.
5. Acknowledgement s
The authors thank Prof. Martin Rechsteiner, Department of Biochemistry, University of Utah
School of Medicine, Salt Lake City, UT, for the generous gift of Proteasome antibodies and
Dr. Lawrence I. Gilbert and Dr. James T. Warren, Department of Biology The University of North
Carolina at Chapel Hill, NC, for the generous gift of 20E. The financial support of the CSIR and DST,
New Delhi for gratefully acknowledged.
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