Apoptosis

APOPTOTIC AND IMMUNOLOGICAL STUDIES ON
SILKWORM (Bombyx mori L.) INFECTED WITH
STAPHYLOCOCCUS AUREUS
By
A.Harinatha Reddy, M.Sc, Ph.D.
Department of Microbiology
Sri Krishnadevaraya University
Ananthapuramu- 515005

Introduction
 Apoptosis is a physiological cell death, also known as “Programmed cell death”.
 The term programmed cell death was introduced in 1964, eventually the term Apoptosis had
been coined in order to describe the morphological processes leading to controlled cellular
self-destruction.
 Apoptosis is of Greek origin, having the meaning “ falling off or dropping off ” as leaves
from a tree.
 The apoptotic mode of cell death is an active and defined process which plays an important
role in the development of multicellular organisms and in the regulation and maintenance of
the cell populations in tissues and pathological conditions.
 Apoptotic processes are of widespread biological significance, being involved in development,
differentiation, regulation of the immune system and in the removal of defect and harmful
cells.

 Involvement of apoptosis in the immune system: Several millions of B and T cells were
generated every day and the majority (>95%) of the cells die during maturation.
 Dysfunction or deregulation of the apoptotic program is implicated in a variety of pathological
conditions.
 Defects in apoptosis can result in cancer, autoimmune diseases and spreading of viral
infections.
 Apoptotic cells can be recognized by stereotypical morphological changes: cell shrinks, shows
deformation and looses contact to its neighboring cells.
 Its chromatin condenses and marginates at the nuclear membrane, the plasma membrane is
blebbing or budding, and finally the cell is fragmented into compact membrane-enclosed
structures, called “apoptotic bodies” which contain cytosol, the condensed chromatin, and
organelles.

 The apoptotic bodies are engulfed by macrophages and thus are removed from the tissue
without causing an inflammatory response.
 Those morphological changes are a consequence of characteristic molecular and
biochemical events occuring in an apoptotic cell, most notably the activation of
proteolytic enzymes which mediate the cleavage of DNA into oligonucleosomal fragments
.
 Apoptosis can be triggered by various stimuli from outside or inside cell. E.g. by ligation
of cell surface receptors, DNA damage as a cause of defects in DNA repair mechanisms,
treatment with cytotoxic drugs or IR-radiation, or by a lack of survivals, or by
developmental death singnals.

Insects
 The insects are the most diverse and important group on land, more than one million species
of insects have been reported.
 Insects are members of a larger group called arthropods, all arthropods have a rigid
exoskeleton and legs that are jointed (arthropod means "jointed foot"). In order to grow,
arthropods have to shed their whole exoskeleton all at once this is called "molting".
 Among the insects, silkworm (Bombyx mori L.) is the domesticated and an economically
important insect. It is, being a primary producer of silk. Silkworm preferred food is mulberry
leaves. It is entirely dependent on humans for its reproduction and does not occur naturally in
the wild.

Silkworm information
 Phylum - Arthropoda
 Class - Insecta
 Order - Lepidoptera
 Family - Bombycidae
 Genus - Bombyx
 Species - Bombyx mori L.
Morphology:
 Larvae are worm-like with a short anal horn.
 Three distinct body parts: head, thorax, abdomen.
 Adult moths has four wings covered with scales, adult females are larger and less active than
males.

The life cycle of the silkworm (Bombyx mori)
 The silkworm life cycle consists of four stages and considered one of the most advanced forms of
metamorphosis.
 Silkworm life cycle is very short and simple. The various stages that it goes through during its short
life span are simply mesmerizing.
 Which includes the embryo (Egg), larva, pupa and adult moth.
Stage:1-Embryo:
 Newly laid eggs are a creamy yellow colour, after a few days the fertile live eggs will be gray.
The silkworm eggs will hatch after 2 weeks.
Stage:2-Larva: 27 days (5 instars):
 After hatching from the egg, larva go through four molts . During each molt, the old skin remove and
a new large one is produced. The larva life is divided into five instars, separated by four molts.
 After hatching the tiny larvae grow the best if they are feed on the soft leaves of the mulberry tree.

Stage:3-Pupa: 7-8 days:
 The silkworm will spin a silk cocoon as protection for the pupa. It produced from raw silk
secreted by salivary glands of the silkworm. Cocoons are white or yellow in colour. After a final
molt inside the cocoon, the larva change into the brown pupa. Further changes inside the pupa
result in an emerging moth.
Stage:4-Adult silk moth: 3-4 days:
 An adult silk moth emerges form the cocoon about two weeks. This is the adult stage of
silkworm, (Bombyx mori).
 The body of the moth is covered in short fine hairs and wings are creamy white. Moth can not fly
or consume nutrition. Females are larger and less active than the males. Male moth move about
beating their wings seeking females.
 If adults copulate , the female moth will lay eggs within 24 hours.

Advantage of silkworm as animal model
 In spite of its appearance, silk worm takes after human in lot of ways such as analogous
tissues and organs, similar sensitivities to pathogens and comparable effects of drugs.
 It is low in cost, no ethical problems and no danger of biohazard.
 Good availability , easy to handle and easily to collect haemolymph from silkworm,
when compare to other insects.

Apoptosis in insects
 Metamorphosis very important phase in the development of holometabolous insects, during
which the larval body is completely reorganized. The larval organs undergo remodeling or
completely degenerate before the final structure of the adult insect. Apoptosis and autophagy
that occur in larval organs of lepidoptera insects during metamorphosis.
 In invertebrates, programmed cell death plays a important role in development, control of
DNA damage, and defense of pathogens.
 Caspases are required for apoptosis in insects, like that in mammals. However, the mechanisms
by which caspases are activated still unclear. On the basis of sequence similarity and
biochemical activity, seven caspases have been identified in Drosophila melanogaster .

Immune system in insects
 The defense mechanism in insects is broadly classified into two broad groups. The first one is
non-specific immunity, which consists of structural and passive barriers like cuticle and gut
physio-chemical properties.
 The second one is specific immune system, which includes the activation of phenol oxidase
cascade, phagocytosis, nodulation (hemocyte aggregation) and encapsulation especially with
reference to bacteria, fungi, protozoa and the synthesis of antimicrobial proteins.
 Antimicrobial proteins appear to be multi components of the innate mechanisms existing in
insects, most of which are produced in the fat body and haemocytes released into the
haemolymph of the insect.

Staphylococcus aureus
 Staphylococcus is a genus of Gram positive, nonspore-forming cocci and recognized as one of the
most important bacteria that cause disease in humans. It is the leading cause of skin and soft tissue
infections and cause serious infections such as bloodstream infections (Bacteremia), pneumonia
and bone and joint infections.
 There are five organisms to consider as potential human pathogens in this genus:
S. aureus, S. epidermidis, S. saprophyticus, S. haemolyticus, and S. hominis, but the first three are
the most common isolates. S. aureus is often considered to be the most problematic than four
pathogens.
 Mannitoal salt agar (MSA) media is commonly used for the isolation of S. aureus.
It contains a high concentration of salt (NaCl), making it selective for gram positive bacterium
Staphylococci. S. aureus produce yellow colonies with yellow zones, whereas other Staphylococci
produce small pink or red colonies

 Among the pathogens S. aureus, one of the most common gram-positive bacterial pathogen in
humans, induces apoptosis. Apoptosis in response to S. aureus infection can be triggered by a
soluble factor α toxin, which mediates caspase activation via the mitochondrial apoptosis pathway.
 In the present study the pathogenicity of S. aureus has been investigated by using silkworm as a
model organism. Mechanisms of bacteria-induced apoptosis employing the host machinery will be
discussed in the case of Pseudomonas aeruginosa, Neisseria meningitidis, Haemophilus influenza,
Mycobacterium tuberculosis and Streptococcus pneumoniae have been shown to trigger
apoptosis.
 However, we would like to focus on Staphylococcus aureus induced apoptosis and the activation of
host cell machinery immune system by using silkworm as a model.

 Most strains of S. aureus are sensitive to the more commonly used antibiotic penicillin and infections can be
effectively treated. Some S. aureus bacteria are more resistant. Those resistant to the antibiotic methicillin
are termed Methicillin Resistant Staphylococcus aureus (MRSA) and often require different types of
antibiotic to treat them. Those that are sensitive to methicillin are termed Methicillin Susceptible
Staphylococcus aureus (MSSA). MRSA and MSSA only differ in their degree of antibiotic resistance, other
than that there is no real difference between them.
 Methicillin-resistant Staphylococcus aureus (MRSA) identified over 4 decades ago, has undergone rapid
evolutionary changes becoming a dominant pathogen (Stan Deresinski, 2005).
 S. aureus (MRSA) infections, are a major cause of illness and death and impose serious economic costs on
patients and hospitals. During this period (1999-2012), the estimated number of
S. aureus related hospitalizations increased from 2,94,570 to 4,77,927, and the estimated number of MRSA
related hospitalizations more than doubled, from 1,27,036 to 2,78,203 (Eili Klein et al., 2007).

Number of death cases of Staphylococcus aureus and MRSA during the
period, 1993 to 2012
Source: Office for National Statistics

Objectives of the present study
 To study cell death in silkworm in final instar larvae
 To identify pathogenicity of S. aureus and its ability induce apoptosis in
invertebrate system.
 To examine the alterations in antioxidant system by studying glutathione,
catalase, superoxide dismutase and phenol oxidase levels in silkworm
haemolymph.
 To determine changes in acid phosphatase activity.
 To estimate malondialdehyde concentration in the silkworm haemolymph.

 To examine histopathological changes in midgut and silk gland.
 To analyse haemolymph by means of SDS-PAGE.
 To analyse DNA ladder by agarose gel electrophoresis.
 To study antimicrobial activity of silkworm haemolymph.
 To study in silico molecular docking of moricin and cecropin on S. aureus
drug target proteins.

Rearing of Bombyx mori. L. larvae
Fifth instar larvae of Bombyx mori. of the type CSR2 were
obtained from Regional Sericultural Research Station,
Rapthadu, Anantapur (District) and reared in the laboratory
by the improved method of rearing technique
(Krishnaswami, 1978). Various stages of the silkworm was
maintained on mulberry leaves at a temperature of 27 ºC. The
life span of the silkworm under these conditions was 28-32
days.

Culturing of S. aureus
 Bacterial sample obtained form Basavatarakam Indo-American Cancer Hospital and Research
Institute, Hyderabad.
 Mannitol salt agar (MSA) medium used for the selective isolation and identification of
S. aureus.
 S. aureus bacterial sample was streaked on MSA slants under aseptic conditions in a laminar air
flow chamber with the help of streaking loop. After streaking the slants were incubated at 37ºC
overnight. After 24 hr, the bacterial growth was noticed and further it was sub cultured.
 Inoculation of bacteria in Luria broth (LB):
 A loopful of bacteria was taken with the help of a loop and streaked onto Luria broth
(LB 1000 ml) and incubated at 37ºC overnight. The inoculated LB sample was centrifuged for
15 min at 4000 rpm. By discarding the supernatant, pellet sediment at the bottom of the tube was dissolved
in 100 ml of distilled water. The number of bacteria cells in the culture suspension was calculated by
colony-forming unit (CFU/ml).

 Silkworm larvae were reared at 27ºC room temperature with relative humidity 70-80%.
Immediately after the fourth moult larvae of 5th instar CSR-2 silkworm strains were infected
with the bacterium (3 × 106 cfu/ml to 1 × 108 cfu/ml) by smearing/spraying the bacterial
solution onto the leaf surface and fed to larvae. A similar number of larvae were fed with
distilled water smeared mulberry leaves and considered as a control. Both control and
infected larvae were reared under room temperature. The time of infection was recorded and
the haemolymph was collected from the infected and control group larvae at 24 hr post
infection and stored in eppendorf tubes.
Infection of silkworm larvae with bacterial strain (Suparna et al., 2011).

Preparation of Mannitol salt agar (MSA) medium and Staphylococcus
aureus bacterial sample was streaked
Inoculation of bacteria in Luria
broth (LB1000 ml)
Incubated at 37 ºC overnight
Centrifuged for 15 min at 4000 rpm
Sediment was dissolved in distilled water
(100ml)
Silkworm larvae was infected with the bacteria by
spraying/smearing the bacterial solution onto the
leaf surface (3 × 106 cfu/ml to 1 × 108 cfu/ml)

Experimental Design
In the present study 5th instar silkworm (Bombyx mori. L.) larvae were used and divided into two
groups.
Group 1 : Normal (Control/Healthy)
Group 2: Staphylococcus aureus infected larvae
Fifth instar silkworm larvae divided in to two groups, each group consisting of 20 to 30 larvae.
One group was maintained as control (normal and healthy ones) and the other group was infected
with the bacterium by smearing/ spraying the bacterial solution onto the leaf surface and fed to
larvae. A strict hygiene was maintained and bed clearing was followed strictly.

Collection of hemolymph from silkworm
 Silkworms were collected and chilled on crushed ice for 10 min and then swabbed with 70%
alcohol. A small cut was made on the proleg cuticle and the haemolymph was collected in sterile
vials containing 0.5 g phenol thiourea. Which prevents the melanization of the hemolymph and can
be stored for longer time intervals. Haemolymph was centrifuged at 3000 rpm for 5 min to remove
haemocytes.
Determination of haemocyte viability/ Trypan blue exclusion test
 0.2 ml of the haemocyte suspension was mixed with 0.3 ml of PBS and 0.5 ml of trypan blue in a
small test tube. An aliquot is then placed on hemocytometer and count number of viable cells under
the microscope. The plasma membrane of the viable cells does not permit the entry of electrolyte
dye substance. This phenomenon is used to distinguish dead cells from living haemocytes.
Percentage of viability was calculated by the following formula.

Estimation of protein form hemolymph
 For the estimation of amount of protein present in the samples, Folin-Lowry method (Lowry et al.,
1951) was used. To estimate total protein content 0.1 ml haemolymph was treated with 0.9 ml of
the alkaline solution. Further, 5 ml of alkaline copper sulphate solution was added and allowed to
stand for 10 minutes at room temperature. Then 0.5 ml of Folin’s reagent was added, mixed
thoroughly and allowed to stand for 30 min for colour development. Absorbance was measured at
750 nm in a Spectrophotometer. Bovine serum albumin was used as standard.
Glutathione (GSH) assay
 The haemolymph homogenate (0.5 ml) was treated with 3.5 ml of 5% TCA (Trichloroacetic acid).
The precipitate was removed by centrifugation. To 0.5 ml of supernatant, 3.0 ml of phosphate
buffer and 0.5 ml of Ellman's reagent (5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were added.
The yellow color developed was read at 412 nm. A series of standards along with a blank
containing 3.5 ml of buffer. Values are expressed as µg of GSH/ml haemolymph.

Acid phosphatase assay
 Two ml of PNPP (p-nitrophenyl phosphate) solution was taken in a test tube and added
0.1 ml of citrate buffer followed by 4 ml NaOH and mixed well. Then 0.4 ml of
haemolymph extract was added and extinction was measured at 405 nm against blank.
The activity of the enzyme was expressed as nano moles of p-nitrophenol
released/hr/ml.
Lipid peroxidation assay
 To 0.5 ml of the homogenate (hemolymph) 0.5 ml of buffer, 2 ml of Trichloroacetic
acid (TCA) were added followed by 4 ml of Thirobarbatic acid (TBA). The system
were heated in a water-bath at 100 ºC for 20 min. After cooling and centrifugation, the
absorbance of the supernatant was read at 535 nm. A reagent blank was prepared using
water instead of homogenate. The extend of lipid peroxidation was expressed as n mole
of MDA formed/mg protein

Phenol oxidase assay
 The phenoloxidase activity was determined by the method of Horowitz and Shen, (1952). The
reaction mixture consisted of 1ml of 0.02 M, 4-di hydroxyphenylalanine (DOPA), 3.9 ml of 0.1M
phosphate buffer, pH 6.0 and 0.1 ml of enzyme solution. After incubation at 30o C for 5 minutes
the color intensity of dopachrome was measured at 490 nm. One unit of enzyme was defined as
the amount causing increase in absorbance of 0.01 under the above condition .
Glutathione peroxidase assay
 The assay mixture contained 0.02 ml enzyme source, 1.78 ml PBS buffer, 0.06 ml
glutathione (GSH), 0.02 glutathione reductase and 0.06 ml NADPH. The reaction was
initiated by the addition of 0.06 ml of H2O2. The change in absorbance was recorded at 1 min
intervals at 340 nm and the specific activity was calculated using extinction co-efficient of
6.22 cm-2/μmol for NADPH. The activity was expressed as n mole NADPH oxidized/min/mg
protein.

Superoxide dismutase (SOD) assay
 Superoxide dismutase activity was measured based on the ability of the enzyme to inhibit the
autooxidation of pyrogallol according to the method of Marklund and Marklund (1974).
 The assay system contained 2.1 ml of phosphate buffer, 0.02 of enzyme source and 0.86 ml of
distilled H2O. The reaction was initiated with 0.02 ml of pyrogallol and change in absorbance
was monitored at 420 nm. The percent inhibition was calculated on the basis of blank assay
system. One unit of SOD was defined as the amount of enzyme required to inhibit the
autooxidation of pyrogallol. The specific activity was expressed as unit/min/mg protein.
Catalase assay
 The assay system contained 1.98 ml PBS buffer 0.02 ml enzyme source and 1.0 ml H202. The
change in absorbance was monitored at 240 nm and specific activity was calculated using a
molar absorbance index for H202 of 43.6. The specific activity was expressed as moles of H202
decomposed/min/mg protein.

Separation of proteins by SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel
electrophoresis)
 After estimation of protein concentration, SDS-PAGE was carried out by using 12% separation gel
and 5% stacking gel. The gels were stained with Coomassie brilliant blue.
 The separating gel consisted of 10%(W/V) acrylamide, N,N-methylene bis acrylamide, 0.375 M,
Tris-HCL (pH 8.8) and 0.1% SDS. It was chemically polymerized with 0.05% (W/V) ammonium
persulphate, 0.05%(V/V)TEMED.
 The stacking gel containing 4%(W/V) acrylamide 0.12M, Tris-HCl (pH 6.8), 0.1% SDS, 0.05%
(W/V) ammonium persulphate, 0.05%(V/V)TEMED.

DNA extraction
 Silk glands of B. mori are collect and incubate in 1 ml lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1M
EDTA, 5g/ml SDS) for 1h. The lysates treat with proteinase K (100 µg/ml) at 37ºC overnight. The
samples centrifuge at 7000 rpm for 10 min. The supernatants mix with an equal volume of
Phenol:Chloroform and centrifuge at 7000 rpm for 10 min. The resulting supernatants transfer to
new tubes and mixed with an equal volume of Chloroform: Isoamyl alcohol and centrifuge at 7000
rpm for 10 min. The supernatant dilute with an equal volume of isopropanol and kept at -20ºC for 1
h, and centrifuge at 15000 rpm for 15 min. DNA pellets wash with 70% alcohol and then dry at
room temperature. DNA dissolve with an appropriate volume of 1×TE buffer. RNA contamination
was remove by treatment with 25µg/ml Rnase at 37ºC for 1h.
DNA Ladder analysis
 After purification, 10 µl of DNA from each sample was mixed with 5 µl of loading buffer and
0.5 µg of genomic DNA load per lane. The banding pattern of the DNA samples was detected on
1% agarose gel and visualized by staining with ethidium bromide.

Examination of histopathological changes in mid-gut and silk gland
Tissue preparation for histology:
 The midgut and silk gland of the larvae of the final instar, prepupae, and adults was excise and
immediately fix in 10% fomalin for 24hrs, then washed under tap water for 15 min. After fixation
they are dehydrated in 80%, 90%, and 100% solutions of alcohol and acetone for 2 hrs and cleared
in xylene for three times for 30 min. The tissues are impregnated in paraffin wax at 50º-60ºC over
night and sections of 3-4 microns are made using microtome. The prepared sections are dried in oven
for 1hr and later transferred to xylene solution.
 Sections are deparaffinised by repeated transferring of the sections in to xylene for three times for
every 10 min each. Then the sections are hydrated in absolute alcohol followed by 90% 80%, 70%
and 50% alcohol respectively for 5 min each. They were then washed under running tap water for 5
min, processed in hematoxylline for 15min.
 The stained sections were mounted on slide by using DPX mounting fluid which provides a high
refractive index for microscopy.

Giemsa staining
 Silkworms were dipped into hot water (60˚C for 1 to 10 minutes) then cut the posterial leg and
place a drop of haemolymph directly over a sterilized glass slide and subsequently draw a cover
slip over it to made a thin film. The film is air dried before staining. Air dried film was immersed
in Giemsa solution for 20 minutes to 2 hours. Rinse the slide in distilled water and then immersed
briefly in water to which few drops of lithium carbonate have been added. Further the thin film of
slide was rinsed in distilled water again and to which a few drops of diluted HCL have been added.
Immediately the hemocytes present on thin film appear as blue particles. Again rinse the slide in
distilled water blot the slide dried and mounted in Canada balsam.
Measurement of silk gland weight (Yoshiyasu et al., 2008)
 The anesthesia silkworms were placed on a cork board, and the head and tail were pinned to it. A
small cut was made in the tail, from which the abdomen was opened longitudinally by running
ophthalmological scissors toward the head, and the silk gland, midgut, located in the middle of the
abdomen, was removed. The wet weight of the silk gland was measured using a balance just after
removal.

Preparation of haemolymph extract
 0.5 ml of hemolymph was collected form infected group and diluted with 0.5 ml of tris-HCL
(40 mM ) and mixed thoroughly. The precipitated proteins were pelleted by centrifugation at 1000
rpm for 10 min. The obtained supernatant was collected , freeze dried and stored at -20ºC until
used.
Antimicrobial activity of haemolymph extract
 The assay was done using the classical well diffusion method. The antimicrobial activity was
evaluated by the growth-inhibition zone on nutrient agar media. Four concentrations of the
haemolymph extract (10, 20, 30 and 40 µl ) were used to test antimicrobial activity. Standard
Streptomycin containing discs (30 μg/ml) were placed in each plate.

In silico molecular docking of Moricin and Cecropin with S. aureus drug
target proteins
Ligand preparation
 The 3D Structure of the moricin (PDB: 1KV4), cecropin(PDB: 2LA2) peptides was retrieved
from PDB (Protein data bank) and prior to initiating the docking simulations, all non-protein
molecules were removed from the PDB.
Protein preparation
 The crystal structures of Glycerol phosphate lipoteichoic acid synthase (PDB: 2W5Q), Dipeptide
ABC transporter-PG110 (PDB: 1P99) and DNA Gyrase (PDB: 2XCO) from S. aureus were
retrieved from the Protein Data Bank (Three different drug target proteins of S. aureus )
In our present work, we have used Hex docking software for protein-protein docking studies.
It requires both ligand PDB files and receptors PDB files.

Preparation of sericin silver nanoparticles
 To isolate sericin protein, the cocoons of silkworm cut into small pieces and boil in the presence of
0.02 M Na2Co3 (Sodium Carbonate) for half an hour. The crude extracts of sericin is use for
experiments.
 5 ml of crude extracts of sericin was added to a conical flask containing 5 ml of 3 mM aqueous
AgNO3 (Silver nitrate) solution and incubate at room temperature.
 The conversion of solution color showed the formation of silver nano particles by observing color
change from colorless to brown color. These resulting colloidal solution shows strong absorption
between 400 and 420 nm.
Antimicrobial activity of silver nano particles
 The antibacterial assays were done on human pathogenic E. coli and S. aureus by standard well
diffusion method. Mueller Hinton (MH) agar medium was used to cultivate bacteria. Fresh
overnight cultures were taken and 100 μl of inoculum were spread on the MH agar plates. Wells
(1 mm diameter and about 2 cm a part) were made in each of these plates using sterile cork borer.
About 40 μl sericin silver nanoparticle solution were added and allowed to diffuse. Standard
Streptomycin containing discs (30 μg/ml) were placed in each plate.

Definition and description of developmental stages of the Bombyx mori L.
Stage Definition
L5D1-L5D6 Fifth larval instar day 1 to 6
SD1 Spinning stage day 1
SD2 Spinning stage day 2
PP Prepupal stage
PD1-PD7 Pupal stage day 1 to 7

0
10
20
30
40
50
60
70
80
90
100
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 SD1 SD2 PP
%
Viable
Haemocytes
Fifth instar larvae developmental stages
Fig : Total no. of viable haemocyte count of 5th instar silkworm larvae. Data are
represented as mean ± S.E

Estimation of protein form haemolymph
Fig : Protein concentration in the larval haemolymph during developmental stages
0
10
20
30
40
50
60
Day1 Day2 Day3 Day4 Day5 Day6 SD1 SD2 PP
Amount
of
protein
(mg/ml)
Fifth instar larvae developmental stages

Fig :Analysis of acid phosphatase activity in the haemolymph of fifth instar silkworm larvae:
0
10
20
30
40
50
60
70
80
Day 4 Day 5 Day 6 SD1 SD2 PP
Acid
phosphatase
activity
µg
pi/gm/hr
5th instar silkworm larvae developmental stages

Fig .Glutathione levels in the haemolymph of fifth instar silkworm larvae:
0
1
2
3
4
5
6
7
Day 4 Day 5 Day 6 SD1 SD2 PP
µg
of
GSH/ml
hemolymph
5th instar larvae developmental stages

Separation of haemolymph proteins by SDS-PAGE
Fig :Haemolymph protein profiles of final instar silkworm larvae
Variations in protein bands were observed in SDS-PAGE of silkworm haemolymph during larval to pupal
metamorphosis. The bands corresponding to molecular weight of 37 kDa, 25 kDa and 18 kDa protein bands were
completely disappear in spinning stage and prepual stage, whereas bands corresponding to 45 kDa, 40kDa,
31 kDa and 21 kDa were found in all stages

FIG : Formaldehyde preserved Bombyx mori larvae, pupa and adult moth

Fig :Histological sections of the silkworm midgut. General aspects of the midgut: Lumen (Lu); Peritrophic membrane
(Pm); Vacuoles (V); Secretary cells (Sc); Columnar cells (C); Dead cells (Dc). Midgut sections of silkworm during
larval to pupal transformation. (A,B). Midgut sections of 5th instar day 6 larva and spinning day 1 larva. (C,D)
Midgut sections of spinning day 2 larva and prepupal stage of silkworm (magniﬁcation of the sections 200X).
Histological studies of midgut
(L5D6 ) (SD1)
(SD2) (PP)

 Silkworm larval midgut changes are very deep during larval to pupal metamorphosis.
 Histological studies confirms the occurrence of extensive modifications in the
midgut epithelium.
 During L5D6 midgut epithelial cells are large and oval shape.
 Degradation of the larval midgut starts from SD1.
 In SD1 SD2 midgut became shorter and epithelial cells were condensed.
 In PP stage all midgut epithelial cells are form apoptotic bodies of various sizes.

Fig : Formaldehyde
preserved Bombyx mori
larvae silk glands (Fifth
instar day 4, 5, 6, spinning
day 1, spinning day 2 and
prepuapl larvae silk glands.

Fig : Histological sections of
the middle silk gland of the
silkworm (Bombyx mori)
during the larval to pupal
metamorphosis: LD4: Day
four larva, LD5: Day five
larva, LD6: Day six larva,
SD1: Spinning day 1 larvae,
SD2: Spinning day 2 larvae,
PP: Prepupal stage (Lu:
Lumen, Ss: Secretary
substance, Epi: Epithelium.
F: Fibroin. (magniﬁcation of
the sections 200X)

DNA ladder assay
Fig : Electrophoretic detection
of DNA fragmentation in the
total genomic DNA isolated
from fifth instar larvae silk
gland.
Strong DNA fragmentation
with the appearance of low-
molecular-weight DNA
fragments is observed during
larval to pupal metamorphosis
(M: DNA marker).

 Apoptotic features were observed during spinning and prepupal phase, the majority of
the cells died, indicates that apoptosis is actually responsible for cell death and for the
disappearance of larval midgut and silk gland .
 Elevated levels of the marker enzyme acid phosphatase, changes in the expression of
proteins and DNA fragmentation are commonly regarded as hall marks of apoptosis.
 The present study clearly indicated that apoptosis responsible for larval to pupal
metamorphosis.
 From the above study it is concluded that silkworm as a good model to study apoptosis
and to test pathogenicity of bacteria.
 The fifth instar larvae is the best suitable for bacteria inoculation and collection of
haemolymph.

0
10
20
30
40
50
60
70
80
90
100
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
%
Viable
Haemocytes
Fifth instar silkworm larvae
Control
Infected
Fig : Changes of total no. of viable haemocytes count in the healthy (control)
and infected groups.

Estimation of protein in the haemolymph
0
10
20
30
40
50
60
70
Protein
mg/ml
Control
infected
Fig : Changes of protein concentration in the in the healthy (control) and infected
groups.

0
1
2
3
4
5
6
7
µg
of
GSH/
ml
Control
infected
Fig: Changes of gluthathione levels in the healthy (control) and infected groups.

0
5
10
15
20
25
30
35
µg
pi/gm/hr
Control
infected
Fig : Changes of acid phosphatase activity in the healthy (control) and infected
groups.

Lipid peroxidation assay
Fig : Changes of lipid peroxidation activity in the healthy (control) and S. aureus
infected groups.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
MDA
µ
mol/ml
Control
Infected

Superoxide dismutase (SOD) assay
Fig: Changes of superoxide dismutase (SOD) activity in the healthy (control) and S. aureus infected
groups.
0
1
2
3
4
5
6
7
mU
mg
protein
Control
Infected

Catalase assay
Fig : Changes of catalase activity in the healthy (control) and S. aureus infected
groups
0
1
2
3
4
5
6
μ
moles/
mg
protein/
min/ml
Fifth instar silworm larvae
Control
Infected

Phenol oxidase assay
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
µ
moles/
mg
protein/min/ml
Control
infected
Fig : Changes of Phenol oxidase activity in the healthy (control) and S. aureus infected
groups

Glutathione peroxidase assay
Fig : Changes of glutathione peroxidase activity in the healthy (control) and S. aureus
infected groups
0
10
20
30
40
50
60
NADPH
Oxidised
nmol/min/ml)
Fifth instar sikworm larvae
Control
Infected

Total No. of viable haemocytes/ml hemolymph
Fifth instar
silkworm
larvae
Day I Day II Day III Day IV Day V Day VI
Control 58.5± 0.56 63.75±2.50 72.25±1.71 78.5±0.96 81.75±2.58 83.5±4.25
Infected 42.25**±0.12 57.5**±1.29 60.5*±2.25 64.5***±2.16 70.5*±3.11 71.25*±1.27
Concentration of total proteins (mg/ml) in hemolymph
Control 19.2±0.25 25.55±2.16 31.34±1.2 33.27±0.2 39.63±1.20 47.21±1.5
Infected 23.35*±3.50 28.74**±1.2 34.5*±3.33 42.13***±1.3 48.6*±2.25 54.46*±1.2
Glutathione estimation in hemolymph (µg of GSH/ ml)
Control 2.18±3.9 4.21±4.0 4.5±1.4 5.26±0.15 5.89±1.38 6.13±0.12
Infected 1.96**±1.5 3.53***±3.5 3.9**±0.45 4.39*±2.0 4.53*±0.25 5.67***±1.6
Acid phosphatase activity (µg pi/gm/hr)
Control 22.63±1.3 20.36±0.25 18.63±2.6 16.15±0.14 16.56±1.8 19.32±0.14
Infected 27.32*±2.5 25.05*±0.17 23.96±3.5 24.23**±0.55 22.87**±2.6 26.98**±1.3
Lipid peroxidation (MDA µ mol/ml)
Control 3.14±0.12 2.82±0.78 2.86±1.5 1.63±0.055 2.25±1.75 2.53±1.45
Infected 3.75***±0.35 3.21*±2.1 3.4***±0.05 2.23*±0.86 2.75**±2.63 3.25**±0.36

Superoxide dismutase (SOD) activity (mU /min/mg protein)
5th instar Day I Day II Day III Day IV Day V Day VI
Control 5.72±0.12 4.82±1.89 3.52±0.68 2.87±0.44 1.54±0.11 1.15±0.07
Inoculated 4.98**±0.36 3.32*±0.19 2.41***±0.45 1.92**±0.44 0.97*±0.16 0.51*±0.22
Catalase activity (μ moles H2O2/ mg protein/ min/ml)
Control 4.24±2.0 3.25±3.1 2.55±0.45 1.94±2.9 1.74±0.12 1.26±0.15
Inoculated 3.96***±1.7 2.97*±1.7 1.83*±1.45 1.61***±4.0 1.25**±0.15 0.95*±1.7
Phenoloxidase activity (µ moles/ mg protein/min/ml )
Control 052±0.042 0.49±0.013 0.41±0.028 0.38±0.013 0.36±0.020 0.25±0.016
Inoculated 0.62**±0.012 0.57**±0.018 0.52*±0.020 0.45*±0.018 0.42***±0.010 0.36**±0.013
Glutathione peroxidase (nmol/min/ml)
Control 28.6±1.5 32.5±3.4 35.7±2.9 42.2±5.5 48.3±8.2 38.3±1.3
Inoculated 23.3**±2.3 26.6***±1.6 30.5*±1.7 36.6*±4.5 42.5**±1.2 35.2***±2.6
Values are mean ± SE from 8 silkworms in each group; Values with different superscripts within
the rows are significantly different at *P≤0.05, **P≤0.01, ***P≤0.001.

SDS –PAGE of silkworm haemolymph
Fig 21: Haemolymph protein profiles of the (A) Healthy (control) and (B) S. aureus infected groups.

Histological studies of mid gut
Fig 22: Mid gut sections from
day 1 to day 6th of fifth instar
silkworm larvae (Control
group). General aspects of
the midgut: Lumen (Lu);
Vacuoles (V); Columnar cells
(C); Dead cells (Dc);
Intracellular space (ie);
Epithelium (Ep); Microvilli
(Mv). 200x

Fig 23: Mid gut sections from day 1
to day 6th of fifth instar silkworm
larvae (Infected group). General
aspects of the midgut: Lumen (Lu);
Vacuoles (V); Columnar cells (C);
Dead cells (Dc); Intracellular space
(ie); Epithelium (Ep); Microvilli
(Mv). 200x

Infected group
Mid gut sections of Control group

Giemsa staining
Fig : Viable haemocytes from the healthy silkworm (Bombyx mori. L) larvae (100x).
Fig : S. aureus induced haemocyte aggregation in infected silkworm (100x)

Mortality:
5th instar silkworm larvae Number of
larvae tested
Mortality
Day 1 Control 20 0
Day 1 Infected 20 12
Day 2 Control 20 0
Day 2 Infected 20 8
Day 3 Control 20 0
Day 3 Infected 20 5
Day 4 Control 20 0
Day 4 Infected 20 4
Day 5 Control 20 0
Day 5 Infected 20 2
Day 6 Control 20 0
Day 6 Infected 20 2
Table: Relationship
between S. aureus
pathogenicity and
silkworm larvae.

Measurement of the silk gland weight
Fifth instar silkworm
larvae
Control group silk
gland weight in grams
(Mean values)
Infected group silk
gland weight in grams
(Mean values)
Day 1 0.17 0.12
Day 2 0.48 0.35
Day 3 0.57 0.42
Day 4 1.28 0.85
Day 5 1.42 1.20
Day 6 2.21 1.74
Table: Measurement of silk gland weight of the control and infected larvae.

Antimicrobial activity of haemolymph extract
Tested microorganisms 40 µl 30µl 20µl 10µl
Bacillus subtilis ++ + + -
Staphylococcus aureus ++ ++ + +
Escherichia coli + + - -
Salmonella typhi + + - -
Vibrio Cholerae + + - -
Aspergillus fumigatus ++ + + +
Candida albicans + + - -
+ : inhibition value 0.1-0.5 cm
++ : inhibition value 0.6-1 cm
Table : Antimicrobial activity of silkworm haemolymph extract

In silico molecular docking of Moricin and Cecropin with S. aureus
drug target proteins
Fig : The 3D structures of Moricin (A) and Cecropin (B) peptides from Bombyx mori.

Fig : 3D structures of (A) Glycerol phosphate lipoteichoic acid synthase (B) Dipeptide ABC
transporter and (C) DNA Gyrase from S. aureus

Target protein Peptides (ligands)
Moricin Cecropin
Glycerol phosphate lipoteichoic acid
synthase
-406.69 -576.67
Dipeptide ABC transporter -538.54 -634.57
DNA gyrase -702.13 -639.39
Table : HEX Docking energy (kcal/mol) for Moricin and Cecropin peptides
with target proteins in S. aureus

Target protein Interacting amino acids
In target protein
Amino acids involved in H-bonding
Moricin Cecropin
Glycerol phosphate
lipoteichoic acid
synthase
Lys 597,Gln 231,Glu 522
and
Leu 322,Tyr 417,Asn 482
Asn 33,Lys 36,Lys
38
Arg 16,Glu
9,Gln 31
Dipeptide ABC
transporters
Gly 172,Asn 144,Gln 193
and Lys 6
Arg 20,Asn 23,
Asp 30
Val 210
DNA gyrase Lys 581,Arg 1048,Glu
1156,Gln 1368 and Ala
509,Met 1029,Gly
1178,Thr 1181
Val 15,Arg 20,Asn
23,Ala 42
Glu 9,Arg
16,Ala 33,Lys
37
Table : Amino acids involved in H-bonding between target protein and ligands.

Fig : Docking conformation of Glycerol Phosphate Lipoteichoic Acid Synthase with (A) Moricin
and (B) Cecropin. The interacting amino acid are represented in sticks and the protein is
displayed in cartoon conformation. Moricin and Cecropin cartoons represented in magenta
colour.
A B

A B
Fig : Docking conformation of dipeptide ABC transporter-PG110with (A) Moricin and (B) Cecropin.
The interacting amino acid is represented in sticks and the protein is displayed in cartoon conformation.
Moricin and Cecropin cartoons represented in magenta colour and yellow color respectively.

A B
Fig : Docking conformation of DNA Gyrase with (A) Moricin and (B) Cecropin. The interacting amino
acid are represented in sticks and the protein is displayed in cartoon conformation. Moricin and
Cecropin cartoons represented in magenta and grey white color respectively.

Synthesis of sericin silver nanoparticles
Fig : (A) Silkworm cocoons (B) The sericin
colloid solution color change from
colorless to brown color after 24 h of
incubation with AgNO3.

Fig : (A). UV-Visible spectroscopic absorption peak of sericin colloid solution at 276 nm
(B). UV-Visible spectroscopic absorption spectra of sericin silver nanoparticles.

Fig : Antimicrobial activity of sericin silver nanoparticles on (A) S. aureus and (B) E. coli.
Zone of inhibition for the 40 μl Concentration sericin silver nanoparticles
Staphylococcus aureus: 9 mm
E. coli: 12 mm

Conclusion
 The present study clearly indicated that apoptosis responsible for larval to pupal
metamorphosis.
 Silkworm as a good animal model to study apoptosis and test pathogenicity of bacteria.
 Silkworms are a promising infection model to study apoptosis and for the identification and
evaluation of virulence factor of pathogenic microorganisms
 S. aureus have ability to induce apoptosis in silkworm by altering antioxidant system and
cause death by damaging the midgut through the exotoxins.
 The immune response induced against S. aureus in response to infection by the synthesis of
antimicrobial peptides in the silkworm haemolymph.
 The isolated haemolymph extract from infected silkworm larvae consists of peptides shows
antimicrobial activity on S. aureus and other pathogenic bacteria.

Applications of research work
 This work is helpful to understand the mechanism of metamorphosis in insects.
 To understand ability of bacteria to induce apoptosis and immune system in
invertebrates system
 This work useful to find biological therapeutic functions of insect extractions, which
include anti-tumor and anti-bacterial activities.

A C K N O W L E D G M E N T S
 I express my profound feeling of gratitude and indebtedness to my teacher and research supervisor
Prof. B. Venkatappa, Chairman, Board of Studies in Microbiology, Sri Krishnadevaraya University, Ananthapuramu,
for assigning this research topic, invaluable guidance, constant encouragement and valuable suggestions throughout my
research work.
 I am indebted to the faculty of the Department of Microbiology, Prof. K. Venkateswarlu (Rtd.),
Prof. R. Ramanjaneyulu (Rtd.), Prof. P.B.B.N. Charyulu (Rtd.), Prof. B. Rajasekhar Reddy (Head) and
Prof. V. Rangaswamy, for their encouragement, cooperation and providing the necessary facilities to carry out research
work.
 I would like to gratefully acknowledge DST, New Delhi for the award of INSPIRE Fellowship for the year 2011 and
supporting me with financial assistance.
 I am thankful to my senior research scholars and co-scholars .
 I thank to the nonteaching staff of the Department of Microbiology.
 I am thankful to Dr. M. Raghupathi, Regional Sericultural Research Station, Anantapuramu and for provided
silkworms for our research.
 I am thankful to Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad. for provided
bacterial cultures for my research work.
 I would like to acknowledge service rendered by Bioserve Labs, Hyderabad for DNA ladder assay.
 I am thankful to Sri Sathya Sai Institute of Higher Medical Sciences and Sri Venkateswara Veterinary University,
Tirupati for histopathology work.

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