Fish & Shellfish Immunology 57 (2016) 243e251

Contents lists available at ScienceDirect

Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Short communication

The cellular death pattern of primary haemocytes isolated from the

black tiger shrimp (Penaeus monodon)
Kwanta Thansa a, *, Patchari Yocawibun a, b, Hathaitip Suksodsai a
a
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin
Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
b
Center of Excellence for Marine Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: A key to successfully generate the penaeid shrimp cell line is to find out how primary cells died. The most
Received 8 April 2016 suitable period to culture Penaeus monodon haemocytes was in the first 48 h of culture because cells had
Received in revised form normal morphology, high percent of viable cells (65.29 ± 5.43%), low percent of early (11.75 ± 1.30%) and
3 August 2016
late apoptotic cells (15.47 ± 11.71%) determined by Annexin V and TUNEL including constant IAP
Accepted 20 August 2016
Available online 22 August 2016
(0.06 ± 0.01e0.07 ± 0.01) and caspase-3 expression (0.30 ± 0.06e0.39 ± 0.10) by real-time PCR
throughout the experiment. Moreover, adding 50 and 250 mM of the cell permeable pan caspase inhibitor
Z-VADeFMK produced some melanised cells since the 48th hour, while percent of viable cells was
Keywords:
Penaeus monodon
decreased since the 24th hour with no difference in percent of early and late apoptotic cells compared to
Cell culture control at each time point. No difference of IAP and caspase-3 expression level in both Z-VADeFMK
Z-VADeFMK groups was found compared to control and vehicle groups at each time point, excluding caspase-3 in
Sodium fluoride 250 mM Z-VADeFMK at the 24th hour was higher than control and vehicle. Supplementing sodium
Apoptosis fluoride (NaF) induced cell membrane damage and cellular shrinkage of primary haemocytes within 2 h.
IAP Even percent of viable cells was reduced down to zero and percent of late apoptotic cells was increased
Caspase-3 by 2 h of incubation in 25 and 50 mM NaF, IAP and caspase-3 in all NaF groups was not different from
control. These results indicate that a number of primary haemocytes derived in this study die through
the apoptotic process.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction Apoptosis is an active form of cell death identified by cellular

To succeed in the establishment of permanent cell lines is one of
the key factors for increasing the knowledge of developmental
biology, molecular biology and biotechnology in animals [1]. In
crustacean, no continuous cell line has been established even many
approaches e.g. improving the culture conditions, transformation
and cellular fusion have been made [2]. If the map of cell death
mechanism of each type of primary cells could be drawn clearly, it
would further help in finding the way to generate continuous cell
lines in the shrimp. In this study, haemocyte, which plays an
important role in immunological system and disease prevention
[3], was selected to be the model for investigation of cell death
mechanism.
* Corresponding author.
E-mail addresses: kwanta.tha@biotec.or.th, kthansa@yahoo.com (K. Thansa),
patchari.yoc@biotec.or.th (P. Yocawibun), hathaitip.suk@biotec.or.th (H. Suksodsai).
shrinkage, nuclear and cytoplasm condensation, chromatin frag-
mentation, cell membrane blebbing without no loss of cellular
integrity and phagocytosis of dying cell. While, necrosis is a passive
process in association with cellular and organelle swelling, plasma
membrane rupture and leakage of cellular contents into the
extracellular compartment. Different kinds of factors may stimulate
either apoptotic or necrotic cell death depending on the type of
cells and severity of injury. Moreover, the death of apoptotic cells
requires the sufficient intracellular energy level and the redox state
compatible with the caspase activation in order to complete the cell
suicidal programme. Therefore, the depletion of ATP or severe
oxidative stress possibly drives the apoptotic cell death to necrosis
[4,5].
In the black tiger shrimp, some well-known key factors regu-
lating the mechanism of apoptotic cell death e.g. caspase-3 and
inhibition of apoptosis protein (IAP) are characterised [6,7], and
morphological changes have been examined by some staining
markers e.g. propidium iodide (PI), Hoechst, 40 ,6-diamidino-2-

http://dx.doi.org/10.1016/j.fsi.2016.08.045
1050-4648/© 2016 Elsevier Ltd. All rights reserved.
244 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251
phenylindole (DAPI), Annexin V and terminal deoxynucleotidyl
transferase-mediated deoxy-UTP nick-end labelling (TUNEL) assay,
including the analysis of DNA fragmentation, and the investigation
of caspase-3 expression, level and activity [6e12]. However, the
determination of cellular death mechanisms with primary cell
cultures has not been reported in the penaeid shrimp before. Some
experiments dealing with cell death mechanisms were conducted
in the isolated haemocytes incubated with nitrite for up to 3 h and
lipopolysaccharide (LPS) for up to 2 h before they were then taken
to investigate the apoptotic cell ratio by flow cytometry in
P. monodon [10,13].
In this present study, our first experiment was aimed to figure
out the most suitable culture period of primary haemocytes iso-
lated from P. monodon by examining the cellular death character-
istics of primary haemocytes cultures at different time points
before other chemical substances e.g. N-Benzyloxycarbonyl-Val-
Ala-Asp-[O-methyl]-fluoromethyl ketone (Z-VADeFMK), a cell-
permeant pan caspase inhibitor that irreversibly suppresses the
function of caspase proteases and subsequently in the inhibition of
apoptotic induction in many mammalian cell types both in vitro and
in vivo [14e16], in association with being known as a chemical-
induced necrotic cell death via the kinases of the receptor inter-
acting proteins [17e20], which is called necroptosis [21], was used
to test its effect with primary haemocytes in the second experi-
ment. Whereas, fluoride, another chemical substance abundant on
the surface of earth that can be found as small amounts in water, air,
plants, and animals [22], was selected to determine its effect with
primary haemocytes in the third experiment. Fluoride is also well-
known as the element producing a variety of cellular and metabolic
changes e.g. glycolysis inhibition, changes in membrane receptors
and apoptotic induction. Supplement of NaF as the main source of
fluoride to mammalian cells can induce apoptosis both in vitro and
in vivo via the reactive oxygen species (ROS)-dependent, caspase-
mediated and JNK signalling pathways [4,22e24]. Morphological
changes and analysis of Annexin V, a well-known early apoptotic
detection marker [25], and TUNEL, the outstanding detection assay
of late apoptosis [9,26], including the investigation of the expres-
sion level of IAP and caspase-3 genes by real-time PCR technique,
were undertaken to indicate the cellular death pattern of primary
haemocytes in this study.
2. Materials and methods
2.1. Chemicals
Most of chemical substances and reagents used in this study
were purchased from Sigma-Aldrich.
2.2. Animals
Both males and females of juvenile black tiger shrimp at the age
of 4e6 months weighing between 15 and 20 g were purchased
from Shrimp Genetic Improvement Center, Surat Thani, Thailand,
they were kept at Shrimp Biotechnology Business Unit, Pathum
Thani, Thailand, before use.
2.3. Preparation of haemocyte suspension
Haemolymph was withdrawn into a syringe half filled with 10%
sodium acetate and then centrifuged at 3500 rpm for 5 min at 4  C.
The cell pellet was roughly washed with 10% sodium acetate twice
before being washed once with culture medium [2 Libovitz L-15, 5%
(v/v) lactalbumin solution, 100 units/ml penicillin, 100 mg/ml strep-
tomycin, 1% fungisone (v/v) and 10% each of FCS and shrimp meat
extract]. Then, culture medium was used to resuspend the pellet and
cells were seeded at the density of 0.8e1 x 105 cells/well of 96-well
plate. Trypan blue staining was the first method used to determine
cell viability of primary haemocytes [27]. If the positive result of try-
pan blue staining of primary haemocytes was less than 5%, cells iso-
lated from that shrimp would be further used in the experiments.
2.4. Experimental designs
There were three experiments conducted in this study. The first
one was to find out the most suitable culture period of primary
haemocytes by determining the death characteristics of primary
haemocytes cultured in the medium at 24, 48, 72 and 96 h of cul-
ture. The second one was aimed to test the effect of Z-VADeFMK at
the dosage of 50 and 250 mM on primary haemocyte cultures
whether it can prolong primary haemocytes in the culture or not,
while the last experiment was to examine the effect of NaF at the
concentrations of 5, 25 and 50 mM on primary haemocytes. In the
second experiment, no addition of Z-VADeFMK in the culture
medium was used as the control group, while supplement of 0.2%
DMSO in the medium was represented as the vehicle control. In
case of the third experiment, no NaF supplemented in the culture
medium was served as the control group. Culture medium was
changed half daily and cells were roughly determined for the
apoptotic and necrotic characteristics by using the morphological
changes. The analysis of apoptotic cell death was performed using
Annexin V and TUNEL assays, together with examining the level of
IAP and caspase-3 genes by real-time PCR technique.
2.5. Detection of cellular morphological changes by Annexin V assay
Samples obtained from primary haemocytes seeding at the
density of 0.8e1 x 105 cells/well of 96-well plate in the culture
medium for 24, 48, 72 and 96 h, together with samples collected
from primary haemocytes treated with different dosages of Z-
VADeFMK for 24 and 48 h including those added with different
concentrations of NaF for 10 min, 2 and 24 h were analysed with
Annexin V assay modified from the manufacturer's instructions
(catalog no. ab66108, abcam). Briefly, cells were washed twice with
cold PBS (3) and then followed by Annexin-binding buffer (1)
once. The combination of 50 ml Annexin-binding buffer (1) and
2.5 ml Annexin V conjugate was added and incubated with cells at
room temperature for 30 min in the dark before 1 ml of 100 mg/ml PI
working solution was applied to incubate with cells at room tem-
perature in the dark for 5 min. Then, samples were washed 3 times
with Annexin-binding buffer (1). Finally, cells were observed
under an Olympus inverted fluorescence microscope (Olympus
Corporation, Tokyo, Japan). The excitation of 494 nm and the
emission of 518 nm were undertaken for detecting Annexin V in-
tensity, and the excitation of 535 nm and the emission of 617 nm for
PI. Apoptotic cells showed Annexin V staining of the cellular
membrane, while the late stage of apoptosis or necrosis showed
both membrane staining by Annexin V and strong nuclear staining
from the red PI, or only PI positive staining. The percent of viable
cells was calculated by this equation, {[(Numbers of total cells -
early apoptotic cells e late apoptosis/necrosis)/numbers of total
cells] x 100, the percent of early apoptosis was calculated by this
equation, {[(Numbers of Annexin V-positive cells - merge cells)/
numbers of total cells] x 100} and the percent of late apoptosis/
necrosis was calculated by this equation, {[(Numbers of PI-positive
cells)/numbers of total cells] x 100}. In general, 2e4 representative
fields of at least 100 cells were captured and scored. These pictures
were taken using a magnification of 640.
 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251
2.6. Detection of cellular morphological changes by TUNEL assay
Samples used to examine with TUNEL assay were similar to
those tested with Annexin V. The procedure of TUNEL detection
used in this study was modified based on the manufacturer's in-
structions (catalog no. V13242, Molecular Probes®, Inc., Eugene,
OR). In brief, samples were fixed with 4% (w/v) paraformaldehyde
and left on ice for 15 min. Then, cells were washed 3 times with cold
PBS (3). Ice-cold 70% (v/v) ethanol was added to samples and let
cells stand for a minimum of 30 min in the freezer before cells were
washed twice with wash buffer. Then, samples were covered with
200 ml equilibration buffer and 50 ml of TUNEL reaction mixture for
5 and 60 min at 28  C respectively. The reaction was stopped by
washing the samples 3 times in the rinse buffer. Thereafter, samples
were counterstained with PI/RNase A solution at room temperature
for 30 min in the dark and finally mounted with Vectashield H-100.
Then, samples were observed and captured under an Olympus
inverted fluorescence microscope (Olympus Corporation, Tokyo,
Japan). The excitation of 495 nm and the emission of 519 nm were
undertaken for detecting FITC-labeled cell intensity, and the exci-
tation of 535 nm and the emission of 617 nm for PI. Generally, 2e4
representative fields of at least 100 cells were scored for TUNEL-
positive cells, which were represented as late apoptotic cells. The
late apoptotic cells showed the strong nuclear staining with FITC.
The percent of late apoptosis was calculated by this equation,
{[(Numbers of TUNEL-positive cells)/numbers of total cells] x 100}.
These pictures were taken using a magnification of 640.
2.7. Total RNA extraction, first strand cDNA synthesis and
determining the expression level of apoptotic-related genes by real-
time PCR analysis
TRI reagent (Molecular Research Center) was used to extract
total RNA contents of primary haemocyte cultures and DNase-free
DNase I (Qiagen, Germany) was undertaken to remove the residual
genomic DNA. Total RNA (1 mg) was reverse transcribed to cDNA
using PrimeScript™ first strand cDNA synthesis kits according to
the manufacturer's protocols. The set of primers of caspase-3 and
IAP were designed from the previous works [6,28]. The elongation
factor-1a (EF-1a) was served as a house-keeping gene in this study.
Each set of the following pairs of primers used to determine the
level of apoptotic-related genes was composed of the accession
number, forward and reverse primers and the product size as listed;
caspase-3181: JQ241180.1, F: 50 -CGGTTCAGACGGACAGCATT-30 , R: 50 -
GGTCGTGGAGAAGATAAGGCA-30 , 181 bp; IAP120: EF114675.1, F: 50 -
GACCTGACTGCCCCTTTGC-30 and R: 50 -AGTCAATGAAAGTCTCCAATC
GCT-30 , 120 bp; and EF-1a214: DQ021452, F: 50 -TTCGTCTTCC
CCTTCAGGACGTC-30 and R: 50 -CTTTACAGACACGTTCTTCACGTTG-30 ,
214 bp. These genes were investigated in each group of this
experiment.
The range of 103-108 copies of recombinant plasmids of caspase-
3181, IAP120 and the internal control EF-1a214 was used to plot its
own standard curve. These genes were examined in each experi-
mental group of these three experiments. The amplification reac-
tion volume of 10 ml in total was composed of 5 ml of 2  LightCycler
480 SYBR Green I Master (Roche), 50 ng of first strand cDNA tem-
plate and 0.3 mM of each primer. The thermal profile for quantita-
tive real-time PCR of EF-1a214 was 95  C for 10 min followed by 40
cycles of 95  C for 30 s, 58  C for 30 s and 72  C for 30 s, for caspase-
3181 was 95  C for 10 min followed by 40 cycles of 95  C for 15 s,
62  C for 30 s and 72  C for 30 s, and for IAP120 was 95  C for 10 min
followed by 40 cycles of 95  C for 15 s, 65  C for 30 s and 72  C for
20 s. Real-time PCR of each sample was done in duplicate. The re-
sults were represented as the relative expression level calculated
using the copy number of target genes e.g. caspase-3181 or IAP120/
245
that of EF-1a214 in the same sample.
2.8. Statistical analysis
Results are presented as mean ± standard error of the mean
(S.E.M.). The data were analysed for significance by one-way
ANOVA and then followed by Duncan multiple comparison tests
using SPSS Software (SPSS Inc., USA). The test value of p < 0.05 was
considered as the significant result.
3. Results
3.1. Determining the apoptotic and necrotic characteristics of
primary haemocytes in the culture medium
Primary haemocytes derived in this study had a variety of
shapes e.g. round (5e10 mm), spherical (10e15 mm) and elongated
(20 mm) (Fig. 1A). Some of them attached to the bottom surface of
the tissue cultured dish within an hour. These intact primary cells
could be maintained for up to 2e4 days without microbial
contamination and melanisation. When these primary cells were
cultured for longer than a week, elongated cells were mainly found
in the culture with a lot of cell debris (Fig. 1B). Consequently, clump
colonies were formed (Fig. 1C) and melanised cells were finally
observed (Fig. 1D).
To figure out the pattern of cell death mechanism of primary
haemocyte cultures, Annexin V, a well-known early apoptotic
marker, and TUNEL, the outstanding late apoptotic marker, were
used in this study. It was found that the percent of viable cells of
primary haemocytes cultured in the medium for 24 and 48 h were
higher than those at 72 and 96 h of incubation, but no difference
was found between the percent of viable cells of primary haemo-
cytes cultured in the medium at 24 and 48 h of cultures determined
by Annexin V assay (Fig. 2A and Table 1). Also, the percent of viable
cells of primary haemocytes cultured in the medium for 72 h was
higher than those at 96 h of incubation. While, the percent of early
apoptotic cells of primary haemocytes cultured in the medium for
24 and 48 h were lower than the 96 h of incubation, but no dif-
ference was found among the percent of early apoptotic cells of
primary haemocytes cultured in the medium at 24, 48 and 72 h of
cultures including no difference between the percent of early
apoptotic cells of primary haemocytes cultured in the medium at 72
and 96 h of incubation examined by Annexin V assay (Fig. 2A and
Table 1). Moreover, the percent of late apoptotic cells at 72 and 96 h
of incubation were higher than those at 24 h, but there was no
difference between these two groups and those at 48 h of culture
investigated by TUNEL assay (Fig. 2B and Table 1). Furthermore, the
analysis of real-time PCR demonstrated that there was no statistical
difference in the expression level of IAP120 and caspase-3181 in
primary haemocytes cultured at any time point (Data is not
shown).
3.2. Determining the apoptotic and necrotic characteristics of
primary haemocyte cultures supplemented with different dosages of
Z-VADeFMK
Various morphological characteristics of primary haemocytes
described in the result of section 3.1 were also observed in all
groups of this experiment. Since 48 h of culture, some melanised
cells were found in both Z-VADeFMK treated groups. However,
most primary haemocytes in all experimental groups remained
intact for up to 48 h. Thereafter, a number of primary cells in all
experimental groups were lost their good appearance since 72 h of
culture; the elongated ones tended to survive in the culture longer
than others.
246 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251

Fig. 1. Morphology of primary haemocytes derived in this study. (A) Primary haemocytes have a variety of shape e.g. round, spherical and elongated with intact cell membrane
indicated as good quality of primary haemocytes. (B) Elongated cells are presented with cell debris. (C) Clump colonies are formed in the culture as circled. (D) Melanised cells are
indicated as arrowed. (E) Shrinkage cells. Scale bar ¼ 20 mm.

Fig. 2. Apoptotic detection of primary haemocytes cultured in culture medium for 24, 48, 72 and 96 h. (A) Annexin V staining is used to detect early apoptotic cells; cell
membranes of this apoptotic cells are only stained with Annexin V-FITC conjugates. While the late stage of apoptosis or necrosis could have both membrane staining by Annexin V-
FITC and strong nuclear staining from the red PI, or only PI positive staining (n ¼ 5). (B) Late apoptotic cells show nuclear staining with FITC by TUNEL assays (n ¼ 3). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1 Moreover, it was shown that the percent of viable cells in 50 and
Percent of viable, early and late apoptotic cells of primary haemocytes cultured for 250 mM Z-VADeFMK treated groups were less than the control and
24, 48, 72 and 96 h in culture medium analysed by Annexin V and TUNEL assays. vehicle groups at 24 h of incubation (Table 2). By 48 h of incubation
Hours Viable cells (%) Early apoptotic cells (%) Late apoptotic cells (%) the percent of viable cells in 50 and 250 mM Z-VADeFMK treated
24 69.29 ± 3.34c
13.62 ± 1.54a
2.57 ± 0.99a
groups were only less than the control group. There was no dif-
48 65.29 ± 5.43c 11.75 ± 1.30a 15.47 ± 11.71ab ference between the percent of viable cells in its own experimental
72 51.68 ± 4.43b 18.99 ± 3.99ab 32.10 ± 4.67b group at 24 and 48 h of culture, except the vehicle group showing
96 37.21 ± 3.54a 23.82 ± 2.50b 40.15 ± 8.81b that the percent of viable cells at 24 h of incubation was higher than
Data are represented as mean ± S.E.M. (n ¼ 5) and different alphabet represents the those at 48 h. Whereas, the percent of early apoptotic cells in 50 and
statistically significant difference (p < 0.05, one-way ANOVA, Duncan multiple 250 mM Z-VADeFMK treated groups were less than vehicle at the
comparison test).
24th hour (Table 2), but there was no difference between these two
groups and the control at 24 h of culture. No significant difference
 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251 247

Table 2
Percent of viable, early apoptotic and late apoptotic cells of primary haemocytes treated with 50 and 250 mM Z-VADeFMK for 24 and 48 h by Annexin V and TUNEL assays.

Hours Groups Viable cells (%) Early apoptotic cells (%) Late apoptotic cells (%)

24 Control 62.03 ± 3.81c 11.77 ± 3.00ab 9.58 ± 3.54a

Vehicle 62.42 ± 1.35c 18.49 ± 2.34b 9.77 ± 2.48a
50 mM 50.86 ± 4.19ab 7.66 ± 1.42a 15.42 ± 4.48ab
250 mM 44.87 ± 3.87ab 7.23 ± 1.42a 14.76 ± 1.48ab
48 Control 54.56 ± 4.64bc 17.63 ± 2.46b 23.70 ± 5.14ab
Vehicle 48.70 ± 3.75ab 15.83 ± 3.11b 23.87 ± 7.06ab
50 mM 41.22 ± 0.90a 13.96 ± 2.66ab 27.07 ± 7.94ab
250 mM 42.54 ± 2.93a 18.70 ± 2.25b 29.30 ± 6.77b

Data are represented as mean ± S.E.M. (n ¼ 4) and different alphabet represents the statistically significant difference (p < 0.05, one-way ANOVA, Duncan multiple comparison
test).

in the percent of early apoptotic cells was also found among the
experimental groups at 48 h of culture. Moreover, there was no
difference between the percent of early apoptotic cells in its own
experimental group at 24 and 48 h of culture, except 250 mM Z-
VADeFMK treated group demonstrating that the percent of early
apoptotic cells at 24 h of incubation was less than those at 48 h. In
case of the percent of late apoptotic cells, no difference was found
in its own experimental group at 24 and 48 h of culture (Table 2),
excluding that the percent of late apoptotic cells of 250 mM Z-
VADeFMK treated group at 48 h of culture was higher than the
control and vehicle groups at the 24th hour. To determine the level
of the apoptotic-related genes using real-time PCR method, it
revealed that the level of IAP120 in 250 mM Z-VADeFMK treated
group was less than the 50 mM Z-VADeFMK group at 24 h of the
experimental period, but not different from those in the control and
vehicle (Fig. 3). Also, the expression level of IAP120 was not different
among control, vehicle and 50 mM Z-VADeFMK groups at 24 h of
culture. In addition, no statistical difference in the relative
expression level of IAP120 was found among these four groups at
the 48th hour. Moreover, there was no significance between the
level of IAP120 in its own group at 24 and 48 h of culture, except the
50 mM Z-VADeFMK group that the expression level of IAP120 at the
48th hour was less than its own group and vehicle at the 24th hour.
Even the relative level of caspase-3181 in 250 mM Z-VADeFMK
treated group at 24 h of culture was higher than the control and
vehicle groups, it was not different from the 50 mM Z-VADeFMK
treated group at the same period of culture. Additionally, the level
of caspase-3181 was not different among control, vehicle and 50 mM
Z-VADeFMK groups at 24 h of culture. At the 48th hour, the
expression of caspase-3181 was found no significant difference
among these four experimental groups. Yet, the level of this gene
tended to increase from the value around 0.336e1.477 at 24 h of
incubation to be 1.305e1.655 at 48 h of the experiment. Moreover,
no difference between the expression level of caspase-3181 gene
was revealed in its own group at 24 and 48 h of the experimental
period, while the level of this gene in the group of 50 mM Z- Fig. 3. The relative expression level of IAP120 and caspase-3181 determined by real-
VADeFMK treatment at the 48th hour and 250 mM Z-VADeFMK time PCR technique in primary haemocytes added with 50 and 250 mM Z-
VADeFMK for 24 and 48 h. These results are presented as mean ± S.E.M. (n ¼ 4) and
treated group at 24 and 48 h of culture were higher than the control
different alphabet states the significant difference (p < 0.05, one-way ANOVA, Duncan
and vehicle at the 24th hour. multiple comparison test).

3.3. Determining the apoptotic and necrotic characteristics of

primary haemocyte cultures supplemented with different dosages of
NaF
A variety of primary haemocyte shapes e.g. round, spherical and
elongated ones as previously found in the result of section 3.1 and
3.2 were observed in the control group for up to 72 h without
forming clump colonies and melanised cells. A lot of cell mem-
branes of primary haemocytes derived from culture medium sup-
plemented with 50 mM NaF were damaged and cells shrank within
10 min of culture (Fig. 1E), while those in the control, 5 mM and
25 mM NaF treated groups still looked good. Within two hours of
culture, a number of cell membranes of primary haemocytes in the
group of 5 mM and 25 mM NaF were damaged and cells shrank.
Since 24 h of incubation, lots of melanised cells were observed in
the group of 25 mM NaF, while a number of primary haemocytes
formed as groups in 5 mM NaF treated group were seen in the
culture at 48 h of cultivation and a lot of melanised cells were
detected since the 72nd hour.
Regarding the result of viable cells of primary haemocytes treated
with NaF, it was found that the percent of viable cells in 25 mM and
248 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251

50 mM NaF treated groups were less than the control and 5 mM NaF Table 3B
treated group at 2 h of incubation; the percent of viable cells of these Percent of late apoptotic cells of primary haemocytes treated with 5, 25 and 50 mM
sodium fluoride for 2 and 24 h by TUNEL assay.
two groups were zero (Table 3A). Whereas, the percent of viable cells
in 5 mM NaF treated group was less than the control at 48 h of Hours Groups Late apoptotic cells (%)
culture. Additionally, the percent of viable cells in the control and 2 Control 1.05 ± 0.27a
5 mM NaF treated group at the 48th hour were less than those in its 5 mM 2.57 ± 0.57a
own group at 2 h of incubation. In case of the percent of early 25 mM 54.81 ± 5.44bc
50 mM 56.64 ± 10.93bc
apoptotic cells, no difference was found among all experimental
24 Control 24.34 ± 20.90a
groups (Table 3A), excluding that the percent of early apoptotic cells 5 mM 17.38 ± 12.55a
in the groups of 25 mM and 50 mM NaF were significantly lower 25 mM 81.18 ± 8.90c
than the result of 5 mM NaF treated group at 2 h of culture, together 50 mM 66.46 ± 8.89c
with the higher percent of early apoptotic cells in the control at 24 Data are represented as mean ± S.E.M. (n ¼ 3) and different alphabet represents the
and 48 h of incubation than its own group at the 2nd hour. statistically significant difference (p < 0.05, one-way ANOVA, Duncan multiple
As it was shown that the percent of viable cells was zero in each comparison test).

group of 25 mM and 50 mM NaF at 2 h of incubation; therefore

primary haemocytes treated with 25 mM and 50 mM NaF were not
further cultured in order to be used for Annexin V examination at
the 24th and 48th hour. They were also prepared in cultures for only
2 and 24 h to test with TUNEL assay and for 10 min, 2 and 24 h to
determine the expression level of IAP and caspase-3 genes by real-
time PCR technique, as previously seen that no viable cell was
found in those two high doses of NaF since 2 h of culture. It was
revealed that the percent of late apoptotic cells in 25 mM and
50 mM NaF treated groups were higher than the control and those
treated with 5 mM NaF at 2 and 24 h of incubation (Table 3B). No
difference in the percent of late apoptotic cells was found between
the groups of primary haemocytes treated with 25 mM and 50 mM
NaF at each time point. The quantitative real-time PCR showed that
no significant difference in the expression of IAP120 in all experi-
mental groups of the NaF experiment was found in both 10 min and
2 h of incubation (Fig. 4). At the 24th hour, the level of IAP120 in the
25 mM NaF group was higher than other groups, while this level in
50 mM NaF treated group was lower than 5 and 25 mM NaF treated
groups. No difference in this expression level was revealed between
control and the 5 mM NaF group at this time point. The level of
IAP120 in 25 mM NaF treated group at the 24th hour was higher than
its own expression level at 10 min and 2 h of culture, and higher
than the level in other experimental groups at all time points. In
case of the relative expression level of IAP120 in the 5 mM NaF group
at 24 h of incubation, it was found higher than the level in its own
group at 10 min of the culture period. At this time point, the relative
expression level of IAP120 in this 5 mM NaF group was also higher
than this level in the control at 10 min and 2 h of the culture period,
the 25 mM NaF group at 10 min and the 50 mM NaF group at all
window periods. No difference in the relative expression level of
IAP120 in the control and 50 mM NaF treated group was shown in its
own group among treatment periods. Furthermore, no significant
difference of the relative expression level of caspase-3181 was
Fig. 4. The relative expression level of IAP120 and caspase-3181 examined by real-
demonstrated in any experimental group at any time point. time PCR method in primary haemocytes tested with various dosages of sodium
fluoride at different time points. These results are demonstrated as mean ± S.E.M.
(n ¼ 4) and different alphabet represents the statistical difference (p < 0.05, one-way
ANOVA, Duncan multiple comparison test).
Table 3A
Percent of viable and early apoptotic cells of primary haemocytes tested with 5, 25
and 50 mM sodium fluoride at different time points by Annexin V technique.
4. Discussion
Hours Groups Viable cells (%) Early apoptotic cells (%)

2 Control 78.53 ± 6.63c 6.01 ± 1.76ab Many patterns of cell death have been reported in animals;
5 mM 53.02 ± 13.36bc 12.01 ± 3.67bc apoptosis and necrosis are two major forms of death basically
25 mM 0.00 ± 0.00a 0.61 ± 0.28a
described in the cell. Apoptosis plays an important role in normal
50 mM 0.00 ± 0.00a 0.65 ± 0.52a
24 Control 65.61 ± 1.92bc 16.35 ± 3.33c development, homeostatic maintenance and defense mechanism.
5 mM 51.81 ± 20.06bc 16.34 ± 3.46c It is activated by extrinsic receptor-mediated and intrinsic
48 Control 46.59 ± 9.59b 13.69 ± 2.57c mitochondria-mediated signalling pathways leading to changes of
5 mM 0.69 ± 0.69a 16.40 ± 1.76c
cellular morphology and biochemical substances e.g. chromatin
Data are represented as mean ± S.E.M. (n ¼ 4) and different alphabet represents the condensation, DNA fragmentation, apoptotic body formation and
statistically significant difference (p < 0.05, one-way ANOVA, Duncan multiple membrane alteration including caspases-mediated pathway
comparison test).
 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251
activation, thereby triggering phagocytosis. In contrast, necrosis is
identified by the swelling of cells and organelles, cell membrane
rupture and leakage of cellular contents into the extracellular ma-
trix. The fate of cell death pattern depends on each cell type and the
degree of cellular injury [4,5].
In the penaeid shrimp, the determination of cellular death
mechanism in primary cell cultures has not been reported before.
Some experiments on cell death were conducted in isolated hae-
mocytes incubated with nitrite for up to 3 h and lipopolysaccharide
(LPS) for up to 2 h before they were then taken to investigate the
apoptotic cell ratio by flow cytometry in P. monodon [10,13]. Also,
some experiments were examined in live shrimps infected with
some viruses e.g. WSSV and yellow head virus, knockdown of an
important gene playing a role in haemocyte homeostasis during
WSSV infection including the dissection slide of infected tissue by
using Annexin V, TUNEL, caspase activity assay, protein localisation
method, DNA fragmentation and molecular analysis [6e9,11,29]. On
the contrary, some recent experiments prefer to manage 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay principally used to assess cellular metabolism and 5-Bromo-
20 -deoxyuridine (BrdU) incorporation assay basically detected DNA
synthesis including the examination of organisation of actin cyto-
skeleton and telomerase activity to test with their primary cells
isolated for example from lymphoid organs, ovarian tissues and
haemocytes in order to claim the cellular proliferation evidence
[30e32].
Considering primary haemocytes derived in this study, a variety
of shapes e.g. round, spherical and elongated have been observed in
all experiments, which are similar to the morphology of primary
haemocytes of P. monodon previously isolated and cultured in vitro
[33,34]. In our first experiment, intact primary haemocytes could
be maintained in the culture medium for up to 4 days. While, other
previous study reported that primary haemocytes cultured in their
invented medium could survive for 7e13 days and some isolated
from lymphoid organs and ovaries were alive in vitro for more than
50 days [30]. These possibly indicate that major parameters affect
the survival period of primary haemocytes between Jayesh's and
our groups the most are the difference in chemical components of
culture medium and temperature used to derive primary cells, as
other factors e.g. the isolation method used to collect haemocytes,
cell seeding density, pH and osmolarity of the culture used in
Jayesh's and our groups are not much different. Additionally, the
inadequate stimulation of telomerase activity in primary cell cul-
tures is proposed to be the cause of failure in the production of cell
line establishment and spontaneous in vitro transformation in the
shrimp [32].
Even some primary haemocytes derived in the first experiment
started dying through the apoptotic process since 24 h of culture
investigated using Annexin V and TUNEL assays, but the relative
expression level of IAP and caspase-3 genes, key markers control-
ling apoptotic cell death mechanism in animals [4], was detected
constantly throughout the incubation period by the quantitative
real-time PCR analysis. Also, the percent of viable cells of primary
haemocytes significantly decreased by the 72nd hour, while the
percent of early apoptotic cells at the 96th hour and the percent of
late apoptotic cells since 72 h of incubation increased when
compared to those at the starting time point. These results mean
that primary haemocytes cultured in the medium for up to 48 h are
suitable to be used for testing with other chemical substances and
pathogens as previously mentioned [33]. It is because these pri-
mary haemocytes do not significantly die through the apoptotic
mechanism during the first 48 h of culture.
According to the supplement of Z-VADeFMK to the cultures, it
does not affect the morphological changes of primary haemocytes
for up to 48 h of culture. Also, supplement of Z-VADeFMK could
249
reduce the percent of viable cells, but no difference was found in
the percent of early and late apoptotic cells since 24 h of incubation
when compared to the control at each time point. Only the percent
of late apoptotic cells in 250 mM Z-VADeFMK treated group at 48 h
of culture was higher than the control and vehicle groups at the
24th hour. In addition, both Z-VADeFMK treated groups showed no
difference in the relative expression level of IAP and caspase-3
when compared to control and vehicle groups at each time point,
except that 250 mM Z-VADeFMK treated group had a higher level of
the relative expression level of caspase-3 than the control and
vehicle at the first 24 h of culture, together with the higher level of
this gene in both Z-VADeFMK treated groups at 48 h of the
experimental period than control and vehicle groups at the 24th
hour. Taken together, these indicate that some primary haemocytes
treated with Z-VADeFMK die through the apoptotic process since
24 h of culture even the morphology of primary haemocytes
treated with Z-VADeFMK were still intact for up to 48 h. This
conclusion is in agreement with the previous result reporting that
even the activation of caspases plays an important role in the
effective execution of apoptosis, a cell-permeant pan caspase in-
hibitor Z-VADeFMK still could not inhibit cell death completely due
to the continuing death signalling process through basal caspase or
caspase-independent pathway [35]. However, it is possible that
some primary haemocytes treated with Z-VADeFMK in this study
die through other cell death pattern e.g. necroptosis as previously
documented [17e20]. It also seems that the potency of 50 and
250 mM Z-VADeFMK on cellular death pattern is not much different
from each other, as the pattern of their results is quite similar in this
study. Only the difference between these Z-VADeFMK treated
groups is found in the relative expression level of IAP at the 24th
hour, but the fold change difference of this gene between these two
groups is quite low. Moreover, it is found that 0.2% DMSO used to
dissolve Z-VADeFMK does not have a huge effect on the cellular
death pattern of primary haemocytes, as shown that no difference
was found in the percent of viable, early apoptotic and late
apoptotic cells including the relative expression level of IAP and
caspase-3 between vehicle and control groups at each time point,
which is similar to the previous work done in mammalian species
[36].
Regarding the addition of NaF, a number of cell membranes of
primary haemocytes were damaged and the cellular shrinkage
was observed in all NaF treated groups within 2 h of culture. The
addition of 25 and 50 mM NaF to the medium used to culture
primary haemocytes could reduce the percent of viable cells
down to zero by 2 h of incubation. The percent of early apoptotic
cells in these two groups were not different from the control, but
lower than the 5 mM NaF treated group at 2 h of the experi-
mental period. These two NaF treated groups also had higher
percent of late apoptotic cells than the control and 5 mM NaF
treated group at the 2nd and 24th hour. No difference in percent of
viable, early apoptotic and late apoptotic cells were found be-
tween the control and 5 mM NaF treated group at any time point,
excluding that the percent of viable cells in 5 mM NaF treated
group was lower than the control at 48 h of incubation and its
value was dropped down nearly zero. No difference in the relative
expression level of IAP and caspase-3 was found between all NaF
treated groups and the control at any window period, excluding
that the relative expression level of IAP in 25 mM NaF was
slightly higher than other groups at the 24th hour including a
scantly higher level of this gene in 5 mM NaF than the 50 mM NaF
at the same time point. Altogether, these results indicate that
primary haemocytes treated with 25 and 50 mM NaF remarkably
die through the apoptotic pattern since 2 h of culture, while the
lower dose 5 mM NaF possibly induces a number of primary
haemocytes die through the apoptotic pattern since 48 h of
250 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251
culture. These mean that NaF has a potent effect to drive a
number of primary haemocytes dying through the apoptotic
process, possibly mainly via the intracellular ROS accumulation,
not the effect of caspase-3/7 activities in a dose-dependent
manner as previously reported in the mouse [4], as seen that
there is no remarkable elevation in the relative expression level
of IAP and caspase-3 in all NaF treated groups in this current
study. It is also found that around 10% of cells treated with
2e5 mM NaF for 24 h die through the necrotic cell death pattern
in this previous work, suggesting that some primary haemocytes
treated with NaF in our study probably die through necrosis also.
Furthermore, it is obviously seen that addition of NaF in a range
between 5 and 25 mM could induce melanised cells in this study.
Therefore, it is possible that these dosages of NaF affect the
cellular process involved in the stimulation of prophenoloxidase
(proPO) system, which plays an important part in the inverte-
brate defensive mechanism [3,37], to PO, which turns L-3,4-
dihydroxyphenylalanine (L-DOPA) to quinones resulting in the
production of the black pigment, melanin [38]. It is well-
published that this phenomenon is motivated by the endoge-
nous activating system and exogenous factors e.g. chemicals and
microbes [37,39,40]. Whereas, the highest dose of NaF used in
this study could not generate any melanised cell possibly because
this dose has fatal effect on primary haemocytes.
It is seen that caspase-3 gene used in this study is not the same
accession number reported [7], but they have similar activity. No
huge difference of IAP and caspase-3 level was found in each
experiment, together with no observation of obvious correlation
between IAP and caspase-3 in all experiments in this study, sug-
gesting that these apoptotic cells were possibly induced via
caspase-independent pathway as mentioned above. If these cells
died through caspase-dependent pathway as seen in those infected
with WSSV, the level of caspase-3 and IAP must be elevated [6,7,41].
Anyway, the information on the regulation of apoptotic mechanism
in the penaeid shrimp is still limited. One group recently showed
that the BIR2 domain of IAP was able to inhibit caspase-3 activity in
the black tiger shrimp. Also, the binding between the IBM peptide
of high temperature requirement A2 (PmHtrA2) and the BIR2 of IAP
could activate caspase-3 activity [42]. While, another team stated
that each isoform of IAPs responded to WSSV infection induced
apoptosis differently in the whiteleg shrimp [43]. These indicate
that many more studies on the apoptotic cell death pattern are
needed to be done in order to draw a whole picture of apoptotic cell
death mechanism in the penaeid shrimp.
In conclusion, many primary haemocytes derived from these
three experiments die through the apoptotic cell death pattern.
Like other animals, morphology of apoptotic cells and percent of
viable, early and late apoptotic cells determined by Annexin V and
TUNEL assays including the expression level of apoptotic-related
genes examined by real-time PCR technique could be used to
indicate the apoptotic cell death pattern in primary haemocytes of
the giant black tiger shrimp.
Acknowledgements
This research was supported by Director Initiative (P1010980),
BIOTEC, Thailand and Platform Technology (P1201182), NSTDA,
Thailand. Also, we would like to express our gratitude and appre-
ciation to our mentor Dr.Pikul Jiravanichpaisal for her kind support
and help. In addition, special thanks also go to Prof. Prapon Wilairat,
Dr.Sirawut Klinbunga and Dr.Piti Amparyup for their kind sugges-
tions and comments. Finally, we would like to extend our sincere
thanks to Miss Somjai Wongtripop for her kind help to continu-
ously provide us the black tiger shrimp to be used in this study.
References
[1] J.Y. Toullec, Crustacean primary cell culture: a technical approach, Methods
Cell Sci. 21 (1999) 193e198.
[2] P. Jayesh, J. Seena, I.S. Singh, Establishment of shrimp cell lines: perception
and orientation, Indian J. Virol. 23 (2012) 244e251.
[3] K. Soderhall, L. Cerenius, Role of the prophenoloxidase-activating system in
invertebrate immunity, Curr. Opin. Immunol. 10 (1998) 23e28.
[4] T.D. Nguyen Ngoc, Y.O. Son, S.S. Lim, X. Shi, J.G. Kim, J.S. Heo, et al., Sodium
fluoride induces apoptosis in mouse embryonic stem cells through ROS-
dependent and caspase- and JNK-mediated pathways, Toxicol. Appl. Phar-
macol. 259 (2012) 329e337.
[5] W.X. Zong, C.B. Thompson, Necrotic death as a cell fate, Genes Dev. 20 (2006)
1e15.
[6] J.H. Leu, Y.C. Kuo, G.H. Kou, C.F. Lo, Molecular cloning and characterization of
an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus mon-
odon, Dev. Comp. Immunol. 32 (2008) 121e133.
[7] K. Wongprasert, P. Sangsuriya, A. Phongdara, S. Senapin, Cloning and char-
acterization of a caspase gene from black tiger shrimp (Penaeus monodon)-
infected with white spot syndrome virus (WSSV), J. Biotechnol. 131 (2007)
9e19.
[8] A.H. Sahtout, M.D. Hassan, M. Shariff, DNA fragmentation, an indicator of
apoptosis, in cultured black tiger shrimp Penaeus monodon infected with
white spot syndrome virus (WSSV), Dis. Aquat. Organ 44 (2001) 155e159.
[9] K. Khanobdee, C. Soowannayan, T.W. Flegel, S. Ubol, B. Withyachumnarnkul,
Evidence for apoptosis correlated with mortality in the giant black tiger
shrimp Penaeus monodon infected with yellow head virus, Dis. Aquat. Organ
48 (2002) 79e90.
[10] J.A. Xian, Y.T. Miao, B. Li, H. Guo, A.L. Wang, Apoptosis of tiger shrimp (Penaeus
monodon) haemocytes induced by Escherichia coli lipopolysaccharide, Comp.
Biochem. Physiol. A Mol. Integr. Physiol. 164 (2013) 301e306.
[11] A.S.S. Hameed, M. Sarathi, R. Sudhakaran, G. Balasubramanian, S.S. Musthaq,
Quantitative assessment of apoptotic hemocytes in white spot syndrome vi-
rus (WSSV)-infected penaeid shrimp, Penaeus monodon and Penaeus indicus,
by flow cytometric analysis, Aquaculture 256 (2006) 111e120.
[12] J.H. Leu, H.C. Wang, G.H. Kou, C.F. Lo, Penaeus monodon caspase is targeted by
a white spot syndrome virus anti-apoptosis protein, Dev. Comp. Immunol. 32
(2008) 476e486.
[13] J.A. Xian, A.L. Wang, X.M. Hao, Y.T. Miao, B. Li, C.X. Ye, et al., In vitro toxicity of
nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow
cytometric analysis, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 156 (2012)
75e79.
[14] J.P. Seery, V. Cattell, F.M. Watt, Cutting edge: amelioration of kidney disease in
a transgenic mouse model of lupus nephritis by administration of the caspase
inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(beta-o-methyl)-fluo-
romethylketone, J. Immunol. 167 (2001) 2452e2455.
[15] E.A. Slee, H. Zhu, S.C. Chow, M. MacFarlane, D.W. Nicholson, G.M. Cohen,
Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) in-
hibits apoptosis by blocking the processing of CPP32, Biochem. J. 315 (Pt 1)
(1996) 21e24.
[16] J.S. Long, K.M. Ryan, New frontiers in promoting tumour cell death: targeting
apoptosis, necroptosis and autophagy, Oncogene 31 (2012) 5045e5060.
[17] D. Vercammen, R. Beyaert, G. Denecker, V. Goossens, G. Van Loo, W. Declercq,
et al., Inhibition of caspases increases the sensitivity of L929 cells to necrosis
mediated by tumor necrosis factor, J. Exp. Med. 187 (1998) 1477e1485.
[18] S. He, L. Wang, L. Miao, T. Wang, F. Du, L. Zhao, et al., Receptor interacting
protein kinase-3 determines cellular necrotic response to TNF-alpha, Cell 137
(2009) 1100e1111.
[19] N. Holler, R. Zaru, O. Micheau, M. Thome, A. Attinger, S. Valitutti, et al., Fas
triggers an alternative, caspase-8-independent cell death pathway using the
kinase RIP as effector molecule, Nat. Immunol. 1 (2000) 489e495.
[20] D.W. Zhang, J. Shao, J. Lin, N. Zhang, B.J. Lu, S.C. Lin, et al., RIP3, an energy
metabolism regulator that switches TNF-induced cell death from apoptosis to
necrosis, Science 325 (2009) 332e336.
[21] A. Degterev, Z. Huang, M. Boyce, Y. Li, P. Jagtap, N. Mizushima, et al., Chemical
inhibitor of nonapoptotic cell death with therapeutic potential for ischemic
brain injury, Nat. Chem. Biol. 1 (2005) 112e119.
[22] J. Gutierrez-Salinas, J.A. Morales-Gonzalez, E. Madrigal-Santillan, J. Esquivel-
Soto, C. Esquivel-Chirino, M.G.L.Y. Gonzalez-Rubio, et al., Exposure to sodium
fluoride produces signs of apoptosis in rat leukocytes, Int. J. Mol. Sci. 11 (2010)
3610e3622.
[23] N.V. Bogatcheva, P. Wang, A.A. Birukova, A.D. Verin, J.G. Garcia, Mechanism of
fluoride-induced MAP kinase activation in pulmonary artery endothelial cells,
Am. J. Physiol. Lung Cell Mol. Physiol. 290 (2006) L1139eL1145.
[24] A.G. Wang, Q.L. Chu, W.H. He, T. Xia, J.L. Liu, M. Zhang, et al., Effects on protein
and mRNA expression levels of p53 induced by fluoride in human embryonic
hepatocytes, Toxicol. Lett. 158 (2005) 158e163.
[25] S.H. Kim, R.P. Sharma, Mercury-induced apoptosis and necrosis in murine
macrophages: role of calcium-induced reactive oxygen species and p38
mitogen-activated protein kinase signaling, Toxicol. Appl. Pharmacol. 196
(2004) 47e57.
[26] K. Wongprasert, K. Khanobdee, S.S. Glunukarn, P. Meeratana,
B. Withyachumnarnkul, Time-course and levels of apoptosis in various tissues
of black tiger shrimp Penaeus monodon infected with white-spot syndrome
 K. Thansa et al. / Fish & Shellfish Immunology 57 (2016) 243e251
virus, Dis. Aquat. Organ 55 (2003) 3e10.
[27] P. Graidist, K. Fujise, W. Wanna, K. Sritunyalucksana, A. Phongdara, Estab-
lishing a role for shrimp fortilin in preventing cell death, Aquaculture 255
(2006) 157e164.
[28] Yen H.T., Oanh K.T.P., Tuan P.A., Hai N.V. Penaeus monodon caspase-3 mRNA,
protein submitted to NCBI, 2011.
[29] K. Apitanyasai, P. Amparyup, W. Charoensapsri, S. Senapin, A. Tassanakajon,
Role of Penaeus monodon hemocyte homeostasis associated protein
(PmHHAP) in regulation of caspase-mediated apoptosis, Dev. Comp. Immunol.
53 (2015) 234e243.
[30] P. Jayesh, S. Jose, R. Philip, I.S. Bright Singh, A novel medium for the devel-
opment of in vitro cell culture system from Penaeus monodon, Cytotech-
nology 65 (2013) 307e322.
[31] P. Jayesh, J. Seena, P. Rosamma, S. Bright, Cellular and molecular markers in
monitoring the fate of lymphoid cell culture from Penaeus monodon Fabricius
(1798), Fish Shellfish Immunol. 47 (2015) 893e901.
[32] P. Jayesh, S. Vrinda, P. Priyaja, R. Philip, I.S. Singh, Impaired telomerase activity
hinders proliferation and in vitro transformation of Penaeus monodon
lymphoid cells, Cytotechnology 68 (4) (2016) 1301e1314.
[33] S. Jose, P. Jayesh, A. Mohandas, R. Philip, I.S.B. Singh, Application of primary
haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and
genotoxicity of heavy metals and pesticides, Mar. Environ. Res. 71 (2011)
169e177.
[34] S. Jose, A. Mohandas, R. Philip, I.S.B. Singh, Primary hemocyte culture of
Penaeus monodon as an in vitro model for white spot syndrome virus titra-
tion, viral and immune related gene expression and cytotoxicity assays,
J. Invertebr. Pathol. 105 (2010) 312e321.
251
[35] L. Egger, J. Schneider, C. Rheme, M. Tapernoux, J. Hacki, C. Borner, Serine
proteases mediate apoptosis-like cell death and phagocytosis under caspase-
inhibiting conditions, Cell Death Differ. 10 (2003) 1188e1203.
[36] Y.A. Kim, D. Xiao, H. Xiao, A.A. Powolny, K.L. Lew, M.L. Reilly, et al., Mito-
chondria-mediated apoptosis by diallyl trisulfide in human prostate cancer
cells is associated with generation of reactive oxygen species and regulated by
Bax/Bak, Mol. Cancer Ther. 6 (2007) 1599e1609.
[37] L. Cerenius, K. Soderhall, The prophenoloxidase-activating system in in-
vertebrates, Immunol. Rev. 198 (2004) 116e126.
[38] A.J. Nappi, E. Vass, Melanogenesis and the generation of cytotoxic molecules
during insect cellular immune reactions, Pigment. Cell Res. 6 (1993) 117e126.
[39] L. Cerenius, B.L. Lee, K. Soderhall, The proPO-system: pros and cons for its role
in invertebrate immunity, Trends Immunol. 29 (2008) 263e271.
[40] B. Duvic, K. Soderhall, Purification and characterization of a beta-1,3-glucan
binding protein from plasma of the crayfish Pacifastacus leniusculus, J. Biol.
Chem. 265 (1990) 9327e9332.
[41] A. Phongdara, W. Wanna, W. Chotigeat, Molecular cloning and expression of
caspase from white shrimp Penaeus merguiensis, Aquaculture 252 (2e4)
(2006) 114e120.
[42] A. Saleeart, K. Mongkorntanyatip, P. Sangsuriya, S. Senapin, T. Rattanarojpong,
P. Khunrae, The interaction between PmHtrA2 and PmIAP and its effect on the
activity of Pm caspase, Fish Shellfish Immunol. 55 (2016) 393e400.
[43] P.-H. Wang, D.-H. Wan, Z.-H. Gu, W. Qiu, Y.-G. Chen, S.-P. Weng, et al., Analysis
of expression, cellular localization, and function of three inhibitors of
apoptosis (IAPs) from Litopenaeus vannamei during WSSV infection and in
regulation of antimicrobial peptide genes (AMPs), PLOS One 8 (8) (2013)
e7259.