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I.J.A.B.R, VOL. 5(1) 2015: 58-61
ISSN 2250 – 3579
STUDIES ON THE TOTAL AND DIFFERENTIAL HAEMOCYTE COUNT IN
SOME BREEDS OF SILKWORM, (Bombyx mori L.)
a
Nisar A. Ganie, bAfifa S. Kamili, aBaqual, M.F., aSharma, R.K., aDar, K.A. & Masarat Bashir
aTemperate
Sericulture Research Institute, S.K. University of Agricultural Sciences & Technology of Kashmir,
Shalimar, Srinagar, J&K, 190 025 (India)
bDirectorate of Extension, S.K. University of Agricultural Sciences & Technology of Kashmir, Shalimar, Srinagar, J&K, 190 025 (India)
ABSTRACT
In the present study haemocytes isolated from the larval haemolymph of silkworm, Bombyx mori L. were classified into five
types viz., prohaemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Micrometric measurements revealed
that prohaemocyte was the smallest cell type observed while plasmatocytes were amongst the most polymorphic and prominent
types. Multivoltine breeds registered significantly maximum haemocyte count during both the seasons and Nistari recorded the
maximum THC value of 10550 cells mm-3 of haemolymph during spring and 11600 cells mm-3 of haemolymph during summer
season. Among the bivoltine breeds, SKUAST-28 with a THC value of 9500 cells mm-3 of haemolymph during spring and
9150 cells mm-3 of haemolymph during summer season was found to be a better breed. During summer, multivoltine breeds
were found to register comparatively higher cell counts, while the trend was reverse in case of bivoltine breeds.
KEY WORDS: Bombyx mori, Haemocytes, Prohaemocytes, Spherulocyte, Granulocyte
of haemocytes (Lavine and Strand, 2002). The total and
differential haemocyte count may indicate the susceptibility
status of the insect but since no such information is available
with respect to breeds reared in temperate climatic
conditions of Jammu and Kashmir, therefore, the need for
the present study was felt.
INTRODUCTION
Insects have an open blood system with the blood occupying
the general body cavity known as the ‘haemocoel’. This
blood or haemolymph of insects consists of liquid plasma
and numerous blood cells or haemocytes (Pandey and
Tiwari, 2012).The haemocytes of insects comprise of several
types of mesodermal cells which circulate within the
haemolymph and sometimes attach loosely to other tissues
(Kerenhap et al., 2005). There are three well defined types
of haemocytes in most of the insects namely prohaemocytes,
plasmatocytes and granulocytes and one or more of other
types in some insects which include coagulocytes,
spherulocytes, adipocytes and oenocytoids (Al-Robai et al.,
2002). Balavenkatasubbaiah et al. (2001) studied the
haemocytes in adult, Bombyx mori L. and classified its blood
cells into six types viz., prohaemocytes, plasmatocytes,
granulocytes, spherulocytes, imaginal spherulocytes and
oenocytes. Zahedi (1993) identified three basic types of
haemocytes namely plasmatocytes, cystocytes and
prohaemocytes in Armigeres subalbatus. Haemocytes are
very vital components of insect immune system. Insects
show defense response through cellular and humoral
components (Gupta, 1986). Humoral reactions involve slow
synthesis of anti-bacterial and anti-viral principles and
require several hours for full expression. Cellular responses
are direct interactions between circulatory haemocytes and
invading non-self material. The interaction is immediate and
includes phagocytosis, nodule formation and encapsulation.
Synthesis and transport of nutrients and hormones for proper
growth and wound healing are the other important functions
MATERIALS & METHODS
Different bivoltine breeds of the silkworm viz., NB4D2, SH6,
SKAU-R-6 and SKUAST-28 and multivoltine breeds viz.,
Pure Mysore and Nistari were used in the present study. The
stock breeds were received from the Germplasm bank of
TSRI, Mirgund and CSGRC, Hosur, Tamil Nadu. Rearing of
all these breeds was carried out as per the standard package
of practices (Raja, 2000). The experiment was laid out in a
completely randomized block design with four replications
for each treatment. Each replication comprised of 200
silkworms of uniform age and size. Haemolymph was
obtained by puncturing the abdominal legs with sterilized
needle/blade. The haemolymph thus bled was collected in
pre-cooled tubes containing a few crystals of phenyl thiourea
@ 1mg/sample. Phenyl thiourea was used to avoid the
activity of prophenol oxidase followed by melanization of
the haemolymph samples (Takeda et al., 1996). The samples
were stored at -20oC till further use. The total and
differential haemocyte counts were estimated using
haemocytometer following standard procedure (Jalali and
Salehi,2008). Total haemocyte counts (THC) were
determined mL-1 of haemolymph and THC per mm3 was
estimated according to the formula suggested by Jalali and
Salehi (2008).
58
Total and differential haemocyte count in some breeds of silkworm
Haemocytes in five 1mm2 squares x Dilution x depth factor of the chamber
Number of squares counted
Where,
Dilution
= 20 times
Depth factor of the chamber =
10 (constant)
Number of squares counted
=
05
cells mm-3 of haemolymph), SKAU-R-6 (7900 cells mm-3 of
haemolymph) and SKUAST-28 (9000 cells mm-3 of
haemolymph. During summer 2012, the same trend was
repeated with Nistari recording the significantly highest
haemocyte count of 12600 cells mm-3 of haemolymph
followed by Pure Mysore (10100 cells mm-3 of
haemolymph), SKUAST-28 (9300 cells mm-3 of
haemolymph), NB4D2 (8100 cells mm-3 of haemolymph),
SKAU-R-6 (7600 cells mm-3 of haemolymph) and SH6 (7100
cells mm-3 of haemolymph). SKUAST-28 was found to
record significantly higher THC value and it differed
significantly from SKAU-R-6, SH6 and NB4D2. While
pooling the data of summer seasons of 2011 and 2012, it was
observed that Nistari was the significantly superior breed
with a THC value of 11600 cells mm-3 of haemolymph,
whereas SH6 proved to be the poor performer with respect to
this parameter with a THC value of 6900 cells mm-3 of
haemolymph. The observations of high THC in the
multivoltine breeds of silkworm, Bombyx mori L. may be
attributed to their high haemolymph content which inturn
contributes to their higher survival under adverse climatic
conditions, while in case of bivoltine breeds, the high THC
values during spring are attributed to higher feeding
efficiency coupled with quality mulberry leaf during the
same season. These results are supported by the findings of
Chapman (1982) who reported that density of haemocytes
(total haemocyte count) in insects generally depends upon
the blood volume of the insects. The present findings are
also in conformity with the findings of Paul et al. (1992)
who revealed that feeding efficiency of the larvae increases
the haemocyte count in insects. Similar results were also
obtained by Ling et al.(2005) in different life stages of
Mediterranean flour moth, Ephestia kukniella wherein he
reported that the THC may normally vary greatly with
amount of haemolymph, stages of development and
physiological status of the insect. Another reason that could
be assigned to the higher THC values in multivoltine breeds
is probably the release of more haemocytes from the
haematopoetic organs as haematopoetic tissue produces
haemocyte population to a large extent in the form of
prohaemocytes, plasmatocytes and these basic haemocytes
are pluripotent and the main source for other cell types.
Differential haemocyte count (DHC) was estimated by
counting different haemocytes from a haemocyte population
of 200. Different haemocytes were identified based on the
morphological features described by Al-Robai et al.
(2002).The haemocytes were measured with the aid of a grid
eye piece and the values obtained were checked with a
micrometric ruler.
RESULTS & DISCUSSION
Total Haemocyte counts (THC)
The total haemocyte count estimated for different breeds of
silkworm, Bombyx mori L. showed significant differences
among the breeds. During spring, 2011 THC was found least
in SH6 which recorded total haemocyte population of 7300
cells mm-3 of haemolymph and it was found significantly low
from the haemocyte count of Pure Mysore, Nistari, SKAU-R6 and SKUAST-28. Multivoltine breeds viz., Nistari and Pure
Mysore recorded high THC values of 9900 and 9300 cells
mm-3 of haemolymph respectively during the same season
(Table-1). The total haemocyte count was found to be
significantly high in Nistari (11200 cells mm-3 of
haemolymph) which was followed by Pure Mysore (9900
cells mm-3 of haemolymph), SKUAST-28 (9700 cells mm-3
of haemolymph), SKAU-R-6 ( 9200 cells mm-3 of
haemolymph), NB4D2 (8900 cells mm-3 of haemolymph) and
SH6 (7800 cells mm-3 of haemolymph) during the same
season of 2012. Pooled analysis of the spring data revealed
that multivoltine breeds i.e Nistari and Pure Mysore with
their respective THC values of 10550 and 9600 cells mm-3 of
haemolymph are better as compared to bivoltine breeds viz.,
NB4D2, SH6, SKAU-R-6 and SKUAST-28 with THC values
of 8600, 7550, 8950 and 9500 cells mm-3 of haemolymph
respectively. The present investigations revealed that during
summer, the total haemocyte count showed an increased
trend in case of multivoltine breeds, however there was a
decline in the total haemocyte population of both tropical
bivoltine and temperate bivoltine breeds. Nistari recorded
the highest THC value of 10600 cells mm-3 of haemolymph
whereas SH6 recorded the lowest THC value of 6700 cells
mm-3 of haemolymph (Table-1). The total haemocyte count
recorded in other breeds during summer, 2011 include: Pure
Mysore (9800 cells mm-3 of haemolymph), NB4D2 (8200
59
I.J.A.B.R, VOL. 5(1) 2015: 58-61
ISSN 2250 – 3579
TABLE 1: Total haemocyte count (THC) in different breeds of silkworm, Bombyx mori L
THC mm-3 of haemolymph
Breeds
2011
2012
Pooled
Spring
Summer
Spring
Summer
Spring
Summer
Pure Mysore 9300
9800
9900
10100
9600
9950
Nistari
9900
10600
11200
12600
10550
11600
NB4D2
8300
8200
8900
8100
8600
8150
SH6
7300
6700
7800
7100
7550
6900
SKAU-R-6
8700
7900
9200
7600
8950
7750
SKUAST-28 9300
9000
9700
9300
9500
9150
C.D(p≤0.05)
1201.820 1901.554
928.247 1037.211 543.231
1218.284
Each value represents a mean of four replications
Each value indicates the average performance of ten individuals (insects)
relatively smaller than other haemocyte types with variable
sizes. Micrometric measurements reveal that prohaemocytes
are 5-12µm wide and 8-13 µm long (Table-2). These cells
were characterized by their small size, spherical shape and
large round nucleus which occupied most of the cytoplasm.
Differential Haemocyte counts (DHC)
The results of the present study revealed that the
haemolymph of the silkworm, Bombyx mori L. contained
five types of haemocytes namely prohaemocytes,
plasmatocytes, granulocytes, spherulocytes and oenocytoids.
Prohaemocytes were found to be round in shape and
Haemocytes observed in silkworm, Bombyx mori L.
Plasmatocytes were amongst the most polymorphic and
prominent types. Their shapes ranged from oval, elliptical to
spindle with very pointed ends (fusiform) and have a large
centrally placed nucleus. The longer axis of elliptical forms
ranged from 11 to 22 µm and in fusiform cells, it varied in
between 16 to 33 µm.The smaller axis measured at cells
broadest point ranged from 9 to 17 µm in elliptical forms,
while in fusiform cells, this axis was 7-16 µm (Table-2). The
calculated area of the elliptical cells ranged from 99 to 374
µm2 and of fusiform cells from 112 to 528 µm2.
Granulocytes are spherical, oval or irregular cells which
were found to vary considerably in size and were
characterized by the presence of a small nucleus and large
amount of different sized granules. The nucleus was found
to be centrally located and the cytoplasm was
characteristically granular, the granules being spherical,
ovoid, elongate or irregularly polygonal. The longer axis of
the granulocytes ranged in between 11µm to 24 µm. The
short axis length ranged between 8 µm to 22 µm (Table-2).
TABLE 2: Morphological characteristics of larval haemocytes of silkworm, Bombyx mori L.
Size (µm)
Nature of
Type
Shape
Position of nucleus
cytoplasm
Width Length
Prohaemocytes Round or spherical
5-12
8-13
Central
Basophilic
Elliptical
9-17
11-22
Generally central
Basophilic
Plasmatocytes
Fusiform
7-16
16-33
Central
Basophilic
Granulocytes
Spherical or oval
8-22
11-24
Central or eccentric
Slightly acidophilic
Spherulocytes
Round to oval
6-12
11-27
Generally eccentric
Basophilic
Oenocytoids
Rounded
13-24 13-24
Eccentric
Acidophilic
60
Total and differential haemocyte count in some breeds of silkworm
Spherulocytes were found to be round, oval or irregular in
shape. The longer axis of spherulocytes ranged from 11µm
to 27 µm and the small axis ranged 6 µm to 12 µm. The
shape of the nucleus was mostly found to be irregular and
eccentric in position (Table-2).
Oenocytoid cells were characterised by their spherical or
ellipsoidal shape, large amount of cytoplasm and small
nuclei. The oenocytoid length measured in between 13 µm
to 24 µm in dimensions (Table-2). Shape and structure of the
insect haemocytes is also very important because these
parameters, separately or in combination, have been reported
to be very helpful in the characterisation of the haemocytes
of different insect orders (Wigglesworth, 1959). In the
present study, two types of plasmtocytes, namely, elliptical
and fusiform were observed in the silkworm breeds under
study. The results are in conformity with the findings of
Akai and Sato (1973), who while working on the
ultrastructure of the haemocytes of the silkworm, Bombyx
mori L. found that prohaemocytes are spherical in shape,
plasmatocytes were fusiform with elongated nucleus,
granulocytes to be polymorphic in size and shape,
spherulocytes were characterised by their oval shape and
oenocytoids were large cells characterized mostly by round
shape. Jalali and Salehi (2008) observed that plasmatocytes
are pleomorphic cells and are accordingly rounded, fusiform
or spindle shaped. Variable shapes of the plasmatocytes have
also been reported with special emphasis on fusiform type
by Patil and Shah (2011) and Sanjayan et al. (1996). These
findings are in agreement with the present findings. Shape
and structure of the haemocytes observed in the present
study was found to be same in all the bivoltine and
multivoltine breeds, which is in line with the findings of
Lavine and Strand (2002), who has reported that there is not
much variation in the shape and structure of haemocytes of
different insect species falling within the same order.
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