Journal of Entomology and Zoology Studies 2013; 1 (6): 92-96
ISSN 2320-7078
JEZS 2013; 1 (6): 92-96
© 2013 AkiNik Publications
Received 17-11-2013
Accepted: 03-12-2013
Essawy M. M
Economic entomology Department,
Faculty of Agricultural, Kafr ElSheikh University, Egypt.
Email: [email protected]
Saad I. A. I
Department of Sericulture, Plant
Protection Institute, Agricultural
Research Center, Giza, Egypt.
Effect of potassium, sodium and its mixture
supplementation on 1- Blood volume, total and absolute
haemocyte counts in mulberry silkworm Bombyx mori (L.)
Essawy M. M., Saad I. A. I
Abstract
The current studies were carried using two biosalt minerals, potassium sulphate, sodium sulphate and
a mixture of both biosalts at concentration of 1,2,4,8 and 16% were added to the mulberry leaves of
silkworm B. mori L., to evaluate their effect on the blood volume (BV) and total haemocyte counts
(THC). The data revealed that larvae treated with potassium sulphate and biosalt mixture at 1%
concentration positively enhanced blood volume to reach its maximum peak at 10th day of feeding
period in comparison to the control peak which gained at 9th day. While, Larvae treated with sodium
sulphate positively enhanced blood volume during the feeding period from the 4th day until 8th day.
Potassium sulphate at concentration of 4% at 5th day of the feeding period increased the total
haemocyte counts/mm3 (THC) of larvae up to 23958 cell/mm3 in comparison to 11542 cell/mm3 of
control. While, sodium sulphate at 2% increased the THC value up to 25417 cell/mm3 in comparison
to 11542 cell/mm3 of control during the 5th day of feeding period. The biosalt mixture at 2%
concentration also enhanced the THC during the feeding period, the maximum peak was observed at
7th day.
Absolute haemocyte counts (AHC) reached its maximum peak (4990758 cell/larva) at 9th day of
larval period for larvae treated with potassium sulphate at concentration of 2% in comparison to
(3342625 cell/larva) of control. Larvae treated with sodium sulphate at 1% concentration during 8th
and 9th days showed two peaks of AHC (4393750 and 4659375 cell/larva respectively) in comparison
to (2145000 and 3342625 cell/larva) of untreated respectively. Larvae treated with a biosalt mixture
at 1% concentration positively enhanced the AHC value during the obligatory feeding period, and
mature larvae stage.
Keywords: Bombyx mori (L.), blood volumw (BV), Total haemocyt counts (THC), Absolut haemocyte
counts.
Correspondence:
Essawy M. M
Economic entomology Department,
Faculty of Agricultural, Kafr ElSheikh University, Egypt.
Email: [email protected]
Tel: 00201200001416
1. Introduction
The insect body cavity (haemocoel) contains haemolymph, which serves a function
analogous to blood in mammals in that it transports nutrients, waste products and several
micro-and macromolecules. In addition, several types of haemocytes circulate in insects
haemolymph, originated from mesodermally derived stem cells that differentiate into
specific lineages[1]. Haemocytes have been studied mostly in Lepidoptera, Hymenoptera,
Coleoptera, and Diptera[2]. The haemocytes perform various physiological functions in the
body of insect. They direct nutrients to various tissues and store them. They perform
phagocytosis[3], encapsulation of foreign bodies in the insect body cavity, wound healing[4],
coagulation to prevent loss of blood, nodule formation, transport of food materials, hormones
and detoxification of metabolites and biological active materials[5,6]. The mulberry silkworm,
Bombyx mori L. is an economically important insect in the silk industry. The development of
the worm is depending on metamorphosis process which is a dynamic biochemical process
[7]
. As a new trend some workers[8, 9, 10, 11] tried to study the changes in the haemolymph of
the mulberry silkworm Bombyx mori L. after different treatment to increase the production
of silk. Any changes in total haemocyte counts of particular insect directly or indirectly
affect the insect. The present study is primarily concerned with the total haemocyte counts
and blood volume of mulberry silkworm fed on some mineral supplement during the last
larval instar.
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Journal of Entomology and Zoology Studies
2. Materials and methods
2.1 Insects
Chinese hybrid (9F7X) of mulberry silkworm Bombyx mori L.,
imported from China was used during the present study. Larvae
were reared at Kafr El-Sheikh Faculty of Agriculture under the
laboratory hygrothermic conditions of 25±2 0C and 65-70 R.H.,
according to the conventional method in trays and provided with
suitable amounts of fresh mulberry leaves[12]. All larvae which
molted to the last instar at the same time were grouped and used in
the experiments. The determination of the last larval instar was
determined by (0 hour) from the last larval ecdysis[13].
day more than control. There is also a sharp decrease in BV
(91±41ul) gained at the last day of treated larvae by potassium
sulphate at concentration of 2% compared with other
concentrations and also compared with untreated (165±8ul). Larvae
treated with sodium sulphate at concentrations of 2, 4, 8, and 16%
presented low blood volume compared to untreated one during the
feeding period (from the first day until the end of 8th day). Larvae
treated with sodium sulphate at concentration of 1% showed an
gradual increase in BV higher than those of control during the
period from the 4th day until the 8th day (mature larvae) in
comparing to(156±14 ul) of control , then reached
2.2. Biosalt minerals compounds as food additives.
The mineral salt compounds potassium sulphate anhydrous
(k2SO4), sodium sulphate anhydrous (Na2SO4) (99%), provided
from El Nasr Pharmaceutical Chemicals Co. (ADWIC), Egypt. A
mixture of both sodium and potassium sulphate (1:1) were also
used as supplementary nutrient. The bio salt crystals were
dissolved in distilled water and diluted to five concentrations, i.e.,
1, 2, 4, 8 and 16%. Fresh mulberry leaves were soaked for 15
minutes in each concentration then left at the room temperature to
dry. Then treated mulberry leaves were offered to silkworm larvae
at the first day of last larval instar for one day only at three times.
While during the next days until the end of last instar fed on
untreated leaves. Control larvae were fed on mulberry leaves
soaked in distilled water only for one day also.
The principal experiments of nutrient supplementation included
five treatments and control. Each treatment and control was
replicated three times. Each replicate contained 20 larvae of 5th
instar larvae (were reared in plastic boxes 20x20x15 cm).
2.3. Blood volume
The blood volume were calculated daily by cutting a proleg of
larvae, blood volume were calculated as in[14, 3].
2.4. Total haemocyte counts (THC).
Five larvae were killed daily in a hot–water bath (56-58 0C).
Haemolymph was withdrawn by cutting a proleg with fine scissors,
and blood was quickly drawn into a Thoma white blood cell pipette
and diluted with 2% versene plus a trace of methylene blue or
crystal violet[15, 16, 8]. Pipette was shaken gently for several minutes,
and transferred to a Naubauŕ double lined haemocytometer and
haemocytes were counted in squares situated at the four corners of
the calibrated area (each square consists of 16 small squares, each
one of them with volume of 1/10 x 1/20 x 1/20 mm). THC's were
calculated according to[17].
2.5. Absolute haemocyte counts (AHC).
The absolute haemocyte counts (AHC) was calculated from the
THC and BV according to[18, 4, 3, 8].
3. Results and Discussion
3.1. Blood volume (BV)
As shown in Fig (1) blood volume of untreated larvae showed a
gradual increase started from the first day and continued to reach
maximum peak at the 9th day (the end of feeding period) then a
decrease after the 9th day was observed and continued until the end
of the last instar. Treated larvae showed a similar trend of normal
BV except larvae treated with 1% of potassium sulphate as it
prolonged the peak of BV (243±4ul) by 24 hour, at the 10th day.
This increase period is related to an increase of feeding period one
approximately the same maximum value of control but at the 9th
day. Then a fluctuation decrease were noticed after the 8th day, as
shown in (Fig 2).
Larvae treated with a biosalt mixture (potassium and sodium
sulphate) at low concentration of 1% positively increased the BV
during the middle of feeding period (4th day) and a significant peak
was observed compared to control at 8th of mature larvae. A lower
BV was also observed when larvae treated with a biosalt mixture at
concentrations of 2, 4, 8 and 16% comparing to untreated larvae.
The concentration of 1% gave also another peak (225±27 ul) in BV
at 10th day higher than those of control (185±19 ul) due to the
prolongation of feeding period of treated larvae.
Blood volume was studied during the different developmental
periods of the mulberry silkworm B. mori.[13], BV presented a
gradual increase until the end of the obligatory feeding period , the
maximum value was noted at 10th day , however during prepupal
period and pupation BV was decreased. In his study[13] on the
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Journal of Entomology and Zoology Studies
silkworm Bombyx mori indicated that both treated larvae with
cholesterol and untreated showed the same trend of BV during the
developmental periods. The same trend also was found by[11] after
treatment with thyroxine. In their studies, the maximum differences
in the BV was observed on the 9th day ( the end of feeding period),
and BV was higher in treated larvae than untreated then a gradual
decline was observed in both treated and untreated larvae during
the prepupal period. Present study showed also the same
observations as the above mentioned results in both treated and
untreated larvae.
3.2. Total haemocyte counts (THC)
The total number of haemocytes was calculated in order to
determine quantitative changes in treated larvae with different
biosalts. It was been reported that the numbers of haemocytes may
vary according to the physiological state in each stage of B.
mori.[19, 8].
Fig (4) showed that THC of normal larvae recorded (6000±1231
cells/mm3) at first day and continued to increase gradually until
reached to maximum value (21250±7604 cells/mm3) at 7th day,
then declined at the 8th day. A slight increase of THC was observed
at 9th day before it declined again during the prepupal stage to
reach their minimum value (11292±3527 cells/mm3) at 11th day.
Treated larvae with potassium sulphate showed the same trend of
THC during the last larval instar. Generally, the high
concentrations of 8 and 16% caused a reduction in THC during the
last larval instar except a noticeable increase of THC was occurred
at 3rd day at concentration of 8%. While the low concentrations of
1, 2 and 4% treated larvae were positively enhanced THC and were
highly increased than the control larvae. The higher peak of THC
was showed when larvae treated with concentration of 4%, it was
(23958±955 cells/mm3) gained at 5th day of feeding period
comparing to (11542±1688 cells/mm3) of untreated larvae at the
same day, and also when compared to those maximum THC of
control (21250±7604cells/mm3) gained at 7th day as shown in (Fig
4). Larvae treated with sodium sulphate at 2% showed the
maximum higher peak of THC (25417±1909 cells/mm3) during the
feeding period when compared to (11542±1688 cells/mm3) of
control at 5th day (Fig 5).
Also, larvae treated with low concentration 1% gained another
peak of THC at 8th day (23750±1250 cells/mm3) when compared to
untreated one (13750±2734 cells/mm3). Larvae treated with a
biosalt mixture of potassium and sodium sulphate exhibited
fluctuation trend of THC during the last larval instar. THC was
higher in treated than untreated one during the most days of feeding
period until 5th day whereas a decline was noticed in THC. Only a
maximum high peak of THC (27083±2602 cells/mm3) was noticed
at the 7th day at 2% concentration in comparison to untreated
(21250±7604 cells/mm3) (Fig 6).
From the above results THC was increased in treated larvae than
untreated ones during the feeding period with all biosalts, then
declined at the end of feeding period and increased again during the
beginning of the cocoon spinning period. It has been reported that
[8, 13, 11]
in Bombyx mori last larval instar THC reached its peak
during the early spinning period, while an decrease was observed
during the prepupal period. In most insects the THC increased prior
to ecdysis, declines sharply at ecdysis and soon afterwards increase
again[20].
3.3 Absolute haemocyte counts (AHC)
As showed in Fig (7) larvae treated with potassium sulphate at
concentrations of 1, 2 and 8%, showed a gradual increase in AHC
from the beginning of last instar and until reached to first peaks
(1885788 cell/larva), (2016630 cell/larva) and (2041634 cell/larva)
at 3rd day respectively comparing to that of control (1010592
cell/larva). Another peak (2825122 cell/larva) could be observed at
6th day of 1% concentration comparing to (2377960 cell/larva) of
control, and at 8th day in the concentrations of 1, 2 and 4%
appeared an increase in AHC, (2887500, 2540356 and
3263274cell/larva) comparing to (2145000 cell/larva) of untreated
respectively. The maximum increase in AHC's was observed in
concentration of 2% (4990758 cell/larva) followed by 8%
(3987500 cell/larva) at 9th day comparing by control (3342625
cell/larva). Another increase in AHC was observed in concentration
of 1% (4050081 cell/larva) at 10th day comparing to (2775000
cell/larva) of untreated ones. Treated larvae with sodium sulphate
presented several peaks at AHC during the feeding period at
different concentrations as shown in Fig (8). Small peak was
gained (1283724 cell/larva) when larvae treated with concentration
of 1% which was higher than other treatments and control (522500
cell/larva) at 2nd day. Larvae treated with sodium sulphate at 4th day
with concentrations of 1 and 4% showed high value of AHC's
about (2137536 and 1898781 cell/larva) respectively, more than
control (1094375 cell/larva). At 5th day the concentrations of 1, 2, 4
and 8% increased the AHC value, (3044756, 3151708, 2015794
and 1968324cell/larva) more than control (1408124 cell/larva). At
8th day the concentrations of 2, 4 and 8% increased the AHC to
(3071288, 4393750, and 3282994 cell/larva respectively) higher
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Journal of Entomology and Zoology Studies
than the control which gave (2145000 cell/larva).
hemocyte density was increased in the molting stage and spinning,
and then decreased after the ecdysis[21]. An increase of BV, THC
and AHC after treatment could be attributed to the indirect effect
on the hormonal balance which in turn affects the BV and THC.
The AHC in circulation depends on both THC and BV[22]. While,
an inverse relationship was advocated between THC and BV in B.
mori larvae[19]. Differential haemocyte counts (DHC) and absolute
differential haemocyte counts (ADHC) will be the next part of this
work.
As shown in (Fig: 9) larvae treated with a biosalt mixture of
potassium and sodium sulphate at 2, 4, 8, and 16% showed a
negative effect on AHC's value during the last larval instar except
only the low concentration of 1% positively affected the AHC. The
concentration of 1% increased the AHC during the 4th day of
feeding period to (1776462 cell/larva) when compared to (1094375
cell/larva) of untreated. Another peak (4406175 cell/larva) was also
observed during the 10th day when compared to (2775000
cell/larva) of control, which was also higher than the control at the
9 th day.
From the above results, AHC indicated a positive effect during the
early and late feeding period when larvae treated with low
concentration of potassium sulphate and sodium sulphate. In
Bombyx mori L.[8] two peaks of THC were reported during the
feeding period of last larval instar at 8th and 10th day, also noted an
increase of AHC during the prepupal stage. In Philosamia ricini
(Boisd.) AHC showed a gradual increase in control larvae until
reached its maximum peak at 9th day, while treated larvae with IKI
at 0.5 ppm significantly increased AHC about 47% than control [10].
A positive correlations were found between BV, THC and AHC in
both untreated and treated larvae by cholesterol during the feeding
period from the 1st day until 9th day (the end of feeding period and
beginning of spinning)[13]. It was noted that in B. mori. the
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