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Annals of Biological Research, 2012, 3 (7):3518-3532

(http://scholarsresearchlibrary.com/archive.html)
ISSN 0976-1233
CODEN (USA): ABRNBW

Analysis of correlation between haemolymph and midgut tissue amylase with

commercial characters of silkworm Bombyx mori L.
Farshid Ghasemi Kasmaei* and H. B. Mahesha

Department of Sericulture, Yuvaraja’s College, University of Mysore, Mysore-570 005, India

*Department of Veterinary, Sahneh Branch, Islamic Azad University, Sahneh, Iran
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ABSTRACT

Four mulberry silkworm races viz., Pure Mysore, Nistari, NB4D2 & CSR2 and two hybrid (Pure Mysore ×
CSR2 and Nistari × NB4D2 ) silkworms were selected for the present study. The specific activity of amylase in
the haemolymph as well as midgut tissue was estimated. The qualitative analysis of amylase was carried out by
Native-PAGE. The commercial characters viz., fecundity, larval weight, larval duration, cocoon weight, shell
weight, shell ratio, filament length, denier and renditta were selected. The results of quantitative analysis were
subjected for statistical analysis against selected commercial characters to know the level and kind of correlation
between them. The results of statistical analysis clearly showed that haemolymph amylase has highly positive
correlation with selected commercial characters except larval duration. In contrast, the midgut amylase indicated
highly positive correlation with larval duration only. The zymograms of amylase also exhibited variation among the
selected silkworm varieties.

Keywords: Bombyx mori, haemolymph, midgut, amylase, Native -PAGE, commercial characters.
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INTRODUCTION

Ever since its inception, Sericulture is playing an important role in the economic life of man [1]. To enhance the
vertical as well as horizontal productivity, many attempts are being made to improve the silkworm stocks through
conventional breeding programmes. However, the traditional breeding approaches have to overcome some
limitations. Recent advances in genetics and molecular biology have offered a number of alternative strategies to
overcome the limitations [2]. Silkworm breeders mainly concentrating on protein polymorphism in different races so
as to provide insight into genetic variability between races to step up further hybridization programme to breed the
best [3]. Telebi et. al. reported that the protein polymorphism gives a clue on the heterosis expression for selected
traits and can be used as an index in silkworm breeding[4]. Thus, the study of polymorphic proteins of Bombyx mori
is significantly important for selection and hybridization [5].

The α-amylases (α-1, 4-glucan-4-glucanohydrolases; EC 3.2.1.1) are the hydrolytic enzymes and are one of the key
enzymes involved in digestion and carbohydrate metabolism in insects [6,7]. Research activities concerning
different digestive enzymes in the silkworm Bombyx mori were initiated after the pioneering works of Matsumura
[8]. Of the various enzymes analyzed, amylase is the most well worked out because of its association with economic
characters of silkworm Bombyx mori [9]. Hirata and Yosuo [10] found that the silkworm strains having more
amylase activity, showed better cocoon weight, shell weight, shell percentage and cocoon productivity. Gamo [11]
reported that the B. mori strains with high amylase activity showed higher growth, economic traits, and survival than
the low activity strains. The correlation between yield and biochemical parameters [12] and genetic variability for
egg characters [13] were reported. However, correlation studies combining biomolecules like amylase with
commercial characters of silkworm Bombyx mori are rather scarce. Hence, the present investigation was undertaken.

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MATERIALS AND METHODS

Four mulberry silkworm races viz., Pure Mysore, Nistari, NB4D2 & CSR2 and two hybrid (Pure Mysore × CSR2
and Nistari × NB4D2) silkworms were used for the present investigation. The silkworm rearing was conducted in
the laboratory following the method described by Krishnaswamy [14, 15]. All experimental batches were
maintained in triplicate. In each replication 500 larvae were kept after third moult. The economic traits selected for
present study included fecundity, weight of fifth instar larva, larval duration, single cocoon weight, single shell
weight, shell ratio, filament length, denier and renditta.

The larvae from first day of fifth instar were collected daily with a regular interval of 24h till the end of fifth instar.
The haemolymph was collected, centrifuged at 3000 rpm for 5 minutes in a cooling centrifuge at 5°C [9, 16] and
preserved in a deep freezer at -20°C as stock and it was used whenever required.

The midgut tissue was obtained from five larvae of fifth instar by dissecting the larvae in ice cold water and the gut
contents were removed. The tissue was thoroughly washed in distilled water. A 10 % (w/v) homogenate of the
midgut tissue was prepared in pre cooled distilled water using mortar and pestle. The homogenate was centrifuged
at 3000 rpm for 10 minutes in a cooling centrifuge at 5°C. The clear supernatant was used for the enzyme analysis.

The total soluble protein present in the haemolymph and midgut tissue was estimated by following the method of
Lowry et al. [17]. Bovine Serum Albumin was used as standard protein. Quantitative analysis of amylase activity
was done in haemolymph and midgut tissues following the method of Noelting and Bernfeld [18] using 3, 5-
dinitrosalicylic acid reagent, as modified by Ishaaya and Swirski [19]. The activity of the enzyme was expressed as
µ moles of glucose generated/mg protein/min at 37ºC.

The experimental data were statistically analyzed through SPSS by one way ANOVA, two way ANOVA [20],
Scheffe’s post hoc test [21] and linear regression analysis [22] wherever they were applicable.

The qualitative analysis of amylase isozymes was carried out in Native Poly Acrylamide Gel Electrophoresis with
the discontinuous buffer system. The vertical slab gel apparatus was used. The gels, soon after the removal, washed
in running distilled water followed by the incubation in 1% starch in 40 mM phosphate buffer pH 7.0 at 37ºC in
rotary shaker for 30 min. After incubation, the gel was placed in Iodine solution (5 mM I2 - KI in distilled water) for
15 min or until negative bands appeared [23]. Then the gels were scanned, analyzed and photographed in a gel
scanner (Vilber Laurmat Bioprofil image analysis system).

RESULTS

The summary of the studied commercial characters are presented in the table1. From the table it is clear that
the two bivoltine races are superior for productivity traits in all the seasons when compared to multivoltines. The
hybrids showed mid parental values. The results of Two way ANOVA revealed that the variation in all commercial
characters among the experimental batches are all significant at 0.1 % (P<0.001). The specific activity of amylase in
haemolymph and midgut tissue samples is shown in the tables 2 & 3 respectively. The Specific activity of amylase
in haemolymph and midgut tissue samples showed significant changes in their levels at every 24 hours till the end of
fifth instar. Almost similar trend was observed in both the tissues of all the experimental batches. The highest
Specific activity of amylase in haemolymph was observed in CSR2 (0.156 µM/mg/min at 37°C was the average
during fifth instar), followed by NB4D2 (0.151 µM/mg/min at 37°C ), PMXCSR2 ( 0.117 µM/mg/min at 37°C) ,
Pure Mysore ( 0.116 µM/mg/min at 37°C) , Nistari X NB4D2 ( 0.112 µM/mg/min at 37°C) and Nistari ( 0.109
µM/mg/min at 37°C). The results of statistical analysis revealed that the variation among the experimental batches
are all found to be significant at 0.1 % (P<0.001). In the case of midgut tissue, the highest activity of amylase was
observed in Pure Mysore (0.066 µM/mg/min at 37°C), followed by CSR2 (0.064 µM/mg/min at 37°C), PMXCSR2
(0.062 µM/mg/min at 37°C), Nistari (0.062 µM/mg/min at 37°C), NB4D2 and Nistari X NB4D2 (0.058 µM/mg/min at
37°C). The results of statistical analysis revealed that the variation among experimental batches are all significant at
0.1 % (P<0.001). The results of regression analysis between the haemolymph amylase activity levels and
commercial characters are presented in figures 1-9. From the results of statistical analysis, it is very clear that the
activity of amylase in haemolymph with shell ratio (R2=0.813), shell weight (R2=0.770), fecundity (R2=0.700),
larval weight (R2=0.615), cocoon weight (R2=0.549), filament length (R2=0.535), denier (R2=0.532) and renditta
(R2=0.511) showed strong positive relationships; whereas larval duration (Y= -315.6X+630.8) and renditta
(R2=0.029) showed week and moderately strong negative relationships respectively. In the case of midgut tissue
amylase and commercial characters, the results of regression analysis are presented in figures 10-18. From the
results of statistical analysis, it is very clearly that the larval duration (R2=0.631) showed strong positive
relationship. On the other hand, denier (R2=0.018) and fecundity (R2=0.014) showed weak positive relationships.

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Table 1: Mean values ± SD of nine commercial characters in six verities of silkworm, Bombyx mori
LARVAL LARVAL SINGLE COCOON SINGLE SHELL SHELL FILAMENT
RACES FECUNDITY DENIER RENDITTA
WEIGHT (g) DURATION (h) WEIGHT (g) WEIGHT (g) RATIO (% ) LENGTH (m)
PURE MYSORE 467.22±10.96 2.01±0.06 660±10.39 1.02±0.75 0.12±0.01 12.57±0.49 426.44±19.83 1.77±0.09 11.77±0.82
NISTARI 485.11±5.30 2.83±0.06 564.88±10.01 1.14±0.71 0.15±0.01 13.41±0.87 435.66±17.21 1.78±0.07 13.26±0.24
CSR2 509.10±16.58 4.07±0.05 578.88±6.45 1.81±0.47 0.43±0.01 24.02±0.18 1011.99±12.34 2.93±0.22 5.78±0.23
NB4D2 520.55±16.65 4.16±0.05 576.67±11.08 1.76±0.30 0.35±0.01 20.27±0.15 1020±29.96 2.48±0.06 8.34±0.47
PURE MYSORE X CSR2 466.66±11.52 2.68±0.07 610±11.10 1.67±0.23 0.28±0.01 17.29±0.21 910±18.74 2.75±0.06 7.64±0.12
NISTARI X NB4D2 490.77±6.81 3.46±0.04 557±10.21 1.47±0.22 0.23±0.01 16.06±0.85 805.99±12.36 1.83±0.02 9.22±0.85
F 89.775 4210.79 853.92 3570.99 3898.36 1484.63 65.17 311.48 28230.47
Values are the mean± SD of Pre monsoon, Monsoon and post monsoon observations.
The variation between the races is statistically significant at 0.1 % (P<0.001).

µ moles of glucose generated/mg protein/min at 37ºC) in haemolymph during fifth instar development (Days)
Table 2: Amylase activity levels (µ
RACES 1st Day 2nd Day 3rd Day 4th Day 5th Day 6th Day 7th Day 8th day AVERAGE
0.117 0.119 0.120 0.119 0.115 0.111 0.108
PM 0.119 0.116
(-1.68) (+1.70) (+0.84) (-0.83) (-3.36) (-3.47) (-2.70)
0.125 0.127 0.103 0.089 0.096
NISTARI 0.115 - - 0.109
(+8.69) (+1.60) (-18.89) (-13.59) (+7.86)
0.188 0.169 0.126 0.102 0.101
CSR2 0.253 - - 0.156
(-25.69) (-10.10) (+25.44) (-19.04) (-0.98)
0.182 0.167 0.131 0.102 0.100
NB4D2 0.229 - - 0.151
(-20.52) (+0.54) (-21.55) (-22.13) (-0.01)
0.127 0.134 0.112 0.113 0.106 0.103
PM× CSR2 0.125 - 0.117
(+1.60) (+5.51) (-16.41) (+0.89) (-6.19) (-2.83)
0.103 0.104 0.113 0.127 0.119
NISTARI× NB4D2 0.106 - - 0.112
(-2.83) (+0.97) (+8.65) (+12.38) (-6.29)
The variation between the races is statistically significant at 0.1 % (P<0.001).
Values within parentheses represent per cent change over previous day.

µ moles of glucose generated/mg protein/min at 37ºC) in midgut during fifth instar development (Days)
Table 3: Amylase activity levels (µ
RACES 1st Day 2nd Day 3rd Day 4th Day 5th Day 6th Day 7th Day 8th day AVERAGE
0.046 0.053 0.061 0.075 0.082 0.081 0.085
PM 0.049 0.066
(-6.12) (+15.21) (+15.09) (+22.95) (+9.33) (-1.21) (+4.93)
0.051 0.054 0.063 0.070 0.072
NISTARI 0.052 - - 0.060
(-1.92) (+5.88) (+16.66) (+11.11) (+2.85)
0.054 0.059 0.067 0.077 0.076
CSR2 0.054 - - 0.064
(0) (+9.25) (+13.55) (+14.92) (-1.29)
0.050 0.054 0.059 0.065 0.072
NB4D2 0.051 - - 0.058
(-1.96) (+8.00) (+9.25) (+10.16) (+10.76)
0.057 0.060 0.056 0.063 0.071 0.080
PM× CSR2 0.052 - 0.062
(+9.61) (+5.26) (-6.66) (+12.50) (+12.69) (+12.67)
0.053 0.057 0.055 0.066 0.066
NISTARI× NB4D2 0.052 - - 0.058
(+1.92) (+7.54) (-3.50) (+20.00) (0)
The variation between the races is statistically significant at 0.1 % (P<0.001).
Values within parentheses represent per cent change over previous day.

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Larval weight (Y= -134.1X+16.6) gave moderately negative relationship, whereas filament length (Y=-
25738X+2346), cocoon weight (Y= -29.75X+3.303), shell weight (Y= -4.50X+0.536) and shell ratio (Y=-
88.87X+22.72) showed weak negative relationships.

A number of qualitative and quantitative variations were observed in the zymogram of haemolymph and midgut
tissue amylase (figures 19-26). In the case of multivoltines, the haemolymph amylase isozymes exhibited almost
same pattern of banding in all the experimental sets. However, in the case of Nistari from 1st to 3rd day the bands are
more prominent in addition to appearance of five diffused bands with R.F 0.184, 0.346, 0.573, 0.680 and 0.915.
Also, the R.F. values of all the bands are slightly more than Pure Mysore. Among the bivoltines, two bands with
R.F. 0.305 and 0.359 are appeared from 3rd to 6th day and showed gradual increment in their intensities only in
NB4D2 silkworms. Also, two more minor bands with R.F 0.093 and 0.173 appeared during later ages of fifth instar.
Whereas in CSR2 silkworms one band with R.F. 0.388 appeared on 1st day and the same band gradually decreased as
the age advances. In case of hybrids, two negative bands of 0.035 R.F. and 0.262 R.F. are prominent in Nistari X
NB4D2 worms, whereas in PMXCSR2 silkworms two bands of 0.027 R.F. and 0.318 R.F are prominent. In the case
of midgut amylase isozyme profiles of Pure Mysore, the intensity of all the bands gradually decreased from 1st day
to 8th day. Interestingly, reverse trend was noticed in case of Nistari silkworms. In the case of bivoltines, the isozyme
profiles of NB4D2 follows Pure Mysore pattern. However, there was no much variation in case of CSR2 silkworms.
Among the hybrids, two bands with R.F. 0.335 and 0.448 are prominent in Nistari X NB4D2 silkworms, whereas in
PMXCSR2 silkworms two bands with R.F. 0.322 and 0.435 are prominent. Among the silkworm varieties the R. F.
values are different with each other.

530
520
510
Fecundity

500
490
480 y = 869.8x + 378.8
470 R² = 0.700
460
0.1 0.11 0.12 0.13 0.14 0.15 0.16
Activity of amylase in haemolymph (µM/mg/min at 37ºC)
.

Figure 1: Correlation between haemolymph amylase and fecundity

4.5
Larval Weight (g)

4
3.5
3
2.5
2
1.5
1 y = 31.7x - 0.818
0.5 R² = 0.615
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

.

Figure 2: Correlation between haemolymph amylase and larval weight

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680
y = -315.6x + 630.8

Larval Duration (h)

660
R² = 0.029
640
620
600
580
560
540
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

Figure 3: Correlation between haemolymph amylase and larval duration

2
1.8
1.6
1.4
Cocoon Weight (g)

1.2
1
0.8
0.6 y = 11.76x - 0.013
0.4
R² = 0.549
0.2
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

.

Figure 4: Correlation between haemolymph amylase and cocoon weight

0.5
0.45
0.4
Shell Weight (g)

0.35
0.3
0.25
0.2
0.15 y = 4.966x - 0.369
0.1 R² = 0.770
0.05
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

.

Figure 5: Correlation between haemolymph amylase and shell weight

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30
25

Shell Ratio
20
15
10
y = 186.0x - 6.328
5 R² = 0.813
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

.

Figure 6: Correlation between haemolymph amylase and shell ratio

1200
Filament Length (m)

1000

800
600

400
y = 9546.x - 442.9
200
R² = 0.535
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

Figure 7: Correlation between haemolymph amylase and filament length

3.5
3
2.5
Denier

2
1.5
1 y = 18.42x - 0.079
0.5 R² = 0.532
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in hamolymph (µM/mg/min at 37ºC)

.

Figure 8: Correlation between haemolymph amylase and denier

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14
12
10

Renditta
8
6
4
y = -94.07x + 21.26
2 R² = 0.511
0
0.1 0.11 0.12 0.13 0.14 0.15 0.16

Activity of amylase in haemolymph (µM/mg/min at 37ºC)

Figure 9: Correlation between haemolymph amylase and renditta

530
520 y = 1.4x + 484.2
R² = 0.014
510
Fecundity

500
490
480
470
460
0 1 2 3 4 5 6 7

Activity of amylase in midgut (µM/mg/min at 37ºC)

Figure 10: Correlation between midgut amylase and fecundity

4.5
4
Larval Weight (g)

3.5
3
2.5
2
1.5
1 y = -134.1x + 11.42
0.5 R² = 0.268
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

.

Figure 11: Correlation between midgut amylase and larval weight

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680
660

Larval Duration (h)

640
620
600
580
y = 9362.x + 16.6
560 R² = 0.631
540
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37 ºC) .

Figure 12: Correlation between midgut amylase and larval duration

2
1.8
1.6
Cocoon Weight (g)

1.4
1.2
1
0.8
0.6
0.4 y = -29.75x + 3.303
0.2 R² = 0.085
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

.

Figure 13: Correlation between midgut amylase and cocoon weight

0.5
0.45
0.4
Shell Weight (g)

0.35 y = -4.5x + 0.536

0.3 R² = 0.015
0.25
0.2
0.15
0.1
0.05
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

Figure 14: Correlation between midgut amylase and shell weight

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30

25

Shell Ratio
20

15

10
y = -88.87x + 22.72
5
R² = 0.004
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

Figure 15: Correlation between midgut amylase and shell ratio

1200
Filament Length (m)

1000

800

600

400

200
y = -25737x + 2346
R² = 0.095
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

.

Figure 16: Correlation between midgut amylase and filament length

3.5
3
2.5
Denier

2
1.5
1
y = 21.87x + 0.915
0.5 R² = 0.018
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

.

Figure 17: Correlation between midgut amylase and denier

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14
12
10

Renditta
8
6
4
y = -14.62x + 10.23
2 R² = 0.000
0
0.056 0.058 0.06 0.062 0.064 0.066 0.068

Activity of amylase in midgut (µM/mg/min at 37ºC)

.

Figure 18: Correlation between midgut amylase and renditta

Figure 19: Native PAGE analysis of haemolymph amylase of Pure Mysore silkworms. Lanes: 1-8 days in fifth instar.

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Figure 20: Native PAGE analysis of haemolymph amylase of Nistari silkworms.

Lanes: 1-6 days in fifth instar.

Figure 21: Native PAGE analysis of haemolymph amylase of NB4D2 and CSR2 silkworms. Lanes: 1-6 days in fifth instar.

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Figure 22: Native PAGE analysis of haemolymph amylase of Nistari X NB4D2 silkworms. Lanes: 1-6 days in fifth instar.

Figure 23: Native PAGE analysis of haemolymph amylase of Pure Mysore X CSR2 silkworms. Lanes: 1-7 days in fifth instar.

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Figure 24: Native PAGE analysis of midgut amylase of Pure Mysore and Nistari silkworms. Lanes: 1-8
1 days in fifth instar.

Figure 25: Native PAGE analysis of midgut amylase of NB4D2 and CSR2 silkworms. Lanes: 1-66 days in fifth instar.

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Figure 26: Native PAGE analysis of midgut amylase of Nistari X NB4D2 and Pure Mysore X CSR2 silkworms. Lanes: 1-7
1 days in fifth
instar.

DISCUSSION

Quantitative analysis of amylase clearly indicated three types of correlation viz.,., positive correlation or negative
correlation or neutral correlation between haemolymph and midgut amylase activity levels with commercial
characters. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), there are two sources of α‐ amylases, the
digestive fluid and the hemolymph [24].
[24] However, the role of haemolymph amylase is not yet known [25].
According to Wyatt [26],, it may participate in the degradation of glycogen in haemolymph. Hirata and Yosuo [10]
found that the silk worm strains which have more digestive amylase activity had better cocoon
co weight, shell weight
and shell percentage. Chatterjee et al.
al [12] also, reported the importance of digestive amylase activity for the
survival of the silkworm. Our results are also in accordant with the resultsesults of mentioned researchers as the
haemolymph amylase activity showed highly
high positive correlation with all the selected commercial characters (except
larval duration), such as shell ratio (R2=0.813), shell weight (R2=0.770), fecundity (R2=0.700), larval weight
(R2=0.615), cocoon weight (R2=0.549), filament length (R2=0.535), denier (R2=0.532) and renditta (R2=0.511).

Observation on the amylase isozyme pattern has revealed that the banding pattern differs between pure races,
between hybrids and between pure races and hybrids. The zymogram indicated the t variation in R.F. and
volume/intensity of the bands among the experimental silkworm varieties. The zymogram of amylase indicated five
major bands in all the silkworm varieties and 2-3 additional minor bands in NB4D2 and Nistari silkworms
silk only. The
qualitative analysis of amylase indicated six types of changes i.e., the intensity of the bands either more or less,less
besides, some of the bands either present or absent. Some of the bands increased or decreased in their intensity as
the age advances in addition to altered R.F. value. Presence or absence of protein bands indicates either the non
production or utilization or degradation of protein contents [9] when the studies are restricted within a particular
race/breed. However, when the studies
udies are concentrated between the races,
races it directly targets the genetic material as
they are exactly determined by the genetic material of the organism. The variations in the activity levels and
isozyme pattern clearly showed differences between the races.
race Therefore, by studying the silkworm amylase with
commercial characters, it is possible to have a clear picture of the kind and degree of correlation between them. An
understanding of such correlations will help us to identify and exploit the marker molecule
molecule during breeding of new
races of silkworm Bombyx mori with improved commercial characters.

CONCLUSSION

The present results clearly indicated that haemolymph amylase has highly positive correlation with selected
commercial characters except larval duration. On the other hand the midgut amylase indicated highly positive

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Farshid G Kasmaei et al Annals of Biological Research, 2012, 3 (7):3518-3532
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correlation with larval duration only. Hence, by studying the silkworm proteins with commercial characters, it is
possible to have a clear picture of the correlation between them.

Acknowledgments
Authors wish to thank University of Mysore for extending the facilities to carry out this work.

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