INTRODUCTION:

In India, Uttar Pradesh is a developing mulberry silk producing state where the Tarai Belt is gaining a leading status in the extension of sericulture industry. Bombyx mori race nistari is a resistant variety of multivoltine mulberry silkworm which contributes up to a great extent in the commercial production of cocoon in varying ecological conditions in our country. The larval stages of silkworm are monophagous, since a diet of which the chemical content is completely known, both quantitatively and qualitatively. The ultimate aim of sericulture industry is the production of quality seed cocoons i.e. raw silk as per demand. Plant extracts and their essential oils are interesting as sources of natural products for decades (Burt, 2004). Many kinds of essential oils have been screened for their potential uses for food preservation, aromatherapy and fragrance industry (Bakkali et al., 2008). Beneficial effects of Aloe vera (Hindi- Gikanvar or Ghrita kumari) in human and laboratory animals are contributed to the promotion of immune system, analgesic, anti-inflammatory, wound healing and anti-tumor activities as well as antiviral, antibacterial and antifungal properties (Reynolds and Deweck, 1999).

Aloe vera (L) contains over 75 nutrients and 200 active compounds, including vitamins, enzymes, minerals, sugars, lignin, anthraquinones, saponins, salicilic acid and amino acids (Park and Jo, 2006). Aloe vera products are also used in medicine folk, cosmetics, supplement and food material (Eshun and He, 2004). Preparations of Aloe are, therefore, used both topically and as dietary supplement (Buenz, 2008). The variation in protein content in haemolymph influences the life cycle pattern and production of cocoon. In recent years efforts have been made in sericulture to study the effect of 20-hydroxyecdysone hormone (Prasad and Upadhyay, 2012), phytoecdysteroid (Upadhyay and Pandey, 2012), plant volatile (Fatma et al., 2013 and 2014), Aloe vera essential oil (Tiwari et al.; Singh et al., 2014) and linseed and hemp oil (Zah et al., 2011) on the performance of Bombyx mori. The effect of temperature were observed on amino acids content (Gupta et al., 2005), nucleic acid content (Upadhyay et al., 2007) and protein content (Upadhyay et al., 2014) of silkworm. Exposure of magnetic field (Upadhyay and Tripathi 2005; Prasad and Upadhyay 2011a; Tripathi, 2012), phytoecdysteroid (Srivastava and Upadhyay, 2013) and phytojuvenoid (Srivastava and Upadhyay, 2014) influences the protein content of B. mori. The amino acid content also changed (Prasad and Upadhyay 2011 b and c; Tripathi et al., 2012; Srivastava and Upadhyay, 2014) in the tissues of silkworm. Keeping this in view, an attempt has been made to investigate the effect of Aloe vera essential oil treated mulberry leaves on the total protein content in haemolymph of Bombyx mori larvae.

MATERIALS AND METHODS:

The seed cocoons of multivoltine mulberry silkworm (Bombyx mori nistari) were obtained from the silkworm grainage. Directorate of sericulture, Behraich Uttar Pradesh and were maintained in the plywood trays (23 x 20 x 5cm) under the ideal rearing conditions in the silkworm laboratory, Department of Zoology, DDU Gorakhpur University Gorakhpur. The temperature and relative humidity were maintained at 26 ± 1°C and 80 ± 5% RH, respectively till the emergence of moths from the seed cocoons. The newly emerged moths were quickly picked up and kept sex-wise in separate trays to avoid copulation. The male moths were smaller in size but more active than the female months which were comparatively larger and less active. The whole grainage operation was performed as per description given by (Krishnaswamy et al., 1973).

Moths have a tendency to pair immediately after emergence and, therefore, the female moths required to copulate with the male moths, were allowed their mates for copulation. Sufficient pairs, each containing one male and one female from newly emerged moths were allowed to mate at 26±1°C and 80±5% RH in 12 hour/day dim light condition. After four hours of mating, the paired moths were decoupled manually by holding the female moths between the thumb and middle finger gently and pushing the male away by the fore finger. The male moths were discarded while the female moths were allowed to lay eggs. After 24 hours of egg laying, the female moths were individually examined for their disease freeness.

The disease free layings (D.F.L’s), thus prepared, were treated with 2% formaline for 15 minutes to increase the adhesiveness of eggs on the paper sheet and surface disinfection. Thereafter, the egg sheets, with egg laid on, were thoroughly washed with running water to remove formaline and the eggs were dried in shade. The dried eggs were transferred to the incubator for hatching. After hatching, the larvae were reared on the mulberry leaves given as food in the trays. Further, the 3rd instar larvae were taken for experiment.

Experimental Design:

To observe the influence of Aloe vera essential oil on the total protein content in haemolymph of Bombyx mori larvae, the experiment was performed with different doses of Aloe vera essential oil with respect to the treatment of 3^rd, 4^th and 5^th instar larvae. Aloe vera essential oil purchased from the Katyani Exports Delhi, India. Four dose of Aloe vera essential oil viz, 0.25, 0.50, 0.75 and 1.00 ml were uniformly sprayed over mulberry leaf separately by sprayer for 10 minutes before given for feeding to the larvae as 100 gm mulberry leaves/100 larvae. Three sets of experiment were designed viz, single, double and triple treatment of larvae. All the experiments were conducted in the BOD incubator. The experiment was conducted on normal rearing condition i.e. 26 ±1⁰C temperature, 80±5% relative humidity and 12±1 hour photoperiod a day.

Single Treatment:

Single treatment of larvae was performed with the 5th instar larvae just before two days of the beginning of larval spinning. One hundred larvae were taken out from the BOD incubator and the mulberry leaf treated with 0.25 ml of Aloe vera essential oil was given as food. Further, the treated larvae were given normal mulberry leaf for food.

Double Treatment:

Double treatment of larvae was started from the final stage of 4^th instar larvae. In the first treatment, one hundred larvae of 4^th instar were treated just before two days of 4^th moulting, by providing treated mulberry leaf as food with 0.25 ml of Aloe vera essential oil. The treated larvae then transferred in BOD incubator for further rearing and development. Further, second treatment for the same larvae was given at the final stage of 5^th instar larvae i.e. just before two days of spinning. Thus, in double treatment, 4^th and 5^th instar larvae were treated.

Triple Treatment:

For triple treatment, the 3^rd instar larvae just before 3^rd moulting were separated from BOD incubator. In the first treatment, one hundred larvae of 3^rd instar were treated by providing treated mulberry leaf and kept in BOD incubator for rearing. The second treatment of same larvae was done just before two days of 4^th moulting i.e. at the final stage of 4^th instar larvae and transferred in BOD incubator for further rearing. Third treatment was given to 5^th instar larvae, two days before the start of spinning by providing mulberry leaf treated with 0.25 ml of Aloe vera essential oil as food. Thus, in the triple treatment 3^rd, 4^thand 5^th instar larvae were treated. Similar experiments were performed by 0.50, 0.75 and 1.00 ml of Aloe vera essential oil. A control set was always maintained with each set of experiment.

Total Protein Content:

For the estimation of total protein content in haemolymph of Bombyx mori larvae, the fifth instar larvae were dissected in distilled water at the initial and final stage of spinning and 0.5 ml (0.62mg) haemolymph was taken. Protein content was estimated according to Lowery et al. (1951) as modified by Singh and Agrawal (1989). In above tissues added 4.0 ml of 10% T.C.A. and prepared the homogenate separately. The homogenate, thus obtained, was centrifuged at the top speed (20,000 rpm) for 10 minutes. Further, the supernatant was discarded, washed the precipitate with 5% T.C.A., again centrifuged for 10 minutes and discarded the supernatant. The precipitate was again washed with 10% T.C.A., centrifuged and discarded the supernatant. The precipitate, thus obtained, was dissolved in 4 ml of 10 NaOH. Now in 1 ml diluted supernatant, 0.5 ml, of freshly prepared alkaline copper solution (Reagent C) was added. Reagent C was prepared by the addition of 50.9 ml, reagent A (2% sodium carbonate in 0.1 N NaOH) and 1 ml reagent B (1% of sodium potassium tartrate, 0.5% copper sulphate, mixed in 1:1 ratio at the time of experiment). The reaction mixture was kept for 10 minutes at room temperature, and then 0.5 ml folin ciocalteu reagent (diluted 1:2 ratio with distilled water at the time of experiment) was added and mixed thoroughly. Thirty minutes after this the blue colour developed which was measured at 600 nm. Standard curves were prepared with different concentration of Bovin serum albumen. The value of total protein has been expressed as µg/ml of respective tissues. Six replicates of each experiment were made.

Statistical Analysis:

Results were subjected to analysis of variance by Two-Way ANOVA (Sokal and Rohlf, 1973) to detect significant changes and Post-hoc test for determining the significant group difference with the help of MS Excel.

RESULTS:

Total protein content in the haemolymph of larvae at the initial stage of spinning:

The data given in Table-1a clearly indicates that change in the Aloe vera essential oil amount and the number of larval treatment both influenced the total protein content in the haemolymph of larvae at the initial stage of spinning. With the increase in number of larval treatment by Aloe vera essential oil from one to three times, the total protein content in the haemolymph of larvae at the initial stage of spinning increased in case of 0.25, 0.50 and 0.75 ml of Aloe vera essential oil treatment. In case of larval treatment with 1.00 ml Aloe vera essential oil, the total protein content in the haemolymph of larvae at the initial stage of spinning slightly increased in single treatment of larvae but further increase in the number of the larval treatment caused decline in the total protein content in the haemolymph of larvae in the initial stage of spinning. Increase in the total protein content in the haemolymph of larvae at the initial stage of spinning, with the increase in number of larval treatment has been recorded to be almost of similar trend in case of 0.25, 0.50 and 0.75 ml Aloe vera essential oil treatment. The maximum total protein content in the haemolymph of larvae at the initial stage of spinning was recorded to be 30.245±0.423 µg/ml (19.21% increased as compare to control) in the triple treatment of larvae by 0.75 ml of Aloe vera essential oil and that was minimum 22.217±0.210 µg/ml in case of triple treatment of larvae by 1.00 ml Aloe vera essential oil.

Two-way ANOVA indicates that change in the Aloe vera essential oil amount significantly (P₁<0.01) influenced the total protein content, while the number of larval treatment has no significant influence on the total protein content in the haemolymph of larvae at the initial stage of spinning (Table-1a). The Post-hoc test (Table-1b, HSD=2.928) shows significant group difference in the total protein content in the haemolymph of larvae at the initial stage of spinning in between 0.50 and 1.00 ml, and 0.75 and 1.00 ml Aloe vera essential oil in case of double treatment of larvae. In the triple treatment of larvae, significant group difference in the total protein content in the haemolymph of larvae at the initial stage of spinning was noticed in between control and 0.75 ml, control and 1.00 ml, 0.25 and 0.75 ml, 0.25 and 1.00 ml, 0.50 and 1.00 ml, and 0.75 and 1.00 ml of Aloe vera essential oil treatment. In case of single treatment there was no significant group difference.

Table 1a: Effect of Aloe vera essential oil treatment on the total protein content (µg/ml) in the haemolymph of Bombyx mori larvae at the initial stage of spinning.

Stage of treatment (larval instar)	Aloe vera essential oil applied (ml)
Stage of treatment (larval instar)	Control (X₁)	0.25 (X₂)	0.50 (X₃)	0.75 (X₄)	1.00 (X₅)
Single	25.372±0.283	25.618±0.281	25.913±0.146	26.794±0.225	26.067±0.253
( 5^th )	(100)	(100.97)	(102.13)	(105.60)	(102.74)
Double	25.372±0.283	26.018±0.239	26.821±0.233	28.063±0.404	23.425±0.216
( 4^th -5^th )	(100)	(102.55)	(105.71)	(110.61)	(92.33)
Triple	25.372±0.283	26.463±0.133	27.729±0.393	30.245±0.423	22.217±0.210
( 3^rd -5^th )	(100)	(104.30)	(109.29)	(119.21)	(87.57)
· F₁ = 17.9405 (n₁=4, n₂=38), P < 0.01; F₂ = 0.7619 (n₁=2, n₂=38), not significant. · Each value represents mean ± S.E. of six replicates. · X₁, X₂, X₃, X₄and X₅are the mean values of total protein content in control, 0.25, 0.50, 0.75 and 1.00 ml Aloe vera essential oil treatment, respectively. · Figures in parentheses indicate percent value when control was taken as 100%.

Table 1b: Post-hoc test showing effect of Aloe vera essential oil treatment on the total protein content (µg/ml) in the haemolymph of Bombyx mori larvae at the initial stage of spinning.

Mean difference in between groups	Stage of treatment
Mean difference in between groups	Single	Double	Triple
X₁ ~ X₂	0.246	0.646	1.091
X₁ ~ X₃	0.541	1.449	2.357
X₁ ~ X₄	1.422	2.691	*4.873
X₁ ~ X₅	0.695	1.947	*3.155
X₂ ~ X₃	0.295	0.803	1.266
X₂ ~ X₄	1.176	2.045	*3.782
X₂ ~ X₅	0.449	2.593	*4.246
X₃ ~ X₄	0.881	1.242	2.516
X₃ ~ X₅	0.154	*3.396	*5.512
X₄ ~ X₅	0.727	*4.638	*8.028

MSE = Mean Square Error from ANOVA table

q = Value from studentized range table

n = No. of replicates per treatment

* = Shows significant group difference

X₁, X₂, X₃, X₄ and X₅ are mean values of protein content in control, 0.25, 0.50, 0.75 and 1.00 ml Aloe vera essential oil treatment, respectively.

Total protein content in the haemolymph of larvae at the final stage of spinning:

The data presented in Table-2a clearly indicates that change in the Aloe vera essential oil amount and the number of larval treatment influenced the total protein content in the haemolymph of larvae at the final stage of spinning. With the increase in number of larval treatment by Aloe vera essential oil from one to three times, the total protein content in the haemolymph of larvae at the final stage of spinning increased in case of 0.25, 0.50 and 0.75 ml of Aloe vera essential oil treatment. In case of larval treatment with 1.00 ml Aloe vera essential oil, the total protein content in the haemolymph of larvae at the final stage of spinning slightly increased in single treatment of larvae but further increase in the number of the larval treatment caused decline in the total protein content in the haemolymph of larvae in the final stage of spinning. The trend of increase in the total protein content in the haemolymph of larvae at the final stage of spinning, with the increase in number of larval treatment has been recorded to be almost similar in case of 0.25, 0.50 and 0.75 ml Aloe vera essential oil treatment. The maximum total protein content in the haemolymph of larvae at the final stage of spinning was recorded to be 23.023±0.204 µg/ml (26.20% increased as compare to control) in the triple treatment of larvae by 0.75 ml of Aloe vera essential oil and that was minimum 15.553±0.280 µg/ml in case of triple treatment of larvae by 1.00 ml Aloe vera essential oil.

Two-way ANOVA indicates that variation in the Aloe vera essential oil amount caused significant (P₁<0.01) influence on the total protein content but the number of larval treatment did not cause significant influence on the total protein content in the haemolymph of larvae at the final stage of spinning (Table-2a). The Post-hoc test (Table-2b, HSD=2.671) shows significant group difference in the total protein content in the haemolymph of larvae at the final stage of spinning in between control and 0.75 ml, 0.50 and 1.00 ml, and 0.75 and 1.00 ml Aloe vera essential oil in case of double treatment of larvae. In the triple treatment of larvae, significant group difference in the total protein content in the haemolymph of larvae at the final stage of spinning was noticed in between control and 0.50 ml, control and 0.75 ml, control and 1.00 ml, 0.25 and 0.75 ml, 0.25 and 1.00 ml, 0.50 and 1.00 ml, and 0.75 and 1.00 ml of Aloe vera essential oil treatment. In case of single treatment there was no significant group difference.

Table 2a: Effect of Aloe vera essential oil treatment on the total protein content (µg/ml) in the haemolymph of Bombyx mori larvae at the final stage of spinning.

Stage of treatment (larval instar)	Aloe vera essential oil applied (ml)
Stage of treatment (larval instar)	Control(X₁)	0.25(X₂)	0.50(X₃)	0.75(X₄)	1.00(X₅)
Single	18.243±0.212	18.631±0.230	19.437±0.304	20.488±0.302	18.981±0.326
( 5^th )	(100)	(102.13)	(106.54)	(112.31)	(104.05)
Double	18.243±0.212	19.217±0.358	20.325±0.226	21.917±0.343	16.662±0.243
( 4^th -5^th )	(100)	(105.34)	(111.41)	(120.14)	(91.33)
Triple	18.243±0.212	19.822±0.318	21.273±0.365	23.023±0.204	15.553±0.280
( 3^rd -5^th )	(100)	(108.66)	(116.61)	(126.20)	(85.25)
· F₁ = 29.8415 (n₁=4, n₂=38), P < 0.01; F₂ = 0.6313 (n₁=2, n₂=38), not significant. · Each value represents mean ± S.E. of six replicates. · X₁, X₂, X₃, X₄and X₅are the mean values of total protein content in control, 0.25, 0.50, 0.75 and 1.00 ml Aloe vera essential oil treatment, respectively. · Figures in parentheses indicate percent value when control was taken as 100%.

Honesty significant difference (HSD) =

MSE = Mean Square Error from ANOVA table

q = Value from studentized range table

n = No. of replicates per treatment

* = Shows significant group difference

X₁, X₂, X₃, X₄ and X₅ are mean values of protein content in control, 0.25, 0.50, 0.75 and 1.00 ml Aloe vera essential oil treatment, respectively.

DISCUSSION:

The protein content in the haemolymph of Bombyx mori larvae at the initial and final stage of spinning influenced due to variation in the Aloe vera essential oil amount and the number of larval treatment. The total protein content in the haemolymph of larvae at the initial and final stage of spinning gradually increased with the increasing number of larval treatment by Aloe vera essential oil from one to three times in case of 0.25, 0.50 and 0.75 ml of Aloe vera essential oil treatment. The total protein content in the haemolymph of larvae at the initial and final stage of spinning slightly increased in single treatment of larvae when leaves treated with 1.00 ml Aloe vera essential oil, but further increase in the number of the larval treatment up to triple treatment caused decline in the total protein content. The rate of increase in the total protein content in the haemolymph of Bombyx mori was rapid in the larvae than in other stages (Doira, 1968). The change in the protein metabolism during insect development has been studied with reference to the changes in the amino acid spectrum, net synthesis of protein and kinetics of certain enzymes associated with the synthesis and degradation of amino acid and protein in Drosophila melanogaster (Dingle and Smith, 1969). The decreased protein content in the haemolymph was recorded in Rhodnius prolixus at high temperature regimes (Okasha, 1964). Protein level in the haemolynph increased due to cold adaptation in ectoderm (Precht et al., 1973), while the cold-stress (Velide, 2012) influenced the protein level in haemolymph of Anthereae mylitta. The protein content in the haemolymph towards the end of the last instar was attributed to the increased rate of protein biosynthesis in the fat body of Galleria mellonella (Collins and Doene, 1970). The variation in the protein and related components in the haemolymph during insect development is directly related to the spinning process (Beament et al., 1975). The moulting hormone regulates the metabolism of protein synthesis and plays a significant role in the nucleic acid metabolism in the silkworm (Murakoshi et al., 1972; Dai et al., 1985). The tropical application of methoprene (Bharthi and Miao, 2001 and 2003), juvenile hormome (Chowdhary et al., 1986; Nair et al., 2009; Uranli et al., 2011) and phytojuvenoid (Srivastava and Upadhyay, 2014) also influenced the total protein level in the haemolymph of larvae in Bombyx mori. The application of magnetic field caused an increase in the enzyme (Carboxymutase and Catalase) activities in the biological system (Young, 1969). The total protein content in the haemolymph increased due to magnetization of eggs (Upadhyay and Tripathi, 2005) and larvae (Tripathi, 2012) in B. mori. The protein content was increased in haemolymph with potasium bromide treated groups in all CSR₂xCSR₄ crossbreed race of the silkworm (Kochi and Kaliwal, 2006). The highest concentration of haemolymph protein was observed in PMXCSR₂ (Kasmaei and Mahesha, 2012) and hybrid races (Mahesha et al., 2013) of silkworms. Nucleic acids are the responsible factors for secretion of proteins quantitative estimation of DNA and RNA at different stages of silkworm (Brindha et al., 2012). The drastic difference in protein profiles was noticed in haemolymph of infected silkworm compared to control (Rajitha and Savithri, 2013).

On the basis of present observation and above information it may be concluded that variation in the Aloe vera essential oil amount and the number of larval treatment, increasing the food intake of silkworm larvae, causing such physiological and biochemical changes which may have influenced the synthesis and utilization of total protein content in tissue of Bombyx mori larvae at initial and final stage of spinning. But treatment of larvae with high amount of Aloe vera essential oil generated stress response may cause general decline in the rate of protein synthesis.

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Received on 22.12.2014 Modified on 01.02.2015

Research J. Science and Tech. 7(1): Jan.-Mar. 2015; Page 29-34

DOI: 10.5958/2349-2988.2015.00006.6

Mean difference in between groups	Stage of treatment
Mean difference in between groups	Single	Double	Triple
X₁ ~ X₂	0.388	0.974	1.579
X₁ ~ X₃	1.194	2.082	*3.030
X₁ ~ X₄	2.245	*3.674	*4.780
X₁ ~ X₅	0.738	1.581	*2.690
X₂ ~ X₃	0.806	1.108	1.451
X₂ ~ X₄	1.857	2.700	*3.201
X₂ ~ X₅	0.350	2.555	*4.269
X₃ ~ X₄	1.051	1.592	1.750
X₃ ~ X₅	0.456	*3.663	*5.720
X₄ ~ X₅	1.507	*5.255	*7.470