Improvement in cocoon yield induced by phytojuvenoid on the multivoltine mulberry silkworm ( Bombyx mori Linn . )

Impact of phytojuvenoid on commercial parameters in Bombyx mori, a monophagous insect, was studied. The variation in the phytojuvenoid concentration significantly influenced the length of cocoon and the number of larval treatment did not cause significant influence on the length of cocoon of B. mori. The length of cocoon increased from 2.88 cm (control) to the maximum level of 3.65 cm in 30% phytojuvenoid concentration triple treated larvae and the volume of cocoon increased with increasing the number of larval treatment from single to triple in 10, 20 and in 30% phytojuvenoid concentration and the volume was highest (3.48 ml) in 30% phytojuvenoid concentration at triple treated larvae. The results show that topical application of bioactive phytojuvenoid improved the commercial parameters in B. mori.


INTRODUCTION
Silk, the natural fiber that spells splendor lusture and elegance, has been an inseparable part of Indian culture and tradition, over thousands of years.Mulberry sericulture in India is a commercially attractive and sustainable farm based economic enterprise positively favoring the rural poor in the unorganized sector.Nistari is a resistant variety of multivoltine mulberry silkworm (Bombyx mori) which contributes up to a great extent in the commercial production of cocoon.The efforts are being made to evolve new technologies that are effective, labour saving and eco-friendly.In order to increase, the production of silk, efforts have been made to study effect of ecological factor, photoperiod, artificial diet (Iwanvat and Ono, 1969), X-rays (Kanarev and Cham, 1985) etc. on the performance of silkworm.The Magnetization of eggs influences silk producing potential and incubation period of eggs (Upadhyay and Prasad, 2010b) and larval performance (Prasad and Upadhyay, 2011).
The phytoecdysteroid has been noticed to influence the *Corresponding author.E-mail: dr.rolisri@gmail.comAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License development, growth, silk producing and reproductive potential of B. mori (Srivastava and Upadhyay, 2012a, b).The juvenile hormone (JH) analogue also has been noticed to influence the reproductive and commercial potential of B. mori (Srivastava and Upadhyay, 2013a, b, c;Nair et al., 2003).The JH analogues and mimics have been reported to have some hormonal influence on the growth of B. mori and cocoon production (Nair et al., 2006).However, the response to such treatment varies depending on the dosage of compounds showing duration and number of applications (Chowdhary et al., 1990).
The more food ingested during this period, the more it gets converted and in turn contributes to silk protein.Delay in moulting is probably due to the inhibitory action of JH on ecdysone synthesis in B.mori (Sakurai et al., 1986;Trivedy et al., 1997).JH is claimed to inhibit protein synthesis in early treated larvae with later on region protein synthesis resulting in bigger silk gland and the result is improvement of cocoon shell weight (Garel, 1983).Some plants like Pinus longifolia, Abies balsomea, Psorelea corylifolia and Azadiracta indica act on B. mori larvae as bioactive juvenoid compounds (Nair et al., 1999).Keeping this in view, an attempt has been made to study the topical effect of bioactive phytojuvenoid on the improvement in the commercial parameters in this monphagous insect (B.mori), which is the aim of the present investigation.

MATERIALS AND METHODS
The seed cocoons (pupa enclosed in silken case) of multivoltine mulberry silkworm (B.mori nistari), a native of West Bengal in India, were obtained from the silkworm grainage, Directorate of sericulture, Behraich Uttar Pradesh and were maintained in the plywood trays (23 x 20 x 5 cm) under the ideal rearing conditions (Krishnaswamy et al., 1973) in the silkworm laboratory, Department of Zoology D.D.U.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 moths emerged generally in the morning at around 4 A.M. The tray in which seed cocoons were kept, was suddenly illuminated by light in the morning at 4 O'clock on 9 th and 10 th day of spinning.The newly emerged moths were quickly picked up and kept sexwise in separate trays to avoid copulation.The male moths were smaller in size but more active than the female moths which were comparatively larger and less active.The whole grainage operation was performed as per description given by Krishnaswamy et al. (1973) and Jolly (1983).

Copulation
Moths have a tendency to pair immediately after emergence, 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 h / day dim light condition.After 4 h of mating, the paired moths were detached 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 for egg laying.

Oviposition
The gravid females laid eggs on the sheet of paper in the dark condition at 26  1°C and 80  5% RH.The egg laying moths were covered by open plastic cellules to prevent intermixing of egg masses deposited by different moths.After 24 h of egg laying, the female moths were individually examined for their disease freeness.The females were crushed individually in mortar with pestles and blood smears were examined by microscope under 15 x 45 magnifications for the detection of bacterial and protozoan pathogens.

Incubation of eggs and hatching
The disease free layings (D.F.L's), thus prepared, were treated with 2% formaline for 15 min to increase the adhesiveness of eggs on the paper sheet and surface disinfection.Thereafter, the egg sheets with eggs 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.

Rearing of larvae
After two consecutive days of hatching, the silkworm larvae were collected with the help of feather of birds and reared to maintain a stock culture in the silkworm laboratory at 26  1°C and 80  5% RH and 12  1 h light a day.Four feedings of the small pieces of fresh and clean leaves of Morus alba were given to the larvae and care was taken that food always remained in excess in the rearing trays.These larvae were taken for the purpose of experiments.
After completion of fifth instar, the ripe worms ceased feeding and ready for spinning.Now small mountages were provided to the ripe worms.The ripe worms soon begin the mounting which was completed within three days.Thus, sufficient number of cocoons was obtained from the silkworm larvae reared in our laboratory.

Design of experiment
For extraction of phytojuvenoid, the needle of Pinus longifolia were collected, washed thoroughly with distilled water and dried in incubator at 37°C.The dried materials were powdered separately with the help of mechanical device.Further, 50 g powder was subjected to extraction separately through soxlet apparatus with 250 ml distilled water for 40 h.After 40 h of extraction, a little amount of concentrated solution of plant extract was obtained.The concentrated solution was dried and 6.45 g material was obtained in powdered form.The dried powder thus obtained, was dissolved in distilled water as 5 g in 25 ml water and this solution was used for further experiment, as 100% concentration of phytojuvenoid.For further experiment, the suitable narrow ranges of Pinus phytojuvenoid concentrations viz. 10, 20, 30 and 40% were taken.Thus, four phytojuvenoid concentrations were applied topically by spraying as 1 ml on to 100 larvae separately.Three sets of experiments were designed viz., single, double and triple treatment of larvae.

Single treatment of larvae
Single treatment of larvae was performed at the initial stage of fifth instar larvae just after fourth moulting.One hundred larvae of fifth instar at the initial stage were taken out from the BOD incubator and treated with 1 ml of 10% concentrated solution of Pinus needle extract by sprayer.

Double treatment of larvae
Double treatment of larvae was started from the initial stage of fourth instar larvae.In the first treatment, 100 larvae of fourth instar were treated by 1 ml of 10% concentrated solution of Pinus needle extract by spraying.The treated larvae were then transferred in BOD incubator for rearing and development.Further, similar second treatment for the same larvae was given at the initial stage of fifth instar larvae.Thus, in double treatment, fourth and fifth instar larvae were treated.

Triple treatment larvae
For triple treatment, the third instar larvae in the initial stage were separated from BOD incubator.In the first treatment 100, third instar larvae, were treated by 1 ml of 10% concentrated solution of Pinus needle extract by sprayer and kept in BOD for rearing.The second treatment of same larvae was done just after third moulting that is at the initial stage of fourth instar larvae and transferred in BOD incubator for rearing.Third treatment was given at the initial stage of fifth instar that is just after fourth moulting of the same treated larvae as earlier.Thus, in the triple treatment third, fourth and fifth instar larvae were treated.Similar experiments were performed by 20, 30 and 40% concentrations of phytojuvenoid obtained from Pinus needle extract.A control set was always maintained with each set of experiment.All the data obtained by the experiment were analyzed statistically by two-way ANOVA and Post-hoc test.

Length of cocoon
The cocoon obtained by the experiment was measured for length.The length of cocoon was taken by cutting the cocoon from the middle in length.The average length of cocoon (three batches of 10 cocoons in each batch) was recorded for each replicate.Three replicates of each experiment were made.

Volume of cocoon
To observe the volume of cocoon, healthy cocoons were tooken and cut slightly at the top end and the pupae was removed.The empty cocoon was filled with water with the help of pipette and the volume of required water was measured in milliliter.For the average volume of cocoon, 30 cocoons (three batches of 10 cocoons in each batch) were filled with water for each replicate.Three replicates of each experiment were made.

Length of cocoon
It is clear from the data given in the Table1a that the phytojuvenoid concentration and number of larval treatment caused notable influence on the length of cocoon.With the increasing number of larval treatment with 10, 20 and 30% phytojuvenoid concentration, the length of cocoon increased gradually and reached the maximum level of 3.65±0.02cm in the case of triple treated larvae with 30% phytojuvenoid concentration.In the case of larval treatment with 40% phytojuvenoid concentration, the length of cocoon increased in single treated larvae but further increase in the number of larval treatment caused decline in the length of cocoon which reached to the minimum level of 2.78±0.01cm in triple treated larvae.The trend of increase in the length of cocoon was almost of same fashion in 10, 20 and 30% phytojuvenoid concentration in relation to the number of larval treatment.Two-way ANOVA indicates that variation in phytojuvenoid concentration significantly (P1 < 0.01) influenced the length of cocoon.The Post-hoc test (Table1b) shows significant group difference in the length of cocoon in all group combinations in single treated larvae except in between control and 10%, 10 and 40% and 20 and 30% phytojuvenoid concentration.In the double and triple treated larvae, all the group combinations showed significant group difference in the length of cocoon except in between 20 and 30% phytojuvenoid concentration.

Volume of cocoon
The data presented in Table 2a clearly indicates that the phytojuvenoid concentration and the number of larval treatment of larvae caused notable influence on the volume of cocoon.With the increasing number of larval treatment with 10, 20 and 30% phytojuvenoid concentration, the volume of cocoon increased gradually and reached to the maximum level of 3.48±0.02ml in the case of triple treated larvae with 30% phytojuvenoid concentration.In the case of larval treatment with 40% phytojuvenoid concentration, the volume of cocoon increased in single treated larvae but further increase in the number of larval treatment caused decline in the volume of cocoon which reached to the minimum level of 2.69±0.02ml in triple treated larvae.The trend of increase in the volume of cocoon was almost of same pattern in 10, 20 and 30% phytojuvenoid concentration in relation to the number of larval treatment.
Two-way ANOVA indicates that variation in the phytojuvenoid concentration significantly (P1 < 0.01) influenced the volume of cocoon.The Post-hoc test (Table 2b) shows significant group difference in the volume of cocoon in between control and 20% control and 30%, 10 and 20%,10 and 30% and 20 and 40% at in case of single treated larvae.In the double and triple treated larvae, all the group combinations showed significant group difference in the volume of cocoon except in between control and 40% and 20 and 30% phytojuvenoid concentration.

Length of cocoon
The length of B. mori cocoon was influenced due to change in the phytojuvenoid concentration and the number of larval treatment.With the increasing number of larval treatment from single to triple, the length of cocoon increased in case of 10, 20 and 30% phytojuvenoid concentration while in 40% concentration, the length of cocoon increased in single treatment and further decreased with the increasing number of larval treatment.The reserpine of Ranwolfia serpentina plays a stimulative role in an increase in the length of cocoon (Sujatha and Rao, 2002b).The silkworm strains have been classified on the basis of cocoon length and other variables (Nakada, 1989(Nakada, , 1992a(Nakada, , 1992b)).The cocoon length and width are important variables on account of the evolutionary aspects of the silkworm (Nakada, 1991(Nakada, , 1994)).The variability existed in the polyvoltine germplasm stocks with regard to cocoon length (Rao and Nakada, 1998).The number of genes with regards to the expression of cocoon length has been identified and it was found that not many genes are involved in this (Gamo et al., 1985).In the present investigation, the length of cocoon is increased with increasing the application of phytojuvenoid concentrations up to 30% while the cocoon length decreased with 40% concentration of phytojuvenoid showing that the response is largely dose dependent.The higher phytojuvenoid concentration either resulted in the formation of vulnerable larvae or in pupal mortality.This seems to be due to the total disturbance in the endogenous hormone titre and concombinent disarrangements in the tissue metabolic activities.The lower phytojuvenoid concentrations may have influenced the metamorphic rhythm as well as economic traits as spinning process.

Volume of cocoon
The variation in the phytojuvenoid concentration and number of larval treatment influenced the volume of B. mori cocoon.The minimum volume of cocoon was noticed in case of the larvae treated with 40% phytojuvenoid concentration -triple treated larvae, whereas the maximum volume of cocoon was recorded in the case of 30% phytojuvenoid concentration -triple treated larvae (Table 2a).The topical application of the juvenile hormone or of a structural analog is able to increase the silk production (Akai et al., 1971).
The reserpine of Ranwolfia serpentine plays a stimulative role in an increase in volume of cocoon (Sujatha and Rao, 2002a, b).Cocoon volume and width variables are important on account of the evolutionary aspects of the silkworm (Nakada, 1991(Nakada, , 1994) ) and the variability existed in the polyvoltine germplasm stocks with regard to cocoon volume (Rao and Nakada, 1998).The genes with regards to expression of cocoon volume have been identified (Gamo et al., 1985).Twenty-five eco-races of Antheraea mylitta alone reveal interesting variability in volume of cocoon (Rangaswami et al., 1987).The females spin larger cocoon than the males and thus the female cocoon volume is more than for male (Rangaswami et al., 1987).Thus, with the increasing phytojuvenoid concentration from 10 to 30%, the volume of cocoon increased.It seems that the increase in the volume of cocoon may be due to the conversion of additional leaf consumed during the extended period into the silk material and the direct stimulatory effect of phyojuvenoid compound on the protein synthesis in silk gland.

Table 1a .
Effect of phytojuvenoid treatment on the cocoon length (cm) in Bombyx mori.Non significant.Each value represents mean+ S.E. of three replicates; X1, X2, X3, X4 and X5 are the mean values of the cocoon length (cm) in control, 10, 20, 30 and 40 % phytojuvenoid concentration respectively.

Table 2a .
Effect of phytojuvenoid treatment on the cocoon volume (ml) in Bombyx mori.

Table 2b .
Post -hoc test showing effect of phytojuevnoid treatment on the cocoon volume (ml) in Bombyx mori.are the mean values of cocoon volume (ml) in Bombyx mori in control, 10, 20, 30 and 40 per cent phytojuvnoid concentration respectively.