International

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Innovative Research International Academic Journal of Innovative Research Vol. 3, No. 10, 2016, pp. 1-31.

ISSN 2454-390X

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International Academic Institute for Science and Technology

The Pattern of Chitin Deposition in the Integument of Fifth

Instar Larvae of Silkworm for Topical Application of Acetone

Solution of Triterpene Compounds

Vitthalrao B. Khyade Malegaon Sheti Farm, Agricultural Development Trust Baramati, Shardanagar, (Malegaon Khurd) Post Box No.- 35 Tal. Baramati.

Dist. Pune 413 115 Maharashtra, India.

Residence: “Dr. APIS”, Shrikrupa Residence, Teachers Society, Malegaon Colony (Baramati) Dist. Pune

Abstract

The exogenous metabolites are serving a lot to take pause in the progression of insect metamorphosis

through arresting some of the biochemical reactions including chitin synthesis or accelerating progression

through other biochemical pathways in the larval body of insects. The ten microliters of various

concentrations of acetone solution of Fernasol Methyl Ether (FME) and each selected triterpene

compounds (Squalene; Polypodatetraene; Malabaricane; Lanostane; Hopane and Oleanane) were used for

topical application to individual larval instars of silkworm, Bombyx mori (L) (Race: PM x CSR2) at 48

hours after the fourth moult. The body wall integument chitin of untreated control larvae; acetone treated

control; FME treated larvae and triterpene treated larvae was estimated at 120 hours after the fourth

moult. Topical application of selected concentrations of acetone solutions of FME and selected

triterpenes to fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2) was found reflected

into the reduction in the deposition of chitin in the larval body wall. The reduction in body wall chitin was

found ranging from zero to hundred percent.

Topically applied acetone solution of triterpenes may act through binding to the “Ex-JHA-Receptors”

(EJHAR) in the epidermal cells of integument of the fifth instar larvae of silkworm, Bombyx mori (L)

(Race: PM x CSR2 ). This may further associate with DNA of epidermal cells as heterodimer. This

association may alter the configuration of EJHAR, which affects the working of DNA. It may either

induce or repress transcription of a nearby genes, responsible for reduction in the deposition of chitin in

the integument of the fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2 ). “Ex-JHA-

Receptors” (EJHAR) in the integument of the fifth instar larvae of silkworm, Bombyx mori (L)(Race: PM

x CSR2 ) may be responsible to mediate transcription of different sets of genes controlling differentiation

of epidermal cells through change in the pattern of chitin deposition.

Keywords: Squalene; Polypodatetraene; Malabaricane; Lanostane; Hopane; Oleanane

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Introduction

Human culture regard certain insects as pests, and attempt to control them using insecticides and a host of

other techniques. Few insects damage crops by feeding on sap, leaves or fruits. Some parasitic species

are pathogenic. The study of phytophagous insect pests has been very important in agriculture. Possibly

from ancient times, humans have selected varieties of crop plants that are minimally attacked by insects,

and in the last 100 years breeding programs have been important in specifically increasing plant

resistance. The life stages of insect obtain their nutrients and growth promoting biocompounds from the

variable or specific flora available for them. The flora on earth is the richest source of metabolites

including juvenile hormone analogues for leaf eating insects like silkworm, Bombyx mori (L) (Vitthalrao

B. Khyade, et al2015). The concentration of the juvenile hormone (JH) &the moulting hormone (MH)

serves a lot to orchestrate the progression of metamorphosis in the insects, like silkworm, Bombyx mori

(L). The insect neuroendocrine system is a mechanism involving secreted signals that allows distal cell–

cell communication and that is found in all species with a nervous system. The secreted signals,

hormones, are typically produced in endocrine glands and regulate a range of cell and tissue types,

including neurons, through which they can have a substantial influence on behavior of the insect. The

Juvenile Hormone (JH) is synthesized in the corpora allata, which is a pair of tissues in the insect brain

that function as endocrine glands. The JH has a well-characterized role in regulating worker behaviour in

honeybees, and this seems to be conserved in ans.The treatment of larvae from several species of the

dimorphic myrmicine genus Pheidole with methoprene (which is a chemical analogue of JH) is sufficient

both to induce major-destined workers and to activate a conserved but latent developmental trajectory that

expresses a super-soldier caste. Similarly, application of methoprene to larvae induces development of

majors in the formicine ant Camponotus floridanus (D.F.S., unpublished observations) and development

of queens in many ant and bee species, including Apis mellifera and Harpegnathos saltator (Vitthalrao B.

Khyade, 2016).The corpora allata belong to cephalic region of insect body secrete JH. Inhibition of

morphogenetic programme at predetermined and group specific ontogenetic positions is the

distinguishing feature of JH (Zaoral Slama, 1970). There are many compounds of plant derived, animal

derived and synthetic that exhibit the biochemical properties of natural juvenile hormone of the insects.

Such compounds are termed as “Juvenoids (Williams,1956). Prolongation of larval age seems to be the

significant influence of the exogenous topical application of acetone solutions of the juvenoids. Further,

the juvenoids are found plant material through suitable solvent exhibiting potent activity through massive

turnover, alteration of constituency of metabolites like proteins, lipids, carbohydrates, amino acids, fatty

acids & chitin too Gopakumar et al (1977); Slama (1979); Khyade et al (2002); Khyade et al ( 2003)&

Khyade( 2004). The juvenile hormone (JH) and juvenile hormone analogues(JHA or juvenoids ) are well

known to prolong the larval life; improve the physiological status of larval body of insects and therefore,

they have been tried for qualitative improvement of silk Grenier & Grenier (1983) ; Kamimura & Kikichi

(1998) ; Ratnasen (1988) ; Mamatha et al (1999) & Khyade (2002, 2003 & 2004). Gopakumar, et al

(1977) reported the juvenomimetic activity of extractives of some of the South Indian plants. This

attempt leads to imagine the probability of occurrence of juvevenomimetic action in other plants. The

larval instars of insects especially phytophagus, use to manage the titre of JH in their body and juvenoid

contents received from the host plants. This is the prime requirement of phytophagous insects for

metamorphosis to proceed. Sclerotized proteins and chitin contribute for rigidity of cuticle. This

contribute for limited capacity for keeping the pace for the growth of insect body. For the purpose of

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growth and development to proceed, the body of insects replace the old cuticle through ecdysis or

moulting. The process of ecdysis deserve periodicity and therefore, exert significant influence. The

newly deposited cuticle contribute for nonsclerotized integument present below the older cuticle. This

nascent integument exhibit strong furrows and have a capacity to expand during the process of ecdysis or

moulting. The apolysis is the metamorphic event that initiates the ecdysis. Separation of epidermal cells

from the older cuticle through the supportive action of moulting fluid and formation of ecdysial

membrane are the significant features of apolysis. Reynolds and Samuels ( 1996) reported the presence

of enzymes, like protease and chitinase in the moulting fluid, integrated action of which is responsible

for digestion of constituents of the older cuticle during the apolysis. It has been supposed that, shortly

before ecdysis, the molting fluid, which hasaccumulated in the apolysial space, get reabsorbed. And this

is for allowing the recycling of the individual constituents of older cuticle. The proteins of cuticle and

chitin fibres through the apical membranes of epidermal cells, get secreted, which is responsible for

opening the ecdysial space. Firstly, the proteins and chitin forms patches of cuticullin. This get followed

by formation of so called the outer epicuticle. The procuticle get formed below the outer epicuticle. The

inner epicuticle get deposited and seals the epidermis. This seems essential for prevention of protection

of cuticle from the digestive enzymes in moulting fluid. Before hardening or sclerotization of chitin, the

body of insect get expand, which leads to release 9 or to shed) the older envelope in the form of exuvia.

According to Carlson and Bentley ( 1977) , the release of older cuticle during moulting in insects is

through distinct motor programmes and through increasing body pressure. The behavior pertaining pre-

ecdysis and ecdysis in insects are controlled by the action of moulting hormones, such as eclosion

hormone. This eclosion hormone is secreted in response to falling the titre of ecdysteroid, which in it‟s

turn causes the release of pre-ecdysis-triggering-hormone and ecdysis hormone (Truman and Riddiford,

1970; Kingan and Adams, 2000). The juvenile hormone and juvenoids regulate the quality of the moult

(Ratnasen, 1988; Khyade, et al , 2003 and Khyade, 2004). In the last larvalstadium of holometabolous

insects like silkworm, Bombyx mori (L), reduction in the titer of juvenile hormone (JH) in haemolymph is

essential event for the initiation and metamorphosisand to change into the pupa (Mamatha, et al, 1999).

Bioassay of activity of juvenile hormone and it‟s analogues (Juvenoid) have been amongst exclusively

based on the evaluation of heterochronic deviations caused in insect metamorphosis. The favourite

objects of evaluation of juvenoid effects have always been partly adult mosaic intermediates generally

known as adultoids. Since the effects of juvenoids mostly involve inhibition of metamorphosis through

change in the rate of biochemical reactions including the chitin deposition it become easier to express the

content ration (dose) of juvenoid content, topically applied in specific terms (units). The juvenoid activity

of exogenous compounds is expressed in terms of units of percent reduction of chitindeposited in the

body wall of larval stadia ( Khyade, 2011 and Jagtap, 2014). It refers to the titre or dose or concentration

of exogenous juvenoid compound topically applied „ responsible for percent inhibition of chitin

deposition in the body wall of larval instars of insects, like silkworm, Bombyx mori (L). The terpenes are

a large and diverse class of organic compounds, produced by a variety of plants. The terpenes are also

produced by some insects such as termites or swallowtail butterflies, which emit terpenes from their

osmeteria. They are often strong-smelling. They may protect the plants that produce them by deterring

herbivores and by attracting predators and parasites of herbivores. The biochemical actions of natural

insect juvenile hormone and terpenes and terpenoid compounds are similar. That is to say, the terpenes

mimics the actions of natural “Insect Juvenile Hormone”.

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Triterpenes are a class of chemical compounds composed of three terpene units with the molecular

formula C30H48; they may also be thought of as consisting of six isoprene units. Animals, plants and fungi

all create triterpenes, with arguable the most important example being squalene as it forms the basis of

almost all steroids. Triterpenes exist in a huge variety of structures with nearly 200 different skeletons

known from natural sources or enzymatic reactions.[1]

These may be broadly divided according to the

number of rings present; although in general pentacyclic structures (5 rings) tend to dominate. The

triterpenes includes: Squalene (with zero rings); Polypodatetraene (with two rings); Malabaricane (with

three rings); Lanostane (with four rings); Hopane (with five rings) and Oleanane (with six rings).The

difference between terpenes and terpenoids is that terpenes are hydrocarbons, whereas terpenoids contain

additional functional groups. Screening the plant extractives for juvenoids seems to be well established

attempt. To proceed on the same line, the present attempt on screening the acetone solution of selected

triterpene compounds has been planned.

MATERIAL AND METHOD

The experimentation was divided into seven steps, which include: Rearing of larval instars of silkworm,

Bombyx mori (L); Daily bioassay of body wall chitin of fifth instar larvae; Preparation of acetone

solutions of selected triterpenes; Grouping the fifth instar larvae and topical application of acetone

solution of triterpene; Bioassay of body wall chitin at 120 hours after the fourth moult; Statistical analysis

of the data and Plotting the “Punyamayee Baramati Dose Response Curves” for the compounds used for

topical application.

(A). Rearing of larval instars of silkworm, Bombyx mori (L):

The disease free layings (DFL) of polyvoltine, crossbreed race (PM x CSR2) of silkworm, Bombyx mori

(L) were procured. They were processed for incubation through black boxing for 48 hours. The larvae

were reared in laboratory condition on the leaves of mulberry (M-5 variety). Standard Methods of rearing

(Krishnaswami, et al, 1978 and Vitthalrao B. Khyade, 2004).

(B). Daily bioassay of body wall chitin of fifth instar larvae:

The chitin content of body wall was estimated at zero (soon after the fourth moult) ; 24; 48; 72; 96 and

120 hours after the fourth moult. The method followed for chitin estimation was volumetric (Baishya and

Hazarika, 1996; Vitthalrao Khyade, et al, 2006). Twenty larvae for each time were selected randomly and

anaesthetized with little quantity of chloroform soaked cotton pad. They were dissected in insect saline.

The abdominal fat bodies and visceral organs were removed carefully. After removing all the organ

systems, trachae and adhering fat bodies the part remained was designated as integument. The integument

of each larva was blotted and weighed on electronic balance. The integument piece of individual larva

was placed in separate test tube containing 50 ml. of 30 percent potassium hydroxide (KOH) solution. All

the test tubes in a group were placed in separate water bath. The contents of test tube were allowed for

boiling for thirty minutes. After treating the integument with boiling potassium hydroxide solution, it was

subsequently washed with distilled water; two times in ninety six percent ethanol and two times in ether.

Treated pieces of integument (body wall) were weighed accurately on electronic balance. The weight of

integument (body wall) after potassium hydroxide treatment corresponds to the quantity of chitin

(mg/gm).

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(C). Preparation of acetone solutions of selected Triterpenes:

The triterpene compounds (Squalene; Polypodatetraene; Malabaricane; Lanostane; Hopaneand Oleanane)

and Farnesol methyl etherwere procured through the local chemical suppliers. Based on preliminary

studies, known quantity of FME was dissolved in known volume of acetone so as to get desired

concentration. Various concentrations of acetone solution of FME include: 00.010 to 00.160 mg/ml.

Likewise, each triterpene compound was dissolved in acetone to get desired concentrations (00.150 to

00.320 mg/ml for Squalene; 00.160 to 0.340 mg/ml for Polypodatetraene; 00.240 to 0.400 mg/ml for

Malabaricane; 00.380 to 0.550 mg/ml for Lanostane; 00.400 to 00.560 mg/ml for Hopaneand 00.430 to

00.600 mg/ml). FME was used as a “standard Insect Juvenoid Compound” for comparison. Various

concentrations (00.005 to 00.160 mg/ml) of FME were prepared by dissolving it‟s appropriate quantity in

acetone.

(D). Grouping the fifth instar larvae and topical application of acetone solution of triterpene compounds:

Soon after the fourth moult, the larvae of fifth instar were grouped into control (Untreated and acetone

treated, each one) groups and experimental groups ( 7 x 30 ), each with fifty individuals. Ten microliters

of each concentration of acetone solution of FME (as a standard Insect JHA); Squalene;

Polypodatetraene; Malabaricane; Lanostane; Hopaneand Oleanane were topically applied with

micropipette separately to the individual fifth instar larvae at 48 hours after the fourth moult. The larvae

of all groups were maintained according to usual schedule.

( E ). Bioassay of body wall chitin at 120 hours after the fourth moult:

Body wall chitin contents of fifth instar larvae at 48 hours after the fourth moult, that is to say before

treatment was carried. The weight of chitin before treatment (at 48 hours after the fourth moult) was

considered as, “Initial Body Wall Chitin Content”(A). The Body wall chitin contents of fifth instar larvae

( Untreated Control group; Acetone Treated Control group; FME Treated group and Triterpene Treated

groups) was carried out at 120 hours after the fourth moult.The weight of chitin before treatment (at 48

hours after the fourth moult) was considered as, “Final Body Wall Chitin Content” (B). The reading

“Initial Body Wall Chitin Content”(A) was subtracted from the reading “Final Body Wall Chitin

Content” (B), which yields the quantity of chitin deposited in the body wall from 48 hours to 120 hours.

The method followed for chitin estimation was volumetric (Baishya and Hazarika, 1996; Vitthalrao

Khyade, et al, 2006). Twenty larvae from each group were selected randomly and anaesthetized with little

quantity of chloroform soaked cotton pad. They were dissected in insect saline. The abdominal fat bodies

and visceral organs were removed carefully. After removing all the organ systems, trachae and adhering

fat bodies the part remained was designated as integument. The integument (body wall) of each larva was

blotted and weighed on electronic balance. The integument (body wall) piece of individual larva was

placed in separate test tube containing 50 ml. of 30 percent potassium hydroxide (KOH) solution. All the

test tubes in a group were placed in separate water bath. The contents of test tube were allowed for

boiling for thirty minutes. After treating the integument with boiling potassium hydroxide solution, it was

subsequently washed with distilled water; two times in ninety six percent ethanol and two times in ether.

Treated pieces of integument were weighed accurately on electronic balance. The weight of integument

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after potassium hydroxide treatment corresponds to the quantity of chitin (mg/gm) in the integument of

fifth instar larvae at 120 hours after the fourth moult.

(F). Statistical analysis of the data:

The experimentations were repeated for three times for the consistency in the results. Data was collected

and subjected for statistical analysis ( mean, standard deviation and student “t” test for knowing the

significant level of treatment) (Norman and Baily, 1955). Soon after the fourth moult (zero hour) and 120

hours after the fourth moult were considered as initial and final quantity of chitin respectively.

Subtraction of initial quantity from final quantity give the quantity of chitin deposited in body wall of the

fifth instar larvae for 120 hours after the fourth moult ( 5 days of fifth instar larvae ). Quantity of chitin

(mg/gm) deposited in the treated group was subtracted from the quantity of chitin deposited in the control

group. This figure was divided by quantity of chitin deposited in control group. The quotient, thus

obtained was multiplied by hundred to know percent reduction in the chitin in the integument of larvae of

treated groups.

(G). Plotting the “Punyamayee Baramati Dose Response Curves” for the compounds used for topical

application:Dose response curve for each compound was plotted (Fig. 1). The scale for plotting the graph,

for X- axis was 1 = 00.010 mg/ml concentration of acetone solution. And that for Y- axis, the scale was 1

= 5 percent. Dose response curve for each compound was plotted (Fig. 1). The x- co-ordinate, that

corresponds to the value of fifty on y-axis in dose response curve was designated as: Baramati

Concentration of Acetone Solution of Triterpene responsible for fifty percent reduction in chitin

deposition ( BCAS50) in the integument (Body Wall) of the Fifth Instar Larvae of

multivoltine Crossbreed Race (PM x CSR2) of Silkworm, Bombyx mori (L). Thus, BCAS50 value for each

compound in the study was calculated through the use of respective dose response curve. The plot of

dosages of acetone extractives of selected compounds and percent change in the body wall chitin of larval

instars of silkworm, Bombyx mori (L) is to be recognized as “Punyamayee Baramati Dose Response

Curve”.

RESULTS AND DISCUSSION

The results are summerised in Table – 1 to 8 and Fig.1 and Fig.2. The amount of chitin( mg/ gm)

deposited in the body wall of the fifth instar larvae at 0.00;24; 48;72;96 and 120 hours after the fourth

moult were found measured as: 19.774 (±1.087); 19.779 (±1.143); 19.786 (±2.057); 20.679(±1.789);

26.823(±3.018) and 38.186(±3.632) units respectively (Table-1 and Fig.2).

Table – 1 : Chitin content in the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2).

Serial No. Hour After the Fourth Moult Body Wall Chitin Content

(mg/Gm)

1 000.000 19.774 (± 1.087 )

2 024.000 19.779 ( ± 1.143 )

3 048.000 19.786 ( ± 2.057 )

4 072.000 20.679 ( ± 1.789 )

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5 096.000 26.823 ( ± 3.018 )

6 120.000 38.186 ( ± 3.632 )

- Each figure is the mean of three replications.

- Figures with ± sign in parentheses are the standard deviations.

- Chitin Deposition for Untreated Control Larvae = Chitin content at 120 hours after the fourth

moult – Chitin content at 48 hours after the fourth moult ( 18.4 = 38.186 – 19.786 ).

Fig.2 :Daily Chitin content (mg/Gram) in the body wall of the fifth instar larvae of silkworm,

Bombyx mori (L) (Race: PM x CSR2).

In the untreated and acetone treated groups, the body wall chitin at 120 hours after the fourth moult was

38.186 (±3.632) and at 48 hours after the fourth moult was 19.786 (±2.057). Subtraction of chitin content

at 48 from 120 hours gives the amount of chitin deposited during the experimental period (38.186 –

19.786 = 18.400 ).During the early age ( up to 48 hours) of fifth instar larvae of silkworm, Bombyx mori

(L), the titer of juvenile hormone (JH) in the haemolymph is maintained at significant detectable level

(Shi-Hong Gu andYein Shing Chow, 1996). Rate of chitin deposition during this period seems to be non

significant. Thereafter, the juvenile hormone (JH) in the larval haemolymph get decreased rapidly. The

most possible reason for this includesaccelerative rate activity of esterase after 48 hours after the fourth

moult (Ajami andRiddiford, 1973; Khyade, 2004).

Topical application of ten microlitres of FME and selected triterpenes was found reduction in

chitin deposition in the body wall (integument). And the pattern

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was exhibiting significant response with reference to chitin deposition pattern in the body wall of fifth

instar larvae of silkworm, Bombyx mori (L) ( Race: PM x CSR2). The reduction in body wall chitin was

found ranging from zero to hundred percent. The plot of concentrations of acetone solutions of FME and

triterepene compounds and percent reduction in the body wall chitin was found exhibiting a characteristic

Sigmoid form of displacement, which herewith titled as “Punyamayee Baramati Dose Response Curve”

(Fig.1). The FME was found with lower concentration of it‟s acetone solution for reduction in chitin

deposition in the body wall of fifth instar larvae of silkworm, Bombyx mori (L) ( Race: PM x CSR2). The

concentrations namely, 00.000; 00.500; 01.00; 01.500; 02.000; 02.500; 03.000; 03.500; 04.000; 04.500

and 05.00 ppm (mg/ml) of FME were found with non-significant reduction in chitin deposition. The

concentrations such as 05.400; 06.000; 07.000; 08.000; 09.000; 10.000 and 10.500 ppm (mg/ml) of FME

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were found with significant reduction in chitin deposition. Higher concentrations of FME (from 11.000

ppm and above ) were with most significant reduction in the body wall chitin deposition ( they found to

yield maximum possible reduction in chitin deposition).

The sigmoid curve of pattern of percent reduction in chitin deposition and concentrations of

acetone solutions of FME and triterpenes topically applied at 48 hours after the fourth moult to the larval

instars of silkworm, Bombyx mori (L) (Race: PM x CSR2) in the study seems to reflect three groups of

concentration of acetone solutions topically, which include: Non-significant; Significant and the most

significant. The non-significant concentrations of acetone solutions of Squalene; Polypodatetraene;

Malabaricane; Lanostane; Hopaneand Oleananein the study include: 00.000 to 00.195; 00.000 to 00.225;

00.000 to 00.285; 00.000 to 00.435; 00.445 and 00.000 to 00.470 mg/ml respectively.

The significant concentrations of Squalene; Polypodatetraene; Malabaricane; Lanostane;

Hopaneand Oleanane include: 00.205 to 00.260; 00.230 to 00.280; 00.285 to 00.340; 00.440 to 00.4950;

00.450 to 00.505and 00.475 to 00.535 mg/ml respectively. That is to say, the percent reduction of chitin

deposition of these concentrations occupy the steeper region of the sigmoid curve.

Table – 2: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of Fernasol

Methyl Ether (FME) at 48 hours after the fourth moult.

X Concentration

of Acetone

Solution (ppm)

Body Wall

Chitin

(mg / gm)

Chitin

Deposition

( mg / gm )

Percent

Reduction

Y

00.000 00.000 38.186

( ± 4.673)

18.400 000.000 000.000

00.500 00.005 38.002 *( ±

4.651)

18.216 01.000 000.200

01.000 00.010 37.910 *

(± 4.397)

18.124 01.500 000.300

01.500 00.015 37.823*

(± 4.089)

18.037 02.000 000.400

02.000 00.020 37.726*

(± 3.391)

17.940 02.5000 000.500

02.500 00.025 37.634*

(± 3.906)

17.848 03.000 000.600

03.000 00.030 37.542*

(± 4.289)

17.756 03.500 000.700

03.500 00.035 37.266*

(± 3.258)

17.483 05.000 001.000

04.000 00.040 36.990 *

(± 4.078)

17.204 06.500 01.300

04.500 00.045 36.346 *

(± 3.966)

16.560 10.000 02.000

05.000 00.050 35.610* 15.824 14.000 02.800

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(± 4.023)

05.400 00.054 34.966* *

(± 3.843)

15.180 17.500 03.500

06.000 00.060 35.586* *

(± 4.143)

13.800 25.000 05.000

07.000 00.070 31.286 * *

(± 4.518)

11.500 37.500 07.000

08.000 00.080 28.986 * *

(± 3.513)

09.200 50.000 10.000

09.000 00.090 26.686* *

(± 3.795)

06.900 62.500 12.500

10.000 00.100 24.386* *

(± 3.786)

04.600 75.000 15.000

10.500 00.105 23.236* *

(± 3.897)

03.450 81.250 16.250

11.000 00.110 22.362* * *

(± 3.841)

02.576 86.000 17.200

11.500 00.115 21.718* * *

(± 4.948)

01.932 89.500 17.900

12.000 00.120 21.258* * *

(± 4.013)

01.472 92.000 18.400

12.500 00.125 20.798* * *

(± 3.427)

01.012 94.500 18.900

13.000 00.130 20.522* * *

(± 3.734)

00.736 96.000 19.200

13.500 00.135 20.246* * *

(± 3.964)

00.460 97.000 19.500

14.000 00.140 20.062* * *(±

3.687)

00.276 98.500 19.700

14.500 00.145 19.878* * *

(± 3.789)

00.092 99.500 19.900

15.000 00.150 19.786 * * *

(± 3.881)

00.000 100.00 20.000

15.500 00.155 19.786 * * *

(± 3.963)

00.000 100.00 20.000

16.000 00.160 19.786* * *

(± 3.794)

00.000 100.000 20.000

1. Each figure is the mean of three replications;

2. Figures in parenthesis with ± sign are the standard deviations.

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3. *=P < 0.005 ; * *=P < 0.01 And * * *=P < 0.001

4. Scale: 0.01 mg/ml acetone solution of Triterpene Compound = 1 Unit of : “X”. : 5 Percent

Reduction in chitin Deposition = 1 Unit of : “Y”.

The higher concentrations of acetone solutions of Squalene; Polypodatetraene; Malabaricane;

Lanostane; Hopaneand Oleanane (00.265 and above; 00.285 and above; 00.345 and above; 00.500 and

above; 00.510 and above and 00.540 mg/ml and respectively) resulted into the most significant reduction

in the chitin deposition. During the early age (up to 48 hours) of fifth instar larvae of silkworm, Bombyx

mori (L), the titer of juvenile hormone (JH) in the haemolymph is maintained at significant detectable

level . Rate of chitin deposition during this period seems to be non significant. Thereafter, the juvenile

hormone (JH) in the larval haemolymph get decreased rapidly. The most possible reason for this include

accelerative rate activity of esterase after 48 hours after the fourth moult Ajami & Riddiford (1973);

Khyade, (2004). The present study demonstrate to decrease in chitin deposition in the body wall of fifth

instar larvae of silkworm, Bombyx mori (L) (Race : PM x CSR2) recipient of the exogenous juvenoid

material in the form of acetone extractives of selected plants. The significant feature of exogenous

juvenoids is to slows down the rate of chitin synthesis in the body of insects. The appreciable

sclerotization before spinning seems to be prerequisite for metamorphosis to proceed Omana Joy (1983).

The titer of juvenile hormone in the haemolymph of fifth instar larva in late age ( last three days) is to be

maintained at insignificant, undetectable level for the purpose to proceed metamorphosis through

accelerate rate of metabolism including chitin deposition. Delay in the maturation for spinning in the

larvae treated with FME and terpenes ( let us label them “Silkworm Juvenoids”), as observed in the

present study, may be to resume normal rate of chitin deposition.

Table – 3: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of Squalenene

(Triterpene) at 48 hours after the fourth moult.

X Concentration

of Acetone

Solution (ppm)

Body Wall

Chitin

(mg / gm)

Chitin

Deposition

( mg / gm )

Percent

Reduction

Y

15.000 00.150 38.186*( ±

4.729)

18.400 00.000 00.000

15.500 00.155 38.186*( ±

4.337)

18.400 00.000 00.000

16.000 00.160 38.094*

( ± 3.899)

18.400 00.000 00.000

16.500 00.165 38.0094*( ±

4.107)

18.308 00.500 00.100

17.000 00.170 38.002*( ±

4.786)

18.216 01.000 00.200

17.500 00.175 37.818*( ±

4.517)

18.032 02.000 00.400

International Academic Journal of Innovative Research,

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12

18.000 00.180 37.542*

( ± 3.583)

17.756 03.500 00.700

18.500 00.185 37.266*

( ± 4.404)

17.480 05.000 01.000

19.000 00.190 37.082*

( ± 3.651)

17.296 06.000 01.200

19.500 00.195 36.714*

( ± 3.793)

16.928 08.000 01.600

20.000 00.200 36.116* *

( ± 4.761)

16.330 11.250 02.250

20.300 00.203 35.926* *

( ± 4.583)

15.640 15.000 03.000

20.500 00.205 34.3506* *( ±

4.188)

14.720 20.000 04.000

21.000 00.210 33.816* *

( ± 3.919)

14.030 23.750 04.750

21.500 00.215 32.666* *

( ± 4.724)

12.880 30.000 06.000

22.000 00.220 31.516* *

( ± 4.592)

11.730 36.250 07.250

22.500 00.225 30.366* *

( ± 3.798)

10.580 42.500 08.500

23.000 00.230 29.216* * *

( ± 4.478)

09.430 48.750 09.750

23.500 00.235 28.066 (

± 4.076)

08.280 55.000 11.000

24.000 00.240 26.916* * *

( ± 3.877)

07.130 61.250 12.250

24.500 00.245 25.766* * *

( ± 3.813)

05.980 67.500 13.500

25.000 00.250 24.662* * *

( ± 3.845)

04.876 73.750 14.750

25.500 00.255 23.466* * *

( ± 2.892)

03.680 80.000 16.000

26.000 00.260 22.316* * *

( ± 2.883)

02.530 86.250 17.250

26.500 00.265 21.442* * *

( ± 4.729)

01.656 91.000 18.200

27.000 00.270 20.899* * *

( ± 3.071)

01.104 94.000 18.800

27.500 00.275 20.222* * * 00.736 96.000 19.200

International Academic Journal of Innovative Research,

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13

( ± 2.984)

28.000 00.280 20.246 * * *

( ± 3.715)

00.460 97.500 19.500

28.500 00.285 20.062* * *

( ± 2.946)

00.276 98.500 19.700

29.000 00.290 19.878* * *

( ± 3.246)

00.092 99.500 19.900

29.500 00.295 19.832* * *( ±

3.847)

00.046 99.750 19.950

30.000 00.300 19.786* * *

( ± 3.351)

00.000 100.000 20.000

The present study demonstrate the titer of exogenous juvenoid material get reflect into various

conditions of juvenility ( in the form of decreased amount of chitin in the body wall) of fifth instar larvae

of silkworm, Bombyx mori (L) ( Race : PM x CSR2). Reduction in the deposition of chitin in body wall of

treated larvae( irrespective of acetone solution of FME and trioterpenes; and their concentrations too)

recorded in the study, establish a positive effect, which seems to be in agreement with results obtained

through the use of Juvenoids compounds in silkworm larvae (Akai and Kobayashi ,1971; Sharad Jagatap ,

2007 ; Vitthalrao Khyade , 2009). Selected doses of selected of triterpenes may be utilized for the purpose

to sustain the larval age, which is essential to uplift the time required for eating mulberry leaves and

amount of mulberry leaves eaten. If the maximum possible juvenoid effect in the form of reduction in

body wall chitin in the fifth instar larvae of silkworm considered as hundred percent reduction in the

chitin content, it has been found that, successive percent reduction from zero to hundred appear to be

proportional to the topically applied concentration (dosage) within some narrow range .The relationship

between titer (concentration) of exogenous juvenoid material (acetone solutions of selected FME and

triterpenes) & intensity of chitin deposition in the body wall of larvae appear to be in the form sigmoid

curve, which, herewith entitled as “Punyamayee Baramati Dose Response Curve”. These curves seems to

exhibit a characteristic S-form (sigmoid) displacement across the scale of concentration (mg/ml) of FME

and triterpenes. The change from zero to hundred percent effect commonly exhibited over 10-50 fold

change in the dose topically applied.

Table – 4: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of

Polypodatetraene (Triterpene) at 48 hours after the fourth moult.

X Concentration

of Acetone

Solution (ppm)

Body Wall

Chitin

(mg / gm)

Chitin

Deposition

( mg / gm )

Percent

Reduction

Y

International Academic Journal of Innovative Research,

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14

18.000 00.180 38.186*

( ± 4.786)

18.400 00.000 00.000

18.500 00.185 38.094*

( ± 4.758)

18.308 00.500 00.100

19.000 00.190 38.002*

( ± 4.877)

18.216 01.000 00.200

19.500 00.195 37.910*

( ± 4.647)

18.124 01.500 00.300

20.000 00.200 37.818*

( ± 4.673)

18.032 02.000 00.400

20.500 00.205 37.634*( ±

4.696)

17.848 03.000 00.600

021.000 00.210 37.358*

( ± 3.756)

17.572 04.500 00.900

21.500 00.215 36.990 *

( ± 3.938)

17.204 06.500 01.300

22.000 00.220 36.622*( ±

4.088)

16.836 08.500 01.700

22.500 00.225 36.070* *( ±

4.413)

16.284 11.500 02.300

23.000 00.230 35.196 (

± 3.836)

15.410 16.250 03.250

23.500 00.235 34.046* *( ±

4.273)

14.260 22.500 04.500

24.000 00.240 32.896* *

( ± 3.781)

13.110 28.750 05.750

24.100 00.241 32.666* *( ±

4.024)

12.880 30.000 06.000

24.500 00.245 31.746* *

( ± 3.791)

11.960 35.000 07.000

25.000 00.250 30.596* *( ±

4.333)

10.810 41.250 08.250

25.500 00.255 29.446* *( ±

4.526)

09.660 47.500 09.500

26.000 00.260 28.296* * *

( ± 3.589)

08.510 53.750 10.750

26.500 00.265 27.146* * *

( ± 3.019)

07.360 60.000 12.000

27.000 00.270 25.996* * *

( ± 3.326)

06.210 66.250 13.250

27.500 00.275 24.846* * * 05.060 72.500 14.500

International Academic Journal of Innovative Research,

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15

( ± 3.089)

28.000 00.280 23.696* * *( ±

3.581)

03.910 78.750 15.750

28.500 00.285 22.546* * *

( ± 3.334)

02.760 85.000 17.000

29.000 00.290 21.626* * *( ±

2.789)

01.840 90.000 18.000

29.500 00.295 21.166* * *

( ± 3.061)

01.380 92.500 18.500

30.000 00.300 20.706* * *

( ± 2.926)

00.920 95.000 19.000

30.500 00.305 20.430* * *

( ± 2.911)

00.644 96.500 19.300

31.000 00.310 20.246* * *

( ± 3.091)

00.460 97.500 19.500

31.500 00.315 20.154* * *

( ± 2.517)

00.368 98.000 19.600

32.000 00.320 19.97* * *

( ± 2.645)

00.184 99.000 19.800

32.500 00.325 19.878* * *

( ± 2.853)

00.092 99.500 19.900

33.000 00.330 19.786

( ± 3.326)

00.000 100.00 20.000

Table – 5: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of Malabaricane

(Triterpene) at 48 hours after the fourth moult.

X Concentration

of Acetone

Solution (ppm)

Body Wall

Chitin

(mg / gm)

Chitin

Deposition

( mg / gm )

Percent

Reduction

Y

24.000 00.240 38.186*

( ± 4.817)

18.400 00.000 00.000

24.500 00.245 38.094*

( ± 4.801)

18.308 00.500 00.100

25.000 00.250 38.002*( ±

4.678)

18.216 01.000 00.200

25.500 00.255 37.910*( ±

4.732)

18.124 01.500 00.300

26.000 00.260 37.726*( ±

4.789)

17.940 02.500 00.500

International Academic Journal of Innovative Research,

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16

26.500 00.265 37.542*( ±

4.757)

17.756 03.500 00.700

27.000 00.270 37.174*

( ± 5.842)

17.388 05.500 01.100

27.500 00.275 36.714*

( ± 4.863)

16.928 08.000 01.600

28.000 00.280 36.070* *

( ± 5.781)

16.284 11.500 02.300

28.500 00.285 35.426* *

( ± 5.291)

15.640 15.000 03.000

29.000 00.290 34.276* *

( ± 5.045)

14.490 21.250 04.250

29.500 00.295 33.126* *

( ± 4.893)

13.340 27.500 05.500

30.000 00.300 32.022* *

( ± 4.923)

12.236 33.750 06.750

30.500 00.305 30.826* *

( ± 4.811)

11.040 40.000 08.000

31.000 00.310 29.676* *

( ± 4.845)

09.890 46.250 09.250

31.500 00.315 28.526* *

( ± 4.759)

08.740 52.500 10.500

32.000 00.320 27.376* * *

( ± 3.813)

07.590 58.750 11.750

32.500 00.325 26.206* * *

( ± 4.321)

06.440 65.000 13.000

33.000 00.330 25.076* * *

( ± 3.062)

05.290 71.250 14.250

33.500 00.335 23.926* * *

( ± 4.562)

04.140 77.500 15.500

34.000 00.340 22.776* * *

( ± 4.181)

02.990 83.750 16.750

34.500 00.345 21.626* * *

( ± 4.393)

01.840 90.000 18.000

35.000 00.350 21.534* * *

( ± 3.678)

01.748 90.500 18.100

35.500 00.355 20.798* * *

( ± 4.639)

01.012 94.500 18.900

36.000 00.360 20.614* * *

( ± 4.223)

00.828 95.500 19.100

36.500 00.365 20.338* * * 00.552 97.000 19.400

International Academic Journal of Innovative Research,

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17

( ± 4.514)

37.000 00.370 20.154* * *

( ± 3.559)

00.368 98.00 19.600

37.500 00.375 20.062 (

± 4.035)

00.276 98.500 19.700

38.000 00.380 19.970* * *

( ± 4.418)

00.184 99.000 19.800

38.500 00.385 19.878

( ± 3.786)

00.092 99.500 19.900

39.000 00.390 19.786

( ± 4.021)

00.000 100.00 20.000

Table – 6: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of Lanostene

(Triterpene) at 48 hours after the fourth moult.

X Concentration

of Acetone

Solution (ppm)

Body Wall

Chitin

(mg / gm)

Chitin

Deposition

( mg / gm )

Percent

Reduction

Y

09.500 00.095 38.186*( ±

5.093)

18.400 00.000 00.000

39.000 00.390 38.186*( ±

4.956)

18.400 00.000 00.000

39.500 00.395 38.186*( ±

5.789)

18.400 00.000 00.000

40.000 00.400 38.094*( ±

4.886)

18.308 00.500 00.100

40.500 00.405 38.002*( ±

4.857)

18.216 01.000 00.200

41.000 00.410 37.818*( ±

5.856)

18.032 02.000 00.400

41.500 00.415 37.542*( ±

4.832)

17.756 03.500 00.700

42.000 00.420 37.358*( ±

5.847)

17.572 04.500 00.900

42.500 00.425 36.990*( ±

5.165)

17.204 06.500 01.300

43.000 00.430 36.622* *

( ± 4.817)

16.836 08.500 01.700

43.500 00.435 36.070* *( ±

4.858)

16.284 11.500 02.300

International Academic Journal of Innovative Research,

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18

44.000 00.440 35.426* *( ±

4.991)

15.640 15.000 03.000

44.500 00.445 34.276* *( ±

4.924)

14.490 21.250 04.250

45.000 00.450 33.126* *

( ± 4.817)

13.340 27.500 05.500

45.500 00.455 31.976* *

( ± 4.817)

12.190 33.750 06.750

46.000 00.460 30.826* *( ±

5.871)

11.040 40.000 08.000

46.500 00.465 29.976* * *( ±

5.371)

09.890 46.250 09.250

47.000 00.470 28.526* * *( ±

4.897)

08.740 52.500 10.500

47.500 00.475 23.376* * *( ±

3.367)

07.590 58.750 11.750

48.000 00.480 26.226* * *( ±

5.093)

06.440 65.000 13.000

48.500 00.485 25.076* * *( ±

4.951)

05.290 71.250 14.250

49.000 00.490 23.926* * *( ±

3.897)

04.140 77.500 15.500

49.500 00.495 22.776* * *( ±

4.556)

02.990 83.750 16.750

50.000 00.500 21.994* * *( ±

3.896)

02.208 88.000 17.600

50.500 00.505 21.350* * *( ±

4.226)

01.564 91.500 18.300

51.500 00.515 20.614* * *( ±

4.521)

00.828 95.500 19.100

52.000 00.520 20.338* * *( ±

4.669)

00.552 97.000 19.400

52.500 00.525 20.154* * *( ±

3.997)

00.368 98.000 19.600

53.000 00.530 19.970* * *( ±

3.613)

00.184 99.000 19.800

53.500 00.535 19.878

( ± 3.814)

00.092 99.500 19.900

54.000 00.540 19.786

( ± 3.333)

00.000 100.00 20

International Academic Journal of Innovative Research,

Vol. 3, No. 10, pp. 1-31.

19

Table – 7: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of Hopane

(Triterpene) at 48 hours after the fourth moult.

X Concentration

of Acetone

Solution (ppm)

Body Wall

Chitin

(mg / gm)

Chitin

Deposition

( mg / gm )

Percent

Reduction

Y

40.000 00.400 38.186*

( ± 4.951)

18.400 00.000 00.000

40.500 00.405 38.002*( ±

5.033)

18.216 01.000 00.200

41.000 00.410 387.956*( ±

4.487)

18.170 01.250 00.250

41.500 00.415 37.910*( ±

5.112)

18.124 01.500 00.300

42.000 00.420 37.818*( ±

4.982)

18.032 02.000 00.400

42.500 00.425 37.726*( ±

5.891)

17.940 02.500 00.500

43.000 00.430 37.542*( ±

5.278)

17.756 03.500 00.700

43.500 00.435 37.266*( ±

5.294)

17.480 05.000 01.000

44.000 00.440 36.989* *( ±

4.686)

17.112 07.000 01.400

44.500 00.445 36.254* *( ±

4.187)

16.468 10.500 02.100

45.000 00.450 35.656* *( ±

4.436)

15.870 13.750 02.750

45.500 00.455 34.506* *( ±

5.873)

14.720 20.000 04.000

46.000 00.460 33.356* *( ±

4.764)

13.570 26.250 05.250

46.500 00.465 32.206* *( ±

4.928)

12.420 32.500 06.500

47.000 00.470 31.056* *( ±

4.193)

11.270 38.750 07.750

47.500 00.475 29.906* * *( ±

4.826)

10.120 45.000 09.000

48.000 00.480 28.756* * *( ±

4.959)

08.970 51.250 10.250

International Academic Journal of Innovative Research,

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20

48.500 00.485 27.606* * *( ±

5.294)

07.820 57.500 11.500

49.000 00.490 26.456* * *( ±

3.393)

06.670 63.750 12.750

49.500 00.495 25.306* * *( ±

3.748)

05.520 70.000 14.000

50.000 00.500 24.156* * *( ±

5.614)

04.370 76.250 15.250

50.500 00.505 23.006* * *( ±

3.789)

03.220 82.500 16.500

51.000 00.510 22.086* * *( ±

4.441)

02.300 87.500 17.500

51.500 00.515 21.534* * *( ±

5.136)

01.748 90.500 18.100

52.000 00.520 20.982* * *( ±

4.297)

01.196 93.500 18.700

52.500 00.525 20.706 (

± 3.978)

00.920 95.000 19.000

53.000 00.530 20.522 (

± 4.137)

00.736 96.000 19.200

53.500 00.535 20.246

( ± 4.053)

00.460 97.500 19.500

54.00 00.540 20.062

( ± 4.053)

00.276 98.500 19.700

54.500 00.545 19.970

( ± 4.199)

00.184 99.000 19.800

55.000 00.550 19.924* * *

( ± 4.345)

00.138 99.250 19.850

55.500 00.555 19.878

( ± 4.392)

00.092 99.500 19.900

56.000 00.560 19.786

( ± 3.885)

00.000 100.00 20.000

Table – 8: Chitin content of the body wall of the fifth instar larvae of silkworm, Bombyx mori (L) (Race:

PM x CSR2) recipient of topical application of various concentration of acetone solution of Oleanane

Hopane(Triterpene) at 48 hours after the fourth moult.

X Concentration

of Acetone

Body Wall

Chitin

Chitin

Deposition

Percent

Reduction

Y

International Academic Journal of Innovative Research,

Vol. 3, No. 10, pp. 1-31.

21

Solution (ppm) (mg / gm) ( mg / gm )

43.000 00.430 38.186*

( ± 4.951)

18.400 00.000 00.000

43.500 00.435 38.094*

( ± 5.033)

18.308 00.500 00.100

44.000 00.440 38.002*

( ± 4.487)

18.216 01.000 00.200

44.500 00.445 37.910*

( ± 5.112)

18.124 01.500 00.300

45.000 00.450 37.726*

( ± 4.982)

17.940 02.500 00.500

45.500 00.455 37.542*

( ± 5.891)

17.756 03.500 00.700

46.000 00.460 37.266*

( ± 5.278)

17.480 05.000 01.000

46.500 00.465 36.989*

( ± 5.294)

17.112 07.000 01.400

47.000 00.470 36.346* *

( ± 4.686)

16.560 10.000 02.000

47.500 00.475 35.656* *

( ± 4.187)

15.870 13.750 02.750

48.000 00.480 34.506* *

( ± 4.436)

14.720 20.000 04.000

48.500 00.485 33.356* *

( ± 5.873)

13.570 26.250 05.250

49.000 00.490 32.206* *

( ± 4.764)

12.420 32.500 06.500

49.500 00.495 31.056* *

( ± 4.928)

11.270 38.750 07.750

50.000 00.500 29.906* *

( ± 4.193)

10.120 45.000 09.000

50.500 00.505 28.756* * *

( ± 4.826)

08.970 51.250 10.250

51.000 00.510 27.606* * *

( ± 4.959)

07.820 57.500 11.500

51.500 00.515 26.456* * *

( ± 5.294)

06.670 63.750 12.750

52.000 00.520 25.306* * *

( ± 3.393)

05.520 70.000 14.000

52.500 00.525 24.156* * *

( ± 3.748)

04.370 76.250 15.250

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22

53.000 00.530 23.006* * *

( ± 5.614)

03.3220 82.500 16.500

53.500 00.535 21.856* * *

( ± 3.789)

02.070 88.750 17.750

54.000 00.540 21.074* * *

( ± 4.441)

01.288 93.000 18.600

54.500 00.545 20.614* * *

( ± 5.136)

00.828 95.500 19.100

55.000 00.550 20.430* * *

( ± 4.297)

00.644 96.500 19.300

55.500 00.555 20.246

( ± 3.978)

00.460 97.500 19.500

56.000 00.560 20.154

( ± 4.137)

00.368 98.000 19.600

56.500 00.565 19.970

( ± 4.053)

00.184 99.000 19.800

57.00 00.570 19.786

( ± 4.053)

00.000 100.00 20.000

57.500 00.575 19.786

( ± 4.199)

00.000 100.00 20.000

58.000 00.580 19.786* * *

( ± 4.345)

00.000 100.00 20.000

58.500 00.585 19.786

( ± 4.392)

00.000 100.00 20.000

56.000 00.560 19.786

( ± 3.885)

00.000 100.00 20.000

FOOT NOTE for Table: 2; 3; 4; 5; 6; 7 and 8: -Each

figure is the mean of three replications;

-Figures in parenthesis with ± sign are the standard deviations.

- *=P < 0.005 ; * *=P < 0.01 And * * *=P < 0.001

- Scale: 0.01 mg/ml acetone solution of Triterpene Compound = 1 Unit of : “X”. : 5 Percent Reduction in

chitin Deposition = 1 Unit of : “Y”.

The concentrations (dosages) of acetone solutions of FME and triterpenes in the study, on steeper

slope of curves, seems to be most significant in the percent reduction in the body wall chitin. Therefore,

the dosages of acetone solutions of FME and triterpenes on the steeper slope of “Punyamayee Baramati

Dose Response Curve” may be called as effective dosages.The effects of juvenoids involve inhibition of

insect metamorphosis, significantly through reduction in chitin deposition Slama (1974). It has been

proposed to express the concentration (dosage) of acetone solutions (Juvenoid) topically applied in terms

of BCAS50 ( Baramati Concentration of Acetone Solution ) of Triterpene responsible for fifty percent

reduction in chitin deposition in the integument (Body Wall) of the Fifth Instar Larvae of multivoltine

International Academic Journal of Innovative Research,

Vol. 3, No. 10, pp. 1-31.

23

Crossbreed Race (PM x CSR2) of Silkworm, Bombyx mori (L). According to Slama et al (1974), the

BCAS50 unit of juvenoid material (in microgram)(orAcetone Solution of Triterpene ) , which reduce fifty

percent chitin deposition in the body wall of insect larvae.

The concentrations (mg/ml) of acetone solutions of FME and triterpenes in the study, that inhibit

the chitin deposition in the body wall of larvae by fifty percent can be calculated by the use of

“Punyamayee Baramati Dose Response Curves”. Accordingly, the BCAS50 ( Baramati Concentration of

Acetone Solution ) values for FME; Squalene; Polypodatetraene; Malabaricane; Lanostane; Hopane and

Oleanane were found calculated 00.080; 00.231; 00.257; 00.313; 00.468; 00.479 and 00.504 units

(mg/ml) respectively. Ten microlitres out of thousand microlitres of each acetone solution was utilized for

topical application on individual larva in each group.

Topically applied acetone solution of triterpenes may act through binding to the Exogenous

Juvenile Hormone Analogue (JHA) receptors in the epidermal cells of integument of the fifth instar larvae

of silkworm, Bombyx mori (L) (Race: PM x CSR2 ). Let label these receptors as: “Ex-JHA-Receptors”

(EJHAR). This may further associate with DNA of epidermal cells as heterodimer. This association may

alter the configuration of EJHAR, which affects the working of DNA. It may either induce or repress

transcription of a nearby genes, responsible for reduction in the deposition of chitin in the integument of

the fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2 ). “Ex-JHA-Receptors” (EJHAR)

in the integument of the fifth instar larvae of silkworm, Bombyx mori (L)(Race: PM x CSR2 ) may be

responsible to mediate transcription of different sets of genes controlling differentiation of epidermal

cells through change in the pattern of chitin deposition.

The “Punyamayee Baramati Dose Response Curves” in the study may form baseline platform for

estimation of BCAS50 ( Baramati Concentration of Acetone Solution ) values of any compounds (plant

derived; animal derived and synthetic compounds). The present study tried its best to establish

preliminary work on screening the acetone solutions of FME and selected triterpenes for juvenoid activity

in the fifth instar larvae of silkworm, Bomby xmori (L)(Race: PM x CSR2). Farnasol Methyl Ether (FME)

or acetone like solvents may serve the purpose to know intensity of juvenoids in any compound. The

triterpenes deserve many more cellular and molecular activities that could potentially underlie their

juvenomimetic index with reference to the phytophagous insects like, silkworm, Bombyx mori (L). The

present attempt is going to help to establish maximum tolerated dose of triterpene to be used for future

trials in which the efficacy of triterpenes will be tested for qualitative improvement of silk spinned by

mature fifth instar larvae of silkworm, Bombyx mori (L). If the efficacy is seen in larval developmental

setting, it will likely trigger future development and testing the triterpenes for the fortified health of larval

instars, that could spin the qualitative silky cocoon. The triterpenes are thus an example of the

development of agents that will bridge the areas of sericulture. The Baramati attempt of use of terpenes

for topical application to the larval instars of silkworm, Bombyx mori (L) hope more efficiently

benefitting the areas of both the areas of sericulture and juvenoid research. And the “Punyamayee

Baramati Dose Response Curves” in the present attempt may open a new avenue in the field of Juvenoid

research, especially, for quantification of juvenoid activity in given compound.

ACKNOWLEDGEMENT:

The research work is dedicated to the memories of Dr. D. G. Alias Appasaheb Pawar , who like a titan

raised the “Global Agro-academic Culture”. Author would like to express his gratitude towards the

International Academic Journal of Innovative Research,

Vol. 3, No. 10, pp. 1-31.

24

dignitaries of International Academic Journal of Innovative Research for kind co-operation and

encouragement, which served a lot to orchestrate the progression of publication of present research work.

The present research work is the part of trials on improvement of silk yield at Malegaon Sheti Farm,

Agricultural Development Trust Baramati, Shardanagar, (Malegaon Khurd) Post Box No.- 35 Tal.

Baramati.

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(L) (Race: PM x CSR2). Journal of Association of Zoologists, India. Vol. 2 (1): 11 – 21.

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of Association of Zoologists, India. Vol. 2 (1): 39 – 47.

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Vitthalrao B. Khyade; Poonam M. Patil; Sharad G. Jagtap; Sunanda V. Khyade and Jeevan P. Sarawade

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fifth instar larvae of silk worm, Bombyx mori (L) (Race : PM x CSR2). Research Journal of

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gut homogenate of fifth instar larvae of silk worm, Bombyx mori(L) (Race : PM x CSR2) fed

with herbal drug (Kho Go) treated mulberry leaves. Research Journal of Recent Sciences Vol. 1

(2): 49 – 55. www.isca.in ISSN 2227 – 2502.

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Nitrate Reductase In the Leaves of mulberry, Morus alba(L). Journal of Recent Sciences Vol.1

(5): 13 – 21. www.isca.in ISSN 2227 – 2502.

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amylase in the mid gut homogenate of fifth instar larvae of Bombyx mori L. (PM x CSR2) fed

with herbal drug(Kho-Go) treated mulberry leaves. International Journal of Science and Nature

Vol.3 (3): 526 – 530 www.scienceandnature.org ISSN 2229 – 6441.

Vitthalrao B. Khyade and Anil N. Shendage (2012). Influence of Aloe vera (L) Herbal formulation on

Larval Characters and Economic Parameters of silkworm, Bombyx mori (L)(Race : PM x CSR2).

The Ecoscan Special Issue Vol. 1 (121): 321 – 326. www.theecoscan.in ISSN: 0974 – 0376.

Vitthalrao B. Khyade and Babita M. Sakdeo ( 2012 ). Influence of foliar spray on the quality of mulberry

leaves. International Journal of Bioassays ( IJB ) 01 (12): 166 – 169. www.ijbio.com ISSN:

2278 – 778X.

Vitthalrao B. Khyade ( 2012). Isolation of glycoside from the seed powder of Syzigium cumini (L).

International Journal of Bioassays (IJB) 01 (12): 207 – 209 www.ijbio.com ISSN: 2278 – 778X.

Rajkumar Bapurao Deshmukh and Vitthalrao B. Khyade (2013). Utilization of Aloe vera (L) herbal tonic

for treating mulberry leaves before feeding the fifth instar Larvae of silkworm, Bombyx mori

(L)(Race : PM x CSR2). International Journal of Bioassays 02 (01): 281 – 285. www.ijbio.com

ISSN: 2278 – 778X.

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Vitthalrao B. Khyade and Jiwan P. Sarawade ( 2013). Glycosides from the seed powder of Syzigium

cumini (L). Annals of Plant Sciences 2 (1): 46 – 48. www.annalofplantsciences.com ISSN: 2287

– 688X.

Gauri U. Kadam and Vitthalrao B. Khyade ( 2013 ): Effect of age and sex on the activity of protease in

the mid gut and integument of fifth instar silk worm, Bombyx mori (L) (Race: PM x CSR2 ).

International Journal of Advanced Biological Research ( Society for Science and Nature ).Vol. 3

(2) 2013 : 188 - 190.ISSN 2250 – 357.www.scienceandnature.org

hyade V. B. ; Gaikwad D. R. and Thakare U. G. ( 2012). Utilization of Aloe vera (L) Herbal Tonic for

Treating Mulberry Leaves before feeding the Fifth Instar Larvae of Silkworm, Bombyx mori(L)

(Race: PM x CSR2) ( Editor: Dr. A. R. Tuwar and Dr. M. J. Shaikh Dept. of Life Sciences, Arts

and Science College, Sonai Tal. Newasa, Dist. Ahmednagar – 414105 India): 37 – 40.

Jagtap S. G. and Khyade V. B. (2012). The pattern of chitin deposition in the body wall / Integument of

fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2 )recipient of acetone

extractives of some non – mulberry plants. Biodiversity; Biotechnology and Climate Change (

Editor: Dr. A. R. Tuwar and Dr. M. J. Shaikh Dept. of Life Sciences, Arts and Science College,

Sonai Tal. Newasa, Dist. Ahmednagar – 414105 India): 105 – 109. www.sonaicollege.com

ISBN: 978 – 93 – 81921 - 23 – 4.

Vitthalrao B. Khyade and Jiwan P. Sarwade ( 2013 ): Utilization of Digoxin, the herbal product for

treating the mulberry leaves and feeding the fifth instar larvae of silkworm, Bombyx mori (L)

(Race: PM x CSR2). 2013 International Journal of Multidisciplinary Research (IJMR) Vol. I /

Issue 12 (III): 38-42. ISSN: 2277 – 9302.

Vitthalrao B. Khyade and Jiwan P. Sarwade ( 2013 ): Analysis of Sterol Contents in the Eggs of

Silkworm, Bombyx mori (L).2013 . Journal of Association of Zoologists India Vol. 6 No. 1

(March 2013):41 – 45.

Vitthalrao B. Khyade and Jiwan P. Sarwade ( 2013 ): Glycoside from the seed powder of Syzigium

cumini(L). Journal of Association of Zoologists India Vol. 6 No. 1 (March 2013): 91 - 96.

Vitthalrao B. Khyade and Vivekanand V. Khyade ( 2013 ): Plants: The Source of Animal Hormones.

“Frontiers in Life sciences”, the book published by Science Impact Publication, Ahmedpur

(Latur ) – 413515 ( India ) : 151 – 168. Editor: Dr. Sayyed Iliyas Usman( Poona College, Camp

Pune). ISBN : 978 – 93 – 5067 – 394 – 2.

Vitthalrao B. Khyade and Jiwan P. Sarwade ( 2013 ): Utilization of Retinol through the topical

application to the fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2) for

qualitative improvement of the economic parameters. International Journal of Advance Life

Sciences Vol. 6 Issue 5 November, 2013. www.ijals.comhttp://ijals.com/wp-

content/uploads/2014/01/19.-Utilisation-of-Retinol-through-the-topical.pdf

Vitthalrao B Khyade* and Vivekanand V Khyade (2013). The Phytocompounds of Animal Hormone

Analogues. Annals of Plant Sciences Vol. 2 (5): 125 – 137.

http://annalsofplantsciences.com/index.php/aps/issue/view/10 ISSN: 2287 – 688X

Vitthalrao B. Khyade ( 2014 ): Influence of Lanoxin Treared Mulberry Leaves on the contents of proteins

in the fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2). 2014. (Page: 8 –

17). Proceeding, Two day UGC sponsored National seminar on, “Recent Trends in Cell Biology,

Biotechnology and Bioinformatics”, Organized by Department of Zoology, Balwant College,

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29

Vita Tal. Khanapur, Dist. Sangli 415311 (India) (6 and 7 September, 2013 ). Editor: Prof. (Smt.)

U. H. Shah (Department of Zoology, Balwant College, Vita ). ISBN 978 – 81 – 927211 – 3 – 2.

Sucheta S. Doshi ; Anil N. Shendage and Vitthalrao B. Khyade ( 2014 ): Utilization of Digixin the herbal

product for treating the mulberry leaves and feeding the fifth instar larvae of silkworm, Bombyx

mori (L) (Race: PM x CSR2 ). Standard Global Journal of Scientific Research Vol. 1 (2) : 020 –

024 March

2014.http://www.standardglobaljournals.com/journals/SGJSR/2014/march/Doshi%20et%20al.ht

ml

Vitthalrao B. Khyade (2014).THE ACTIVITY OF PROTEASE IN THE FIFTH INSTAR SILKWORM,

Bombyx mori (L) ( RACE : PM X CSR2 ). Biolife April – June Vol. 2 (2) 2014:

Vitthalrao B. Khyade, Vivekanand V. Khyade and Amar H. Kadare (2014): Influence of Acetone

Extractive of Oroxylum indicumon Cocoon characters; Silk Filament Characters and the

Electrophoretic patterns of esterase activity of silk worm Bombyx mori (L.)(Race: PM x CSR2).

Research Journal of Recent Sciences Vol. 3(IVC-2014), 1-5 (2014) ISSN 2277-2502 .

www.isca.in , www.isca

Vitthalrao B. Khyade; Vivekanand V. Khyade and Rhidim D. Mote (2014). Influence of Acetone

extractive of Oroxylum indicum (L) on cocoon characters, silk filament character and

electrophoretic patterns of esterase activity of silkworm, Bombyx mori (L) (Race: PM x CSR2).

Recent Trends in Zoology(Pages: 12-22). Editor: Dr. R. K. Kasar ; Publisher: Dr. L. S. Matkar

(Principal, New Arts, Commerce and Science College, Shevgaon Dist. Ahmednagar – 414502

(M.S.) India. ISBN: 978-93-84916-68-8.

V. B. Khyade and K. Slama (2014). Changes in the Pattern of Chitin Deposition in The Integument of

Fifth Instar Larvae of Silkworm, Bombyx mori (L) (Pm X Csr2) Topically Applied With Various

Concentrations Of Acetone Solution Of Retinol. Journal of Biodiversity and Ecological Sciences

Vol. 4, Issue 4: 159 – 167.ISSN: 2008-9287.

Vitthalrao B.Khyade ; Vivekanand V. Khyade and Randy Wayne Schekman (2015). Utilization of the

topical application of Limonene to the fifth instar larvae of the silkworm, Bombyx mori (L)

(Race: PM X CSR2) for the parameters of Larvae, Cocoon and Silk filament. International

Journal of Bioassay 4 (02): 3632 – 3635.ISSN: 2278-778Xwww.ijbio.com

Vitthalrao Khyade, Edvard Moser and May – Britt Moser (2015). INFLUENCE OF AQUEOUS

MACERATIVES OF SEED POWDER OF SYZIGIUM CUMINI (L) ON THE MID GUT

ENZYME ACTIVITY IN THE FIFTH INSTAR LARVAE OF SILK WORM, Bombyx mori (L)

(Race: PM x CSR2). World Journal of Pharmaceutical Research Volume 4, Issue 6:997 – 1008.

(ISSN 2277– 7105).www.wjpr.net

Vitthalrao B. Khyade and Karel Slama (2015). SCREENING OF ACETONE SOLUTION OF FME AND

SELECTED MONOTERPENE COMPOUNDS FOR JUVENILE HORMONE

ACTIVITYTHROUGH CHANGES IN PATTERN OF CHITIN DEPOSITION IN THE

INTEGUMENT OF FIFTH INSTAR LARVAE OF SILKWORM, Bombyx mori (L) (PM x

CSR2). IJBRITISH Vol. 2 Issue 3 (May – June 2015): 68 – 90. ISSN 2349-

9419www.ijbritish.com

Vitthalrao B. Khyade and Rajkumar B. Deshmukh (2015). Mid gut protease and amylase activity in the

fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2) fed with mulberry leaves

International Academic Journal of Innovative Research,

Vol. 3, No. 10, pp. 1-31.

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treated with aqueous solution of stevia inulin powder. Proceedings, U G C Sponsored National

Conference on Recent Trends in Life Sciences (10 - 11, July, 2015), organized by Department of

Zoology, S. M. Joshi College, Pune.Page : 95 – 106. ISBN 978-93-5235-362-0.

Vitthalrao B. Khyade; Karel Slama; Rajendra D. Pawar and Sanjay V. Deshmukh (2015). Influence of

Various Concentrations of Acetone Solution of Retinol on Pattern of Chitin Deposition in the

Integument of Fifth Instar Larvae of Silkworm, Bombyx mori (L) (PM X CSR2).Journal of

Applicable Chemistry. 2015, 4 (5): 1434 – 1445. www.joac.info

Vitthalrao B. Khyade; Karel Slama; Rajendra D. Pawar and Sanjay V. Deshmukh (2015). Influence of

Various Concentrations of Acetone Solution of Retinol on Pattern of Chitin Deposition in the

Integument of Fifth Instar Larvae of Silkworm, Bombyx mori (L) (PM X CSR2).Journal of

Medicinal Plants Studies. Volume 3 Issue 5 Part C : 124 – 131.

http://www.plantsjournal.com/archives/?year=2015&vol=3&issue=5&part=C

Vitthalrao B. Khyade, Sivani C. Bhosale; Vishakha R. Kakade and Jiwan P. Sarawade (2015). Pattern of

Chitin Deposition in The Integument of Fifth Instar Larvae of Silkworm, Bombyx mori(L) (PM x

CSR2) Treated with Acetone Solution of Selected Monoterpene Compounds and Fernasol

Methyl Ether (Fme).Journal of Basic Sciences, 2015, Special Issue on BioIPPF, 34-

40.www.skpubs.com

Sharad G. Jagtap; Vitthalrao B. Khyade and Santoshrao G. Mali (2015). Influence of Treating the

mulberry leaves with aqueous maceratives of seed powder of Syzigium cumini(L) on the

activities of digestive enzyme in the fifth instar larvae of silkworm, Bombyx mori(L) (Race: PM

x CSR2). Elixir International Journal .Applied Zoology / Elixir Appl. Zoology 85 (2015) 34140-

34144. www.elixirpublishers.com(Elixir.International.Journal.Applied.Zoology

Vitthalrao B. Khyade and Abhilasha C. Bhunje (2015).Efficient use of acetone extractive of Oroxylum

indicum for the improvement of quality of silk in silkworm Bombyx mori (L.) (Race: PM x

CSR2). Malaya Journal of Biosciences 2015, 2(4):185-190 ISSN 2348-6236 print /2348-3075

online http://www.malayabiosciences.com/

Sucheta S. Doshi ; Anil N. Shendage and Vitthalrao B. Khyade (2016).The monoterpene compounds for

juvenile hormone activity through changes in pattern of chitin deposition in the integument of

fifth instar larvae of silkworm, Bombyx mori (L) (PM x CSR2). World Scientific news 37

(2016): 179-201. www.worldscientificnews.com .

Vitthalrao B. Khyade and Atharv Atul Gosavi (2016).Utilization of mulberry leaves treated with seed

powder cowpea, Vigna unguiculata (L) for feeding the fifth instar larvae of silkworm, Bombyx

mori (L) (Race: PM x CSR2). World Scientific news 40 (2016): 147-162.

www.worldscientificnews.com .

Vishakha S. Chape; Abhilasha C. Bhunje and Vitthalrao B. Khyade (2016). Efficient Use of Extractive

of Oroxylum indicum for the improvement of Quality of Silk in Silkworm, Bombyx mori (L)

(Race: PM x CSR2 ). International Conference on “Plant Research and Resource Management”

And 25th

APSI Silver Jubilee Meet 2016 at T. C. College Baramati 11, 12 and 13 February,

2016. Pages: 304 – 308.

Vitthalrao B. Khyade; Vrushali D. Shinde and Shraddha S. Maske (2016).Influence of the diterpenoids

(Retinol and Phytol) (Race: PM x CSR2) on the cocoon and silk parameters in silkworm,

International Academic Journal of Innovative Research,

Vol. 3, No. 10, pp. 1-31.

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Bombyx mori (L) (Race: PM x CSR2). World Scientific news 42 (2016): 1-12.

www.worldscientificnews.com .

Madhuri Anil Shivpuje; Hanumant V. Wanve and Sadashiv N. Belpatre (2016). Influence of magnetic

energy on protein contents in the fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x

CSR2). World Scientific news 42 (2016): 73-86. www.worldscientificnews.com .

Vitthalrao B. Khyade, Kajal D. Gokule, Sunanda Rajendra Pawar, Rajkumar B. Deshmukh

(2016).Utilization of the Retinol and Phytol for the quality improvement of cocoon and silk fibre

spinned by fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2). World

Scientific News 42 (2016): 167-181. www.worldscientificnews.com .

Vitthalrao B. Khyade and Dhanashri R. Gaikawad (2016). Insect Juvenile Hormone. World Scientific

News 44 (2016): 216-239. www.worldscientificnews.com.

Vitthalrao B. Khyade (2016). Utilization of mulberry leaves treated with seed powder of

cowpea, Vigna unguiculata (L) for feeding the fifth instar larvae of silkworm,

Bombyx mori (L) (Race: PM x CSR2). Journal of Medicinal Plants Studies 2016; 4(3): 182

- 188. http://www.plantsjournal.com/archives/2016/vol4issue3/PartC/4--2-33-339.pdf