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Biochemical analysis of Acraea terpiscore: (Linnaeus, 1758)

Synonymus: Acraea violae (Fabricius)

Common name: Tawny Coster.

Introduction:

The Acraea terpiscore (Tawny Coster) is a small leathery winged butterfly

which is common in grassland and scrub habitats. It belongs to the

Nymphalidae or Brush- footed butterfly family. It has a weak fluttery flight. It

is avoided by most insect predators. This species and the Yellow Coster

(Acraea issoria) are the only two species found in India. It is a butterfly of

both hilly as well as plains. It is plentiful in the monsoon and pre-monsoon

period and becomes scarce later on.

The butterfly exudes an oily and smelly yellow liquid when handled and is

unpalatable to birds and most insects. Thus they are well protected, have a

slow and weak flight, and are frequently visiting flowers. There are no mimics

of this species, in India.

The butterfly breeds on plants of family Loganiaceae and species of

Passiflora, many of which contain toxins that are sequestered by the

caterpillars. The female butterfly deposits clusters of white eggs on the

surface of Passiflora leaves. The tiny first instar caterpillars hatch about 4-5

days later, from the eggs and start feeding on the leaves of Passiflora.

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It is cylindrical, slender; with six longitudinal rows of fine branched spines. It

is reddish brown in colour with an oily gloss. Its head, second and last

segment are pale in colour than the remaining body.

Due to its unwholesome look it is well protected from its predators. After the

caterpillar hatches, its first meal is the eggshell itself. It lives its entire larval

life on the lower side of the leaf. During the first few days it has a very

interesting manner of feeding. It will take up spot on the underside of the leaf

and nip a complete circle around itself in the lower cuticle of the leaf. By

doing this it stops the poisonous sap of its host plant from flowing into the

area inside the circle. It then proceeds to eat the lower surface of this area,

leaving the upper cuticle intact. As it grows, it eats both the lower and upper

cuticle of the circle thus leaving smallish circular holes in the leaves of its

host plants. When its mandibles are large enough it eats the complete leaf by

gnawing at the edges.

The caterpillar is uniformly cylindrical. Its body is covered with bands of

black and white interspersed with thick, dorso-lateral spots. The most striking

characteristics are the 3 pairs of long and black tentacle-like appendages.

The first pair is moveable and also the longest. The tentacles are present on

the 3rd, 6th, and 12th segments. The head is shiny, smooth and has

alternating black and white semicircular bands. The legs and prolegs are

black and the prolegs have white bands at their bases.

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Pupa is white, long, slender, and smooth; with two lateral angles on

the thorax. It hangs perpendicularly, freely from the support. It is widest at the

7th abdominal segment where it has a double chain of extremely tiny golden

and black beads. There are tiny golden spots on the shoulders and wing-

cases.

Being a non pest on agricultural crop plants, no economical losses are

being caused by A. terpiscore, hence this species has been largely neglected

by researchers. Except the study of life cycle, no other basic information

related to biochemical, molecular or a genetical aspect is available. Hence in

the present study, biochemical analysis of different stages of A. terpiscore

during metamorphosis is done.

Materials and methods :

Rearing of butterflies:

To initiate the process of rearing, eggs and the larvae of Acraea terpiscore

were collected from Nehru Garden, Nasik. Larvae were reared under

laboratory conditions at 27°c ± 2°c temperature and 70% ± 5% relative

humidity, in the glass petri plates kept in a wooden box (20”x20” x20”) with

wire-netted sides and top.

The larvae were fed on fresh leaves of Passiflora. The completely

grown larvae, about to pupate, were sorted and placed in a separate glass

dish at room temperature. Some of these larvae were used for biochemical

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analysis, while others were use further reared for pupation and adult

emergence. The adults were reared out door, in natural conditions, further

allowed to mate and deposit eggs on the leaves of Passiflora. The leaves

containing the clusters of eggs were collected and kept under controlled

conditions of temperature and humidity as mentioned above. The eggs

hatched to produce first instar larvae, and the rearing was thus continued.

Haemolymph collection:

Haemolymph was drawn from the acclimatized last instar larvae, pupae and

newly emerged adults of the Acraea terpiscore.

Before the haemolymph sample was collected, the body weight (in mg), of

each stage was measured.

Haemolymph (2-4µl) was drawn by inserting the calibrated capillary in the

pseudo legs of the last instar larvae, and anterior end of the pupae. It was

exuded immediately in separate appendrof tubes, kept in an ice box, each

containing saturated 1-phenyl 2-thiourea and glutathione (G.R.Wyatt,

M.L.Pal; 1978), to prevent melanization. About 40-50 pre-pupal larvae and

20-25 pupae were sacrificed for each experiment. The haemolymph from the

0-24 hrs old adults was withdrawn by inserting the calibrated capillary in the

ocular lenses and in thoracic region. The haemolymph was stored in the

same manner as mentioned in case of larva and pupa.

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The collected haemolymph was then centrifuged for 15 min at 18,000 rpm to

separate haemocytes and plasma (Wyatt, and Pal, 1978; Nursel and Cevat,

2002). The cell-free haemolymph was then collected in 1ml aliquots and

stored at -40ºc till further use. Haemolymph thus obtained was used for total

protein, carbohydrate, lipid, Uric acid and trehalose estimation.

The last instar larvae were also dissected in insect saline solution to remove

the fat bodies. These fat bodies were dried, weighed, homogenized in saline

solution (1gm/1ml) and used for further biochemical estimation.

Biochemical assays:

Protein estimation by Lowry method:

The total protein content was estimated according to modified Lowry –

Protein Assay (Lowry et al, 1951), using Bovine Serum Albumin as

standard. Following reagents were prepared for this estimation.

• Alkaline copper reagent:

20gm of sodium carbonate was dissolved in 260 ml distilled water;

0.4gm cupric sulfate was dissolved in 20 ml distilled water, and 0.2

gm sodium potassium tartarate was dissolved in 20 ml distilled

water. All the three solutions were mixed to prepare the copper

reagent.

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• SDS Solution: 100ml of 1% solution of Sodium Dodecyl Sulfate

(SDS) was prepared by dissolving 1gm of SDS in 100 ml water.

• NaOH Solution: 1M solution of NaOH was prepared by dissolving

4gm of NaOH in100ml water.

• Lowry Reagent: 2x Lowry concentrate were prepared by mixing 3

parts of copper reagent with 1 part SDS and 1 part of 1M NaOH.

The three solutions were kept separately and mixed just before use.

• Folin Reagent: 0.2 N Folin reagents were prepared by mixing 10ml

2N Folin reagent with 90ml water.

• 30% trichloroacetic acid.

• 0.1 N NaOH.

Total protein contents were measured from the fat body of larvae, gonads of

adults and haemolymph of larvae, pupae, and adult stages of Acraea

terpiscore. The tissue protein was precipitated by adding 1 ml of 30%

trichloroacetic acid solution. The samples were then centrifuged at 3000rpm,

for 30 minutes (Roe, 1955). The precipitate was dissolved in 1ml of 0.1N

sodium hydroxide.

2µl each of, larval, pupal, and adult haemolymph protein precipitate

was taken in three separate appendrof tubes. Similarly, 2µl of larval fat body

extract was taken in two separate appendrof tubes. The volume in each tube

was raised up to 400 µl by adding distilled water. To this, 400 µl of 2x Lowry

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reagent was added. The tubes were incubated for 10 minutes at room

temperature. After incubation 200 µl of 0.2 N Folin reagent was added very

quickly and vortexed immediately. This mixture was incubated for 30 minutes

at room temperature. Absorbance was measured at 750nm on UV

spectrophotometer. Amount of protein was calculated from standard curve.

Each assay was done in three to five replicates. Data was further subjected

to statistical analysis.

Standard graph for determination of Total Proteins :

Protein standards were prepared from 0.25 mg/ml bovine serum albumin

(BSA). From this, aliquots of 40-400 micro liters were added to the test tubes.

Distilled water was added accordingly to make the total volume of 400 micro

liters per tube. A reference of 400 micro liters was prepared by using water.

400 micro liters of 2x Lowry reagent was added to each test tube, mixed

thoroughly and incubated at room temperature of 10 minutes. After

incubation 200 micro liters of 0.2N Folin reagent was added very quickly and

vortexed immediately. This mixture as incubated for 30 minutes at room

temperature. Absorbance was read at 750 nm. A standard curve of protein

concentration against optical density was plotted. (Fig. 1)

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Fig.1. Sta ndard graph for proteins

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Carbohydrate estimation by Modified Anthrone method :

The total haemolymph carbohydrates were measured by the modified

Anthrone method (Beck and Bibby, 1961). Following Reagents were

prepared for the estimation:

• Glucose (stock solution):10mg/100ml i.e. 0.1mg/ml

• 2% Anthrone Reagent: Prepared in ethyl acetate.

• Conc. Sulphuric acid.

• Distilled Water.

• Deprotenized reagent: 5% Trichloroacetic acid.

Standard graph for determination of Total Carbohydr ates:

Glucose standards were prepared from 0.1mg/ml stock solution of Glucose,

by adding 0ml (0 µg) to 2ml (200µg) to the test tubes. The volume was

raised by adding Distilled Water to make the total volume of 2ml per tube.

0.5ml Anthrone reagent was added to each tube. 5ml of conc. Sulphuric

acid was then added through the sides of the tube. The tubes were

incubated for 10 min. Further incubation was done at 100º C for10 minutes,

followed by 4º C for 5minutes, and to prevent condensation of moisture on

the optical tubes, the test tubes were kept in water at 20ºC for 5minutes.

Absorbance was read at 630nm against distilled water-reagent-sulphuric

acid blank.

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The haemolymph and fat body was deprotenized using 5% trichloroacetic

acid, in the proportion 1:10 (Bhattacharya and Kaliwal, 2005).The tissues

were then centrifuged at 3000rpm for 30 minutes. The protein free

supernatant was further used for total carbohydrate estimation. 2µl of

deprotenized haemolymph and fat body sample was diluted by adding

distilled water so as to make the volume in the tube 2ml. Further additions

were made as mentioned above. Absorbance was read at 630nm. Standard

curve of glucose concentration against optical density was plotted. Glucose

concentration of the sample was determined from the standard curve (Fig. 2).

The assay was done in replicates of three to five. Data obtained was

subjected to statistical analysis.

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Fig.2. Standard graph for Glucose

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Lipid estimation by Zoellner and Kirsch method:

Reagents required:

• Phospho-Vanillin reagent: (13mM vanillin and 11.8 M phosphoric acid)

0.6 gm of vanillin was dissolved in 10ml of absolute ethanol. It was

further diluted to 100ml with distilled water. This solution was mixed

with 400ml of concentrated phosphoric acid, with constant stirring. It

was stored in a dark bottle at room temperature.

• Concentrated sulphuric acid

• Cholesterol- 1gm dissolved in 100ml of absolute ethanol.

Standard graph for determination of total lipids:

Total lipid concentrations from haemolymph, were measured by using

Sulpho-Phospho-Vanillin reagent (Zoellner and Kirsch method, 1962). Lipid

standards were prepared from 1gm/100ml stock solution of cholesterol. 5ml

concentrated sulphuric acid was added to a test tube containing 0.1ml of

stock solution. The test tube was heated for 10minutes in a boiling water bath

and then cooled. Aliquots of 0.1 to ml were prepared from this solution. The

volume of each aliquot was raised up to 0.7ml with concentrated sulphuric

acid. 6ml of phospho-vanillin reagent was added to each tube. The tubes

were incubated in dark for 30 minutes. Absorbance was read at 540nm.

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0.7

Fig.3. Standard graph for Lipids

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A standard curve of cholesterol concentration against optical density was

plotted (Fig.3).

20µl of haemolymph was mixed with 200µl of concentrated sulfuric acid and

heated to 100ºc for 10 min. Samples were allowed to cool. Upon cooling,

50µl was removed and mixed with 1.25 ml of Sulpho-phospho-vanillin

reagent. After 30 minutes of incubation in dark, the optical density was

measured at 540 nm with a spectrophotometer.

Estimation of Uric acid:

Uric acid estimation was done by enzymatic methods, using a commercial

kit, obtained from BIO ERA, a chemical Laboratory in Pune. (Fig.4)

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Fig.4. Standard graph for uric acid.

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Qualitative analysis of Proteins by SDS-PAGE:

The qualitative analysis of total soluble protein was done in haemolymph by

using the Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis

(SDS-PAGE) as described by Laemmii Nursel G.L, Cevat Ayvali; (2002)

using a 5% (w/v) acrylamide stacking gel and a 12% (w/v) acrylamide

separating gel.

Following solutions were prepared for the stacking and separating gels:

Solutions for 12% separating gel: 18x16 cm, 1.5 mm thick gel (40 ml)

• Distilled water- 15.9 ml

• 30% Acrylamide solution- 13.3ml

• 1.5M Tris (pH 8.8)- 10.0ml

• 10% SDS- 0.4ml

• 10% Ammonium per sulphate - 0.4ml (freshly prepared)

• TEMED - 0.016 ml

Solutions for 5% stacking gel -18x16 cm, 1.5 mm thick gel (8ml)

• Distilled water- 5.5ml

• 30% Acrylamide solution- 1.3ml

• 10M Tris (pH 6.8)- 1.0ml

• 10% SDS- 0.08ml

• 10% Ammonium per sulphate - 0.08 ml (freshly prepared)

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• TEMED- 0.008ml

Tris- glycine electrophoresis running buffer:

• 25mM Tris

• 250mM glycine (pH8.3)

• 10% SDS

A 5X stock solution of electrophoresis running buffer was prepared by

dissolving 15.1gm of Tris base and 94 gm of glycine in 900ml of distilled

water. To this, a 50 ml of 10% SDS solution was added and volume was

adjusted to 1000ml with distilled water.

1X SDS sample loading buffer: (40 ml)

• Distilled water-16ml

• 0.5M Tris (pH-6.8)- 5ml

• 50% Glycerol- 8ml

• 10% SDS- 8ml

• 2-β mercaptoethanol- 2ml (add immediately before use)

• 0.1% Bromophenol blue- 1ml

Gel Fixing Solution- 100ml

• 10% Acetic acid- 25ml

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• 12% trichloroacetic acid solution- 25ml

Gel staining solution:

• Coomassie Brilliant Blue R-250- 0.25 gm

• Methanol: Acetic acid stock solution- 1000ml

Mix 900ml methanol: H2O (500ml of Methanol and 400ml of H2O) and

100ml of glacial acetic acid. A staining solution was prepared was dissolving

0.25gm of Coomassie Brilliant Blue R-250 in 100ml of methanol: acetic acid

solution. The solution was filtered through Whatman No.1 filter paper to

remove the particulate matter, if any.

A vertical gel apparatus from Bangalore Genie was used. The glass plates,

spacers and the apparatus was cleaned and dried properly. Glass plates and

spacers were assembled together using bulldog clips. Then, 2% agar was

applied around the edges of the spacers to hold them in proper place and to

seal the chamber between the glass plates. 40ml of separating gel mixture

was prepared. After adding ammonium per sulphate and TEMED, the

solution was mixed gently and carefully. The solution was then poured in the

chamber between the glass plates. The gel was allowed to polymerize for 30-

45minutes. 5% stacking gel was prepared as per the above contents, and

poured above the separating gel. The comb was placed in the stacking gel

and the gel was allowed to polymerize for 30-45 minutes. The comb was

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then removed without distorting the shape of the wells. The gel was installed

carefully, after removing the clips and agar, in the gel electrophoresis

apparatus. The upper and lower chambers of the apparatus were filled with

1X electrophoresis running buffer.

The haemolymph proteins from larva pupa and adult were

dissolved in sample loading buffer and heated for 30 min at 60º c. This

ensured the complete interaction between proteins and SDS. The sample

solutions were cooled and a uniform quantity of protein (100µg) from

haemolymph of larvae, pupae and adult was loaded to slots of the gel. The

gel was calibrated with broad range molecular markers

A mixture of broad range Molecular weight markers such as myosin

(205,000DA), phosphorylase-b (120,000DA), β-galactosidase (116,000DA),

bovine serum albumin (84,000DA), albumin (66,000DA), ovalbumin

(45,000DA), carbonic anhydrase (29,000DA), trypsin inhibitor (20,000DA),

and lysozyme (18,000DA), were loaded in one slot to compare the molecular

weight of the proteins separated from that of the samples. Then 10-15mA

current was applied for initial 15-20 minutes i.e. until the samples travelled

through the stacking gel. The current was the raised at 30mA till the

Bromophenol blue reached the bottom of the gel (about 3hours).

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After electrophoresis, the gel was removed from between the glass

plates and fixed in 12% trichloroacetic acid for 30 minutes, and then in 10%

acetic acid for another 30 minutes. The gel was stained in 0.25% Coomassie

brilliant blue R-250, overnight, with uniform shaking. Proteins absorbed the

Coomassie brilliant blue dye. After appropriate staining, it was destained in a

mixture of distilled water, methanol and acetic acid (9:6:1 v/v). The dye that

was not bound to proteins was thus removed. The destainer was changed

frequently (30 minutes) particularly during initial periods, until the background

of the gel was colourless and bands could be distinctly seen. The gel was

then photographed.

Trehalose estimation by Anthrone Method :( Roe, 199 5)

Reagents required:

• 2% Sodium hydroxide – 100ml

• 0.05% Anthrone reagent prepared in 70% sulphuric acid

The estimation of trehalose from haemolymph and fat body was carried out

according to the method of Roe (1995). In each test tube, 0.1ml haemolymph

was collected and 0.5ml of 2% of sodium hydroxide was added to each test

tube. The test tubes were shaken thoroughly and then kept in boiling water

bath (100°C) for 10minutes. Later the tubes were cooled in an ice box (4°C).

Then 5ml of Anthrone reagent was added to the tubes, and they were again

kept in boiling water bath (40°C) for 15 minutes, for the development of

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colour. Then the tubes were cooled to room temperature. Then the colour

intensity was read on spectrophotometer at 620nm. For the reference

standard trehalose was used. Anthrone positive carbohydrate in the

haemolymph is considered as trehalose.

Total haemocyte count:

Solutions Required:

• Toisson’s solution: NaCl- 1.0gm

Na2SO4- 8.0gm

Neutral glycerine- 20ml

Methyl violet-0.025 gm

Distilled water-160ml.

For the total haemocyte count (THC), the haemolymph was directly

drawn into a Thoma white blood cell pipette up to the 0.5 mark, by pricking

the pseudolegs of larvae by sharp needle. It was diluted up to the 11 mark

with Toissons fluid (Mohmood and Yousaf, 1985). The pipette was shaken

for several minutes and the first three drops were discarded. A drop of diluted

haemolymph was then placed near the edge of the cover slip of the Neubaur

ruling hemocytometer and the chamber was automatically filled by capillary

action. The hemocytometer was left undisturbed for 5minutes, to let the

haemocytes settle down. The haemocytes were then counted in the four

corners and one control (1 mm2) ruled squares. The number of circulating

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hemocytes per cubic millimeter (mm3) was calculated using the following

formula of Jones (1962):

THC = Haemocytes in five 1 mm2 × Dilution × Depth factor of chamber

No. of squares counted

Where, Dilution = 20 times, No. of squares counted = 5.

Depth factor of the chamber = 10 (constant)

Differential haemocyte count:

The haemolymph was fixed by glacial acetic acid vapours for 5 - 10 min in a

small desiccator at 40oC. One of the thoracic legs of last instar larva was

pricked by needle and the exuding haemolymph was drawn in to Thoma

white blood cell diluting pipette. Similarly the haemolymph was drawn from

early pupa and adult too. A small drop of this blood from each stage was

placed on separate clean white grease free microscope slide and smear was

made by drawing second slide across the first one at 45oangle. The smear

was air dried and stained by Geimsa stain for four min. A freshly prepared

buffer solution (Na2HPO4 = 3.8 g, KH2PO4 = 5.47 g and distilled water 1 L) of

pH 6.6 was applied for 15 min to neutralize the haemocyte contents for

differential staining. Differential counting of haemocytes was under oil

immersion phase microscope (10 x 100 X). Each time 100 cells were

counted and the percentage of various classes was computed (Mahmood &

Yousaf, 1985).

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Results:

Results in Table 1.reveal that the levels of organic biomolecules in the

haemolymph and fat bodies of Acraea terpiscore were significantly different at

different stages of development.

Total Protein concentration in haemolymph and fat b odies:

The protein levels in haemolymph were found to be significantly lower

(1.56mg/ml) in adult stage, when compared to haemolymph protein levels of

pre pupal larvae (3.24mg/ml) and early pupae (2.20mg/ml). The protein

concentrations in fat bodies of larvae were low (1.14mg/ml) when compared

to the haemolymph protein levels of the same stage of development.

Total Carbohydrate concentration in haemolymph and fat bodies:

In Larval haemolymph, total carbohydrate levels were higher

(6.00µg/ml) than the pupal (5.20µg/ml) and adult (4.50µg/ml) haemolymph

carbohydrate levels. The total carbohydrate levels in fat bodies of larvae

(2.40µg/ml) were significantly low when compared to the haemolymph

carbohydrate levels of all the stages of development.

Trehalose concentration in haemolymph and fat bodie s:

Trehalose concentration in haemolymph of pre pupal larvae and early

pupal stage were found to be almost constant and higher (4.33µg/ml and

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4.00 µg/ml respectively), when compared to haemolymph trehalose levels in

0-24 hrs old adults (3.12µg/ml). On the contrary, trehalose levels in pre pupal

larval fat bodies were found to be significantly low (0.056µg/ml), when

compared to the trehalose levels of pre pupal larval haemolymph.

Total lipid concentration in haemolymph and fat bod ies:

Lipid concentration in haemolymph of all the stages of development

was found to be almost constant and lower than the lipid level in fat bodies

(0.09µg/ml), when compared with larval fat bodies.

Total lipid concentration in haemolymph and fat bodies was also found

to be least among all the organic biomolecules studied.

Uric acid concentration in haemolymph and fat bodie s:

Uric acid contents in pre pupal haemolymph (30.00µg/ml) of

A.terpiscore were more than uric acid contents of pupal haemolymph

(19.5µg/ml).While in adult haemolymph they were lowest (6.90µg/ml). In

larval fat body the uric acid contents (31.00µg/ml) were at par when

compared with the contents in pre pupal haemolymph.

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Table 1 : Biochemical changes in the haemolymph of larvae pupae and adult

of Acaria terpiscora

Haemolymph µg/ml Fat body µg/ml

Biomolecules Larva Pupa Adult Larva

Total Proteins

3.24 ± 0.045**

2.20 ± 0.085**

1.56 ± .071**

1.14 ± 0.5**

Total Carbohydrates

6.00 ± 0.40**

5.2 ± 0.24**

4.5 ± 0.37**

2.40 ± 0 .67**

Total Lipids

0.07 ± 0.003**

0.05 ±.0.013**

0.045± 0.12**

0.09 ± 0 .04**

Trehalose

4.33 ± 0.323**

4.0 ± 0.20**

3.12± 0.14**

0.056 ± 0.01**

Uric acid

30.00 ± 0.54*

19.5 ± 0.33**

6.90 ± 0.89**

31.00 ± 0.72*

∗∗- Significant at P< 0.05

∗N.S–Non-significant

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Protein estimation by SDS-PAGE : (Fig.5)

SDS PAGE result in Acraea terpiscore reflects six protein bands at larval

stage, whose molecular weight is in the range between 120kDa to 20kDa. At

pupal stage five protein bands are seen with molecular weight almost in the

same range as in larva. Where as at adult stage the number of protein bands

is reduced to four, whose molecular weight ranges from 118kDa to 29kDa.

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Fig.5 Protein determination by SDS-PAGE, A. terpiscore

1. Haemolymph of Adults – 4 bands

2. Haemolymph of pupae – 5 bands

3. Haemolymph of Larvae – 6 bands

120kDA

45kDA

20kDA

205kDA

66kDA

116kDA

84kDA

29kDA

18kDA

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Total Haemocyte count :( Table 2.)

The total haemocyte count in the fourth instar pre pupal stage was (8997.0).

It decreased in the next stage i.e. fifth instar, up to 8016.0. The decrease

continued in early pupal stage to 4567.0. It declined further in the late pupal

stage (660.0) and in adult stage (420.0).

Differential haemocyte count: (Table 3.)

Present results reveal that there are six types of haemocytes identified in

Acraea terpiscore, as are largely met in Lepidoptera (Ribeiro and Berhelin,

2006). They are the prohaemocytes (PRs), plasmatocytes (PLs),

granulocytes (GRs), spherulocytes (SPs), oenocytoids (OEs), and

adipohaemocytes (ADs).

The PRs appeared mostly small, and rounded in shape. Their percentage

was high in last instar larva (4.2%), decreased further in pupal stage (3.8%),

and adult stage (3.5%).

The PLs were pleomorphic cells, appear rounded, fusiform or spindle

shaped. They appeared large in shape with a relatively smaller nucleus.Their

population ranged from 13.1 percent (last instar) to 25.1 percent (Adult).

The GRs appeared usually rounded to ovoid in shape. They were smaller in

size than PLs but almost equal to PRs. The population of these cells was

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lowest in last instar stage (15.7%), increased further in pupal stage (18.7%),

and reached its peak in the adult stage (22.4%).

The SPs were also rounded to ovoid in shape. They were larger than GRs,

and had centrally located nucleus. Their population was least in last instar

stage (15.9%), steadly increased in early pupal stage (20.3%) and in adult

stage (26.6%).The surface appeared rough due to accumulation of spherules

in the cytoplasm.

The OEs were rounded, small to large cells, with a small eccentric nucleus.

The population of these cells was high in the larval stage (19.6%) but

lowered in the pupal stage (9.9%), and further in the adults (12.5%).

The Ads were rounded to ovoid in shape with centrally located nucleus. Their

population steadly decreased from 30.5% in larval stages to 25.5 % in pupal

stage and further steeply lowered in the adult stage (13.5%).

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Table 2. THC changes during the post embryonic deve lopment of A.

terpiscore

Instar stage

Insect no.

THC /mm3 ± SE

IV

V

Early Pupa

Late Pupa

Adult

05

05

05

05

05

8997.0 ± 80.72

8016.0 ± 65.80

4567.0 ± 322.0

660.0 ± 32.0

420.0 ± 29.8

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Table 3. DHC changes during the post embryonic dev elopment

A. terpiscore

Instar

stage

Insect no. Percentage haemocyte types ± SE

PR PL GR SP AD OE

Last instar

Early pupa

Adult

03

03

03

4.2± 0.3 13.1±2.8 15.7± 1.0 15.9 ±2.3 30.5±1.1 19.6± 0.5

3.8± 0.7 20.2 ±2.9 18.7±1.9 20.3±3.5 25.5±3.2 9.9 ± 0.9

3.5± 0.6 25.1± 1.8 22.4± 0.7 26.6±2.9 13.5±1.9 8.7± 2.6

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DISCUSSION:

The above results reveal that various changes occur in the total

protein, carbohydrate, trehalose and lipid contents in haemolymph of pre

pupal larvae, early pupae and 0-24 hrs old adults, as well as in the fat body

of the pre pupal larvae, during metamorphosis. These changes indicate that

various tissue specific changes occur at different developmental stages. In

the present investigation protein levels are higher (3.24 µg/ml) in the

haemolymph of pre pupal larvae when compared with that of haemolymph of

early pupae (2.20µg/ml) and freshly emerged adult (1.56µg/ml).

The increase in protein levels of haemolymph during larval

development was due to synthesis of new proteins by the tissues, particularly

fat bodies and their release into haemolymph. The present results agree with

the report of Martin, M.D (1990). He observed that during early stages of last

instar larvae in Calliphora stygia there was an increase in the soluble

proteins and this increase was correlated to high rate of protein synthesis by

the fat bodies.

Nagata and Kobayashi (1974) have also reported an increase in

protein synthesis during feeding stage in Bombyx mori. Hurlimann and Chen

(1969) have reported that the concentration of protein in haemolymph

increases progressively during larval development and reaches maximum in

the late fifth instar larvae but declines in the pupal stage. The results of the

present investigation also support the earlier stated report.

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In the present study, the SDS- PAGE results reveal that the number of

protein bands obtained at pre-pupal stage is more than those at the adult

stage. The variation in the number of protein bands and specificity of protein

bands during various stages of development could be due to the fact that the

proteins in the haemolymph vary quantitatively during development of A.

terpiscore. Fluctuations in haemolymph protein during development of

Bombyx mori have been reported earlier by Heller, 1924. Certain bands were

found to be common through in all the stages of development, while certain

bands disappeared or changed in adult stage. Disappearance of bands

indicates that either those proteins are not produced during the adult stage or

may be absorbed to maintain the amino acid concentration in the

haemolymph. This hypothesis is in agreement with the results of Beadle and

Shaw (1950) who reported the hydrolysis of proteins during the larval life of

Bombyx mori for the maintenance of amino acid concentration in the

haemolymph.

Haemolymph carbohydrate levels are also under hormonal control. It

is well known that extracts of corpora cardiaca contain hyperglycemic factors

which regulate the haemolymph carbohydrate levels. Daily fluctuations in

haemolymph sugar levels have been reported in Periplanata americana.

In the present investigation results of carbohydrate estimation of

haemolymph of A. terpiscore indicate that carbohydrate contents in

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haemolymph at pre pupal larvae are more (6.00µg/ml), than that in the

haemolymph of early pupae (5.20 µg/ml) and 0-24 hrs old adults (4.50µg/ml),

which is in agreement with the earlier data.

According to Wyatt G.R (1961) and Wyatt G.R, G.F. Kalf (1957),

Trehalose usually is the main sugar found in the haemolymph of many

insects. Although trehalose is absent in certain stages of several species, it

has been found in at least one stage of almost all insects studied so far

(G.N.Somero.1995). In the present investigation, trehalose was present in

the haemolymph of all studied stages.

The trehalose level in haemolymph of pre pupal larva and that of early

pupa was almost constant (4.33µg/ml and 4.00µg/ml respectively), when

compared with the trehalose level in haemolymph of adults (3.12 µg/ml). The

results indicate that more trehalose is utilized as a source of energy during

the pupal-adult transformation than larval-pupal transformation. The

trehalose level in the fat body of pre-pupal larva was drastically decreased

(0.056µg/ml) when compared with its level in haemolymph. A similar

reciprocal relationship between haemolymph trehalose and fat body

glycogen levels was reported by Ivanovic, J., M. et al. (1992), in the larvae of

Morimus funereus when exposed to high temperature i.e. 35°C.

According to Mullins, D. E., (1985), haemolymph lipid levels in insects

may fluctuate under a variety of conditions, such as muscular activity,

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development, starvation and disease. In the present investigation, the lipid

levels in haemolymph of prepupal larvae are more compared to the

haemolymph of early pupae and 0-24 hrs old adults. This indicates that the

lipid reserves that are stored during prepupal feeding stage are utilized

during the non-feeding pupal stage and highly active adult stage,

subsequently showing lesser lipid contents in these stages.

Insect Haemolymph contains high amount of end products of nitrogen

metabolism such uric acid, allantoin, allantoic acid, urea, and ammonia.

Being concentrated or saturated, very often, it is found in the form of crystals

in haemolymph. The uric acid was detected in the haemolymph of all the

stages of development of A.terpiscore. Its concentration was highest in the

haemolymph and fat bodies of pre-pupal stage and declined in the further

stages of development. This stage being the highly voracious stage, feeds on

the leaves of Passiflora. The high nitrogenous contents of the leaves may be

the cause of high uric acid contents in haemolymph and fat bodies of the pre-

pupal larvae.

The pattern of total haemocyte count (THC) changes during

development is very much similar to what has been reported in the earlier

literature on holometabolous insects. THC increases during the larval stages;

it attains its peak at the last instar stage, and further declines in the pupal

and adult stage. The probable reason behind this may be that the larval

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period is a period of active growth, during which the rate of mitosis is

elevated and also the number of haemocytes is increased as per our

observation.

Insect haemocytes are also known to have a role in intermediary

metabolism such as protein synthesis, transport of nutrients, phenol

metabolism, growth stimulation, etc. Larval period, being a period of active

growth, the rate of intermediary metabolism is higher. Therefore there is a

need of large number of haemocytes during this period of development. The

maximum THC at the prepupal may be due to the effect of ecdysone

hormone. The steep decline in the haemocyte count in the pupae of Acraea

terpiscore species, in the present investigation, agrees with the results of

earlier reports.

The results of differential haemocyte count; in Acraea terpiscore reveals that

the haemocytes can be classified into six major classes on the basis of

distinctive morphological and cytological features revealed by Light (LM) and

Phase contrast microscopy (PCM). They are the Prohaemocytes (PRs),

Plasmatocytes (PLs), Granulocytes (GRs), Spherulocytes (SPs),

Oenocytoids OEs and Adipohaemocytes (ADs). Similar results have been

reported for various lepidopteran insects (Sabri, M.A., 1995, and Jalal Jalali,

2008).

PRs are considered to be the stem cells (Arnold, 1952; Srivastava and

Richards, 1965; Lai-fook,1973 and Beeman et al., 1983) which give rise to

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other types of haemocytes, such as SPs and ADs (Yeager,1946;

Jones,1959), or the PLs,GRs, and SPs, ( Arnold,1970). In A.terpiscore, the

PRs remain low in number throughout the larval development except in the

first instar stage where their percentage is high (Jalal Jalali, 2008). As their

population declines during the later stages of development, the percentage of

PLs and GRs increases.This suggests that the PRs might be getting

converted into PLs and GRs.Similar reports have been reported by Arnold

and Sohi,1974; and Arnold and Hinks,1976). Present study reports are in

agreement with the above reports.

The PLs have been reported in all insect orders (Gupta, 1985a). In

A.terpiscore, PLs were pleomorphic either rounded or spindle- shaped

(without pseudopodia).

The GRs are considered to be plesiomorphic haemocytes, and are the only

haemocyte types that have been reported in all major arthropod groups and

Onychopohora (Gupta, 1985a). A phagocytic role has been assigined to

them by several authors (Crossley, 1964; Arnold, 1970; Akai and Sato, 1978,

1979). In A.terpiscore, the relative percentage of GRs increases and that of

ADs correspondingly decreases, as the developmental stage progresses

towards adults.

The SPs are easily identified by their conspicuous spherules present in the

cytoplasm.In the present study the Phase contrast microscopy gave the best

confirmatory results by revealing clearly their morphological structure.Some

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authors (Gupta and Sutherland, 1966; Arnold and Salkeld, 1967) consider

SPs to be derived from GRs.In A.terpiscore, as the population of both these

cell types tends to increase simultaneously, during last instar onwards, the

chances of interconversion of GRs into SPs are ruled out.

The ADs, and OEs, in A.terpiscore decrease in number in the non feeding

and adult stage, suggesting their magnificient role only during pre-pupal

stage.