Biochemical analysis of Ariadne merione Cramer Common name: Castor Butterfly

Transcription

1 Biochemical analysis of Ariadne merione Cramer Common name: Castor Butterfly 126

2 Introduction: Ariadne merione, is also known as common castor butterfly. It is an orange butterfly with brown lines. The larvae of this butterfly feed exclusively on leaves of castor plant, Ricinus communis. It belongs to the Nymphalidae family. Common Castors are also known as castor butterflies. They are very active but their wings are very weak. Castor butterflies are often found flying gracefully as if sailing through the air among the castor plants or any dense vegetation. They are fond of resting on leaves at the top canopy, keeping the wings slowly moving sideways and are always nearer to host plants. They are found through the year, more densely populated during the rainy and summer season. Upper side of the Common Castor butterfly is reddish brown with transverse black wavy lines. In females, these wavy lines are broader in the form of distinct bands. The summer season butterfly has a wider wingspan as compared to rainy season species. It takes about 20 days for maturity of reproductive organs in chrysalis. The green coloured caterpillar has dorsal longitudinal brown stripe and two dorsal and two lateral rows of short branched-spines. There is a pair of long, straight branchedspines on the head. The caterpillar turns reddish-brown when matured. Ricinus communis is the host plant of the Common Castor butterfly (Ariadne merione). It is an important non-edible oilseed crop. It is a large plant belonging to family Euphorbiaceae. It is cultivated around the world primarily 127

3 because of the commercial importance of its oil in pharmaceutical industries. It is also used in landscaping because of its handsome, giant, 12-lobed, palmate (fanlike) leaves. Its fruits are attractive but often are removed before they mature because the poison ricin gets concentrated in their beanlike seeds. It is also the host plant of Eri silkmoth (Samia cynthia ricini), and the Castor Semi-Looper moth (Achaea janata). It is also used as a food plant by the larvae of some other species of Lepidoptera including Hypercompe hambletoni and the Nutmeg (Discestra trifolli). Materials and Methods: Rearing of butterflies: Eggs and the larvae of Ariadne merione were collected from NDMVP Samaj s, KTHM College, Nasik, campus area. Larvae were reared under laboratory conditions in the glass petri plates kept in a wooden box (20 x 20 x20 ) wire-netted from sides and top. The temperature was maintained at 27 c ± 2 c and relative humidity at 70% ± 5%. The larvae were fed on fresh leaves of castor plant (Ricinus communis). The completely grown larvae, about to pupate, were sorted out and placed in a separate glass dish at room temperature, for pupation. The required number of larvae was used for biochemical analysis, while others were allowed to pupate at room temp. The required number of the pupae 128

4 was used for biochemical investigation while others were allowed to grow into adults. The adults were also captured from the nature, acclimatized under laboratory conditions for 24 hours and then used for biochemical analysis. Haemolymph collection: Haemolymph was drawn from the last instar larvae, pupae and adult of the Ariadne merione. About 4-5 µl of haemolymph was drawn by inserting the capillary in the pseudolegs of the last instar larvae, anterior end of the pupae and from the thoracic region of the adult. 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. The collected haemolymph was then centrifuged for 15 min at 18,000 rpm to separate haemocytes and plasma (G.R.Wyatt, M.L.Pal, 1978; Nursel G.L, Cevat Ayvali; 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, total carbohydrate, total lipid trehalose and uric acid estimation. The separated haemocytes were used for total haemocyte count. 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 and used for further biochemical estimation. 129

5 Biochemical assays: Estimation of total proteins was done by Modified Lowry method; Total Carbohydrates were estimated by Modified Anthrone method. Lipid estimation was done by Zoellner and Kirsch method; Estimation of Trehalose was done by Anthrone Method while, qualitative analysis of proteins was done by SDS- PAGE. Details of all the assays are given in chapter two. Total haemocyte count was also done by the same method as given in chapter two. Differential haemocyte count: Chemicals: Glacial acetic acid: 5ml Geimsa stain Buffer solution: Na2HPO4 = 3.8 g, KH2PO4 = 5.47 g added to 1 L distilled water, ph adjusted to 6.6 Protocol: The haemolymph was fixed by glacial acetic acid vapours for 5-10 min in a small desiccator at 40 o C. One of the thoracic legs was pricked by needle and the exuding haemolymph was drawn in to Thoma white blood cell diluting pipette. A small drop of this blood was placed on the clean white grease free microscope slide and smear was made by drawing second slide across the 130

6 first one at 45 o angle. 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). Results: Results in table 1 reveals that the levels of Organic biomolecules in the haemolymph and fat bodies of Ariadne merione were significantly different at different stages of development. Total Protein concentration in haemolymph and fat bodies: (Fig.1) The protein levels in haemolymph were found to be significantly lower (780µg/ml) in adult stage, when compared to haemolymph protein levels of larvae (1240µg/ml) and pupae (1210µg/ml). The protein concentration in fat bodies of larvae was low (255µg/ml) when compared to the haemolymph protein levels of the same stage of development. Total Carbohydrate concentration in haemolymph and fat bodies: (Fig.2) 131

7 In Larvae, haemolymph total carbohydrate levels were higher (36.27µg/ml) than the pupae (31.27µg/ml) and adult (23.7µg/ml) haemolymph carbohydrate levels. The total carbohydrate levels in fat bodies of larvae were significantly low (25.33µg/ml) when compared to the haemolymph carbohydrate levels of all the stages of development. Trehalose concentration in haemolymph and fat bodies: Trehalose concentration in haemolymph of larvae(29.00µg/ml) and pupae were found to be almost constant and higher when compared to haemolymph trehalose levels in adults. On the contrary trehalose levels in larval fat bodies were found to be significantly low when compared to the trehalose levels of larval haemolymph. Total lipid concentration in haemolymph and fat bodies: (Fig.3) Lipid concentration in haemolymph of all the stages of development was found to almost constant and lower than the lipid level in fat bodies, when compared with larval fat bodies. Total lipid concentration in haemolymph and fat bodies were also found to the least among all the organic biomolecules studied. Total uric acid concentration in haemolymph: (Fig.4) 132

8 Uric acid contents in pre pupal haemolymph (45.21mg/ml) of Ariadne merione were lower than uric acid contents of pupal haemolymph (28.79µg/ml).While in adult haemolymph the uric acid contents were the least (5.67µg/ml).In larval fat body the uric acid contents (6.89µg/ml) were slightly higher than the uric acid contents of adult haemolymph. Total Haemocyte count: (Table 2.) The total haemocyte count in the fourth instar pre-pupal stage was (8762.0), it increased in the next stage i.e. fifth in star, up to and drastically decreased in early pupal stage to of Ariadne merione. It declined further in the late pupal stage (1612.0) and in adult stage (598.0). Differential Haemocyte count: (Table 3.) Present results reveal that there are six types of haemocytes identified in Ariadne merione, 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). Two additional types, vermicytes (VE) and podocytes (PO), were reported by Jalal Jalali and Rasoul Salehi (2008). These two types were not identified in Ariadne merione. 133

9 The PRs appeared mostly small, and rounded in shape. Their percentage was high in last instar larva (4.5%), decreased further in pupal stage (2.9%), and adult stage (1.7%). The PLs were pleomorphic cells, appeared rounded, fusiform or spindle shaped. They appeared large in shape with a relatively smaller nucleus.their population ranges from 26.0 percent (Last instar larva) to 34.7 percent (Adult). The GRs appeared usually rounded in shape. They were smaller in size than PLs but almost equal to PRs. The population of these cells was least in last instar stage (25.6%) steeply increased further in pupal stage (35.96%), and in the adult stage (43.5%). The SPs were also rounded to ovoid in shape. They were larger than GRs, and had centrally located nucleus. Their population was highest in last instar stage (15.8%); declined in early pupal stage (10.8%) and further in adult stage (5.3%).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 (13.5%) but lowered in the pupal stage (8.8%), and further in the adults (5.2%). The Ads were rounded to ovoid in shape with centrally located nucleus. Their population ranged from 13.8% in larval stages to 8.7% in pupal stage and was the least in the adult stage (6.8%). 134

10 Protein estimation by SDS PAGE: (Fig.5) Protein estimation by SDS- PAGE reveals that the number of bands obtained from the haemolymph of larval stage is maximum. Total eighteen protein bands are obtained at larval stage, but few bands are not distinct, instead are discrete. Two to three bands (MW 120kDa, 84kDa and 20kDa) are distinct. The number of protein bands is reduced in pupal haemolymph to eight. But increased to at adult stage. Two to three bands are very distinct at adult stage, while others are of low density. 135

11 Table 1: Biochemical changes in the haemolymph of larvae and pupae of Ariadne merione Biomolecules Haemolymph µg/ml Fat body Larva Pupa Adult Larva Total Proteins 1240±1.419** 1210±0.76** 80±1.25** 255±1.565** Total Carbohydrates 36.27±0.046** 31.27±0.45** 23.7 ± 0.06* 25.33± 0.05* Total Lipids 2.063±0.022* 1.743± * 0.527± 0.12* 3.25± 0.02* Trehalose 29.00±0.371** 27.33±0.20** 15.70± 0.23** 37.98± 0.07** Uric acid ±0.55** 32.09±0.36** 24.78± 0.57** 6.48±0.29** - Significant at P< 0.05 N.S Non-significant 136

12 Fig 1. Standard curve for Proteins 137

13 Fig 2. Standard curve for Carbohydrates 138

14 Fig.3 Standard curve for Lipids. 139

15 Fig.4 Standard curve for Uric acid 140

16 Table 2: THC Changes in the haemolymph of Ariadne merione during the postembryonic development Instar stage Insect no. THC /mm3 ± SE IV V Early Pupa Late Pupa Adult ± ± ± ± ±

17 Table 3. DHC changes during the post embryonic development of Ariadne merione: Instar stage Last instar Early pupa Adult Insect no Percentage haemocyte types ± SE PR PL GR SP AD OE 4.5± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.3 Marker Proteins 142

18 205kDA 120kDA 116kDA 84kDA 66kDA 45kDA 29kDA 20kDA 18kDA Fig. 5. Protein determination by SDS PAGE, in A.meriodne 1. Haemolymph of adult bands 2. Haemolymph of pupa- 8 bands 3. Haemolymph of larva- 18 bands Discussion: 143

19 Ariadne merione, a castor butterfly, undergoes various biochemical changes during its development. The results in the present investigation reveal that the rate of synthesis of all the biomolecules viz. proteins, lipids, carbohydrates, trehalose and uric acid is maximum in the larval stage. The results of total protein estimation show that protein concentration is high in larval stage (1240µg/ml), slightly low in pupal stage (1210µg/ml) and drastically low in adult stage (80µg/ml). Larval fat bodies have proteins less than the larval haemolymph. Since larval stage is a stage of rapid growth and development, various new proteins are synthesized particularly by fat bodies and then released into haemolymph. Increase in protein concentration during feeding stage has also been reported by Hurlimann and Chen (1974), Nagata and Kobayashi (1990) and Martin M.D. (1969), which supports the data of the present investigation. Results of Qualitative estimation of proteins by SDS PAGE, show that maximum number of protein bands is obtained in larval stage. There are 16 protein bands seen at larval stage, ranging from 120kDa to 18kDa.The number of protein bands is reduced from 16 to 9 in pupa and further reduced to 6, in adults.but the bands become more distinct in pupal and adult stage. This indicates that relative intensity of band changes strikingly in proportional to growth ( G.R.Wyatt and M.l.Pan; 1978). The variation in the number of protein bands during various stages of development, suggests that the 144

20 proteins in haemolymph vary quantitatively during various stages of development of A.merione. Heller (1924), has earlier reported similar fluctuations in haemolymph proteins during development of Bombyx mori. Development stage specific alterations in tissue proteins have also been earlier reported in lepidopterans by Chippendale and Kilby (1969), Chippendale (1970 a and b). The results of total carbohydrate estimation, reveals that the carbohydrate concentration decreases during development. Reduction in carbohydrate concentration during development may be due to utilization of energy for tissue formation. 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 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 145

21 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 A. merione species, in the present investigation, agrees with the results of earlier reports. The results of differential haemocyte count; in A. merione reveals that the there are six major classes of haemocytes, similar to the other three species investigated earlier. They are the Prohaemocytes (PRs), Plasmatocytes (PLs), Granulocytes (GRs), Spherulocytes (SPs), Oenocytoids OEs and Adipohaemocytes (ADs). In A. merione, 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 satges of development, the percentage of PLs and GRs also 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. PLs were pleomorphic, either rounded or spindle- shaped (with or 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, 146

22 1979). In A. merione, 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 authors (Gupta and Sutherland, 1966; Arnold and Salkeld, 1967) consider SPs to be derived from GRs.In A. merione, 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. merione decrease in number in the non feeding and adult stage, suggesting their magnificent role only during pre-pupal stage. General Discussion: 147