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Arif et al.

Archives of Insect Biochemistry and Physiology 66:32–44 (2007)

Significance of the 19-kDa Hemolymph Protein HP19 for the Development of the Rice Moth Corcyra cephalonica: Morphological and Biochemical Effects Caused by Antibody Application
Abul Arif,1,2 Damodar Gullipalli,1 Klaus Scheller,3 and Aparna Dutta-Gupta1*
The hemolymph protein HP19 of the rice moth, Corcyra cephalonica, mediates the 20-hydroxyecdysone (20E) -dependent acid phosphatase (ACP) activity at a nongenomic level. Affinity-purified polyclonal antibody against HP19 (αHP19-IgG) was used in the present study to understand the role of HP19 during the postembryonic development of Corcyra. In the in vitro studies, HP19 action was blocked either by immuno-precipitation using αHP19-IgG, prior to its addition to the fat body culture or by the addition of the antibody directly to the culture, along with 20E and hemolymph containing HP19. The αHP19-IgG blocked the HP19-mediated 20E-dependent ACP activation. In the in vivo studies, the αHP19-IgG was injected into the fully developed last (final/Vth) instar larvae of Corcyra, to complex the HP19 in vivo, in order to block the action of HP19. The injection of αHP19-IgG resulted in defective development of larvae, which grew either into non-viable larvae or larval-pupal/pupaladult intermediates relative to the effect of pre-immune IgG injected controls. The present study shows that HP19 plays an important role in controlling the metamorphosis of Corcyra by regulating the 20E-dependent ACP activity. Coupled with the earlier findings, the ecdysteroid hormone regulates this action at a nongenomic level. Arch. Insect Biochem. Physiol. 66:32–44, 2007. © 2007 Wiley-Liss, Inc.
KEYWORDS: Corcyra cephalonica; 20-hydroxyecdysone; acid phosphatase; fat body culture; hexamerins; nongenomic ecdysteroid action

INTRODUCTION
The role of ecdysteroids controlling the postembryonic development of insects is well established (Trumann and Riddiford, 2002). The hormones regulate a wide variety of functions including initiation of breakdown of larval structures during

metamorphosis (Gilbert et al., 1996) and uptake of hexamerins (Burmester and Scheller, 1999). Programmed cell death is crucial for normal development and occurs mostly by apoptosis of individual cells and autophagy of cell groups. Ecdysteroid triggered regulation of autophagy is well demonstrated in Drosophila (Lee and Baehriecke, 2001;

1 2 3

Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio Department of Cell and Developmental Biology, Biocentre of the University, Wurzburg, Germany

Contract grant sponsor: DST, Govt. of India; Contract grant sponsor: University Grants Commission, India (UGC); Contract grant sponsor: Council for Industrial and Scientific Research (CSIR), India. Abbreviations used: αHP19-IgG = IgG fraction of polyclonal antibody against HP19 raised in rabbit; αhexamerin-IgG = IgG fraction of polyclonal antibody against hexamerin raised in rabbit; ACP = acid phosphatase; LLI = late-last instar larvae; PMSF = phenylmethylsulfonylfluoride; PNP = p-nitrophenol; 20E = 20-hydroxyecdysone. *Correspondence to: Prof. Aparna Dutta-Gupta, Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India. E-mail: apdgsl@uohyd.ernet.in Received 15 May 2006; Accepted 17 February 2007

© 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20195 Published online in Wiley InterScience (www.interscience.wiley.com)

Archives of Insect Biochemistry and Physiology

September 2007

doi: 10.1002/arch.

Acid phosphatases occur in multiple forms and different isozymes in almost all organisms (Konichev et al. 1979. were reared on coarsely crushed sorghum seeds at 26 ± 1°C. it can be suggested that some additional factor(s) mediate the 20E regulated stimulation of ACP activity in vivo. However. and 1 mM PMSF as described in Arif et al. 2001). and ACP assay after estimating the protein content using bovine serum albumin (fraction V) as standard (Bradford. the larval structures degenerate at the beginning of metamorphosis (Lockshin and Beaulton. The last instar larvae (=Vth) were further classified into different stages on the basis of their body weight and head capsule size as described by Lakshmi and Dutta-Gupta (1990). From these results. 1991). diluted (1:20) with 10 mM Tris-HCl (pH 7. Ashok and Dutta Gupta 1988. 1976). We later identified this hemolymph factor as a 19-kDa protein (HP19) that mediated the 20E-stimulated ACP activity at a nongenomic level (Arif et al. In the present study. The activity of the enzyme was expressed as n moles of PNP released/h/µg fat body protein. 1988). pH 7...1 mM CaCl2. The required energy and metabolic fuel are provided by the fat body. Sass and Kovacs. Similar observations were also reported in Manduca sexta. 1991). and 1 mM PMSF) were homogenized in buffer containing 10 mM Tris-HCl. and reorganization. cellular destruction. 0. Corcyra cephalonica (Stainton).. The increase in the lysosomal activity is governed by an increase of the 20E titer (Verkuil.4) and spun for 3 min at 1. 1982. Assay of Acid Phosphatase (ACP) This was carried essentially according to the method of Henrickson and Clever (1974) as described in Ashok and Dutta-Gupta (1991) using pnitrophenyl bisodium phosphate as a substrate. The hemolymph samples were checked immediately for their effect on ACP activity. Acid phosphatase is one of the commonly used marker enzymes to study lysosomal activity in insects (Verkuil. Kutuzova et al. In holometabolous insects. mainly the late-last instar (LLI) larvae were used. Freshly dissected fat bodies in cold insect Ringer (130 mM NaCl.000g to remove haemocytes and debris. Western blotting. 0.4.. . 1979. 1974). (2003) and used for SDSPAGE. 1991). Such a factor was identified in the hemolymph of late-last instar larvae of Corcyra because only when the fat body culture was supplemented with hemolymph could a stimulation of the ACP activity by 20E could be observed (Ashok and Dutta-Gupta. The LLI larvae were thorax-ligated behind the first pair of prolegs by slipping a loop of silk thread around the head of the larvae as described earlier (Ashok and Dutta-Gupta. MATERIALS AND METHODS Insects and Thorax Ligation The larval forms of the rice moth.025% phenylthiourea. Ashok and Dutta-Gupta. The administration of exogenous 20E stimulates the ACP activity in ligated larvae of Spodoptera litura (Sridevi et al. 1991). we report that the ACP activity is required for the normal metamorphosis and HP19 plays a critical role in controlling the metamorphosis of Corcyra by regulating the 20E-dependent ACP activity. 2004). Preparation of Hemolymph Samples and Fat Body Homogenates The prolegs of LLI larvae were cut and the oozing hemolymph was collected into tubes pretreated with 0. 1988). cephalonica 33 Thummel. 1980.1% Triton X-100. In the present study. Lysosomal enzymes play an important role in the histolysis of larval organs. where the ACP activity remained unchanged in fat body cultures in response to 20E (Caglayan.1002/arch. Autophagic process or the lysosomal activity in whole animal as well as in the fat body exhibits a specific pattern during postembryonic development. 5 mM KCl.Role of HP19 in C. 1990). Archives of Insect Biochemistry and Physiology September 2007 doi: 10. 60 ± 5% relative humidity and 14:10 h light:dark photoperiod. the addition of 20E alone to the larval fat body culture of Corcyra did not alter the ACP activity (Ashok and Dutta-Gupta. 1987) and Corcyra cephalonica (Ashok and Dutta-Gupta. 1991). 1980. tissue remodeling.

At the end of incubation.05% ethanol). 1979). MO). The HP19 protein band obtained after ultrafiltration and gel filtration was resolved on 12% SDS-PAGE and was electroeluted using a model-422 electroeluter (BioRad). In another experiment.1% stacking and 12% resolving gel (Laemmli. After rinsing. St. Production of Polyclonal Antibodies The αHP19-IgG antibody was produced as described in Arif et al. 1970) and the resolved proteins were visualized by silver staining (Blum et al. Louis. rinsed in insect Ringer. 10 mM Na2HPO4 and 1. . Functional Assay of α HP19-IgG to Check Its Specificity Against HP19 To test the specificity of αHP19-IgG against HP19. the HP19 present in the hemolymph of LLI larvae was first immunoprecipitated using αHP19-IgG followed by addition of either the precipitate (immuno-complex) or the resulting immunodepleted supernatant (termed immuno-supernatant) to the fat bodies kept in culture along with 80 nM 20E for 4 h at 25°C. hemolymph proteins resolved on SDSPAGE were transferred to nitrocellulose membrane (Towbin et al. MO) in TBST containing 3% BSA for 1 h at room temperature. Inc. the tissue was removed.. 1987). and used for ACP assay.34 Arif et al. The blocked membrane was incubated with αHP19-IgG diluted (1:1.05% ethanol) was added to the control cultures.000 dilution of anti-rabbit IgG coupled with alkaline phosphatase (SigmaAldrich. homogenized. the membrane was incubated with 1:1.. The immunoprecipitation of HP19 from a fixed dilution (1:20) of total hemolymph protein was carried out with various dilutions of αHP19-IgG using protein-A-agarose (Boehringer Mannheim) as described in the manufacturer’s protocol. the tissue was transferred to fresh 200 µl culture medium and 80 nM 20E (in 0.000) in TBST containing 3% BSA for 2 h at room temperature. Fat Body Culture Studies The ribbon-shaped visceral fat bodies from 24 h post-ligated LLI larvae were dissected under sterile conditions in cold insect Ringer and transferred to 100 µl of TC-100 insect culture medium (JRH Biosciences.. diluted (1:20) hemolymph from LLI larvae.4). The detection of specific cross-reactivity of αHP19-IgG with HP19 in total hemolymph protein was carried with nitroblue tetrazolium chloride/5-bromo-4-chloro-3indolyl phosphate color reaction. and 0. functional assays were performed in two different ways. St.1% Tween-20) for 1 h at room temperature. Then membrane was blocked with 3% bovine serum albumin (BSA) in TBST (10 Injection of Antibodies for Immunocomplexing HP19 In Vivo The LLI larvae received injections of αHP19-IgG (15 µg in 5 µl phosphate buffered saline. The electroeluted protein was used as antigen to generate antibody against HP19 in 3-month-old male rabbit (New Zealand variety). the diluted hemolymph (1:20) or purified hemolymph protein HP19 (Arif et al. In one experiment. 130 mM NaCl.) with 1 µg of streptomycin sulfate (Sigma-Aldrich. 2. Electrophoresis and Western Blotting Tris-glycine SDS-PAGE was performed using 2. For Western analysis. and different dilutions of αHP19-IgG for 4 h at 25°C. (2004). mM Tris-HCl. the fat bodies kept in culture were incubated with 80 nM 20E (in 0. The cultures were finally incubated for 4 h at 25°C with gentle shaking. 150 mM NaCl.05% ethanol) was added while an equal volume of carrier solvent (0.5 mM Archives of Insect Biochemistry and Physiology September 2007 doi: 10. 2004) was added to the fat body culture in the presence or absence of 80 nM 20E.5 mM KCl. The IgG fraction was purified by protein-A agarose chromatography (Bio-Rad) according to the manufacturer’s protocol.4. pH 7. In case of studies with hemolymph. Louis. The 1:10 dilution of αHP19-IgG contained 10 µg of IgG from which the antibody was serially diluted with 10 mM Tris-HCl (pH 7. Thereafter.1002/arch.

05. For histological studies.4] with 0. 1a. behavioral. hence. Statistical Analysis All data were statistically analyzed by one-way analysis of variance followed by comparisons of means by Tukey multiple comparison test using Sigma Stat software (Jandel Corporation). were analyzed on different days after αHP19-IgG injection. The results showed no cross-reactivity against 19 kDa in the total hemolymph protein (data not presented). These larvae together with additional controls such as uninjected and phosphate buffered saline (5 µl) –injected larvae were placed on a diet (crushed sorghum) and allowed to grow under normal conditions. among control groups. Various parameters such as morphological. 6:3:1) for 4 h at room temperature. The values were considered significantly different from each other when *P < 0. HP19 is present in the hemolymph in a very low concentration and is not detected as distinct protein band in the total hemolymph protein preparation (Fig. pH 7.Role of HP19 in C. 1a. The immuno-com- . lane 1) and of purified electroeluted HP19 (Fig. the 19-kDa band is clearly seen (Fig. dependent on its concentration. αHP19-IgG was used for immunoprecipitation of HP19 from total hemolymph. 2) revealed that αHP19-IgG. 7.4. in the Western blot. and biochemical changes like fat body ACP activity. 2001) for 24 h at 4°C with gentle shaking. αHP19-IgG were added in different dilutions to the fat body cultures together with hemolymph containing active HP19 and 20E. the deparafinized tissue sections were first treated with blocking solution (2% BSA and 1% non-immune goat serum in TBS [10 mM Tris-HCl pH 7. Control insects received injections of pre-immune IgG (15 µg in 5 µl phosphate buffered saline/insect).1% azide in 0. The washing after each step was done with three changes of TBS. Twenty-five LLI larvae were used for each group studied. The results obtained (Fig. lane 2).5% gelatin. significantly blocked the potentiation of 20E-mediated ACP activity. the sections were deparafinized and stained in hematoxylin/eosin. paraffin embedded.1% Triton X-100 for 1 h at 4°C. pH 7. per insect) through the dorsal surface using a microsyringe. The tissue was processed.4. cephalonica 35 KH2PO4. and 0..1002/arch. only the data of pre-immune-IgG injected larvae are presented. 150 mM NaCl. In another experiment. 1b. lane 2). The specificity of antibody was also confirmed by a protein adsorption assay in which the functional purified HP19 was preincubated with αHP19-IgG to form antigen-antibody complex and was then used for Western blot as described in Figure 1b. 1a. Histological and Immunohistochemical Studies The fat bodies from αHP19-IgG-injected larvae and pre-immune IgG-injected larvae were fixed in Carnoy’s fixative (ethanol:chloroform:acetic acid. These slides were finally processed for staining using nitroblue tetrazolium chloride/5-bromo4-chloro-3-indolyl phosphate and mounted in glycerol gels (50% glycerol. Functional Test of α HP19 IgG to Check the Specificity Against HP19 In order to confirm whether anti-HP19 antibody is capable of influencing the HP19-mediated 20E-dependent ACP activity. The electroeluted HP19 was used for antibody production. 1b. lane 1). followed by treatment with αhexamerin-IgG (Arif et al. Archives of Insect Biochemistry and Physiology September 2007 doi: 10. RESULTS Specificity of the Antibody Against HP19 The results presented in Figure 1a show the SDSPAGE profile of hemolymph proteins (Fig. The specificity of the antibody cross-reaction was checked by parallel processing of the tissue sections with pre-immune-IgG.1 M TBS). the specificity of the αHP19-IgG was found to be high without any nonspecific cross-reactions (Fig. The results with all control groups were identical. Furthermore. However. A comparison was made between the αHP19-IgG and various control groups of larvae. 5-µm-thick sections were cut and mounted on glass slides. For the immuno-histochemical localization of HP19. lane 1). The slides were then treated with anti-rabbit IgG coupled with alkaline phosphatase for 1 h.

which when added along with 20E could stimulate the ACP activity. . hence the protein. HP19. Specificity of αHP19-IgG. 2004). pupae. a: Silver-stained SDSPAGE showing the purified HP19. lane M: protein markers (kDa). The HP19 protein band obtained from ultrafiltration and gel filtration of total hemolymph protein of Corcyra was resolved on 12% SDSPAGE and electroeluted (Arif et al. αhexamerin-IgG. 3). Use of another antibody. which were supplemented with the immuno-complex that act as a control for this experiment (Fig. Effect of α HP19-IgG Injection on Larval Growth and Development To get more insight into the role of HP19 for the postembryonic development of Corcyra. physiological functions of the protein will be at least partly suppressed possibly resulting in altered growth and differentiation of the larvae. At the end of incubation. Under such circumstances. lane 2: electroeluted HP19 (3 µg).000) was insufficient to completely precipitate HP19 in the immuno-complex and thus. the culture with a very high dilution of antibody (1:10.36 Arif et al. The presence of αHP19-IgG rendered HP19 unavailable to mediate the 20E-dependent ACP activity stimulation in low antibody dilutions. Each value is the mean ± SD of four independent determinations and for each assay fat body from two LLI larvae was pooled. These studies suggest that the αHP19-IgG is complexed with HP19. which was injected to LLI larvae. whereas the presence of αhexamerin-IgG had no effect. The protein was injected to rabbit for antibody production. 1. not aimed to complex HP19 had no effect on the ability of HP19 to potentiate the fat body ACP activity. The fat bodies kept in culture were incubated in the presence of 80 nM 20E and 10 µl of 1:20 diluted hemolymph along with different dilutions of αHP19-IgG and αhexamerin-IgG antibodies for 4 h at 25°C. 2. we studied the effect of αHP19-IgG. lane 2: 20 µg of total hemolymph protein. Fig. b: Western blot demonstrating the specificity of αHP19-IgG. plex as well as the immuno-supernatant (see Materials and Methods) was added to the fat body cultures. The significant stimulation in the fat bodies kept in culture in the presence of 20E and HP19 containing hemolymph. *. Lane 1: 10 µg of total hemolymph protein.1002/arch. However. The Archives of Insect Biochemistry and Physiology September 2007 doi: 10. was unavailable to mediate the 20E-dependent ACP activation. Lane 1: Total hemolymph protein (10 µg). Fig. Functional test of αHP19-IgG to check the specificity against HP19. The 20E-stimulated ACP activity could not be detected in all the fat body cultures. the immunodepleted supernatant (immuno-supernatant) still contained HP19. and adults. the fat bodies were assayed for ACP activity.

4m. 5a). 0 day post-injection). Effect of α HP19-IgG Injection on Fat Body ACP Activity The ACP activity profile in LLI that received injection of αHP19-IgG shows the suppression of HP19 function. 7b. all adults abnormally developed. However.l). which had received αHP19-IgG injections developed either in nonviable larvae (Fig. . nonviable pupal-adult intermediate Fig. however. except in wounded controls where pupation was delayed. Functional test of αHP19-IgG to check the specificity against HP19. The control group showed a gradual and significant increase in ACP activity. pupa. abnormally developed nonviable larval-pupal intermediate Delayed metamorphosis. The immuno-complex and the supernatant thus obtained were added to the fat bodies kept in culture in the presence of 80 nM 20E and incubated for 4 h at 25°C. Effect of α HP19-IgG Injection on Fat Body and Implications on Hexamerin Uptake The results obtained from morphological and histological studies suggest that HP19 plays a role in hexamerin sequestration. Figures 4 and 5 show that the larvae.j). Histological studies reveal the presence of a large number of darkly stained granules in the fat bodies of both the control and αHP19-IgGinjected larvae on the day of injection (Fig.Role of HP19 in C. 6). or in nonviable larval-pupal intermediates (Fig. we observed significant morphological and behavioral changes (Table 1). cephalonica TABLE 1. 10 days post-injection). was well developed Emergence of well-developed adults in all controls after 21–23 days αHP19-IgG injected larvae 37 15% mortality Reduced silk secretion Delayed reduction in body and head capsule size after 11 days Pupation normally. The ACP activity did not increase and remained fairly low after 4. whereas the immuno-supernatant obtained from a very high dilution of αHP19-IgG immunoprecipitation had a negligible effect. 7b. 7b. 5b– d) compared to the normally growing control larvae (Figs. 10. 4k. and 14 days postantibody injection. Each value is the mean ± SD of four independent determinations and for each assay fat body from two LLI larvae was pooled. 7a). or in non-viable pupal-adult intermediates (Figs. The immunoprecipitation was carried using serially diluted αHP19-IgG and αhexamerin-IgG with a fixed dilution (1:10) of the hemolymph on a protein-A agarose support. which is a 20E-dependent process. which in turn is responsible for blocking the increase in the fat body ACP activity (Fig.1002/arch. 4b–h. The control group of larvae injected with an equal quantity of pre-immune-IgG developed into normal adults clearly indicating that the effect caused by αHP19-IgG was not due to the adventitious effect of protein injection. 7. Morphological and Behavioral Changes Upon αHP19 IgG Injection to Final (=Vth) Instar Corcyra Larvae Control larvae 10% mortality Normal silk secretion Reduction in body and head capsule size from 8 days onward Pupation after 13–15 days. Addition of immunoprecipitated HP19 (immuno-complex) and the immunodepleted HP19 (immunosupernatant) from the total hemolymph proteins to check their ability in mediating the ACP activity of fat bodies kept in culture. when results indicated that although the mortality rate was more or less the same in the antibody-treated larvae compared to the control larvae. The presence of αhexamerin-IgG had no effect. 3. 14 days post-injection). Both immuno-complex and immunosupernatant significantly blocked the potentiation of fat body ACP activity in low αHP19-IgG dilutions. There was a decline in the number of cytoplasmic granules in the fat body sections of both control and αHP19-IgG-injected larvae (Fig. The whole mount preparations of fat bodies from control (pre-immune-IgG injected) and αHP19-IgG-injected larvae exhibit a clear difference in the morphology and are more pronounced in larvae 10 and 14 days post-injection (Fig. 4i . after 13–15 days. the number of granules increased significantly in the control (Fig. Archives of Insect Biochemistry and Physiology September 2007 doi: 10.

.1002/arch. Figure 4 Figure 5 Archives of Insect Biochemistry and Physiology September 2007 doi: 10.38 Arif et al.

Each value is mean ± SD of 4 independent experiments and for each assay a fat body from 2–3 insects was pooled. 1996). ergy and amino acid building blocks for imaginal tissues have to be provided.Role of HP19 in C. . Changes in the ACP activity in Corcyra larvae after different days of αHP19-IgG injection. 6.” the hexamerins are metabolized by lysosomal enzymes. 1974. the activity of acid phosphatases increases in the fat body and causes the death of larval tissues (Lockschin and Beaulton. Fig. 4. Thummel. Fig. This increase is mainly due to the sequestration of hexamerins from the hemolymph. Each arrow in the control group indicates the gradual and normal development of the last (=Vth) instar larvae into a healthy adult. Immunohistochemical studies using αhexamerin-IgG substantiate the histological findings. The photographs were taken after 30 days of αHP19-IgG (b–d) or pre-immuneIgG injection (a). These storage proteins whose major fraction accounts for the hexamerins are taken up by receptor-mediated endocytosis by the fat body shortly before pupation (Burmester and Scheller. 14 days post-injection). Lee and Baehriecke. 2001. cephalonica 39 compared with the αHP19-IgG-injected larvae (Fig. Intense immunostaining was observed only in the control due to the sequestration of hexamerins but it was significantly reduced in the fat bodies of αHP19-IgG-injected larvae (Fig. enFig. 1999). Effect of αHP19-IgG injection in last instar larvae of Corcyra. DISCUSSION During the development of holometabolous insects. Injection of αHP19-IgG resulted into abnormal development of larvae as compared to the control. Twenty-five larvae were used for each group studied. 14 days post-injection).1002/arch. As a part of cell remodeling during metamorphosis. Effect of αHP19 IgG injection in last instar larvae of Corcyra. Development of non-viable pupal-adult intermediates (b–d) as compared to normal adult shown in control (a) upon injection of αHP19-IgG to the last (=Vth) instar larvae of Corcyra (Fig. Results obtained from other control groups were identical to that of the pre-immune-IgG-injected group. 7b. Archives of Insect Biochemistry and Physiology September 2007 doi: 10. and uninjected larvae. 7c. many determinate larval cells undergo cell death at the end of the ultimate larval stage to ensure metamorphosis and the subsequent emerging of an adult insect from the pupa. Haunerland. Furthermore. The increase in ACP activity was negligible in αHP19-IgG-injected larvae when compared with control larvae where a gradual increase is seen. 2001). The last instar (=Vth) larvae were injected with αHP19-IgG (15 µg in 5 ml phosphate buffered saline/larvae) and were allowed to grow on crushed sorghum diet together with the various control groups including pre-immune-IgG (15 µg in 5 µl phosphate buffered saline/insect) injected. Storage proteins that are dense protein granules and serve as the reserve pool of amino acids are the major source of energy during metamorphosis (Levenbook. The photographs for control (pre-immune-IgG injected) and αHP19-IgG-injected larvae group were taken as indicated. After having been included in “coated vesicles. phosphate buffered saline (5 µl) injected. 1985. 4a). A comparison for morphological changes between the αHP19-IgG-injected and various control groups of insects show a developmental arrest of the αHP19-IgG-injected larvae. 5.

September 2007 doi: 10. paraffin embedded. and processed for hematoxylin/eosin staining and immunostaining with αhexamerin-IgG .40 Arif et al. The whole mount fat body was immediately photographed after fixation. 7. Archives of Insect Biochemistry and Physiology Fig. histological.1002/arch. Morphological. The fat bodies were fixed. and immunohistochemical changes in the αHP19-IgG-injected larvae. . The αHP19-IgG-injected larvae showed significantly reduced hexamerin sequestration as evident from a lack of darkly stained cytoplasmic granules and immunostaing of hexamerins in fat body sections obtained from 10 and 14 days post-injection when compared with pre-immune-IgG-injected controls.

silk secretion. 1980. Ashok and Dutta-Gupta. universal mechanism can account for the hormonal control of histolysis. resulted in a decreased oviposition (Del Pino et Archives of Insect Biochemistry and Physiology September 2007 doi: 10. no single. in antibody-injected insects this increase was totally suppressed and remained more or less the same throughout 14 days post-injection. Thus. However. The present study was designed to get further insights into the role of HP19. However. 2004). Numerous experiments with different species have shown that insect metamorphosis is under the control of ecdysteroids and a few of the studies indicate that some events necessary for the larval-pupal-adult transition are controlled by ecdysteroid hormone at a nongenomic level (Verkuil.. In another study. Kutuzowa et al. nongenomic actions have been reported (Losel and Wehling. studies on these mechanisms are limited to a few experimental systems like the activation of lysosomal enzymes including ACP or the activation of the hexamerin receptors. Burmester and Scheller. al. However. or pupal-adult intermediates.. 1979. Previous studies have shown that ACP activity gradually increases during the postembryonic development of Corcyra and reaches a peak value at the pupal stage when the larval organs undergo histolysis (Ashok and Dutta-Gupta. 1984. and some of them could metamorphose into adult but gave rise to non-viable pupal-adult intermediates. Verkuil. 1980. 1979.. Boophilus microplus. but almost nothing is known about the molecular mechanism of the hormone action governing this process. The mortality rate was more or less the same in the αHP19-IgG-injected and control groups. To date. like the steroid hormones in vertebrates.. One approach was to deactivate or suppress the function(s) of the protein with the help of specific antibodies. 1988. which convert the hormonal stimulus into a transcriptional response (White and Parker. larval-pupal. act on gene transcription by interacting with nuclear receptors. 2003).. 2004).Role of HP19 in C. 1988). in order to understand the role of HP19 in insect growth and development. Beyond it. the control group showed similar ACP activity pattern. 1997. several mechanisms for rapid. Although the duration required by αHP19-IgG-injected larvae for pupation was identical to that of control group of larvae. Further analysis on various parameters including mortality rate. We have recently found that the hemolymph protein HP19 is required to mediate the 20E-stimulated ACP activity in Corcyra and this process is controlled by the hormone at a nongenomic level (Arif et al. . body and head capsule size revealed significant developmental changes in αHP19-IgGinjected larvae when compared with the developmental pattern of control group of larvae. Our results suggest that the injected antibodies suppressed the physiological action of the protein possibly by interfering with the HP19 molecule and caused the development of either nonviable larval. The use of antibodies to understand the role of a molecule in the physiological processes has been demonstrated in several species of invertebrates including insects. 1998). 2003). Ueno and Natori. the inoculation of antibodies against β-N-acetylhexosaminidase of the bovine tick. Our results further strengthen the view that the ACP plays an important role in insect development by regulating the histolysis of larval organs. 1974. Sass and Kovacs. Hiraoka and Hayakawa (1990) reported that a monoclonal antibody against apolipophoriin II in Locusta migratoria inhibited the diacylglycerol uptake into the fat body. 1991). the protein is unable to mediate the 20E-dependent action. the protein was immunocomplexed in vivo. cephalonica 41 Mounting evidence shows that the ACP activity in the fat bodies of last instar larvae is stimulated by ecdysteroid hormones (Lockshin and Beulton. It is widely accepted that ecdysteroids. Scheller et al. larvae that received exogenous antibodies showed reduced salivation. 1998. Nijhout and Grunert (2002) showed that specific antibodies against a bombyxin-like protein completely removed the growth-promoting activity in the hemolymph that is required by 20E to regulate the normal growth of imaginal disks in the butterfly Precis coenia. Hence. We infer that blockage of HP19 by specific anti-HP19 antibody resulted in the blockage of the stimulation of ACP activity. and reduced head capsule size (Table 1). most of the larvae developed into abnormal non-viable larvae or larval-pupal intermediates upon antibody injection. delayed reduction in body length.1002/arch. Arif et al. In the present study.

The immunohistochemical analysis further confirmed that HP19 antibody injection to last instar larvae resulted in improper sequestration suggesting that HP19 plays an important role in the process of 20E-regulated hexamerin sequestration besides its effect on ACP activity. Dutta-Gupta A (Ray). The formation of coated vesicles. Nagamanju et al. hence. Dutta-Gupta A. Arif and G.-G. Marx. J Biol Chem 279:28000–28008. Govt..D. 1990. Beier H. 2004). followed by the uptake of hexamerins into storage granules in the fat bodies. and (2) this action is regulated at a nongenomic level (Arif et al. The insect hemolymph protein HP19 mediates the nongenomic effect of ecdysteroids on acid phosphatase activity. Corcyra cephalonica (Lepidoptera). Effect of 20-hydroxyecdysone on acid phosphatase activity in the larval fat body of Manduca sexta. DNA in polyacrylamide gels. 1976. of India. Naturwissenchaften 86:468–474. Ligand and receptors: common theme in insect storage protein transport. Ashok M. Hemolymph factor(s) potentiate the 20-hydroxyecdysone mediated stimulation of acid phosphatase activity in larval fat body of rice moth. very little or no hexamerin was sequestered. Hexamerins are quantitatively the most prominent proteins in the larvae of many holometabolous insects. in the fat body of 14-day post-antibody-injected larvae. Ecdysteroid mediated fat body acid phosphatase activity during larval development of rice moth. Scheller K. Caglayan SH. Hansen IA. Blum H.. whose functions. 1968. 1988. respectively. India. Arif A. mission (UGC) and Council for Industrial and Scientific Research (CSIR). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein dye binding. Burmester T. Entomon 26:1–11. has been reported for dipteran as well as lepidopteran insects (Locke and Collins. As in all insect species investigated so far. Dutta-Gupta A (Ray). Corcyra cephalonica. for financial support through a direct fellowship. A rapid and reproducible protocol for the purification of insect storage protein. 2003). 2001. 1999). sanctioned to A. Henriques JAP. The hexamerins are later taken back via a receptor-mediated endocytosis by the non-feeding prepupal or pupal fat body cells to meet energy requirements (Haunerland. Scheller K. Dewes Archives of Insect Biochemistry and Physiology September 2007 doi: 10. LITERATURE CITED Arif A. 1999. 1997. Dutta-Gupta A. 1991. Bradford M. Our results show that HP19 did not interfere with hexamerin synthesis but played a distinct role during its sequestration. Vasanthi M. There was a significant reduction in the number of cytoplasmic granules in the fat body of antibody-injected insects compared with the controls. suggesting that hexamerins were not sequestered in these insects. Scheller K. Biochem Int 17:1087–1091. 2003. Tyrosine kinase mediated phosphorylation of the hexamerin receptor in the rice moth Corcyra cephalonica by ecdysteroids. we cannot decide to date whether or how the pathways by which HP19 controls the ecdysteroid-regulated hexamerin uptake and acid phosphatase activity are connected. 1996. 1987. Dutta-Gupta A. RNA. and biosynthesis are well known (Haunerland. Improved silver staining of plant proteins. A. the hexamerins of Corcyra are synthesized by the fat bodies of the actively feeding larvae and released into hemolymph (KiranKumar et al. Developmentally controlled cleavage of the Calliphora arylphorin receptor and posttranslational action of the steroid hormone 20-hydroxyecdysone. Damodar thank the University Grants Com- . 1983. 1996. 1985). Arif A. Ashok M. Brandelli A. Burmester and Scheller. Burmester and Scheller. Biochem Int 20:511–518. 1999). Gonzales JC.. Insect Biochem Mol Biol 33:921–928. 2004. Although we know that (1) HP19 mediates the ecdysteroid hormone action. Invertebr Reprod Dev 20:159–165. ACKNOWLEDGMENTS The work was partly supported by a grant from DST. Gross HJ. Del Pino FAB. 1997. physico-chemical structure. Levenbook. Eur J Biochem 247:695–702. Burmester T. Electrophoresis 8:93–99. Anal Biochem 72:248–254. Vasanthi P.1002/arch.42 Arif et al. Scheller K.

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