hp19 antibody injection

8/2/2019 HP19 Antibody Injection

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32 Arif et al.

Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.

Archives of Insect Biochemistry and Physiology 66:3244 (2007)

2007 Wiley-Liss, Inc.DOI: 10.1002/arch.20195

Published online in Wiley InterScience (www.interscience.wiley.com)

Significance of the 19-kDa Hemolymph Protein HP19for the Development of the Rice Moth Corcyra

cephalonica: Morphological and Biochemical EffectsCaused 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 ac idphosphatase (ACP) activity at a nongenomic level. Affinity-purified polyclonal antibody against HP19 (HP19-IgG) was usedin the present study to understand the role of HP19 during the postembryonic development ofCorcyra. In the in vitro studies,HP19 action was blocked either by immuno-precipitation using HP19-IgG, prior to its addition to the fat body culture or bythe addition of the antibody directly to the culture, along with 20E and hemolymph containing HP19. The HP19-IgG blocked

the HP19-mediated 20E-dependent ACP activation. In the in vivo studies, the HP19-IgG was injected into the fully devel-oped last (final/Vth) instar larvae ofCorcyra, to complex the HP19 in vivo, in order to block the action of HP19. The injectionofHP19-IgG resulted in defective development of larvae, which grew either into non-viable larvae or larval-pupal/pupal-adult intermediates relative to the effect of pre-immune IgG injected controls. The present study shows that HP19plays an important role in controlling the metamorphosis ofCorcyraby regulating the 20E-dependent ACP activity. Coupledwith the earlier findings, the ecdysteroid hormone regulates this action at a nongenomic level. Arch. Insect Biochem. Physiol.66:3244, 2007. 2007 Wiley-Liss, Inc.

KEYWORDS: Corcyra cephalonica; 20-hydroxyecdysone ; acid phosphatase; fa t body culture; hexamerins;nongenomic ecdysteroid action

1Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India2Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio

3Department of Cell and Developmental Biology, Biocentre of the University, Wurzburg, GermanyContract grant sponsor: DST, Govt. of India; Contract grant sponsor: University Grants Commission, India (UGC); Contract grant sponsor: Council for Industrial andScientific Research (CSIR), India.

Abbreviations used: HP19-IgG = IgG fraction of polyclonal antibody against HP19 raised in rabbit; hexamerin-IgG = IgG fraction of polyclonal antibodyagainst 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: [email protected]

Received 15 May 2006; Accepted 17 February 2007

INTRODUCTION

The role of ecdysteroids controlling the postem-

bryonic development of insects is well established

(Trumann and Riddiford, 2002). The hormones

regulate a wide variety of functions including ini-

tiation of breakdown of larval structures during

metamorphosis (Gilbert et al., 1996) and uptakeof hexamerins (Burmester and Scheller, 1999).

Programmed cell death is crucial for normal de-

velopment and occurs mostly by apoptosis of indi-

vidual cells and autophagy of cell groups. Ecdysteroid

triggered regulation of autophagy is well demon-

strated in Drosophila (Lee and Baehriecke, 2001;


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Role of HP19 in C. cephalonica 33


Thummel, 2001). In holometabolous insects, the

larval structures degenerate at the beginning of

metamorphosis (Lockshin and Beaulton, 1974).

Lysosomal enzymes play an important role in the

histolysis of larval organs, tissue remodeling, cel-

lular destruction, and reorganization. The requiredenergy and metabolic fuel are provided by the fat

body. Acid phosphatase is one of the commonly

used marker enzymes to study lysosomal activity

in insects (Verkuil, 1979, 1980; Ashok and Dutta

Gupta 1988, 1991). Acid phosphatases occur in

multiple forms and different isozymes in almost

all organisms (Konichev et al., 1982; Kutuzova et

al., 1991).

Autophagic process or the lysosomal activity in

whole animal as well as in the fat body exhibits a

specific pattern during postembryonic develop-ment. The increase in the lysosomal activity is gov-

erned by an increase of the 20E titer (Verkuil, 1979;

Sass and Kovacs, 1980; Ashok and Dutta-Gupta,

1988). The administration of exogenous 20E stimu-

lates the ACP activity in ligated larvae ofSpodoptera

litura (Sridevi et al., 1987) and Corcyra cephalonica

(Ashok and Dutta-Gupta, 1988). However, the ad-

dition of 20E alone to the larval fat body culture

of Corcyra did not alter the ACP activity (Ashok

and Dutta-Gupta, 1991). Similar observations were

also reported inManduca sexta, where the ACP ac-tivity remained unchanged in fat body cultures in

response to 20E (Caglayan, 1990). From these re-

sults, it can be suggested that some additional

factor(s) mediate the 20E regulated stimulation of

ACP activity in vivo. 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 stimula-

tion of the ACP activity by 20E could be observed

(Ashok and Dutta-Gupta, 1991). We later identi-

fied this hemolymph factor as a 19-kDa protein

(HP19) that mediated the 20E-stimulated ACP ac-

tivity at a nongenomic level (Arif et al., 2004). In

the present study, we report that the ACP activity

is required for the normal metamorphosis and

HP19 plays a critical role in controlling the meta-

morphosis ofCorcyra by regulating the 20E-depen-

dent ACP activity.

MATERIALS AND METHODS

Insects and Thorax Ligation

The larval forms of the rice moth, Corcyra

cephalonica (Stainton), were reared on coarselycrushed sorghum seeds at 26 1C, 60 5% rela-

tive humidity and 14:10 h light:dark photoperiod.

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). In the present

study, mainly the late-last instar (LLI) larvae were

used. 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, 1991).

Preparation of Hemolymph Samples and

Fat Body Homogenates

The prolegs of LLI larvae were cut and the ooz-

ing hemolymph was collected into tubes pretreated

with 0.025% phenylthiourea, diluted (1:20)with10 mM Tris-HCl (pH 7.4)and spun for 3 min at

1,000g to remove haemocytes and debris. The

hemolymph samples were checked immediately fortheir effect on ACP activity. Freshly dissected fat

bodies in cold insect Ringer (130 mM NaCl, 5 mM

KCl, 0.1 mM CaCl2, and 1 mM PMSF) were ho-

mogenized in buffer containing 10 mM Tris-HCl,

pH 7.4, 0.1% Triton X-100, and 1 mM PMSF as

described in Arif et al. (2003) and used for SDS-

PAGE, Western blotting, and ACP assay after esti-

mating the protein content using bovine serum

albumin (fraction V) as standard (Bradford, 1976).

Assay of Acid Phosphatase (ACP)

This was carried essentially according to the

method of Henrickson and Clever (1974) as de-

scribed in Ashok and Dutta-Gupta (1991) usingp-

nitrophenyl bisodium phosphate as a substrate.

The activity of the enzyme was expressed as n

moles of PNP released/h/g fat body protein.


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34 Arif et al.


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 l of TC-100 insect culture medium (JRH Bio-sciences, Inc.) with 1 g of streptomycin sulfate

(Sigma-Aldrich, St. Louis, MO). After rinsing, the

tissue was transferred to fresh 200 l culture me-

dium and 80 nM 20E (in 0.05% ethanol) was

added while an equal volume of carrier solvent

(0.05% ethanol) was added to the control cultures.

In case of studies with hemolymph, the diluted

hemolymph (1:20) or purified hemolymph pro-

tein HP19 (Arif et al., 2004) was added to the fat

body culture in the presence or absence of 80 nM

20E. The cultures were finally incubated for 4 h at

25C with gentle shaking. At the end of incuba-

tion, the tissue was removed, rinsed in insect

Ringer, homogenized, and used for ACP assay.

Production of Polyclonal Antibodies

The HP19-IgG antibody was produced as de-

scribed in Arif et al. (2004). The HP19 protein

band obtained after ultrafiltration and gel filtra-

tion was resolved on 12% SDS-PAGE and waselectroeluted using a model-422 electroeluter (Bio-

Rad). The electroeluted protein was used as antigen

to generate antibody against HP19 in 3-month-old

male rabbit (New Zealand variety). The IgG frac-

tion was purified by protein-A agarose chromatog-

raphy (Bio-Rad) according to the manufacturers

protocol.

Electrophoresis and Western Blotting

Tris-glycine SDS-PAGE was performed using2.1% stacking and 12% resolving gel (Laemmli,

1970) and the resolved proteins were visualized

by silver staining (Blum et al., 1987). For Western

analysis, hemolymph proteins resolved on SDS-

PAGE were transferred to nitrocellulose membrane

(Towbin et al., 1979). Then membrane was blocked

with 3% bovine serum albumin (BSA) in TBST (10

mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1%

Tween-20) for 1 h at room temperature. The

blocked membrane was incubated with HP19-IgG

diluted (1:1,000) in TBST containing 3% BSA for

2 h at room temperature. Thereafter, the membrane

was incubated with 1:1,000 dilution of anti-rabbitIgG coupled with alkaline phosphatase (Sigma-

Aldrich, St. Louis, MO) in TBST containing 3% BSA

for 1 h at room temperature. The detection of spe-

cific cross-reactivity ofHP19-IgG with HP19 in

total hemolymph protein was carried with nitro-

blue tetrazolium chloride/5-bromo-4-chloro-3-

indolyl phosphate color reaction.

Functional Assay of HP19-IgG to Check ItsSpecificity Against HP19

To test the specificity of HP19-IgG against

HP19, functional assays were performed in two dif-

ferent ways. In one experiment, the fat bodies kept

in culture were incubated with 80 nM 20E (in

0.05% ethanol), diluted (1:20) hemolymph from

LLI larvae, and different dilutions ofHP19-IgG

for 4 h at 25C.In another experiment, the HP19

present in the hemolymph of LLI larvae was first

immunoprecipitated usingHP19-IgG followed by

addition of either the precipitate (immuno-com-plex) or the resulting immunodepleted superna-

tant (termed immuno-supernatant) to the fat

bodies kept in culture along with 80 nM 20E for 4

h at 25C.The immunoprecipitation of HP19 from

a fixed dilution (1:20) of total hemolymph pro-

tein was carried out with various dilutions of

HP19-IgG using protein-A-agarose (Boehringer

Mannheim) as described in the manufacturers pro-

tocol. The 1:10 dilution ofHP19-IgG contained

10 g of IgG from which the antibody was serially

diluted with 10 mM Tris-HCl (pH 7.4).

Injection of Antibodies for Immunocomplexing

HP19 In Vivo

The LLI larvae received injections ofHP19-IgG

(15 g in 5 l phosphate buffered saline; 130 mM

NaCl, 2.5 mM KCl, 10 mM Na2HPO4 and 1.5 mM


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KH2PO4, pH 7.4, per insect) through the dorsal sur-

face using a microsyringe. Control insects received

injections of pre-immune IgG (15 g in 5 l phos-

phate buffered saline/insect). These larvae to-

gether with additional controls such as uninjected

and phosphate buffered saline (5 l) injectedlarvae were placed on a diet (crushed sorghum)

and allowed to grow under normal conditions.

Twenty-five LLI larvae were used for each group

studied. Various parameters such as morphologi-

cal, behavioral, and biochemical changes like fat

body ACP activity, were analyzed on different days

after HP19-IgG injection. A comparison was

made between the HP19-IgG and various con-

trol groups of larvae. The results with all control

groups were identical; hence, among control

groups, only the data of pre-immune-IgG injectedlarvae are presented.

Histological and Immunohistochemical Studies

The fat bodies from HP19-IgG-injected larvae

and pre-immune IgG-injected larvae were fixed in

Carnoys fixative (ethanol:chloroform:acetic acid.

6:3:1) for 4 h at room temperature. The tissue was

processed, paraffin embedded; 5-m-thick sections

were cut and mounted on glass slides. For histo-

logical studies, the sections were deparafinized andstained in hematoxylin/eosin. For the immuno-his-

tochemical localization of HP19, the deparafinized

tissue sections were first treated with blocking so-

lution (2% BSA and 1% non-immune goat serum

in TBS [10 mM Tris-HCl pH 7.4, 150 mM NaCl,

pH 7.4] with 0.1% Triton X-100 for 1 h at 4C,

followed by treatment with hexamerin-IgG (Arif

et al., 2001) for 24 h at 4C with gentle shaking.

The slides were then treated with anti-rabbit IgG

coupled with alkaline phosphatase for 1 h. The

washing after each step was done with three changes

of TBS. These slides were finally processed for stain-

ing using nitroblue tetrazolium chloride/5-bromo-

4-chloro-3-indolyl phosphate and mounted in

glycerol gels (50% glycerol, 7.5% gelatin, and 0.1%

azide in 0.1 M TBS). The specificity of the antibody

cross-reaction was checked by parallel processing of

the tissue sections with pre-immune-IgG.

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). The val-ues were considered significantly different from

each other when *P< 0.05.

RESULTS

Specificity of the Antibody Against HP19

The results presented in Figure 1a show the SDS-

PAGE profile of hemolymph proteins (Fig. 1a, lane

1) and of purified electroeluted HP19 (Fig. 1a, lane

2). The electroeluted HP19 was used for antibody

production. HP19 is present in the hemolymph

in a very low concentration and is not detected as

distinct protein band in the total hemolymph pro-

tein preparation (Fig. 1a, lane 1). However, in the

Western blot, the 19-kDa band is clearly seen (Fig.

1b, lane 1). Furthermore, the specificity of the

HP19-IgG was found to be high without any non-

specific cross-reactions (Fig. 1b, lane 2). The speci-

ficity of antibody was also confirmed by a protein

adsorption assay in which the functional purified

HP19 was preincubated with

HP19-IgG to formantigen-antibody complex and was then used for

Western blot as described in Figure 1b. The results

showed no cross-reactivity against 19 kDa in the

total hemolymph protein (data not presented).

Functional Test of HP19 IgG to Check theSpecificity Against HP19

In order to confirm whether anti-HP19 anti-

body is capable of influencing the HP19-mediated

20E-dependent ACP activity, HP19-IgG were

added in different dilutions to the fat body cul-

tures together with hemolymph containing active

HP19 and 20E. The results obtained (Fig. 2) re-

vealed thatHP19-IgG, dependent on its concen-

tration, significantly blocked the potentiation of

20E-mediated ACP activity. In another experiment,

HP19-IgG was used for immunoprecipitation of

HP19 from total hemolymph. The immuno-com-


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36 Arif et al.


Fig. 1. Specificity ofHP19-IgG. a: Silver-stained SDS-

PAGE showing the purified HP19. The HP19 protein band

obtained from ultrafiltration and gel filtration of total

hemolymph protein ofCorcyra was resolved on 12% SDS-

PAGE and electroeluted (Arif et al. 2004). The protein was

injected to rabbit for antibody production. Lane 1: Totalhemolymph protein (10 g); lane 2: electroeluted HP19

(3 g); lane M: protein markers (kDa). b: Western blot

demonstrating the specificity of HP19-IgG. Lane 1: 10

g of total hemolymph protein; lane 2: 20 g of total

hemolymph protein.

Fig. 2. Functional test ofHP19-IgG to check the speci-

ficity against HP19. The fat bodies kept in culture were

incubated in the presence of 80 nM 20E and 10 l of 1:20

diluted hemolymph along with different dilutions ofHP19-IgG and hexamerin-IgG antibodies for 4 h at

25C. At the end of incubation, the fat bodies were as-

sayed for ACP activity. Each value is the mean SD of

four independent determinations and for each assay fat

body from two LLI larvae was pooled. *, The significant

stimulation in the fat bodies kept in culture in the pres-

ence of 20E and HP19 containing hemolymph. The pres-

ence ofHP19-IgG rendered HP19 unavailable to mediate

the 20E-dependent ACP activity stimulation in low anti-

body dilutions, whereas the presence ofhexamerin-IgG

had no effect.

plex as well as the immuno-supernatant (see Ma-

terials and Methods) was added to the fat body

cultures. The 20E-stimulated ACP activity could not

be detected in all the fat body cultures, which were

supplemented with the immuno-complex that act

as a control for this experiment (Fig. 3). However,

the culture with a very high dilution of antibody

(1:10,000) was insufficient to completely precipi-

tate HP19 in the immuno-complex and thus, the

immunodepleted supernatant (immuno-superna-

tant) still contained HP19, which when added

along with 20E could stimulate the ACP activity.

These studies suggest that the HP19-IgG is

complexed with HP19, hence the protein, HP19,

was unavailable to mediate the 20E-dependent

ACP activation. Use of another antibody, hexa-

merin-IgG, not aimed to complex HP19 had no

effect on the ability of HP19 to potentiate the fat

body ACP activity.

Effect of HP19-IgG Injection onLarval Growth and Development

To get more insight into the role of HP19 for

the postembryonic development ofCorcyra, we stud-

ied the effect ofHP19-IgG, which was injected to

LLI larvae. Under such circumstances, physiological

functions of the protein will be at least partly sup-

pressed possibly resulting in altered growth and dif-

ferentiation of the larvae, pupae, and adults. The


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results indicated that although the mortality rate was

more or less the same in the antibody-treated lar-

vae compared to the control larvae, we observed sig-

nificant morphological and behavioral changes

(Table 1). Figures 4 and 5 show that the larvae,

which had receivedHP19-IgG injections developed

either in nonviable larvae (Fig. 4i ,j), or in non-

viable larval-pupal intermediates (Fig. 4k,l), or in

non-viable pupal-adult intermediates (Figs. 4m, 5b

d) compared to the normally growing control lar-

vae (Figs. 4bh, 5a). The control group of larvae

injected with an equal quantity of pre-immune-IgG

developed into normal adults clearly indicating that

the effect caused byHP19-IgG was not due to the

adventitious effect of protein injection.

Fig. 3. Functional test ofHP19-IgG to check the speci-

ficity against HP19. Addition of immunoprecipitated HP19

(immuno-complex) and the immunodepleted HP19 (immuno-

supernatant) from the total hemolymph proteins to checktheir ability in mediating the ACP activity of fat bodies

kept in culture. The immunoprecipitation was carried us-

ing serially diluted HP19-IgG and hexamerin-IgG with

a fixed dilution (1:10) of the hemolymph on a protein-A

agarose support. The immuno-complex and the superna-

tant thus obtained were added to the fat bodies kept in

culture in the presence of 80 nM 20E and incubated for 4 h

at 25C. Each value is the mean SD of four independent

determinations and for each assay fat body from two LLI

larvae was pooled. Both immuno-complex and immuno-

supernatant significantly blocked the potentiation of fat

body ACP activity in low HP19-IgG dilutions, whereasthe immuno-supernatant obtained from a very high dilu-

tion ofHP19-IgG immunoprecipitation had a negligible

effect. The presence ofhexamerin-IgG had no effect.

TABLE 1. Morphological and Behavioral Changes Upon HP19 IgGInjection to Final (=Vth) Instar Corcyra Larvae

Control larvae HP19-IgG injected larvae

10% mortality 15% mortality

Normal silk secretion Reduced silk secretion

Reduction in body and head capsule Delayed reduction in body and head

size from 8 days onward capsule size after 11 daysPupation after 1315 days, except Pupation normally, after 1315 days,

in wounded controls where pupation abnormally developed nonviable

was delayed; pupa, however, was larva l-pupal intermediate

well developed

Emergence of well-developed adults Delayed metamorphosis, all adults

in all controls after 2123 days abnormally developed, nonviable

pupal-adult intermediate

Effect of HP19-IgG Injection onFat Body ACP Activity

The ACP activity profile in LLI that received in-jection ofHP19-IgG shows the suppression of

HP19 function, which in turn is responsible for

blocking the increase in the fat body ACP activity

(Fig. 6). The ACP activity did not increase and re-

mained fairly low after 4, 7, 10, and 14 days post-

antibody injection. The control group showed a

gradual and significant increase in ACP activity.

Effect of HP19-IgG Injection on Fat Body andImplications on Hexamerin Uptake

The results obtained from morphological and

histological studies suggest that HP19 plays a role

in hexamerin sequestration, which is a 20E-depen-

dent process. The whole mount preparations of fat

bodies from control (pre-immune-IgG injected)

and HP19-IgG-injected larvae exhibit a clear dif-

ference in the morphology and are more pro-

nounced in larvae 10 and 14 days post-injection

(Fig. 7a). Histological studies reveal the presence

of a large number of darkly stained granules in

the fat bodies of both the control and HP19-IgG-

injected larvae on the day of injection (Fig. 7b, 0

day post-injection). There was a decline in the

number of cytoplasmic granules in the fat body

sections of both control and HP19-IgG-injected

larvae (Fig. 7b, 10 days post-injection). However,

the number of granules increased significantly in

the control (Fig. 7b, 14 days post-injection), when


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38 Arif et al.


Figure 5

Figure 4


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compared with the HP19-IgG-injected larvae (Fig.

7b, 14 days post-injection). This increase is mainly

due to the sequestration of hexamerins from the

hemolymph. Immunohistochemical studies using

hexamerin-IgG substantiate the histological find-

ings. Intense immunostaining was observed onlyin the control due to the sequestration of hexa-

merins but it was significantly reduced in the fat

bodies ofHP19-IgG-injected larvae (Fig. 7c, 14

days post-injection).

DISCUSSION

During the development of holometabolous in-

sects, many determinate larval cells undergo cell

death at the end of the ultimate larval stage to en-sure metamorphosis and the subsequent emerging

of an adult insect from the pupa. Furthermore, en-

ergy and amino acid building blocks for imaginal

tissues have to be provided. Storage proteins that

are dense protein granules and serve as the reserve

pool of amino acids are the major source of en-

ergy during metamorphosis (Levenbook, 1985;

Haunerland, 1996). 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, 1999). After having been included in

coated vesicles, the hexamerins are metabolized

by lysosomal enzymes. As a part of cell remodel-

ing during metamorphosis, the activity of acid

phosphatases increases in the fat body and causes

the death of larval tissues (Lockschin and Beaulton,

1974; Lee and Baehriecke, 2001; Thummel, 2001).

Fig. 6. Changes in the ACP activity in Corcyra larvae af-

ter different days ofHP19-IgG injection. The increase in

ACP activity was negligible in HP19-IgG-injected larvae

when compared with control larvae where a gradual in-

crease is seen. Each value is mean SD of 4 independent

experiments and for each assay a fat body from 23 in-sects was pooled.

Fig. 4. Effect ofHP19-IgG injection in last instar lar-

vae of Corcyra. The last instar (=Vth) larvae were injected

with HP19-IgG (15 g in 5 ml phosphate buffered sa-

line/larvae) and were allowed to grow on crushed sor-

ghum diet together with the various control groups

including pre-immune-IgG (15 g in 5 l phosphate buff-

ered saline/insect) injected, phosphate buffered saline (5

l) injected, and uninjected larvae. Twenty-five larvae wereused for each group studied. A comparison for morpho-

logical changes between the HP19-IgG-injected and vari-

ous control groups of insects show a developmental arrest

of the HP19-IgG-injected larvae. The photographs for con-

trol (pre-immune-IgG injected) and HP19-IgG-injected lar-

vae group were taken as indicated. Results obtained from

other control groups were identical to that of the pre-im-

mune-IgG-injected group. Injection ofHP19-IgG resulted

into abnormal development of larvae as compared to the

control. Each arrow in the control group indicates the gradual

and normal development of the last (=Vth) instar larvae into

a healthy adult.

Fig. 5. Effect ofHP19 IgG injection in last instar larvae

ofCorcyra. Development of non-viable pupal-adult inter-

mediates (bd) as compared to normal adult shown in

control (a) upon injection ofHP19-IgG to the last (=Vth)

instar larvae of Corcyra (Fig. 4a). The photographs were

taken after 30 days ofHP19-IgG (bd) or pre-immune-

IgG injection (a).


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Mounting evidence shows that the ACP activity

in the fat bodies of last instar larvae is stimulated

by ecdysteroid hormones (Lockshin and Beulton,

1974; Verkuil, 1979, 1980; Sass and Kovacs, 1980;

Ashok and Dutta-Gupta, 1988; Kutuzowa et al.,

1991), but almost nothing is known about themolecular mechanism of the hormone action gov-

erning this process. To date, no single, universal

mechanism can account for the hormonal control

of histolysis. It is widely accepted that ecdysteroids,

like the steroid hormones in vertebrates, act on

gene transcription by interacting with nuclear recep-

tors, which convert the hormonal stimulus into a

transcriptional response (White and Parker, 1998;

Scheller et al., 2003). Beyond it, several mechanisms

for rapid, nongenomic actions have been reported

(Losel and Wehling, 2003). Numerous experimentswith different species have shown that insect meta-

morphosis is under the control of ecdysteroids and

a few of the studies indicate that some events nec-

essary for the larval-pupal-adult transition are con-

trolled by ecdysteroid hormone at a nongenomic

level (Verkuil, 1979; Ueno and Natori, 1984;

Burmester and Scheller, 1997; Arif et al., 2004).

However, 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. We have recently foundthat 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., 2004).

The present study was designed to get further

insights into the role of HP19. One approach was

to deactivate or suppress the function(s) of the pro-

tein with the help of specific antibodies. The use

of antibodies to understand the role of a molecule

in the physiological processes has been demon-

strated in several species of invertebrates includ-

ing insects. Hiraoka and Hayakawa (1990) reported

that a monoclonal antibody against apolipophoriin

II in Locusta migratoria inhibited the diacylglycerol

uptake into the fat body. In another study, the in-

oculation of antibodies against -N-acetylhexo-

saminidase of the bovine tick, Boophilus microplus,

resulted in a decreased oviposition (Del Pino et

al., 1998). Nijhout and Grunert (2002) showed

that specific antibodies against a bombyxin-like

protein completely removed the growth-promot-

ing activity in the hemolymph that is required by

20E to regulate the normal growth of imaginal

disks in the butterflyPrecis coenia. Hence, in orderto understand the role of HP19 in insect growth

and development, the protein was immuno-

complexed in vivo. Thus, the protein is unable to

mediate the 20E-dependent action. Our results sug-

gest that the injected antibodies suppressed the

physiological action of the protein possibly by in-

terfering with the HP19 molecule and caused the

development of either nonviable larval, larval-pu-

pal, or pupal-adult intermediates. Further analysis

on various parameters including mortality rate, silk

secretion, body and head capsule size revealedsignificant developmental changes in HP19-IgG-

injected larvae when compared with the develop-

mental pattern of control group of larvae. Themortality rate was more or less the same in the

HP19-IgG-injected and control groups. However,larvae that received exogenous antibodies showed

reduced salivation, delayed reduction in bodylength, and reduced head capsule size (Table 1).

Although the duration required byHP19-IgG-in-jected larvae for pupation was identical to that of

control group of larvae, most of the larvae devel-oped into abnormal non-viable larvae or larval-pu-

pal intermediates upon antibody injection, andsome of them could metamorphose into adult but

gave rise to non-viable pupal-adult intermediates.Previous studies have shown that ACP activity

gradually increases during the postembryonic de-velopment ofCorcyra and reaches a peak value atthe pupal stage when the larval organs undergo

histolysis (Ashok and Dutta-Gupta, 1988). In the

present study, the control group showed similar

ACP activity pattern. However, in antibody-injectedinsects this increase was totally suppressed and re-mained more or less the same throughout 14 dayspost-injection. We infer that blockage of HP19 by

specific anti-HP19 antibody resulted in the block-age of the stimulation of ACP activity. Our results

further strengthen the view that the ACP plays animportant role in insect development by regulat-

ing the histolysis of larval organs.


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42 Arif et al.


Hexamerins are quantitatively the most promi-

nent proteins in the larvae of many holometab-

olous insects, whose functions, physico-chemical

structure. and biosynthesis are well known (Hauner-

land, 1996; Burmester and Scheller, 1999). As in

all insect species investigated so far, the hexamerinsofCorcyra are synthesized by the fat bodies of the

actively feeding larvae and released into hemo-

lymph (KiranKumar et al., 1997; Nagamanju et al.,

2003). 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, 1996; Burmester and

Scheller, 1999). Our results show that HP19 did

not interfere with hexamerin synthesis but played

a distinct role during its sequestration; hence, in

the fat body of 14-day post-antibody-injected lar-vae, very little or no hexamerin was sequestered.

The immunohistochemical analysis further con-

firmed that HP19 antibody injection to last instar

larvae resulted in improper sequestration suggest-

ing that HP19 plays an important role in the pro-

cess of 20E-regulated hexamerin sequestration

besides its effect on ACP activity.

The formation of coated vesicles, followed by

the uptake of hexamerins into storage granules in

the fat bodies, has been reported for dipteran as

well as lepidopteran insects (Locke and Collins,1968; Marx, 1983; Levenbook, 1985). There was a

significant reduction in the number of cytoplas-

mic granules in the fat body of antibody-injected

insects compared with the controls, suggesting that

hexamerins were not sequestered in these insects.

Although we know that (1) HP19 mediates the

ecdysteroid hormone action, and (2) this action is

regulated at a nongenomic level (Arif et al., 2004),

we cannot decide to date whether or how the path-

ways by which HP19 controls the ecdysteroid-regu-

lated hexamerin uptake and acid phosphatase

activity are connected.

ACKNOWLEDGMENTS

The work was partly supported by a grant from

DST, Govt. of India, sanctioned to A.D.-G. A. Arif

and G. Damodar thank the University Grants Com-

mission (UGC) and Council for Industrial and Sci-

entific Research (CSIR), India, respectively, for fi-

nancial support through a direct fellowship.

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