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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
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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.
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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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Role of HP19 in C. cephalonica 35
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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.
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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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Role of HP19 in C. cephalonica 37
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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.
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
Figure 5
Figure 4
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Role of HP19 in C. cephalonica 39
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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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Role of HP19 in C. cephalonica 41
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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.
Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch.
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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