Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B
j ourna l homepage: www.e lsev ie r .com/ locate /cbpbCloning and characterization of a riboflavin-binding hexamerin from the
larval fat body of a lepidopteran stored grain pest, Corcyra cephalonicaV. Venkat Rao a, Thuirei Jacob Ningshen b, R.K. Chaitanya c, B. Senthilkumaran a, Aparna Dutta-Gupta a,⁎
a Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India
b Department of Biotechnology, Manipur University, Canchipur, Manipur 795003, India
c Centre for Animal Sciences, Central University of Punjab, Bhatinda, Punjab 151001, India⁎ Corresponding author at: Department of Animal B
University of Hyderabad, P O Central University, Hyderab
E-mail addresses: apdgsl@uohyd.ernet.in, aparnadutta
(A. Dutta-Gupta).
http://dx.doi.org/10.1016/j.cbpb.2016.01.008
1096-4959/© 2016 Elsevier Inc. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:
Received 14 August 2015
Received in revised form 29 December 2015
Accepted 25 January 2016
Available online 27 January 2016In the present study, a riboflavin-binding hexamerin (RbHex) was cloned and characterized from the larval fat
body of Corcyra cephalonica. The complete cDNA (2121 bp) encodes a 706-amino acid protein with a molecular
mass ~82 kDa. Expression of RbHex 82 was predominant in fat body among larval tissues. Further, it is promi-
nently expressed during the last instar larval development. Homology modeling and docking studies predicted
riboflavin binding site of the hexamerin. Spectrofluorimetric analysis further confirmed riboflavin release from
the hexamerin fraction. Quantitative RT-PCR studies demonstrated hormonal regulation of RbHex 82. 20-
Hydroxyecdysone (20HE) had a stimulatory effect on its transcription whereas JH alone did not show any effect.
However, JH in the presence of 20HEmaintains the RbHex 82 expression which indicates the JH's role as a status
quo factor. This study is the first to report the characterization of riboflavin-binding hexamerin in a lepidopteran
pest. Further, the possibility of RbHex 82 as a pest control target is discussed.
© 2016 Elsevier Inc. All rights reserved.Keywords:
Fat body
Hexamerin
20-Hydroxyecdysone
Juvenile hormone
Riboflavin
Stored grain pest management1. Introduction
Hexamerins belong to an arthropod protein superfamily, which in-
cludes hemocyanins, dipteran hexamerin receptors and phenoloxidases
(Decker and Terwilliger, 2000; Burmester, 2002). These proteins with a
nativemolecularmass of about 500 kDa exist as either homo- or hetero-
hexamer subunits ranging 70–90 kDa (Telfer and Kunkel, 1991;
Burmester and Scheller, 1999). Insect hexamerins are predominantly
synthesized and expressed in the fat body and secreted into the hemo-
lymph (Burmester, 1999; Telfer and Kunkel, 1991; Wang and
Haunerland, 1991).
Hexamerins are primarily known to function as storage proteins that
provide energy for non-feeding periods (Munn et al., 1967; Munn and
Greville, 1969) but recent evidence demonstrate them as a versatile
molecule. Hexamerins may transport hormones such as ecdysteroids
(20HE) (Enderle et al., 1983) and juvenile hormone (JH) (Braun and
Wyatt, 1996). In termites, hexamerins play a major role in caste deter-
mination in cooperation with JH (Zhou et al., 2006). Few studies impli-
cate hexamerins' involvement in immune response (Beresford et al.,
1997; Ma et al., 2005; Phipps et al., 1994; Poopathi et al., 2014).
Hakim et al. (2007) have reported that aryphorin hexamerins haveiology, School of Life Sciences,
ad 500046, India.
gupta@gmail.commitogenic effect and they stimulate stem cell proliferation in vitro.
Recently, Martins et al. (2011) demonstrated the intranuclear localiza-
tion of a hexamerin, Hex70a, in the ovaries and testes of the honeybee,
and suggested that the hexamerin might have tissue specific role.
Hexamerins may also bind to small organic metabolites like riboflavin
(Magee et al., 1994) and biliverdin (Miura et al., 1994) with high
affinity.
Five distinct types of hexamerins have been identified in lepidop-
terans, which differ in terms of amino acid composition and evolutionary
history: i) the arylphorins, which are rich in aromatic amino acids (∼20%
phenylalanine and tyrosine), ii) the distantly related arylphorin-like
hexamerins, iii) the methionine-rich hexamerins, iv) the moderately
methionine-rich hexamerins, and v) the riboflavin-binding hexamerins
(RbHex) (Burmester, 2015). While the specific role of other hexamerin
types can be easily associated with the accumulation of certain amino
acids, the function of RbHex is obscure. So far, RbHex has only been iden-
tified and characterized in the giant saturniid silk moth, Hyalophora
cecropia, as a protein that binds to riboflavin (vitamin B2) (Magee
et al., 1994).
The present study is the first to clone and characterize a riboflavin
bindinghexamerin from the fat body of last instar larvae of a lepidopter-
an stored grain pest, Corcyra cephalonica. Further, RbHex being
lepidopteran-specific (Burmester, 2015), and most major stored grain
and agricultural pests belonging to the same order, its molecular eluci-
dation could provide cues for exploitation as a potential target for pest
management.
V.V. Rao et al. / Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64 592. Materials and methods
2.1. Insect rearing and maintenance
The eggs of C. cephalonica were procured from National Bureau of
Agriculturally Important Insects (NBAII, ICAR), Bengaluru. The eggs
were allowed to hatch in culture troughs containing coarsely crushed
sorghum seedswithmultivitamin tablets. The culturesweremaintained
at 26 ± 1 °C, 60 ± 5% relative humidity and 14:10 h light:dark (L:D)
photoperiod. The larval development proceeds through five instars
and is completed in about 45–50 days followed by a short prepupal
stage (4–5 days), and a pupal stage, which lasts for 7–8 days. The
adult moths normally survive for 8–10 days. For the present study, all
the stages of insect i.e. embryo, larvae (1st, 2nd, 3rd, 4th and 5th instar),
prepupa, pupa, and adult were used. Further, 5th instar larva were clas-
sified as early-late (ELI), mid-late (MLI) and late-last (LLI) instar larvae.
These larval instars were distinguished based on their body weight and
the size of the head capsule (Lakshmi and Dutta-Gupta, 1990).
2.2. Full-length cDNA cloning of RbHex 82 using rapid amplification of
cDNA ends (RACE) strategy
Total RNA was isolated from the fat body using Trizol reagent
(Sigma, USA). Fivemicrograms of total RNAwas used for cDNA prepara-
tion using Superscript™ III first strand synthesis system (Life Technolo-
gies, USA). Degenerate primers were designed based on the alignment
of known lepidopteran hexamerin sequences (which were neither
arylphorins nor methionine-rich) from GenBank, (AF032397.1,
EF646282.1, AY661710.1, M57443.1, L21997.1, EU366905.1). A partial
clone of 800 bp corresponding to RbHex 82 was obtained. The partial
fragment obtained was further confirmed using gene specific primers.
For obtaining the full-length cDNAof RbHex 82, 5′ and 3′RACE reactions
were carried out using RACE kit (Clontech Laboratories Inc., USA) ac-
cording to the manufacturer's protocol. The 5′ RACE was carried out
with gene specific reverse primer (GSP) and universal primer A mix
(UPM), while 3′ RACEwas performedwith gene specific forward primer
and universal primerAmix. All the PCR conditionswere programmedas
specified in the manufacturer's protocol. The amplified products were
cloned into p-GEM-T easy vector (Promega, USA) and sequenced. All
the primers used in the study are listed in Supplementary Table 1.
2.3. Quantitative RT-PCR to study tissue specificity, developmental expres-
sion and hormonal regulation of RbHex 82
The purity and quantity of RNAwere assessed using Nanodrop spec-
trophotometer (ND-1000). Threemicrograms of total RNAwas convert-
ed to cDNA using Superscript III™ first strand synthesis kit. All the RNA
samples were treated with DNase I prior to cDNA synthesis to eliminate
any possible genomic DNA contamination. Real-time PCR was per-
formed on an ABI Prism® 7500 fast thermal cycler (Applied Biosystems,
USA). Each sample was run in triplicate in a final volume of 20 μl con-
taining 0.3 μl of cDNA (1:10 dilution), 10 pmol of each primer and
10 μl of Power SYBR® Green PCR master mix (Applied Biosystems,
USA). PCR conditions were optimized to generate N95% PCR efficiency.
Dissociation curve analysis was performed after the last cycle to confirm
amplification of a single product. Quantitative RT-PCR results were
expressed as change in expression relative to control using target
gene. Ct values were normalized to that of internal control gene Ct
values (mention internal control gene) based on the 2 (−ΔΔC(T)) method
(Livak and Schmittgen, 2001). All the quantitative RT-PCR primers used
are listed in Supplementary Table. 1.
2.4. Homology modeling of RbHex 82
The 3D model of the RbHex 82 was built by homology modeling
based on high-resolution crystal structure of homologous proteins.The crystal structure of the closest homolog of C. cephalonica RbHex
82 available in the Brookhaven Protein Data Bank was searched by de-
termining the sequence similarity aided by a BLAST (NCBI) search. The
results pointed to the crystal structure of Antheraea pernyi arylphorin
(APA) with a resolution of 2.43 Å as a suitable template (PDB ID:
3GWJ). The identity score was 29% and E value was 2e − 79. Hence,
the coordinates of crystal structure of the APA were used as a template
to build themodel bymultiple sequence alignment usingClustalW soft-
ware. The 3D model of C. cephalonica RbHex 82 was generated by the
homology modeling tool Modeller (Marti-Renom et al., 2000). The
steepest descent energy minimization using the Gromos96 43a1 force
field was performed to regularize the protein structure geometry.
2.5. Molecular docking studies
Automated docking analysis was carried by using Autodock 4.0
(Morris et al., 2009). The three-dimensional structure of riboflavin
was built and its geometry was optimized through Discovery Studio
3.1 software package (accelrys). To recognize the binding sites in
RbHex 82, blind docking was carried out essentially, the grid size set
to 126, 126 and 126 along X-, Y-, and Z-axis with 0.525 Å grid spacing.
The docking parameters used were GA population size: 150; maximum
number of energy evolutions: 250,000. During docking, a maximum
number of 10 conformers were considered, and the rms (root-mean-
square) cluster tolerancewas set to 2.0 Å. One of the lowest energy con-
formations was used for further analysis.
2.6. Sequence and phylogenetic analysis
Homology search of sequences was carried out by BLAST to get the
putative ortholog sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
ClustalW was used for multiple alignment of the RbHex 82 sequence
with the other similar lepidopteran hexamerins to show the residual
similarity (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Expasy (http://
www.expasy.org/) tools were used for in silico characterization of de-
duced amino acid sequence, such as signal peptide prediction, putative
N- and O-glycosylation sites, putative phosphorylation sites detection,
amino acid composition, theoretical pI andmolecularweight determina-
tion. Phylogenetic analysis and tree rendering were done using PhyML
and TreeDyn available at Phylogeny.fr.
2.7. Purification of hexamerins
The hexamerins were purified from the hemolymph of 4th and 5th
instar larvae of C. cephalonica. The hemocyte-free diluted hemolymph
(1 mg protein/50 μl) was passed through a Sephadex G-100 column
(1.5 × 60 cm) equilibrated with 10 mM Tris–HCl (pH 7.4) at room tem-
perature. The protein was eluted with the same buffer at a flow rate of
1 ml/2 min till the absorbance of the elutants at 280 nm reached a
value of 0.002. Fractions containing protein were checked by SDS-
PAGE. The fractions that contained hexamerinswere pooled and loaded
on to ion exchange DEAE Sephacel column (1.25 × 25 cm) pre-
equilibrated with 10 mM Tris–HCl (pH 7.4). The bound hexamerins
were eluted with a linear gradient of 0–0.5 M NaCl. The peak fractions
were pooled and analyzed on 7.5% resolving SDS-PAGE for purity and
probed with hexamerin antibody for confirmation (Bradford, 1976;
Laemmli, 1970; Towbin et al., 1979).
2.8. Spectrofluorimetric analysis to determine the release of bound
riboflavin
Equal concentrations of hexamerin fraction (in 50 mM Tris–Cl,
pH 7.4) from 4th and 5th instar larvae (4th instar hexamerin fraction
contains two hexamerins [except RbHex82] and 5th instar hexamerin
fraction contains all the three hexamerins [includes RbHex 82]) were
subjected to heat treatment at 85 °C for 5min that resulted in unfolding
60 V.V. Rao et al. / Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64and precipitation of the proteins simultaneously thus releasing the
bound riboflavin into buffer. This released riboflavin in the supernatant
was separated from the precipitate by centrifugation at 10,000 rpm for
10min. The supernatant obtained was used to detect riboflavin by fluo-
rescence spectroscopy (excitation maxima of 450 nm and emission
maxima of 525 nm).2.9. Thorax ligation and hormone treatments
Thorax-ligation of last instar larva depleted the endogenous hor-
mone levels (Dutta-Gupta and Ashok, 1998). In brief, the larvae were
narcotized for 10 min on ice. Ligation was done behind the first pair of
prolegs by slipping a loop of silk thread around the head of the larvae.
The loop was adjusted behind the first pair of prolegs and gradually
tightened. The region anterior to ligature was cut with sterile scissors
andwoundwas dressedwith traces of antibioticmixture (1:1, penicillin
and streptomycin) and phenylthiourea (0.025%). Finally, it was sealed
with wax (paraffin and beeswax, 10:0.1). These ligatures (isolated ab-
domen) were kept in petri dishes covered with moist filter paper to
maintain humidity and prevent desiccation. Stock solutions of 20HE
and JH analog, methoprene (hereafter referred as ‘JH’ in the text) were
prepared by dissolving 1mg of hormone ormethoprene in 100 μl of eth-
anol and acetone respectively, whichwere diluted to 1ml with distilled
water. The last instar larvae were administered with either 5 pg/μl of
20HEor JH or both. JHwas topically appliedwhereas 20HEwas injected.
Control insects received equal volume of carrier solvents. The insect
tissue was collected after the required time points (1, 3, 6, 12 and
24 h) of hormone treatment.3. Results
3.1. Analysis of full-length RbHex 82 isolated from fat body
The total mRNA length of C. cephalonica RbHex 82 was found to be
2.5 kb with an ORF of 2121 bases that code for a polypeptide of 706
amino acids with a theoretical molecular mass of 77.6 kDa. However,
on SDS-PAGE, the molecular mass of the protein corresponds to
82 kDa, which could be due to the post-translational modifications as
evident by the putative N- and O-glycosylation and phosphorylation
sites in the sequence obtained. The 5′ UTR region consists of 128 bp,
while 3′ UTR region consists of 256 bp with a polyadenylation signal.
The theoretical pI of RbHex 82 is 6.04. The amino acid composition re-
veals low percentage of both, aromatic amino acids (phenylalanine
and tyrosine, 10.4%) and methionine (1.7%). The deduced amino acid
sequence also contains the characteristic hemocyanin N (33–156),
hemocyanin M (260–439) and hemocyanin C (445–693) domains.
Further, the putative 16-amino acid signal peptide in the N-terminus
could be essential for secretion into hemolymph (Supplementary
Fig. 1). Sequence homology of RbHex 82 shows substantial amino acid
sequence identity (71.25%) with Galleria mellonella hexamerin (neither
arylphorin nor methionine-rich) followed by Helicoverpa armigera
(52.59%), Spodoptera exigua (49.79%), H. cecropia (46.97%) and
Trichoplusia ni (42.96%) hexmerins. Further, the putative riboflavin
binding amino acid residues are also conserved among the compared
species (Fig. 1). The nucleotide sequence was submitted to GenBank
under accession number KF984196.1. Phylogenetic analysis further in-
dicates that both C. cephalonica and G. mellonella hexamerins fall
under the same clade which is likely due to the close evolutionary rela-
tion, as both of them belong to same family, Pyralidae (Supplementary
Fig. 2).Fig. 1. Multiple alignment of deduced amino acid sequence of C. cephalonica RbHex 82 with
GenBank. (Accession numbers: AF032397.1 (H. cecropia), EF646282.1 (S. exigua), AY66
(C. cephalonica). Predicted riboflavin-binding amino acids in the alignment was denoted in red3.2. Tissue-specific and developmental expression of RbHex 82
It was observed that the transcript for RbHex 82was predominant in
the fat body. The expression was negligible in the other tissues exam-
ined (i.e. gut, brain, Malpighian tubules, salivary glands and hemocytes)
(Fig. 2a). Further, the quantitative RT-PCR analysis demonstrates specif-
ic expression of RbHex 82 during thefinal instar (5th instar). The expres-
sion gradually increased from ELI to LLI larval stage during fifth instar.
Thereafter, the expression declined during the wandering prepupal
and pupal stages. It is noteworthy that RbHex 82 was not expressed in
the embryo, early larval (1st, 2nd, 3rd and 4th) instars and adult stage
of development (Fig. 2b).
3.3. Homology modeling and molecular docking analysis
The 3D structure of C. cephalonica RbHex 82was comparedwith that
of APA to find out the structural identity. The overall fold of the two
hexamerin structures was found to be conserved. The amino acid se-
quence alignment revealed 29% identity between the C. cephalonica
RbHex 82 and APA. The Ramachandran plot for RbHex 82 showed that
approximately 97.6% of all amino acids residues were within the gener-
ously allowed region and 1.4% of residueswere in the disallowed region
(Fig. 3a). Sequence identity scores and Ramachandran plot statistics for
the generated RbHex 82 model clearly indicate its identity with amino
acid sequence of the template, APA, used in the present analysis. The
binding constant and free energy change revealed riboflavin as a poten-
tial substrate due to the tight-fit in to the active site of hexamerin. The
riboflavinmoleculewas locatedwithin the binding pocket, and adjacent
to hydrophobic residues Asp 176, Ile 634, and Leu 659,which contribute
to the formation of hydrogen bonds between the Hex-riboflavin com-
plexes (Fig. 3b). Further, the binding affinity of riboflavin with the
other two types of hexamerins characterized from C. cephalonica
(arylphorin andmethionine-rich)was found to beweak (Supplementa-
ry Fig. 3) indicating that riboflavin binds specially to RbHex 82.
3.4. Release of bound riboflavin from RbHex 82
To further substantiate the structural studies, stage-specificity of the
RbHex 82 in C. cephalonicawas exploited to demonstrate the riboflavin
release. Fig. 4a shows the fluorescence spectrum of riboflavin from
hexamerin protein fractions obtained from 4th and 5th larval stages.
The green line of spectrum corresponds to pure riboflavin in the buffer.
Hexamerin fraction from the 5th instar larval stage (+RbHex 82) indi-
cated by blue line shows the release of the bound riboflavin. However,
hexamerin fraction from 4th instar larvae (−RbHex 82) did not show
any release of riboflavin (represented in yellow). This supports the
specific riboflavin binding property of RbHex 82. Further, the ribofla-
vin release was directly proportional to the increase in RbHex 82
concentration (Fig. 4b). The different colors in the spectra indicate
different concentrations of hexamerin fraction from the 5th instar
larval stage compared to control.
3.5. Effect of 20E and JH on RbHex 82 expression
20HE injected insect groups showed significant upregulation of
RbHex 82 expression. The increase in expression was gradual and
time-dependent. However, towards the end, at 24 h time point RbHex
82 expression declined which might be due to the short circulatory
half-life of 20HE (Fig. 5a). On the contrary, JH treatment did not show
any effect on the RbHex 82 expression (Fig. 5b). However, upon simulta-
neous treatment with both 20HE and JH, hexamerin expression in-
creased in a time dependent manner up to 12 h (Fig. 5c).the other known hexamerins (non-arylphorin and non-methionine-rich) present in the
1710.1 (H. armigera), M57443.1 (T. ni), L21997.1 (G. mellonella) and AHL24706.1
boxes.
V.V. Rao et al. / Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64 61
62 V.V. Rao et al. / Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64
Fig. 2. a) Tissue specific expression of RbHex 82 in C. cephalonica b)Developmental expression pattern of RbHex 82 in C. cephalonica during 0 day, 4 day, 1st instar, 2nd instar, 3rd instar, 4th
instar, early-last instar, mid-last instar, late-last instar larvae, pre-pupa, pupa and adult stages. Ribosomal protein, rS7 was used as internal control. The results were obtained from three
independent experiments (n = 3).4. Discussion
Earlier from our laboratory, an arylphorin (Nagamanju et al., 2003)
and a methionine-rich hexamerin (Damara and Dutta-Gupta, 2010a)
were cloned and characterized from C. cephalonica. In the present
study, we cloned a hexamerin, which is neither aromatic amino acid-
rich (Telfer et al., 1983; Scheller et al., 1990) nor methionine-rich
(Tojo et al., 1980; Telfer and Kunkel, 1991; Chandrasekhar et al., 2007)
from the fat body,whichhas amolecularweight of 82 kDa and is smaller
than the reported sizes of Hex 84 and Hex 86. Phylogenetic analysis
clearly shows that it belongs to a separate class. Further, C. cephalonica
RbHex 82 showed 46.97% sequence identity with Hylophora cercopia ri-
boflavin binding hexamerin protein (Magee et al., 1994). Hence, we fur-
ther characterized this putative ‘riboflavin-binding hexamerin (RbHex
82)’.
During C. cephalonica larval development, the metabolically active
fat body (Arrese and Soulages, 2010) predominantly expresses
hexamerins, including RbHex 82 as observed in the present study. How-
ever, the stage-specific expression of C. cephalonica hexamerins varies.Fig. 3. a) Ramachandran plot of RbHex82 depicting 97.6% of amino acid residues within the gen
helical and yellow are beta-pleated sheets, molecular docking of RbHex 82 with riboflavin molExpression of Hex 84 was observed from 2nd larval instar, while Hex
86 transcripts were detected from 4th larval instar. However, RbHex
82 was predominantly expressed during the last larval instar. Studies
in different insect families, Pyralidae (Memmel et al., 1994), Noctuidae
(Miller and Silhacek, 1992) and Saturniidae (Magee et al., 1994), report-
ed the presence of flavin-binding hexamerins during late larval and
pupal stages of development. The differential ontogenic profiles of
hexamerins probably have a functional significance. Hex 84 is known
to be involved in cuticle deposition (KiranKumar et al., 1997), immune
response (Arif et al., 2003) and xenobiotic binding (Budatha et al.,
2007; Ningshen et al., 2013). Owing to its diverse functions, Hex 84 ex-
pression could be essential from the early larval stages.Methionine-rich
hexamerins are mostly female-specific and essential for egg develop-
ment in lepidopterans (Tojo et al., 1980; Telfer and Kunkel, 1991;
Chandrasekhar et al., 2007). Hence, Hex 86 gradually accumulates in
C. cephalonica from fourth larval instar to probably support and facilitate
reproductive development and functions (Ismail and Dutta-Gupta,
1991). Expression of RbHex 82 during the last instar larval development
might be associated with some specialized function, which needs to beerously allowed region, b) 3D structure of RbHex82. Region represented in red are alpha-
ecule, clearly reveals presence of an active binding site.
V.V. Rao et al. / Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64 63
Fig. 4. a) Riboflavin release from 4th and 5th instar hexamerin fractions (blue: 5th instar; yellow: 4th instar) determined by emission wavelength b) concentration-dependent release of
riboflavin from hexamerin fraction (only 5th instar). The inset picture is the graphical representation showing the proportional increase in the fluorescence intensity of riboflavin release.
Black line in the spectrum represents blank.ascertained. Being a putative riboflavin binding protein, it may bind to
small molecules such as riboflavin or its derivatives that are crucial for
regulating important physiological functions during larval–pupal–
adult transition. Bioinformatics studies predicted the highest binding
constant (7.45 × 103/M) for RbHex 82. On the contrary, Hex 84 and Hex
86 exhibited weak binding affinities with Rb (2.13 × 103/M &
3.52 × 103/M respectively). Further, spectrofluorimetric analysis demon-
strated the release of bound riboflavin from RbHex 82 upon heating. To-
gether, these studies confirm the identity of Hex 82 as RbHex 82.
We also showed the effect of morphogenetic hormones, 20HE and
JH, on the RbHex 82 expression. 20HE had a stimulatory effect on
RbHex 82 as observed in the case of other two hexamerin types,
arylphorin, Hex 84 (Damara et al., 2010b) and methionine-rich
hexamerin, Hex 86 (data not shown), in this species. On the contrary,
JH alone did not show any effect on the RbHex 82 expression. However,Fig. 5. a)Effect of 20HE onRbHex 82 expression.b)Effect of JH analog,methoprene onRbHex 82m
as internal control. X-axis: time points in hours after hormone treatment; Y-axis: relativemRNA
denotes significance (P b 0.05).JH in the presence of 20HE induced the expression yet again indicat-
ing its role as a ‘status quo’ factor, well demonstrated in earlier stud-
ies (Riddiford, 1996) as well as in our insect model (Chaitanya et al.,
2011).
As of now, the role of RbHex 82 is obscure and remains to be inves-
tigated. But as a putative carrier of riboflavin molecule, which serves as
cofactor for flavin mononucleotide (FMN) and flavin adenine dinucleo-
tide (FAD) involved in several mitochondrial oxidation–reduction en-
zymes like pyruvate dehydrogenase complex (PDH) and succinate
dehydrogenase (SDH) participating in energymetabolism, further stud-
ies ascertaining its role could be important. Also, with RbHex 82 being
lepidopteran-specific (Burmester, 2015) and the ability of hexamerins
to bind to insecticides known (Haunerland and Bowers, 1986), it is a
practically feasible idea to design and test riboflavin-like small insecti-
cide molecules in the laboratory.RNA levels. c) Effect of 20HEandmethoprene on RbHex 82 expression. 18S rRNAwasused
expression levels. The results are obtained from three independent experiments (n=3). *
64 V.V. Rao et al. / Comparative Biochemistry and Physiology, Part B 194–195 (2016) 58–64Acknowledgements
Central equipment facilities of school of life sciences generated with
DBT (CREBB), DST (FIST) and UGC (UPE) were used for the present
study. Venkat Rao V acknowledges Department of Biotechnology
(DBT), India for awarding junior and senior research fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.cbpb.2016.01.008.
References
Arif, A., Scheller, K., Dutta-Gupta, A., 2003. Tyrosine kinase mediated phosphorylation of
the hexamerin receptor in the rice moth Corcyra cephalonica by ecdysteroids. Insect
Biochem. Mol. Biol. 33, 921–928.
Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Annu.
Rev. Entomol. 55, 207–225.
Beresford, P.J., Basinski, J.M., Chiu, J.K., Chadwick, J.S., Aston, W.P., 1997. Characterization
of hemolytic and cytotoxic gallysins: a relationship with arylphorins. Dev. Comp.
Immunol. 21, 253–266.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem.
72, 248–254.
Braun, R.P., Wyatt, G.R., 1996. Sequence of the hexameric juvenile hormone-binding pro-
tein from the hemolymph of Locusta migratoria. J. Biol. Chem. 271, 31756–31762.
Budatha, M., Meur, G., Kirti, P.B., Dutta Gupta, A., 2007. Characterization of Bacillus
thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body
of the moth Spodoptera litura. Biotechnol. Lett. 29, 1651–1657.
Burmester, T., 1999. Evolution and function of the insect hexamerins. Euro. J. entomol. 96,
213–225.
Burmester, T., 2002. Origin and evolution of arthropod hemocyanins and related proteins.
J. Comp. Physiol. B. 172, 95–107.
Burmester, T., 2015. Expression and evolution of hexamerins from the tobacco horn-
worm,Manduca sexta, and other Lepidoptera. Insect Biochem. Mol. Biol. 62, 226–234.
Burmester, T., Scheller, K., 1999. Ligands and receptors: common theme in insect storage
protein transport. Naturwissenschaften 86, 468–474.
Chaitanya, R.K., Sridevi, P., Senthilkumaran, B., Dutta-Gupta, A., 2011. 20-
Hydroxyecdysone regulation of H-fibroin gene in the stored grain pest Corcyra
cephalonica during the last instar larval development. Steroids 76, 125–134.
Chandrasekar, R., Suganthi, L.M., Krishnan, M., 2007. Intra-ovarian synthesis and tissue
distribution of hexameric storage protein-1 in the ovary of red hairy Caterpillar
Amsactaal bistriga. J.Asia-Pacific Entomol. 10, 1–10.
Damara, M., Dutta-Gupta, A., 2010a. Identification of 86 kDa protein as methionine rich
hexamerin in the ricemoth, Corcyra cephalonica. Comp. Biochem. Physiol. B, Biochem.
Mol. Biol. 157, 229–237.
Damara, M., Gullipalli, D., Dutta-Gupta, A., 2010b. Ecdysteroid-mediated expression of
hexamerin (arylphorin) in the rice moth, Corcyra cephalonica. J. Insect Physiol. 56,
1224–1231.
Decker, H., Terwilliger, N., 2000. Cops and robbers: putative evolution of copper oxygen-
binding proteins. J. Exp. Biol. 203, 1777–1782.
Dutta-Gupta, A., Ashok, M., 1998. A comparative study on the ecdysteroid titre in the nor-
mal, decapitated and thorax-ligated larvae of stem borer, Chilo partellus and rice
moth, Corcyra cephalonica. Entomon 23, 245–250.
Enderle, U., Kauser, G., Renn, L., Scheller, K., Koolman, J., 1983. Ecdysteroids in hemolymph
of blowfly are bound to calliphorin. In: Scheller, K. (Ed.), The Larval Serum Proteins of
Insects: Function, Biosynthesis, Genetic. Thieme, Stuttgart, pp. 40–49 New York.
Hakim, R.S., Blackburn, M.B., Corti, P., Gelman, D.B., Goodman, C., Elsen, K., Loeb, M.J.,
Lynn, D., Soin, T., Smagghe, G., 2007. Growth and mitogenic effects of arylphorin
in vivo and in vitro. Arch. Insect Biochem. Physiol. 64, 63–73.
Haunerland, N.H., Bowers, W.S., 1986. Binding of insecticides to lipophorin and
arylphorin, two hemolymph proteins of Heliothis zea. Arch. Insect Biochem. Physiol.
3, 87–96.
Ismail, P.M., Dutta-Gupta, A., 1991. In vitro uptake of larval haemolymph proteins by male
accessory reproductive glands of the stem borer, Chilo partellus. Inv. Rep. Dev. 20,
193–199.
KiranKumar, N., Ismail, S.M., Dutta-Gupta, A., 1997. Uptake of storage protein in the rice
moth Corcyra cephalonica: identification of storage protein binding proteins in the
fat body cell membranes. Insect Biochem. Mol. Biol. 27, 671–679.Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of
bacteriophage T4. Nature 227, 680–685.
Lakshmi, M., Dutta-Gupta, A., 1990. Juvenile hormone mediated DNA synthesis during
larval development of Corcyra cephalonica (insecta). Biochem. Int. 22, 269–278.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-
time quantitative PCR and the 2(−Delta Delta C (T)) method. Methods 25, 402–408.
Ma, G., Roberts, H., Sarjan, M., Featherstone, N., Lahnstein, J., Akhurst, R., Schmidt, O., 2005.
Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagula-
tion reaction in the gut lumen of resistant Helicoverpa armigera larvae? Insect
Biochem. Mol. Biol. 35, 729–739.
Magee, J., Kraynack, N., Massey, H.C., Telfer, W.H., 1994. Properties and significance of a
riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia. Arch. Insect
Biochem. Physiol. 25, 137–157.
Martins, J.R., Anhezini, L., Dallacqua, R.P., Simoes, Z.L., Bitondi, M.M., 2011. A honey bee
hexamerin, HEX 70a, is likely to play an intranuclear role in developing and mature
ovarioles and testioles. PLoS One 6, e29006.
Marti-Renom, M.A., Stuart, A., Fiser, A., Sanchez, R., Melo, F., Sali, A., 2000. Comparative
protein structure modeling of genes and genomes. Ann. Rev. Biophys. Biomol. Struct.
29, 291–325.
Memmel, N.A., Trewitt, P.M., Grzelak, K., Rajaratnam, V.S., Kumaran, A.K., 1994. Nucleotide
sequence, structure and developmental regulation of LHP82, a juvenile hormone sup-
pressible hexamerin gene from the waxmoth, Galleria mellonella. Insect Biochem.
Mol. Biol. 24, 133–144.
Miller, S., Silhacek, D., 1992. Binding of riboflavin to lipophorin and a hexameric protein in
the haemolymph of HeIiothis virescens. Insect Biochem. Mol. Biol. 22, 571.
Miura, K., Nakagawa, M., Chinzei, Y., Shinoda, T., Nagao, E., Numata, H., 1994. Structural
and functional studies on biliverdin associated cyanoproteins from the bean bug
Riptortus clavatus. Zool. Sci. 11, 537–545.
Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., Olson, A.J.,
2009. AutoDock4 and AutoDockTools4: automated docking with selective receptor
flexibility. J. Comput. Chem. 30, 2785–2791.
Munn, E.A., Greville, G.D., 1969. The soluble proteins of developing calliphora
erythrocephala, particularly calliphorin, and similar proteins in order insects.
J. Insect Physiol. 15, 1935–1950.
Munn, E.A., Feinstein, A., Greville, G.D., 1967. A major protein constituent of pupal of the
blowfly, Calliphora erythrocephala. Biochem. J. 102, 5–6.
Nagamanju, P., Hansen, I.A., Burmester, T., Meyer, S.R., Scheller, K., Dutta-Gupta, A., 2003.
Complete sequence, expression and evolution of twomembers of the hexamerin pro-
tein family during the larval development of the rice moth, Corcyra cephalonica. In-
sect Biochem. Mol. Biol. 33, 73–80.
Ningshen, T.J., Chaitanya, R.K., Hari, P.P., Vimala Devi, P.S., Dutta-Gupta, A., 2013. Charac-
terization and regulation of Bacillus thuringiensis Cry toxin binding aminopeptidases
N (APNs) from non-gut visceral tissues, Malpighian tubule and salivary gland: com-
parison with midgut-specific APN in the moth Achaea janata. Comp. Biochem. Phys-
iol. B Biochem. Mol. Biol. 166, 194–202.
Phipps, D.J., Chadwick, J.S., Aston, W.P., 1994. Gallysin-1, an antibacterial protein isolated
from hemolymph of Galleria mellonella. Dev. Comp. Immunol. 18, 13–23.
Poopathi, S., Thirugnanasambantham, K.,Mani, C.,Mary, K.A.,Mary, B.A., Balagangadharan, K.,
2014. Hexamerin, a novel protein associated with Bacillus sphaericus resistance in Culex
quinquefasciatus. Appl. Biochem. Biotechnol. 172, 2299–2307.
Riddiford, L.M., 1996. Juvenile hormone: the status of its “status quo” action. Arch Insect.
Biochem. Physiol. 32, 271–286.
Scheller, K., Fischer, B., Schenkel, H., 1990. Molecular properties, functions and develop-
mentally regulated biosynthesis of arylphorin in Calliphora vicina. In: Hagedorn,
H.H. (Ed.), Molecular Insect Science. Plenum Press, New York, pp. 155–162.
Telfer, W.H., Kunkel, J.G., 1991. The function and evolution of insect storage hexamers.
Annu. Rev. Entomol. 36, 205–228.
Telfer, W., Keim, P., Law, J., 1983. Arylphorin, a new protein from Hyalaphora cecropia:
comparisons with calliphorin and manducin. Insect Biochem. 13, 601–613.
Tojo, S., Nagata, M., Kobayashi, M., 1980. Storage proteins of the silk worm, Bombyx mori.
Insect Biochem. 10, 289–303.
Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from poly-
acrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl.
Acad. Sci. U. S. A. 76, 4350–4354.
Wang, Z., Haunerland, N.H., 1991. Ultrastructural study of storage protein granules in fat
body of the corn earworm, Heliothis zea. J. Insect Physiol. 37, 353–363.
Zhou, X., Oi, F.M., Scharf, M.E., 2006. Social exploitation of hexamerin: RNAi reveals a
major caste-regulatory factor in termites. Proc. Natl. Acad. Sci. U. S. A. 103,
4499–4504.