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Short
Views on Insect Molecular Biology, Vol. (1), 2009
Short Views on Insect Molecular Biology

Review Article
Review Article

Vol. (1)., 00 Vol.

00, 2009
(1), 49 68, 2009

Chapter 3

Hexamerin Storage Proteins: Biosynthesis, Utilization and Evolution

1

Chandrasekar R1*., Muthu Meenaskhi P2., and Luiz Paulo AM3

Division of Applied Life Science, Geyongsang National University, Jinju, South Korea.
2
Department of Eco-Biotechnology, Bharathidasan University, India.
3
Departamento de Gentica e Biologia Evolutiva, Instituto de Biociencias,
Universidade de So Paulo Cidade Universitria 05508-900, Sao Paulo, Brazil,
Abstract

The transformation from a lepidopteran caterpillar to a non-feeding pupa and adult moth involves
complete remodeling and restructuring of the insect and its organs. The synthesis of hexameric storage
proteins by the fat body, secretion into the larval hemolymph and reuptake by the fat body shortly before
pupation seemes to be a common process in the life cycle of all holometabolous insects. During
pupation, proteins are initially stored in protein granules and later proteolytically broken down to supply
amino acid resources necessary for the completion of adult development and also for reproduction.
Hexamerins are evolutionary related with arthropod hemocyanins and prophenoloxisases.
Key words: Hexameric storage protein; hemocyanin; peripheral fat body; perivisceral fat body;
endocytosis; storage protein receptor; metamorphosis
*For Correspondence (email: koreanchandru@yahoo.com )

Overview
1. Introduction
2. Organization of the fat body
3. Regional and functional differentiation of
fat body tissue
4. Developmental differences
5. Types of Storage Proteins (SP)
a) Arylphorin
b) Methionine-rich storage proteins
c) Juvenile hormone-suppressible proteins
d) Tyrosine-rich proteins
e) Riboflavin-binding proteins
6. Biosynthesis, secretion and storage of
storage proteins (SP)
7. Role of storage proteins
______________________________________

a) Nutrient storage
b) Binding of hormones and other small
organic compounds
c) Involvement of hexamerins in cuticle
formation
d) Possible role of hexamerins in humoral
immune defense
e) Other possible role of storage proteins
8. Hexamerins uptake and Receptors
9. Gene regulation
10. Evolution
11. Conclusion
12. Acknowledgements
13. References

*Present Address: College of Agriculture Entomology,

University of Kentucky, S-225 Agricultural Science Center
Building North, Lexington, KY, USA.

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1. Introduction
A common means of these reservoir amino acids in insects is Storage Proteins (Wheeler et al.,
2000). These larval storage proteins have a molecular masses of about 500 kDa and are hexamers
composed of six subunits of approximately 72 ~ 80 kDa which may or may not be identical. They are
called hexamerins. Hexameric storage proteins appear to undergo special adaptation to insect molting,
metamorphosis, and cyclic reproduction and have no known analogue in vertebrates. The transformation
from larva to non-feeding pupa and adult involves a complete remodeling and restructuring of the insect.
The insect fat body is a complex multifunctional tissue which participates in multiple biochemical and
physiological functions and is a major site for synthesis and storage of carbohydrates, lipids, proteins and
nitrogenous components (Keeley, 1985; Haunerland et al., 1990). In most cases, the descriptions of
regional and temporal variations in the fat body have not been clearly illustrated expect for some
dipterans and lepidopterans. Fat body heterogeneity in structure and function has been shown in
Hyalophora cecropia (Tojo et al., 1978), G. mellonella (Bean and Silhacek, 1989), H. zea (Wang and
Haunerland, 1992), C. vicina (Hansen et al., 2002), B. mori (Vanishree et al., 2005), and Amsacta
albistriga (Chandrasekar et al., 2008). In lepidopterans hexamerins are synthesized abundantly during
the larval active feeding period in the peripheral fat body (PF) and subsequently released into the
haemolymph. During the larval-pupal transformation, these proteins are sequestered by the perivisceral
fat body (PVF) and stored in crystalline form until they are utilized during pupal and adult development
(Haunerland and Shirk, 1995; Chandrasekar et al., 2008). Hexamerins are important for insect
development and, therefore, numerous studies concerning their structure, biosynthesis, regulation and
evolution have been conducted during recent year (Haunerland, 1996; Burmester 1999, 2001;
Chandrasekar et al., 2008).
Although the synthetic, secretary and storage ability of fat body tissues insects are well
documented, the concept of heterogeneity in lepidopterans remains ambiguous as structural changes
takes place rapidly during development stages (Haunerland et al., 1990; Wang and Haunerland, 1992).
Storage proteins are a powerful model system to study genes that are proximally controlled by hormones
and distally regulated by photoperiod, gender, and nutrition (Telang et al., 2002). Because of their
physiological significance and patterns of developmental control, insect hexamerins have become
attractive for the study of gene regulation. With regard to their function, development, and evolution,
storage proteins are a complex family whose analysis promises to be very highly useful for the study of
many basic problems in insect biology.
2. Organization of the fat body
Generally, fat body tissue is organized in thick lobes of
highly tracheated tissue that is suspended in the haemolymph.
The tissue was described as leaf like bladder (Hydropsyche
sp., Buys, 1924), lace-like (Rhodnius prolixus, Wigglesworth,
1967), flat sheets or folded lobes (Calliphora erythrocephala,
Thomsen and Thomsen, 1974) and ribbon like lobes (G.
mellonella, Miller and Silhacek, 1982). In this form, the fat
body has ready to access nutrients, proteins, and hormones that
are transported the haemolymph. The fat tissue close to the
epidermis is called the sub-cuticular or peripheral fat body
(PF), while the central region that surrounds the gut is known
as the perivisceral (Fig. 1) or visceral fat body (PVF). Fat
body is normally, composed mainly of adipocytes or
trophocytes (Keeley, 1985). Fat body color varies different
insect species; in most cases, the fat body is a white tissue, but
it be yellow, tan brown, green, or blue. Color differences
within the individual insects have been reported in P.
interpunctella (Shirk and Malone, 1989) and Helicoverpa zea
(Haunerland et al., 1990).
50

Cu

PF
PVF
Gut

PVF

Fig.1. Cross sections of paraffinembedded A. albistriga stained with

HE/mercuric bromophenal blue, as
showing the location of the PF and
PVF tissues. Cu- cuticle; G- gut.

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3. Regional and functional differentiation of fat body tissue

Structural and functional differences between regions of the fat body have been described in
Diptera (Rizki, 1978, Hansen, 2002). The regionally differentiated fat body tissues were consequently
shown to be distinct in their ultra structure, biochemistry and gene expression pattern. In most cases,
however, protein expression has only been studied in the entire fat body, but not in the separate areas of
the tissue. Although some proteins are undoubtedly expressed in all regions, many genes may be
expressed exclusively or in specific parts of the fat body. Lauverijat (1977) observed that the cells of
peripheral and perivisceral regions of Locusta migratoria, show different responses to ovariectomy and
allatoectomy. Schin et al. (1977) observed that the sub-epidermal fat body is green while perivisceral fat
body is creamy white in Chironomus thummi. Regional differences in the color of the fat body were
noticed in H. zea. In this species, while most of the abdominal fat body become blue during pre-pupal
stage, (Haunerland and Bowers, 1986) during larval and pupal life it is creamy white in the peripherial
region. In this case, difference in color was shown to differential uptake of a protein that contained a blue
chromophore (Haunerland et al., 1990). In the study, Haunerland and colleagues observed that
perivisceral tissue become visible as it expan rapidly, turning brightly blue different from the peripheral
fat body that remain colorless and attached to the integument. Similar results were reported by Tojo and
Yoshiga (1994); Yoshiga et al.(1998) and Meenakshi, (2005) in Spodoptera litura, where the color of the
periviceral body is blue due to the biliverdin in the hemolymph. PVF tissue is principally involved in
pinocytosis of haemolymph proteins, including injected ferritin. The Indian meal moth, Poldia
interpunctella, has functionally and morphologically differentiated tan and white fat bodies were
observed (Shirk and Malone, 1989). De Loof and Lagasse (1970) reported that PF in Leptinotarsa
decemlineata, contains more glycogen than the PVF tissue, and therefore, may serve as a storage tissue.
Since the storage protein arylphorin is stored only in the perivisceral fat body, the author speculated that
a specialized perivisceral storage organ also occurred in other insect species that lack a colored storage
protein marker like the H. zea chromoprotein. During development the structure of the fat body changes
drastically, although just few studies investigate how differences during development changes fat body
functions.
4. Developmental differences
In many insects that undergo complete metamorphosis, the fat body dissociates into single cells
before it reaggregates into the adult form or the tissue is completely histolyzed and the new fat body
differentiates from stem cells. As the main biosynthetic and storage tissue of insects, the fat body is
crucial in all stages of insects life. During metamorphosis of holometabolous insects, virtually all organs
and tissues change and adult-specific proteins are expressed, while production of larval and pupalspecific proteins is terminated. These changes in the fat body are usually immediately visible upon
observation of the gross anatomy: larval tissue is frequently arranged in sheets, whereas the adult fat
body has nodular clusters. The ultra structural data support that the biosynthetic function of PF in the
larvae: mitochondria, rough endoplasmic reticulum (RER) and plasma membrane reticular systems
(PMRS) are abundant in PF of actively feeding larva (day1 5) of last instar. After SP synthesis has
ceased and the proteins have been released into the haemolymph, PF gradually disappears. Autophagic
vacuoles dominate its structure at the end of the last larval instar, the cell membrane and other (RER,
mitochondria) organelles are completely hydrolyzed during spinning stage (Wang and Haunerland, 1992;
Muller et al., 2004). At this time, peripheral fat body tissue consists in fragments of cell remainders.
Thus, PF fat body therefore seems to be a larval specific tissue, which is not needed in the adult stage.
Fat body remodeling has been extensively examined in C. ethlius (Dean et al., 1985) and it has been
confirmed in the other species of Lepidoptera such as Phylosamia Cynthia ricini (Walker, 1966); A.
kuhniella (Colln, 1973); G. mellonella (Dutkowski, 1974); H. cecropia (Tojo et al., 1978); B. mori (Tojo
et al., 1980); P. rapae (Kim et al., 1989b) and H. zea (Wang and Haunerland, 1992). Differences
between the PF and PVF are also obvious in the cellular ultra structure (Dean et al., 1985), particularly
in Lepidoptera and Diptera species. The appearance of electron-dense structures are noted only in the
PFV during the pre-pupal period. On the other hand, in day2 pre-pupal stage, the perivisceral fat body
has cytoplasm filled with numerous protein granules, many of which were crystalline or partly
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crystalline, and mitochondrial and RER content increases during the larval-pupal transformation as well.
In the mid pupal and late pupal stage (day 6-8) crystalline protein granules a hydrolyzed and the amino
acid pool is utilized for adult structure formation and reproduction. Two fundamentally different
mechanisms have been suggested to explain these changes: (a) the complete destruction of larval fat
body and simultaneous synthesis of adult fat body from undifferentiated stem cells, and, (b) a process
called cells remodeling in which larval fat body tissue is dissociated to isolate cell that re-associate to
form the adult fat body (Larsen, 1976; Haunerland and Shirk, 1995; Lorenz and Anand, 2004).
5. Types of storage Proteins (SP)
The life cycle of insects is divided into distinct stages that are variously specialized for feeding,
dispersal and reproduction. Lepidoptera is a good example of that. Essential nutrients obtained in one
stage but needed in another must be sequestered and carried across stages until they are mobilized.
Similarly, if one specialized stage does not feed or restricts its diet; its activities must be supported by
nutrient intake during the stage that does feed. A common means of reserving amino acids in insects is
storage proteins (Wheeler et al., 2000).
Pupal

Adult cuticle

Silk gland
SP2

SP2

SP
SP1 & SP2

Peripheral
fat body

Haemolymph

Biosynthesis

Accumulation

SP

SP

Adult fat body

Pervisceral
fat body
tissues
Storage

SP1 & SP2

Haemolymph

SP1

Ovary

Release
SP1

Larval cuticule

SP1

SP1

Eggs

Reproductive
gland

Reproductive
accessory gland

Fig. 2. Schematic diagram showing the hexameric storage protein (SP) bio-synthesis, and its distribution in
Amsacta albistriga.

The term storage proteins implies uptake of SP from the haemolymph and their storage in fat
body tissue, which serves to storage the pool of amino acids resources for metamorphosis (Fig.2). In
holometabolous insects, storage proteins represent a major protein component of the larval haemolymph
(Levenbook, 1985; Roberts, 1987; Pan and Telfer, 2001; Hahn and Wheeler, 2003; Chandrasekar et al.,
2008). These proteins have been identified in a wide range of insects and consist of high molecular
weight hexamers composed of homologous or heterologous subunits with an average molecular weight
of 80kDa (Tysell and Butterworth, 1978; Kanost et al., 1990; Telfer and Kunkel, 1991; Burmester et al.,
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1998; Burmester, 1999). SP are synthesized in large quantities by the larval fat body tissues during the
active feeding period and released into the haemolymph. Nevertheless, high hemolymph concentration is
found only during the last larval instar (Chrysanthis et al., 1981) and storage protein uptake occurs only
during a brief time period shortly before and after pupation, and this uptake is an endocytic process
(Ueno and Natori, 1987; Pan and Telfer, 1992; Haunerland et al., 1996; Burmester and Scheller, 1997a).
According to their biochemical properties, hexamerin subunits can be categorized in one of the
three major classes: 1) the arylphorins (74 and 76kDa) or proteins rich in aromatic amino acids (Fujii,
1989; Willott et al., 1989); 2) the female-specific methionine-rich proteins (SP 1, 82kDa) which contain
more than 4 % methionine (Ryan et al., 1985; Sakurai et al., 1988; Seo et al., 1998) typical in certain
Lepidoptera; and 3) specific hexamerins binding small organic compounds, such as riboflavin or juvenile
hormone (Telfer and Massy, 1987; Jones et al.,1990; Haunerland, 1996).
a) Arylphorin
In general, the arylphorin contains 1to 3% methionine and 16 -21% aromatic amino acids and
utilized for insect metamorphosis. Arylphorin has been positively identified in crystalline protein
granules in the fat body and it appears that these granules are gradually, but not completely, broken
down during the pupal stage (Levenbook and Bauer, 1985); in fact, many protein granules have been
detected in adult fat body and it has been suggested that hexamerin mainly serves as an amino acid
source for yolk protein production (Wang and Haunerland 1991).
b) Methionine-rich storage proteins
Methionine-rich proteins contain more than 4% methionine. In contrast to arylphorin, SP 1
proteins are not glycosylated and found in several, but not all, lepidopteran species investigated. In some
species, such as M. sexta and H. cecropia, two or more isoforms of methionine-rich proteins have been
found (Tojo et al., 1978; Wang and Hanuerland, 1992). Storage protein synthesis was reported in early
larval instar in M. Sexta, B. mori and G. mellonella (Riddiford and Hice, 1985; Ray et al., 1987; Fujii et
al., 1989). It is interesting to note, however, that in contrast to all other classes of storage proteins and
hexamerins, methoinine-rich proteins (82kDa) at least in M. sexta, are much more abundant in female
than in male larvae (Ryan et al., 1985). Subburathinam and Krishnan (1998); Chandrasekar et al. (2007)
reported that supplementation of hydrolyzed soy protein (p-soyatose) increased the accumulation of
storage protein and yolk proteins in the haemolymph of Bombyx mori. Recently we observed that SP1 of
A. albistriga is actively taken up as protein granules by the developing oocytes and serves as a yolk
protein during egg formation. It has long been suggested that the female-specific SP1 of B. mori supply
amino acids for the formation of the egg yolk protein precursor vitellogenin. (Engelmann, 2000; Ogawa
and Tojo, 1981). The SP1 of H. cunea has a high methionine content (6.0%) and low aromatic amino
acid content (8.5%) (Cheon et al., 1998). The relatively high methionine content of the B. mori SP1 may
be metabolized to cystine for chorion formation (Inokuchi, 1972; Zhu et al., 1986; Cheon et al., 2001
Chandrasekar, 2006). Cystathionine and lanthionine, which are metabolic intermediates from methionine
to cystine, remain low in males but increase prominently in silkworm females (Sumioka and Yoshitake,
1974). Both metabolic intermediates are finally incorporated into the egg shell protein (Inokuchi, 1972;
Shinbo, 1978), the major sulfur amino acid of which is cysteine (Kawasaki et al., 1971; Inokuchi, 1972;
Sumioka and Yoshitake, 1974).
c) Juvenile hormone-suppressible proteins
Expression of storage proteins is mostely confined to the last instar, a period where juvenile
hormone titers are low, and its has long been suspected proteins. Indeed, JH has been shown to suppress
the expression of some, but not all storage proteins. In addition to the above mentioned basic juvenile
hormone suppressible proteins, an acidic hexamerin has been characterized in T. ni (Jones et al., 1990)

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and G. mellonella (LSP-82, Memmel et al., 1994). Further studies needed for fat and exact function of
these storage proteins.
d) Tyrosine-rich proteins
Tyrosaturins and related tyrosine-rich storage proteins are proteins with up to 27% tyrosine found
in coleopteran fat body (Delobel et al., 1992). Their synthesis and accumulation in fat body occurs
shortly before pupation, and these proteins are deposited in protein granules of pupal fat body. Later in
development these granules are partially broken down, suggesting that the tyrosine residues of
tyrostaurins are used for the biosynthesis of cuticular structures of the pharate adult. These poorly
soluble proteins appear to be deposited into protein granules immediately after being synthesized. So, far
no structural or sequence data are available for these interesting proteins.
e) Riboflavin-binding proteins
This proteins are glycosylated
and appear to contain a high
concentration of histidine and arginine.
These proteins are expressed strongly
only in the last larval stage, they are
not actively sequestered by fat body
before pupation and do not accumulate
in pupal fat body. Their hemolymph
concentration
diminishes
rapidly
during the pupal-adult eclosin, but it is
not known where these proteins are
simply hydrolyzed or taken up by fat
body, ovaries or any other tissue. To
date, no sequence information is
available for these proteins.
6. Biosynthesis, secretion and storage
of storage proteins (SP)
In many insect species, fat body
was shown to be the principle site for
biosynthesis of proteins during larval
stage. In the active feeding period, the
weight of the fat body tissues is
directly proportional to weight of the
larva. The synthetic activity of the fat
Fig. 3. Electron micrograph of shows a few hours before
body reflects the cyclical nature of
ecdysis to the pupa of Amsacta albistriga, the protein has
growth and development. A few hours
begun to crystallize (0.5 m - 2m). (A) MVB leads to
after ecdysis the cells are small, with
formation of storage protein crystalline (4m), (B), (C) & (D)
little cytoplasm, in which the
Storage protein crystalline (smaller & larger), (E) Ellipsoid
mitochondrias are conspicuous, while
shape crystalline protein granule. (From: Raman
little RER and GCs are small with only
Chandrasekar, 2006).
a few transition vesicles and an
incomplete stack of saccules lacking
secretary vesicles (SV). During the first 2 days after ecdysis the cells become basophilic with many free
ribosome, an increasing amount of RER and larger GCs with saccules. Also lipid droplets arises and
glycogen appear. It was further shown that this preparatory phase was characterized by RNA synthesis.
Tysell and Butterworth (1978) showed in the larvae of D. melanogaster that RNA synthesis, RER
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proliferation and mitochondrial division increased prior to heightened storage protein synthesis and
secretion. Time of peak synthesis of protein in the fat body coincided with steeply rising heamolymph
protein concentrations (Hahn and Wheeler, 2003). Furthermore, it was demonstrated that larval fat body
mRNA directed the synthesis of storage proteins in Lucilia sercata (Thomson et al., 1976); Calliphorin
sp. (Sekeris and Scheller 1977); D. melanogaster (Roberts et al., 1977); H. cecropia (Tojo et al., 1978);
G. mellonella (Ray, 1987) and C. vicina (Pau et al., 1979). It has been suggested that the subepidermal
(peripheral) part of the fat body is the site of the most active protein synthesis and there is no part in
sequestration of protein granules as they are programmed to undergo cell death during larval-pupal
transformation (Keeley, 1985; Haunderland et al., 1990; Wang and Haunerland, 1992; Muller et al.,
2004), while the main function of PVF in larvae and pre-pupae is storage of nutrients needed for later
development. To evaluate this proposal, localization and fate of the endocytosed storage protein and
storage protein granules in A. albistriga were examined by electron microscope, which indicated that the
storage proteins were crystallized in different forms as protein granules in PVF (Fig. 3). The reliability
of the identification of the storage protein granules was confirmed by immunogold labeling
(Chandrasekar et al., 2008a,b).
In addition to storage proteins, B. mori and M. sexta have a group of structurally related proteins
with a molecular weight of 30 kDa, known as micro vitellogenin, which accounts for 30% of total egg
protein, and are not stage or sex specific (Chen and Yamashita, 1990). The fat body of both sexes
initiates the synthesis of these proteins on day 2 of the 5th instar larvae. Like vitellogenin, micro vitellin
is also synthesized by the fat body, secreted into the haemolymph, and then taken up by the maturing
eggs (Kawooya et al., 1986; Sato and Yamashita, 1991). Micro vitellin is a single polypeptide, which
contains no lipids, carbohydrates or phosphate and plays a role in embryogenesis (Kawooya et al., 1986).
It was presumed that 30 kDa proteins contributed to storage function of haemolymph (Izumi et al., 1980,
1981; Tojo et al., 1981, Zhong 1999). Yolk proteins are degraded and used as raw materials for new
organ formation and as a source of energy during embryo development. Yolk proteins are synthesized by
fat body and released into hemolymph and sequestered by the growing ovary where Vg is transformed
into Vtn (Indrasith et al., 1987; Sappington and Raikhel, 1995; Chen et al., 2004).
7. Role of storage proteins
Storage proteins are the source of amino acids and energy for protein synthesis during
metamorphosis and reproduction (Ogawa and Tojo, 1981; Levenbook and Bauer, 1984; Roberts, 1987;
Chandrasekar et al., 2008a,b). They contribute to an array of functions for sustaining the life of insects
(Fig.4).
a) Nutrient storage
In insects, storage hexamers are secreted into larval hemolymph, where they can attain
extraordinary concentrations. The correlation between the appearance of fat body granules and the
disappearance of storage proteins from the hemolymph has been demonstrated repeatedly (Dean et al.,
1985; Burmester and Scheller, 1995; Wheeler et al., 2000; Chandrasekar et al., 2007, 2008a,b). In the
mid-pupal and late pupal stages (days 68), crystalline protein granules were hydrolysed and utilized as
an amino acid pool for adult structure formation (Kinnear and Thomson, 1975) and reproduction (Telfer
and Pan, 2003). In an earlier study over 99% of injected M-MtH was cleared from the hemolymph of
pharate pupae, compared with only 35% of ArH (Pan and Telfer, 1992). A consequence of this in both
Luna and Cecropia pupae is that the MtHs are the most prominent proteins of fat body extracts while
ArH is the most prominent protein of hemolymph (Pan and Telfer, 1996, and Tojo et al., 1978). There
can also be differences in the disposition of proteins within the fat body, for in Cecropia the hexamerin
storage granules contain protein crystals embedded in an amorphous material (Tojo et al., 1978).
Differences in accessibility provide a plausible speculation on how V- and M- MtH, but not ArH, might
survive metamorphosis in sufficient amounts to support post-eclosion egg formation. A specific storage
function of hexamerins is also the most likely explanation for caste-specific accumulation in colony
founding and adult development of the ants (Martinez and Wheeler, 1994).
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b) Binding of hormones and Other small

organic compounds
The expression of storage proteins is
mostly confined to the last larval instar, a
period where JH titers are low. In fact JH
has been shown to suppress the expression
of some, but not all, SPs. In addition to the
above-mentioned basic JH suppressible
proteins, an acidic hexamerin has been
characterized in Trichoplusia ni (Jones et
al., 1990).
Some hexamerins bind to small
metabolites like biliverdin (Miller and
Silhacek, 1992) or riboflavin (Magee et al.,
1994) with high affinity. It has been
suggested that these hexamerins serve as
specific transporters for these molecules, but
the exact physiological role of these
interactions is uncertain. Biliverdin was also
found associated with Vg, but its function
remains unclear (Maruta et al., 2002). For
the cockroach, L. maderae and P.
Americana, two major JH-binding proteins
have been identified: lipophorin (Lp) and
vitellogenin (Vg) (Englemann and Mala,
2000). There is a general non-specific
affinity of this protein to small organic
compounds, or some role of hexamerins in
the detoxification of xenobiotics.

Fig.4 Evolution of hexamerin function. Known functions

of the hexamerins and hemocyanins are compiled and
arranged according to the phylogeny of these proteins
(From Burmester, 1999).

c) Involvement of hexamerins in cuticle formation

Tyrostaurins and related tyrosine-rich storage proteins. The tanning of the insect cuticle involves
the incorporation of proteins into a complicated matrix and the subsequent cross-linking of these
proteins by various diphenols (Anderson, 1979). In the Diptera, the epidermis does not synthesize
hexamerins; therefore, their presence can be attributed to specific incorporation (Schenkel and
Scheller, 1986). Arylphorins play a role in the sclerotizing system during cuticle formation (Scheller et
al., 1980).
d) Possible role of hexamerins in humoral immune defense
The protection of the insect from infections and parasites is essential for the survival of the
animal. In recent years, several lines of evidence show that the arylphorins of the lepidoptera may be
specifically involved in immune protection and may act as cytotoxic effectors, which are specifically
induced by bacterial infections (Beresford et al., 1997).
e) Other possible role of storage proteins

56

The methionine-rich storage proteins provide amino acids for the developing adults
especially for chorion proteins and vitellogenin (Ogawa and Tojo, 1981; Chandrasekar
et al., 2008).

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The arlyphorins bind and serve as carrier proteins for ecdysteroids (Reum et al., 1982).
The presence of lipids and aromatic amino acids led to the conception that hydrophobic
pockets may function in the binding and transport of ligands.

8. Hexamerins uptake and Receptors

The passage of macromolecules through biological membranes is an essential process for all
multicellular organisms. Insects have developed a mechanism different from that known for all other
eukaryotes investigated so far. Insect pupae do not feed during metamorphosis. Therefore, they depend
on materials that have been accumulated during the larval life. At the end of larval period, shortly
before pupariation, a rise in the ecysteriod hormones induces the incorporation of large fraction of a
storage protein (hexamerins) from the body fluid into the fat body cells. Hexamerin uptake has been
shown to be receptor-mediated endocytosis (Fig. 5).

Peripheral fat body

Perivisceral fat body

Fig.5. Schematic diagram shows the receptor-mediated endocytotic uptake of storage proteins and
their utilization. (From: Haunerland, 1996)
A- protein granules; VHDL- very heavy density lipophroin; R receptor; PG Protein granules,
MVB- multivesicular bodies, BM- basal membrane

The presence of storage protein granules in larval tissue has been already observed by Bishop
(1922) in Apis mellifera, and later in D. melanogaster (Butterworth, 1965). Specific receptors were
postulated to explain the stage-specific uptake of hemolymph proteins by the insect larval perivisceral
fat body. It was also speculated that 20-hydroxyecdysone could stimulate the binding of storage
proteins to their receptors. It was subsequently demonstrated (Ueno et al., 1983) that the storage
protein receptor (SPR) is a protein of molecular range 125kDa, which is converted to active molecules
of 120kDa under the influence of 20HE (Burmester and Scheller, 1995, 1997b, 1999). The specificity
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and efficiency of very high-density lipoprotein (VHDL; one of the storage proteins) uptake by
perivisceral fat body (PVF) of H. zea suggested a distinct endocytotic pathway involving a VHDL
receptor (Wang and Haunerland 1993; Burmester and Scheller 1999; Persaud and Haunerland 2004).
Such pathways have been demonstrated so far in the Lepidoptera (Wang and Haunerland, 1994a;
Kirankumar et al., 1997) and Diptera (Ueno et al., 1983; Burmester and Scheller 1995; Hansen et al.,
2003; Chandrasekar et al., 2008b) (Fig.6). Surprisingly, storage protein receptor also belongs to the
members of the hexamerin superfamily (Burmester and Scheller, 1999; 2001).

Fig. 6. Electron Micrograph of formation of coated pits in PVF, which shows the sequestration of storage
proteins via the receptor-mediate endocytotic pathway (From: Chandrasekar et al., 2008a).

9. Gene regulation
Several authors well documented the haemolymph proteins and their involvement in insect
development and reproduction. Storage proteins allow the study of genes that are proximally controlled
by hormones and distally regulated by photoperiod, gender, and nutrition. The classes of hexameric
storage proteins include JH suppressible, methionine rich, and arylphorin. The mechanisms that control
expression of developmentally regulated genes in holometabolous insects have been the subject of
research for many years, mostly because in these systems metamorphosis clearly separates the sets of
larval and adult specifically-expressed genes. The exact molecular mechanism behind the stage specific
expression of storage proteins in insects attracted a great deal of attention and the first investigation in
this direction was made by Kim et al. (1989b) in S. peregrina.
In B. mori and M. sexta, the upstream region of a sex specific storage protein (SP 1) gene has
regions of sequence similar to an EcRE, and the first intron has sequence similar to a viral (SV40)
enhancer (Sakurai et al., 1988). The upstream region of an arylphorin storage protein (SP 2) gene also
has a sequence similar to the SV40 enhancer and another sequence associated with fat body-specific
expression in D. melanogaster (Fujii et al., 1989). A larva-specific B.mori storage protein (BmLSP)
gene has a TGATAAA heptamer (Fujiwara and Yamashita, 1992) that is typically found in the upstream
regions of the storage protein genes (Willott et al., 1989). The late larval mRNA developmental profiles
from a female-specific storage protein gene and a gender-undifferentiated storage protein gene were
determined in M. sexta (Riddiford and Hice, 1985). Allatectomy, used in conjunction with
administration of JH analog, demonstrated that female-specific storage protein (SP 1) gene expression
was specifically suppressed by JH (Webb and Riddiford, 1988). The D. melanogaster and L. migratoria
yolk protein genes are expressed in the fat body and ovarian follicle cells and are under control of
regulatory sequence responsive to tissue-specific factors, 20HE, and nutritional-mediated signals
(Bownes, 1994). Therefore, different sensitivity of storage protein genes to JH during insect
development, suggested the stage and tissue-specific and hormonal regulation on the expression of
storage protein in insects.
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The nucleotide sequences at the 5-end of the translation initiation site of TATA contain typical
promoter elements and putative regulatory sequences (Cherbas and Cherbas 1993; Kutach and
Kadonaga, 2000). Promoters of several genes expressed specifically in the fat bodies of insects,
including those encoding the D. melanoagastor and Ades atropalpus hexamerins, contain binding sites
for the GATA factors (Abel et al., 1993; Benes et al., 1996; Zakharkin et al., 2001; Attardo et al., 2003).
The exon/intron composition of SP 2 gene is remarkably similar to that of SP 1 gene. They observed
that the deuced primary structures of SP 1 and SP 2 exhibit nearly 30% homology, implying that two SP
genes of B. mori silkworm might have evolved from a common gene (Fig.7).
10. Evolution
The origin and the evolutionary
Spodoptera litura
relationships of this taxon is one of the
(0.16220)
most debated issues in evolutionary
H.cercopia
(0.33333)
biology (Fortey and Thomas, 1997;
G.mellonella
Corpuz et al., 1991; Jones et al., 1993).
(0.36310)
Hyphantria cunea
Recently, in Musca domestica,the
(0.15923)
Manduca sexta
predicated secondary structure of the
(0.24219)
polypeptides encoded by SP 1 (MdHex
Panulirus interruptus Hc
L1) and SP 2 (MdHe F2) revealed that
(2.2439)
Amsacta albistriga
the positions of several-helixes and
(0.00)
sheets. Also, three domains orginally
Partial sequences
described for proteins belonging to
Bombyx mori
hemocyanin superfamily are highly
(0.11240)
conserved in the house fly hexamerins
(Moreira et al., 2004). Hemocyanins
and hexamerins are the members of the
D.Melanosgaster
Aedes agypti
same superfamily and it has been
(0.45536)
(0.41235)
estimated that hemocyanins from
primitive crustaceans diverged more
than 360 million years ago giving rise
Fig..7 Radial tree of phylogenic analysis from Bombyx mori
to the insect hexamerins (Naumann and
(SP) and other species. Lengths of branches along the axis are
Scheller, 1991; Beintema et al., 1994;
proportional to evolutionary distances calculated from the
Burmester
and
Scheller,
1996;
pair-wise amino acid identity matrix.
Burmester, 1999, 2001). The entire
hemocyanin gene family-hemocyanin,
cryptocyanin, proph-enoloxidase and hexamerins-may participate to varying degrees in these two vital
functions of molting animals (oxygen binding and molting). Structural similarities between hemocyanins
and hexamerins also were observed for C. vicina LSP1 (Markl et al., 1992), D. melanogastor LSP1 and
LSP2 (Massey et al., 1997; Mousseron-Grall et al., 1997) and Aedes aegypti and Anopheles gambiae
LSP1 and LSP2 (Gordadze et al., 1999). Although hexamerins and hemocyanins are structurally similar,
the organization of the genes that encode these proteins are different (Markl et al., 1992). For example,
genes that encode arachnidan hemocyanins have eight introns that correspond to 96% of the total gene
nucleotides, while insect hexamerins have fewer and smaller introns. The genes that encode hexamerins
of the Lepdioptera B. mori and M. sexta contain four introns each and these correspond to 60% of the
total number of base pairs (Burmester and Scheller, 1996; Burmester, 2001, 2002). This hypothesis is
supported by the fact that the position of aromatic amino acid residues are not more conserved in
arylphorins than in the order hexamerins; in fact, when all insect hexamerins are aligned with the
Clustral W multiple aligment program, phenylalanine and tyrosine residues are common in many
positions in all hexamerins (Table 1). Much of this sequence conservation is located in regions that
have been shown in hemocyanin to form contacts between subunits and to lie near the beginnings
and ends of alpha-helical segments. In all three of the sequenced storage hexamers, only one of
the six copper- binding histidines is conserved. The divergence of the hemocyanin from
59

Short Views on Insect Molecular Biology, Vol. (1), 2009

Vertabrates

Craniata
75,000 species
Scorpion, mites, spider
(Arachnids)

Amphioxus

Chelicerata
Tunicata

Myriapoda

13,000 species
Centipedes, millipeds

ta

nonrespiratory serum proteins that have

very similar size and composition may
have occurred as early as 600 my BP, a
currently agreed upon minimal age of the
arthropods. Nonrespiratory serum proteins
are known from several crustaceans and
arachnids, and the insect hexamerins may
be related more directly to these than to
the hemocyanins.

Review Article

da

Hexapoda

6,000 species
Echinoderms

Ch

or

950,000 species
Insects

Crustacea
40,000 species Cray fish,
losters, crabs, shrimp

dy

od

n
oa

70,000 species
Mollusks

op

soz

Annelids
12,000 species
Earthworm, leeches
Platyhelminthes

Nematodes

t hr

Ec

Ar

A leading phylogenetics (Fig. 8)

hypothesis indicates a close relationship
between insects and crustaceans (Hwang et
al., 2001; Giribet et al., 2001) suggesting
that fundamental changes in the mechanisms
of respiration must have accompanied the
diversification of insects from crustaceans
and their invasion of terrestrial and aerial
environments. During evolution, hexamerins
diversified according to divergence of the
insect orders, within the orders, there is a
notable structural diversification of these
proteins, which probably reflects specific
functions.

Ascaris, hookworms

20,000 species
Liver fluks, tapeworm
Coelenterata
10,000 species
Hydrozoa, Scyphozoa and Antozoa

Diploblastic

Sponge
10,000 species

Fig.8 Schematic diagram shows the evolutionary tree of

animal kingdom.

11. Conclusion
In the recent years, studies have focused on the biochemistry and physiology of lepidopteran
storage proteins, including the most intriguing problem of why the fat body first secretes storage proteins
into the hemolymph before sequestering them, apparently unchanged. It has been proposed that a
functional shift of the fat body takes place at the end of the last larval stage, from biosynthetic organ to
storage organ. The uptake of hexamerins is an important process that allows holometabolous insects to
survive during the non-feeding pupal period. In addition, the dynamics between sequestration and release
of amino acids from storage proteins in the pupa/adult stage may be important feature enabling many of
their diverse strategies of reproduction. This preliminary approach plays a pivotal role on insect
development and egg production and offers a promising tool to curtail the synthesis of storage protein or
preventing them from to reach the respective tissues. This novel strategy can apply to arrest the insect
pests physiology and development, there by controlling the pest population in a natural way with out
using toxic pesticides, which could be economical and eco-friendly.

60

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Table. 1 Sequence information of insect hexamerin storage proteins.

Species

Accession
Number

Protein Name

Reference

Aedes aegypti (Yellowfever

mosquito)
Anopheles gambiae (African
malaria mosquito)
Aedes aegypti

Hexamerin 1 gamma
(Fragment)
Hexamerin Precursor

P90663

Caccone et al., 1998

Q17020

Zakharkin, 1997

Hexamerin 2

P90664

Gordadze et al., 1999

Aedes aegypti

Hexamerin 1

U86079

Gordazde et al., 1999

Anopheles arabiensis

Hexamerin A (Fragment)

O76210

Caccone et al., 1998

Aedes aegypti
(Yellow fever mosquito)
Anopheles arabiensis

Arylphorin-like hexamerin-1

Q5D0X2

Scharf M.E., 2005

Hexamerin A

AF020873

Caccone et al., 1998

Anopheles merus

Hexamerin A

AF020875

Apis mellifera
(Honey bee)
Apis mellifera

Riboflavin binding
hexamerin
Hexamerin 2

Q86G31

Schaefer, 2001

Aprona germari

Hexamerin SP

AY29387

Kim et al., 2004

Amsacta albistriga (groundnut

pest)

Storage Protein-1

Chandrasekar et al., 2008

Bracon hebetor

Hexamerin

N-terminal amino
acid sequence
SwissProt #
P84632
125974

Bombxy mori

Sex-specific SP1

P09179

Sakurai et al., 1988

Bombyx mori

Sex-specific SP2

P20613

Fuji et al., 1989

Blaberus discoidalis (Tropical

cockroach)
Camponotus fetinatus

Hexamerin

U31328

Jamroz et al., 1996

Hexaermin 2

Martinez & Wheeler .1994

Calliphora vicina
(Blue blowfly)
Calliphora vicina

LSP-1/Arylphorin

P28513

Naumann & Scheller, 1991

LSP-1/Arylphorin (partial)

X63340-X63344

Fischer & Scheller, 1991

Corcyra cephalonica
(Rice moth)
Corcyra cephalonica

Juvenile hormone binding

protein
Allergen Cr-PI

Q9GQ56

Braun and Wyatt, 1996

Q9U5Y8

Kort and Koopmanschap 1995

Camponotus festinatus

Cyanoprotein protein

Q94607

Miura, 1998

Choristoneura fumiferana

SP-1

AAC35428

Palli et al., 1998

Choristoneura fumiferana

SP-2

AAC35429

Palli et al., 1998

Drosophila melanogaster (fruit

fly)
Drosophila melanogaster

LSP-1 (partial)

X03872

Delaney et al., 1986

LSP-1

U63556

Massey et al., 1997

Drosophila melanogaster

LSP-1 (partial)

AF016033

Bauer & Aquadro, 1997

Drosophila melanogaster

LSP- 2

X97770

Mousseron-Grall et al. 1997

Drosophila melanogaster

Fat body protein 1

Q04691

Maschat et al., 1990

Eurypelma californicum

Hemocyanin a

P02242

Voit and Feldmater-Fuchs

1990
Sehartau et al., 1983

Caccone et al., 1998

Burmester, 1998

Quistad & Leisy, 1996

Eurypelma californicum

Hemocyanin d

P02241

Eurypelma californicum

Hemocyanin c

S06701

Galleria mellonella
(Wax moth)
Galleria mellonella

Larval Hemolymph Protein

82
Arylphorin

L21997

Voit and Feldmater-Fuchs

1990
Memmel et al., 1994

M73793

Memmel et al., 1992

Hyalophora cercopia

Riboflavin-binding
hexamerin

AF032397

Massey, 1995

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Hyalophora cercopia

Review Article
AF032398

Hyalophora cercopia

Moderately Met-rcih
hexamerin
Very Met-rcih hexamerin

AF032399

Massey, 1995

Hyalophora cercopia

Arylphorin

AAB86647

Burmester et al., 1998

Hyphanteria cunea

SP-1

U60988

Mi et al., 1998

Hyphanteria cunea

SP-2

U60988

Mi et al., 1998

Helicoverpa armigera (cotton

bollworm)
Locusta migratoria

Arylphorin-like hexamerin

Q86G31

Kim, 2003

Cyanoprotein beta subunit

Q25461

Miura, 1998

Leptinotarsa decemelineata

LSP-1

Q24995

Koopmanschap et al., 1995

Manduca sexta

Arylphorin

P14296

Willot et al., 1989

Manduca sexta

Arylphorin

P14297

Willot et al., 1989

Manduca sexta

L07609

Wang et al., 1993

Manduca sexta

Methionine-rich storage
protein
Prophenoloxidase

L42556

Hall et al., 1995

Musca domestrica

LSP-1

U72651

de Capurro et al., 1997

Periplaneta americana

Hexamerin

AAB09629

Wu et al., 1996

Perla marginata (Stone fly)

Hexamerin 4

A4Q991

Hagner-Holler 1007

Perla marginata

Hexamerin 70c

A4Q004

Martin 2007

Plutella xylostella (Diamon black

moth)
Panulirus interruptus

Hexamerin 2

Q56DL4

Ashfaq et al. , 2005

Hemocyanin a

Po4254

Bak and Benitema 1987

Panulirus interruptus

Hemocyanin b

P10787

Jekel et al., 1988

Plodia interpunctella (Indianmeal

moth)
Plodia interpunctella (Indianmeal
moth)
Ochlerotatus atropalpus (rockpool mosquito)
Reticulitermes flavipes (Eastern
subterranean termite)
Reticulitermes flavipes

LSP-1

AF356842

Zhu et al., 2002

LSP-2

AF356843

Zhu et al., 2002

Hexamerin

AF430247

Jinwal et al., 2006

Hexamerin SP-1

EG325041

Zhou et al., 2006

Hexamerin SP-2

EG327184

Zhou et al., 2006

Riptortus clavatus (Bean bug)

Cyanoprotein

D87272

Miura et al., 1996

Riptortus clavatus

Cyanoprotein

D87273

Miura et al., 1996

Scarcophaga peregrina

LSP-1/Arylphorin

A24941

Matsumoto et al., 1986

Sesamia nonagrioides

DQ147770

Spyliotopoulos et al., 2008

Spodoptera litura

Methionine-rich storage
protein (SP2)
SP-1

CAB55603

Zheng et al., 2000

Spodoptera litura

SP-2

CAB55604

Zheng et al., 2000

Trichoplusia ni
(cabbage looper)
Trichoplusia ni

Acidic JH-suppressible
protein
Basic JH-suppressible
protein
Basic JH-suppressible
protein
Hexamerin storage protein

P22327

Jones et al., 1990

Q06342

Jones et al., 1993

Q06343

Jones et al., 1993

--

Cho et al., 1999

Trichoplusia ni
Tenebrio molitr
(yellow meal worm)

SP1- storage protein 1; SP2- storage protein 2; LSP 1 Larval Serum Protein 1;
LSP 2- Larval Serum Protein 2

62

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12. Acknowledgements
The authors are grateful to Dr. Immo Hansen (Biology Dept., New Mexico State University) for
critical reading of this article. The author thanks the Department of Science and Technology, New Delhi
(MK. No. Lr. No. SP/SO/C24/99 DE 08/01/2002) for partial financial support and APMC9-South Korea,
APSERI-08 Nagoya University, Japan for providing travel grant for fruitful discussion with
Prof. Seo Seook Jae, Prof. M. Kobayashi and Prof. S. Tojo.
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