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Biological Effects of Ecdysteroids and Their Non Steroidal Agonist
Bisacylhydrazines A Review
RESHMA JOHN , RASHMI P.A, LINU MATHEW
*
School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala Pin-686560
*Corresponding author email: [email protected], mob: +919447505690
Abstract
Ecdysteroids are polar steroid hormones controlling arthropod development and
reproduction. Ecdysteroids in plants are called phytoecdysteroids, their concentration in
plants are higher than that in insects. The isolation of phytoecdysteroids from plants has
initiated fruitful research leading to the discovery of new phytoecdysteroids. Ecdysteroids
possess a number of pharmacological, medicinal and agricultural applications. Ecdysteroids
agonist and antagonists are potential insect control agents and valuable tool for studying
ecdysteroid action. New agents to control insect pest species, interference with ecdysteroid
action is an attractive, and little exploited target. The purpose of the review is to summarise
the biological effects of ecdysteroids and its agonist and antagonists.
Key words: Ecdysteroids, agonist, antagonists, plant defences, bisacylhydrazines.
Introduction
Insect development is driven by the action of two hormone classes, the ecdysteroids
and the juvenile hormones (Riddiford, 1994; Gilbert et al., 2000; Thummel, 2002) and they
are primarily involved in the regulation of postembryonic development, which is
characterized by moulting and metamorphosis. Ecdysteroids are produced in the prothoracic
glands in larvae and in the reproductive organs in adults (ovaries and testis), and in the
abdominal integument of some insects (Hoffmann & Gerstenlauer 1997). In the larval stages,
20-hydroxyecdysone initiates the moulting and metamorphosis process in larvae, and
Juvenile hormone regulates these changes during the commitment period. Ecdysone, the first
ecdysteroid was isolated by Butenandt and Karlson in 1954 (Dinan, 2001), and its structure
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was identified by Huber and Hoppe (Hoppe & Huber 1965). Insects sporadically shed their
rigid external exoskeleton and then replace it to grow. This process is called ecdysisis or
molting (Gilbert, 2004). Insect molting is regulated via binding of the molting hormone (20-
hydroxyecdysone: 20E) to its receptor protein, the heterodimer of ecdysone receptor (EcR)
and ultraspiracle (USP). The 20E-EcR/USP complex binds to the ecdysone response element
in the promoter of target genes and regulates their transcription. The molting process is
initiated by an increase in the titer of 20E, the larva stops feeding and apolysis of the
epidermis from the old cuticle takes place leaving an ecdysial space that is filled with molting
fluid containing inactive chitinolytic enzymes. Meanwhile massive protein synthesis takes
place in the epidermal cells for deposition of a new cuticle (Riddiford, 1994;Dhadialla et
al.,1998). Decline in the titer of 20E leads to the activation of enzymes for the digestion of
procuticle underlying the old cuticle. After completion of this process moulting fluid is
reabsorbed and pre ecdysal tanning of the new cuticle takes place (Reynolds, 1987). Finally,
eclosion hormone and ecdysis-triggering hormone are released, when 20E titer is declined to
the basel level and these events lead to the ecdysis of the larva leaving behind the remnants of
the old cuticle (Truman et al., 1983; Dhadialla et al.,1998; Zitnanova et al., 2001). With the
completion of ecdysis, feeding resumes and endocuticular deposition continues during the
intermolt period. Every moult corresponds to the end of one growth stage and and the
beginning of another (Fig 1).
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Fig 1 The ecdysteroid titer in Drosophiladevelopment (Kozlova & Thummel ,
2000)
Phytoecdysteroids
Phytoecdysteroids are analogues of invertebrate steroid hormones (zooecdysteroids) that
occur in a wide variety of plant species (Bergamasco & Horn, 1983) (Table 1). Within a few
years of the isolation of ecdysone from insects, an array of compounds having structural
similarity to insect moulting hormones has been extracted from plants and because of plant
origin these compounds are called phytoecdysteroids. Subsequent research has revealed that
phytoecdysteroid occurrence is widespread (Dinan, 2004a) and their concentration in plants
is higher than that in insects. It appears that 56% of terrestrial plant species contain
significant levels of ecdysteroids ( Dinan, 2001). Most crop species do not contain detectable
levels of ecdysteroids, spinach (Spinaciaoleracea) and quinoa (Chenopodium quinoa) being
notable exceptions (Bthory et al.,1982; Grebenoket al.,1991;Dinan, 1995,Dinan ,2001). A
range of evidence exists in support that ecdysteroid concentrations are highest in tissues
which are most important for the survival of the plant or, in the case of annuals, of the species
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into the next generation (Dinan,2001).The concentration of phytoecdysteroids in plants are
increased by mechanical damage,insect herbivory or methyljasmonate treatment(Schmelz et
al.,1998,1999,2002). Phytoecdysteroids are apparently non toxic to mammals and have a
number of beneficial and pharmacological and medicinal applications (Dinan, 2001; Slama &
Lafont, 1995) which explain the usage of ecdysteroids containing plants in medicine. These
plant species are primarily represented byLeuzea carthamoides, Rhaponticum uniflorum, and
Serratula coronate, Cyanotis vaga, Ajuga turkestanica, Pfaffia paniculata, P.
iresinoides(Lafont & Dinan ,2003).
Table 1 Medicinal plants containing phytoecdysteroids
Plant species Family Parts containing
phytoecdysteroids
Concentration Reference
Achyranthes fauriei
Ajuga iva
Cyanotis vaga
Pfaffia iresinoides
Serratula tinctoria
Sida rhombifolia
Tinospora cordifolia
Amaranthaceae
Laminaceae
Commelinaceae
Amaranthaceae
Asteraceae
Malvaceae
Menispermaceae
Roots
Aerial parts
Leaves
Roots
Aerial parts
Aerial parts
Aerial parts
0.41.2mg/gdw
4.5 mg/g dw
7 mg/g dw
6.2 mg/g dw
1020 mg/g dw
0.82 mg/g dw
0.25 mg/g dw
Ogawa et al., 1974
Wessneret al., 1992
Santos et al., 1970
Nishimoto et al.,
1987
Rudel et al., 1992
Jadhav et al., 2007
Pathaket al., 1995
Chemical structure
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Ecdysteroid carbon skeleton is termed as cyclopentano-perhydro-phenanthrene with a
beta side chain at C 17,which is the product of terpene biosynthesis through mevalonic acid
,cholesterol and related sterols(Bathori & Pongracz, 2005). The whole side chain is kept in
the case of C27-C29 ecdysteroids. C19,C21 and C24are formed by the cleavage of the side
chain. Ecdysteroids contain cis A-B ring junction, a 7-en-6-one chromophore in the B
ring,addition of -hydroxyl group at C14 and -hydroxyl group at C3.The B-C and C-D ring
junctions are always trans. A-B ring junction is normally cis(5-H)and only rarely trans(5-
H). The methyl groups at C10 and C13 have a configuration(Adler & Grebenok
1999;Dinan, 2001;Bathori & Pongracz 2005; Reginaldo etal.,2008). The 14-hydroxy-7-en-
6-one chromophore result in a characteristic ultra violet absorption with max at 242 nm in
methanol(Dinan, 2001).Usually two to eight hydroxyl group are present on ecdysteroids. Any
carbon atom other than the C7, C15, and C18 carbons may be hydroxylated. Additional and
alternative double bonds may exist between the4-5, 8-9,9-11,12-13,14-15,24-25,25-26,24-28
carbons. A second oxo group may be located on C2, C3,C12,C17,C20,C22. Numerous
ecdysteroids exist with 5-OH,1-OH and 11-OH groups and all of these are phytoecdysteroids.
Ecdysteroids hydroxylated at C9,C12,C17,C19,C21,C24,C28 and C29 occur in plants.
Phytoecdysteroids may be found conjugated with both organic and inorganic acids,organic
alcohol and glycosides(Bathori & Pongracz, 2005). Both the phytoecdysteroid level and
structural diversity of plant ecdysteroids are surprising(Fig 2) . The number, location and the
position of the hydroxyl substituents explain the structural diversity of phytoecdysteroids.
Plants are capable of biosynthesising phytoecdysteroids from mevalonic acid. In many cases,
the biosynthesis of phytoecdysteroids has been demonstrated to proceed via cholesterol
and/or lathosterol (Adler & Grebenok, 1995). Current knowledge about the biosynthetic
pathway(s) for ecdysteroids in plants is limited.
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Fig 2 Structure of some Ecdysteroids
Role of phytoecdysteroids in plants
Phytoecdysteroids as Plant hormones
20-Hydroxyecdysone in nanomolar concentrations affected metabolic processes, including -
amylase activation, retardation of leaf senescence, having synergistic effect with auxin on
coleptile elongation, antagonizing the effects of gibberelic acid (Golovatskaya, 2004). 20HE
also altered germination, shoot growth, and root elongation in tomato (Bakrim et al., 2007).
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Exogenous phytoecdysteroids produced weak gibberilin like activity in dwarf mung bean and
rice bioassay(Drier &Tower,1988). There are examples of ecdysteroid induced growth and
proliferation, but there is very little evidence supporting to consider ecdysteroids as plant
hormone.
Phytoecdysteroids in Defence
Phytoecdysteroids may play a role in plant defense. Ecdysteroids (predominantly 20E) have
been shown to have a range of detrimental effects on the development and survival of a
number of insect species (Bombyx mori (Kubo et al., 1983),Pectinophora gossypiella (Kubo
et al., 1981, 1983), Spodoptera frugiperda (Kubo et al., 1981), Acrolepiopsis assectella
(Arnault & Slma, 1986; Harmatha, 2000), Agrius convolvulus (Tanaka & Naya, 1995),
including inhibition of growth, supernumerary larval instars, death without moulting, death
associated with promoted moulting and prothetely(Dinan et al., 2009). Detoxification of
ecdysteroids occur in certain species by conjugating it with fatty acid and blocking the C-22
hydroxyl group, which is important for the biological activity of ecdysteroids (Dinan &
Hormann, 2005; Dinan et al., 2009). Insect species like Heliothis virescens, H. armigera,
Locusta migratoria,Manducasexta, Spodoptera littoralis,Lacanobia olereaceae,Acherontia
atropos are remarkably tolerant to ecdysteroids in their diet (Dinan, 1998; Dinan et al.,
2009), while others are partially tolerent(e.g. Cynthia cardui, Tyria jacobaeae; Blackford &
Dinan, 1997b), but higher levels are toxic to them. Ecdysteroid sensitive species are deterred
from eating an ecdysteroid-containing diet that they die of hunger rather than consume
ecdysteroid-containing food (e.g.Inachis io; Blackford & Dinan, 1997b).
Extraction and isolation of phytoecdysteroids
Ecdysteroid extraction and purification is complicated due to their polar nature, poor
crystallisation properties and the purification strategy comprises of multistep procedure
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including extraction, prepurification and combination of one or more chromatographic steps.
Dried plant parts were extracted exhaustive with large volumes of methanol. Ten fold excess
of solvent usually methanol was used in this first step. The removal of both polar and apolar
contaminants can be done using fractional precipitation and solvent-solvent partition.
Fractional precipitation was done using acetone which removes the compound more polar
than ecdysteroids. Solvent- solvent partition was done using aqueous methanol and hexane,
the ecdysteroids remain in the aqueous methanolic phase, this process removes both lipohilic
and hydrophilic contaminants. Next step is solid phase extraction(SPE) which removes
certain compounds from ecdysteroids with similar polarity but difference in having their
aromatic ring. Usually polyamides is used as stationary phase in SPE ( Bathori , 2002).
Multistep chromatographic methods such as adequate combination of adsorption
chromatography, partition chromatography, size exclusion and reverse phase chromatography
are generally done for the final purification of ecdysteroid( Bathori ,1998). Ecdysteroid
content of plant samples was generally determined by the use of bioassay, radio
immunoassay (RIA), spectrophotometry, thin-layer chromatography or HPLC(Bathori et
al.,2000). HPLC is generally used in the last step of isolation of ecdysteroids for ,UV,MS
and NMR analysis. Fig 3 explain method for extraction and isolation of ecdysteroid from
Silence viridiflora( Toth & Bathori,2008). Phytoecdysteroid levels have been quantified by
an ecdysteroid-specifc radioimmunoassay (RIA) which detects compounds structurally
similar to ecdysteroids (Dinan, 1995a). Bioassay is dependent on biological activity detects
compounds having ecdysteroid-like (agonist) activity (Clemment & Dinan, 1991; Clemment
et al., 1993). Combination of these two assays provides a powerful tool for the analysis of
phytoecdysteroids.
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al., 1977). The Drosophila Kc cell line is one of the most well documented 20E-responsive
cell lines currently available and has been used in numerous studies examining the effects of
20E. The epithelial cell line from Chironomus tentans established by Wyss (1982), is
considered as a simplified model to study early events in ecdysteroid induced tissue
differentiation (Spindler-Barth et al., 1989, 1995; Spindler-Barth, 1991, 1993; Lammerding-
Ktippel et al., 1994) and cuticle formation. Chironomus cells do not synthesise moulting
hormone but metabolise added ecdysteroids very slowly. TheDrosophila imaginal disc cell
lines (Cottam &Milner1997) and Spodoptera frugiperda Sf9 (Lynn & Oberlander 1983;
Vaughn et al.,1997) cell line was also used to study and compare the effect of ecdysteroids
and bisacylhydrazenes. Dinan and co-workers (2001a) used D. melanogaster BII tumorous
blood cells to test different ecdysteroids and extracts with ecdysteroid agonistic or
antagonistic activities. This BII cell line did not metabolize ecdysone, (Dinan et al., 1985).
Changes in cell morphology and a reduction in cell density occur in the presence of
ecdysteroid agonist that can be assessed turbidometrically.
I n vitroproduction of phytoecdysteroids
Cell culture technique seems to be a good alternative for production of biologically active
ecdysteroids. The ability of several plant species to produce phytoecdysteroids in vitro was
first demonstrated in callus cultures ofAchyranthes fauriei and Trianthema portulacastrum
(Hikino et al., 1971, Ravishankar & Mehta, 1979). Phytoecdysteroids have also been isolated
from the culture filtrates of Ajuga turkestanica (Lev et al., 1990), A. reptans var.
atropurpurea (Matsumoto & Tanaka, 1991), Pteridium aquilinum (McMorris & Voeller,
1971) and Serratula tinctoria (Corio-Costet et al., 1996). Callus and suspension cultures of
A. turkestanica, synthesised 20E and turkesterone, the 20E concentration in the cells being
several times higher than that in the roots and leaves of the plants, whereas the turkesterone
concentration was somewhat lower than in the intact plant (Lev et al., 1990). Hairy root
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cultures ofAjuga reptans var. artropurpurea, Achyranthes fauriei, Pfaffia iresinoides and
Vitex strickeri (Matsumoto & Tanaka, 1991) produced phytoecdysteroids. A. reptans hairy
root clones were shown to synthesize 20E, norcyasterone, cyasterone and isocyasterone,
which are characteristic ecdysteroids of intact plant roots. The cell culture that produced
secondary metabolites are considered interesting model system for studies of biosynthesis
and physiological role of phytoecdysteroids, the available information on which is scanty.
Commercial availability of phytoecdysteroids
Today more than 140 different ecdysteroid-containing preparations are available on the
market(Fig 4). They ususally contain 20E, sometimes in combination with other ecdysteroids,
and they are proposed in particular for use by bodybuilders (Lafont & Dinan, 2003). These
preparations contain ecdysteroids isolated from just a few plant species, mainly Leuzea
carthamoides, Cyanotis arachnoidea ,Pfaffia sp.
Fig 4 Examples of commercially available preparations containing phytoecdysteroids and their Web
address.(http://www.myprotein.com/sports-nutrition/beta-ecdysterone/10529949.html
,http://www.rocksolidbodybuilding.com/Beta-Ecdysterone-supplement.html, http://astronutrition.com/sni-20-beta-hydroxy-120-
caps.html,http://www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-
Ecdysterone,www.nutrend-supplements.com)
http://www.myprotein.com/sports-nutrition/beta-ecdysterone/10529949.htmlhttp://www.myprotein.com/sports-nutrition/beta-ecdysterone/10529949.htmlhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://astronutrition.com/sni-20-beta-hydroxy-120-caps.html,http:/www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/Beta-Ecdysterone,www.nutrend-supplements.comhttp://www.myprotein.com/sports-nutrition/beta-ecdysterone/10529949.html7/28/2019 Finaly Revew for Book
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Applications of phytoecdysteroids
Phytoecdysteroids are apparently non toxic to mammals and have a number of
beneficial and pharmacological and medicinal applications (Dinan, 2001; Slama & Lafont,
1995). Phytoecdysteroids display a wide range of biological activities which explain the
usage of ecdysteroids containing plants in medicine (Table 2).
Table 2 Medicinal applications of phytoecdysteroids
Pharmacological
effects
Effect of ecdysteroids Species/
cells
Reference
Protein synthesis Stimulatory effect on protein synthesis,
ingested ecdysteroids(5mg/Kg) increased
mRNA translation efficiency
Mice Otaka et al.,1968,
Dinan & Lafont ,2006
Antidiabetic effects 20E reduced induced
hyperglycemia,stimulates glucose
incorporation into glycogen and enhance
glucose utilisation by tissues.
Antidiabetic effects are known for
ecdysteroid containing plants used in
traditional medicine.
In vitro experiments demonstrate thatecdysteroids increase glucose consumption
in an insulin independent fashion.
Rat
Mouse
HumanHepatocytes
Uchiyama & Yoshida,
1974,Yoshida et al., 1971
Kutepova et al.,2001,
Wessneret al.,1992,Chen
et al., 2006.
Dinan & Lafont ,2006
Dinan ,2009
Hypocholesterolaemic
effects
Ecdysteroids reduce cholesterol level by
reducing biosynthesis,enhancing catabolism,
and promoting its conversion into bile acid.
Rats Uchiyama & Yoshida,
1974, Dinan ,2009,
Lupien et al., 1969,
Dinan & Lafont ,2006
Wound healing Ecdysteroid containing liposome promote
wound healing, 20E stimulate keratinocyte
differentiation.
In vitro,it has psoriasis inhibiting activity
Humans Detmaret al.,1994,
Lafont &Dinan,2003
,Dinan & Lafont ,2006
Growth and cell
proliferation
Ecdysteroids have been shown to increase
growth in a wide variety of animals
20E (5mg/Kg) administered accelerates
healing process after experimental bone
fracture.
Stimulates proliferation of human umbilical
vein endothelial cells
Stimulates erythropoiesis
Mice,Rats
Sheep,Pigs
Quails
Rats
Rats
Rats
Feldman et al.,2008
Lafont &Dinan , 2003
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Gene switches
The heterodimer of nuclear receptors,ecdysteroid receptor(EcR) and ultraspiracle(USP) an
homologue of the vertebrate retinoid X receptor considered as the functional ecdysteroid
receptor. The homologue of USP in animals and humans is the retinoid X receptor (RXR) .
EcR/USP complex interacts with specific ecdysteroid responsive elements in the promoter
region of ecdysteroid responsive gene to alter gene transcription activity( Dinan & Lafont
,2006). Ecdysteroid receptor is not a natural component of vertebrate cell,ecdysteroids are
nontoxic to vertebrates and ecdysteroids do not activate vertebrate steroid hormone receptors
even at very high concentrations. Ecdysteroids are successfully developed as an effective
inducer forEcR based gene expression system and are suitable technologies for tight control
transgene expression in mammalian cells(Lafont & Dinan ,2003). EcR based gene switches
have been successfully applied in animal models (Karzenowski et al.,2005,Galimi et
al.,2005,Hoppe et al.,2000), in functional genomics(Takeda et al.,2000), disease
models(Albanese et al.,2000) and are candidate systems for switch controlled human gene
therapy(Palli et al.,2005,Toniatti etal,2004,Goverdhana et al.,2005,) and feasibility has been
demonstrated for two ecdysteroid ligands: ponasterone A and muristerone A(Lapenna&
Dinan,2009).The non steroidal ecdysteroids agonist bisacylhydrazene are considered as an
alternative ligand to generate an ecdysteroid inducible gene switch systems.
Bisacylhydrazines
Disturbances in the normal course of molting and metamorphosis under the action of
exogenous ecdysteroids or substances mimicking their biological activity usually lead to the
insect death. Ecdysteroids has limited application in controlling arthropod pest because of its
chemical and metabolic instability, they are too polar to penetrate insect cuticle and too
expensive to synthesise on large scale (Dinan, 198;Lafont & Dinan 2009).
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Bisacylhydrazines are non steroidal agonist of 20E and exhibit their insectisidal activity via
interaction with the ecdysteroid receptor proteins. Bisacylhydrazines are more metabolically
stable in vivo than ecdysteroids and because they are true ecdysone agonists at the receptor
level, ingestion of bisacylhydrazines creates hyperecdysonism in susceptible insects, thus
inducing effects and symptomology of a molt event (Dhadialla et al.,1998, 2005). The larvae
ultimately die as a result of their inability to complete a molt, starvation, and desiccation due
to hemorrhage. Four potent analogs (tebufenozide, methoxyfenozide, halofenozide and
chromofenozide) are currently on the market (Fig 5, Table 3) as safer insecticides with
reduced mammalian toxicity. These insecticides have very good ecotoxicological profile,
having virtually no impact on most non target organisms including beneficial insects and
pollinators, (bees, predators and parasitoids), birds, fish and terrestrial invertebrates
(Dhadialla et al., 2005). The BAH insecticides also have low-toxicity to mammals making
them reduced risk materials for humans handling the products.
tebufenozide
methoxyfenozide
Chromofenozide
halofenozide
Fig 5 Chemical structure of bisacylhydrazine insecticides
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Table 3 Bisacylhydrazine ant their pest spectrum.
Common name Registered
names
Industry Pest spectrum
Tebufenozide
( RH-5992)
MIMIC,
CONFIRM
,ROMDAN
Rohm and Haas
Co.,a
DowAgroSciencesLLC
b
Lepidoptera specific
Chilo suppressalis,Cnapholocrocis medinalis
Lymantria dispar, Plusia spp.,
Pseudoplusia includes, Spodoptera spp.
Trichoplusia ni
Methoxyfenozide
(RH-2485)
INTREPID
RUNNER
PRODIGYFALCON
Rohm and HaasCo.,
aDow
AgroSciencesLLC
b
Lepidoptera specific
Heliothis spp.,Helicoverpa zea
Chromofenozide
(ANS-118,CM-001)
MATRIC
KILLAT
Nippon Kayaku,
Saitane,Japan and
Sankyo,Ibaraki, Japan
Lepidoptera specific
Spodoptera littura, Spodoptera exigua
Spodoptera littoralis ,
Cnaphalocrosis medinalis,Heliothis virescens
Chilo suppressalis
Halofenozide
(RH-0345)
MACH 2 Rohm and HaasCo.,a DowAgroSciencesLLCb
Lepidoptera, Coleoptera specific
Agrotis ipsilon,Spodoptera frugiperda
Popillia japonica
Ecdysterone antagonists
Ecdysterone antagonists inhibit the effect of ecdysterone. The compound azadirachtin from
the seeds of the tree Azardirachta indica,is a feeding inhibitor and growth disrupting
compound for most insect orders. Treatment of insects with azadirachtin frequently elicits a
delay or permanent block of molting. Molting failures are due to reduced ecdysteroid titre,(
Redfern et al., 1982) , and azadirachtin inhibits ecdysteroid secretion from the prothoracic
glands ( Sieber & Rembold (1983). Azadirachtin may also affect the metabolism of
ecdysteroids, for example by hindering the conversion of ecdysone into the hormonally more
active 20-hydroxyecdysone (Smith & Mitchell, 1988). Imidazole compounds, KK-42 (1-
benzyl-5-[(E)-2,6-dimethyl-1, 5-heptadienyl] imidazole) and KS-175 (4-
phenoxyphenoxypropyl imidazole), have been reported to inhibit ecdysteroid synthesis,
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leading to disrupted development( Smagghe ,2007). Cucurbitacins B and D, isolated from
seeds ofIberis umbellata (Cruciferae) and shown to be responsible for the antagonistic
activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-
induced morphological changes in the Drosophila melanogaster BII permanent cell line
(Dinan et al.,1997). Other compounds being considered for anti-ecdysteroid effects include
the brassinosteroids, , limonoids and triterpenes (Richter & Koolman 1991; Decombel et
al.,2005; Dinan & Hormann, 2005). Antagonists of ecdysteroid action in invertebrate systems
will be useful as biochemical probes for the investigation of the control of gene expression by
ecdysteroids as well as lead compounds for the development of new insect pest control agents
(Dinan, 1995; Keckeis et al., 2000).
Conclusion
Phytoecdysteroids are structural analogues of insect molting hormone ecdysone. They exert a
number of beneficial pharmacological effects on mammals and hundreds of ecdysteroid
containing anabolic preparations are available in the markets. Some ecdysteroids may provide
promising alternative to anabolic androgenic steroids in therapy, but their mode of action is
completely unknown. So it would be worthwhile to perform detailed clinical trials to evaluate
its importance as a medicine. In vitro culture with high potential activity seems to be a good
source for the production of biologically active phytoecdysteroids. Bisacylhydrazenes are non
steroidal agonist of 20-hydroxyecdysone and exhibit their activity via interaction with
ecdysteroid receptor proteins. These insecticides are safe to beneficial insects and have
benign ecotoxicological properties,they are selective in their mode of action and potentially
act only on target species.The effect of insect hormone ecdysterone analogues or agonists has
been widely studied to understand the regulation of insect development and recent
developments in these technologies should help in developing novel pest management
methods.
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References
1. Adler, J.H. and Grebenok, R.J. 1995. Biosynthesis and distribution of insect-molting
hormones in plantsa review,Lipids 30:257262.
2. Adler, J.H. and Grebenok R.J. 1999. Occurrence, biosynthesis, and putative role of
ecdysteroids in plants, Crit. Rev. Biochem. Mol. Biol. 34: 253-264
3. Albanese,C., Reutens,T.A., Maofu Fu,B.B., Damico,M., Link,T., Nicholson,R.,Depinho,A.R. and Pestell,G.R.2000. Sustained mammary gland-directed,
ponasterone A-inducible expression in transgenic mice,The FASEB Journal 878
4. Arnault C. and Slama K.1986.Dietary effects of phytoecdysone in the leek-moth,Acrolepiopsis assectella Zell. (Lepidoptera: Acrolepiidae), J Chem Ecol 12: 1979
1986.
5. Bakrim, A., Lamhamdi, M., Sayah F. and Chibi F. 2007. Effects of plant hormones
and 20-hydroxyecdysone on tomato (Lycopersicum esculentum) seed germination and
seedlings growth,African Journal of Biotechnology. 6(24):2792-2802.
6. Bathori, M. 2002.Phytoecdysteroid effects on mammalians ,isolation and analysis,
Minireviews in medicinal chemistry2:285-293.
7. Bthori, M., Gergely B.A., Kalsz ,H.C., Nagy,G. A., Dobos, A.A. and Mth
A.I.2000. Liquid Chromatographic Monitoring Of Phytoecdysteroid Production Of
Serratula Wolffii,Liq. Chrom. & Rel. Technol 23(2): 281294
8. Bathori, M.1998.Purification and characterisation of plant ecdysteroids of Silence
species, Trends in analytical chemistry 17:6
9. Bathori, M.and Pongracz Z. 2005. Phytoecdysteroidsfrom isolation to their effects
on humans, Curr. Med. Chem. 12: 153-172
10.Bthory, M., Toth, I., Szendrei K. and Reisch J. 1982. Ecdysteroids in Spinacia
oleracea and Chenopodium bonus-henricus,Phytochemistry21: 236238
7/28/2019 Finaly Revew for Book
19/30
11.Bergamasco, R. and Horn D. H. S. 1983. Distribution and role of insect hormones inplants. In:Downer R. G. H. and Laufer H,eds., Endocrinology of Insects. Liss, New
York, pp. 627654
12.Blackford, M. and Dinan L.1997b. The effects of ingested 20-hydroxyecdysone on
the larvae ofAglais urticae, Inachis io, Cynthia cardui (Lepidoptera: Nymphalidae)
and Tyria jacobaeae (Lepidoptera: Arctiidae),J Insect Physiol43: 315327.
13.Chen, Q., Xia Y. and Qiu Z. 2006. Effect of ecdysterone on glucose metabolism in
vitro,Life Sciences 78 :11081113.
14.Cherbas, P., Cherbas, L. and Williams C.M. 1977. Induction of acetylcholinesterase
activity by ecdysone in aDrosophila cell line, Science197:275277
15.Cho, G. and Itami T. 2004. Plant extract as cholesterol substitute in shrimp, Aqua
Feeds:Formulation &Beyond1: 1617.
16.Chow, W S.and Lu, H S .1980. Growth regulation and silk production in Bombyx
mori L from Phytogenous ecdysterone.In : Hoffman, J. A. eds., Progress in Ecdysone
Research .Elsevier, North Holland, pp. 281-297.
17.Clemment, C. Y., Bradbrook, D. A., Lafont, R. and Dinan, L., 1993. Assessment of a
microplate-based bioassay for the detection of ecdysteroid like or antiecdysteroid
activities,Insect Biochem. Molec. Biol. 23:187-193.
18.Clemment, C. Y.and Dinan, L. 1991. Development of an assay for ecdysteroid like
and antiecdysteroid activities in plants. In: Hrdy, I. eds., Insect Chemical Ecology.
Academia. Prague, pp. 221-226.
19.Corio-Costet, M.F., Chapuis L. and Delbecque J.P. 1996. Serratula tinctoria L.
(Dyers Savory): in vitro culture and the production of ecdysteroids and other
secondary metabolites. In: Bajaj Y.P.S.,eds., Biotechnology in Agriculture and
7/28/2019 Finaly Revew for Book
20/30
Forestry 37: Medicinal and Aromatic Plants IX . Springer, Berlin/Heidelberg, pp 384
401.
20.Cottam, D. M. and Milner M. J. 1997. The effects of several ecdysteroids andecdysteroid agonists on twoDrosophila imaginal disc cell lines ,Cell. mol. life sci. 53
:600603
21.Courgeon, A.M. 1972. Action of insect hormones at the cellular level. Morphological
changes of a diploid cell line ofDrosophila melanogaster,Exp Cell Res74:327336
22.Decombel, L., Tirry L. and Smagghe G .2005. Action of 24-epibrassinolide on cell
line of the beet armyworm, Spodoptera exigua,Arch Insect Biochem Physiol58:145
156
23.Detmar, M., Dumas, M., Bonte, F., Meybeck A. and Orfanos C.E. 1994. Effects of
ecdysterone on the differentiation of normal human keratinocytes in vitro,European
Journal of Dermatology4: 558569.
24.Dhadialla,S.T., Carlson R.G. and Le,P.D. 1998.New Insecticides With Ecdysteroidal
And Juvenile Hormone Activity, Annu. Rev. Entomol. 43:54569
25.Dhadialla,T.S., Retnakaran, A. and Smagghe,G. 2005. Insect Growth- and
Development-Disrupting Insecticides.In: GilbertI.L., KostasI and Gill,S.S,eds.,
Comprehensive insect molecular science, volume 6 , Pergamon ; Elsevier, Oxford,
UK,pp. 55-116
26.Dinan L. 1995. Distribution and levels of phytoecdysteroids within individual plants
of the species of the Chenopodiaceae, Eur. J. Entomol. 92: 295300.
27.Dinan L. 2001. Phytoecdysteroids: biological aspects,Phytochemistry 57:325-339
28.Dinan L. 2004a. Literature reports occurrence of ecdysteroids. In: Lafont R.,
Harmatha J., Marion-Poll F., Dinan L., Wilson I.D. eds., The Ecdysone Handbook.
3rd ed. On-line http://ecdybase.org
https://biblio.ugent.be/publication?q=author%3D%22Gilbert%2C+Lawrence+I*%22+or+%28type+exact+bookEditor+and+editor%3D%22Gilbert%2C+Lawrence+I*%22%29https://biblio.ugent.be/publication?q=author%3D%22Kostas%2C+Iatrou*%22+or+%28type+exact+bookEditor+and+editor%3D%22Kostas%2C+Iatrou*%22%29https://biblio.ugent.be/publication?q=author%3D%22Gill%2C+Sarjeet+S*%22+or+%28type+exact+bookEditor+and+editor%3D%22Gill%2C+Sarjeet+S*%22%29https://biblio.ugent.be/publication?q=parent+exact+%22Comprehensive+insect+molecular+science%2C+volume+6+%3A+control%22https://biblio.ugent.be/publication?q=publisher+exact+%22Pergamon+%3B+Elsevier%22http://ecdybase.org/http://ecdybase.org/https://biblio.ugent.be/publication?q=publisher+exact+%22Pergamon+%3B+Elsevier%22https://biblio.ugent.be/publication?q=parent+exact+%22Comprehensive+insect+molecular+science%2C+volume+6+%3A+control%22https://biblio.ugent.be/publication?q=author%3D%22Gill%2C+Sarjeet+S*%22+or+%28type+exact+bookEditor+and+editor%3D%22Gill%2C+Sarjeet+S*%22%29https://biblio.ugent.be/publication?q=author%3D%22Kostas%2C+Iatrou*%22+or+%28type+exact+bookEditor+and+editor%3D%22Kostas%2C+Iatrou*%22%29https://biblio.ugent.be/publication?q=author%3D%22Gilbert%2C+Lawrence+I*%22+or+%28type+exact+bookEditor+and+editor%3D%22Gilbert%2C+Lawrence+I*%22%297/28/2019 Finaly Revew for Book
21/30
7/28/2019 Finaly Revew for Book
22/30
38.Dinan, L.2009. The karlson lecture. Phytoecdysteroids: what use are they?,Archives
Of Insect Biochemistry And Physiology72( 3): 126141
39.Dinan,L., Harmatha,J., Volodin,V. and Lafont,R.2009. Phytoecdysteroids: Diversity,
Biosynthesis and Distribution. In: G. Smagghe ,eds., Ecdysone: Structures and
Functions, Springer Science , Business Media B.V.pp.3-45
40.Dinan,L.1992. The association of phytoecdysteroids with flowering in fat hen,
Chenopodium album, and other members of the Chenopodiaceae, Experientia 48:
305308
41.Dreier S.I and Tower G.H.N.1988.Activity of ecdysterone in selected plant growth
bioassays,J.plant Physiol.132:509-512.
42.Feldman,G.J., Maclean,D., Ilic,N., Poulev,A., Lila,A.M., Cheng,D. and Raskin
I.2008. Phytoecdysteroids Increase Protein Synthesis in Skeletal Muscle Cells,J.
Agric. Food Chem. 56:35323537
43.Galimi, F., Saez, E., Gall, J.G., Hoong, N., Cho, G., Evans, R.M., and Verma I.M.
2005. Development of ecdysone-regulated lentiviral vectors, Molecular Therapy11:
142-148.
44.Gilbert L.I. 2004. Halloween genes encode P450 enzymes that mediate steroid
hormone biosynthesis inDrosophila melanogaster,Mol. Cell. Endocrinol. 215: 1-10
45.Gilbert, L.I., Granger N.A. and Roe, R.M. 2000. The juvenile hormones: historical
facts and speculations on future research directions, Insect Biochem. Mol. Biol. 30:
617644.
46.Golovatskaya I.F. 2004. Effect of ecdysterone on morphological and physiological
processes in plants,Russ. J. Plant Physiol. 51: 407-413
47.Goverdhana,S., Puntel,M., Xiong, W. , Zirger, J. M. , Barcia, C., Curtin, J.F., Soffer,
E. B.,Mondkar,S., King, G. D., Hu, J., Sciascia, S.A., Candolfi, M., Greengold, D.S.,
7/28/2019 Finaly Revew for Book
23/30
Lowenstein. P.R and Castro,M.G. 2005.Regulatable Gene Expression Systems for
Gene Therapy Applications: Progress and Future Challenges ,Molecular Therapy 12,
No. 2,
48.Grebenok, R.J., Ripa P. V. and Adler J.H. 1991. Occurrence and levels of
ecdysteroids in spinach,Lipids26: 666668
49.Harmatha J .2000. Chemo-ecological role of spirostanol saponins in the interaction
between plants and insects. In: Oleszek W, Marston A, Kluwer, Dordrecht,eds.,
Saponins in Food, Feedstoffs and Medicinal Plants. Proceedings of the
Phytochemistry Society of Europe, vol 45, pp 129141B11.
50.Hikino, H., Jin H. and Takemoto T.1971. Occurrence of insect-moulting substances
ecdysterone and inokosterone in callus tissues ofAchyranthes radix. Chem Pharm
Bull19: 438439.
51.Hoffmann, K.H. and Gerstenlauer B. 1997.Effects of ovariectomy and allatectomy on
ecdysteroid synthesis and ecdysteroid titers during larval-adult development of
Gryllus bimaculatus deGeer (Ensifera: Gryllidae), Arch Insect Biochem Physiol
35:149158
52.Hoppe,C.U., Marban E. and Johns C.D.2000. Adenovirus-Mediated Inducible GeneExpression in Vivoby a Hybrid Ecdysone Receptor, molecular therapy 1(2)
53.Huber, R. and Hoppe W. 1965.On the chemistry of ecdysone. VII. Analysis of the
crystal and molecular structure of the molting hormone in insects, ecdysone, using the
automized folding molecule method, Chem Ber98:24032424
54.Imai, S., Toyosato, T., Sakai, M., Sato, Y., Fujioka, S., Murata E. and Goto M. 1969.
Screening results of plants for phytoecdysones, Chem Pharm Bull17: 335339.
7/28/2019 Finaly Revew for Book
24/30
55.Jadhav, A.N., Rumalla, C.S., Avula B. and Khan I.A.2007. HPTLC method for
determination of 20-hydroxyecdysone in Sida rhombifolia L. and dietary
supplements, Chromatographia 66:797800.
56.Kaplsnis, L.J.N., Tabor, L. A.,Thompson, J., Robbins W. E. and Shortino T.
J.1996.Assay For Ecdysone (Molting Hormone) Activity Using The House Fly,
Musca Domestica ,Steroids8(5):625-631.
57.Karzenowski, D., Potter D.W and Padidam M. 2005. Inducible control of transgene
expression with ecdysone receptor: gene switches with high sensitivity, robust
expression, and reduced size,BioTechniques39:191-200
58.Keckeis, K., Sarker, S.D. and Dinan, L. 2000. Resveratrol-type oligostilbenes from
Iris clarkei antagonise 20-hydroxyecdysone action in a Drosophila melanogastercell
line, Cell. Mol.Life Sci.57: 333336
59.Kozlova, T. and Thummel S.C. 2000.Steroid Regulation of Postembryonic
Development and Reproduction inDrosophila, TEM 11( 7):276-280
60.Kubo, I., Klocke J.A. and Asano S.1981. Insect ecdysis inhibitors from the East
African medicinal plantAjuga remota (Labiatae),Agric Biol Chem45: 19251927.
61.Kubo, I., Klocke J.A. and Asano S. 1983. Effects of ingested phytoecdysteroids on the
growth and development of two lepidopterous larvae,J Insect Physiol29: 307316.
62.Kutepova, T.A., Syrov, V.N., Khushbaktova Z.A. and Saatov Z. 2001.
Hypoglycemic activity of the total ecdysteroid extract from Ajuga turkestanica,
Pharm Chem J35:608609.
63.Lafont, R.and Dinan L.2009. Innovative and Future Applications for EcdysteroidsIn:
G. Smagghe ,eds., Ecdysone: Structures and Functions. Springer Science , Business
Media B.V. pp-551-572
7/28/2019 Finaly Revew for Book
25/30
64.Lafont, R.and Dinan L. 2003. Practical uses for ecdysteroids in mammals including
humans: and update,Journal of Insect Science, 3:1-30
65.Lammerding-Kippel, M., Spindler-Barth, M. and Drews, U.1994. Ecdysone-induced
morphogenetic movements are forsbadowed and accompanied by expression of an
embryonic muscarinic system in a Chironomus cell line, W. Roux's Arch. Dev. Biol.
203:439-444
66.Lapenna, S. and Dinan,L.2009.HPLC andTLC characterisation of ecdysteroid alkylethers,Journal of chromatography 877:2996-3002
67.Lev, S., Zakirova, R., Saatov, Z., Gorovits M. and Abubakirov N .1990. Ecdysteroids
from cell and tissue culture ofAjuga turkestanica ,Chem Nat Comp 26 (1): 4041
68.Lupien, P.J., Hinse C. and Chaudhary K.D. 1969. Ecdysone as a hypocholesterolemic
agent,Arch Int Physiol Biochim77:206212.
69.Lynn D.E. and Oberlander H.1983. The establishment of cell lines from imaginal
wing discs of Spodoptera frugiperda and Plodia interpunctella, J Insect Physiol
29:591596
70.Matsumoto, T. and Tanaka N .1991. Production of phytoecdysteroids by hairy root
cultures ofAjuga reptans var. atropurupurea,Agric Biol Chem55: 10191025.
71.McMorris T.C. and Voeller B. 1971. Ecdysones from gametophytic tissues of a fern,
Phytochemistry 10: 32533254.
72.Nishimoto, N., Shiobara, Y., Fujino, M., Inoue, S.S., Takemoto, T., Oliveira, F.,
Akisue, G., Akisue, M.K.,Hashimoto, G., Tanaka, O., Kasai, R. and Matsuura H.
1987. Ecdysteroids from Pfaffia iresinoides and reassignment of some 13C NMR
chemical shifts,Phytochemistry26:25052507.
7/28/2019 Finaly Revew for Book
26/30
73.Ogawa, S., Nishimoto N. and Matsuda H. 1974a. Source and availability of
ecdysterones. In: Burdette W.J,eds., Invertebrate Endocrinology and Hormonal
Heterophylly . Springer, Berlin,pp.218232.
74.Otaka, T., Uchiyama, M., Okui, S., Takemoto, T., Hikino, H., Ogawa S. and
Nishimoto N. 1968. Stimulatory effect of insect metamorphosing steroids from
Achyranthes and Cyathula on protein synthesis in mouse liver, Chemical and
Pharmaceutical Bulletin16: 24262429.
75.Palli, S.R., Hormann R.E., Schlattner U. and Lezzi M. 2005a. Ecdysteroid receptors
and their applications in agriculture and medicine, Vitam Horm73:59100
76.Pathak, A.K., Agarwal, P.K., Jain, D.C., Sharma R.P.and Howarth O.W. 1995. NMR
studies of 20-hydroxyecdysone, a steroid isolated from Tinospora cordifolia,Indian
J Chem34B:674676.
77.Ravishankar , G.A.and Mehta A.R. 1979. Control of ecdysterone biogenesis in tissue
cultures ofTrianthema portulacastrum,Nat Prod42: 152158.
78.Redfern, R.E., Kelly, T.J., Borkovec A.B. and Hayes D.K. 1982. Ecdysteroid titers
and moulting abberations in last stage Oncopeltus nymphs treated with insect growth
regulators,Pesticide Biochemistry and Physiology18: 351356.
79.Reginaldo,A.,Festucci, B.,Contim,A.S.L.,Barbosa,A.C.L.,Stuart, J . and
Otoni.C.W.2008.Biosynthesis and potential functions of the ecdysteroid 20-
hydroxyecdysone-a review,Botany86:978-987
80.Reynolds, S.E. 1987. The cuticle, growth and moulting in insects: the essential
background to the action of acylurea insecticides,Pestic. Sci.20:13146
81.Richter, K and Koolman J .1991. Antiecdysteroid effects of brassinosteroids in
insects. In: Cultler H.G, Yokota T, Adam G, eds., Brassinosteroids: chemistry,
bioactivity and applications. ACS Symp Ser, Washington, DC, pp.474:231245
7/28/2019 Finaly Revew for Book
27/30
82.Riddiford, L.M. 1994. Cellular and molecular actions of juvenile hormone.1. General
considerations and premetamorphic actions,Adv. Insect Physiol. 24: 213274.
83.Rudel, D., Bthori, M., Gharbi, J., Giault, J.P., Racz, I., Melis, K., Szendrei K.and
Lafont R. 1992. New ecdysteroids from Serratula tinctoria,Planta Med58:358364
84.Santos, A.C., Chua, M.T., Eufemio N.and Abela C. 1970. Isolation of commisterone,
a new phytoecdysone from Cyanotis vaga,Experientia26:10531054.
85.Schmelz, E.A., Grebenok, R.J., Galbraith D.W. and Bowers W.S. 1998. Damage-
induced accumulation of phytoecdysteroids in spinach: a rapid root response
involving the octadecanoic acid pathway, J. Chem. Ecol. 24: 339-360.
86.Schmelz, E.A., Grebenok, R.J., Galbraith D.W. and Bowers W.S.1999. Insect-
induced synthesis of phytoecdysteroids in spinach, Spinacia oleracea,J. Chem. Ecol.
25 :1739-1757.
87.Schmelz, E.A., Grebenok, R.J., Ohnmeiss T.E. and Bowers W.S.2002. Interactions
between Spinacia oleracea andBradysia impatiens: a role for phytoecdysteroids,
Arch. Insect Biochem. Physiol. 51 :204-221.
88.Sieber, K.P.and Rembold H. 1983. The effects of azadirachtin on the endocrine
control of moulting inLocusta migratoria,Journal of Insect Physiology29: 523527.
89.Slama, K and Lafont R. 1995. Insect hormones - ecdysteroids: their presence and
actions in vertebrate,Eur J Entomol92:355-377
90.Smagghe, G.2007. Insect Cell Lines as Tools in Insecticide Mode of Action Research.
In: Ishaaya,I., Nauen R. and Horowitz,R.A., eds .,Insecticides Design Using
Advanced Technologies . Springer-Verlag ,Berlin, Heidelberg, pp.263-304
91.Smith, S.L. and Mitchell M.J. 1988. Effects of azadirachtin on insect cytochrome P-
450 dependent ecdysone 20-monooxygenase activity, Biochemical Biophysical
Research Communications154:559563.
7/28/2019 Finaly Revew for Book
28/30
92.Spindler-Barth, M., Junger, E.,Spindler, K.D. 1995.The epithelial cell line from
Chironomus tentans: hormonal regulation of tissue differentiation and cuticle
formation.Actes de Colloque18:87-93.
93.Spindler-Barth, M., Kammann, V. and Spindler, K.D.1989. Hormonal regulation of
chitin synthesis in two insect cell lines.In: Skjak-Braek, G., Anthonson, T., Sandford,
P, eds., Chitin and Chitosan. London: Elsevier Applied Science, pp. 279-289.
94.Spindler-Barth, M.1991. Hormonal regulation of acetylcholinesterase in an epithelial
cell line from Chironomus tentans, Z. Naturforseh. 46e:1089- 1093
95.Spindler-Barth, M.1993. Hormonal regulation of chitin metabolism in insect cell
lines. In: Muzzarelli, R. A. A., ed., Chitin enzymology. Ancona, Italy, Eur, Chitin
Soc, pp.75~32.
96.Takeda,T., William Y. Go., Orlando,A.R. and Farquhar,G.M.2000. Expression ofPodocalyxin Inhibits CellCell Adhesion and Modifies Junctional Properties in
Madin-Darby Canine Kidney Cells,Molecular Biology of the Cell11:32193232
97.Tanaka Y. and Naya S .1995. Dietary effect of ecdysone and 20-hydroxyecdysone on
larval development of two lepidopteran species,Appl Entomol Zool30: 285294.
98.Thomson, J.A., Paula Imray F. and Hohn D. H. S. 1970. An Improved Calliphora
Bioassay For Insect Moulting Hormones, Ausl. J. extp. Biol. med. Sci. 48, pp. 32I-
32S
99.Thummel, C.S. 2002. Ecdysone-regulated puff genes, Insect Biochem. Mol. Biol. 32:
113120.
100. Toniatti1,C., Bujard,H., Cortese R. and Ciliberto G.2004. Gene therapy
progress and prospects: transcription regulatory systems ,Gene Therapy11: 649657
7/28/2019 Finaly Revew for Book
29/30
7/28/2019 Finaly Revew for Book
30/30
110. Zitnanova, I., Adams M.E. and Zitnan D. 2001. Ecdysteroid action on the
epitracheal glands and central nervous system preceding ecdysis ofManduca sexta,J.
Exp. Biol. 204: 34833495.