This document summarizes research on the effects of phytoecdysteroids, plant-derived compounds similar to insect molting hormones, on the growth and development of insects. Phytoecdysteroids are found in over 100 plant families and can comprise up to 3% of plant dry weight. They act as toxins and disruptors of insect hormones. Effects include inhibition of digestive enzymes and proteins, precocious pupation, abnormal adult emergence, larval death, cannibalism, reduced fecundity and fertility, and anti-feedant activity. While phytoecdysteroids show potential for insect control, the document concludes they are not a replacement for integrated pest management strategies and more research is still needed.

1. Dietary effects of Phytoecdysteroids on
growth and development of insects
Submitted by: K.V. NAGARJUNA REDDY

2. Introduction
• Ecdysteroids - control moulting and metamorphosis
in insects.
• Regulate biochemical and physiological processes
• Ecdysone (E) first isolated by Butenandt and
Karlson in 1954 from silkworm pupae.
• Subsequently, 20-hydroxyecdysone (20 HE)
was identified from a number of arthropod sources
Structure of a typical ecdysteroid

3. • Phytoecdysteroids - Analogues of ecdysteroids occurring in plant species.
• Nakanishi et al. (1966) isolated three polyhydroxylated steroids
(Ponasterones A, B and C).
• Takemoto’s group isolated 20E and inokosterone from the roots of
Achyranthes fauriei.
• 20 HE found in the wood of Podocarpus elatus (Galbraith and Horn,
1966), the rhizomes of Polypodium vulgare (Heinrich and Hoffmeister,
1967) and in dry pinnae of Pteridinium aquilinum (Kaplanis et al., 1967).

4. Distribution of ecdysteroids in the plant world
• Occur in over 100 terrestrial plant families representing ferns, gymnosperms and
angiosperms.
• Phytoecdysteroids detected in 27 families of the Pteridophyta, 10 families of Gymnospermae
and 74 families of angiosperms.
• Majority of these families are within eleven orders: Liliales, Ranunculales, Urticales,
Malvales, Violales, Capparales, Rosales, Sapindales, Polemoniales and Scrophulariales.
• Phytoecdysteroids occur in relatively high concentration in many plants and comprise 0.001-
3% of the dry weight and have been isolated from all parts of plants in much higher amounts
than those present in arthropods.

5. Table 1. List of few Phytoecdysteroids.
Ponasterone A Ajugasterone B 29-norcyasterone
1-epi-integristerone Dacryhainansterone Rapisterone C
Paristerone Tenuifoliosides A and B 22-oxo-cyasterone
5α-polypodine B 22-deoxy-20,21-dihydroxyecdysone Praemixisterone
Viperidinone Inokosterone Stachysterone A
Turkesterone Makisterone D Stachysterone C
22-dehydro-12 hydroxycyasterone Amarasterone B Decumbesterone A
Sogdisterone Gerardiasterone Cheilanthones A and B
Silenosterone Rapisterone Makisterone A
Poststerone Venustone Makisterone C
4α-hydroxypinnasterol Canescensterone Amarasterone B
Kaladasterone Rubrosteron Sidisterone
Podecdysone B Carthamosterone Poststerone
Carpesterol Taxisterone (22-deoxyecdysterone) 29-norsengosterone

6. Chemistry of phytoecdysteroids
• Phytoecdysteroids - triterpenoids
• Polar steroids with sugar-like solubility properties
• Lack polyhydroxylated side chain- lipophilic.
• (Cyclo pentano- perhydrophenanthrene carbon skeleton)
cholest-7-en-6-one carbon skeleton (C27)
• Hydroxylated with 2-8 hydroxyl groups.
• Over 1,000 possible structures - various analogues differ in the number and site
of hydroxylations, the length and structure of the carbon side chain.

7. Diversity of phytoecdysteroid structures

9. Biological significance of phytoecdysteroids to plants
• Two main hypotheses
• PEs have a hormonal role within the plant - little evidence
• PEs possess insect moulting hormone activity - participate in the defence of plants against non-
adapted phytophagous invertebrates
• PEs - hormonal disruptors and toxins for insects and other invertebrates
• Plants ʻturn onʼ the production of phytoecdysteroids when under stress, animal attack or other
conditions

10. Mechanism of action

11. Effects on growth and development of insects
Inhibition of digestive enzymes:
- Cytotoxic effects on epithelial cells synthesizing digestive enzymes (α- amylase).
- Direct inhibition of enzyme activity
- Indirectly perturbation of neuropeptides (Sulfakinin) involved in alpha-amylase signalling
and regulation.
Test insects References
Tribolium castaneum Ajaha et al. 2019, Jbilou et al. 2008
Plodia interpunctella Bouayad et al. 2012, Rharrabe et al. 2009
Spodoptera eridania Shannag et al. 2015

12. Table: Effect of 20-hydroxyecdysone on alpha-amylase activity of Tribolium
castaneum larvae after 7d of treatment.
Treatment
α-Amylase
(µg of starch consumed /larva)
Control 174.63 ± 1.7a
300 ppm 161.25 ± 2.06b
600 ppm 150.53 ± 2.5b
900 ppm 145.15 ± 2.2c
1,200 ppm 134.95 ± 1.9c
Starved 132.38 ± 2.6c
Means in the same column followed by different letters indicate that the difference between controls and treated or starved larvae
is statistically significant as determined by the Tukey’s HSD test. Between the letters ‘a’ and ‘b’, the effect is significant (P <
0.05). Between the letters ‘a–c’, the effect is very significant (P < 0.01). Between the letters ‘a–d’, the effect is very highly
significant (P < 0.001).

13. Reduction in larval proteins
- Deterioration of the internal tissues of insects
- Strong mobilization of proteins as response to the nutritional stress
Test insect Phytoecdysteroids References
Plodia interpunctella 20-hydroxyecdysone Rharrabe et al. 2008, 2009
Bombyx mori
Phytoecdysteroids from
Radix achyranthes
Miao et al. 2004
Tribolium castaneum 20-hydroxyecdysone Ajaha et al. 2019

14. Table: Effect of 20-hydroxyecdysone on protein activity of Tribolium castaneum larvae after 7
d of treatment.
Treatment Protein (µg/larvae)
Control 233.15 ± 24a
300 ppm 151.32 ± 15b
600 ppm 149.13 ± 5b
900 ppm 110.07 ± 20c
1,200 ppm 95.7 ± 16c
Starved 48.34 ± 7.7d
Means in the same column followed by different letters indicate that the difference between controls and treated or starved
larvae is statistically significant as determined by the Tukey’s HSD test. Between the letters ‘a’ and ‘b’, the effect is
significant (P < 0.05). Between the letters ‘a–c’, the effect is very significant (P < 0.01). Between the letters ‘a–d’, the effect is
very highly significant (P < 0.001).

15. Precocious pupation and adult emergence/ Abnormal adults
- Emerged ones display structural abnormalities- deformed wings, remnants of moult skin,
pupal cover and head capsule).
- The effect on pupation a consequence of hormonal balance disruption with internal levels
of ecdysone.
Test insect References
Tribolium castaneum Ajaha et al. 2019
Leptinotarsa decemlineata Zolotar’ et al. 2001
Plodia interpunctella Rharrabe et al. 2009
Bombyx mori – Inkosterone Shigematsu et al. 1974
Cynthia cardui Blackford and Dinan, 1997
Anticarsia gemmatalis Macedo et al. 2011
Bemisia tabaci, Oligonychus perseae
- Ajuga iva extracts
Radi et al. 2011
Spodoptera littoralis Leena et al. 2020

16. Morphological changes in the larvae, pupae and adults of T. castaneum
Doseof20HE

17. Figure: Adult Bemisia tabaci mortality rates obtained after ecdysterone and cyasterone
extracted from Ajuga iva, compared with a commercial ecdysterone standard and a water
control.

18. Fig : Metamorphosis of control and treated S. littoralis larvae. Pupation of S. littoralis larvae
after 15 days of exposure (feeding for 4 days) on A. iva crude leaf extract. Control (A), 250
µg/µl A. iva crude leaf extract (B) and 250 µg/µl of three fractionated and purified
phytoecdysteroids from A. iva leaf extract fractions (20-hydroxyecdysone, makisterone A and
cyasterone) (C). Deficient development of pupation in (b) and (c) is due to disturbed levels of
the ecdysteroids responsible for molting.

19. Fig : Effect of 20-hydroxyecdysone on adult emergence of Tribolium castaneum. Each point
represents the mean ± SE.

20. Larval death
- Insects die in the juvenile stages with abnormalities.
- Disruption involves a number of effects
- inhibition of growth, induction of supernumerary larval
instars, death without moulting, head capsule apolysis,
uncoordinated writhing side-to-side larval movements unable to
maintain their balance, haemolymph emanation followed by hind-gut
extrusion and death during or after induced moulting.
- Cytotoxicity of phytoecdysteroids on the larvae’s midgut.

21. Test insect References
Brevicoryne brassicae Pavela et al. 2005
Cynthia cardui Blackford and Dinan, 1997
Lobesia botrana Mondy et al. 1997
Anticarsia gemmatalis Macedo et al. 2011
Spodoptera litura, Helicoverpa armigera Balasubramanian et al. 2008
Tribolium castaneum Ajaha et al. 2019
Cryptorrhynchus lapathi Chi De-fu et al. 2002
Spodoptera littoralis Leena et al. 2020
Plutella xylostella Li et al. 2015

22. Fig : Effect of different concentrations of A. iva crude leaf extract on S. littoralis first instar (L1) larval (n = 110)
mortality (mean ± SEM) (a), and larval weight gain (%) (mean ± SEM) of S. littoralis third instar (L3) larvae (b).
Asterisks above columns indicate significant difference (p ≤ 0.05) by t-test (t108 = 6.105, 4.308 and 3.220 for
50, 100 and 250 µg/µl, respectively); p < 0.001 for all treatments, Levene’s test p = 0.326 (a), and by repeated
measures ANOVA (F3.104 =20.334, 17.246 and 13.007 for 50, 100 and 250 µg/µl, respectively); p < 0.001,
Mauchly’s test p = 0.152 (b) between treatments and the control

23. Fig: The morphological changes in diamondback moth larvae and a pupae
caused by ingestion of exogenous dietary 20-hydroxyecdysone.
a. Exosmosis of ecdysial fluid and an additional molt
b. failure to shed the head capsule
c. failure to shed the exuvium
d. bulging of the hindgut
e. deformed pupa

24. Cannibalism
The stressful conditions (high population and scarce food) when phytoecdysteroids are
present in the larval food can induce cannibalism
Test insect Reference
Plodia
interpunctella
Rharrabe et al. 2009

25. Fig: Effect of different phytoecdysteroids, at a concentration of 200 ppm, on
P. interpunctella larval cannibalism. Each data point represents the mean ± standard
error of five replicates

26. Anti-feedant activity
- Phytoecdysteroids - deterrent action
- detected by maxillary and labial palps of the insects
Test insect References
Leptinotarsa decemlineata Khalilova et al. 2002
Popillia japonica, Cerotoma trifurcate,
Diabrotica virgifera virgifera,
Trirhabda canadensis
Jurenka et al. 2017

27. Table: Various concentrations of phytoecdysteroid affected feeding by 3 species of
chrysomelids on preferred host plants in a choice assay. Values represent mean % defoliation
± SEM
BLB = bean leaf beetle (Cerotoma trifurcate) feeding on soybean leaves; WCRW = western corn rootworm (Diabrotica virgifera virgifera)
feeding on maize leaves; GLB = goldenrod leaf beetle (Trirhabda canadensis) feeding on common goldenrod. Letters indicate significant
differences between treatments within a column P<0.05 (ANOVA followed by Fisher’s LSD test). A (F = 21.89; df = 3, 8; P = 0.0003) b (F =
25.69; df = 3, 8; P = 0.0002) c (F = 23.23; df = 1, 21; P = 0.0004) d (F = 37.67; df = 3, 16; P = 0.0001).

28. Reduced fecundity/ fertility.
- Phytoecdysteroids target ovarian development
- disruption in ecdysteroid titres
- egg abortion during oogenesis,
- less fecundity
- mortality of adults (indirect effect)
- less fertility

29. Test insect Phytoecdysteroids References
Brevicoryne brassicae
ajugasterone C (ajuC) and
polypodine B (polyB)
Pavela et al. 2005
Anticarsia gemmatalis Talisine Macedo et al. 2011
Helicoverpa armigera Extracts of C. farinosa Rajkumar et al. 2000
Glossina morsitans Whitehead, 1981
Bemisia tabaci,
Oligonychus perseae
Ajuga iva extracts Radi et al. 2011

30. Effect of ecdysterone and cyasterone from Ajuga iva on Bemisia tabaci fecundity (A) and
fertility (B). The treatments were compared with a commercial ecdysterone standard and a
water control.

31. Improvement of silk yields
- Larvae of the silkworm (4th and 5th instars)
- Enhance synchronous development
- Co-administration with juvenile hormone
analogue elevate silk yield.
- Posterior division of the silk gland and the
fat body

32. CONCLUSION
- Renewed attention
- Phytoecdysteroids - defensive substances against phytophagous insects
- Are phytoecdysteroids feasible for crop protection ?
- Not an alternative
- Integrated pest management strategies

33. THANK YOU