Physiological Bases of Using Insect Hormone Analogs for. Use of tebufenozide against the spruce budworm, Pest Management. Choristoneura fumiferana

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1 Physiological Bases of Using Insect Hormone Analogs for Pest Management Use of tebufenozide against the spruce budworm, Choristoneura fumiferana Arthur Retnakaran, Daniel Doucet, Basil M. Arif, Great Lakes Forestry Centre, Sault Ste. Marie, Canada

2 Overview The molting cycle Interference of the molting cycle by diacylhydrazines Use of tebufenozide against the spruce budworm (Choristoneura fumiferana): effectiveness during diapause Conclusion

3 Hormonal regulation of molting and metamorphosis Hormonal regulation of molting and metamorphosis Spruce budworm Lateral NSC (PTTH) Proprioreceptive signal Critical size Ecdysis th th Instar 6 th Instar

4 20-Hydroxyecdysone and Ecdysone Agonists 20-hydroxyecdysone RH-5849 (original analog) RH-0345 (Halofenozide ( Halofenozide) RH-2485 (Methoxyfenozide) RH-5992 (Tebufenozide ( Tebufenozide)

5 Phenotypic effects of diacylhydrazines Feeding inhibition within 3-14h + synthesis of new cuticle, apolysis of new cuticle from old one. Head capsule slippage New cuticle not tanned or sclerotized

6 Mode of Action of RH-5992: early molecular events EcRE EcRB EcRE EcRB EcRE EcRA EcRE EcRA EcRE CHR EcRE CHR EcRE CHR EcRE CHR DDC LCP14 MOLT INCOMPLETE MOLT RH 20E EcR

7 Molecular Mode of Action of RH-5992 Age in Days Hydroxyecdysone Equivalents Instar V Instar VI Ecdysis RH-5992 (ng/2ml) Molting Cycle Early Late Intermolt 20E(+) (+)Cleared(-) Absent(-) 20E EcR ERE CHR3 DDC LCP14 OC ED CU EP ES CU EP EN Molt Events: RH-5992 Cell Division Cuticulin & Epicuticle Ecdysial Fluid Muscle Release Apolysis Head Capsule Slippage EC EC EC DDC For Tanning Precursor Exo, Endocuticle Chitin Muscle Attachement Eclosion Hormone Release (ecdysis) Bursion Release (waterproofing)

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9 Diapause 1. Developmental arrest to overcome seasonal adversity. 2. Obligatory or facultative. Embryonic, Larval Pupal or Adult. 3. Onset & termination controlled by temp and or photoperiodism. 4. Photoperiodic clock regulated by several genes, period and timeless ess accumulate during scotophase. 5. Induction ahead of diapause for preparation - storage proteins, glycerol, sorbitol. 6. Types of diapause: a) Embryonic a) Embryonic - Diapause hormone - Silk worm b) Larval, Pupal - Shut down Brain c) Adult - no JH Shut down Brain ( PTTH), Prothoracic gland (20E)

10 Diapause in Insects Diapause in Insects Stage Environmental Signal Egg (embryonic) LD + High Temp Endocrine Regulation Diapause Hormone Example Silkworm Early Larval SD ± Low Temp No 20E + JH(?) Spruce Budworm Last Larval SD ± Low Temp No 20E JH Corn borer Pupal SD ± Low Temp No 20E Flesh Fly Adult SD ± Low Temp No JH White Pine Weevil

11 Life cycle of the spruce budworm Life cycle of the spruce budworm Choristoneura fumiferana 6 th Instar Pupa 5 th Instar Open Feeding 4 th Instar Needle mining 3 rd Instar 2 nd Instar Eggs in hibernaculum Non Non-feeding feeding 1 st Instar Adult

12 Glycerol and Trehalose Levels in First and Second Instars Glycerol and Trehalose Levels in First and Second Instars Glycerol Trehalose Concentration of sugars (mg/kg body weight) st Instar (days) Prediapause (weeks) Diapause (weeks) Postdiapause (days)

13 Expression Profiles of Some mrnas and Proteins During Prediapause, Diapause and Postdiapause of First and Second Instars 1 st Instar Prediapause (16ºC) 2 nd Instar Diapause (2ºC) 2 nd Instar Postdiapause (20ºC) 2 nd Instar DAP1 RNA DAP1 Protein DAP2 RNA DAP2 Protein Defensin m RNA GST RNA GST protein PTP RNA HSP70RNA Ubiquitin RNA

14 Cf rrna Prediapause (weeks) Diapause (weeks) Post diapause (weeks) 2 nd nd Instar (Days) 3 rd rd Instar (Days) 4 th Instar (Days)

15 Some Ecdysone Related mrna Expression Profiles of Choristoneura Fumiferana CfEcRA CfEcRB Cf CHR75A CHR75B CHR3 CfJHE rrna Prediapause (weeks) Diapause (Weeks) Postdiapause (Days) 2nd Instar (Days)

16 EcR CHR75 CHR3 1 st Instar Second Instar - Diapause (30 weeks, 2 C) 2 2 nd Instar

17 Hydroxyecdysone Profile 1 st Instar Spruce Budworm Age in Days Relative 20-Hydroxyecdysone Level (ng/ml)

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19 Slipped 2 nd instar head capsule Frass pellets 3 rd instar head capsule Effect of RH-5992 on 2 nd instar larva 1 st instar head capsule

20 First instar budworm feed! Untreated control 1 st instar CfMNPV-GFP polyhedra fed 1 st instar

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23 Ingestion of Diet and CHR3 Expression in First Instar Spruce Budworm Diet Ingestion CHR3 Level CHR3 Expression level ( 32 P CPM x 10 3 )v Diet Ingested (micrograms/insect) Age of First Instar (hours)

24 Summary 1. RH RH-5992 binds strongly, is not cleared, has to be ingested before 20E 2 peak and is carried over to the next instar incomplete because the down-regulated events do not occur. instar.. The precocious molt induced is The spruce budworm is univoltine, has 6 larval instars and diapauses as a 2 nd instar for 8 months. 3. Glycerol and trehalose are high; DAP 1 and 2, Defensin and GST are a highly expressed but JHE peak is conspicuously absent. and EcR are present making the 2 nds responsive to 20E. 4. The effect of RH-5992 on diapausing 2 nds was discovered serendipitously during spray trials. First instars are less sensitive because they t consume critical doses after the 20E peak.