Mass rearing lepidoptera with persistent baculovirus infections. Insect population dynamics are impacted by lethal and chronic infections

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1 Mass rearing lepidoptera with persistent baculovirus infections Helen Hesketh & Rosie Hails Natural Environment Research Council (NERC) Centre for Ecology & Hydrology (CEH) Wallingford, UK Natural Environment Research Council (NERC) Centre for Ecology & Hydrology (CEH) Wallingford, UK Baculoviruses Double stranded DNA viruses Nucleopolyhedroviruses (NPV s) & Granuloviruses (GV s) Principally infect Lepidoptera (larval stage) Systemic, acute infection leads to death Cypoviruses (CPV s) RNA viruses Chronic infections Common in rearing of: Trichoplusia ni Cydia pomonella Orygia pseudotsugata Spodoptera exigua Insect population dynamics are impacted by lethal and chronic infections (Anderson & May, 1981) Phil. Trans. Roy. Soc. B. 291 (1054):

2 Healthy Infected Ingestion or contact Pathogen transmission 2 Case studies Dead Horizontal transmission NPV, fungi, bacteria, some protozoans Faeces or regurgitate Infective propagules?? Triggered to full infection Vertical transmission Cabbage moth (Mamestra brassicae) Supplying laboratory experimental units Codling moth (Cydia pomonella) Large scale sterile insect release programme CPV, most protozoans, small RNA viruses Persistent NPV infections Cabbage moth (Mamestra brassicae) Infected by Mamestra brassicae nuclear polyhedrosis virus - MbNPV L - + B Ov SE A laboratory stock of this insect reared in culture for over 25 years has been shown to harbour a persistent low-level infection of MbNPV since at least 1993 kb Cabbage moth (Mamestra brassicae) A persistent infection gene expression occurs at low levels and transmitted vertically from adult to offspring Hosts appear healthy & minimal fitness costs are noted Reactivated virus is identical to type strain

3 RFLP of purified MbNPV DNA What turns a low level persistent infection into a lethal pathogenic one? Environmental stresses overcrowding / high population densities diet quality immuno-suppression of host temperature? Pathogen Invasion Baculoviruses Other insect viruses Fungi Parasitoids Activated by exposure to PfNPV or AcMNPV to fully overt infection - horizontal transmission? Rearing facility at CEH Rearing protocol 5-10mins in 0.75% Sodium Hypochlorite (w/vol of 12% solution) rinsed in sterile, distilled water

4 Rearing protocol Anecdotal precautions If reared past 3 rd instar in large groups, there is potential for the virus to outbreak >1 larva per pot results in prepupal mortality Reared on autoclaved, artificial diet containing antibioitics and steroids Separated at 2 nd instar to individual pots Pupae allowed to eclose in flight cages Codling Moth, Cydia pomonella 1 Sterile insect release programme rearing >15 million moths/week Colony contaminated with cpgv Tested routes of entry: Codling Moth, Cydia pomonella GV present in egg wash water (> 1 st than 3 rd washes) 40% of pre-neonates contained GV 31% of 10-day old larvae contained virus (no mortality) Air filtration system (filters) Rearing room walls Diet waste bins Spent diet blog.landscapedesign.co.nz % of swabs +ve in PCR for cpgv Diet waste bins positioned below air duct caused greater contamination 1 Cossentine et al. (2005) J. Invertebr. Pathol. 90:

5 Summary 1) baculoviruses Summary 2) rearing - Persistent infections may be symptomless and incur minimal, if any fitness costs to hosts - Persistent infections have the potential to become overt infections - The link between triggering an overt infection and host density is complex & lacks empirical testing - Rigorous investigation of entry routes when designing colony management strategies -Diagnosis of pathogens by microbiologists will assist in designing appropriate rearing strategies - Use of molecular techniques will be essential in detecting persistent, endemic infections - Insect pathogens will play a role in the quality of any mass reared insects Acknowledgements Bob Possee John Burden Steve Sait Tim Carty