To Obtain Approval for New Organisms in Containment

APPLICATION FORM Containment

To obtain approval for new organisms in containment

Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.

Application Number

APP202587

Date

www.epa.govt.nz 2

Application Form Approval for new organism in containment

Completing this application form

1. This form has been approved under section 40 of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers importing, development (production, fermentation or regeneration) or field test of any new organism (including genetically modified organisms (GMOs)) in containment. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 2. If your application is for a project approval for low-risk GMOs, please use the Containment – GMO Project application form. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application including providing advice on any consultation requirements. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise.

Application Form Approval for new organism in containment

Commercially sensitive information

10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available. 12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions

Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation

Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment

Any organism in which any of the genes or other genetic material:  Have been modified by in vitro techniques, or Genetically Modified  Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques

A new organism is an organism that is any of the following:  An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998;  An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation;  An organism for which a containment approval has been given under the HSNO Act; New Organism  An organism for which a conditional release approval has been given under the HSNO Act;  A qualifying organism approved for release with controls under the HSNO Act;  A genetically modified organism;  An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand;  An organism present in New Zealand before 29 July 1998 in contravention of

Application Form Approval for new organism in containment

the Animals Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because:  It is subject to a conditional release approval; or  It is a qualifying organism approved for release with controls; or  It is an incidentally imported new organism

An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal

Application Form Approval for new organism in containment

1. Applicant details

1.1. Applicant

Company Name: The New Zealand Institute for Plant and Food Research Ltd.

Contact Name: Angelene Holton/John Charles

Job Title: National Biosafety & Environmental Compliance Manager/ Senior Scientist Physical Address: 120 Mt Albert Road, Sandringham, Auckland

Postal Address: Private Bag 92169, Auckland 1142

Phone (office and/or mobile): 064 9 925 7285 or 064 21 226 8285

Fax: 064 9 925 7001

Email: [email protected]

1.2. New Zealand agent or consultant (if applicable)

Company Name:

Contact Name:

Job Title:

Physical Address:

Postal Address (provide only if not the same as the physical):

Phone (office and/or mobile):

Fax:

Email:

Application Form Approval for new organism in containment

2. Information about the application

2.1. Type of containment activity Tick the box(es) that best describe your application

Application type Type of new organism

☐ GMO Import into containment  Non-GMO ☐ Develop in containment i.e. regeneration, fermentation GMO or genetic modification ☐ Non-GMO

☐ GMO Field test in containment ☐ Non-GMO

2.2. Brief application description Approximately 30 words about what you are applying to do

Import Trissolcus japonicus (a tiny parasitoid wasp that attacks Brown Marmorated Stink Bug eggs) into containment to evaluate its suitability as a biocontrol agent for Brown Marmorated Stink Bug in New Zealand.

2.3. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it

We propose to import Trissolcus japonicus within BMSB eggs into containment at Plant and Food Research (PFR). Trissolcus japonicus is a tiny wasp that is harmless to humans. It attacks and kills the eggs of the Brown Marmorated Stink Bug (BMSB). The wasp is the key natural enemy of BMSB in Asia, the region of origin of both species. BMSB is a major threat to New Zealand’s economy. It is frequently intercepted at New Zealand’s borders, but has not yet been recorded from within New Zealand. Should BMSB establish, none of the natural enemies already present in New Zealand will control BMSB. Biocontrol by new natural enemies will be the only long-term, sustainable means to control the pest, and T. japonicus is acknowledged to be the primary candidate biocontrol of the pest in the USA and Europe where it has recently established. We plan to import T. japonicus into containment from laboratory populations in the USA. Eggs of New Zealand’s stinkbugs will be exposed to adult female T. japonicus to determine the potential host range of the parasitoid. We do not expect the parasitoid to be able to survive on these non-target

Application Form Approval for new organism in containment

hosts, and so plan for multiple (estimated 10-20) shipments over two-three years to complete the host-range testing programme. The goal of the project is to support a classical biocontrol programme against BMSB in preparedness for its possible establishment in New Zealand.

2.4. Background and aims of application This section is intended to put the new organism(s) in perspective of the wider activities that they will be used in. You may use more technical language but all technical words must be included in a glossary

The Brown Marmorated Stinkbug (BMSB), Halyomorpha halys (Stål 1855) (Hemiptera: Pentatomidae), is a significant threat to New Zealand’s economy and environment (Duthie 2012, Charles 2015). Its overwintering strategy, powers of dispersal and increasingly common interception at the border have led to an acknowledged moderate to high risk of entry and establishment (Duthie 2012). There are limited tools for monitoring and surveillance of BMSB in the field, and its discovery in New Zealand may not be made until there is a breeding population of a size that precludes our ability (technically and/or economically) to eradicate it. If (or when) BMSB does establish in New Zealand, then a classical biocontrol programme (i.e. the introduction of new-to-New Zealand natural enemies) will become a major strategy for reducing populations everywhere in the country, and for obtaining long-term, environmentally sustainable control.

Trissolcus japonicus (1.3 – 1.8 mm long) is a solitary endoparasitoid in the eggs of BMSB and, like its host, also overwinters as an adult. It often parasitises 70% of BMSB egg clusters in its native range in Asia where it is considered to be one of the most effective natural biocontrol agents of BMSB (Yang et al. 2009, Lee et al. 2013).

BMSB is now well established the USA, where it has caused severe economic damage to horticultural crops, especially in the mid-Atlantic States (Leskey et al 2012, Rice et al. 2014). There are many specialist natural enemies of perhaps hundreds of species of native Pentatomidae within the USA, but none has shown any ability to control BMSB. Research in containment laboratories across several States has shown that a few species of native Pentatomidae are attacked by T. japonicus, and research to further define the host range and non-target impacts of T. japonicus is ongoing prior to any application to release the parasitoid. In the meantime, a wild population of T. japonicus has recently (January 2015) been discovered in Beltsville, Maryland, and the extent of its establishment is currently under investigation (Talamas et al. 2015b). This wild parasitoid population probably originated from BMSB eggs on ornamental plants imported for the garden industries, as there are no laboratory cultures close to the recovery sites.

In New Zealand, there are only 16 species of pentatomid bugs, compared with about 5000 species globally. As a consequence, there are few specialist natural enemies of

Application Form Approval for new organism in containment

these bugs in New Zealand, and our knowledge of these species predicts that they will not be able to control BMSB. Only one of the species of Pentatomidae in New Zealand occurs in the USA (the cosmopolitan pest Nezara viridula), so the host-range data from the USA cannot be directly transferred to New Zealand. Consequently, the host range of T. japonicus on New Zealand’s pentatomids should be examined prior to any application to release.

Globally, all species in the genus Trissolcus only attack species in the superfamily Pentatomoidea, so no other fauna in New Zealand are at risk. All non-target testing to date shows that T. japonicus is restricted to hosts in the families Pentatomidae and Scutelleridae. Scutelleridae do not exist in New Zealand, so containment studies will be focussed on species in the Pentatomidae.

From what is known about T. japonicus, it is expected that it will not form sustainable populations on any existing pentatomid species in New Zealand. However, the main purpose of this application is to test this hypothesis through laboratory experiments in containment.

3. Information about the new organism(s)

3.1. Name of organism Identify the organism as fully as possible

Non-GMOs - Provide a taxonomic description of the new organism(s).

GMOs – Provide a taxonomic description of the host organism(s) and describe the genetic modification.

Both -  Describe the biology and main features of the organism including if it has inseparable organisms.  Describe if the organism has affinities (e.g. close taxonomic relationships) with other organisms in New Zealand.  Could the organism form an undesirable self-sustaining population? If not, why not?  How easily could the new organism be recovered or eradicated if it established an undesirable self- sustaining population?

Taxonomy:

The name is Trissolcus japonicus (Ashmead)(Hymenoptera: Platygastridae; Telenominae). Recent taxonomic studies have clarified and stabilised the identity of the taxon (Talamas et al. 2013; 2015a).

Application Form Approval for new organism in containment

The parasitoids used for experiments in New Zealand will be supplied by Dr Kim Hoelmer (USDA – ARS), from laboratory populations held in containment in Delaware. The T. japonicus individuals will be free of any hyperparasitoids, diseases, or unwanted associated organisms.

Biology:

Trissolcus japonicus is an egg endoparasitoid of BMSB. Female wasps lay a single egg in each BMSB egg. A single parasitoid larva hatches and completes development within the BMSB egg, killing it in the process. The emerging adult parasitoid then mates and seeks BMSB eggs to parasitise. Female Trissolcus wasps have evolved to recognise a few chemical cues which they use to locate and identify suitable host eggs. As a consequence, BMSB is the dominant field host of T. japonicus in its native range.

Affinities with NZ fauna:

There are two existing species of Trissolcus in New Zealand. T. maori is endemic (but closely related to T. oedipus from Tasmania). Its hosts are restricted to the Acanthosomatidae. It has been reared only from field collected, parasitised eggs of Rhopalimorpha obscura, R. lineolaris and Oncacontias vittatus (Cumber 1964, Valentine 1964). In laboratory choice tests, T. maori attacked only Acanthosomatidae eggs and none of several species of Pentatomidae eggs (Cumber 1964).

The other species is Trissolcus oenone. It is an Australian native, where it is extremely common throughout the country. It is also considered to be a New Zealand native (Johnson 1991), but it is not known when it arrived. It has been reared from seven species of Pentatomidae in New Zealand and Australia, including both phytophagous and predatory species. Cumber (1964) carried out a series of host-testing experiments that included both field collections and choice-tests in the laboratory. He showed that T. oenone attacked and successfully developed in Monteithiella humeralis, Cermatulus nasalis nasalis, Cuspicona simplex, Dictyotus caenosus and Glaucias amyoti, but did not attack the pest Nezara viridula, the green vegetable bug (GVB). However, GVB is apparently a host for T. oenone in Australia (Johnson 1991). By 2014, GVB had been exposed to attack by New Zealand native parasitoids for 66 years, but T. oenone has not yet been recorded from it. It is predicted that T. oenone may attack a few BMSB eggs, but will not to be able to provide control.

Ability to form sustainable populations in the event of an escape:

From what is known about T. japonicus, it is expected that it will not form sustainable populations on any existing pentatomid species in New Zealand.

Eradication:

As with any parasitoid, if T. japonicus did escape and establish a self-sustaining population, it would probably be difficult or impossible to eradicate. Eradication attempts

Application Form Approval for new organism in containment

would likely require broad scale application of insecticides, with the associated risk to human and environmental health.

3.2. Regulatory status of the organism

Is the organism that is the subject of this application also the subject of:

An innovative medicine application as defined in section 23A of the Medicines Act 1981?

☐ Yes  No

An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?

☐ Yes No

4. Information about the containment

4.1. For field tests: The nature and method of the field test

Describe the nature and method of the field test and the experimental procedures to be used

N/A

4.2. Proposed containment of the new organism(s) (physical and operational)

Describe how you propose to contain the new organism(s) after taking into account its ability to escape from containment (i.e. the possible pathways for escape)

Trissolcus japonicus will be imported to New Zealand by air cargo, as immature life- stages developing within eggs of BMSB. The BMSB egg rafts will be supplied from a USDA laboratory in Maryland, where the parasitoid is maintained continuously for host-testing on US native pentatomid bugs. There will not be any other parasitoids in the shipment.

All eggs in an egg mass of BMSB are usually parasitised by one T. japonicus female. Skilled technical personnel within the Maryland laboratory will first select healthy BMSB egg rafts for for shipment to New Zealand from their laboratory culture. They will then expose them to ovipositing T. japonicus females for a sufficient length of time that there is a very high probability that all will be parasitized. The parasitized eggs will then be immediately packaged for shipment to New Zealand.

Application Form Approval for new organism in containment

The insects will be packaged and despatched from Maryland by air, according to standard IATA requirements for shipping live insects. On arrival in New Zealand, and once cleared for importation, the unopened package will be transferred directly to PFR’s Transitional and Containment Facility for Invertebrates (TCFI) at MARC, preferably by PFR personnel or by courier if directed by MPI.

Containment in New Zealand is achieved by (i) identifying possible pathways for escape and then (ii) applying procedures to prevent escape via these pathways. These procedures have been developed into ‘best practice’ over the past 30 years or so by Biocontrol specialists in the CRIs and preceding organisations. These procedures are detailed in PFR’s containment manual for this facility.

On arrival in New Zealand, there are the following possible pathways for escape:

 During transport from the border to the TCFI.

 By accidental/ unobserved adult flight from the TCFI.

 On contaminated laboratory equipment from the TCFI.

 By accidental/ unintentional or deliberate removal from the TCFI by human intervention.

 Following a natural disaster (e.g. earthquake) or accident (e.g. flooding or fire) that breaches the physical fabric of the TCFI.

These pathways will be mitigated as follows:

Escape of organisms during On arrival at Auckland airport, the package with T. japonicus transport to containment remains sealed until it arrives in the designated containment facilities facility, and so the insects are contained within the triple layer detailed by IATA requirements. Border security agencies are instructed not to open the package. The package is, by preference, collected by PFR staff and carried directly to the containment facility at Mt Albert; or it is couriered to PFR at Mt Albert if so directed by MPI border staff. Accidental/unintentional Authorised persons permitted entry have received training in removal by authorised containment protocols to reduce the risk of escape. All project personal staff will be required to wear protective clothing in the containment facility which is to be removed before leaving. The four levels of confinement as described above along with other measures such as sticky tape traps will further reduce the likelihood of escape via this pathway. Deliberate removal by Security measures in place mitigate the risk of unauthorised unauthorised personal personell gaining access to the facility. This includes: swipe cards only being issued to approved facility users. Escape from contaminated Sterilisation of waste reduces the risk of organisms escaping. All laboratory equipment and solid rubbish and the small quantities of liquid waste generated waste by the insects in the secure rooms is triple-bagged and autoclaved. The facility is kept in a clean and tidy condition with no unnecessary rubbish. Floors are mopped regularly. All rearing containers are sterilised between uses. The room is disinfected, thoroughly cleaned, and sterilised between projects.

Application Form Approval for new organism in containment

Escape due to a failure of There is inbuilt redundancy in the physical structure of the containment barriers containment design that reduces the risk of fracture during earthquakes. In the event of devastating fire, biohazard labelling on the facility will allow the insects to be incinerated rather than risk their escape.

Once within the TCFI, the insects will be managed by implementing procedures detailed in PFR’s management plan for T. japonicus.

For the insects arriving from the USA, each BMSB egg raft will be isolated from all others in the shipment until all parasitoids have emerged. Any remaining unhatched eggs, and any emerging BMSB nymphs will be killed and autoclaved according to the management plan protocols.

In addition to the standard containment procedures that form an integral part of the ongoing registration of the TCFI with MPI, PFR has developed a management plan which details how T. japonicus will be held in containment, and how the possible pathways for escape are blocked (see Appendix 1).

5. Māori engagement

Discuss any engagement or consultation with Māori undertaken and summarise the outcomes. Please refer to the EPA policy ‘Engaging with Māori for applications to the EPA’ on our website (www.epa.govt.nz) or contact the EPA for advice.

Tangata whenua have been consulted about the use of the containment facility in Auckland for biological control, but have not been directly approached about importation of this specific parasitoid into containment. Nationwide consultation with Maori will be undertaken before any application is made for T. japonicus to be released in New Zealand.

We forsee no negative effects on the relationship of Maori and their culture, traditions and ancestral lands. If T. japonicus were to be released, then it will be beneficial to both the environment and to many commercial horticultural ventures in which Maori have interests.

6. Risks, costs and benefits

Provide information of the risks, costs and benefits of the new organism(s).

Application Form Approval for new organism in containment

These are the positive and adverse effects referred to in the HSNO Act. It is easier to regard risks and costs as being adverse (or negative) and benefits as being positive. In considering risks, cost and benefits, it is important to look at both the likelihood of occurrence (probability) and the potential magnitude of the consequences, and to look at distribution effects (who bears the costs, benefits and risks).

Consider the adverse or positive effects in the context of this application on the environment (e.g. could the organism cause any significant displacement of any native species within its natural habitat, cause any significant deterioration of natural habitats or cause significant adverse effect to New Zealand’s inherent genetic diversity, or is the organism likely to cause disease, be parasitic, or become a vector for animal or plant disease?), human health and safety, the relationship of Māori to the environment, the principles of the Treaty of Waitangi, society and the community, the market economy and New Zealand’s international obligations.

You must fully complete this section referencing supporting material. You will need to provide a description of where the information in the application has been sourced from, e.g. from in-house research, independent research, technical literature, community or other consultation, and provide that information with this application.

Benefits:  Improved knowledge and understanding of the biology of Trissolcus japonicus and its possible non-target impacts in New Zealand.

 Skills in the development of insect rearing and managing the biological relationship between T. japonicus and its potential hosts.

 Discoveries that will underpin a likely request, in due course, for release of T. japonicus into New Zealand for BMSB biocontrol.

 Development of strategies by New Zealand’s horticultural industries and Plant & Food Research for improved sustainable pest management and enhanced biological control of existing and invasive exotic pests.

Adverse effects: The only conceivable adverse effect of T. japonicus on New Zealand’s environment, in the unlikely event of its escape from containment, is the potential to adversely affect a native insect in the superfamily family Pentatomoidea. This is considered unlikely because:

 Global evidence indicates that the parasitoid will only rarely attack species other than BMSB. Potential attacks elsewhere are limited to species in the Pentatomidae and Scutellaridae, and the family Scutellaridae is not present in New Zealand. Most of the New Zealand species of Pentatomidae originated from other countries, and even if T. japonicus can potentially attack an endemic insect, evidence from other countries suggest that it is very unlikely to have an impact on the population or the species’ survival. Other ecological consequences (such as intra- specific competition with other parasitoids) are also likely to be negligible.

Application Form Approval for new organism in containment

 The purpose of importing T. japonicus into containment is to determine how small these risks and consequences are.

Application Form Approval for new organism in containment

7. Alternative methods and potential effects from the transfer of genetic elements This section is for developments of GMOs that take place outdoors and field tests of GMOs only

 Discuss if there are alternative methods of achieving the research objective.  Discuss whether there could be effects resulting from the transfer of genetic elements to other organisms in or around the site of the development or field test.

N/A

8. Pathway determination and rapid assessment This section is for the imports of GMOs only

Under section 42B of the HSNO Act your application may be eligible for a rapid assessment. The pathway for your application will be determined after its formal receipt, based on the data provided in this application form. If you would like your application to be considered for rapid assessment (as per the criteria below), we require you to complete this section.

8.1. Discuss whether the GMO(s) to be imported fulfil the criteria The criteria are:  The host organism(s) are Category 1 or 2 host organisms as per the HSNO (Low Risk Genetic Modification) Regulations  The genetic modifications are Category A or B modifications as per the HSNO (Low Risk Genetic Modification) Regulations and the modifications are not listed in the Schedule of these Regulations  The minimum containment of the GMO(s) will be as per the HSNO (Low Risk Genetic Modification) Regulations (PC1 or PC2 as per AS/NZS2243.3:2002)

N/A

9. Other information

Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application.

Appendix 1.

PFR management plan for holding Trissolcus japonicus in containment in New Zealand. Purpose. The purpose of this management plan is to provide appropriate controls for the containment of egg parasitoids of Brown Marmorated Stink Bug (BMSB). The controls are written specifically for Trissolcus japonicus, but, due to the similar size and biological characteristics of all egg parasitoids of Pentatomidae, they will also apply to any other egg parasitoids of stink bugs that are imported into containment in the future.

Application Form Approval for new organism in containment

Trissolcus japonicus parasitizes BMSB eggs, and is a potential biological control agent. The small size of the insects creates specific risks during transport and handling in containment which are addressed in this management plan. All measures described below are designed to ensure containment at each of these risk stages.

At the time of writing, BMSB is presumed absent from New Zealand. Our goal is to determine the threat posed to New Zealand’s pentatomid fauna by exposing eggs of these species to T. japonicus and recording their fate. In the absence of BMSB we do not expect to be able to maintain a reproducing culture of the parasitoids in containment, and plan for multiple importations of parasitoids to provide sufficient adults for these trials. The adult parasitoids are relatively long-lived and are expected to be available for host-range testing experiments for 1-2 months.

Requirements for Containment. The parasitoids will only be held in PFR’s invertebrate containment facilities at Mt Albert which is approved under the MPI/EPA Standard 154.02.08 (Transitional and Containment Facilities for Invertebrates). They will be held for research purposes only. Any insects not required for research will be destroyed. All staff and students conducting scientific research using the parasitoids will be trained annually on the containment procedures outlined in the facility’s Quality Assurance Operating Manual (QAOM) and this management plan.

Specific procedures and controls to be followed are outlined below.

Requirements for containment facility. The parasitoids will be held at all times in PFR’s Mt Albert invertebrate containment facility. Construction and specifications of the facility and containment rooms used to house the parasitoids are described in the QAOM.

Detailed daily plans for managing the adult parasitoids will be developed as their biology is discovered. Daily management plans and appropriate responses to unexpected or emergency events will be prominently displayed on a wall of each room in which the parasitoids are held.

Maintaining Containment in the Invertebrate Containment Facility. All insects will be held behind at least 4 containment barriers within the facility: (1)The individual tubes within boxes, or cages in which the BMSB eggs, immature and later adult parasitoids develop; (2) the room in which they are held; (3) the corridor into which the room opens; and (4) the baffle chamber linking the corridor with the external (non- containment) laboratory.

All staff will wear personalised protective clothing in the facility at all times when working with live insects. The protective clothing is removed before leaving the containment facility and stored in a dedicated deep-freezer within the facility between uses to kill any insects.

No live material (either T. japonicus, or pentatomid eggs) will leave the Invertebrate Containment building. Any live surplus material will be killed by placing it into the containment freezer (-20oC) and holding for a minimum of 24 hours. All dead insects or used surplus material (host plant material etc.) will be placed in triple walled rubbish bags within the containment room. The bags will be tied and taped shut and then autoclaved within the IQF service laboratory. Re-useable rearing containers will be placed into the containment freezer after use for a minimum of 24 hours. The containers will then be washed and treated in the IQF washroom either with alcohol or appropriate disinfectant as detailed in the QAOM.

Application Form Approval for new organism in containment

Managing insects in the rooms. Two or three yellow sticky traps will be hung permanently in each room and monitored daily for any adult parasitoids. Parasitoids are attracted to the colour yellow, and so the traps will remove individual parasitoids that have breached the first containment layers (from individual tubes, boxes or cages) into the room. In the event of an accidental escape during handling, escapees will be recaptured or killed. A spray bottle of 70% alcohol will also be available in each room, and used when flying insects are observed. They are killed when hit by a spray of alcohol.

No parasitoids should be trapped when they are not being worked with. If parasitoids are consistently trapped on the yellow cards – indicating an unexpected breach – the source of egress (e.g. a hole in a cage) will be located and repaired.

In the event of accidental breakage of a cage or container, from which an unknown number of wasps are freed into the room, then the room-temperature will be raised to c.35oC overnight to kill them. In such an event, the discoverer will attempt to recover as many parasitoids as possible before leaving the room to raise the temperature. Such parasitoids will be transferred to secure boxes and placed in a single plastic bag. The plastic bag will be checked to ensure that no insects are present on the outside, and will be transferred rapidly to an empty containment room (to be designated), and placed in a perspex box. The bag will remain there until any insects in the original room are dead. Then it will be transferred back to the original room.

Response to insects in the corridor. Yellow sticky traps will also be permanently placed in the inner corridor of the facility and regularly monitored. Parasitoids are attracted to the colour yellow, and so the traps will remove individual parasitoids in the corridor. Any not caught in the trap will soon die through lack of water and food – probably within 24h.

Any parasitoids caught in the corridor will trigger a full review of these management plans.

Response to insects in the baffle chamber. The baffle chamber contains a light trap that is permanently illuminated and holds a yellow sticky trap. The primary purpose is to monitor for any insects that enter the facility from the service laboratory. Any insect caught in this trap is immediately identified. If the insect is a parasitoid from containment, then a full review and internal audit of the management plans for insects in the rooms and corridor will be carried out to identify how it arrived there, and to prevent a re-occurrence.

The review and corrective actions will be completed within 24h of the insect being detected.

Response to insects outside the containment facility

Contingency plans for the accidental release or escape of the parasitoids will be held at the entrance to the facility, together with all other documentation.

In the unlikely event of an escape from the facility to the service laboratory, or the discovery of the parasitoid outside the containment facility, the CSM and PFR’s Containment Response Team will be immediately contacted. MPI will be informed within 24 hours of the event. Further action will be implemented in agreement with MPI, recognising that eradication may not be feasible or possible, and that a response is likely to be limited.

Application Form Approval for new organism in containment

In the absence of BMSB from New Zealand, the threat posed to the environment by an escape of T. japonicus egg parasitoids is considered low, as it is predicted that New Zealand’s pentatomid fauna will not be significantly attacked. Over time, our experiments in the facility will support or refute this hypothesis. In addition, the New Zealand fauna is likely to be temporally isolated from the threat from T. japonicus between February and October. This is because, as far as is known, the native species of Pentatomidae are univoltine in the wild (have only one generation per year). They lay their eggs between October and December, and it is predicted that they have all hatched by the end of January.

Emergency contact details are provided in the QAOM page 9-10 and Appendix 8; and on the whiteboard immediately outside the baffle chamber.

Limiting access to the Containment Facility Measures for access control to the facility are identified in the QAOM (page 13) including swipe card access issued to authorise trained staff only. Visitors to the facility must be accompanied by authorised staff.

Entry is through one primary point into the facility. Procedures for receiving specimens into the facility are outlined in Appendix 9.2 and 9.3 of the QAOM.

A checklist for each visit to the facility is followed as outlined in QAOM 9.4.

Requirements for staff training Staff using the Invertebrate Containment Facility attend annual containment training modules provided by the Containment Sector Manager and Facility Manager at Mt Albert. A competency based questionnaire is provided to staff and students at the conclusion of each training session with a minimum pass mark (85%). A lesser pass mark means repeat training is required. All project staff using the facility are aware of all the species held in the facility at any one time and are able to identify them should they be found trapped in ‘common’ areas of containment (such as the corridor or baffle chamber).

Only staff with training specific to T. japonicus and the BMSB biocontrol programme will enter the containment rooms holding egg parasitoids.

Site visitors and maintenance staff (cleaners etc) are not permitted entry unless they have completed these training modules or unless accompanied at all times by a staff member who has completed them.

In addition specific Quarantine Training Course training is provided (QAOM Appendix 3) by the Containment Facility Manager and records and dates of training are maintained.

Measures to ensure freedom from associated organisms, and to prevent escape during importation, while held in containment, and if appropriate, while transferring between containment facilities. The insects will be couriered by air from laboratories in the USA, initially from the laboratory of Dr Kim Hoelmer (USDA-ARS, Newark, Delaware), where research into biocontrol of BMSB by egg parasitoids is underway. The cultures are presumed to be healthy and free of associated organisms, because that is a requirement of the research in the USA. Parasitoids will be shipped as immature stages growing within eggs of BMSB. Each BMSB egg cluster contains about 28 BMSB eggs, each of which will contain a developing T. japonicus larva. It is expected that each egg cluster will be shipped within a separate plastic tube.

On arrival in New Zealand, and after biosecurity clearance, the shipment will be taken to the Plant & Food Research Invertebrate Containment Facility, Mt Albert, Auckland. The

Application Form Approval for new organism in containment

preferred option for transportation is to have trained Plant & Food Research staff pick up the egg packages from the border and transport them directly to the containment Facility in Mt Albert.

On arrival within the Facility, the tubes will be unpacked, with careful visual examination for any live BMSB life-stage within the packaging, and the packaging destroyed by autoclaving. All BMSB eggs will be visually re-inspected. Any eggs showing symptoms of disease infection (e.g. fungal, bacterial, viral, etc) will be destroyed by first freezing for >24h (at -20oC) and then by autoclaving.

All BMSB eggs, with developing T. japonicus, will be held in a different room to any New Zealand insect or plant material. On arrival, each BMSB egg cluster will be transferred to a clean tube and held at 21-250C and a 16h photoperiod in a room (designated ‘room 1’) until adult parasitoids emerge. Any BMSB nymphs that emerge from unparasitised eggs will be isolated within a tube and frozen. They will be offered to MPI or NZAC staff for curation as reference material.

When all parasitoids have emerged from eggs in room 1, the remnant insect material will be triple-bagged and destroyed by autoclaving within the facility.

Emerged adult parasitoids (male and female) will be transferred to vented, secure, holding containers, and stored in the dark at 13oC until required, with a food supply of honey to maximise their longevity. When required for host-range testing, individual, mated, female parasitoids will be aspirated from a holding container into a glass vial (c. 75 x 25mm with a secure lid) containing a fresh raft of pentatomid eggs (attached to a labelled card) and a small smear of honey. The pentatomid eggs will all be from species already present in New Zealand. Cultures of the New Zealand pentatomid species will be held in non-containment laboratories and egg-rafts will be brought into containment for the purpose of host-range testing. In a typical test, an egg raft will be exposed to a single female adult parasitoid for 24 hours at 20oC, a 16h photoperiod and 61% RH). The parasitoid will then be removed and returned to a holding container, and the egg raft (inside the test vial) will be incubated for a further 45 days under the same physical environment. Test vials from any one day will be bundled together and the bundles of test vials will be held on trays within a secure Perspex container for the incubation period. Vials will be checked every 2-3 days and any pentatomid nymphs and/or parasitoid adults that emerge will be recorded and removed from the test vial. After identification, the emerged nymphs and parasitoids will be destroyed by freezing for at least 24 hours. At the end of the test period the egg raft will be frozen for at least 24 hours then dissected to determine the fate of unhatched eggs.

It is envisaged that most testing will be done in glass vials described above. For some more detailed experiments the parasitoids may also be held in framed cages with nylon gauze or muslin covers with mesh sufficiently fine to prevent egress of individual adults. ‘Standard’ mesh size will be c.0.3mm2. The suitability of any alternative mesh size (for example from a new commercial gauze supplier) will be determined by holding c 10 parasitoids in a small trial cage (a few cm2), held within a Perspex box. If parasitoids remain contained within the trial cage, then the new mesh will be considered suitable for larger, experimental or containment cages.

To confirm freedom from insect pathogens, up to 15 adult parasitoids per shipment will be selected and killed in a -20oC freezer for >24h. They will be examined microscopically by Dr Louise Malone (a recognized insect pathologist at PFR) to confirm absence of any diseases. If any disease or unwanted micro-organisms are found, further testing will isolate the infected egg batches in the shipment, which will be destroyed. If the disease

Application Form Approval for new organism in containment

is such that it cannot be removed, then the entire shipment of parasitoids will be destroyed.

To confirm the identity of the parasitoids, at least 20 voucher specimens (from each laboratory source received) will be curated and deposited at the NZAC, Landcare Research, Tamaki (send to Dr Darren Ward, curator of Hymenoptera).

Treatment of waste, killing organisms before disposal Procedures will be followed for treatment of waste and insects not required for research purposes as outlined in the QAOM.

Inspection of the containment facility and access by MPI; inspection following events, remedial requirements and structural modifications. Internal auditing of the facility is conducted every 6 months as outlined in the QAOM (page 4). The CFM and CSM will inspect the facility as soon as possible after any Act of God, or unauthorised attempt to enter the facility. Any compromise to containment as a result of such event will be remedied as soon as possible, and reported to the Operator (or nominated Manager) and the Facility’s MPI Supervisor.

Remedial containment requirements identified by the approval holder or MPI (as applicable) will be completed as soon as possible, including such interim measures as are necessary to mitigate the risk of breach of containment.

Additional independent audits of the facility may be conducted by PFR on behalf of the Facility Operator. The results of all inspections, audits and reviews are recorded in the ‘Regulations’ folder and held for 5 years.

Internal and external MPI audits are monitored by the Nominated Manager and overall trends reported to the Containment Sector, Facility Managers and Biosafety Advisory Committee if significant issues are identified.

Prior to significant work being undertaken on a containment facility which may result in structural modifications, a risk assessment is completed by the CSM or CFM with the contractor and a ‘Permit to Work’, reviewed by the Nominated Manager, and provided to MPI for approval before any work commences on the facility.

Application Form Approval for new organism in containment

10. Checklist This checklist is to be completed by the applicant

Application Comments/justifications All sections of the application form completed ☐ Yes ☐ No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)

Confidential data as part of a separate, ☐ Yes ☐ No identified appendix

Supplementary optional information attached:

 Copies of additional references ☐ Yes ☐ No

 Relevant correspondence ☐ Yes ☐ No

Administration Are you an approved EPA customer? ☐ Yes ☐ No If Yes are you an: Applicant: ☐ Agent: ☐

If you are not an approved customer, payment of fee will be by:  Direct credit made to the EPA bank ☐ Yes ☐ No account (preferred method of payment) ☐ Payment to follow Date of direct credit:

 Cheque for application fee enclosed ☐ Yes ☐ No ☐ Payment to follow

Electronic, signed copy of application e-mailed ☐ Yes to the EPA

Application Form Approval for new organism in containment

Signature of applicant or person authorised to sign on behalf of applicant

☐ I am making this application, or am authorised to sign on behalf of the applicant or applicant ＊ organisation.

＊ I have completed this application to the best of my ability and, as far as I am aware, the ☐ information I have provided in this application form is correct.

8 Sept 2015

Signature Date

Request for information waiver under section 59 of the HSNO Act

I request for the Authority to waive any legislative information requirements (i.e. concerning the ☐ information that has been supplied in my application) that my application does not meet (tick if applicable).

Please list below which section(s) of this form are relevant to the information waiver request:

Application Form Approval for new organism in containment

Appendices and referenced material (if any) and glossary (if required)

List of References and further reading: Charles JG. July 2015. Preparing for classical biological control of Brown Marmorated Stinkbug in New Zealand: why CBC will be necessary, and the potential environmental impact of an exotic egg parasitoid, Trissolcus japonicus. A Plant & Food Research report prepared for B3. Milestone No. 58918. Job code: P/321021/03. SPTS No. 11538. 30pp.

Cumber RA 1964. The egg-parasite complex (Scelionidae: Hymenoptera) of shield bugs (Pentatomidae, Acanthosomatidae: Heteroptera) in New Zealand. New Zealand journal of science 7 (4): 536-554.

Duthie C 2012. Risk analysis of Halyomorpha halys (brown marmorated stinkbug) on all pathways. MPI report November 2012. 51p.

Johnson NF 1991. Revision of Australasian Trissolcus species (Hymenoptera: Scelionidae). Invertebrate Taxonomy, 5: 211–239.

Lee D-H, Short BD, Joseph SV, Berch JC, Leskey TC 2013. Review of the biology, ecology and management of Halyomorpha halys (Hemiptera: Pentatomidae) in China, Japan, and the Republic of Korea. Environmental Entomology 42(4): 627-641.

Leskey TC, Hamilton GC, Nielsen AL, Polk DF, Rodriguez-Saona C, Bergh JC, Herbert DA, Kuhar TP, Pfeiffer D, Dively GP, Hooks CRR, Raupp MJ, Shrewsbury PM, Krawczyk G, Shearer PW, Whalen J, Koplinka-Loehr C, Myers E, Inkley D, Hoelmer K, Lee D-H, Wright SE 2012a. Pest status of the brown marmorated stink bug, Halyomorpha halys in the USA. Outlooks on Pest Management 23(5): 218-226.

Rice KB, Bergh CJ, Bergmann EJ, Biddinger DJ, Dieckhoff C, Dively G, Fraser H, Gariepy T, Hamilton G, Haye T, Herbert A, Hoelmer K, Hooks CR, Jones A, Krawczyk G, Kuhar T, Martinson H, Mitchell W, Nielson AL, Pfeiffer DG, Raupp MJ, Rodriguez-Saona C, Shearer P, Shrewsbury P, Venugopal PD, Whalen J, Wiman NG, Leskey TC, Tooker JF 2014. Biology, Ecology, and management of brown marmorated stink bug (Hemiptera: Pentatomidae) Journal of Integrated Pest Management 5(3): 1-13.

Talamas EJ, Buffington M, Hoelmer K (2013). New synonymy of Trissolcus halyomorphae Yang. Journal of Hymenoptera Research 33:113-117.

Talamas EJ, Johnson NF, Buffington M 2015a. Key to Nearctic species of Trissolcus Ashmead (Hymenoptera, Scelionidae), natural enemies of native and invasive stink bugs (Hemiptera, Pentatomidae). Journal of Hymenoptera Research 43: 45 – 110.

Talamas EJ, Herlihy MV, Dieckhoff C, Hoelmer KA, Buffington ML, Bon M-C, Weber DC 2015b. Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae) emerges in North America. Journal of Hymenoptera Research 43: 119-128.

Valentine EW 1964. A note on the Asolcus spp. (Scelionidae: Hymenoptera) parasitic upon shield bug eggs (Pentatomidae, Acanthosomatidae: Heteroptera) in New Zealand. New Zealand Journal of Science 7(4): 643.

Yang ZQ, Yao YX, Qiu LF, Li ZX 2009. A new species of Trissolcus (Hymenoptera: Scelionidae) parasitizing eggs of Halyomorpha halys (Heteroptera: Pentatomidae) in

Application Form Approval for new organism in containment

China with comments on its biology. Annals of the Entomological Society of America 102(1): 39-47.

Glossary: Egg Parasitoid: A parasitoid is an insect that develops on or within their host (usually another insect or arthropod). Egg parasitoids develop only within the eggs of their hosts. Although the host egg may not initially show any effects, it is eventually killed by the parasitoid (unlike a parasite, which requires its host to survive).

Endoparasitoid: A parasitoid that develops inside the host insect. A single T. japonicus completes its entire development within a host’s egg, and emerges as an adult wasp.