APPLICATION FORM Release

To obtain approval to release new organisms (Through importing for release or releasing from 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 APP203853

Date 28 June 2019

www.epa.govt.nz

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Application Form Approval to release a new organism

Completing this application form

1. This form has been approved under section 34 of the Hazardous Substances and New Organisms (HSNO) Act 1996. It covers the release without controls of any new organism (including genetically modified organisms (GMOs)) that is to be imported for release or released from containment. It also covers the release with or without controls of low risk new organisms (qualifying organisms) in human and veterinary medicines. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency, conditional release or containment), a different form will have to be used. All forms are available on our website. 2. 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. 3. 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. 4. 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. 5. Please add extra rows/tables where needed. 6. 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. 7. Information about application fees is available on the EPA website. 8. All application communications from the EPA will be provided electronically, unless you specifically request otherwise. Commercially sensitive information

9. 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. 10. 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.

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Application Form Approval to release a new organism

11. 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 maybe 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

As defined in section 3 of the Medicines Act 1981 Medicine http://www.legislation.govt.nz/act/public/1981/0118/latest/DLM53790.html?src= qs

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; • An organism for which a conditional release approval has been given under the HSNO Act; New Organism • 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 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

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Application Form Approval to release a new organism

• It is an incidentally imported new organism

Qualifying Organism As defined in sections 2 and 38I of the HSNO Act To allow the organism to move within New Zealand free of any restrictions Release other than those imposed in accordance with the Biosecurity Act 1993 or the Conservation Act 1987

As defined in section 2 of the Biosecurity Act 1993 Unwanted Organism http://www.legislation.govt.nz/act/public/1993/0095/latest/DLM314623.html?src =qs

As defined in section 2(1) of the Agricultural Compounds and Veterinary Medicines Act 1997

Veterinary Medicine http://www.legislation.govt.nz/act/public/1997/0087/latest/DLM414577.html?se arch=ts_act%40bill%40regulation%40deemedreg_Agricultural+Compounds+a nd+Veterinary+Medicines+Act+_resel_25_a&p=1

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Application Form Approval to release a new organism

1. Applicant details

1.1. Applicant

Company Name: (if applicable) New Zealand Forest Research Institute Limited (Trading as Scion)

Contact Name: Stephanie Sopow

Job Title: Entomologist

Physical Address: Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua

Postal Address (provide only if not the same as the physical): Private Bag 3020, Rotorua 3046

Phone (office and/or mobile): 07 343 5877 / 027 727 2090

Fax: 07 348 0952

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:

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Application Form Approval to release a new organism

2. Information about the application

2.1. Brief application description

Approximately 30 words about what you are applying to do

We are applying to release a parasitoid wasp, Pauesia nigrovaria (Provancher) (Hymenoptera: Braconidae: Aphidiinae), from containment as a biological control agent to provide long-term sustainable control of the invasive giant willow aphid, Tuberolachnus salignus (Gmelin) (Hemiptera: Aphididae: Lachninae).

2.2. Summary of application

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

This application is for permission to release Pauesia nigrovaria as a biological control agent of the giant willow aphid (GWA). Pauesia nigrovaria has been thoroughly assessed for its impact against potential non-target hosts in a containment facility. GWA is an exotic pest causing numerous problems throughout New Zealand, particularly for the apiculture sector. Impacts from GWA include willow tree dieback, compromised honey and increased pest social wasp populations that result in bee colony losses.

Options for sustainable control of GWA in New Zealand are limited. The use of chemicals is both undesirable and impractical as aphids are widespread throughout the country, in both rural and urban environments. Even for isolated cases, for example to protect iconic trees, spot spraying an entire large canopy would be difficult. Systemic insecticides applied by injection or soil application pose unacceptable risks to other organisms. Systemic insecticides ingested by GWA would be expelled in honeydew before they die and could harm honeydew feeders, e.g., bees and some native birds.

Control of pest willows in wetlands (e.g. Bodmin and Champion 2010), is not a practical option to control the giant willow aphid. Pest willows (Salix fragilis and S. cinerea) poisoned or removed from wetlands will only reduce giant willow aphid populations in very localised areas. However, non-pest willow species are widespread and abundant throughout the New Zealand landscape and are highly susceptible to GWA. These species are highly valued for slope stabilisation, the protection of stream banks from erosion, crop and livestock shelter, the provision of fodder, basket weaving, and spring bee nutrition (pollen and nectar) when other resources are scarce. The introduction of natural enemies from a pest’s place of origin (termed classical biological control) can be a very effective and environmentally sustainable option for reducing the impact of a pest. The greatest risk of classical biological control is that the

20 imported agent has unforeseen impacts on native or beneficial organisms. For this reason, selecting a highly specific parasitoid (rather than a generalist predator) is the best option. Furthermore, the chosen organism must be rigorously assessed for its potential to impact non-target organisms.

Parasitoid wasps have been observed attacking GWA in eastern Asia and the United States. We collected, reared and completed a safety assessment of Pauesia nigrovaria, a GWA parasitoid known only from California. Almost all species of Pauesia are known to specifically attack aphids in the subfamily Lachninae. The few exceptions primarily attack species of lachnines, but have been recorded on rare occasions also attacking other aphid species on the same host plants as their main host species (Žikić et al. 2016). Some of these records may be spurious as the most polyphagous Pauesia species also happen to be the most common and well-studied European species, such as P. pini (Völkl & Novak 1997), P. unilachni (Völkl & Kraus 1996), and P. picta (Völkl 2000). The more information added to plant/herbivore records, the less reliable the information tends to be, since there is a tendency for marginal host records and mistaken identifications to become included. There are no native Lachninae in New Zealand, only GWA and seven other invasive pest species from three genera. Pauesia nigrovaria is an excellent candidate for biological control of GWA in New Zealand because of its host specificity and our lack of closely related native aphids.

2.3. Background and aims of application

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

GWA in New Zealand and the proposed biological control project

The giant willow aphid (GWA), Tuberolachnus salignus (Gmelin) (Hemiptera: Aphididae: Lachninae), was first recorded in New Zealand in 2013 (Gunawardana et al. 2014). Since then it has spread rapidly throughout the country, with over 50 species and hybrids of willow (Salix spp.) now confirmed as GWA hosts. In addition, some poplars (Populus spp.), apple trees (Malus spp.), pear trees (Pyrus spp.) and Coprosma macrocarpa subsp. minor (coastal karamu), a New Zealand native, are known hosts (Sopow et al. 2014). As the common name suggests, giant willow aphids are the largest species of aphid in the world, reaching up to 6 mm in length. They reproduce entirely through parthenogenesis, with females giving birth to live clones of themselves. They can give birth to approximately four offspring per day at 20°C (S. Sopow, pers. obs.). Males are unknown. Individual aphids live for about a month, taking around two weeks to grow through four nymphal instars (stages) before reaching adulthood. At cooler temperatures this lifecycle can extend to three months. GWA are very scarce in the spring, often only found in ones or twos. Larger colonies do not start to form until well into summer, with peaks in abundance occurring in early to mid-autumn. Most adults are wingless, but as winter approaches, more winged adults (alates) are produced. It is presumed that GWA overwinters as winged adults, perhaps by

21 hiding in crevices or underground as they await warmer weather. Colonies on host trees persist into winter and begin to disappear from June onwards. Giant willow aphid has significant negative impact on willow tree health, growth and survival (Sopow et al. 2017). This is of concern because of the important role of willows in rural land stabilisation and the prevention of soil erosion along river banks (Isebrands et al. 2014). Willows are extremely important to New Zealand’s thriving apiculture industry as their flowers are an important early spring source of pollen and nectar that builds colony strength when few other resources are available (Newstrom-Lloyd et al. 2015). Honeydew produced by GWA feeding on willows is also collected by bees and results in crystallised ‘cement honey’ that is difficult to extract from hives and unmarketable (Sopow et al. 2017). Furthermore, GWA honeydew availability supports an exponential increase in the vespid wasp (Hymenoptera: Vespidae) population in our urban and rural environments (analogous to the devastating beech honeydew wasp system, Beggs 2001) that has significant environmental, public health and nuisance impacts (Sopow et al. 2014). Since willows are widespread throughout New Zealand, in both rural and urban areas, there are virtually no parts of New Zealand that are exempt from the negative impacts of GWA. Some predatory invertebrates (including several species of ladybird beetles (Coccinellidae), and lacewing larvae (Neuroptera)) have been observed feeding on GWA in New Zealand (Sopow et al. 2017). Most are generalists and feed on a wide variety of species. There are no quantitative data on the efficacy of any of the predators feeding on GWA in New Zealand, however none have shown any observable impact on GWA populations to date. Moreover, generalist predators can have highly detrimental effects on non-target species including native or rare species (Rand et al. 2016). A prime example of this is the recent arrival of the harlequin ladybird, Harmonia axyridis. This large ladybird has been observed feeding on GWA and may exert some population level control in the future (no data are currently available), however as a generalist predator, it feeds on a wide variety of soft- bodied insects. Once touted as a potential biological control agent for pest aphids in Europe and North America, the harlequin ladybird is now considered a very destructive invasive species because of its impacts on native insect populations, and its propensity to congregate in inappropriate overwintering shelters, such as bee hives and human dwellings (https://www.cabi.org/ISC/datasheet/26515). In New Zealand, giant willow aphid appears to be a favoured food source for the harlequin ladybird (Bennett, pers. comm.). Pauesia nigrovaria will reduce GWA numbers and therefore potentially reduce the abundance of harlequin ladybirds is areas with high numbers of willows. However, reductions in GWA may have little impact on harlequin ladybird abundances at larger spatial scales, since they are generalist feeders. Parasitoids are typically more specialised than predatory insects (Le Ralec et al. 2010; Žikić et al. 2016). Thus, they exert a continuous measure of control and have less, if any, direct impacts on non-target species (see Paynter & Teulon 2019). Unfortunately, no parasitoids of GWA are present in New Zealand. In its native range, GWA is attacked by two species of parasitoid wasp, Pauesia salignae (Hymenoptera: Braconidae: Aphidiinae) in East Asia and Pauesia nigrovaria (Provancher) in North America. Very little is known about the

22 biology of either species. For the biological control effort detailed here, we selected P. nigrovaria because we confirmed its presence in field populations first.

Pauesia

The biology of all Pauesia species, and indeed all members of the subfamily Aphidiinae, is very similar. They are endoparasitoids that feed internally on nymphs and adults of their host aphid species, killing them in the process. Most aphidiine parasitoids attack only a few species of aphids, and Pauesia species are among the most specialised. Of the 75 described Pauesia species, 69 have been recorded only attacking aphids in the subfamily Lachninae (Žikić et al. 2016). Of these, 58 are specialised to single genera (mostly conifer aphids from the genus Cinara), and 39 are known from a single host species (Žikić et al. 2016). Even the few generalist Paeusia species may not be what they seem (see Carver 1984; Höller 1991). A recent molecular analysis of apparently generalist species within the Aphidiinae found that most are actually species complexes, comprised of multiple, morphologically indistinguishable parasitoids, each specific to a particular host (Derocles et al. 2015). The evolution of parasitoid host specificity is the result of intense selection pressure on aphids to avoid parasitism, and in turn by parasitoids to overcome the defences of their aphid hosts (see Le Ralec et al. 2010). As this arms race intensifies the parasitoids lose their ability to recognise, and thus parasitise, other aphid species. It is highly unlikely that P. nigrovaria would evolve an interest in the chemical cues of unrelated aphid species since closely-related species are more likely to be similar in the composition of chemicals on the cuticle or released into the air that the parasitoid uses for host recognition. If host confusion were to occur, in all probability it would only be with one of the very close relatives of GWA, such as a species of Cinara. Aphidiine parasitoids search for their target aphids through diminishing spatial scales from long distances to physical contact with the aphids themselves. Searching at different spatial scales utilises different, mostly chemical, stimuli (Völkl 2000). At long range, parasitoids often track host plant volatiles and not the chemical cues emitted by the aphids themselves (Powell et al. 1998). This is due to obvious differences in the quantity of volatiles given off by large host trees versus small aphids. Pauesia species are highly specialised to the plants on which their host aphids feed (Žikić et al. 2016), strongly suggesting that many, if not all, Pauesia utilise host plant volatiles to locate aphids. This phenomenon has been documented repeatedly for different species of aphidiine wasps (Powell et al. 1998; Gols et al. 2012). Once a host tree is located, the parasitoid then locates aphid colonies using airborne cues, such as honeydew, sex or alarm pheromones (Micha & Wyss 1996), or indirectly by detecting other aphid-associated species, such as honeydew- feeding ants (Völkl & Novak 1997). Some species seem to be incapable of remotely detecting aphids within host trees and simply search methodically until they encounter aphids by chance (Völkl & Kraus 1996). Finally, once an aphid is located, Pauesia species use their antennae to make physical contact. Parasitoids are able to detect chemical cues present on

23 the cuticle of the aphid to confirm its species and then trigger acceptance or rejection of the aphid based on its potential quality for supporting a developing parasitoid larva (size, general health, whether that aphid has already been parasitised, etc.) (Mackauer et al. 1996). We currently cannot confirm how Pauesia nigrovaria locates GWA over medium to large spatial scales. In an attempt to answer this question, we carried out laboratory experiments using olfactometer and host plant choice tests, but the results were inconclusive. At close range, we have observed P. nigrovaria touching aphids with their antennae, presumably to assess their quality and determine their species before accepting or rejecting that individual as a host. The behavioural observations we conducted are detailed in Section 5, but are summarized briefly here. In the case of C. fresai, a closely related pest aphid in the same subfamily as GWA (Lachninae), P. nigrovaria almost always rejected the pest aphid as a host after physical contact and did not attack it. Of 24 mated female P. nigrovaria, introduced to 5 individual C. fresai sequentially, only a single individual attacked (two) aphids. It is likely that this P. nigrovaria female simply made a host error. Dissection of these aphids showed no signs of oviposition (i.e. no egg(s) present within the body cavity) despite the apparent oviposition behaviour observed. In the case of B. persicae, which belongs to the aphid subfamily Aphidinae and is therefore less closely- related to GWA than C. fresai, no attacks were ever observed. The widespread use of host plant volatiles in the long-range tracking of target aphid species among Pauesia spp. (Völkl 2000) suggests that non-target aphid species feeding on other host plants are unlikely to be encountered by Pauesia. The use of aphid chemical cues to track target aphid species within host plants, and for the recognition and acceptance of host aphids after physical contact (Mackauer et al. 1996; and observed here), provides further assurance that non-target aphid species are unlikely to be attacked by Pauesia, even if they share a host plant with the target aphid species. Lastly, widespread suppression of GWA populations is also reliant on P. nigrovaria being tolerant of the New Zealand climate. Its natural distribution range (California) experiences a similar climate to New Zealand, occupying a near-identical latitudinal range with similar temperatures and rainfall (e.g. see https://weather-averages.co.uk/compare-climate). Our colonies of P. nigrovaria were created from specimens collected between the equivalent latitudes of Auckland and Wellington. As a specialist on GWA it is also expected that the natural range of P. nigrovaria should match that of its host, which extends far northward and southward in North America (from at least southern British Columbia to southern California), and from sea level to approximately the same elevation as Mount Cook Village (for example, GWA has been found near Las Vegas, Nevada) (https://www.inaturalist.org/taxa/354036- Tuberolachnus-salignus). Since GWA is clearly well adapted to the New Zealand climate, having rapidly spread throughout the country, it is expected that P. nigrovaria will also thrive here and eventually be found wherever GWA occurs. We have not done any modelling to predict the potential impact of climate change on populations of GWA and P. nigrovaria in New Zealand. However, assuming a change towards a warmer and drier climate, we would expect that GWA would persist, as long as its host trees continued to survive. It has been

24 recorded in Tucson, Arizona on iNaturalist, which has an average temperature in July of 38°C, with extremes into the mid-40s most years (https://www.currentresults.com/Yearly- Weather/USA/AZ/Tucson accessed 11 July 2019). We also expect that P. nigrovaria would continue to thrive since it is present in hot and dry areas such as the Los Angeles basin. It is possible that P. nigrovaria, as opposed to the alternative species P. salignae that we located in Japan, was the better choice for long term management of GWA in the face of climate change.

The vulnerability of New Zealand aphids to Pauesia nigrovaria

New Zealand is home to about 130 species of aphids (Hemiptera: Aphididae), most of which (>115 spp.) are introduced pests of exotic plants (Teulon & Stufkins 2002; Von Dohlen & Teulon 2003; MacFarlane et al. 2010). Our native aphid fauna consists of no more than 15 known species, all of which specialise on a small number of native plants (Von Dohlen & Teulon 2003; Teulon et al. 2013). Our native aphid fauna is taxonomically limited with all species belonging to just three different genetic lineages (Von Dohlen & Teulon 2003; Teulon et al. 2013). There are no New Zealand native species in the same subfamily as GWA (the Lachninae) (Table 1).

Lachninae Molecular analyses suggest that the subfamily Lachninae represents an early branch in the phylogeny of aphids (Ortiz-Rivas and Martinez-Torres 2010; Figure 1) and is therefore not closely related to any other living aphid group. Most Lachninae species are relatively large and feed on various species of conifers. They typically have robust, rounded bodies and short, pore-like siphunculi located on cone-like protuberances. It is through these siphunculi that P. nigrovaria oviposits (S. Sopow pers. obs.). Most other aphids have tube-like siphunculi, which may hinder oviposition by P. nigrovaria if a female made such an attempt. New Zealand has no native Lachninae species, but eight exotic pest species have become established here, including GWA. All except GWA are pests of exotic conifers and include two pests of pines, Essigella californica (Essig) and Eulachnus brevipilosus (Börner), and five Cinara spp. that are pests of Cupressus and Juniperus spp.. Both E. californica and E. brevipilosus are relatively small (<2.5 mm) and are thus unlikely to support the development of P. nigrovaria. Most known Pauesia species attack various species of Cinara, some of which are relatively large (>4 mm). Given their size and that they are hosts of various other Pauesia species, a large species of Cinara is therefore the most likely non-target of P. nigrovaria in the confines of an artificial laboratory experiment. In the field, it is predicted that P. nigrovaria would seldom encounter a Cinara species based on how distantly related their host plants are (willows versus conifers). In any case, attacks on Cinara species, although unlikely, could be viewed as an unexpected benefit since all Cinara species in New Zealand are exotic pests.

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Eriosomatinae This subfamily is represented in New Zealand by 11 introduced pest species. Most are gall formers or feed on plant roots (willows are not host plants), and are therefore very unlikely to encounter P. nigrovaria if it was introduced. Phylogenetically the Eriosomatinae are only distantly related to Lachninae (Ortiz-Rivas and Martinez-Torres 2010; Figure 1), hence they are very unlikely to be attacked by P. nigrovaria.

Hormaphidinae Just two exotic species are present in New Zealand from this subfamily. Both are very small (<2 mm) (Blackman & Eastop 1994) and attack monocotyledons (orchids, palms and grasses). The combination of small body size, very different host plant species and distant- relatedness to GWA (Ortiz-Rivas and Martinez-Torres 2010; Figure 1), strongly indicates that these species would not be encountered or attacked by P. nigrovaria.

Neophyllaphidinae Our only representatives of this subfamily are two closely-related endemic species that feed on tōtara (Podocarpus spp.): Neophyllaphis totarae Cottier and an undescribed Neophyllaphis species on Podocarpus nivalis Hook.. This genus displays a Gondwanan distribution and is therefore considered to be an ancient lineage within the subfamily. In the phylogeny of the aphids, this and the remaining subfamilies below that are present in New Zealand are part of various clades in the family Aphididae that are distantly related to the Lachninae, (Figure 1). While its status as an endemic species belonging to an ancient lineage means it is important to include N. totarae in our host-testing trials, its small size, very different host plant and distant phylogenetic relationship to GWA suggest it is highly unlikely to encounter, be attacked by, or support the development of P. nigrovaria.

Taiwanaphidinae The endemic southern beech aphid, Sensoriaphis nothofagi Cottier, is our only representative of this subfamily in New Zealand. Like N. totarae above, S. nothofagi belongs to an ancient lineage with a Gondwanan distribution. It was therefore deemed a priority to test this species against P. nigrovaria. However, like N. totarae, S. nothofagi is very small (<1.5 mm), lives on a completely unrelated host tree and is distantly related to GWA. It is therefore highly unlikely to be at risk from P. nigrovaria.

Phyllaphidinae The only species in this subfamily in New Zealand is the introduced beech aphid, Phyllaphis fagi (Linnaeus). It is considerably larger than other native aphids (up to 3.2 mm in length) but still substantially smaller than heavy-bodied adult GWA (5-6 mm long). P. fagi specialises on European beech (Fagus spp.), which are not closely related to willows. The biology of this species strongly suggests that P. nigrovaria is not likely to encounter or attack it, while its exotic pest status means that it was not considered to be an important species to include in host-testing trials.

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Saltusaphidinae A single introduced species, Thripsaphis foxtonensis Cottier, is present in New Zealand. Though described from New Zealand and once thought to be native, it is actually an introduced species from North America. It is a relatively small species (2.5 mm long) that feeds on sedges (Carex spp.). Consequently, this species is neither very likely to encounter or be attacked by P. nigrovaria, nor is it considered an important species to test.

Calaphidinae Thirteen exotic species from this subfamily have established themselves in New Zealand. All are pests of exotic plants, including various birches, oaks, chestnuts, alder and bamboo. One, Therioaphis trifolii (Monell), is a pest of alfalfa. Most of these species are small, with only the silver birch aphid, Euceraphis betulae (Koch), being larger than 3 mm in length (up to 4.2 mm). With no native species, small body sizes, and being found on unrelated host plants, species from this subfamily were not considered important to test or likely to be vulnerable to P. nigrovaria and were therefore not included in host- testing trials.

Chaitophorinae The two exotic species from this subfamily in New Zealand are pests of maples and sycamores (Acer spp.). Like many other subfamilies, these species were considered to be too small, distantly related and unimportant to include in host-testing trials.

Drepanosiphinae The introduced sycamore aphid, Drepanosiphum platanoidis (Schrank), is the only species in New Zealand from this subfamily. It comes relatively close to GWA in body size, reaching up to 4.3 mm in length, though it is not as robust. Its host plant (sycamore) is also distantly related to those of GWA, resulting in a very low chance that P. nigrovaria would encounter the sycamore aphid in the field. Despite that, and its comparable size, this species was considered too distantly related and unimportant to include in host-testing trials.

Aphidinae: Aphidini The subfamily Aphidinae includes more than half of all described aphid species. The two biggest tribes (Aphidini and Macrosiphini) are well represented in New Zealand and will be treated separately here. The Aphidini is the third and last taxonomic group to include endemic species. Our native Aphidini fauna includes 11 known species in four genera: six species of Aphis (including two that are undescribed), two species of Paradoxaphis, two undescribed species of Schizaphis, and an undescribed species of Casimira (Teulon et al. 2013). Two further Aphis species are known from single collections, but due to a lack of specimens and other data their status as native or introduced species has yet to be determined (Teulon et al. 2013). All our native species are relatively small (<2.5 mm in length) and mostly specialised to single native plant genera (one species, Paradoxaphis

27 plagianthi, is known from two) (Teulon et al. 2013). The Coprosma aphid (Aphis coprosmae) is reported to share a host plant with GWA, as there is a single record of a GWA colony on Coprosma macrocarpa. It was therefore deemed necessary to include an endemic Aphis species in our P. nigrovaria host testing trials. However, given their small size and distant phylogenetic relationship with GWA, it is predicted that the risk to any member of this tribe from P. nigrovaria will be very low.

Aphidinae: Macrosiphini This tribe is represented in New Zealand by over 60 introduced pest species. One small species (1.6-2.0 mm in length) on Carmichaelia in Canterbury is possibly native (Megoura stufkensi Eastop), but has not been seen since 2006. Since the likelihood of locating this potentially native species was considered to be very low, the most prudent action was deemed to use an appropriate proxy species that was a similar size to M. stufkensi rather than one of the larger species in the tribe. While most Macrosiphini are small, at least two species (the pea aphid (Acyrthosiphon pisum) and the blackberry aphid (Amphorophora rubi)) can exceed 4 mm in length, though they are more slightly built than lachnines and all are bright green in colouration. Since GWA is dark grey, preference was given to using a species that was also dark in colouration. A few species in this tribe are known to feed on willows and poplars, and therefore may be found in close proximity to GWA. However, their relative small size and distant phylogenetic relationship to GWA strongly indicate that no Macrosiphini species will be vulnerable to P. nigrovaria.

Table 1. The taxonomic composition of aphids in New Zealand and the potential vulnerability of each group to an introduction of P. nigrovaria. Subfamily Tribe No. of Vulnerability Reasons species of non- in NZ target (native) species to P. nigrovaria

Lachninae 8(0) Moderate to No native species. none Several relatively large (>4 mm) species. Most other Pauesia spp. attack Cinara spp. aphids. Eriosomatinae 11(0) Very No native species. low/none Mostly very small (<3 mm). No shared host plants. Mostly gall formers and/or root feeders.

28

Hormaphidinae 2(0) Very No native species. Very low/none small (<2 mm). No shared host plants. Neophyllaphidinae 2(2) Very Very small (<2 mm). low/none No shared host plants. Taiwanaphidinae 1(1) Very Very small (<1.5 mm). low/none No shared host plants. Phyllaphidinae 1(0) Very No native species. low/none Relatively small (3.2 mm). No shared host plants. Saltusaphidinae 1(0) Very No native species. Very low/none small (2.5 mm). Attacks sedges (Carex spp.). Calaphidinae 13(0) Very No native species. low/none Mostly very small (1 species >3 mm). No shared host plants. Chaitophorinae 2(0) Very No native species. Both low/none species relatively small (<4 mm). No shared host plants. Drepanosiphinae 1(0) Very No native species. No low/none shared host plants. Aphidinae Aphidini 28(11) Very Both native and exotic low/none species. All small (<3 mm). One native species potentially shares a host plant.

Macrosiphini 61(1?) Low to none Possibly no native species. Some host plant overlap. A small number of species are larger (>4 mm) and may be vulnerable, but these are exotic pests.

Selecting non-target aphid species for host-testing

29

Phylogenetic relatedness (of the non-target compared to the target pest) remains the best predictor of host utilisation by parasitoids in the field (Hoddle 2004). Sharing important ecological similarities (i.e., the same feeding niche on the same host plant) is also considered important (Kuhlmann et al. 2006). It is known that some parasitoid species target hosts present in a certain ecological niche, regardless of their taxonomic affinities (Messing 2001), whereas others, especially koinobiont parasitoids like P. nigrovaria (where the host continues to develop and is only killed when the parasitoid reaches maturity), are strongly host-specific (see Žikić et al. 2016). Body size of both the host and the parasitoid is also important. For instance, some parasitoids may not be able to fully develop on very small host species. This is an important issue in the current application since GWA is the largest aphid species in the world and P. nigrovaria is also correspondingly large at 3-4 mm in length. Most aphid species are much smaller than this (all our native species for instance are <2.5 mm in length) and are therefore highly unlikely to be attacked by, or physiologically support the development of, P. nigrovaria larvae. Occasionally, both the target pest and the co-evolved natural enemy species being considered for importation are so different from any native fauna in New Zealand that there is no scientific justification for any host testing (Charles 2012). Since the New Zealand aphid fauna lacks any native species in the same subfamily as GWA (Lachninae), there were no obvious choices for host-testing trials. Ideally, we would test all of our native aphid species to ensure no risk is posed to any of them from an introduction of P. nigrovaria. However, most of our native aphid species are poorly known, have restricted geographical ranges, and are very difficult to find. Since our fauna is so depauperate, selecting representatives of each of the three aphid lineages that naturally occur here (Taiwanaphidinae, Neophyllaphidinae and Aphidinae: Aphidini), and possibly a fourth (Aphidinae: Macrosiphinie) was deemed the most appropriate course of action, and a conservative one. Taiwanaphidinae is represented in New Zealand by a single species (Sensoriaphis nothofagi), so this was selected to represent this lineage. Neophyllaphidinae is represented by two closely-related native congeneric species; one common and widespread and the other restricted to mountainous areas. The common species (Neophyllaphis totarae) was deemed the most appropriate as it occurs more frequently in the landscape with willows. Of our five known native Aphis species, our test subject Aphis cottieri is the largest and therefore probably the best to test. All are patchily distributed in time and space and difficult to find. Furthermore, a molecular analysis of the phylogeny of our native Aphidini showed that our native Aphis species form a distinct clade of closely-related species (von Dohlen and Teulon 2003). We know less about the remaining native species in this tribe (2 species of Paradoxaphis and 3 or 4 undescribed species that are tentatively placed in the genera Casimira and Euschizaphis). None of these species are at all common or widespread and would therefore have been very difficult to locate and rear (location data for most are decades old and may no longer be reliable). All are small however, and of course belong to the same tribe as Aphis. Given the small size and patchy distributions of our native Aphidini, using the largest of those species (A. cottieri) as a proxy for the others was therefore deemed the most appropriate course of action. Testing exotic species was also considered important to ascertain the potential host

30 range limits of P. nigrovaria. In addition to GWA there are seven other adventive species in the subfamily Lachninae present in New Zealand: Cinara fresai Blanchard, C. juniperi (de Geer), C. louisianensis Boudreaux, C. pilicornis (Hartig), C. tujafilina (Del Guercio), Essigella californica (Essig) and Eulachnus brevipilosus Börner (Macfarlane et al. 2010). These are all pests of exotic conifers (Blackman & Eastop 1994; Watson & Appleton 2007). The most appropriate species for host testing amongst these was a larger Cinara species, such as C. fresai or C. pilicornis, as these species come closest to GWA in body size, and most other known Pauesia species attack Cinara spp. aphids. The last non-target species selected was a representative of the large aphidine tribe Macrosiphini, as it is the most species-rich group of aphids (nearly half of our aphid fauna) present in New Zealand and possibly includes one native species. Since our only possible native species of Macrosiphini is very rare (Megoura stufkensi has not been seen since its description in 2006), a similarly sized proxy species from the same tribe had to be used instead. By using a substitute species of a similar size we were able to simultaneously test the same taxonomic group and body size range of M. stufkensi against P. nigrovaria. Table 2 summarises the non-target aphid species selected for host testing.

Table 2. Summary of the non-target aphid species used in host-testing trials against P. nigrovaria showing their taxonomic placement relative to giant willow aphid (GWA), adult body size (length in mm) and host plants. Note the difference in size ranges between GWA and the native non-target species. Target? Aphid species Subfamily: Tribe Native Adult body Host plants to NZ? size Yes Tuberolachnus Lachninae No 5.0-5.8 mm Salix spp. and Populus salignus spp. (Salicaceae), Malus spp. and Pyrus spp. (Rosaceae)* No Cinara fresai Lachninae No 2.5-4.2 mm Cupressus spp. and Juniperus spp. (Cupressaceae) No Brachycaudus Aphidinae: No 1.5-2.2 mm Prunus spp. (Rosaceae) persicae Macrosiphini No Aphis cottieri Aphidinae: Yes 2.0-2.5 mm Muehlenbeckia spp. Aphidini (Polygonaceae) No Neophyllaphis Neophyllaphidinae Yes 1.5-2.0 mm Podocarpus spp. totarae (Podocarpaceae) No Sensoriaphis Taiwanaphidinae Yes 1.3 mm Fuscospora spp. nothofagi (Nothofagaceae)

Conclusions

A review of the taxonomy and biology of our native aphid fauna and that of the proposed biological control agent, P. nigrovaria, strongly suggests that this parasitoid will pose no risk to our native species, and very little risk to closely-related exotic pest species. Nonetheless, risk has been formally tested against each of the three subfamilies to whichour native species belong, as well as two major groups of exotic species. It is expected that our

31 native aphids are physically too small and distantly related to GWA to facilitate successful physiological development of P. nigrovaria. Furthermore, since only one native species shares a potential host plant with GWA, it is highly unlikely that P. nigrovaria will ever encounter a native species. Indeed, if P. nigrovaria utilises host plant volatiles to locate likely sites to find GWA (like many other species of aphidiines), it probably would not search any potential host plants other than the primary hosts for GWA (Salix and Populus species). Since almost all known Pauesia species only attack aphids in the subfamily Lachninae, we expected that the only test species at risk of attack would be C. fresai. Details of completed host-testing trials conducted with these species are outlined in Section 5 (Risks, costs and benefits).

Figure 1. Unrooted Bayesian inference topology based on the concatenated matrix including 1st and 2nd codon positions. Three main clusters are defined (shaded areas) to facilitate comparison with the Ortiz-Rivas and Martínez-Torres (2010) scheme, and are resolved as a trichotomy in this unrooted analysis. Solid lines refer to monophyletic taxa. Dashed lines designate paraphyletic/polyphyletic taxa. Numbers are posterior probabilities. Reprinted from Novakova et al. (2013).

32

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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? This is a non-GMO organism.

Taxonomic description

Pauesia nigrovaria (Provancher, 1888) belongs to the Hymenoptera family Braconidae (all parasitoids) and the subfamily Aphidiinae (all specialist parasitoids of aphids). Our imported specimens were identified as P. nigrovaria by aphidiine taxonomist Dr Manfred Mackauer, Professor Emeritus, Simon Fraser University, Vancouver, Canada. Pauesia nigrovaria was first described in 1888, as Aphidius nigrovarius Provancher. The type specimen is from Los Angeles, California, USA, and was reared from what was at the time referred to as “Lachnus sp.” This aphid host is presumed to be synonymous with Tuberolachnus salignus (Mackauer, pers. comm.). We have sequenced the COI gene region of P. nigrovaria (and all host-tested aphid species) and these will be available on GenBank in conjunction with a publication of our host specificity testing results. Parasitoids within the genus Pauesia are known for their high degree of host specificity. Most Pauesia are very host specific with species normally parasitizing a single aphid species from the subfamily Lachninae (Žikić et al., 2016). Pauesia nigrovaria is only known to parasitise Tuberolachnus salignus. Collected from California, P. nigrovaria is physically distinct from Pauesia salignae Watanabe which parasitises GWA in eastern Asia. This conclusion was reached by comparison of specimens from both regions by taxonomist Dr Manfred Mackauer, Professor Emeritus, Simon Fraser University, Vancouver, Canada.

Biology and main features

Pauesia nigrovaria is a small parasitoid wasp approximately 3 mm in length (Figure 2) that

34 does not sting humans or animals. Although it is difficult to tell with the naked eye, the colouration is a combination of deep yellow and black. The males are darker overall, and males and females have different numbers of antennal segments (23-24 and 21-22, respectively). Pauesia nigrovaria may be distinguished morphologically from other species of Pauesia by furrows which partially separate the mesopleura and mesosternum, and by the distinctly indicated endings of notaulices near the posterior margin of the mesonotum (Smith, 1944). Scion’s laboratory colony of Pauesia nigrovaria contains no other inseparable organisms. A species of hyperparasitoid was present in the original import material, however these were separated from the colony in the first generation produced and were destroyed.

Figure 2. a) Pauesia nigrovaria female, size is approximately 3 mm in length; b) P. nigrovaria attacking GWA (preparing to oviposit); c) Nearly full-grown P. nigrovaria larva (yellow) dissected from a GWA mummy.

a) c)

b)

35

Could the organism form an undesirable self-sustaining population? If not, why not? No. The intent of this application is to seek approval to release and establish a self- sustaining population of a new organism to be used as a biological control agent for giant willow aphid. This request follows a thorough assessment of the risks associated with the release of this new organism in New Zealand (see Section 5.1 for more information). We do not believe that Pauesia nigrovaria will become undesirable, as the risk to native species and beneficial non-target species has been demonstrated to be very low. The benefits of successful biological control are expected to far outweigh these risks (see Section 5.2 for more information).

How easily could the new organism be recovered or eradicated if it established an undesirable self-sustaining population? The target organism (giant willow aphid) and its host plants (willows and poplars) are widespread in New Zealand. Therefore, if the new organism were to establish an undesirable self-sustaining population, eradication would likely only be possible by widespread, repeated application of broad-spectrum insecticides. For this reason, eradication attempts would be undesirable and futile. Eradication efforts would likely prove to be more environmentally damaging than any undesirable impacts caused by the new organism. Pauesia nigrovaria is relatively poorly known in its natural range and little is known about its natural enemies. We found evidence of hyperparasitism in California populations, but the hyperparasitoids were not identified. It is unknown whether any hyperparasitoid species already present in New Zealand may attack P. nigrovaria. However, like all hyperparasitoids they would not eradicate P. nigrovaria, but may reduce their abundance.

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

36

Application Form Approval to release a new organism

4. 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.

Consultation with Māori

We have taken the opportunity to engage directly and indirectly with Māori. Table 3 outlines specific Māori consultation prior to the submission of this application. Feedback received from Māori individuals, and our responses, are documented in Table 4, and detailed in Appendices 4 & 5.

Table 3. Summarised pre-application consultation with Maori to date:

Maori engagement in relation to giant willow aphid biological control project

Date Name Location/Region Action

15 Blanche & Kaitaia, Northland Posted and emailed consultation April Team Kai Ora letter*, feedback form** and 2019 information leaflet***

15 John Hill / NZ Whakatane, Bay of Posted and emailed consultation April Mānuka Group Plenty letter*, feedback form** and 2019 information leaflet***

15 Laney Hunia / Opotiki, Bay of Plenty Posted and emailed consultation April Whenua Honey letter*, feedback form** and 2019 information leaflet***

15 Owhaoko A Taupo, Taupo Posted and emailed consultation April East & A1B letter*, feedback form** and 2019 Blocks, Māori information leaflet*** Lands Trust

15 Waireka Honey Palmerston North, Posted and emailed consultation April Wanganui letter*, feedback form** and 2019 information leaflet***

35

15 Hauora Honey Johnsonville, Posted and emailed consultation April Wellington letter*, feedback form** and 2019 information leaflet***

15 Victor St Heliers, Auckland Posted and emailed consultation April Goldsmith / letter*, feedback form** and 2019 Ngāti Apiaries information leaflet*** Ltd

15 Ora Honey, Whakatane, Bay of Plenty Posted and emailed consultation April Whakaari letter*, feedback form** and 2019 International information leaflet***

15 Lorraine & Taneatua, Bay of Posted and emailed consultation April Jason Stanley Plenty letter*, feedback form** and 2019 / Golden information leaflet*** Grove Apiaries Honey

15 Poutama Honey Rotorua, Bay of Plenty Posted and emailed consultation April letter*, feedback form** and 2019 information leaflet***

15 Brenda Tahi / Ruatahuna, Bay of Plenty Posted and emailed consultation April Manawa letter*, feedback form** and 2019 Honey information leaflet***

15 Maori Honey Mt Eden, Auckland Posted and emailed consultation April Ltd letter*, feedback form** and 2019 information leaflet***

15 Tahi NZ Whangarei, Northland Posted and emailed consultation April letter*, feedback form** and 2019 information leaflet***

24 Karin Kos, New Zealand wide Requested that ApiNZ email April CEO / consultation letter*, feedback 2019 Apiculture form** and information New Zealand leaflet*** specifically to their Maori apiarist members

36

14 Ngāpuhi Ngāpuhi, Northland Emailed consultation letter*, May HSNO feedback form** and 2019 Komiti information leaflet***

14 Te Ngāi Tahu, South Island Emailed consultation letter*, May Rūnanga o feedback form** and 2019 Ngāi Tahu information leaflet*** HSNO Komiti

17 EPA’s Te New Zealand wide Emailed consultation letter*, May Herenga feedback form** and 2019 Network, information leaflet*** via EPA (Kaupapa Kura Taiao)

18 Te Arawa Lakes Rotorua, Bay of Plenty Presentation at a biosecurity July Trust hui at Toi Ohomai: “Insect 2019 biosecurity hui pests; introducing new parasitoids to Aotearoa to combat invaders” – Toni Withers and Stephanie Sopow (Scion) * Appendix 1, ** Appendix 2, *** Appendix 3

Table 4. Feedback received from pre-application consultation with Maori:

Feedback received as a result of Maori pre-consultation.

Date Who Location/affiliation Action

20 Tui Whangarei Emailed to express concerns May Shortland about the native aphids that 2019 were not tested and cultural impacts. See Appendix 5 for full feedback details and response.

37 20 Hëmi Te Ngä Puhi, Waitaha, Emailed (via EPA) to express May Räkau Rapuwai, Mamoe, concerns about the length of 2019 Ngäti Päkau exposure of the parasitoid to native insects and the possibility of evolutionary adaptation to parasitise native species. See Appendix 6 for full feedback details and response.

Consultation with the general public and scientific community

In addition to consultation with Māori, we have also undertaken extensive consultation with the general public (Table 5) and New Zealand’s scientific community (Table 6) using a wide variety of methods. These include conference presentations, conference posters, radio interviews, magazine articles, newsletters, info sheets (Appendix 4), a survey, letters, emails, personal conversations, and creation of a project website that we keep up-to-date (giantwillowaphid.co.nz).

Table 5. Pre-application consultation with the general public to date:

Public engagement in relation to giant willow aphid biological control project

Date Who Where Action

21 Apiculture NZ Rotorua: during Apiculture Presentation: “Giant Willow June members New Zealand’s annual Aphid in New Zealand: Impacts 2016 conference and Solutions” - by Stephanie Sopow (Scion)

19- Apiculture NZ Rotorua: during Apiculture Poster: “Giant willow aphid: 22 members New Zealand’s annual impacts and solutions” – June conference presented by Stephanie Sopow 2016 (Scion)

Oct New Zealand Various: Apiculture New Article: “Combatting the giant 2016 Beekeeper Zealand members reached willow aphid threat” contributed Magazine via magazine subscription by Barry Foster (Apiculture NZ), published in New Zealand Beekeeper, October 2016, Page 19 - see Appendix 7.

Aug Facebook Northland Regional Council Interview of Toni Withers by 2017 public Facebook page Don Mackenzie on GWA shared subscribers as a 2 min video

38 2017- GWA website Various Management of Giant Willow curre Aphid created and updated nt regularly: https://www.giantwillowaphid.c o.nz/

Feb New Zealand Various: Apiculture New Article: “Giant willow aphid 2017 Beekeeper Zealand members reached research kicks off” contributed Magazine via magazine subscription by Barry Foster (Apiculture NZ), published in New Zealand Beekeeper, February 2017, Page 9 - see Appendix 8.

11 Apiculture NZ Rotorua: during Apiculture Presentation: “Management of July members New Zealand’s annual Giant Willow Aphid” - by 2017 conference Stephanie Sopow (Scion)

Presentation: “RNAi proof of concept experiments and the challenges of rearing GWA in containment” - by Carl Wardhaugh (Scion)

Presentation: “Giant willow aphid research at Plant & Food Research” - by Trevor Jones (Plant & Food Research)

Presentation: “Observations of the Giant Willow Aphid Tuberolachnus salignus (Gmelin) in a willow stand in the Matokitoki Valley, Gisborne” - by John McLean (Apiculture NZ)

18 Radio New Various: broadcast on Radio interview: “Taking on the Aug Zealand Radio New Zealand: Giant” - Stephanie Sopow (Scion) 2017 National Country Life interviewed by Carol Stiles (https://www.rnz.co.nz/national/p rogrammes/countrylife/audio/2018 55271/taking-on-the-giant)

10 Feb Apiculture New Various: received by Presentation: a talk on the GWA 2018 Zealand, attendees of Waikato Hub project and the relationship with Waikato Hub Mini-Conference the harlequin ladybird was presented by Toni Withers (Scion)

39 July New Zealand Various: Apiculture New Article: “Management of Giant 2018 Beekeeper Zealand members reached Willow Aphid” contributed by Magazine – via magazine subscription Stephanie Sopow (Scion) and July issue Trevor Jones (Plant & Food Research) – see Appendix 9. Note that this material is the same as the annual project newsletter for June 2018 (entered in Table 3).

24 Apiculture NZ Blenheim: during Presentation: “Management of Giant July members Apiculture New Zealand’s Willow Aphid” - by Stephanie 2018 annual conference Sopow (Scion)

Presentation: “Impact of the giant willow aphid on willow tree health” by Trevor Jones (Plant & Food Research)

1 Apiculture Various – beekeepers Survey: “How is giant willow Aug NZ throughout NZ aphid affecting you?” - 201 membership represented (55 Apiculturists were polled to 8 recipients) determine current impacts of GWA

29 Radio New Various: broadcast on Radio interview: “Giant willow Nov Zealand Radio New Zealand: Our aphids – a sticky invasive 2018 National Changing World nuisance” – Stephanie Sopow (Scion) interviewed by Alison Balance (https://www.rnz.co.nz/national /programmes/ourchangingworld /20181129)

18 Apiculture NZ Various: Members Email newsletter: Members April membership contacted via weekly email were informed of this EPA 2019 newsletter from Karin Kos, application to release Pauesia nigrovaria, and that public CEO / Apiculture New consultation would be part of Zealand the process. A link to the GWA website was provided (www.giantwillowaphid.co.nz), as well as an info sheet with updated host testing results****

40 12 Apiculture NZ, Christchurch Presentation: An overview of the May Canterbury project and the relationship 2019 Hub, between GWA and the invasive ladybird Harmonia axyridis was Beekeeper’s presented to approximately 60 Day Out beekeepers by Stephen Pawson (Scion)

20 Students at Toi Tauranga Presentation: An overview of the May Ohomai project was provided to 20 first 2019 Institute of year environmental science students, by Roanne Sutherland Technology (Scion)

11 Forest & Bird, Palmerston North Presentation: The Giant Willow June Manawatū Aphid and its Environmental Impact, By Andrea McCormick 2019 Branch (Massey University) and Trevor Jones (Plant & Food Research) – Appendix 10 27 Apiculture NZ Rotorua: during Apiculture Presentation: Management of June members New Zealand’s annual Giant Willow Aphid in New conference Zealand, by Stephanie Sopow 2019 (Scion)

Poster: Pauesia nigrovaria, ready to take on giant willow aphid, presented by Stephanie Sopow (Scion) – included collecting feedback on EPA application - Appendix 11

Presentation: Giant willow aphids affect the spring flowering and growth of willow trees, by Trevor Jones (Plant & Food Research), and other authors

Poster: Seasonal activities of the giant willow aphid in the Gisborne area 2018-2019, presented by John McLean (ApiNZ)

28 Recipients of Envirohub Bay of Plenty Short news story “Biosecurity June digital newsletter Alert: Giant Willow Aphids”, with 2019 newsletter a link to the giant willow aphid project website for more information.

41 Aug New Zealand Various: Apiculture New Article: Content as for 2019 Beekeeper Zealand members reached Management of giant willow aphid annual newsletter #3 (pendi Magazine – via magazine subscription (published by Scion) – Project ng) August 2019 summary and announcement of issue EPA submission with an appeal for public submissions.

****Appendix 4

Table 6. Pre-application consultation with the scientific community to date:

Engagement with the scientific community in relation to giant willow aphid biological control project Date Who Where Action

Jul Scion Various: recipients of Article: “Giant willow aphid 2016 publication newsletter (primarily update” published in Forest forest health and Health News 268, July 2016 - Appendix 12 biosecurity surveyors)

Apr Entomological Wellington – annual Presentation: “Management of 2017 Society of NZ conference, 10 – 13 Giant Willow Aphid: Sustainable April Farming Fund 404830” – by

Carl Wardhaugh (Scion)

Presentation: “Impacts of the g iant willow aphid,

Tuberolachnus salignus

(Gmelin) in the New Zealand agricultural landscape” – by John McLean (ApiNZ)

Apr Scion Various: recipients Article: “Update on giant willow 2017 publication of newsletter aphid management” published (primarily forest in Forest Health News 272, April health and 2017. June Regional biosecurityWellington Presentation: “Biocontrol of 2017 Council’s surveyors) Giant Willow Aphid in New Biosecurity Zealand” – by Toni Withers (Scion) Working June GroupMinistry for Various Article: “Combating the Giant 2017 Primary Willow Aphid” – published in Industries the booklet: 1000 projects and counting – Celebrating the success of the Sustainable Farming Fund 2000-2017 – Appendix 13

42 July Scion Various Article: Management of giant 2017 publication willow aphid annual newsletter #1, published by Scion

7 Aug Department Tauranga Met with Chris to give him an 2017 of overview of the project and Conservation discuss concerns about anti- willow groups (Chris Green) Aug New Zealand Wellington Presentation: “Scion’s current 2017 Biosecurity biological control projects to Institute combat tree pests – tortoise beetle and giant willow aphid” – by Toni Withers (Scion) Dec New Zealand Various: newsletter Article: “Attack of the giant 2017 Ecological received by society willow aphid” –by Bruce Burns, Society members published within “Ecotones – New ecological research” in the Ecological Society of New Zealand Newsletter 162, December 2017, Pp 3-4.

Jan Scion Various: recipients Article: “Trojan aphids used to 2018 publication of newsletter import parasitoids” published (primarily forest in Forest Health News 278, January 2018 – Appendix 14 health and biosecurity surveyors)

Apr Entomological Wanganui – annual Presentation: “Activities of the 2018 Society of NZ conference g iant willow aphid, Tuberolachnus salignus (Gmelin) in the Gisborne-East Coast area last year” – by John McLean (ApiNZ)

June Scion Various Article: “Citizen science saves 2018 publication the day” published in Scion Connections, Issue 28, June 2018 – Appendix 15

June Scion Various Article: Management of giant 2018 publication willow aphid annual newsletter #2, published by Scion

43 11 Wasp Tactical Wellington Presentation: An overview of Group the project was given by Kat June meeting Webb (Scion) 2018

22 Regional Wellington Presentation: An in-depth overview of the project was March Council’s given by Toni Withers, Scion 2019 Biosecurity Working Group

30 Audience of Tauranga Presentation: A summary of the project was provided by May B3 Science Nicolas Meurisse (Scion), as 2019 Partnership part of a broader talk on Forum Scion’s research

26 June Scion Various Article: Management of giant 2019 publication willow aphid annual newsletter #3, published by Scion. Appendix 16

23 Department Chris Green and Informed of the EPA application to release Pauesia June of Rod Hitchmough sp., and provided info sheet 2019 Conservation reached by email with host testing results (first informed Chris Green July 2017)

* Appendix 1

44

5. Risks, costs and benefits

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

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.

5.1 RISKS

5.1.1 Potential risks to the environment and non-target species

Based on the known biology of Pauesia nigrovaria and its relatives, the biology and phylogeny of our native aphids, and the results of host-testing trials and behavioural assays, we are very confident that P. nigrovaria poses little to no risk to New Zealand’s environment, native species, people or economy. The greatest potential risks are probably indirect effects, where P. nigrovaria may have a negative effect on native species through shared natural enemies or by affecting abundances of GWA and willows, which in turn affects other species in the environment. The major potential risks posed by P. nigrovaria to the New Zealand environment are outlined below and summarised in Tables 9 and 10.

Risk 1: P. nigrovaria attacks non-target native aphid species Our native aphid species are too distantly-related to GWA and too small to interest Pauesia nigrovaria. This is strongly supported by the host-testing results (see below), which show P. nigrovaria poses little or no risk to native aphid species. None of the non- target aphid species exposed to P. nigrovaria were parasitised. Behavioural assays showed that two individuals of the exotic pest species Cinara fresai appeared to be attacked, but no larvae developed. Dissections of these individuals revealed no evidence of a parasitoid egg being deposited. Furthermore, since none of our native aphid species attack willows or poplars,

45 they are unlikely to be encountered by P. nigrovaria.

Host specificity of Pauesia nigrovaria and risk to non-target aphids in New Zealand

Methods Host range testing To elucidate the host range of Pauesia nigrovaria, no-choice host-testing trials were conducted with mated female P. nigrovaria and a range of non-target aphid species, with GWA as a control. Newly emerged P. nigrovaria were stored at 12°C in individual glass vials with mesh lids. Each wasp was provided with a drop of honey and a wet piece of paper towel as a water source and left overnight to mature (mating was found to be more successful for one to two-day old wasps than for newly emerged individuals). Mating occurred in vials containing a female/male pair of P. nigrovaria. Males were selected based on size (equal to or larger than the female, as small males were often rejected). Age of males did not appear to have an effect, so it was not considered important. The pair were monitored for up to 30 minutes to ensure copulation occurred. Occasionally, if mating had not occurred a second male was introduced to the vial. After mating, female P. nigrovaria were isolated for 24 hours prior to being used in experiments. Mated naïve female wasps (no prior exposure to aphids) between 2 and 5 days old were released into cylindrical test cages (approximately 40 cm in height and 24 cm in diameter) containing 30 aphids settled for 24 hours on cut stems of their host plant in water. Non- target aphid species were selected for host-testing experiments as described in Section 2.3 above. The female wasp was left in the test cage with the aphids for 24 hours and was then removed and placed in a second cage containing 30 GWA on cut willow stems in water. This was done to confirm the viability of each female wasp using its preferred host. Female P. nigrovaria were left in these viability cages until they died. If no GWA in the viability cages were parasitised, the wasp was considered infertile and the replicate was discarded from the analyses. Following exposure to mated female P. nigrovaria, all test and viability cages were monitored daily (excluding weekends) for mummy formation and general health. Cut stems were changed as required. Any mummies found in test or viability cages were placed in individual gel caps to await eclosion at 20°C. Emerged adult wasps were numbered, sexed, and fed honey and water in preparation for experiments or to start new colonies. Although mummies begin to form around 10 days after oviposition in GWA (with emergence occurring between days 16 and 23), test and viability cages were monitored for four weeks. All remaining non-target aphids were preserved in ethanol and later dissected to check for any signs of parasitism. Between 10 and 13 replicates were completed for four of the non-target species (our target was 10 replicates), but only two replicates could be completed for the beech aphid, Sensoriaphis nothofagi. This species was particularly difficult to rear in quarantine as it performed poorly on cut stems and could only survive on flush foliage of its host southern beech trees (Fuscospora spp.). These trees only flush for a few weeks each year and once

46 the leaves mature, the aphids disappear, presumably entering diapause to await the next flush.

Behavioural assays In addition to traditional no-choice host specificity testing, observational behavioural assays were run to determine whether Pauesia nigrovaria interacted with or attempted to attack non-target aphid species. Non-target species chosen for these trials were Cinara fresai, which is the closest relative to GWA and most similar body size among the species tested, and Brachycaudus persicae, which was the next largest aphid species tested. Trials were conducted in pairs, with one non-target species replicate and one GWA replicate run concurrently in random order. Individual aphids were chosen based on consistency in body size, both within each species (i.e. same instar) and with the other species in the trial pair (e.g., 4th instar C. fresai and 2nd or 3rd instar GWA). A single aphid (either a non-target species or GWA) was placed in a petri dish lined with filter paper. A mated female Pauesia between 2 and 7 days old was then introduced to the same dish and observed for five minutes. Behaviours recorded were i) antennal contact, ii) probing with the ovipositor, and iii) oviposition. The behavioural response (kicking, jerking, running, etc.) of the aphids was also recorded. The number of times each of these behaviours occurred within the five- minute trial were recorded. This protocol was repeated five times with each female P. nigrovaria by transferring it to another petri dish with another individual aphid of the same species every five minutes and recording their interactions. Once five assays had been completed with one P. nigrovaria female on one aphid species, a new female P. nigrovaria was then used on the other aphid species in the pair (a non-target species or GWA) using sterilised instruments to prevent odour or volatile transfer between aphid species. For tests with C. fresai, each experiment was replicated 24 times (i.e. 24 individual wasps were exposed to 240 aphids, 120 of each species). For B. persicae, each experiment was replicated 13 times. All aphids stung by P. nigrovaria in these trials were reared for up to three weeks to allow for mummy formation and parasitoid emergence. Occasionally the aphids being reared died unexpectedly and, if this occurred, these aphids were preserved in 70% ethanol for subsequent dissection.

Results (including statistical methods): Host testing No non-target species resulted in the production of any mummies or Pauesia nigrovaria adults; only the GWA control replicates produced mummies and adult P. nigrovaria during host specificity testing. An average of 8 of the initial 30 aphids (or 27%) became mummies after 24 hours of exposure to a P. nigrovaria female. Although these mummies effectively represent killed GWA, not all mummies gave rise to a next generation adult P. nigrovaria (an average of 6.06, or 20% of available GWA) (Table 7).

Table 7. Mean number of mummies and wasps produced from exposure of non-target aphid species and GWA (control) to mated, female Pauesia nigrovaria for 24 hours.

47

The numbers of GWA mummies and wasps approximated a zero inflated Poisson distribution, hence the non-parametric, conservative, Kruskal-Wallis test was used to compare results between aphid species. The numbers of mummies and wasps differed significantly between GWA and the non-target aphids species (P<0.001 for both mummies and wasps). The corresponding non-parametric pairwise Wilcoxon rank sum test was used to calculated pairwise comparisons between group levels, correcting for multiple comparisons using the Benjamini and Hochberg (1995) method. All aphid species, except S. nothofagi (since only two replicates were completed) had significantly fewer mummies and wasps than T. salignus (mummies: A. cottieri P=0.0047, B. persicae P=0.0052, C. fresai P=0.0047, N. totarae P=0.0052, wasps: A. cottieri P=0.0047, B. persicae P=0.0051, C. fresai P=0.0047, N. totarae P=0.0051). A concise summary of the host testing results including details of non-target aphid species is shown in Table 8.

55 Table 8. Pauesia nigrovaria host specificity testing – summary of results Subfamily Status Species Host plants Results Conclusion /Tribe

Most closely related aphids (same subfamily)

Cinara fresai

A ‘giant conifer

aphid’ (Genus

Cinara) No evidence of Various parasitism by Lachninae Exotic Cupressaceae, Not a host P. nigrovaria Cryptomeria (no mummies japonica formed) (Taxodiaceae)

Less closely related aphids (different subfamily, same family)

Aphis cottieri

No evidence of Aphidinae: Native parasitism by Muehlenbeckia spp. Not a host Aphidini P. nigrovaria (no mummies formed)

Brachycaudus persicae

Black peach aphid No evidence of Aphidinae: Exotic parasitism by Prunus spp. Not a host Macrosiphini P. nigrovaria (no mummies formed)

Neophyllaphis totarae

Totara aphid No evidence of

Neophyllaphi parasitism by Native Podocarpus spp. Not a host dinae P. nigrovaria (no mummies formed)

56 Sensoriaphis nothofagi

No evidence of parasitism Taiwanaphid Unlikely to Native Fuscospora spp. by P. inae be a host nigrovaria (no mummies formed)*

*Only two replicates completed

Behavioural assays Cinara fresai Pauesia nigrovaria displayed some attack behaviour when exposed to C. fresai. Primarily, this was touching the body of the aphid using the antennae and, in 3 cases (2 wasps), probing the body of the aphid with the ovipositor. A single Pauesia nigrovaria also appeared to oviposit into two C. fresai during the behavioural assays. However, no mummies were formed from these aphids (and therefore no wasps emerged) (Figure 3). Dissections of these aphids showed no signs of parasitism (eggs or immature larvae).

Figure 3. Pauesia nigrovaria behavioural assay with Cinara fresai (control = T. salignus): mean number of observations of antennal contacts, ovipositor probes, oviposition events, mummies formed, and wasps emerged. For C. fresai, the mean number of oviposition events was 0.033 (a single wasp appeared to oviposit into two aphids), and number of mummies and wasps formed was zero.

57 Data were analysed using the non-parametric Friedman rank sum test, blocked by the individual wasp. Pauesia nigrovaria exhibited significantly more contacts, probes, oviposition events, mummies and wasps emerging for GWA compared to C. fresai (P<<0.0001 for all behaviours). Both aphid species displayed a wide range of behaviour, showing no apparent trends in terms of their reaction to contact by the parasitoid. These aphid behaviours included walking away, ceasing walking, jerking the body, kicking and, rarely, exuding a substance from the siphunculi (presumably a defensive secretion). For the most part, the aphids appeared to be relatively docile when met with this natural enemy.

Brachycaudus persicae During the behavioural assays with Brachycaudus persicae, P. nigrovaria exhibited some antennal contact behaviour with this non-target species and, for a single aphid with a single wasp, also ovipositor probing. However, no oviposition behaviour was observed as occurred with C. fresai (Figure 4). Using the non-parametric Friedman rank sum test, blocking by the individual wasp, P. nigrovaria displayed significantly more contacts, probes, oviposition events, mummies and wasps emerging for GWA compared to B. persicae (contacts P=0.0002, probes P<<0.0001, oviposition events P<<0.0001, mummies P=0.0008, wasps emerging P=0.008). Brachycaudus persicae also displayed a range of behaviours in reaction to contact with the parasitoid, with no obvious trends other than seeming surprisingly passive.

Figure 4. Pauesia nigrovaria behavioural assay with Brachycaudus persicae (control =

S. salignus): mean number of observations of antennal contacts, ovipositor

58 probes, oviposition events, mummies formed, and wasps emerged. For B. persicae, the mean number of oviposition events was zero, and therefore the number of mummies and wasps formed was also zero.

Conclusions

Host testing trials showed that P. nigrovaria is in all likelihood monophagous and will only complete development in the giant willow aphid. None of the native aphid species we tested against P. nigrovaria were parasitised or showed any evidence of attack. With representatives from New Zealand’s three native aphid lineages, we conclude that our native aphid fauna is not at risk of attack by P. nigrovaria. Although we did not test all of our native aphid species, those not tested are highly unlikely to be attacked since all of the species within each lineage are closely-related to each other.

A laboratory study by Cameron et al. (2013) demonstrated that two aphidiine parasitoid species introduced into New Zealand as biological control agents parasitised some native aphids at a low rate. However, these parasitoids were known to be polyphagous species, and the native aphids that were parasitised were close taxonomic relatives of the parasitoid’s previously known hosts. It can also be said that laboratory tests of host specificity tend to overestimate the actual host range of a parasitoid (Cameron et al., 2013). Based on searches over more than 8 years in several locations in New Zealand, no evidence of attack of New Zealand’s native aphids by these introduced parasitoids was found (Bulman, pers. comm.).

On body size alone, it is highly unlikely that any native aphid will be attacked. All our native aphid species are very small (<2.5 mm), while P. nigrovaria are substantially larger at 3-4 mm in length. Sensoriaphis nothofagi, which we could not thoroughly test due to its biology (just two replicates were completed) reaches a maximum size of about 1.3 mm. It is therefore almost certainly too small to facilitate the development of a P. nigrovaria larva. The most likely species to be attacked among those we chose for host testing was the introduced pest Cinara fresai, which belongs to the same subfamily as GWA, is relatively close to it in body size, and is attacked by other species of Pauesia in its native range. The fact that no parasitism was recorded on C. fresai in no-choice tests in the artificial confinements of containment strongly suggests that P. nigrovaria is highly host-specific to GWA. Host-testing trials can exaggerate the host range of parasitoids, since females are confined to small enclosures with non-target species and have no choice but to oviposit on a non-

59 target species, or not lay any eggs at all. Under those conditions, some individuals of most species will eventually accept a closely-related non-target species. P. nigrovaria did not, potentially making it a rare example of a true monophagous species, and therefore an ideal candidate for a biological control agent.

An average of 20% of the test GWA gave rise to adult P. nigrovaria after 24 hours of exposure, however the percentage of GWA killed by P. nigrovaria was at least 27%, as evidenced by the number of mummies that formed. These numbers may appear low at first glance, but these results reflect only the 24 hour periods of exposure of 30 GWA to a P. nigrovaria female. We know that P. nigrovaria often lived for a period of a few weeks in our laboratory setting, and data from rearing cages gives us a better idea of total potential fecundity. We used females aged from 2 days old to create new parasitoid colonies, and observed a maximum of 75 offspring produced from a single female, despite an initial supply of only 40 GWA. It is therefore evident that P. nigrovaria is able to continue to parasitise GWA over a period of several days, while the aphids are continuing to reproduce. We also expect that any figures taken from colony rearing are likely to underestimate total potential fecundity because approximately 30% of the laboratory reared mummies failed to reach eclosion. We suspect this had something to do with our manual handling of each mummy such that we could rear them individually in gel caps, and that greater success would occur in the field without this handling. Some test cage aphids also died of unknown causes, possibly due to the trauma of being attacked by P. nigrovaria. We did not quantify these since we could not distinguish between possible reasons for their deaths. If the aphids were not receiving sufficient nutrition because they were being reared on cut stems, then this in turn may be another reason for considering that parasitism rates ought to be higher in the field. On the flip side, natural enemies in the field can also be expected to negatively impact populations of P. nigrovaria, and these impacts are unquantified. Nonetheless, other species of Pauesia have proven to be successful biological control agents elsewhere, demonstrating an ability to spread rapidly, survive seasons during which their host aphid is scarce, and offer excellent control of the pest aphid. Examples include Pauesia cinaravora Marsh, used to control black pine aphid (Cinara cronartii Tissot & Pepper) in South Africa (Kfir et al., 2003), Pauesia momicola Watanabe & Takada, used to control Cinara todocola Inouye in Japan (Yamaguchi and Takai, 1977), and Pauesia cedrobii Stary, introduced to control Cinara laportei Remaudière in France (Fabré and Rabasse, 1987). In the latter example, only 225 individuals were initially released and yet these were found to have established four years later despite attack by eight species of hyperparasitoids (Fabré and Rabasse, 1987). These authors

The behavioural assays offer further evidence that non-target aphids, including any native species, are unlikely to be at risk. For the two C. fresai individuals that a single wasp appeared to oviposit into, we can conclude that P. nigrovaria either did not actually oviposit into these aphids (despite the apparent behaviour), or the immature stages were unable to develop inside these aphids and were broken down by the host’s immune system. The wasp that probed a B. persicae individual with her ovipositor was a highly active female that had been first exposed to GWA and was therefore no longer naive. She had previously probed 4

60 of 5 GWA and had oviposited into 3, eventually producing 3 progeny. The B. persicae that was probed was the first of the new species and, after this initial aphid, she did not probe any others.

Results of both host specificity testing and behavioural assays strongly indicate that Pauesia nigrovaria is highly host-specific and unlikely to parasitise any other aphid species (or any other types of insects) in New Zealand. The greatest risk would likely be to species closely related to GWA, those in the aphid subfamily Lachninae. All species present in New Zealand that belong to this group are other exotic pests. Therefore, should P. nigrovaria be shown to parasitise any of these species in New Zealand in the future, it would not be a concern and could be considered an additional benefit.

Risk 2: P. nigrovaria is attacked by hyperparasitoids

Hyperparasitoids attack developing parasitoids inside other insect species. They are often rare in the environment but can sometimes be more generalised in their host range than primary parasitoids. There is one hyperparasitoid that attacks P. nigrovaria in its natural range (S. Sopow, pers. obs.), but it has not been identified, and is not known from New Zealand. We cannot rule out the possibility that a hyperparasitoid species already present in New Zealand may shift hosts and attack P. nigrovaria in the future. Hyperparasitoids are a known issue with biological control agents, and may reduce the population size of the agent, thereby limiting their ability to control the target pest species. However, in practise this seems to be a very rare phenomenon. For instance, when the hyperparasitoid Baeonusia albifunicle invaded New Zealand two decades after its host Enoggera nassaui was established as a biological control agent for Paropsis charybdis, researchers feared it would disrupt the biological balance (Mansfield et al 2011). Two decades on E. nassaui continues to persevere in the environment and is still an important egg parasitoid despite on-going hyperparasitism (A. Pugh, T. Withers, unpublished data). As another example, Pauesia cedrobii, introduced to control Cinara laportei in France became well-established despite only a small number of individuals being released, and subsequent attack by eight species of hyperparasitoids (Fabré and Rabasse, 1987).

Risk 3: P. nigrovaria may be attacked by predators

There are two potential negative impacts from predation on P. nigrovaria; firstly they may be unpalatable to predator native species and consumption may induce illness, and secondly P. nigrovaria populations may be reduced, thus reducing impact on GWA. The former impact is unlikely as there is no evidence that any Pauesia species is unpalatable to potential predators. However, predation on

61 adults and developing larvae inside GWA hosts may reduce P. nigrovaria abundance. Predation on GWA that contain developing P. nigrovaria larvae is likely to occur due to foraging ladybirds, especially the harlequin ladybird. These predatory beetles are indiscriminate in their choice of prey and may consume parasitised GWA. However, research has shown they prefer unparasitised aphids to mummified ones, but do not discriminate between newly parasitised and unparasitised nymphs of the soybean aphid (Xue et al. 2012). Since both GWA and the harlequin ladybird are recent invaders, it is unknown what effect GWA is having on the population of the ladybird, and vice versa. Research within greenhouses has suggested harlequin ladybirds can complement aphid biocontrol rather than disrupting control through intraguild predation (Snyder et al. 2004). This supports our informal observations that GWA remains relatively abundant in areas invaded by harlequin ladybirds in New Zealand. We conclude that predation from harlequin ladybirds may not be sufficient to negatively impact P. nigrovaria abundance.

Risk 4: Reduction of GWA by P. nigrovaria causes indirect food-web effects

The ecosystem in New Zealand may be affected at various trophic levels by the release of Pauesia nigrovaria for biological control of GWA. The main concern is that the reduction of GWA as prey available to predators will see a temporary lack of resources for predators, who may then put greater predation pressure on native insects. This risk is short-term and is outweighed by the more significant long-term benefits of fewer harlequin ladybirds and vespid wasps. The seasonal population dynamics of GWA may also largely limit incidences of prey-switching. Most insects emerge in the spring, feed through the summer, reach adulthood and reproduce before dying off in the autumn. GWA reaches peak abundances in the late summer and autumn when most predators have completed their development and therefore have limited capacity to multiply in response to GWA abundances. If GWA were most abundant in the spring and early summer, these indirect effects would likely be much stronger. It should also be noted that since GWA has only been in New Zealand since 2013 that it is still very novel in the New Zealand environment, so it is highly unlikely that any native or beneficial species yet relies on either the aphids or their honeydew for their survival or successful reproduction.

Risk 5: P. nigrovaria causes indirect effects on pest willows by reducing GWA populations

62 The pest willows Salix fragilis and S. cinerea are under active management and eradication in many wetlands. GWA will also be acting to suppress willow growth in wetlands where active management is not undertaken. With reduced GWA populations as a result of the parasitoid, we could expect to see a return of pest willow tree health to levels experienced before the introduction of GWA. However, it should be stated that while GWA is no doubt impacting the health of these pest willows, it is not likely to contribute significantly to their eradication. Moreover, the associated negative impacts of increased wasp abundances on wetland invertebrate communities likely outweigh any benefits from reduced willow vigour.

Risk 6: P. nigrovaria causes indirect effects on native species

As stated above, the introduction of P. nigrovaria to the New Zealand environment may lead to host-switching by established hyperparasitoid species from their current hosts to include P. nigrovaria. In areas with abundant willows, GWA and P. nigrovaria, the population sizes of any hyperparasitoid species capable of attacking Pauesia could increase substantially. These hyperparasitoids may in turn attack other beneficial or native parasitoid species in those environments, reducing their effectiveness to control their host species, which may also be important pests. Indeed, hyperparasitoid species are known from other Aphidiine wasps already established in New Zealand (e.g., Dendrocerus aphidum, Alloxysta victrix) and may host-switch to P. nigrovaria. These sorts of indirect effects are very difficult to quantify, let alone predict prior to the introduction of a new organism. However, there is no documented evidence that any population increase of a hyperparasitoid caused by host switching to a biological control agent had detrimental impacts on other parts of a parasitoid food web. This is theoretically possible but as indicated it has never been documented. It should also be noted that no hyperparasitoid species in New Zealand is known to attack P. nigrovaria. Of those hyperparasitoid species present here, Alloxysta victix is one of the most generalised and is unknown from any Pauesia species (Ferrer- Suay et al. 2014).

Risk 7: P. nigrovaria competes for nectar resources

Like most parasitoids, P. nigrovaria will readily imbibe plant exudates, such as nectar, to prolong their lives and fuel their foraging. In the laboratory, P. nigrovaria that are given honey and water live for approximately 2-3 weeks, while those deprived of these resources typically die in 2-3 days. The potential then exists for competition to occur between P. nigrovaria and other native or

63 beneficial species for nectar resources. However, aphid parasitoids typically feed on the honeydew produced by their host species (as observed for P. nigrovaria and GWA in the laboratory). We therefore expect that P. nigrovaria adults in the wild will feed predominantly on GWA honeydew rather than from flowers or other sources. The very small size of P. nigrovaria also means that individuals consume very little, so they will not exhaust floral resources. Their small size also means they will likely be outcompeted by much larger beneficial species such as honeybees and bumblebees that also visit flowers. Therefore, we conclude there is a very low risk that P. nigrovaria will negatively affect other species through competition for floral resources.

Risk 8: P. nigrovaria hybridises with another aphidiine parasitoid present in New Zealand

In New Zealand there are no other species of Pauesia. In the same subfamily as Pauesia (Aphidiinae) there are 12 species in five genera (Aphidius, Diaeretiella, Ephedrus, Lysiphlebus, Trioxys, MacFarlane et al. 2010). All are either deliberately introduced biological control agents or were accidentally introduced along with their host aphid species. None of these species are considered to be sufficiently related to facilitate natural hybridisation with P. nigrovaria.

Table 9: Summary of potential risks posed to and from Pauesia nigrovaria in the New Zealand environment

Source of the risk Possible reasons for Adverse effect Exposure pathway the risk

1. Female P. Female P. nigrovaria Potential for A female P. nigrovaria may may make host increased mortality nigrovaria encounters attack non-target recognition errors and of native species (as a native aphid while native aphid attack native aphids, a result of wounding searching for GWA. species, even if even though they are from oviposition, or they cannot not attractive hosts. attempted oviposition develop on these reducing their species. abundance).

2. Pauesia larvae One or more species of Decreased population A hyperparasitoid may be attacked hyperparasitoids size of P. nigrovaria species host-switches by (native or exotic) and less control of to P. nigrovaria. hyperparasitoids. already present in New GWA populations. Zealand may attack developing P. nigrovaria larvae.

64 3. P. nigrovaria Many species are Predation may Flying adult P. may be attacked insectivorous, reduce P. nigrovaria nigrovaria may be by predators. including numerous population sizes, taken by birds and native birds and reducing their impact predatory invertebrates. on GWA numbers. invertebrates. Larvae may be eaten while developing in GWA by ladybirds or other predatory species.

4. Reduction of Reduction of GWA may Temporary increased Indirect impacts of

GWA by P. lead generalist mortality of native reduced GWA nigrovaria causes predators like species, but long populations. indirect food-web harlequin ladybird to term lower harlequin ppppprekjashfglkhak effects. predate on native populations result in glhjkj preda insects (more than positive benefits for currently). native species.

5. P. nigrovaria Pest willow species Wetlands see willow Indirect impacts of causes indirect Salix fragilis and S. tree vigour return to reduced GWA effects on pest cinerea may increase pre-GWA introduction populations. willows by in health and vigour. levels. reducing GWA populations.

6. P. nigrovaria Host-switching by Increased A hyperparasitoid causes indirect established hyperparasitism of species host-switches effects on native hyperparasitoids to P. beneficial or native to P. nigrovaria. or beneficial nigrovaria increases parasitoid species, species. the abundance of making them less these species, which effective at then attack other suppressing the native or beneficial abundances of their parasitoids. hosts, which may be pests.

7. P. nigrovaria Parasitoids commonly Reduced quantities of Adult flying competes with drink nectar and water nectar for other parasitoids. other species for to extend their lives or species. nectar resources. mature their eggs.

8. P. nigrovaria P. nigrovaria may Hybridisation, if Individual parasitoids hybridises with hybridise with other successful, could attempt to mate with other species. Aphidiinae parasitoids. create unknown other species. genetic risk.

65 Table 10: Summary of the magnitude of the potential risks posed to and posed by Pauesia nigrovaria in New Zealand.

Source of the risk Adverse effect Likelihood of Magnitude of effect Evaluated adverse level of risk effect occurring

1. Female P. Increased Highly In the highly unlikely event Insignificant nigrovaria may mortality of unlikely. that a female P. nigrovaria . attack non- native attacked a native aphid, target native aphid species, the effect on that non- target species, even if reducing their species would almost they cannot abundance. certainly be minimal since our develop on these native species are too small to species. facilitate the development of P. nigrovaria larvae. Without successful development P. nigrovaria cannot permanently host-switch to one of our native species.

2. Pauesia Decreased Low to Given that numerous species Insignificant to nigrovaria larvae population medium. of hyperparasitoids occur in medium. may be attacked by size of Pauesia New Zealand, it is possible hyperparasitoids. and less that one or more species may control of GWA attack P. nigrovaria. Impacts abundances. are difficult to predict, but range from insignificant to medium, where population sizes may be reduced. The probability of hyperparasitoids eradicating P. nigrovaria is very small.

66 3. Pauesia Pauesia Medium to Some Pauesia will inevitably Insignificant to nigrovaria may be nigrovaria high. be eaten by predatory species low. attacked by may be already present in New predators. distasteful and Zealand. Consumption of predation Pauesia is unlikely to have within GWA direct negative effects unless nymphs may it is more toxic than other reduce insects, which seems unlikely. Pauesia The bigger impact may be a population reduction in Pauesia sizes, reducing abundances, which may their impact reduce their impact on GWA. on GWA The overall impact of numbers. predation will probably be low since predators do not currently seem to be greatly reducing GWA abundances.

67 4. Reduction of Reduced GWA Low to At best any adverse effects Insignificant to GWA by P. prey medium. from prey-switching will be low. nigrovaria causes availability short-term (one season at a indirect food-web leads to given locality). Prey- effects increased switching will also be predation of minimised by the fact that native GWA abundances are highest invertebrates. in autumn, when most other insects have already completed development.

5. P. nigrovaria Vigour of pest Medium. While it is likely that the Low to causes indirect willows in health of some pest willows medium. effects on pest wetlands, will increase, this is likely to willows by reducing return to pre- be outweighed by the positive GWA populations. GWA levels. effects on native insect communities from a reduction in vespid wasp abundances. Furthermore, GWA was never likely to eradicate or significantly reduce willow numbers, so their presence is not exacting any meaningful control on these wilding species.

6. P. nigrovaria Increased Low. With no other Pauesia species Insignificant to causes indirect hyperpara- or the one known low. effects on native or sitism on hyperparasitoid of P. beneficial species. native or nigrovaria present in New beneficial Zealand, the chances of host- parasitoid switching occurring can be species. considered low. If switching does occur, adverse effects would be dependent on P. nigrovaria becoming a major host for the hyperparasitoid, which is unlikely. No evidence for detrimental effects on non- target species from host- switching of hyperparsitoids to new biocontrol agents is known.

68

7. P. nigrovaria Reduced Low. Their small body size and Insignificant to competes with quantities of likely infrequent use of floral low. other species for nectar for resources means that they nectar resources. other species. would have an insignificant impact on other insects that visit flowers.

8. P. nigrovaria Creation of a Negligible. The lack of other species of Insignificant hybridizes with new genotype. Pauesia in NZ suggests this will . other species. not occur.

5.1.2 Potential adverse effects on public health taha hauora

It is highly unlikely that P. nigrovaria would pose any risk to taha hauora, public health. Although they are wasps, they lack venom and do not form colonies. They are too small to pierce human skin with their ovipositors and there is no evidence that they seek out human habitation or are toxic if accidentally swallowed or inhaled. They should be no more of a threat to human health that any of the thousands of other small, harmless insect species present in New Zealand.

5.1.3 Potential adverse effects on Māori

The interaction of parasitoids like P. nigrovaria with its host GWA reflects the eternal struggle between Tane and Whiro, where good is pitched against evil. Māori understand this tension and the story of Tane defeating Whiro illustrates that good can triumph over evil. In general Māori favour using natural methods for managing environmental issues. Therefore, it may be that many Māori will prefer to see willows removed, than to risk the introduction of new species into New Zealand to manage the pest GWA. Māori are likely to be concerned about any negative effects P. nigrovaria could have on culturally significant species, and also on taha wairua. Spiritual health and well being, taha wairua is maintained when there is a balance between nature and the protection of mauri. We hope that in this application we will be able to reassure Māori that P. nigrovaria will in fact help to restore ecological equilibrium by controlling an invasive pest that is impacting negatively on taha hauora through bringing increased vespid wasps into contact with humans, and it will also enhance taha wairua if the important honey industry is protected from the cement honey problem that GWA causes.

5.1.4 Economic risks of not controlling giant willow aphid

Giant willow aphid (GWA) impacts various industries both directly and indirectly. Table 11 shows the main assets impacted by the presence of this pest, divided into regions. The willow trees that the aphids feed on are directly impacted. In a trial conducted by Collins et al.

69 (2001), the biomass of GWA infested willows was < 50% than for control willows. A significant reduction in tree diameter and height growth has been observed by Plant and Food Research NZ (2019, unpublished), where willows infested with the aphid showed a minimum of 20% growth reduction. This growth reduction has implications for carbon sequestration, which is valued at $25 per tonne. Basket weavers who grow willows are impacted and have less material to work with. Total willow and poplar cover in New Zealand is estimated at 223,000 ha. (Jones and McIvor, 2016). In a study by Collins et al. (2001) on hydroponic- cultured willows, aphid infested willows produced 84% less primary roots and leaves than non- infested willows. This reduced root production could have serious implications for erosion control, one of the prime uses for willows. Sediment not lost from the productive environment to erosion has been valued at $3 per tonne (Daigneault et al., 2017). Giant willow aphids feeding on willows produce honeydew, which contains several kinds of sugar: fructose, glucose, sucrose and the trisaccharide melezitose (Mitler, 1958). Honey bees gather this honeydew, resulting in the formation of a hard, crystalised honey in the comb when present above a concentration of ~ 18%. This is caused by the melezitose, which has a low solubility and crystalises out when the moisture content of the honey drops as bees cure the honey. Crystalized honey cannot be extracted using the centrifugal extraction methods employed in New Zealand (it could only be done using high heat, a practice not employed because it degrades the valued properties of the honey). According to the NZ colony loss survey (2018), bee keepers with more than 250 hives had 2.5% of their apiaries lost or compromised due to crystalized honey. Even when the proportion of giant willow aphid honeydew is small and not enough to cause the formation of crystalised honey, it can still be problematic for beekeepers. This is because it has a disproportionate amount of fructose, which is interpreted as possible adulteration with high fructose corn syrup. An Auckland beekeeper recently had an export shipment to China refuse because of this (McLean, pers. comm.). In addition to this, the presence of melezitose can suggests that honey labelled as multifloral honey should in fact be labelled as honeydew honey (McLean, pers. comm.).

The honeydew produced by GWA also drips from trees, covering leaves and stems and anything under the trees, and will drift on the breeze to land on anything nearby such as fruit. Sooty mould grows on this honeydew, resulting in a black furry coating on everything the honeydew lands on. Orchards growing near willow trees or using them as shelter may see a loss of produce due to this sooty mould. Zespri estimates 20%-30% of their kiwifruit losses are due to sooty mould on the fruit (Gould, pers. comm.). GWA contribute to this but passion vine hoppers and cicadas also cause problems. Given that Zespri experience about 1.3% onshore fruit loss (Zespri, 2018), we estimate the impact of GWA on orchards at 0.3% output reduction. The value of kiwifruit exports in 2017 was $1.7 billion NZD, the majority of which is grown in the Bay of Plenty, resulting in potential losses of

$5.1 million (Aitken and Warrington, 2017). This sooty mould may also make willow branches unusable for basket weavers, or at least require extra time and effort to clean.

Pest vespid wasps will also feed on the honeydew produced by GWA, leading to increased numbers of wasps in areas where GWA is present and the numerous issues they bring with

70 them. Wasps attack honey bees, steal their honey and destroy hives. In 2018 about 8.5% of colony losses were attributed to wasps (NZ Colony loss survey, 2018). The total value of lost hives and time and money spent on wasp management by beekeepers is about $9 million per year. A further $9 million is lost each year to wasps directly attacking hives, stealing honey and in some cases destroying the hive (Cunningham and Davies, 2019). Wasps also compete with bees for other food sources, leading to reduced honey production. The total value of this lost production is estimated at $58 million per year (Dixon, 2018. In total, wasps are responsible for about $76 million NZD in losses to the honey industry per year. By interfering with bees, wasps can disrupt pollination. Reduced pollination of clover leads to reduced nitrogen fixation in pasture, leading farmers to increase fertilizer use to maintain production. The estimated cost of this indirect impact is about $62 million per year (Dixon, 2018).

Besides affecting bees and honey production, wasps are also responsible for significant costs in terms human health. Wasps are associated with about $1 million NZD per year in health costs, and a cost further estimated $1.4 million NZD each year by causing traffic accidents (MacIntyre and Hellstrom, 2015).

Wasps also cause a lot of damage to the native New Zealand environment. In areas with high amounts of honeydew, such as South Island beech forests, the invertebrate prey biomass consumed by vespid wasps can match that of all the insectivorous birds combined (Harris 1991). These invasive wasp species have even been observed killing baby birds in the nest (Moller 1990).

Changing temperatures and weather patterns will affect the reproduction rates of giant willow aphid, potentially increasing the scale of their impacts further, but in what way and to what extend is not yet understood. Any bio-control will also be affected by climate change, but how their interaction with GWA is likely to be affected is yet to be determined. As such we have not included any climate change impacts within this analysis, focusing only on the impacts observed to date in the current environment.

Table 11. Main assets impacted by giant willow aphid, by New Zealand region. Mitigate Region Willow Honey Production Average Carbon Carbon d Appl Kiwifruit + Area (tonne/year) Honey Seq Seq Erosion es pipfruit (ha) returns (tonne/ye ($/year) ($/year) (ha) EBIT ($million/ye ar) ar)

Northland/Auckla 2,500 43.5 104 2.0 nd/ Hauraki Plains

Waikato/King 2,857 49.7 144 2.8 country/ Taupo 253 Coromandel/ Bay 3,094 53.8 1668.9 of Plenty /Rotorua/ Poverty Bay

71

Hawke's Bay/ 5,911 102.9 4885 94.9 Wairarapa/Mana watu/ Taranaki/ Wellington

Marlborough/Nels 1,712 29.8 2421 47.0 on/West Coast

Canterbury/Kaiko 2,612 45.5 312 6.1 ura

Otago/Southland 1,199 20.9 427 8.3 2,022,610 50.6 ($25 8,61 NZ Total 223,0 19,855 345.5 per 678.6 5 1831.4 00 tonne) ($3 per tonne)

Yield per hive 29.1 (kg) willow NZ MPI Daigneault, New Dominati NZ.Stat MPI

Poplar

(2018) Source (2012,

Zealand

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Commission

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017)

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-

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2012

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booklet)

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poplar

and

Table 12 summarises the estimated impact of GWA on each of the assets, as described above, as well as the additional costs via increased pest wasp populations. The total impact of GWA is estimated to be nearly $300 million NZD per year. The impact on the honey industry is second only to the loss of erosion protection afforded by willow and poplar trees. As mentioned above, GWA has quite a significant impact on root development. Even if the trees are not dying, their root structure and ability to hold the soil together is greatly impacted.

Table 12: Expected impact of giant willow aphid on Assets (in millions of dollars per year).

Average Pest Carbo Mitigate Kiwifruit Total honey wasp n seq. d + impact returns impacts erosion Clover Pipfruit nitrogen returns fixation

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Expected 2.5 % $ 2.4 20% 20% $62 0.3% GWA reduction + million in reduct reductio million reductio reduction $76 million health ion n due to n % due to pest costs and reduced wasp traffic clover manageme accidents pollinatio nt and n production impacts

Total GWA 84.2 2.4 10.1 135.7 62 5.5 300 impact per year (unmitigate d)

5.2 Benefits

The economic and environmental benefits to New Zealand of introducing Pauesia nigrovaria are numerous and wide-ranging. The high versatility of non-pest willows means they are planted around the country for many purposes, so improved willow health is a direct benefit to thousands of farmers, apiarists, most councils and the general public. Reducing GWA abundance has flow-on effects for the other species in the environment that interact either directly or indirectly with GWA or more commonly, the copious quantities of honeydew they produce. Fewer GWA and less GWA honeydew means fewer social wasps, which reduces predation of native invertebrate species and benefits native birds, bats and lizards. Below are listed in detail the major benefactors from the release of P. nigrovaria and a subsequent reduction in the abundances of GWA and the problems associated with this invasive pest.

It is difficult to separate the environmental and economic benefits of introducing P. nigrovaria. We therefore provide a comprehensive list of benefits, followed by a purely economic analysis.

5.2.1 List of benefits of introducing Pauesia nigrovaria (summarised in Table 13).

Benefit 1: Reduced negative impacts of GWA on willow health

Willows and poplars are planted extensively for slope stabilisation and erosion control. Although there is a growing interest in New Zealand in investigating the inclusion of other tree species for these purposes, particularly New Zealand natives (e.g. Phillips et al. 2011), willows and poplars are fast growing, hardy and easy to plant, have a proven track record and are currently performing these functions in the landscape (Basher 2013). In addition, willows and poplars offer additional benefits such as providing animal fodder (particularly valuable in times of drought) (see New Zealand Herald article published 31 July 2017: https://www.nzherald.co.nz/the- country/news/article.cfm?c_id=16&objectid=11897180) and providing vital resources for bees in spring when there is little else in flower (Newstrom-Lloyd et al. 2015). We therefore foresee willows continuing to be valued well into the future. Stress from GWA is reducing the growth of willows throughout New Zealand and making them more susceptible to other mortality factors (disease, drought, high winds, etc.). Collins et al. (2001) found that willows infested with GWA

73 produced 84% less primary roots and leaves. Severe infestations may kill host trees (T. Jones, unpubl. data). Increased mortality of non-pest willows is likely to result in increased erosion of steep land and river banks, and the loss of soils, and increased downstream sedimentation, which will have numerous detrimental environmental and economic impacts. The introduction of P. nigrovaria will reduce the impacts of GWA on these non-pest willows. Over time we should see an improvement in willow health, with the result of reduced stress and tree mortality and, consequently, less erosion to hillsides and river banks.

Benefit 2: Reduction in abundance of pest vespid wasps

Invasive social wasps are not only a nuisance and health issue to people, they also have a large detrimental effect on our native ecosystems. These wasps harvest carbohydrates in the form of plant exudates or honeydew to fuel their foraging trips for invertebrate prey to feed their developing larvae. In the United Kingdom, Vespula germanica foraging on giant willow aphid honeydew was reported as being up to 6 times more abundant than bumblebees doing the same (Cameron et al., 2019). In New Zealand we have observed strong correlations between pest wasp abundance and the presence of both beech and giant willow aphid honeydew, with localised populations of giant willow aphid often being found only because large wasp swarms around willow trees drew attention. Since wasps reach their highest abundances in areas with abundant honeydew resources, it is likely that this resource is a significant limiting factor for their population growth. Reducing the abundances of GWA should significantly reduce the availability of honeydew in many landscapes, thus leading to a reduction in the abundances of these pest species. Fewer wasps means fewer stings to people, pets and livestock. It also means fewer native insects, birds, bats and lizards are killed by these voracious predators.

Benefit 3: Reduction in formation of cement honey

GWA honeydew is high in the trisaccaride sugar melezitose. When bees gather large quantities of GWA honeydew the resultant honey crystalises in the comb, making it difficult or impossible to extract using the centrifugal extraction methods employed in New Zealand. The honey itself has a bitter taste and is unmarketable. It may not even be able to be used by the bees. The introduction of P. nigrovaria will reduce GWA abundance. This in turn will reduce the incidence of cement honey, increasing the quality of honey produced by apiarists and reducing losses and time spent servicing affected hives.

Benefit 4: Reduction of sooty mould

Sooty mould grows on almost any surface on which honeydew splashes. When it covers leaves it reduces the photosynthetic capacity of the plant, while fruit bearing even small amounts of sooty mould is unmarketable and discarded. The kiwifruit industry is particularly vulnerable to sooty mould contamination as there is no practical method to clean fruit prior to export. As much as 30% of kiwifruit losses are due to sooty mould contamination (Gould, pers. comm.). Although this sooty mould grows predominantly on honeydew from passion vine hoppers (Scolypopa australis) and cicadas (Hemiptera: Cicadidae), GWA contribute to this problem

74 where willows are used as shelterbelt species. Reductions in GWA should reduce the growths of sooty mould since there is a direct relationship between the abundance of honeydew producing insects and the amount of sooty mould growing under infested trees (Wardhaugh et al. 2006).

Benefit 5: Reduction in abundance of harlequin ladybird beetles

Like vespid wasps, the harlequin ladybird is a voracious generalist predator that is undoubtedly having a detrimental impact on native insect species. In addition to the numerous species on which this ladybird feeds, they can also kill other species which feed on them. Harlequin ladybirds carry high loads of microsporidia fungi in their blood (haemolymph). The ladybirds do not seem to be adversely affected by this fungus, but other predators that feed on harlequin ladybirds or their eggs are often killed by this fungus (e.g. Vilcinskas et al., 2013). Reductions in the abundances of GWA, on which harlequin ladybirds regularly feed as both adults and larvae, may reduce the population sizes of these beetles in areas with numerous willows. However, reducing GWA abundances may have little impact on harlequin ladybird numbers at larger spatial scales as they are generalists and will likely switch to alternative food sources.

Table 13. Summary of potential benefits (both economic and environmental) of releasing Pauesia nigrovaria Source of benefit Likelihood of benefit occurring Magnitude of effect

1. Reduced negative All benefits are dependent on the Probably Moderate:

impacts of GWA on ability of P. nigrovaria to Magnitude will invariably depend willow health establish in New Zealand and on the level of control P. negatively impact GWA nigrovaria is able to exert on abundances. If this occurs it is GWA. Increased willow health will Highly likely that willow health improve the performance of will improve. willows and poplars when used for slope stability and erosion control by reducing tree mortality and increasing growth rates.

75 2. Reduction in Likely: Moderate: abundance of pest In New Zealand invasive social The benefits here will depend on vespid wasps wasps reach their highest the abundance of willows in the abundances in areas with landscape, being greatest where abundant sources of honeydew, willows are more abundant or such as South Island beech grown in urban or suburban forests. It appears then that their areas. populations are in large part limited by the availability of a carbohydrate source. By reducing GWA populations the abundance of vespid wasps should also decrease in areas with willow trees.

3. Reduction in Highly likely: Low to moderate: formation of cement Cement honey forms when bees The magnitude of this effect honey use large quantities of GWA depends also on the abundance of honeydew when making honey. willows in the surrounding Reductions in the population landscape. Little impact may be sizes of GWA will mean the seen in areas with few willows, threshold amount of GWA but in areas with abundant honeydew required for willows the potential benefits may crystalisation to occur will be less be much higher. likely to be reached.

4. Reduction of sooty Highly likely: Low to moderate: mould There is a direct relationship Reductions in sooty mould on the between the population size of leaves of host trees should honeydew producing insects and increase their efficiency in the amount of sooty mould photosynthesis, thus increasing growing under and on infested their vigour and health. Benefits trees (Wardhaugh et al. 2006). of reduced sooty mould will Reductions in GWA numbers will depend on what is under infested almost certainly result in less trees. Impacts will be greatest sooty mould on and under host where willows are used as trees. shelterbelt trees in orchards, as fewer fruit will be spoiled.

76 5. Reduction in Possible: Low to moderate, but localised:

abundance of Harlequin ladybirds are As generalists, harlequin harlequin ladybirds generalist predators that feed on ladybirds are probably not almost any smaller invertebrates dependent on GWA to maintain they encounter, including their high abundances in the numerous native species. The landscape. degree to which their However, high levels of abundances will be reduced in parasitism could reduce aphid response to lowered populations abundances at the local scale or of GWA is dependent upon the on individual willow trees to the proportion of GWA in their diet, point where ladybird larvae or the proportion the population starve, or their growth slows, that depends on GWA as a larval thus increasing the probability of and adult food source. This is succumbing to other time-based currently unknown. mortality factors.

5.2.1 Economic benefits of releasing Pauesia nigrovaria

The current aphid population is large, therefore depending on how many Pauesia nigrovaria are released it will take time for the parasitoid population to mitigate GWA impacts. Observations of other Pauesia species used as biological control agents have seen huge reductions in target aphid numbers following release. The black pine aphid abundance in South Africa was reduced from an infestation rate of 99% of trees to ~2% within two to three years after the release of Pauesia cinaravora (Kfir et al., 2003). Figure 5 Illustrates the model used to estimate the effect Pauesia nigrovaria will have on GWA populations, and thus to what extent it will mitigate their impacts. The arrows represent the direction of movement. Nymphs become adults at a set development rate (and for the purpose of the model turn back into nymphs once they pass reproductive age), both adults and nymphs can be attacked by P. nigrovaria and become a mummy at a rate dependent on how many P. nigrovaria and how many aphids there are in the environment at the time. Mummies develop into wasps a constant rate. Individuals die at a natural death rate throughout this process, while nymphs are born at a rate dependent on the number of reproducing aphids present. The results of this model are compared with the results of the model without P. nigrovaria, in order to estimate the influence of the parasitoid.

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Figure 5: An illustration of how the interaction between P. nigrovaria and GWA is modelled. Non- reproducing adults are grouped in with the nymph stage.

Simulations based on observations and best estimates of GWA and P. nigrovaria fecundity suggest we could see a similar outcome here in New Zealand. For the simulations, we assumed a 60% female-biased sex ratio of P. nigrovaria, consistent with our laboratory rearing and with field studies of other Aphidiinae parasitoids (e.g. Mackauer, 1976; Rabasse and Dedryver, 1983). The average number of mummies produced by P. nigrovaria in a 24 hour period, as calculated from host specificity tests using GWA, is higher than the number of GWA offspring produced during the same amount of time. Assuming a homogeneous release of P. nigrovaria relative to the GWA population of about one wasp per 10,000 aphids, using the average observed P. nigrovaria fecundity, we could expect a 1% reduction in aphid numbers within a month. The initial release ratio of parasitoids to aphids is relatively unimportant, the main effect this has is on how long it

78 takes for wasp numbers to catch up to the aphids. Once wasp numbers catch up, we could see aphid population reductions of up to 80%, with an overall mean of 46%. The expected reduction in GWA impacts after one year is 34%. Figure 6 shows the expected value of mitigating GWA impacts, using P. nigrovaria as a biocontrol. Over a 20-year period, assuming average fecundity observed in lab experiments and a 6% discount rate (Treasury New Zealand, 2008), the net present value of mitigated impacts is estimated at $1.5 billion New Zealand dollars. The value is plotted for the mean expected efficacy of the parasitoid, as well as the minimum and maximum outcomes, as estimated by experiments in the lab. At maximum efficacy, applying the higher levels of fecundity seen, the value of releasing P. nigrovaria is estimated at about $2.6 billion New Zealand dollars over a 20-year period, while at minimum efficacy, the value is about $500 million New Zealand dollars over the same length of time.

Figure 6: The cumulative value (in millions of dollars) of using Pauesia nigrovaria to control giant willow aphid over a 20-year period, assuming a 6% discount rate.

Using Monte Carlo simulation, for the parameter ranges and mean values estimated, a distribution of the likely benefits of releasing Pauesia nigrovaria as a biocontrol was estimated. The distribution of likely benefits over a 20-year period is presented in Figure 7. While there is a small chance the benefits will be as low as the minimum presented in Figure 6, it is most likely to fall somewhere between $1.3 billion and $1.7 billion NZD.

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Distribution of Pauesia nigrovaria biocontrol

0.45 benefits

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

Proportion simulations of Proportion 0

500 - - 500 700 - 700 900

900 - - 900 1100

1100 - - 1100 1300 - 1300 1500 - 1500 1700 - 1700 1900 - 1900 2100 - 2100 2300 - 2300 2500 - 2500 2700 20 year benefit in million NZD

Figure 7: The distribution of Pauesia nigrovaria benefits over a 20-year time horizon, based on the range of parameter values estimated.

6. Pathway determination and rapid assessment

Under sections 38I and 35 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 one of the below sections. Fill in the section that is relevant to your application only. 6A. New organism that is or is contained within a veterinary or human medicine (section 38I)

6.1. Controls for organism

Describe the controls you propose to mitigate potential risks (if any). Discuss what controls may be imposed under the ACVM Act (for veterinary medicines) or the Medicines Act (for human medicines)

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6.2. Discuss if it is highly improbable (after taking into account controls if any):

• The doses and routes of administration of the medicine would have significant adverse effects on the health of the public or any valued species; and • The organism could form an undesirable self-sustaining population and have significant adverse effects on the health and safety of the public, any valued species, natural habitats or the environment Do not include effects of the medicine or new organism on the person or animal being treated with the medicine

6B. New organism (excluding genetically modified organisms) (section 35)

6.3. Discuss if your organism is an unwanted organism as defined in the Biosecurity Act 1993

69

Application Form Approval to release a new organism

It is not an unwanted organism.

6.4. Discuss if it is highly improbable, after taking into account the proposed controls, that the organism after release:

• Could form self-sustaining populations anywhere in New Zealand (taking into account the ease of eradication) • Could displace or reduce a valued species • Could cause deterioration of natural habitats, • Will be disease-causing or be a parasite, or be a vector or reservoir for human, animal, or plant disease • Will have adverse effects on human health and safety or the environment

We consider that the organism will form desirable self-sustaining populations throughout New Zealand to control the pest aphid. It is highly improbable that the new organism could displace or reduce valued species or cause deterioration of natural habitats. It is not the nature of the organism to be disease-causing or a causal vector of human or animal diseases. If the target organism, the pest aphid, becomes known as a vector of plant diseases (as some aphids are), then this new organism will help to minimise those effects by reducing populations of the aphid. The new organism will not have adverse effects on human health and safety; instead it will reduce numbers of pest wasps which sting humans and animals. The new organism is not expected to have any adverse effects on the environment; reducing numbers of pest wasps is expected to help to conserve New Zealand’s biodiversity.

7. 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.

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Application Form Approval to release a new organism

8. 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: 24/5/2019

• Cheque for application fee enclosed □ Yes ✓ No □ Payment to follow

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

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Application Form Approval to release a new organism

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.

28 June 2019

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:

72

Application Form Approval to release a new organism

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

List of appendices

Appendix 1. Maori consultation letter (draft for customization)

Appendix 2. GWA biological control consultation response form

Appendix 3. GWA biological control public engagement info sheet

Appendix 4. Maori feedback and response: Tui Shortland

Appendix 5. Maori feedback and response: Hëmi Te Räkau

Appendix 6. GWA biological control public engagement info sheet (expanded):

Appendix 7. GWA article published in New Zealand Beekeeper, October 2016, Page 19.

Appendix 8. GWA article published in New Zealand Beekeeper, February 2017. Page 9.

Appendix 9. GWA article published in New Zealand Beekeeper, July 2018. Pp 23-24.

Appendix 10. Presentation to Forest & Bird 11 June 2019, by Andrea McCormick and Trevor Jones

Appendix 11. Poster presented at Apiculture New Zealand annual conference, 27 June 2019, Rotorua.

Appendix 12. GWA article published in Forest Health News, July 2016, Page 1.

Appendix 13. GWA article published in MPI SFF Booklet, June 2017, Page 46.

Appendix 14. GWA article published in Forest Health News 278, January 2018. Page 2.

Appendix 15. GWA article published in Scion Connections, June 2018. Pp 4-5.

Appendix 16. Third annual project newsletter, June 2019.

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Glossary of technical terms

Aphid mummy – A parasitised aphid that has died and affixed itself to a surface, usually appearing bloated and dry.

Bayesian Inference - A statistical method that updates the probability of a hypothesis as more evidence or information becomes available.

Concatenated matrix – The process of joining two or more matrices to form a new matrix.

Cuticle – The outer layer of the exoskeleton of arthropods.

Eclosion – The process of emerging from a pupal case or hatching from an egg.

Endemic – Species that occur in a single, defined geographical area and nowhere else (e.g., species endemic to New Zealand occur only here and not in any other countries).

Endoparasitoid – A parasitic organism that feeds internally on its host and eventually kills it.

Fecundity – The potential reproductive ability of an organism, most commonly defined as the total number of eggs, either laid or within the female when dissected.

Codon - A sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis.

Gene flow – The exchange of genes between populations.

Gondwanan – Referring to the distribution of a group of organisms. Gondwanan species are typically distributed across landmasses that once constituted the megacontinent of Gondwana (New Zealand, Australia, South America, Africa, and sometimes also India and Antarctica). It is hypothesised that the common ancestors of these groups were distributed across Gondwana, only to radiate into distinct lineages on each, now isolated, landmass. Well-known examples include southern beech trees (Nothofagaceae), and ratites (ostriches, emus, cassowaries, rheas, elephant birds, kiwi and moa).

Honeydew – The carbohydrate-rich excrement of sap-sucking insects.

Host-plant volatiles – Chemicals emitted into the air by host plants, often in reaction to some external stimuli such as feeding by herbivorous insects.

Host specificity – Refers to the number of host species an organism utilises. The more host species utilised, the less host specific an organism is. Species with only one or a few closely- related host species are considered to be highly host specific.

Hyperparasitoid – A parasitoid that parasitises another parasitoid.

Koinobiont – Parasitoids which only kill the host when the developing parasitoid reaches maturity. The host may continue to feed and grow when parasitised.

74

Monophagous – A species that feeds on a single host, which can be either a plant or an animal.

Mesonotum – The dorsal portion of the second thoracic segment. In many Hymenoptera this structure forms most of the dorsal surface of the thorax.

Mesopleura – The lateral part of the second thoracic segment.

Mesosterum – The ventral part of the second thoracic segment.

Non-target organism – An organism that is not the intended target of a biocontrol agent.

Notaulices (singular = notaulix) – The longitudinal furrows or sutures on the mesonotum.

Nymph – The immature stage of an insect that undergoes partial metamorphosis, such as an aphid.

Oviposition – The act of laying eggs.

Paraphyletic – A group of organisms that are all descended from a common ancestor, but does not include all descendant groups. For example, reptiles are considered paraphyletic because they do not include birds, which are descended from theropod dinosaurs.

Parasitoid – A parasitic organism that kills its host during its development.

Pheromone – A chemical substance released by an insect that affects the behaviour of other individuals of the same or different species. These include sex pheromones, which may aid in locating a mate, alarm pheromones, which alert other individuals of an imminent danger, and aggregation pheromones, which attract other individuals to a potential food source.

Phylogeny – A structured family tree depicting how different species or groups of organisms are related. Typically, these are shown as branching graphs. The underlying assumption is that species or groups of organisms that share more physical characters and/or differ less in their genetic makeup are more closely related (and therefore shown closer together) than those that differ more in diagnostic characters and genes.

Polyphagous – Feeding on many different types of food.

Polyphyletic – A set of organisms that have been grouped together but are derived from more than one distinct lineage.

Posterior probabilities – The statistical probability that a hypothesis is true in the light of relevant observations. In Figure 1, numbers closer to 1.0 indicate high statistical probabilities.

Siphunculi – The pair of tube-like or pore-like structures on the dorsal side of the sixth abdominal segments of aphids that emit pheromones and defensive secretions.

Trichotomy – Split into three parts.

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Unrooted topology – A phylogenetic tree that does not show an ancestral root represented by the basal ancestor of that group of organisms. Rooted trees by contrast, indicate the most recent common ancestor of that group of organisms, with branches radiating from that ancestor showing phylogenetic relationships. Unrooted trees also show the relationships, but not the common ancestor.

Vespid wasp – Social wasps of the family Vespulidae. In New Zealand these include the invasive yellow-jackets (Vespula vulgaris and V. germanica), and paper wasps (Polistes chinensis and P. humilis).

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Application Form Approval to release a new organism

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