FORM NOCR Application for Approval to IMPORT for RELEASE OR

FORM NOCR

Application for approval to

IMPORT FOR RELEASE OR RELEASE FROM CONTAINMENT WITH CONTROLS ANY NEW ORGANISM

under section 38A of the Hazardous Substances and New Organisms Act 1996

Application Title: Conditional release from containment the Irish strain of the parasitic wasp Microctonus aethiopoides for biological control of Sitona lepidus (clover root weevil)

Applicant Organisation: AgResearch Ltd

ERMA Office use only

Application Code: Formally received:____/____/____

ERMA NZ Contact: Initial Fee Paid: $

Application Status:

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

ER-AF-NOCR-1 11/03 Application for approval to import for release or release FORM NOCR from containment with controls any new organism under section 38A of the Hazardous Substances and New Page 1 Organisms Act 1996

IMPORTANT

1. Please refer to the associated User Guide when completing this form. If you need further guidance please contact ERMA New Zealand.

2. This application form covers import for release, or release from containment, with controls, of any new organism (including a genetically modified organism) under s38A of the HSNO Act and may be used to seek approvals for more than one organism where the organisms are of a similar nature.

3. If you are making an application to import for release or release from containment any new organism (i.e. full release without controls as opposed to conditional release) you should use Form NOR. If you are making an application for a field test in containment of any new organism you should use Form NO4.

4. You should periodically check with ERMA New Zealand or on the ERMA New Zealand web site for new versions of this and any other forms mentioned. 5. You can talk to an Applications Advisor at ERMA New Zealand who can help you scope and prepare your application. We need all relevant information early on in the application process. Quality information up front will speed up the process and help reduce costs. 6. Any extra material that does not fit in the application form must be clearly labelled, cross-referenced, and included as appendices to the application form.

7. Commercially sensitive information must be collated in a separate appendix. You need to justify why you consider the material commercially sensitive, and make sure it is clearly labelled as such.

8. Applicants must sign the form and enclose the correct application fee (plus GST). The initial application fee can be found in our published Schedule of Fees and Charges. Please check with ERMA New Zealand staff or the ERMA New Zealand website for the latest schedule of fees. We are unable to process applications that do not contain the correct application fee.

9. Unless otherwise indicated, all sections of this form must be completed for the application to be progressed.

10. Please provide an electronic version of the completed application form, as well as sending a signed hard copy. 11. Note: Applications to conditionally release new organisms shall be publicly notified by the Authority (s 53(1)(d) of the HSNO Act) and may go to a hearing (s 60 of the HSNO Act).

You can get more information by contacting us. One of our staff members will be able to help you. ERMA New Zealand 20 Customhouse Quay PO Box 131 Wellington, NEW ZEALAND Telephone: 64-4-916 2426 Facsimile: 64-4-914-0433 E-mail: [email protected] www.ermanz.govt.nz

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Section One – Applicant Details

1.1 Name and postal address in New Zealand of the organisation or individual making the application:

Name > AgResearch Ltd

Postal Address > Ruakura Research Centre Private Bag 3123 Hamilton

Physical Address > Ruakura Research Centre East Street Hamilton

Phone > 07 856 2836

Fax > 07 838 5012

E-mail >

1.2 If application is made by an organisation, provide name and contact details of a key contact person at that organisation This person should have sufficient knowledge to respond to queries and have the authority to make decisions that relate to processing of the application.

Name > Dr Pip Gerard

Position > Senior Scientist

Address > Ruakura Research Centre Private Bag 3123 Hamilton

Phone > 07 838 5103

Fax > 07 838 5073

E-mail > [email protected]

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

1.3 If the applicant is an organisation or individual situated overseas, provide the name and contact details of the agent authorised to transact the applicant’s affairs in relation to the application This person should have sufficient knowledge to respond to queries and have the authority to make decisions that relate to processing of the application.

Name >

Position >

Address >

Phone >

Fax >

E-mail >

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Section Two – Purpose of the Application This form is to be used for an application to import for release or release from containment, with controls, any new organism (i.e. conditional release).

2.1 Give a short summary statement of the purpose of this application (to be used on ERMA New Zealand’s public register) - Maximum of 255 characters (including spaces) Briefly describe the organism(s) to be imported for conditional release or conditionally released from containment and the purpose(s) for which you wish to do so.

To release from containment the Irish strain of the insect Microctonus aethiopoides Loan (Hymenoptera: Braconidae) for biological control of the clover root weevil Sitona lepidus Gyllenhal (Coleoptera: Curculionidae), a major pest of clovers.

2.2 Provide a brief description of the background and aims of the project suitable for lay readers Describe in less than one page the rationale for the overall project these organisms are to be used in so that people not directly connected with the programme can understand why these organisms are being conditionally released.

Since its discovery in 1996, clover root weevil (Sitona lepidus) has become one of the most damaging clover pests found in New Zealand (Willoughby et al. 1999). Both adults and larvae attack clovers all year round, causing significant declines in clover content and quality in pastures. While adults cause significant clover seedling mortality, it is the larval stage that is the most damaging, with the early instars feeding almost exclusively on clover nodules and the older larvae attacking the roots and stolons. Recent results from a pasture impact trial have shown that modest populations of 300 larvae/m2 reduce spring clover dry matter production by 56% even when management conditions are highly favourable for clover, and that erect large leafed white clovers, typical of cultivars used in dairy pastures, are particularly sensitive to attack. White clover is the best quality component of grazed pastures with its high nutritive value and palatability. The loss of clover content in pastures impacts most strongly on live weight gain and milk solids yield.

Perhaps most importantly, the weevil can destroy the nitrogen-fixing capability of white clover for much of the year. White clover was estimated to fix 1.57 million tonnes of nitrogen annually over 13.5 million ha of New Zealand grasslands to a value of $1.5 billion (Caradus et al. 1996). Therefore instead of relying on this free, natural source of nitrogen, most farmers in infested regions must apply high levels of nitrogen fertiliser (> 200kg N/ha) to maintain soil fertility and farm profitability. However, if used incorrectly, these high levels of fertiliser can pollute our waterways causing undesirable growth of plants and algae and ruining the habitat for our native and introduced freshwater aquatic life. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Control of clover root weevil through whole-farm use of conventional insecticides is unacceptable, because of the risks to our environment and exports. Therefore, it is proposed to release a new Irish strain of a small parasitic wasp Microctonus aethiopoides to control this weevil. A Moroccan strain of this parasitoid species is already widespread in New Zealand, having been introduced in 1982 to control the lucerne weevil Sitona discoideus. While ineffective against the clover root weevil (Barratt et al. 1997a), biological control of the lucerne weevil successfully prevents significant losses in lucerne production with savings valued at $5.6-$6.8 million p.a. (Barlow & Goldson 1993). The Moroccan strain was found to reduce adult egg-laying by 87%, which in turn reduced larval numbers to below economically damaging levels (Goldson et al. 1992). One of the key reasons why M. aethiopoides is such an effective biocontrol agent is that it has multiple generations per year (Goldson et al. 1990) and once the parasitoid egg is laid within the weevil‟s body cavity, sterilisation ensues within days (Aeschlimann 1983a).

Biological control of CRW has the ability to achieve similar results. Without needing to change farm management practices, all New Zealand farmers in clover root weevil-infested regions will regain the ability to maintain sustainable and highly productive grass/clover pastures without the use of high levels of nitrogen fertiliser.

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Section Three – Information on the Organism(s) to be Conditionally Released If more than one type of organism is to be conditionally released, this section must be completed separately for each organism.

3.1 State the taxonomic level at which the organism(s) to be conditionally released are to be specified If the taxonomic level is different to “species”, provide reasons for this. The reasons should take account of the need to adequately describe risk.

This application is for a strain of the species Microctonus aethiopoides Loan from the island of Ireland (Republic of Ireland and Northern Ireland), that is reared from Sitona lepidus Gyllenhal and is thelytokous parthenogenetic (reproduces asexually with unfertilised females producing female progeny).

For clarity, it will be referred to as IPMa (Irish parthenogenetic Microctonus aethiopoides) throughout the application document.

For the purposes of this application, „biotype‟ is used to distinguish European M. aethiopoides reared from Sitona lepidus collected in Europe from Moroccan M. aethiopoides reared from Sitona discoideus collected in New Zealand, while „strain‟ is used to identify different populations within the European biotype. Several strains of the European biotype, including the one from Ireland, have been identified from collections of S. lepidus made in Europe (Goldson et al. 2004). A diagram of relationship between species, biotype and strain is shown in Fig 3.1.1.

While the Moroccan biotype successfully controls S. discoideus in New Zealand, it is ineffective against S. lepidus (Barratt et al. 1997a). The European biotype is highly effective against S. lepidus, but Moroccan x European hybrids are less effective against both pest species, indicating that introduction of inappropriate biocontrol strains could put at risk the existing control of S. discoideus (Goldson et al. 2003). However, the Irish strain of the European biotype (IPMa) specified in this application reproduces asexually, eliminating the possibility of hybridisation (Goldson et al. 2005).

The IPMa collection sites are shown in Fig. 3.1.2 and the breeding lines currently in quarantine are as follow:

Athenry, co. Galway 4 lines Solahead co. Tipperary 1 line Oakpark co. Carlow 2 lines New Forge co Down 1 line Crossnacreevy co Down 1 line

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Weevils collected at the above sites were placed in individually labelled rearing containers and incubated in the laboratory. The parasitoids that emerged from these weevils were removed from the rearing containers as pupae and each newly-emerged female arising from these pupae given a unique code. Both the female and her progeny were maintained in isolation from the other breeding lines thereon. A fuller description of the search methods is in Goldson et al. (2004).

Fig. 3.1.1: Diagram of relationship between species, biotype and strain.

Microctonus aethiopoides Attacks weevils in the genera Hypera and Sitona

European biotype Moroccan biotype Attacks Sitona lepidus Attacks Sitona discoideus

Irish strain (IPMa) asexual

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Fig. 3.1.2: Map of Ireland showing where lines of IPMa were collected.

New Forge

Crossnacreevy

Athenry Oakpark

Solahead

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

3.2 Give the unequivocal identification of the organism(s) to be conditionally released Please include details on the following:

Latin binomial, including full taxonomic authority: (e.g. ----- Linnaeus 1753) class, order and family:

Microctonus aethiopoides Loan 1975 (Insecta, Hymentoptera: Braconidae: Euphorinae)

Common name(s), if any:

Type of organism (e.g. bacterium, virus, fungus, plant, animal, animal cell):

Insect

Strain(s) and genotype(s), if relevant:

Irish parthenogenetic (IPMa) strain

Other information (including presence of any inseparable or associated organisms and information on consideration of the organism(s) by other states, countries or organisations):

3.3 Provide unique name(s) for the new organism(s) to be conditionally released

Irish parthenogenetic Microctonus aethiopoides

3.4 Characteristics of the organism(s) to be conditionally released Provide information on the biology, ecology and the main features or essential characteristics of (each) of the organism(s) to be conditionally released. You should also indicate whether the organism is pathogenic or a potential pest or weed.

The genus Microctonus and their hosts The genus Microctonus belongs to the subfamily Euphorinae within the family Braconidae. The Euphorinae are a cosmopolitan group of small koinobiont endoparasitoids that parasitise the adult stage of various beetles, especially the Curculionidae, Chrysomelidae, Carabidae and Tenebrionidae (Shaw 1985, 1988). Some members of the genus are important in agriculture as they have proven effective biological control agents for several weevil pests (Loan 1983; Tobias 1965) including the alfalfa weevil Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) in the

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

USA (Dysart & Day 1976; Kingsley et al. 1993) and Sitona discoideus Gyllenhal (Coleoptera: Curculionidae) in New Zealand (Goldson et al. 1990).

General description of the species Microctonus aethiopoides This species was formerly known as M. aethiops. Loan (1975) reviewed the Haliday species of Microctonus (Wesmael) and found that a number of Microctonus species had been misidentified. Many reports referring to M. aethiops were in fact referring to M. aethiopoides (designated as a new species, Loan 1975) whose hosts were restricted to the adult weevils of the genera Hypera and Sitona. Morphology, adult size, colour, bionomics and host preferences have been used to distinguish M. aethiopoides biotypes (Adler & Kim 1985; Aeschlimann 1983a; Loan & Holdaway 1961; Sundaralingam et al., 2001). Research has suggested that parasitoid morphology is also influenced by the host in which it develops (Phillips et al. 1993), and in some cases specimens categorised as a `biotype' may simply be a result of host-mediated morphology. However, molecular research has shown genetic variation within and between geographic populations of M. aethiopoides (Vink et al., 2003), supporting the concept of geographic and host associated biotypes of M. aethiopoides.

The geographic origin of M. aethiopoides is believed to be Europe and the Mediterranean where the parasitoiud is widely distributed (Aeschlimann 1980; Loan 1975; Goldson et al., 2001). In the Mediterranean, M. aethiopoides was reported as parasitising several Sitona spp. including the lentil leaf weevil (S. crinitus), S. lepidus (= flavescens), the clover root curculio (S. hispidulus), S. humeralis, the pea and bean weevil (S. lineatus), S. punticollis, the clover weevil (S. sulcifrons) and S. tenuis. A Moroccan biotype of Microctonus aethiopoides collected from S. discoideus was responsible for significant mortality of weevil populations in North Africa (Morocco-Algeria-Tunisia) and is the dominant parasitoid in Spain and Southern France (Aeschlimann 1978, 1980).

In the United States, M. aethiopoides was initially introduced from France to control sweetclover weevil Sitona cylindricollis Fåhraeus (Munro & Post 1948), but it appears these introductions were unsuccessful, mainly because not enough attention had been given to the European host from which the parasitoid emerged. Hence there was a mismatch between the parasitoid and the target weevil (S. cylindricollis) in the USA. Following further screening by USDA staff, the parasitoid was reintroduced from France and released in New Jersey against H. postica (Day et al. 1971; Dysart & Day 1976), where it successfully established (Coles & Puttler 1963). The successful establishment and subsequent levels of parasitism proved Microctonus aethiopoides to be one of the most important of a group of parasitoids released to control alfalfa weevil Hypera postica (e.g. Kingsley et al. 1993).

Life cycle and phenology of M. aethiopoides (IPMa)

The general biology of Irish M. aethiopoides is similar to other biotypes and strains of M. aethiopoides. The following descriptions are based on quarantine-based laboratory

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

observations and experimentation at AgResearch Lincoln, but draw on published research on other biotypes of M. aethiopoides.

Microctonus aethiopoides females lay their eggs into the haemocoel of the host through the membranous area at the apex of the abdomen. Prior to oviposition, the female stalks its prey sometimes for a considerable period of time. When ready to oviposit, the female extends her ovipositor under and well in front of her head, and makes a quick thrust at the caudal end of the weevil's abdomen (Fig 3.4.1). IPMa females prefer an active host to a stationary one, behaviour observed in other European (Loan & Holdaway 1961, Fusco & Hower 1973) and Moroccan M. aethiopoides biotypes. This propensity to oviposit in active hosts has also been observed in other Microctonus species (Smith 1952; Loan 1967; Wylie & Loan 1984) and may indicate a selection process for healthy individuals (Jackson 1928), or that it may be easier to penetrate the host abdomen when the weevil is moving.

Fig. 3.4.1: Diagram of a parasitoid ovipositing in a weevil.

(a) Egg stage Eggs are restricted to the abdomen of the host but float free in the haemocoel. When first laid, eggs are long and slender, rounded at the capsule end and tapering gradually to a narrow pedicel. The evolution of a small egg is associated with endoparasitism in mobile hosts and the need for rapid oviposition (Jackson 1928). After deposition in the host, egg development is marked by a rapid expansion in size. For instance, a 2790 fold increase in volume was found for eggs of the biotype parasitising H. postica (Loan & Holdaway 1961).

(b) Larval development M. aethiopoides goes through five instars during development, the final instar emerging from the host. The description of the different instars is based on Loan and Holdaway (1961). The first instar has a tail-like extension and a head capsule with powerful mandibles that can be used to destroy other larvae if the host is superparasitised. Both second and third instars are similar in appearance to the first but lack the head capsule. The fourth instar is unsclerotised and whitish yellow, with film-like cast skins of instars 1-3 adhering to the apex of abdomen. The fifth instar (or prepupa) emerges from the host and is grub-like and pale yellow.

As larvae develop they expand to occupy the whole of the body cavity possibly extending into the thorax. Weevil body organs may become flattened and pushed to the side by the developing larva. The fifth instar emerges from the host by forcing the caudal end of the abdomen through the membrane describing the junction of the apical tergite and sternite. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Development thresholds of the combined egg plus larval stage and pupal stage of Moroccan M. aethiopoides were 9.8oC and 8.2oC, respectively (Goldson et al. 1990). The degree-days required for development of the egg plus larval stage and pupal stage were 144 and 125oD respectively (Goldson et al. 1990). Comparative studies in quarantine suggest that eggs and larvae of IPMa are about 15% slower to develop than the Moroccan biotype. At a mean temperature of 18oC the development of the IPMa larva takes 20-24 days, after which the prepupa emerges from the host, causing host death. At the same temperature, the time taken from egg to the adult stage is 30 days.

(c) Pupae The prepupal larva pupates in soil litter forming a silk cocoon that is yellow-white, thin and pliable. Silk is used to attach the cocoon to available surfaces. Final instar exuviae and eyes of the developing adult are visible through cocoon 48-72 hours after formation at room temperature. The cocoon appears black shortly before adult emergence.

(d) Microctonus aethiopoides adult stage

IPMa is thelytokous parthenogenetic; unfertilised females able to produce female progeny (clones). The females are 2.7-3.0 mm excluding antennae and ovipositor sheaths. They are orange-brown, particularly on the abdomen, with yellow-gold head and pronotum and dark brown ovipositor sheaths. Ovipositional activity can occur almost immediately after emergence from the cocoon. Each female carries an average of 75 (range 59-121) eggs. The actual number of eggs oviposited will depend on environment, host availability, availability of liquid food sources, and condition of the parasitoid female. Adults of the Microctonus genus have been shown to live three times longer when provided honey solution rather than water and to lay up to 88% more eggs depending on the parasitoid strain (Phillips 1998). Females exhibit no host preference on the basis of sex and size, but ovipositional activity and development is influenced by the age of the weevil host. For instance, laboratory studies have found that older S. lepidus are less attractive for parasitism than recently emerged and more active weevils.

The life cycle of IPMa and its host are presented in Fig. 3.4.2.

Host discrimination and gregarious larval development Studies indicate that IPMa strains attacking S. lepidus are poor at discriminating between parasitised and unparasitised hosts, a behaviour observed in other M. aethiopoides biotypes (Loan & Holdaway 1961). Barratt & Johnstone (2001) found that Moroccan M. aethiopoides showed weak discrimination behaviour when parasitising its host S. discoideus. Research on Moroccan M. aethiopoides has shown that in the field, superparasitism is generally infrequent but during periods of high parasitoid activity the incidence is higher (McNeill, AgResearch entomologist, unpublished data). 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Figure 3.4.2: Life cycles of S. lepidus and IPMa

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Generally, M. aethiopoides is considered to be a solitary endoparasitoid with supernumerary parasitoids eliminated by cannibalism or physiological suppression by the primary parasitoid. IPMa is an exception in that under laboratory conditions, larvae can survive gregariously in a host. This is may be linked to its asexual reproduction but the mechanism has yet to be investigated. In four experiments conducted as part of the host range study on Irish M. aethiopoides, prepupal emergence amongst the S. lepidus controls ranged from 108 to 174% of the total numbers of weevils present. Further studies have shown that up to four larvae are able to survive and emerge from a single weevil. While superparasitism may be a result of confinement of parasitoid with a limited number of hosts it does indicate that Irish M. aethiopoides is capable of gregarious development. However, multiple parasitism does appear to have a detrimental effect of host survival, with high rates of premature mortality sometimes observed in weevils carrying multiple larvae.

Worldwide, there are a small number of Microctonus species which are gregarious, with a variable number of larvae emerging from a single host. Microctonus zealandicus Shaw, (Shaw, 1993); M. caudatus (Thompson) (Luff 1976), M. glyptosceli Loan (Loan et al. 1969), M. eleodis (Viereck) (McCulloch 1918), M. vinelandicus Loan (Loan & Holliday 1979), M. disonychae Loan (Loan 1967) and one unidentified Microctonus sp.(Tironi et al. 2004) are known to exhibit this behaviour. Gregarious development is uncommon, and with the exception of M. zealandicaus which parasitises Irenimus. aequalis Broun (Coleoptera: Curculionidae), gregarious development appears to be restricted to large species of Coleoptera (Loan & Holliday 1979).

Effect of parasitism on the host Parasitism by M. aethiopoides does not appear to alter the external physical shape and behavioural activity of the weevil, nor is there any difference in the appearance of the abdomen to that of the non-parasitised weevil. There are, however, important physiological changes occurring in the female host. The parasitoid usually lays one egg in the abdomen of the host adult weevil. Once parasitised, the effect on the female weevil is rapid. Oviposition is reduced almost immediately. Internal examination of the abdomen reveals that the ovaries degenerate and mature eggs shrink in size. In contrast, parasitism does not have any effect on reproductive potency of male weevils, the testes exhibiting no physical changes. Similar observations have been reported for M. aethiopoides (USA) (Loan & Holdaway 1961; Drea, 1968), M. caudatus (Luff 1976) and M. hyperodae (Loan & Lloyd, 1974).

After emergence of the parasitoid fifth instar, the weevil may survive for up to two days, but while mobile does not feed. The rear legs may also be partially paralysed. The abdominal cavity from which the parasite emerged is devoid of any fluid and fat bodies as all host reserves have been consumed by the developing parasitoid larvae, while the trachea are distinct.

Synchrony with host Unlike S. lepidus in New Zealand, most weevil hosts of M. aethiopoides generally undergo an obligatory diapause (winter dormancy) during periods of unfavourable environmental conditions. In the warmer climatic regions this takes the form of aestivatory diapause in 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

summer, whereas in Northern European and North American sites the weevils undergo hibernatory diapause over winter (Aeschlimann 1983a). During these periods the parasitoid undergoes sympathetic diapause as a 1st instar larva inside the host. This synchronisation (with the host) allows the parasitoid to survive the period when the host is inactive and during unfavourable environmental conditions. In weevils conditioned for diapause, parasitoid eggs develop through to the first instar at which stage the diapause state arrests further growth. These larvae are small and active with reduced brain ganglia (e.g. Loan & Holdaway 1961). The ovaries of weevil females are rendered functionless and the male weevil can be partially to completely castrated. This is in contrast with non-diapausing males where the reproductive system is unaffected by parasitism (e.g. Loan & Holdaway 1961).

Parasitism appears to influence the cessation of weevil diapause. For example, S. discoideus parasitised by Moroccan M. aethiopoides breaks aestivation (a summer dormancy) earlier than non-parasitised weevils so that the parasitoid emerges from its host and pupates in the soil before the non-parasitised adult weevils became reproductively active (Goldson et al. 1990). A similar behaviour has been observed in Ontario Canada, with M. aethiopoides larvae in Hypera postica (Gyllenhal) (Abu & Ellis 1976). The biological significance of this behaviour is that parasitoid adults are present in the field when the bulk of the weevils move back into the crop.

With no evidence that either S. lepidus or Irish M. aethiopoides will diapause in New Zealand, the life cycle duration of the host and parasitoid will be determined mostly by temperature. S. lepidus adults are long-lived, especially if temperatures are cool and clover abundant. Therefore adult weevils are present for most of the year. In 2004 in the Hawke‟s Bay there was good overlap of generations, with old and teneral weevil adults present in high numbers during the spring (M. Slay, private consultant pers. comm.). It is likely a similar pattern will hold for Canterbury. In Southland, where the summer is relatively cool and wet, it is predicted that adult weevil survival and egg laying during summer will be greater than in other regions, with some progeny completing development before winter, and the rest in spring. This also may give rise to a good overlap in generations. However, especially during wet winter and early spring conditions in the Waikato and possibly in the Manawatu, there may be poor overlap between the overwintered adults and those emerging in spring. As a consequence there are very low numbers of adults in October. To have successful control in the Waikato, the emergence of parasitoids from the pupal stage in the pasture litter will need to be synchronised with the commencement of adult S. lepidus emergence, and the parasitoid will need to bridge the gap between the old and new weevil generation. This may occur at the pupal stage of the parasitoid. The availability of nectar sources to ensure 2-3 weeks parasitoid longevity may be important at this time.

Information on the Moroccan Microctonus aethiopoides in New Zealand While IPMa has originated directly from Ireland, the Moroccan Microctonus aethiopoides imported into New Zealand quarantine to control the lucerne weevil S. discoideus was from a population released in Australia. The initial introductions of M. aethiopoides into Australia came from Morocco, Greece and France (Cullen & Hopkins 1982; Aeschlimann 1983b) and originated from different hosts. However, only the Moroccan biotype came from S. discoideus. The 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

French ecotype failed in quarantine, and while releases of the Greek ecotype were made (Aeschlimann 1983b), establishment failed (Aeschlimann 1995). The Moroccan biotype was liberated in South Australia with releases in 1977-78 (Cullen and Hopkins 1982) and was the only biotype considered to have established and spread successfully in South Australia and elsewhere (Aeschlimann 1995; D. Hopkins, Australian entomologist, pers. comm.). This is an example of how selection of M. aethiopoides strain (in this case source species) influences biocontrol success. With the IPMa collected solely from S. lepidus in Ireland, the risk of establishment failure has been minimised.

Following the success of the above programme, Moroccan M. aethiopoides was introduced into New Zealand from South Australia with a total of 30,450 parasitised weevils and 2390 adult parasitoids released in October 1982 at 17 sites throughout Canterbury, Central Otago and Malborough (Stufkens et al. 1987). Subsequent monitoring confirmed that M. aethiopoides had established in several lucerne growing areas in the South Island (Stufkens et al. 1987).

Early research on the phenology of the parasitoid in New Zealand ascertained that there were possibly four parasitoid generations in Canterbury (Proffitt & Goldson 1987). S. discoideus adults aestivate in hedgerows and at one of these sites, levels of parasitism were 15% (Goldson & Proffitt 1986), with between 40-55% parasitism in the lucerne paddock (Stufkens et al. 1987). This compared favourably to levels of parasitism recorded in New South Wales, Australia of between 0.04% in aestivation sites and 6.5-49% in lucerne paddocks (Cullen & Hopkins 1982). Initial indications were that where the parasitoid was present, weevil ground densities dropped significantly (Goldson & Proffitt 1986). Further research by Dr Goldson and co-workers determined the phenology of the parasitoid (e.g. Goldson & Proffitt 1986; Goldson et al. 1990) and later reported on the successful reduction in S. discoideus numbers (Barlow & Goldson, 1993; Goldson et al. 1990). A significant feature of this success was attributed to a mean of 3.0% of the infected pre-aestivatory S. discoideus population having continued parasitoid development. This atypical development meant that there were three extra summer generations in the field and continuous parasitoid oviposition activity in the lucerne stand (Goldson et al. 1990). This, in turn, meant that levels of parasitism approached 100% at certain stages of the weevil‟s life cycle (Goldson et al. 1990).

It is anticipated that the IPMa will follow a similar pattern to the Moroccan biotype and reach similar levels of parasitism in field populations. As there is no aestivatory behaviour in the host nor laboratory evidence that the parasitoid enters diapause, oviposition activity by IPMa is likely to be continuous as long as hosts are available and temperatures above the developmental threshold.

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3.5 If the organism to be conditionally released is a genetically modified organism, where applicable provide details on the development of the organism If the organism to be conditionally released is a genetically modified organism, state whether the development of the organism was carried out under a HSNO approval. If this was the case, provide the approval number and translate the relevant details to the headings below. If the genetically modified organism is to be imported for conditional release, also provide this information to the extent possible:

Not applicable.

Identify the category of the host organism (i.e. category 1 or 2) and genetic modification (i.e. category A or B) involved in the development of the organism with reference to the HSNO (Low- Risk Genetic Modification) Regulations 2003. Please explain your characterisation.

Vector system(s) used in the development of the genetically modified organism(s).

Type and source of additional genetic material.

Use of special genetic material (please complete this table by marking the correct box):

Yes No Were native or valued flora or fauna used as the host organism(s)? Was genetic material from native or valued flora and fauna used? If native flora and fauna were involved, were the species concerned indigenous to New Zealand? Was human genetic material involved? Answer Yes if human genetic material in any form was used, i.e. obtained directly from humans (either Māori or non- Māori from a gene bank, synthesised, copied and so on). Was genetic material obtained directly from human beings used? If Yes, provide additional details below.

If the genetic modification involved DNA of human origin, provide details of from where the material was obtained (including provenance and/or informed consent), and whether approval was obtained from an Ethics Committee, and/or whether consultation with Māori has taken place. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Other relevant details (such as what techniques or experimental procedures were used, whether any unusual manipulations were carried out, and how the foreign genetic material is expressed).

Section Four – Proposed Conditional Release Programme and Controls

4.1 Proposed Conditional Release Programme Please provide full details of your intended conditional release programme e.g. size, timing and location(s) of the conditional release etc. Specifically indicate whether or not an expiry date for any approval is expected and give reasons for this expectation.

The method of mass-rearing M. aethiopoides on Sitona lepidus is similar to that described for M. hyperodae on L. bonariensis (McNeill et al. 2002). A two tier approach will be used to mass-rear M. aethiopoides. To maintain a supply of parasitoids for mass-rearing, newly- emerged (<24 h) females are individually confined for 48-72 h at 20oC in 220 mm ×130 mm × 75 mm cages with 40-60 S. lepidus adults. The parasitoids are provided with 5 mm squares of filter paper soaked in fructose-sucrose solution. The weevils are provided with white clover plants grown in 60 mm × 25 diameter soil plugs sealed in 50 mm × 100 mm polythene bags. After removal of the parasitoids, the weevils are maintained for about 3 weeks at 20oC with fresh clover provided 2-3 times weekly. Their cages are fitted with a 5 mm gauze floor and positioned over a lower chamber into which are placed strips of paper towel. This allows the emergent prepupal parasitoids to drop through the gauze and pupate in the paper strips. The pupae are then transferred to 90 mm Petri dishes along with a moistened dental wick until adult eclosion. Petri dishes are checked daily for newly emerged parasitoids. The method for mass-parasitism of S. lepidus by M. aethiopoides for field release will be achieved by exposing weevils to parasitoids for five - eight days in 8 L (34 cm × 25 cm × 23 cm) plastic containers using a parasitoid:weevil ratio of 1:40. Because M. aethiopoides is thelytokous, the absence of males will simplify maintenance of the cultures and bulking-up of lines for each mass-rearing.

During the exposure period, weevils will be supplied with 4-5 bouquets of glasshouse grown white clover. At the end of the exposure period (8 d), parasitoids will be removed and the weevils transferred to either an insectary or a controlled environment room maintained at 8oC and 12:12 (L:D) h. Coinciding with the removal of parasitoids, a subsample of about 20 weevils was taken for dissection to determine the level of parasitism.

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The field release of Irish M. aethiopoides will most likely be accomplished through the release of parasitized weevils, although adults and pupae could be released at the Waikato site where transfer of these fragile life stages from laboratory to the nearby release site can be achieved with negligible insect mortality. The size of each release has yet to be finalised but will be guided by the necessity to ensure establishment and the logistics of rearing parasitoids for each release site. Experience with the nationwide releases of the thelytokous parasitoid Microctonus hyperodae against Argentine stem weevil (Listronotus bonariensis) achieved parasitoid establishment with releases of 2000-5000, but in one case a single release of only 800 parasitised weevils was sufficient for subsequent establishment (McNeill et al., 2002). However, temperature is important in the establishment and build-up of the parasitoid, and in the case of M. hyperodae, larger releases (5000-6000) increased the probability of establishment and the rate of build-up in cooler regions of New Zealand (McNeill et al., 2002). As IPMa is also parthenogenetic, critical mass is not a factor as there is no need to find a mate as happens with sexually reproducing parasitoids. Furthermore, S. lepidus does not aestivate and M. aethiopoides is likely to have three - five generations a year, allowing for faster multiplication of parasitoid abundance over a 12 month period. In conclusion, a conservative approach to ensure establishment would be to release a total of 2000-5000 parasitised weevils at a site with two-three releases spread over a four month period.

Once ERMA approval is attained, AgResearch plans initial releases of the parasitoid at three experimental release sites as soon as new weevil adults from the overwintering larval generation are active. One release site will be in the Waikato at a site near Morrinsville where weevil S. lepidus populations have been regularly monitored since 1998. The site is typical of the situation now found in Waikato pasture, with weevil and clover populations in equilibrium at around the 300 weevils/m2 and a 10% clover content. The other two sites will be in newly infested districts in the Hawkes Bay and Manawatu, where weevil numbers exceed 1000/m2, where it is hoped the newly established parasitoids will build up numbers rapidly. However, with S. lepidus now well spread throughout the North Island, the presence of the parasitoid will probably be too late to halt the spread of the weevil to the South Island.

Establishment and initial rate of spread will be monitored at intervals in 2005-2006 by sampling S. lepidus populations at the release sites and points at known distances radiating out from the release sites and rearing parasitoids out of the weevils in the laboratory. AgResearch‟s subsequent strategy for establishing the parasitoid nationwide cannot be finalised until results from these experimental releases have been analysed, discussed with industry stakeholders and decisions made as to what resources will be available.

To determine if non-target curculionid species are subject to parasitism by M. aethiopoides, non-target species including native weevils and introduced weed biological control agents, found in the S. lepidus samples taken at the release sites will be counted to determine density and dissected for evidence of parasitism. Where parasite material is found, it will be stored for genetic analysis once a rapid and cheap method has been developed to distinguish IPMa from the Moroccan biotype. Non-target weevil species will be sampled four times per year from 2006–2009 from pasture and the wider vicinity of the release site. This 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

information will be important to verify that the results and conclusions regarding the host range testing carried out in quarantine were valid. It will also add to our knowledge regarding non-target impacts in New Zealand.

We are requesting for approval in perpetuity. Once successfully established, it will be impossible to eradicate IPMa. However, even if control is effective, S. lepidus will remain a risk to New Zealand agriculture for the foreseeable future. There may be a need to source fresh lines of IPMa when S. lepidus establishes in the South Island or following abnormal circumstances that may reduce the parasitoid gene pool, such as widespread severe drought.

4.2 Proposed Course of Action if the Conditional Release Approval is Set to Expire Indicate by marking the following table which course of action is preferred if the conditional release approval is set to expire:

Not applicable as applying for approval in perpetuity.

Course of Action Yes No

1. Full (unconditional) release of the organism. Note: you will need to formally apply for a full (unconditional) release at or after the time of applying for a conditional release but prior to its expiry. The full release would then take effect immediately after the expiry of the conditional release approval. 2. Return of the organism into containment through an existing containment approval. Note: give the appropriate approval code and relevant details in the space below. 3. Return of the organism into containment through a new (yet to be gained) approval. Note: you will need to formally apply for a containment approval at or after the time of applying for a conditional release but prior to its expiry. 4. Disposal of the organism. Note: unless you have gained another approval (containment or full unconditional release) prior to the expiry of your conditional release approval you will need to provide information relevant to disposal in section 4.3.

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Please give reasons justifying the selection of the (above) course of action and any other relevant information (e.g. the approval code and details of the appropriate existing containment approval etc.)

Not applicable as applying for approval in perpetuity.

4.3 Disposal of the organism(s) on the expiry of the conditional release approval Section 38F of the HSNO Act requires the organism(s) to be disposed of if and when a conditional release approval expires, unless before expiry another approval (containment or full unconditional release) is granted under the Act. Provide information (preferably in the form of proposed controls) to explain what steps will be taken to ensure that the organism(s) can be identified and located at the expiry of the approval, and the method of disposal intended.

Not applicable as applying for approval in perpetuity.

4.4 Proposed control(s) Please outline the relevant control(s) that you recommend be imposed to deal with any risks that may be posed by the organism(s) to be conditionally released. In doing so make reference to the types of controls listed in section 38D of the HSNO Act. If you do not consider any of these controls to be appropriate explain why. Also provide information on how effective these controls are likely to be in meeting the objective(s) of the control(s).

Imposing obligations on the approval user A conditional release is requested rather than a full release because to date there is no published definition of the parasitoid strain that enables it to be distinguished from other morphologically-identical strains of the same species. The conditions are that the parasitoid to be released meets the description of the Microctonus aethiopoides strain as set out in Section 3.1. That is, it is a strain of the species Microctonus aethiopoides Loan from the island of Ireland (Republic of Ireland and Northern Ireland), that is reared from Sitona lepidus Gyllenhal and is thelytokous parthenogenetic (reproduces asexually with unfertilised females producing female progeny).

The test for parthogenicity will use methodology as prescribed by the Authority. Initially this is likely be based on a method developed by M. McNeill in which representative samples of IPMa females are exposed to Moroccan biotype or European strain males, then allowed to parasitise S. lepidus through two generations. All offspring must be female, especially if any mating is presumed to have occurred. Allozyme markers that can differentiate IPMa from the Moroccan biotype and some of the European strains attacking S. lepidus have been identified (Illine & Phillips, 2003), and these will reduce the time required for testing. However, further allozyme work needs to be carried out to reliably differentiate all known strains of European M. aethiopoides (Phillips, AgResearch entomologist, pers. comm.)

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Appropriateness of the types of controls listed in section 38D of the HSNO Act Listed below are possible controls that the Authority may impose.

1. Controlling the extent and purposes for which organisms could be used 2. Requiring any monitoring, auditing, reporting, and record-keeping 3. Compliance with relevant codes of practice or standards 4. Development of contingency plans to manage potential incidents 5. Limiting the dissemination or persistence of the organism or its genetic material in the environment 6. Requiring the disposal of any organisms or genetic material 7. Limiting the proximity of the organism to other organisms 8. Setting requirements for any material derived from the organism 9. Imposing obligations on the approval user (e.g. training, number of approval users) 10. Specifying the duration of the approval

Of the above list, controls 1, 2, 3, 4, 6, 7, 8 and 10 are not applicable. This application is for approval to release a biological control agent. There is no other known use for IPMa (1), and as the intention is to have it established throughout all S. lepidus-infested areas of New Zealand, monitoring populations in perpetuity is not warranted (2). There are no applicable codes of practice (3). Once released in the environment, it cannot be eradicated (4), there can be no restrictions on disposal (6) proximity to other organisms (7), and what little material is derived from the IPMa (eg pupal cases)(8).

While it may be possible to limit dissemination to offshore islands (5), that would require preventing the establishment of S. lepidus, which is well outside the scope of this application.

Therefore the only applicable control is to limit approval to release IPMa from containment to entomologists within the organisation holding the approval (AgResearch), who have the expertise to ascertain that the organism is IMPa and not any other strain, biotype or species.

4.5 Monitoring of effects Conditional releases may provide an opportunity to collect information related to the occurrence of adverse effects and to the operation or not of associated pathways. The Authority wishes to encourage applicants to take full advantage of the conditional release situation to conduct monitoring which will provide an assurance that risks are being effectively managed through the controls imposed and/or provide information which will assist in the consideration of any future conditional or full release applications. Describe any such monitoring you propose to put in place.

While a level of monitoring will take place to determine initial establishment and rate of spread, monitoring should not be a control. If the release is successful, IPMa will eventually establish wherever S. lepidus-infested pastures are found. However, as S. lepidus has yet to be found in the South Island, there is a high degree of uncertainty when that will eventuate. Therefore, even superficial monitoring of parasitism levels in pastures throughout New 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Zealand may require considerable time commitment over many years. Monitoring non-target effects on this scale would be impossible. In addition, even if adverse effects were identified, there are no viable options to mitigate the risk.

It is the intention of the scientists involved in this proposed release to undertake targeted monitoring of the establishment, spread and beneficial and adverse impacts of this introduction. The nature, level and duration of investigations will be limited by available funding, a factor over which scientists do not have full control. Therefore, we feel that voluntary monitoring that is designed in consultation with interested parties is the most effective way to obtain information which will assist in the consideration of any future conditional or full release applications.

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Section Five – Identification of Risks, Costs and Benefits Risk means the combination of the magnitude of an adverse effect and the probability of its occurrence. Cost means the value of a particular adverse effect expressed in monetary or non-monetary terms. Benefit means the value of a particular positive effect expressed in monetary or non-monetary terms. In this part of the form you are required to identify (in section 5.3) the risks, costs and benefits of the organism(s) in the context of the application. A very broad approach should be taken to this, so that a wide range of possibilities are canvassed. However, it is also important at the end of this exercise but still at the identification stage to identify those risks, costs and benefits which warrant more detailed assessment at a later stage (in section 6). To assist the process of identification this section also requires information to be provided on the ability of the organism(s) to establish a self-sustaining population and on the ease of eradication. Please refer to the ERMA New Zealand Technical Guides “Identifying Risks for Applications” and “Risks, Costs and Benefits for Applications for further information. These are available from the ERMA New Zealand website or in hard copy on request.

5.1 Ability of the organism(s) to establish a self-sustaining population Discuss the ability of the organism(s) to establish a self-sustaining population and the ease with which the organism(s) could be recovered or eradicated if an undesirable self-sustaining population established.

The primary reason for the application is to release Microctonus aethiopoides as a biocontrol agent, and to establish self-perpetuating populations wherever Sitona lepidus is present in New Zealand. Based on laboratory studies, it is predicted is that IPMa will have the same distribution throughout New Zealand as the existing Moroccan biotype of M. aethiopoides.

In the laboratory, IPMa has been shown to be able to develop to the prepupal stage in Irenimus aequalis, Nicaeana cervina, Protolobus porculus, Steriphus variabilis, Exapion ulicis and in one case Catoptes cuspidatus (Goldson et al. 2005). Whilst two of these species are usually regarded as minor pasture pests, the establishment of self-sustaining IPMa populations in other species of native broad nosed weevils in our tussock grasslands would be undesirable. However, to establish a self-sustaining population within communities of these non-target weevils, there must be temporal and spatial synchrony.

Native weevils such as Irenimus spp. and Nicaeana spp. occur in pastoral as well as native habitats and so there is opportunity for exposure to introduced parasitoids. However, no evidence of self-sustaining populations of the Moroccan biotype in native habitats has been found to date. Barratt et al. (2000) compared patterns of adult phenology of Irenimus and Nicaeana species with the main period of Moroccan M. aethiopoides activity at sites in Otago and found in most sampled areas the main period of reproductive activity was in early spring, before peak parasitism occurred. This coincided with the decline of S. discoideus populations and probably indicated utilization of alternative hosts. In native grasslands, the cold winter temperatures might reduce parasitoid development and survival, since parasitism levels of native weevils in those areas has remained relatively low. If so, and if the phenology of IPMa is similar to Moroccan M. aethiopoides it would be unlikely that native weevils found in cool

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higher altitude or native tussock grasslands in cooler regions and higher altitudes will support self-sustaining IPMa populations. The eradication of an undesirable self-sustaining population would be almost impossible. An undesirable self-sustaining population would be one that had established within native grassland weevil populations. With the possibility of further spill-over from populations in pasture weevils, any attempt of eradication would involve repeated use of insecticide over large tracts of land to kill parasitized adult weevils. This would threaten the existence of many native invertebrate and insectivorous bird and reptile species in the spray zone and increase the levels of pesticide residues in the environment and farm produce.

>5.2 Effects of any inseparable organism(s) State whether or not there are any inseparable organisms associated with the organism that is the subject of the application. If there are any effects of inseparable organisms that might need to be considered, state this and then identify them under the relevant headings in section 5.3.

Hyperparasitoids or pathogens that could potentially attack Microctonus aethiopoides were not detected in parasitoids collected between 2000 and 2003, and none have been encountered in breeding colonies in the AgResearch containment facilities at Lincoln. Cultures breeding in containment at Lincoln remain strong and fecund. Virus-like particles present in the female reproductive system of the Moroccan biotype (Barratt et al. 1999) may have a role in determining host range, but these appear to be absent from the IPMa (Barratt, AgResearch entomologist, pers. comm.). Virus-like particles may be important in the suppression of the target weevils immune defence system and as a consequence permit a wider host range for Moroccan M. aethiopoides.

5.3 Identify all the potential risks, costs and benefits of the organism(s) to be conditionally released Please identify all potential risks, costs and benefits whether you consider them to be non-negligible or not. To do this effectively you should identify both the source of the risk (or hazard) and what is at risk (or area of impact). You should also identify the route (or exposure pathway) between the source and the area of impact. An indication of any non-monetary and monetary costs and benefits to be derived from the conditional release of the organism(s) and whether these are direct or indirect should be given. Please cover all of these issues under each of the following headings (areas of impact) which reflect those matters referred to in Part II of the HSNO Act:

A. Effects on the environment (in particular on ecosystems and their constituent parts) As per sections 5(a), 6(a) and 6(b) of the Act, list the risks, costs and benefits associated with the organism(s) to be conditionally released. Also address the ways that these risks, costs and benefits might adversely effect or improve/enhance (in the case of benefits) the New Zealand environment e.g. the life supporting capacity of air, water, soil and ecosystems; the sustainability of native and valued introduced flora and fauna; natural habitats and the intrinsic value of ecosystems; New Zealand‟s inherent genetic diversity; animal or plant health. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Risks and costs:

 Incorrect identification of parasitoid species, biotype and strain may mean inappropriate tests were undertaken to determine adverse effects on environment. This is highly improbable in that experienced experts with specialist knowledge of Microctonus taxonomy have been involved in the science underpinning this application (eg McNeill et al. 1993; Phillips & Baird, 1995; Vink et al. 2003). In addition, when genetic variation among M. aethiopoides was assessed using insect mitochondrial cytochrome c oxidase subunit I gene sequence data, IPMa was found to be genetically distinct from the Moroccan strain already present in New Zealand (100% bootstrap support) (Vink et al. 2003). Molecular techniques also allowed IPMa to be distinguished from other strains of M. aethiopoides attacking S. lepidus (Vink et al. 2003). An allozyme test, which is rapid and less costly, has also been developed to distinguish IPMa from Moroccan M. aethiopoides (Iline & Phillips 2003).

 Parasitoid biotype variability This application has arisen because biotype variability is recognised within M. aethiopoides. The Irish M. aethiopoides is a strain within the European biotype that attacks the target weevil S. lepidus.

 Parasitoid may cause deterioration of natural habitats Adults of the Microctonus genus feed on liquid food such as nectar (Phillips 1998) but no evidence of damage to flowers has been reported. It will have no direct effect on, or provide risk to, air soil or water.

 Potential impacts on non-target hosts leading to population reduction/extinction of one or more non-target species and reduced native biodiversity. Some parasitism of non-target hosts has been observed in the laboratory. A potential non-negligible risk – discussed in Section 6.1

 Introduction of hyperparasitoids or animal or plant pathogens All hyperparasitoids and undesirable pathogens have been eliminated during the importation and quarantine process (eg Goldson et al. 2001).

 Adverse effect on New Zealand’s inherent genetic diversity by interbreeding IPMa is parthenogenetic (Goldson et al. 2005) and all individuals are female. It therefore will not hybridise with the native parasitoid Microctonus zealandicus Shaw.

 Loss of biocontrol of Sitona discoideus through interbreeding of IPMa with the Moroccan strain IPMa does not mate and therefore will not hybridise with the Moroccan strain. However, if parthogenicity in IPMa is not stable, this would become a potential non-negligible risk. Current knowledge on stability of parthenogenicity is discussed in Section 6.1.

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 Competition with or displacement of native natural enemies by parasitoid. In the laboratory IPMa has been shown to attack a common native weevil of minor pest status Irenimus aequalis Broun. This weevil is parasitized by the native parasitoid Microctonus zealandicus Shaw. A potential non-negligible risk – discussed in Section 6.1

 Impacts on organisms at other trophic levels (eg food chain effects). With the abundance of S. lepidus in pastures, larger numbers of predatory arthropods, small mammals and birds may be present in pastures. Birds would predate on the adult stage, while predatory arthropods such as beetles would predate on eggs Control of the weevil by IPMa may cause a reversion of insectivores to previous numbers, but the fact that biological control only reduces densities and does not eliminate the pest means the impact of introducing the parasitoid on insectivores will be negligible.

Benefits

 Reduced possible misuse of nitrogen fertiliser. S. lepidus is one of the reasons why nitrogen fertiliser use has markedly increased in recent years, with farmers in S. lepidus-infested regions using higher levels than those farming in regions, as yet free of S. lepidus. The release of IPMa may mitigate this. A potential non-negligible benefit – discussed in Section 6.1.

 Improved soil fertility Application of N fertilisers is not feasible or economic in some pasture systems and farmers must rely on the nitrogen-fixing capability of clovers. Reduction of S. lepidus populations in these systems would prevent depletion of N reserves in the soil. A potential non-negligible benefit – discussed in Section 6.1

 Improved pasture sustainability Clovers under attack by S. lepidus are more vulnerable to abiotic and biotic stresses. Poor clover vigour and death, opens up pastures, allowing establishment of weed species. This accelerates the deterioration of pasture quality and subsequent need for renovation. A potential non-negligible benefit – discussed in Section 6.1.

 Reduction in pesticide use One pesticide is registered for protection of clover seedlings from S. lepidus adults during pasture re-establishment. Effective biocontrol would lessen the need for this. A potential non-negligible benefit – discussed in Section 6.1

 Reduction in greenhouse gas emissions. Research in New Zealand has shown that sheep have significantly lower methane losses per unit feed intake when feeding on legumes than on grass dominated pastures (Waghorn et al. 2002). Lee et al. (2004) also found that increasing proportions of white clover in the diet of dairy cows resulted in linear increases in dry matter intake and reductions in methane per kg dry matter eaten. However, because total cow consumption level also increased with clover 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

content (i.e. more grass was consumed), the net effect was a small but significant increase in methane production.

B. Effects on human health and safety (including occupational exposure) As per section 6(c) of the Act, list any potential risks and benefits to human health that may be related to the conditional release of the organism(s) in New Zealand.

Risks and costs:

 Allergies associated with mass rearing of M. aethiopoides No adverse risks specifically related to the rearing and release process have been identified.

 Nuisance to general public M. aethiopoides is so small that it is unlikely to be observed. It does not sting or bite.

Benefits:

 Reduction in pesticide use Successful biological control of S. lepidus might reduce the use of insecticide in the community. A potential non-negligible benefit – discussed in Section 6.2

C. Potential effects on the relationship of Māori and their culture and traditions with their ancestral lands, water, sites, wāhi tapu, valued flora and fauna and other taonga (taking into account the principles of the Treaty of Waitangi) As per sections 6(d) and 8 of the Act, list any potential adverse and beneficial effects on the relationship of Māori and their culture and traditions with their ancestral lands, water, sites, wāhi tapu, valued flora and fauna and other taonga (taking into account the principles of the Treaty of Waitangi). In this area it is especially important to indicate the extent to which potential effects reflect the expressed views of the Māori community. However, details on this can be dealt with under the assessment section (section 6).

An outline of the consultation with Māori organisations undertaken by AgResearch in presented in Section 6.3 and copies of the minutes from the consultation meetings are in Appendix 2. Listed below are the issues raised relating to the proposed release of IPMa.

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Risks and costs:  Fundamental opposition to the introduction of species exotic to Aotearoa AgResearch acknowledges and respects this perspective. As guardians New Zealand‟s natural habitats and resources, it is only proper that Māori should make this the initial starting point when a new introduction is proposed.

 Impact of native weevil extinction on native ecosystem function. Concern was expressed on the flow on impacts in the native grassland ecosystems if a native weevil species became extinct. A potential non-negligible risk – current knowledge is outlined in section 6.3.

 Impact of native weevil extinction on native bird species To our knowledge, there is no native bird species that relies on native weevils as a major dietary component.

 Reliability of predictive models for impact on native weevils Questions were asked as to how we gathered and used our data to form our predictions. The methods are presented in Section 6.1.1

 Parasitism of native insect species other than weevils Microctonus aethiopoides is known only to attack weevils and will not attack other insects, invertebrates or plants.

 Change in non-target impacts if parasitoid population exceeds pest population Concern was expressed at what would happen if IPMa populations increased so fast that they outstripped available S. lepidus adult populations. A potential non-negligible risk – discussed in section 6.3

 Clover may be a future weed With the intensification of farming and the increasing use of pure grass swards and specialized forage crops, it was suggested that some future farmers may view clover as a weed and the weevil as a beneficial insect. We consider that this is unlikely to be a view held by the pastoral industry as a whole. While white clover may not be as important in ensuring the competitiveness of New Zealand farmers in international markets as in the past, it is AgResearch‟s view that white clover will remain a valued pasture component in sustainable farm systems.

 Correct identification Assurance was sought that we were sure that it was only the IPMa strain that AgResearch was introducing. Misidentification was considered highly improbable in that experienced experts with specialist knowledge of Microctonus taxonomy have been involved in the science underpinning this application (eg McNeill et al. 1993; Phillips & Baird, 1995; Vink et al. 2003). Each parasitoid collected was assigned a code and even after many years in quarantine, the progeny can be traced back to the

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original females. The methods used to bring in IPMa are described by Goldson et al. (2004).

 Direct harm to people M. aethiopoides is so small that it is unlikely to be observed. It does not sting or bite. No adverse risks, such as allergies, specifically related to M. aethiopoides have been identified in the rearing and release process.

 Risk of unknown effects on ecosystem Representatives of the Māori community voiced concern that even with all our research, there may be unforeseen and unexpected effects on the environment. We acknowledge that this is possible, but consider that the unexpected effects are more likely to negligible than ecological disasters. The research undertaken to support this proposal has been led by scientists that are internationally known for leading-edge research on desirable (Dr Stephen Goldson) and undesirable impacts (Dr Barbara Barratt) of introductions of biological control agents for weevil pests. In addition, the ERMA process ensures that this application is open to scrutiny and peer review by outside agencies.

 Risk to other ecosystems – eg wetlands Questions were asked regarding spill-over effects of IPMa in other ecosystems such as forests and wetlands. Microctonus aethiopoides is known only to attack weevils on legumes such as clovers and lucerne in arable and grassland environments. The combination of wasp searching behaviours and host preferences and low incidence of clovers in these other ecosystems make it highly improbable that weevils in these ecosystems will be put at risk.

 Eradication of undesirable IPMa populations AgResearch was asked if we could eradicate IPMa if we found unexpected negative impacts. The eradication of an undesirable IPMa population would be almost impossible. Any attempt of eradication would involve repeated use of insecticide over large tracts of land to kill parasitized adult weevils. This would threaten the existence of many native invertebrate and insectivorous bird and reptile species and increase the levels of pesticide residues in the environment and farm produce.

Benefits:

 Decrease in nitrogen fertilizer use At all meetings with the Māori community, concern was voiced about nitrogen run-off to our waterways. Control of S. lepidus should help reduce the need for high nitrogen fertilizer applications. A potential non-negligible benefit – discussed in section 6.3

 Contribution to farm production, sustainability and profits

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Māori also understand farming and the pastoral economy. Māori are major landowners, farmers and developers and wish to develop their land without impinging on core values. Control of S. lepidus should make clovers less vulnerable to abiotic and biotic stresses. Good clover vigour and persistence facilitates maintenance of quality pasture and thus animal performance, and lessens the need for pasture renovation and weed control. A potential non-negligible benefit – discussed in Section 6.3.

 Prevent outbreak of S. lepidus in South Island We were questioned by South Island Māori whether the release of IPMa would prevent S. lepidus reaching the South Island. Even if we gain permission to release in 2005, and undertake releases in late spring in the southern North Island districts where S. lepidus populations are still in the “outbreak phase” with climbing population levels, it is unlikely that parasitoids released at the two proposed experimental sites would increase and spread with such rapidity as to eliminate the risk of ongoing weevil spread from populations already established in regions such Manawatu and Wairarapa. It is very possible that the weevil is already in the South Island but at such low initial levels that it is undetectable.

 Comparison with other control options A number of questions were posed around other management options for clover root weevil that may be more acceptable to Māori. These are discussed in Section 6.3.

D. Economic Effects As per section 6(e) of the Act, list the economic risks, costs and benefits that might arise to New Zealand. Also identify who will bear the risks and costs and/or who the beneficiaries are likely to be.

Risks and Costs: There are no economic risks or costs.

Benefits:

 Re-establishes the benefits of clovers to the New Zealand pastoral industries Introduction of a biocontrol agent will help mitigate the loss of clover through root herbivory. A potential non-negligible benefit – discussed in Section 6.4

 Decrease in direct costs associated with use of nitrogen fertilisers While S. lepidus infestation is only one factor in the increased use of nitrogen fertilizer, some of the costs of nitrogen use are attributable to this pest. However, to date we have no firm cost data. It is known that farmers in S. lepidus-infested regions are using higher levels than those yet to be infested. For example, in 2002-2003, Waikato district pastoral farmers used 39% more urea/farm than those in the 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Manawatu-Wanganui district (Statistics New Zealand 2003). The cost of urea application is $640/tonne (including cartage spreading and GST) and in the year ending 30 June 2003, North island farmers used 209,614 tonnes (Statistics New Zealand 2004). This equates to almost $134 million. A potential non-negligible benefit – discussed in Section 6.4 under “Re-establishes the benefits of clovers to the New Zealand pastoral industries”.

E. Cultural, social, ethical and spiritual effects As per section 5(b) of the Act, list any adverse and beneficial impacts on people and communities that might arise and adversely affect or maintain/enhance (in the case of beneficial impacts) their capacity to provide for their own social and cultural wellbeing both now and into the future. Also list any ethical or spiritual risks, costs and benefits that might arise as per section 68(1)(a) of the Act. Indicate what steps have been taken to assist the identification of the effects in this area, for example, was there any community involvement?

Risks and Costs:  Disillusionment with science Should the biocontrol agent fail to live up to farmer expectations and give inadequate control of S. lepidus, scientists may lose credibility with the farming industry, not only for on-farm problems but also for wider issues. However, this is considered a negligible risk.

Benefits:  Reduction in anger and stress in affected rural communities Various print media articles since 1997 to the present have reported the emotive words of frustrated and angry farmers coping with S. lepidus impacts in the worst- affected districts. The stress that underlies these emotions has flow-on effects in the every day lives of these farmers and their families. The successful biocontrol of S. lepidus will alleviate this stress. A potential non-negligible benefit – discussed in Section 6.5

 Reduction in work load for farmers in worst-affected regions The current recommendation to maintain farm productivity in the present of S. lepidus is to put small applications of nitrogen fertilizer on „little and often‟. This is generally 5- 10 units of nitrogen applied after each grazing. Weevil infestations can cause the deterioration of pasture quality such that pasture renewal becomes the only option. The successful control of S. lepidus by the parasitoid would enable farmers to adopt less labour-intensive strategies to manage soil fertility and pasture quality. A potential non-negligible benefit – discussed in Section 6.5

F. Other effects (including New Zealand’s international obligations) List any remaining adverse and beneficial affects not already covered including any effects on New Zealand‟s international obligations (as per section 6(f) of the Act). 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Risks and costs: No adverse effects on New Zealand‟s international obligations are foreseen.

Benefits:  Assist New Zealand farmers in meeting international trade requirements Since the Uruguay round, there has been a significant shift in focus in agricultural policies in most OECD countries with whom we trade. The control of S. lepidus through the successful establishment of Irish M. aethiopoides will assist our farmers to meet environmental goals set by our overseas markets, from major overseas supermarket chains to the EU. A potential non-negligible benefit – discussed in Section 6.6

 Assist New Zealand meet its Kyoto Protocol obligations by reducing greenhouse gas emissions. According to official sources, agriculture is responsible for approximately 55% of New Zealand‟s greenhouse gas emissions. Nitrous oxide is given off from nitrogenous fertilizers. Improved nitrogen fixation by clovers and reduced reliance on nitrogen fertilizer arising from the control of S. lepidus through the successful establishment of Irish M. aethiopoides may assist New Zealand meet its Kyoto Protocol target of 1990 greenhouse gas emissions levels by the end of the first commitment period of 2008-2012. However, the size of emissions attributable to the impact of S. lepidus is unlikely to be significant in comparison to other factors affecting nitrous oxide release.

Section Six – Assessment of Potential Non-negligible Risks, Costs and Benefits This section entails detailed assessment of those effects identified in section 5.3 that you consider to be non- negligible . It should also provide an estimate of the likelihood of occurrence (which may be measured as frequency or probability) and the magnitude of the outcome if the adverse effect should occur. Please state why you consider an effect to be negligible before discounting it from the assessment. You should carry out your assessment firstly in the absence of any controls then in the context of the proposed controls being in place. Evaluating the effectiveness of controls is also part of the assessment process. ERMA New Zealand uses qualitative scales for assessing effects which may be of some use to you in completing this section – please refer to the ERMA New Zealand Technical Guide “ Decision Making: techniques for identifying, assessing and evaluating risks, costs and benefits” for further details. Any uncertainties associated with this assessment should also be discussed. Please cover all of these issues under each of the following headings (areas of impact) which reflect those matters referred to in Part II of the HSNO Act:

6.1 Effects on the environment (in particular on ecosystems and their constituent parts)

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Assess the risks, costs and benefits associated with the organism(s) to be conditionally released and the ways that they might adversely affect or improve/enhance (in the case of benefits) the New Zealand environment e.g. the life supporting capacity of air, water, soil and ecosystems; the sustainability of native and valued introduced flora and fauna; natural habitats and the intrinsic value of ecosystems; New Zealand‟s inherent genetic diversity; animal or plant health.

Risks and costs: Direct effects The greatest direct risk to the New Zealand environment is that IPMa will attack non-target weevil species. Other direct risks to be considered are the possibility of compromising biocontrol of lucerne weevil, should parthogenicity in the Irish stain prove unstable, and displacement of the native parasitoid Microctonus zealandicus Shaw that attacks the common native weevil Irenimus aequalis Broun.

6.1.1 Potential impact on non-target weevil species.

Background It is known that the Moroccan biotype of M. aethiopoides, released between 1982 and 1985 to control Sitona discoideus (Stufkens et al., 1987), parasitizes 11 indigenous and five exotic non-target species in the field (Barratt 2004), especially broad-nosed weevils in the subfamily Entiminae (subfamily classification of Leschen et al., 2003). The non-target parasitism by the Moroccan biotype is occurring both within and outside lucerne, which is the habitat for the target host S. discoideus (Barratt et al. 1997b). The discovery of this unexpected wide host range has contributed to increased concern in New Zealand about the impacts of biological control agents on non-target species (e.g. Barratt et al., 2000).

The host range of IPMa was tested in quarantine and compared with that of the Moroccan M. aethiopoides. A paper by Goldson et al. 2005 gives full account of the methodology employed, and the results. A summary of the methods, results and conclusion pertaining to IPMa is presented below.

Test insects The potential weevil hosts were selected using both phylogenetic and ecological affinities of S. lepidus to endemic and deliberately introduced beneficial species. Sitona lepidus is in the weevil subfamily Entiminae, tribe Sitonini. Sitonini is not represented in the New Zealand fauna but is considered to be phylogenetically very close to the tribe Tropiphorini (Alonso- Zarazaga & Lyal, 1999). Several genera of this tribe are found in New Zealand‟s natural grassland ecosystems and a few, such as Irenimus and Nicaeana, have also become well adapted to agriculturally modified grassland environments (Barratt et al., 1998). The assumption that these genera are most likely to be at risk from non-target parasitism by IPMa is well supported by earlier research on the host range of Moroccan M. aethiopoides (Barratt et al., 1997b). The species chosen for testing against S.lepidus were:

Common endemic grassland species in the tribe Tropiphorini.

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Irenimus aequalis Broun Nicaeana cervina Broun Catoptes cuspidatus Broun

Endemic grassland–dwelling species of limited distribution Protolobus porculus (Pascoe) (tribe Tropiphorini) Steriphus variabilis Broun (tribe Rhytirhinini) (known to have been parasitised by the Moroccan M. aethiopoides in the field)

Exotic weed biocontrol agents parasitised by Moroccan M. aethiopoides Rhinocyllus conicus Froelich Curculioninae: Lixini Trichosirocalus horridus (Panzer) Curculioninae: Ceutorhynchini Exapion ulicis (Forster) Apioninae: Apionini

Exotic weed biocontrol agent proposed for introduction Cleopus japonicus Wingelmüller Curculioninae: Cionini

I. aequalis, N. cervina and R. conicus are parasitized in the field by Moroccan M. aethiopoides. Therefore, field-collected weevils of these species were held for 20-26 days prior to use in experiments to allow any parasitoids to emerge.

The parasitoids used consisted of IPMa (Goldson et al., 2001) and the Moroccan M. aethiopoides biotype originating from the 1982-85 releases (Stufkens et al. 1987). IPMa had been maintained as cultures under quarantine using S. lepidus collected near Hamilton and the Moroccan M. aethiopoides were reared from S. discoideus populations collected from lucerne at Lincoln. Emerged parasitoid adults were stored in a controlled environment cabinet (9 20C, 54 15 %RH and L:D 14:10) in 5 ml vials with a supply of 20% honey: water solution for a minimum one week before use.

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Background on selected weevils The five native weevils tested are representative of New Zealand broad-nosed weevil genera. Little is known of their biology. The adults feed on pollen, seedling plants and foliage of a broad range of plants, and the larvae feed on roots (Barratt & Kuschel 1996). They occur naturally in a wide range of habitats, particularly native grasslands and sub-alpine and alpine herb fields and many have adapted well in developed pasture (Barratt et al. 1998). In their survey of grassland Curculionoidae, Barratt et al. (1998) found that 50 of the 64 native species identified were in the tribe Tropiphorini, and more than half of these were in the genera Irenimus and Nicaeana. I. aequalis was by far the most common native species, with populations recorded of over 100/m2.

The nodding thistle seed-head weevil (Rhinocyllus conicus) was first released in 1973 and is now widespread. The weevil can reduce seed production by as much as 49% in Hawke‟s Bay, and as little as 3% in Canterbury. Murray et al. (2002) recorded parasitism rates by the Moroccan biotype reaching up to 17% albeit with more males (12%) than females (3.7%) found parasitized, but there is no evidence to show that parasitism translates into a reduction of nodding thistle populations nor impacts on the biological control effort against thistle. Furthermore, research into the impact of R. conicus has concluded that it is not an effective biocontrol agent of the thistle in New Zealand (Kelly & McCallum 1995; Shea & Kelly 1998).

The crown-feeding weevil (Trichosirocalus horridus) attacks nodding thistle rosettes, and several releases have been made since 1975. Exapion ulicis Forst. was released in 1931, and currently destroys about 35% of the annual gorse seed crop (Hill et al. 2000). No evidence of parasitism by the Moroccan biotype has been found in the field for either species (Barratt pers comm.).

An application received from the Forest Research Institute to release Cleopus japonicus, or buddleia leaf weevil, for the control of the weed pest buddleia (Buddleja davidii) is currently under consideration by ERMA. The weevil is from China, where buddleia is its natural host plant. A final decision is due 31st March 2006.

Test regimes All experiments were conducted under ambient conditions of mean temperature 18oC and a 14:10 L:D photoperiod, in a quarantine facility at Lincoln.

No-choice host range tests No-choice tests provide maximum estimates of likely host susceptibility to parasitism through exposure of test species to as much pressure as possible from the candidate control agent (e.g. Goldson et al., 1992; Barratt et al., 1997b) and are useful indicators of a parasitoid‟s potential host range under field conditions (Barratt et al., 1997b; Barratt, 2004). Van Driesche and Murray (2004) recently published an overview of the strengths, weaknesses and current thinking on value of various testing regimes. The strengths of no-choice tests is that negative results provide convincing evidence a test species is not likely to be a field host, and larval 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

development can provide further information on host suitability. Conversely, confinement can give “false positives” as test hosts may not be able to be found by the parasitoid in nature (eg behaviour, or out of geographical range or synchrony). Choice tests are of value in that they can reveal if an agent has a preference among potential host species and allow several species to be assessed simultaneously. However, there can be significant risk of false positives or negatives and Van Driesche and Murray (2004) recommended that the importance of these errors be assessed by comparison of choice data with that from no-choice tests.

In each no-choice test, there were three treatments: (1) non-target „test weevils‟ and (2) S. lepidus (S. lepidus control) and (3) „test weevil‟ control. The first two treatments were exposed to two parasitoids for 48 hours and the test weevils control was left unexposed. The two controls provided measures of parasitoid activity and the test weevils‟ inherent survival and vigour during the course of the experiment respectively. Each treatment consisted of 20 weevils placed in plastic cages (180 mm x 160 mm x 70 mm) fitted with fine-gauze lids. There were five or six replicates of each treatment for every species except for P. porculus which was replicated three times. After 48 hours, the parasitoids were removed from the parasitoid-exposed treatments and the weevils transferred to larger parasitoid rearing cages (McNeill et al., 2002). All cages were furnished with appropriate host plant foliage for each weevil species. The no-choice tests using IPMa were replicated five or six times with the exception of P. porculus, which was replicated three times.

Each cage was inspected every two to three days. All dead weevils were counted, removed and dissected to determine the presence and stages of any parasitoid larvae. Parasitoid numbers were recorded and the time that pre-pupal emergence commenced relative to the S. lepidus control was also noted. When emergence had ceased, all remaining weevils were dissected for the presence, numbers and viability of parasitoid larvae. Parasitoid larval viability was determined by the presence of food in the parasitoid gut, evidence of atrophy, melanisation and/or encapsulation. The stage of reproductive development of any parasitoid- exposed but unparasitised weevils was also assessed.

Choice tests. To gain an understanding of host preference, choice tests were conducted on the native weevil species I. aequalis, C. cuspidatus and S. variabilis. A mix of 10 I. aequalis and 10 S. lepidus were exposed to two parasitoids for 48 hours as described for the no choice tests. Because prior testing had showed that I. aequalis was a permissive host for IPMa, the test was terminated after 12 days after which parasitism and reproductive condition were determined by dissection. Similarly, for C. cuspidatus, a choice test looked at potential attack by IPMa and whether the presence of S. lepidus affected rate of attack on C. cuspidatus. However, unlike the first choice test, this experiment was maintained until prepupal emergence from the control weevils had ceased, after which all weevils were dissected. For both choice tests involving I. aequalis and C. cuspidatus there were five replicates of all treatments including the S. lepidus and unexposed test weevil controls.

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Steriphus variabilis was found in very limited numbers in the field and only three replicates were possible. In this experiment surviving weevils were dissected once parasitoid prepupal emergence of control weevils had ceased.

Results

With the exception of Test 14 (29%), the vigour of the Irish M. aethiopoides was excellent with total percentage parasitism of the S. lepidus controls averaging over 70% (Table 6.1.1). However, superparasitism in the S. lepidus controls was also high, averaging around 50%. Therefore parasitism rate was derived from weevil mortality and weevils found parasitized when dissected at the end of the experiment, rather than from prepupal numbers.

Irish M. aethiopoides was able to complete larval development successfully in I. aequalis, N. cervina, C. cuspidatus, P. porculus and S. variabilis, but in all but N. cervina and S. variabilis, total percentage parasitism was significantly lower than that found in S. lepidus in the same test (Table 6.1.1). In the case of the test with S. variabilis the lack of significance was attributed to the small number of replications. In addition, the levels of superparasitim were lower in I aequalis (Test 3, P<0.01) and N. cervina (Test 10, P= 0.07) than in S. lepidus. Catoptes cuspidatus was not superparasitised at all.

No parasitism was detected in three of the four non-target exotic weevils in the no-choice tests. Dissection revealed a low level of parasitism in E. ulicis, but no prepupae emerged.

Of the seven non-target species that were successfully parasitised, only two showed evidence of delayed development. Parasitoid emergence from S. variabilis test weevils commenced three days after the commencement of prepupal emergence from S. lepidus. Total prepupal emergence from the S. lepidus control was 26 versus 2 for S. variabilis. Only two prepupae emerged from C. cuspidatus, one at two and the other at six days after emergence commenced from S. lepidus. Total emergence of prepupae from S. lepidus was 173.

With the exception of N. cervina, parasitism levels by IPMa in these no-choice host range tests were lower than previously recorded in the Moroccan biotype in similar laboratory tests (Table 6.1.1). Two pairs of tests (Tests 3 and 4 (I. aequalis) and Tests 6 and 7 (C. cuspidatus)) were run in conjunction with each other, allowing comparison of parasitism in choice and no-choice conditions. When a mixture of S. lepidus and I. aequalis were offered to IPMa, total parasitism of I. aequalis increased from 20% in the no-choice experiment to 46% (P<0.01) while S. lepidus parasitism remained high at 70% (Table 6.1.1). Similarly, total parasitism of C. cuspidatus increased from 1% in the no-choice test to 13% in the choice test (P<0.05) with negligible change to S. lepidus parasitism.

When given a choice, Irish M. aethiopoides selected S. lepidus over S. variabilis at a rate of approximately 4:1 but with few replicates because of low S. variabilis availability, the difference was not statistically significant. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Table 6.1.1: Comparison of percent parasitism in choice and no-choice experiments of S. lepidus and non-target test weevil adults by IPMa and the Moroccan biotype (published data) of M. aethiopoides. Test weevil Assay Test type Total percent parasitism in assay IPMa Moroccan biotype S. lepidus Test sp. Lab Field peak ( 10 adults) Native weevils 2 I. aequalis No-choice 81* 29 332 3 I. aequalis No-choice 64* 20 471 3-621 4 I. aequalis Choice 70* 46 5 N. cervina No-choice 62* 45 551 311 433 112 6 C. No-choice 75* 1 05 cuspidatus 7 Choice 74* 13 8 P. porculus No-choice 56* 20 621 04 9 S. variabilis Choice 91 23 61 5-82 Exotic weevils 12 R. conicus No-choice 78* 0 381 91 393 175 13 E. ulicis No-choice 80* 8 04 16 T. horridus No-choice 69** 0 21 04 17 C. japonicus No-choice 80** 0 * S. lepidus parasitism levels significantly higher than the test species (P<0.05 or less) 1 Barratt et al., 1997b; 2Barratt et al., 2000; 3 Barratt et al., 2001; 4 Barratt 2004; 5 Murray et al. 2002 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Parasitoid viability The parasitoid larvae did not survive as well in the native weevils as they did in S. lepidus. Moderate-high levels of atrophied and/ or melanised larvae were found in parasitized I. aequalis (40% and 36% of parasitoids in Tests 3 and 4, respectively); N. cervina (32%); P. porculus (50%); S. variabilis (67%) and C. cuspidatus (60%). The percentage of parasitised S. lepidus with non-viable parasitoid larvae ranged throughout the tests from 0-26%.

Multiple larvae were able to complete development in superparasitised S. lepidus. For example 30% more prepupae emerged than there were weevils present in Test 5. Thus it was apparent that more than one IPMa egg can develop to at least the prepupal stage in a single S. lepidus. This was not observed in the other weevil species tested.

Weevil premature mortality While higher levels of premature mortality was found in parasitoid-exposed weevils compared to unexposed weevils prior to the emergence of prepupae in all assays where comparisons could be made, it was only significant for N. cervina and T. horridus (Table 6.1.2). It should be noted that in both cases the comparison was between a low 5% in the exposed weevils and 0% in the unexposed weevils

Reproductive suppression While the host testing assays were not designed to assess reproductive suppression, examination of the state of reproductive organs during dissections did provide sufficient data on four species to make comparisons between weevils exposed and unexposed to IPMa (Table 6.1.3). Of these species, only N. cervina appeared to show a difference between exposed and unexposed weevils in the percentage of females capable of laying eggs. However, the difference was not significant (P>0.05).

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Table 6.1.2: Comparison of percent premature weevil mortality of non-target test weevil adults exposed and unexposed to IPMa in no-choice experiments.

Assay Test weevil Exposed Unexposed

Native weevils

2 I. aequalis 19 11 5 N. cervina 5** 0 6 C. cuspidatus 30 28 8 P. porculus 22 7

Exotic weevils

12 R. conicus 1 0 13 E. ulicis 3 1 16 T. horridus 5* 0 17 C. japonicus 5 4

* denotes P > 0.05; ** P > 0.01

Table 6.1.3: Comparison of percent reproductive females amongst non-target test weevil adults exposed and unexposed to IPMa in no-choice experiments.

Test weevil Exposed Unexposed Native weevils I. aequalis 18 16 N. cervina 43 82 C. cuspidatus 73 61 Exotic weevils E. ulicis 56 53 C. japonicus 32 35

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Discussion The high levels of parasitism in the S. lepidus controls by IPMa in this study demonstrates both the stringency of the testing regimes and why this parasitoid is considered a good potential biocontrol agent for S. lepidus. Comparative levels of 67% parasitism were achieved during similar laboratory testing of Microctonus hyperodae prior to introduction (Barratt et al. 1997b) and on release has had a significant impact on the Argentine stem weevil in the field (e.g. Goldson et al., 1998).

Our results show that the IPMa does parasitise the native weevils I. aequalis, N. cervina, P. porculus, C. cuspidatus and S. variabilis, and the exotic species E. ulicis but that parasitoid viability was compromised in all of the native species attacked and host reproductive condition was not diminished. However, even though IPMa can complete full development in these species in the laboratory, it does not necessarily indicate that it will be self-sustaining on these species in the field. Prediction of the likely impacts of IPMa on the susceptible non- target weevils the field conditions is, by its nature, speculative. Fortunately, New Zealand has considerable experience of Moroccan M. aethiopoides host behaviour, both in the laboratory and field.

By combining the data gathered in this study with our knowledge of Moroccan M. aethiopoides, it is possible to make the following predictions with a reasonable level of confidence.

Irenimus aequalis is likely to support successive generations of IPMa under field conditions but is highly unlikely to be endangered by the release of IPMa. Barratt et al. (1997b) found that the Moroccan biotype had moderately high parasitism levels in the laboratory and reached peak parasitism levels of 62% in I. aequalis collected from Hamilton pastures. In spite of this, I. aequalis remains a minor pest species in improved pastures in New Zealand, with levels of over 100 adults/m2 recorded in the Waikato (Barratt et al. 2000). IPMa parasitism rate on I. aequalis was around half that previously found for the Moroccan biotype in similar no-choice laboratory tests (Table 6.1.1) and therefore it will probably be out- competed by the latter in the field. While S. lepidus is preferred over I. aequalis by the parasitoid, and only about a third of laboratory-exposed I. aequalis produce prepupae, it is possible that when I. aequalis is relatively abundant in some pastures it may act as an alternative host when S. lepidus adult numbers are scarce.

Other more locally distributed species of Irenimus, such as I. stolidus and I. aemulator are attacked by the Moroccan biotype in Otago (Barratt & Kuschel 1996) and are likely to be attacked by IPMa. If the response of IPMa is the same against all species of this genus, the assumption is that this new strain of M. aethiopoides will be less aggressive than the Moroccan strain and may be out-competed by the existing strain rather than add to existing parasitism levels.

For similar reasons it is probable that Nicaeana species would act as hosts for IPMa but unlikely to be endangered. Species of this genus, common in improved pastures south of Oamaru (Barratt et al. 1998) and tussock grasslands, is probably the species that causes the 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

most damage to oversown white clover seedlings in Otago (Barratt et al. 1992 and references therein) with adult populations often exceeding 40/m2 (Barratt et al. 2000). This study showed that in the laboratory, Irish M. aethiopoides parasitised N. cervina at a similar rate as previously found with the Moroccan biotype (Table 6.1.1). Field parasitism by the Moroccan biotype has been recorded to peak at 31-56% in the Strath-Taieri area (Barratt et al. 1997b). Nicaeana spp. appear to be more favoured non-target hosts than Irenimus spp. (Barratt, AgResearch entomologist, pers. comm.)

Catoptes cuspidatus is most unlikely to provide a host for viable populations of the Irish parasitoid biotype as the weevil sustained a very low level of total parasitism in the no-choice test (1%) and three of the five live parasitized weevils at the end of the choice test contained nonviable parasitoids. The Moroccan strain has not been found in C. cuspidatus in the field (Barratt 2004)

Similarly, P. porculus and S. variabilis are probably unlikely to act as hosts for successive generations of IPMa in the field. Both species showed clear signs of a host immune response resulting in poor parasitoid larval viability resulting in their respective prepupal production being 7% and 8% of the S. lepidus hosts offered. Consequently, even if these species are able to support some parasitoid development in the field, it is unlikely that they would be able to sustain vigorous parasitoid populations. This is reinforced by the knowledge that the Moroccan biotype is reported as parasitizing P. porculus at three times the rate of IPMa in the laboratory, yet has not been recovered from the field (Table 6.1.1). S. variabilis is found in South Island pastures, appearing to favour moist low-lying sites where adult populations can exceed 70/m2 and maximum percentage parasitism by the Moroccan biotype reach 20% (Barratt et al. 1997b).

The relatively high level of parasitism of Irenimus aequalis and Catoptes cuspidatus in the presence of S. lepidus compared with the levels found in the no-choice test is probably an artifact of the experimental conditions. With the high density of two weevil species in one cage, the parasitoids may have had difficulty in differentiating between weevil species as oviposition cues emanating from S. lepidus may confuse the parasitoid into ovipositing into the native weevil. As S. variabilis was only assessed under choice test conditions, it can only be ranked against I. aequalis and C. cuspidatus, in being in between these two species in terms of acceptability as a host for IPMa. In the field, the risk to native weevils in the presence of S. lepidus may be reduced because a buildup of weevil volatiles is unlikely and the parasitoid is not confined to a container (i.e. it can move on if there is a scarcity of suitable hosts).

Of the exotic beneficial weevils, only Exapion ulicis was parasitized in the laboratory. Successful development in the field in this species by IPMa is also unlikely. Tests 14 and 15 revealed no difference between the Irish and Moroccan M. aethiopoides attack rates and no parasitism of E. ulicis has ever been recorded in the field, in spite of the Moroccan biotype having been in New Zealand ecosystem for nearly 20 years and its relatively frequent recovery from other non-target species (Barratt et al., 1999b).

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Depending on S. lepidus population abundance and distribution, the establishment of IPMa may result in this strain being more widespread than current Moroccan M. aethiopoides populations in S. discoideus and its non-target host populations. This could mean more widespread contact with non-target species than has occurred hitherto, and even though less aggressive than the Moroccan biotype, the release of IPMa may add to existing parasitism levels. The levels of „spill-over‟ parasitism from the S. lepidus populations can only be speculated on A simple deterministic model developed by Barlow et al. (2004) calculated that a 15% parasitism of a non-target native weevil population by Moroccan M. aethiopoides, led to an 8% reduction in weevil density. When extrapolating this data to higher altitudes, the model predicted that the reduction in population density of a host may be greater if it occurs at higher altitudes, where the intrinsic rate of increase of the host was shown to be lower. However, parasitism levels at higher altitude sites have never been seen to exceed about 10% and at this stage there is no evidence of a reduction in weevil populations. This is still being investigated and one theory is that M. aethiopoides may not survive the winter in the high altitude native grassland areas (B. Barratt, AgResearch entomologist, pers. comm.).

Conclusion

This study indicates that IPMa is less aggressive and more selective in attack on non-target weevil hosts than its Moroccan counterpart. With the exception of N. cervina, IPMa had lower parasitism rates on non-target hosts in no-choice tests than has been reported for the Moroccan biotype. It is possible that the difference in attack rates may be associated with the virus like particles (VLPs) which have been identified in Moroccan M. aethiopoides (Barratt et al. 1999) but not IPMa (Barratt, AgResearch entomologist, pers. comm.). They are also absent from M. hyperode (Argentine stem weevil parasitoid) which has very low non-target impact in the laboratory and field. These VLPs may be important in the suppression of the host weevils immune defence system and their presence may allow a wider host range. Therefore its ecological impacts are likely to be less severe than those already imparted by the Moroccan M. aethiopoides. Of the species tested, only I. aequalis and N. cervina are likely to act as hosts to the new strain, and both species are less preferred to the target host S. lepidus and had higher levels of non-viable larvae. However, T. repens (clover) is more prevalent than M. sativa (lucerne), the latter grown on a more limited scale. This will mean that should IPMa be released and successfully establish in S. lepidus, it will probably be more widely distributed than the Moroccan M. aethiopoides biotype.

6.1.2 Potential loss of biocontrol of Sitona discoideus through interbreeding with Moroccan strain if parthenogenesis is not stable.

IPMa reproduce by parthogenetic thelytoky (males are absent), whereas Moroccan M. aethiopoides are arrhenotokous (mated females produce both males and female offspring). This minimises the potential for inter-biotype mating and the consequent risk of a dilution in the efficacy of the biological control effort. In quarantine studies Moroccan males are weakly 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

attracted to newly-emerged IPMa females. Attraction is demonstrated by pursuit of the female within a range of 1-5mm and fanning of the wings by the male. While the Irish female will vigorously avoid the male, copulation has been observed on three occasions out of 15 attempts to induce mating. However, when the “mated” females were allowed to parasitise S. lepidus (through two generations), the offspring of these putative matings were all female suggesting that there is reproductive incompatibility between the Moroccan and Irish biotypes.

The potential loss of efficacy as a biological control agent when biotypes interbreed was demonstrated by Goldson et al. (2003) using arrhenotokous strain of European M. aethiopoides collected from Wales and parasitising S. lepidus and Moroccan M. aethiopoides parasitising S. discoideus (Figure 6.1.1). In summary, crosses between biotypes resulted in significant (P<0.05) reduction of impact on parasitoid efficacy against both Sitona spp. When the Moroccan M. aethiopoides biotype was crossed with the Welsh biotype, its efficacy as a control agent against S. discoideus was effectively halved. When the Welsh biotype was crossed with that from Morocco, the efficacy of the offspring against S. lepidus did not differ significantly from the pure European strain. However, the efficacy of the hybrid offspring against S. lepidus was reduced by about half compared to that of both the pure Welsh and Welsh by Moroccan biotypes. The attack rate of the pure Welsh biotype against S. discoideus was even lower at 16%.

20 Percentageparasitism

10 (Barratt et al . 1997)

0 M x M M x E E x E E x E E x M Hybrid M x M S. discoideus S. lepidus

Figure 6.1: Histogram showing parasitism levels of Sitona discoideus and Sitona lepidus. M x M = Moroccan strain, M x E = Moroccan European strain cross, E x E = European strain and Hybrid = offspring of E x M, hybrid by hybrid cross. No overlap of confidence limits denotes significant difference at P<0.05.

Of particular significance, this study showed how the introduction of inappropriate biocontrol strains could potentially destroy a useful biological control opportunity, even if subsequently, 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

more appropriate strains are introduced. If the Welsh strain of M. anthropoids had genetically mixed with the Moroccan strain in New Zealand there could have probably been an irreversible loss of efficiency in the suppression of S. discoideus in lucerne (Goldson et al. 1990). Likewise, and for the same reasons, it would seem that the crossing of the Moroccan and Welsh parasitoid strains could severely and permanently compromise the chances of M. anthropoids ever effectively reducing the impact of S. lepidus on white clover.

Although relatively low numbers of parasitoids were collected from several widely separated sites in Ireland, all were female and parthenogenetic. No evidence has been found of endosymbionts such as Wolbachia, which can induce parthenogenicity. Therefore, is presumed that parthenogenicity in IPMa is of genetic origin and therefore stable.

The genetic variations between and within M. aethiopoides biotypes has recently been documented by Vink et al. (2003) and Iline & Phillips (2003). In summary, genetic analyses provided strong support for two M. aethiopoides groupings, one associated with Hypera species and the other with Sitona species. There was also evidence that IPMa was most closely related to European strains obtained from S. lepidus, in comparison to strains reared from Sitona hispidulus and S. discoideus. This confirms that IPMa is not a different cryptic species. However, analysis using 16S sequences suggested that IPMa was in a different clade than all the other sexually- reproductive strains collected from S. lepidus in 10 countries throughout Europe from Finland to Italy.

To date, there has been no evidence that the stability of parthenogenesis in IPMa will be affected by exposure to New Zealand environmental conditions.

6.1.3 Potential displacement of native Microctonus spp. Three native species of Microctonus are indigenous to New Zealand (Shaw, 1993). Microctonus zealandicus Shaw (Hymenoptera: Braconidae) is a gregarious endoparasitoid of adult Irenimus aequalis Broun (Coleoptera: Curculionidae) (Shaw, 1993; McNeill et al. 1993). Microctonus alpinus Shaw has been collected from Coronet Peak (1640 m) and its host is unknown. Microctonus falcatus Shaw was described from a specimen collected on the margins of Nothofagus forest in the Cobb reservoir. Its host is unknown.

Of the three indigenous species, competitive interaction for hosts is most likely to occur for M. zealandicus. Although the biology of the parasitoid has not been studied in detail, M. zealandicus has been recovered from diverse locations across mid-Canterbury, with the parasitoids recovered from I. aequalis collected from pasture through to modified native grasslands (unpublished data). Parasitism levels are generally low (1-5%), although peak levels of 20% have been recorded at roadside verge and pasture site (McNeill et al. 1993).

Host range studies on IPMa have shown that I. aequalis is a permissive host, and therefore there is a potential for competitive displacement of M. zealandicus by Irish M. aethiopoides should release take place. However, results of field collections in Canterbury suggest that the probability of displacement is low. Currently, I. aequalis is already subject to parasitism by Moroccan M. aethiopoides with parasitism levels of up to 35% recorded in Canterbury. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

While both M. zealandicus and Moroccan M. aethiopoides have been recovered from I. aequalis collected from the same location, levels of parasitism by both native and introduced Microctonus species have been generally low (<10%), so it is difficult to conclude that competitive displacement has occurred. Furthermore, any interaction is confounded by the fact that gregarious development by M. zealandicus means that 2-6 adults can result from parasitism of a single weevil. Given that attack of I. aequalis by Moroccan M. aethiopoides is higher than that of IPMa in the laboratory, it is concluded that IPMa will not have a significant impact on M. zealandicus.

6.1.4 Benefits:  Reduced possible misuse of nitrogen fertiliser. The most significant New Zealand legislation for managing the impacts of the farming sector on the environment is the Resource Management Act 1991 (RMA). While the RMA does not yet have a national policy on water quality, a „Water Programme of Action‟ is currently being led by the Ministry of Environment and Ministry of Agriculture and Forestry. In the meantime, regional councils are beginning to regulate fertiliser usage through district plans and the fertiliser manufacturing industry has promoted the development of a „Code of practice for fertiliser usage‟.

The report „Growing for good: Intensive farming, sustainability and New Zealand‟s environment‟ was commenced in 2002 by the Parliamentary Commissioner for the Environment following widespread concerns about the impacts of intensive farming, particularly dairying, on New Zealand‟s waterways. Below are excerpts from that report on the use on synthetic nitrogen fertiliser.

Trends in synthetic nitrogen fertiliser use

P 91, Table 5.2: Kg urea/ha spread, by sector, for the years ending June 1996 and 2002

Sector 1996 2002 % change Sheep and 0.7 5.7 + 670 beef Dairy 38.8 101.5 +160 Deer 2.9 10.1 +240

P 92, Reasons for nitrogen fertiliser increase include: Enables an increase in farm productivity and profitability Tactical use can overcome seasonal feed shortages and ensure steady supply of forage The cost of fertiliser as a percentage of farm income has decreased The loss of clover in pasture due to clover root weevil

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P 6, Nutrient inputs need to be very carefully managed. If excessive amounts are applied to pasture or crops it leaks into the wider environment. Nitrogen in particular is highly mobile and can easily enter streams or leach through the soil into groundwater, eventually ending up in rivers, lakes and coastal waters. This leads to the eutrophication of fresh waters and coastal waters and the deterioration of groundwater quality. Once nitrogen enters the environment there is no effective way to remove it

Parliamentary Commissioner for the Environment 2004 S. lepidus is one of the reasons why in nitrogen fertiliser use has soared dramatically in recent years, with farmers in S. lepidus-infested regions using higher levels than those yet to be infested. For example, in 2002, Waikato district pastoral farmers used 46% more urea/farm than those in the Manawatu district (Statistics New Zealand 2003). Weevil larvae can destroy the nitrogen-fixing capability of white clover (Gerard 2002). Therefore, because farmers cannot rely on the availability of this free, natural source of nitrogen, most farmers in infested regions must apply high levels of nitrogen fertiliser (>200 kg N/ha) to maintain soil fertility and farm profitability. However, if used incorrectly, these high levels of fertiliser can contaminate our environment, and endanger our native species, especially our native freshwater aquatic life through eutrophication of streams and lakes. For example densities of common smelt Retropinna retropinna, an important prey of trout, in North Island lakes are directly related to water clarity (Rowe & Taumoepeau 2004).

Gerard (2002) has shown that while nodule damage increases and percent foliar nitrogen decreases in response to increasing S. lepidus larval numbers in spring, there is no impact on clover dry matter production unless the foliar nitrogen level falls below 4.7%. Goldson et al. (1988) observed the identical relationship with S. discoideus on lucerne and speculated that when this threshold is reached, the clover plant is consuming more nitrogen than it can fix. This hypothesis is supported by farmer observations that clover growth in clover root weevil infested pastures show increased vigour following the application of nitrogen fertiliser.

The release of IPMa should reduce S. lepidus numbers down to levels that can be tolerated by the plant, and allow the plant to maintain sufficient nodule numbers for effective nitrogen fixation year round hence reduce the need for N application.

 Improved soil fertility The following is quoted from Brock (2004). “When N fertiliser is used, the extra dry matter produced (leaf) comes from an increased shoot:root ratio as the roots do not have to explore as large a soil volume as before to find N, particularly with irrigation. This in turn reduces the ability of the pasture to intercept and utilise the higher N returns from the extra animal N and the amount of organic matter cycling through the soil, which must be replaced by more fertiliser N to maintain productivity. There needs to be a better understanding of the many roles clover mediates in terms of soil nutrient cycling and the health of the whole pasture.” The reliance on ever-increasing levels of fertilisers is not desired by any of the major pastoral industries. The release of IPMa should reduce S. lepidus numbers down to levels that allow farmers to choose sustainable pasture management systems that meet both economic and environmental goals. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

 Improved pasture sustainability Based on research in Waikato pasture, the current densities of 300 S. lepidus larvae/m2 and the nature of their feeding behaviour, are such that clover root performance is grossly impaired in infested pastures. A single first instar larva may destroy up to 10 nodules (Gerard 2001). The nitrogen stress this imposes on the plant results in a higher shoot: root ratio and around 44% less carbon assimilated through photosynthesis within 3 weeks of egg hatch (Murray et al. 2002). Even though many of these larvae may not survive to adulthood, the largest hatching of early instars coincides with the spring flush in pasture growth. This results in visible pasture yellowing. The yellowing can be readily rectified by the application of nitrogen fertiliser. However, with the older larvae inflicting further root damage, the clover plant is at a competitive disadvantage to the grasses and may fail to persist if pasture management is poor, or environmental conditions adverse. This opens up pastures and allows weeds to establish; reducing pasture quality and increasing the likelihood of herbicide use or pasture renovation.

The release of IPMa should reduce S. lepidus numbers down to levels that with wise pasture management, farmers will be able to maintain high quality pastures.

 Reduction in pesticide use The insecticide Lorsban 750 WG insecticide (contains 750 g/kg chlorpyrifos in the form of a water dispersable granule) is registered to control S. lepidus adults when sowing clover into an infested pasture. Chlorpyrifos is an organo-phosphate insecticide. It is toxic to bees and other beneficial insects and is degraded slowly in soil to organo-chlorine compounds and carbon dioxide. Its half-life in the environment is 60-120 days (NZ Agrichemical Manual 2001 www.agrichemical.co.nz). Residues of relatively broad-spectrum pesticides such as chlorpyrifos in the pasture ecosystem influence the abundance of non-target invertebrates, particularly predators and parasitoids. The release of IPMa should reduce S. lepidus numbers down to levels that insecticides are not cost-effective when sowing clover into an infested pasture, thus usage should be minimal.

6.2 Effects on human health and safety (including occupational exposure) Assess any potential risks, costs and benefits to human health that may be related to the conditional release of the organism(s) in New Zealand.

Risks and costs:

There are no known risks to human health and safety

Benefits:

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 Reduction in pesticide use The insecticide Lorsban 750 WG insecticide is registered to control S. lepidus adults when sowing clover into an infested pasture. The active ingredient in Lorsban 750 WG is chlorpyrifos, an organo-phosphate insecticide with an acute oral LD50 of 135-163 in rats. It has contact and vapour action, and may be harmful if inhaled, swallowed or absorbed through the skin.

Organo-phosphates affect the nervous system, reducing the level of cholinesterase in the body. Absorption occurs mainly through the skin and a dangerous level of poisoning may be present before symptoms become noticeable. The effects of organo-phosphates are cumulative. Farmers and contractors working with these insecticides must follow instructions for use very carefully and wear correct protective clothing. While these measures significantly reduce risk, there is always the possibility of accidents or failure to correctly follow guidelines and spray contractors may unavoidably have prolonged exposure during the spraying season.

Successful biological control of S. lepidus should reduce the risks to human health and safety associated with use of this insecticide for S. lepidus control.

6.3 Potential effects on the relationship of Māori and their culture and traditions with their ancestral lands, water, sites, wāhi tapu, valued flora and fauna and other taonga (taking into account the principles of the Treaty of Waitangi) Assess any potential adverse and beneficial effects on the relationship of Māori and their culture and traditions with their ancestral lands, water, sites, wāhi tapu, valued flora and fauna and other taonga (taking into account the principles of the Treaty of Waitangi). If there are potentially non-negligible effects to consider in this area, it is expected that consultation will have occurred with Māori. Give details of this in the space provided (see the User Guide for what is required).

Risks and costs: The key area of concern to Māori regarding the proposed release of IPMa was in the environmental area, in particular the impact on indigenous grassland weevils and their habitat.

 Impact of native weevil extinction on native ecosystem function. The native weevils of New Zealand are not well known taxonomically, with many species undescribed and some probably still undiscovered (Barratt 2004). The ecology of the native broad-nosed weevil species at risk from attack by IPMa is not well known (Barratt & Kuschel 1996). Many are flightless, with soil-dwelling, root-feeding larvae. Adults feed on a wide range of host plants and can damage pasture species (Barratt et al. 1992). They occupy a number of habitats naturally, in particular native grasslands and sub- alpine and alpine herb fields. Generally they are univoltine, with adults emerging in winter-spring (Barratt et al. 2000). 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Our research suggested that native grassland weevils will not be subjected to any risk of extinction by the release of IPMa. As outlined in Section 6.1.1, the existing Moroccan biotype is more aggressive and less host specific than IPMa, yet there is no evidence that the Moroccan biotype has any significant impact on native weevil populations. If significant reductions in the density of native weevil populations did occur, then likely scenarios for the ecosystems which they inhabit would be reduced herbivory on seedlings and roots (native and exotic), and possibly reduced pollination of plants. We have no evidence to suggest that native weevils are major pollinators. Some predators such as ground beetles (Coleoptera: Carabidae) and birds (native and exotic) might be subjected to a reduction in available prey, but again we have no evidence to suggest that native weevils form a major part of the diet of any predators.  Change in non-target impacts if parasitoid population exceeds pest population Concern was expressed at what would happen if IPMa populations increased so fast that they outstripped available S. lepidus adult populations.

It is possible that at the end of the winter weevil generation when there are low adult weevil numbers, there will be relatively high number of IPMa females searching for potential hosts. In these circumstances, the parasitoids are more likely to die before laying their full egg complement and thus the parasitoid population would decline sharply (delayed density response). However, as IPMa females age, they may become less discriminating in host selection. With S. lepidus being the preferred host, higher levels of superparasitism may occur at this time as IPMa is capable of gregarious development (more than one individual completes development in a host). Nevertheless, native adult weevils present in S. lepidus-infested pastures in early spring may be more vulnerable to attack than those present when the spring emergence of S. lepidus is underway.

Benefits:

 Decrease in nitrogen fertilizer use Te Mana Taiao stated that “they were a river people, freshwater is crucial to us, it is a taonga tuku iho. Nitrogen poisons our waters, and kills freshwater species”.

There was genuine interest by Māori in how the introduction of IPMa may reduce nitrogen use and particularly nitrogen run-off into our waterways. The potential benefits are outlined in section 6.1.4. While S. lepidus is only one of the contributing factors to the soaring use of nitrogen fertilizers, farmers in the worst affected areas are those most prone to using very high levels and thus have increased risk of accidental misuse. While control of the weevil may not limit total nitrogen in the system, it does give farmers the sustainable option of obtaining nitrogen requirements through natural fixation by clovers.

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 Contribution to farm production, sustainability and profits As well as undertaking the responsibilities and obligations of guardianship of our environment and natural resources, Māori are major landowners and farmers. Thus they are not antidevelopment but want to develop their land without impinging on core values. Māori farmers and trusts contribute 12% of Meat and Wool NZ levies, 15% of Dairy Insight levies. They have ownership of 791,000 effective hectares in pastoral farming with $800 million total out put.

Therefore the benefits accruing to farmers in general, as outlined in section 6.1.4., are also applicable to Māori landowners.

 Comparison with other control options While the consultation meetings were focused on the release of IPMa, Māori wanted to know what other management options were available, in order to assess how important this option was. The current management options are: . Pasture management strategies to reduce stress on clovers . Nitrogen fertilizer application to maintain clover growth . Use of vigorous clover cultivars that can tolerate weevil attack . Use of insecticide if undersowing clover into infested pastures

Future options currently under development . Clover cultivars with increased tolerance or resistance. . A biopesticide for long-term suppression of the weevil in treated pastures

The current management options do not reduce pest numbers but enable farmers to maintain productivity. The biopesticide must be applied to pastures, but once in the system, should prevent high numbers of weevils building up in treated pastures for at least 12 months. The costs associated with the biopesticide may mean it is mainly used in intensive farming systems.

The advantage of IPMa is that once released, it will spread to all infested farms without the farmer needing to change how he or she operates.

The combination of good pasture management, tolerant clovers and both the biopesticide and biocontrol agent form an integrated pest management approach. While individually each component will assist farm productivity, most is gained when several components are combined, with biocontrol underpinning the integrated pest management approach.

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Consultation with Māori: As the application is for nationwide proposed release of the IPMa will impact on all New Zealand, comprehensive consultation with Māori throughout New Zealand was required. The timing of this consultation happened to coincide with Roger Pikia being appointed the first Māori strategist for AgResearch. Therefore AgResearch viewed this consultation process as a great opportunity to build good relationships with Māori so that in future it is easier to work in partnership on issues of mutual interest. The following is a summary of the process AgResearch undertook to ensure the issues of importance to the Māori community were identified and that their opinions and concerns are presented without bias.

 Introductory Mail Out In December 2004, immediately following confirmation that IPMa must have ERMA approval before release, an introductory letter and response form was sent to the 82 Māori iwi contacts supplied by ERMA (Appendix 1). The letter briefly outlined the rationale and key issues pertaining to the proposed release of the IPMa, and the response form (and self- addressed envelope) facilitated the initial replies. A six week response date was set to allow for communication difficulties over the Christmas/New Year period. By the end of January 2005, 13 Māori organisations had replied with most requesting more information and some asking for meetings. Especially pleasing was that the responses included iwi from almost every region in the country. Jason Holland, Apiha Whakaaetanga-A-Rawa Taiao, Resource Consent Officer, Nga Rawa Taiao requested that Te Runanga o Ngai Tahu be sent a draft of the application. At that time (17 January) we were not prepared to release the draft but we did forward the draft of the scientific paper on the non-target host testing as well as the information folder.

 Information Folder An information folder (Appendix 3 and additional available on request from the Biocontrol & Biosecurity Group, AgResearch) had been prepared that summarised current knowledge on Sitona lepidus and its impact on the pastoral community, the risks and benefits that are associated with the introduction of the wasp, in particular the non-target host impacts, and why we needed Māori community input. Also included was a pamphlet entitled “Managing clover in the presence of clover root weevil” prepared by the Waikato Clover Management Group and a handout in Māori showing the life cycles of the parasitoid and weevil. This folder was sent to all those that requested information in the initial mail out and those invited to attend consultation meetings.

 Consultation meetings The number and locality of the initial meetings was based on the responses from the initial mail out (Table 6.3.1). For each meeting, all contacts on the original ERMA mailing list in the region were sent and invitation, a response form with prepaid envelope, and an information pack. Notices of the meetings were placed in the public notices of the Saturday editions of the appropriate newspapers the weekend prior to each meeting.

AgResearch employed Indigenous Corporate Solutions (IC Solutions) to facilitate this consultation process. At each meeting, the IC Solution facilitator assisted AgResearch staff 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

with Māori protocol, facilitated the meeting and recorded the meeting minutes. These were circulated to the meeting attendees to ensure that they truly reflected the views of each speaker. A copy of report by IC Solutions containing the meeting minutes in Appendix 2.

The success of these meetings was mixed. We had no attendees at the Northland and Canterbury meetings, though did receive apologies from individuals for both. Good discussions were held at the other meetings. The notice in the Christchurch Press resulted in Dr Gerard receiving a phone call from an individual who was very aggrieved about the proposal (did not want any new species introduced). It is not known if the caller was Māori, but it was ascertained he strongly distrusted scientists and DoC staff.

Table 6.3.1: Planned and additional Māori Consultation meetings in 2005.

Meeting Locality Date Planned regional meeting Northland Kerikeri 9 March Waikato/ Bay of Plenty Ruakura 16 March Gisborne/ Hawkes Bay Gisborne 23 March Canterbury/Southland Lincoln 31 March Nelson/Blenheim Nelson 6 April Additional meeting Paul Morgan, Federation of Maori Wellington 6 April Authorities Pohara Pā, Te Mana Taiao 13 April Arapuni

 Additional meetings In light of the disappointing turnout at two of the meetings, Willie Te Aho, IC Solutions, arranged that we could make a presentation at Pohara Marae, Arapuni already arranged by their company for consultation regarding Mighty River Power resource consents to sink two test Geothermal bores.

Also, while passing through Wellington when returning from the Nelson meeting, we had the opportunity to discuss the application with Paul Morgan, Deputy Chairman of the Federation of Maori Authorities, and Chairman of Wakatu Inc. >

6.4 Economic effects Assess the potential magnitude and distribution of the economic risks, costs and benefits. Effects on third parties and to New Zealand of the proposed conditional release need to be specifically evaluated. Applicants should provide a cost benefit analysis. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Risks and Costs:

 Cost of introduction and release While the research and application costs are not considered by ERMA as part of the risk/cost benefit analysis, the funders have invested a significant levels of funds in the hope of means to control S. lepidus. By the end of the 2004/05 financial year, the amount invested will have exceeded $2.45 million. If the application is unsuccessful or the release a failure, this would be seen by those funders as a significant loss.

Benefits:

 Re-establishes the benefits of clovers to the New Zealand pastoral industries

The control of clover root weevil will have large economic benefits. New Zealand produces high quality animal products that are internationally competitive despite the distance from major world markets and various trade barriers. This competitive advantage is founded on cheap, high quality feed provided by the combination of favorable climate, high-producing grass and legume cultivars and appropriate fertiliser use. White clover is the key species; it fixes nitrogen, has high nutritive value, complements seasonal growth patterns of grasses and improves animal feed intake and utilisation rates. Clover root weevil has a demonstrated ability to eliminate clover from pastures, or at lower populations, adversely affect persistence, production and N fixation during much of the year.

The soaring use of fertilizer N in the last decade hides the losses in N fixation and total pasture production attributable to clover root weevil. Does New Zealand still need clover? The following is a summary from a report to MAF Sustainable Farming Fund by Brock (2004): Farmers have a lack of knowledge of white clover management and have moved to the use of fertilizer N technology to make feed supply more predictable. It is not a coincidence that the upper limits for N use of close to 200 kg/ha/year is about the same level that is fixed by high producing clover-based pasture. After allowing for marginal costs, the net return from this level of N application is just an extra $70/ha per year. This does not include environmental costs and it is known that continued use of N fertiliser over time reduces the amount of organic matter cycling through the soil, thus increasing the amount of fertilizer required to maintain soil fertility. However, clover still has the benefit of being superior quality herbage that maximizes production compared to grasses, and maintains quality in summer when even the best of ryegrasses lose quality. While high producing farming is currently becoming more intensive as farmers aim for higher productivity, the wholesale reliance on N fertilizers at the expense of managing clover is potentially short sighted. However, nothing is surer than change and should the economics or regulations regarding N use change, then the importance of white clover will re-emerge.

The economic benefits from maintaining clover in pasture are significant. Milk yields are very sensitive to pasture clover content (Harris et. al 1997, Lee et al. 2004) and Clark & Harris (1996) showed that for dairy farmers, maximum gross margins per ha would be achieved 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

with pasture clover contents of 30-40% and nitrogen fertilizer rates of 100-200 kg/ha/year. Most sheep and beef farmers are still reliant on clover for N requirements, despite using only an average 5.7 kg/ha in 2002 compared with over 100 kg/ha by dairy farmers (Statistics NZ 2003). Lamb live weight gains and wool weights are greater on white clover than ryegrass (eg Fraser & Rowarth 1996) and even small increases in the % of clover on which lambs are grazing can have large positive effects (Hyslop et. al. 2000).

Several estimates of economic losses due to the weevil have been made.

Caradus et al (1996) estimated that in 1995, again prior to the arrival of clover root weevil, the annual financial contribution of white clover to the New Zealand economy through fixed nitrogen, forage yield, seed production and honey production was $3.1billion annually. Using this 1996 estimation, Barlow & Goldson (2002) estimated that the annual direct cost of the pest was $300 million a year; using the conservative assumption that clover root weevil reduced pasture clover content by 20%.

An alternative recent estimate was made using clover dry matter production data from a trial established autumn 2003. Sufficient data has been gathered to compare production from April to November in each year. It shows that while there is little difference in year following establishment, in the second year, the pasture with 320 larvae/m2 (a typical density once the invasive phase is past) produced a tonne less clover dry matter per hectare than pasture with 4 larvae/m2 over the same period. The effect on grass production varied with clover cultivar but overall had no net effect. Using this data, an assessment by MAF staff at Wellington (Chris Baddeley, Policy manager for biosecurity, pers. comm.) estimated that this equated to $300 to $500 million per year of lost potential farmer income (assuming all farms in the currently affected area, as far south as the Manawatu, suffer the 1000 kg DM loss).

Biosecurity New Zealand has recently commissioned the New Zealand Institute of Economic Research, Inc to undertake an economic impact assessment for clover root weevil (Project: RFP 76 / 04) and two other studies relating to other related economic assessments are in progress (FRST – cost-benefit of the biological control of S. lepidus: AGMARDT – on-farm impacts of S. lepidus). These will be available by July 2005.

Overall cost benefit analysis:

In a business case put to industry in 2003, a cost benefit analysis was carried out with the following assumptions: Aim of the programme was to prevent losses due to CRW. If the programme is successful then the outcome would be maintaining the status quo It was calculated (MAF 2003) that the financial benefit of white clover to the New Zealand economy is $4bn

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At any one time only 20% of pastures were predicted to be affected by CRW resulting in a 50% reduction in clover (Based on Waikato on-farm measurements of a decline from 20 to 10% clover content following weevil infestation). This equated to $400m at risk. The economic benefit of implementing the programme was 20% ie only 20% of the loss of white clover benefit to pastures would be “recovered” $400m*.2 = $80m) In a worst case scenario it was assumed that the biocontrol introduction will have the above economic impact only on 40% of the affected pastures. ($80m *.4 = $32m) A staged impact was assumed. In the first year of introduction biological control would realize a 10% increase in clover content to 20%, increasing to 30% in year 2 and 40% for the next 4 years. The business case was only considered for the first 6 years post introduction The analysis assumed that additional funding for 3 cycles of mass rearing will be required 2005/2006

The cost benefit analysis predicted that the potential benefit ( ie prevention of losses caused by CRW to the financial contribution of white clover to the New Zealand pastoral economy) of the research investment by industry equated to a Net Present Value calculated using a discount rate of 14.2% of over $39m for the first 6 years following release. This was with the very conservative assumption that only 20% of the clover losses are recovered, and that biocontrol was only effective on 40% of farms.

6.5 Cultural, social, ethical and spiritual effects Assess the magnitude and distribution of any adverse and beneficial impacts on people and communities that adversely affect or maintain/enhance (in the case of beneficial impacts) their capacity to provide for their own social and cultural wellbeing both now and into the future. Also assess any ethical or spiritual risks, costs and benefits that might arise.

Community Consultation: Rural community consultation at programme initiation The New Zealand response to the S. lepidus incursion and consultation undertaken that gave rise to the initiation of the biocontrol programme has been reported by Willoughby et al. (1999) In brief, the following initiatives reached out to the rural community.

 A “Clover Root Weevil Advisory Group” was formed in April 1997 to provide a forum within which affected primary sectors could join with scientists to discuss and prioritise research. Intensive consultation was undertaken through the professional sector organisations that operated within the rural community such as the Livestock Improvement Corporation, Dairy Research Corporation (now Dexcel), Federated Farmers, District Councils, Landcare Trust groups and the NZ Society of Farm Management. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

 A “Farmer Questionnaire” was distributed throughout all the infested areas to learn of on-farm observations. While focused primarily on on-farm impacts of S. lepidus, the excellent response provided a measure of the concern in the farming community.  A “Clover Root Weevil” hotline with a toll-free number operated at time of peak concern.  The “Clover Root Weevil Road Show” was taken to six venues in the infested regions in February 1998. Researchers and industry leaders spoke with the farming community and lively discussion often ensued as information was exchanged.  Sector commitment. In March 1998, representatives of the Dairy, Deer, Meat and Wool Boards and Federated Farmers met with the Ministry of Biosecurity and Minister for Crown Research Institutes. Knowing the recommendations arising from the above activities and the needs of the farmers they represented, the boards agreed to jointly fund research into on-farm management practices, tolerant clovers and the search for biocontrol agents. The pastoral industry has continued to support the biocontrol programme to the present day. The most recent review of the programme was in November 2004.

Other assessments of community social and cultural impacts  Media reports of social effects of clover root weevil. From the initial outbreaks of S. lepidus in 1997 and 1998, newspapers have reported the mood of affected farmers. The clippings have been scanned for statements that reflect the impacts both the pest and proposed wasp release have on the social wellbeing of the rural community.

 Consultation with the Waikato Clover management group This group is a farmer driven and on-farm focussed group formed in response to the need for immediate solutions to maintaining pasture production in the presence of S. lepidus. The group works with the NZ Landcare Trust and has linkages with other rural communities affected by S. lepidus. The Group was approached and agreed to canvas their members and other contacts for the effects the release of the parasitoid may have on the cultural, social, ethical and spiritual wellbeing of themselves and their families.

Risks and Costs: No adverse effects on social and cultural wellbeing are foreseen.

Benefits:  Reduction in anger and stress in affected rural communities Various print media articles since 1997 to the present have reported the emotive words of frustrated and angry farmers coping with S. lepidus impacts in the worst- affected districts (eg “Weevil‟s rapid spread angers farmers” NZ Herald 22.2.1997). The stress that underlies these emotions has flow-on effects in the every day lives of 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

these farmers and their families. The release of Irish strain of M. aethiopoides should boost farmers‟ morale as they will have the hope that help is at hand. Once S. lepidus numbers are down to sub-economic levels, this particular stress should cease to exist.  Reduction in work load for farmers in worst-affected regions The current recommendation to maintain farm productivity in the presence of S. lepidus is to put small applications of nitrogen fertilizer on „little and often‟. This is generally 5-10 units of nitrogen applied after grazing. This involves considerable use of farmer‟s time from travelling to and from fertiliser suppliers to the hours spent spreading the fertiliser.The successful control of S. lepidus by the parasitoid would enable farmers to adopt less labour-intensive strategies to manage soil fertility.  Safer working environment Nitrogen application involves the danger of handling ½ tonne bulk bags, both loading and unloading off trucks and trailers and moving bags on front-end loaders. The successful control of S. lepidus by the parasitoid would enable farmers to reduce their dependence on nitrogen fertiliser to manage soil fertility.  Reduction in pressure to use environmentally unfriendly practices. Farmers are very aware through the media and other sources that high levels of fertilizer nitrogen usage can have negative impacts on the environment. However, many have proof (eg by using soil tests and the OVERSEER nutrient budget model) that with S. lepidus present in their pastures, they must apply these rates of nitrogen if they are to maintain animal production. The farmers feel as though they are portrayed as the “environmental enemies” by the media and this causes increasing breakdown in relationships between rural and urban dwellers, and indeed farmer against farmer. >

6.6 Other effects (including New Zealand’s international obligations) Assess any remaining adverse and beneficial effects not already covered including any effects on New Zealand‟s international obligations.

Risks and costs:

>No adverse effects on New Zealand‟s international obligations are foreseen.

Benefits:

 Assist New Zealand farmers in meeting international trade requirements Internationally, the regulatory environment is broadening to include new standards and restrictions that New Zealand primary producers must meet to retain market access. For example, the European Union is a significant market for New Zealand agricultural products and an influential player in world trade negotiations. The Common

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Agricultural Policy (CAP) of the Union plays a pivotal role. Environmentally sound production was one of the three topmost concerns in the „Agenda 2000‟ CAP reforms. While environmental constraints in Europe could decrease European production and increase the price of dairy products on the international market, it is important to consider what would happen if New Zealand farmers were forced to comply with European water quality standards. Data available suggests that many New Zealand waterways would fail to meet European standards (eg Environment Waikato water quality data www.ew.govt.nz/enviroinfo/water/healthyrivers/otherrivers/regionalrivers/index.htm).

The introduction of IPMa to mitigate the impact of S. lepidus on clovers will assist the New Zealand pastoral sector develop and maintain sustainable farm systems that meet international environmental standards yet remain competitive in world markets.

6.7 Overall evaluation of risks, costs and benefits It is the role of the Authority to decide whether the positive effects (benefits) of the conditional release outweigh the adverse effects (risks and costs) taking into account any control(s) that may be imposed. However, if you have a view on the relative importance of the different risks, costs and benefits and how they should be brought together in the overall evaluation of your application then please state that here.

The core decision the Authority must deliberate is whether the additional impact of IPMa to the existing adverse effects already imposed on native weevil species by the Moroccan biotype outweighs the economic and environmental impacts of Sitona lepidus.

IPMa is likely to parasitise and could potentially sustain successive generations in native weevil species in the genera Irenimus and Nicaeana, particularly those that reach pest levels in improved pastures. However, laboratory testing shows that Irish M. aethiopoides is more selective and less aggressive against native weevil species than the Moroccan M. aethiopoides biotype which was introduced into New Zealand in the 1980s to control Sitona dicoideus in lucerne. Therefore, if hosts were limited, IPMa may well be out-competed by the Moroccan biotype, and not add significantly to existing parasitism levels in native by this species. There has been no evidence to date that the existing Moroccan biotype sustains successive generations in native weevil populations and the parasitism observed could all be “spill-over” from pastoral ecosystems. Against this, the more widespread prevalence of clovers compared to lucerne may result in more „spill-over‟ into non-pastoral ecosystems of IPMa than Moroccan M. aethiopoides.

However, the target host S. lepidus is a serious threat to both the environment and economy. The weevil larvae reduce clover nitrogen fixation and growth. To maintain farm production, farmers in the worst infested districts resort to high application rates of nitrogen fertilizer. This threatens the health of our waterways (and therein indigenous aquatic species), the long-term sustainability of pastoral soils, and our 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

future relationships with major trading partners, while increasing farm fertilizer and labour costs and greenhouse gas release in the form of nitrous oxide.

Irish M. aethiopoides is in our judgment, the most effective biocontrol agent available to control S. lepidus. If this application is unsuccessful, it is exceedingly improbable a search for an alternative parasitoid will be undertaken. With the presence of S. lepidus making clover performance highly unreliable, farmers throughout New Zealand will have to modify farm systems to maintain profitability and reduce risk, with their main options being increased use of fertilizer, and application of insecticides and biopesticides (not yet available) at times of high risk.

Table 6.7.1: Summary of risks, costs and benefits of the release of Irish Microctonus aethiopoides (IPMa).

Risks and costs Benefits

IPMa will sustain successive generations in the IPMa should reduce S. lepidus numbers to below native weevils Irenimus aequalis and Nicaena economic levels in most regions in most years cervina, minor pests of improved pastures.

The widespread prevalence of clover may result Reduced S. lepidus numbers will return to in „more spill-over‟ into native broad-nosed farmers the option of using clovers to maintain weevil natural habitats of IPMa than currently soil fertility and the long-term sustainability of occurs with the Moroccan biotype. farm systems

While IPMa is more selective and less Reduced S. lepidus numbers should lessen the aggressive, it has the ability to parasitise many need for high application rates of nitrogen of the native broad-nosed weevil species fertilizer and associated risks to the environment attacked by the Moroccan biotype.

IPMa will be freely available to all farmers

Reduced S. lepidus numbers should lessen the need for insecticide use and associated risks to the environment and human health.

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Section Seven – Additional Information

7.1 Do any of the organism(s) to be conditionally released need approvals under any other New Zealand legislation or is the application affected by New Zealand’s international obligations? Please indicate whether any of the organism(s) to be conditionally released are subject to other New Zealand legislative requirements e.g. Biosecurity Act 1993, Welfare Act 1999. Also indicate if the organism is subject to any international obligations e.g. CITES.

The population currently in containment was imported under MAF Permit No. 2003019966 which replaced Permit No. 2002016705.

The permit was for multiple consignments from all countries of six adult weevil species (Sitona lepidus, Sitona discoideus, Atrichonotus sordidus, Atrichonotus taeniatulus, Listroderes delaiguei, Listronotus bonariensis, Rhinocyllus conicus) and larvae of Microctonus aethiopoides. The containment facilities specified were the AgResearch Invertebrate Containment Facility, Lincoln Ref: 3122, and Landcare Research NZ Ltd, Lincoln Ref: 644. The permit was issued to Craig Phillips, AgResearch, Lincoln, by Kerry Mulqueen, Authorising Officer, MAF NZ on 18 August 2003.

No other requirements for approvals or international obligations are known.

7.2 Have any of the organism(s) to be conditionally released been previously considered in New Zealand or elsewhere? For example, have the organism(s) been considered for import under the Plants Act? Have the organism(s) been developed as a result of a genetically modified development approval from either ERMA New Zealand or a delegated IBSC, or has it been considered by ERMA New Zealand for field testing?

This strain of Microctonus aethiopoides has not been considered for release in any other country or for release in New Zealand at any other time.

7.3 Is there any additional information that you consider relevant to this application that has not already been included?

As satisfaction of the minimum standards in section 36 of the Act is a requirement for approval a statement has been provided in regards to each subsection below.

 Displacement of native species State (with reasons) whether the new organism is likely to cause any significant displacement of any native species within its natural habitat.

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Previous studies in Canterbury have shown parasitism rates of native weevil species by the introduced Moroccan Microctonus aethiopoides and native Microctonus spp. are both at levels below 10% in natural grassland ecosystems with no evidence of significant displacement of the native parasitoid. It is considered that the introduction of IPMa will not cause any significant increase in the risk of displacement of the native parasitoid (see section 6). Laboratory studies indicate that IPMa will parasitise some native weevil species but is not considered the introduction of IPMa will cause any significant increase in parasitism above that already imposed by the introduced Moroccan Microctonus aethiopoides (see section 6).

 Deterioration of natural habitats State (with reasons) whether the new organism is likely to cause any significant deterioration of natural habitats.

The release of IPMa is not anticipated to have any effect on natural habitats because it attacks no other organisms or plants other than grassland weevils (see section 6). Even in the unlikely event that it did cause a significant reduction in native weevil populations, it is unlikely there would be any observable deterioration of natural habitats.

 Adverse effects on human health and safety State (with reasons) whether the new organism is likely to cause any significant adverse effects on human health and safety.

IPMa is not anticipated to have any significant adverse effects on human health and safety. No adverse risks specifically related to the rearing and release process for IPMa have been identified. It is so small that is unlikely to be observed by the general public. It does not bite or sting.

 Adverse effect to New Zealand’s inherent genetic diversity State (with reasons) whether the new organism is likely to cause any significant adverse effect to New Zealand‟s inherent genetic diversity.

IPMa is parthenogenetic and therefore will not hybridise with any indigenous Microctonus species (see section 3.4)

 Causing disease, being parasitic, or becoming a vector for disease State (with reasons) whether the new organism is likely to cause disease, be parasitic, or become a vector for human, animal, or plant disease. If, however, the purpose of the importation or release is to import or release an organism to cause disease, be a parasite, or a vector for disease all you need to do is state that.

The purpose of this proposal is to release IPMa as a biological control agent for S. lepidus, that is to be parasitic on this weevil species. It does not attack humans, animals other than weevils, or plants and therefore will not be a vector for disease. IPMa has 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

passed through many generations while in quarantine and there is no evidence of pathogenic organisms in the cultures. >

7.4 Provide a glossary of scientific and technical terms used in the application

Aestivation: Summer dormancy in which the insect is physiologically or metabolically dormant during summer or periods of high temperature. Arrhenotokous: A form of parthenogenetic reproduction in which males are produced by unmated females and both females and males produced by mated females. Biocontrol (Biological Control): The use of living organisms, such as predators, parasitoids and pathogens to control pests. Bionomics: The relationship of insects to their environment. Biotype: A genetically cohesive population of insects that demonstrates biological and phenological differences from morphologically identical forms. Used to distinguish European M. aethiopoides reared from clover root weevil S. lepidus from Moroccan M. aethiopoides reared from the lucerne weevil S. discoideus. Caudal: Of or belonging to the tail. Clade: A group of biological taxa or species that share features inherited from a common ancestor. Diapause: A state of suspended animation or dormancy. Eclosion: Hatching from the eggshell or adult from the pupal stage. Endosymbiont: An organism that lives within the cells or tissues of another organism and has a mutually dependent relationship with that organism. Endoparasitoid: a parasitoid insect whose larval stage feeds and develops inside the body of the host. Eutrophication: the process of a body of water becoming more nutrient rich, either naturally or by fertilisation. Exuviae: Portion of the integument of a larva or nymph that is shed from the body during the process of moulting. Facultative: Performing in a particular way sometimes, but not always. Family: A category of scientific classification ranking below order and above genus. Family names end in idae. Fecund: Characterised by having produced or producing many offspring. Feccund Genus (plural - genera): A group of related species Gregarious: Living in groups without a defined social order Habitat: An area in which an organism naturally occurs. Haemocoel: The fluid filled cavity of the adult insect in which the haemolymph (nutritive fluid) flows. Herbivory: Feeding on plants Host: The living organism that serves as food for a parasite, parasitoid, or pathogen (noun). 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Host-specificity: The level of specificity of a parasite, parasitoid, or pathogen to its host. Hyperparasite: A parasitoid that uses another parasitoid as a host. Insectivore: An organism (bird, beetles) that eats insects Instar: The stage in an insect‟s life cycle between successive larval moults Larva: Insect immature stage undergoing complete metamorphosis between egg and pupa. Koinobiont: A parasitoid which does not kill or paralyse the host after oviposition and therefore do not impair further mobility and development of the hosts. Parasitoid: An organism that, during its development, lives in or on the body of a single host individual, eventually killing that host. Parthenogenesis: The production of offspring from unfertilized eggs. Pedicel: The stem or stalk. Phenology: insect life cycle in relation to host, climate and weather changes Pupa: Nonfeeding stage of an insect between the larvae and adult stage in complete metamorphosis Solitary: In the context of parasitoids refers to parasitoid larvae living or existing alone Species: Category of scientific classification ranking below genus which consists of individuals capable of interbreeding and producing fertile offspring Sternite: A ventral part of a hardened (sclerotized) ring of a generalised body segment separated by membrane from sclerotized lateral and dorsal segments. Strain: A subgroup within a species that differs in a particular characteristic from other members of the same species. Supernumerary: In excess of the normal number. In the context for IPMa this is more than one larvae per host from the same or different females. Superparasitism: [preferred term superparasitoidism]: The situation in which more individuals of a parasite species develop in a host than can obtain adequate resources to complete their development. Teratocytes: Type of cell in the in the parasitised egg or body of host attacked by parasitic Hymenoptera. teratocytes are derived from dissociated cells of extra-embryonic tissue. Teratocytes appear to function in secretion, nutrition and immunology. Tergite: A dorsal part of a hardened (sclerotized) ring or part of a segment of a generalised body segment. Teneral: Newly emerged adult, when the cuticle is not hardened or it definitive colour has not been completed. Thelytokous: Pertaining to female offspring produced without fertilisation of their egg stage. Thelytoky: A form of sex-determination in which only diploid female progeny are produced - there are no males. Trophamnion: An envelope surrounding the egg of polyembryonic parasitic Hymenoptera which provides nourishment to the developing embryos contained within the envelope.

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

7.5 List of appendices Appendix 1: Templates of introductory letter and response form used in Mäori consultation Appendix 2: IC Solutions Report on consultation process and meeting minutes (signed hard copy and DVD will be provided) Appendix 3: Information pack

7.6 References Please include a list of the references cited in this application form. Abu, J. F., Ellis, C. R. 1976. Biology of Microctonus aethiopoides, a parasite of the alfalfa weevil, Hypera postica, in Ontario. Environmental Entomology 5 (6): 1040-1042.

Adler, P.H., Kim, K.C. 1985. Morphological and morphometric analyses of European and Moroccan biotypes of Microctonus aethiopoides (Hymenoptera: Braconidae). Annals of the Entomological Society of America 78: 279-283.

Aeschlimann, J.-P. 1978. Heavy infestations of Sitona humeralis Stephens (Col., Curc.) on alfalfa in southern Morocco. Annales de Zoologie-Écologie Animale 10: 221-225.

Aeschlimann, J.-P. 1980. The Sitona (Col.: Curculionidae) species occurring on Medicago and their natural enemies in the Mediterranean region. Entomophaga 25: 139-153.

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Aeschlimann, J.-P. 1983b. Sources of importation, establishment and spread in Australia, of Microctonus aethiopoides Loan (Hymenoptera: Braconidae), a parasitoid of Sitona discoideus Gyllenhal (Coleoptera: Curculionidae). Journal of the Australian Entomological Society 22: 325- 331.

Aeschlimann, J-P 1995. Lessons from post-release investigations in classical biological control: the case of Microctonus aethiopoides Loan (Hym., Braconidae) introduced into Australia and New Zealand for the biological control of Sitona discoideus Gyllenhal (Col., Curculionidae). In: Hokkanen, H.M.T. & Lynch, J.M. (eds) Biological Control: Benefits and Risks. Cambridge University Press, New York. pp. 75-83.

Alonso-Zarazaga, M.A. Lyal, C.H.C. 1999. A world catalogue of families and genera of Curculionoidea (Insecta: Coleoptera). Entomopraxis, Barcelona, Spain.

Barlow, N.D. & Goldson, S.L. 2002. Alien invertebrates in New Zealand. Biological Invasions: economic and environmental costs of alien plant, animal, and microbe species. (Pimental, D., Ed.). CRC Press, Boca Raton, pp. 195-216.

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Barlow, N.D., Goldson, S.L. 1993. A modelling analysis of the successful biological control of Sitona discoideus (Coleoptera: Curculionidae) by Microctonus aethiopoides (Hymenoptera: Braconidae) in New Zealand. Journal of Applied Ecology 30: 165-178.

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Barratt B.I.P, Goldson S.L., Ferguson C.M., Phillips C.B., Hannah D.J. 1999b. Predicting the risk from biological control agent introductions: A New Zealand approach. Nontarget effects of biological control introductions. (Follett P. A. & DUAN J. J., Eds.). Kluwer Academic Publishers, Norwell, Massachusetts, pp. 59-75

Barratt B.I.P., Evans A.A., Ferguson C.M., Barker G.M., McNeill M.R. & Phillips C.B. 1997b. Laboratory nontarget host range of the introduced parasitoids Microctonus aethiopoides and Microctonus hyperodae (Hymenoptera: Braconidae) compared with field parasitism in New Zealand. Environmental Entomology 26: 694-702.

Barratt, B. I. P., Evans, A. A., Stoltz, D. B., Vinson, S. B., Easingwood, R 1999. Virus-like particles in the ovaries of Microctonus aethiopoides Loan (Hymenoptera: Braconidae), a parasitoid of adult weevils (Coleoptera: Curculionidae). Journal of Invertebrate Pathology 73(2): 182-188. Abu, J.F., Ellis, C.R. 1976. Biology of Microctonus aethiopoides a parasite of the alfalfa weevil, Hypera postica, in Ontario. Environmental Entomology 5: 1040-1042.

Barratt, B. I. P., Ferguson, C. M., Jones, P. A., Johnstone, P. D. 1992. Effect of native weevils (Coleoptera: Curculionidae) on white clover establishment and yield in tussock grassland. New Zealand Journal of Agricultural Research , 35 (1): 63-73.

Barratt, B. I. P., Johnstone, P. D. 2001. Factors affecting parasitism by Microctonus aethiopoides (Hymenoptera: Braconidae) and parasitoid development in natural and novel host species. Bulletin of Entomological Research 91 (4): 245-253.

Barratt, B.I.P. 2004. Microctonus parasitoids and New Zealand weevils: comparing laboratory estimates of host ranges to realized host ranges. Assessing host ranges of parasitoids and predators used for classical biological control: a guide to best practice. (van Driesche, R. & Reardon, R. Eds.). United States Forest Service Publication, USA. Pp. 103- 120.

Barratt, B.I.P., Evans, A.A., Ferguson, C.M. 1997a. Potential for control of Sitona lepidus Gyllenhal by Microctonus spp. Proceedings of the 50th New Zealand Plant Protection Conference 37-40.

Barratt, B.I.P., Evans, A.A., Ferguson, C.M., McNeill, M.R., Addison, P. 2000. Phenology of native weevils (Coleoptera: Curculionidae) in New Zealand pastures and parasitism by the introduced braconid, Microctonus aethiopoides Loan (Hymenoptera: Braconidae). New Zealand Journal of Zoology 27: 93-110. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Barratt, B.I.P., Ferguson, C.M., Evans, A.A. 2001. Non-target effects of introduced biological control agents and some implications for New Zealand. In: Balancing Nature: Assessing the Impact of Importing Non-Native Biological Control Agents (An International Perspective). (Lockwood, J. A., Howarth, F.G. & Purcell, M. F., Eds.). Thomas Say Publications. Maryland, pp. 41-53.

Barratt, B.I.P., Kuschel, G. 1996. Broad-nosed weevils (Curculionidae: Brachycerinae: Entimini) of the Lammermoor and Rock and Pillar ranges in Otago, with descriptions of four new species of Irenimus. New Zealand Journal of Zoology 23: 359-374.

Brock, J. 2004. Clover underperforming: A farm based study to identify ways to improve the reliability of establishment and performance of white clover. Client report to MAF Sustainable Farming Fund. 40 pp

Caradus, J.R., Woodfield, D.R., Stewart, A.V. 1996. Overview and vision for white clover. Agronomy Society of New Zealand Special Publication 11. Grassland Research and Practice Series 6: 1-6.

Clark, D. A., Harris, S. L 1996. White clover or nitrogen fertiliser for dairying? Agronomy Society of New Zealand Special Publication. 11, Grassland Research and Practice Series 6:107-114.

Coles, L.W., Puttler, B. 1963. Status of the alfalfa weevil biological control program in the eastern United States. Journal of Economic Entomology 56: 609-611.

Cullen, J.M., Hopkins, D.C. 1982. Rearing release and recovery of Microctonus aethiopoides Loan (Hymenoptera Braconidae) imported for the control of Sitona discoideus Gyllenhal (Coleoptera Curculionidae) in South Eastern Australia. Journal of the Australian Entomological Society 21: 279-284.

Day, W.H., Coles, L.W., Stewart, J.A., Fuester, R.W. 1971. Distribution of Microctonus aethiops and M. colesi parasites of the alfalfa weevil in the eastern United States. Journal of Economic Entomology 64: 190-193.

Drea, J.J. 1968. Castration of male alfalfa weevils by Microctonus spp. Journal of Economic Entomology 61: 1291-1295.

Dysart, R.J., Day, W.H. 1976. Release and recovery of introduced parasites of the alfalfa weevil in Eastern North America. United States Department of Agriculture Production Research Report: 167.

Fraser, T. J., Rowarth, J. S 1996. Legumes, herbs or grass for lamb performance? Proceedings of the New Zealand Grassland Association 58: 49-52

Fusco, R.A., Hower, Jr. A.A. 1973. Host influence on the laboratory production of the parasitoid, Microctonus aethiops. Environmental Entomology 2: 971-975.

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Gerard, P.J. 2001. Dependence of Sitona lepidus (Coleoptera: Curculionidae) larvae on abundance of white clover Rhizobium nodules. Bulletin of Entomological Research 91: 149- 152.

Gerard, P.J. 2002. Nodule damage by clover root weevil larvae in white clover swards. New Zealand Plant Protection 55: 246-251.

Goldson S.L., Proffitt J.R. 1986. The seasonal behaviour of the parasite Microctonus aethiopoides and its effect on Sitona weevil Proceedings of the 39th New Zealand Weed and Pest Control Conference: 122-125.

Goldson, S. L., Jamieson, P. D., Bourdoot, G. W. 1988. The response of field-grown lucerne to a manipulated range of insect-induced nitrogen stresses. Annals of Applied Biology 113: 189-196

Goldson, S. L., McNeill, M.R., Phillips, C.B., Proffitt, J.R. 1992. Host specificity testing and suitability of the parasitoid Microctonus hyperodae (Hym.: Braconidae, Euphorinae) as a biological control agent of Listronotus bonariensis (Col.: Curculionidae) in New Zealand. Entomophaga 37: 483-498.

Goldson, S.L., McNeill, M.R., Gerard, P.J., Proffitt, J.R., Phillips, C.B., Cane, R.P., Murray, P.J. 2004. British-based search for natural enemies of the clover root weevil, Sitona lepidus in Northwest Europe. New Zealand Journal of Zoology 31: 233-240.

Goldson, S.L., McNeill, M.R., Proffitt, J. R., Barratt B.I.P. 2005. Host Specificity Testing and Suitability of a European Biotype of the Braconid Parasitoid Microctonus aethiopoides Loan as a Biological Control Agent Against Sitona lepidus (Coleoptera: Curculionidae) in New Zealand. (in press)

Goldson, S.L., McNeill, M.R., Proffitt, J.R. 2003. Negative effects of strain hybridisation on the biocontrol agent Microctonus aethiopoides. New Zealand Plant Protection 56: 138-142.

Goldson, S.L., Phillips, C.B., McNeill, M.R., Proffitt, J.R., Cane, R.P. 2001. Importation to New Zealand quarantine of a candidate biological control agent of clover root weevil. New Zealand Plant Protection 54: 147-151

Goldson, S.L., Proffitt, J.R. & Baird, D.B. 1998. Establishment and phenology of the parasitoid Microctonus hyperodae Loan in New Zealand. Environmental Entomology 27: 1386-1392.

Goldson, S.L., Proffitt, J.R., McNeill, M.R. 1990. Seasonal biology and ecology in New Zealand of Microctonus aethiopoides (Hymenoptera, Braconidae), a parasitoid of Sitona spp. (Coleoptera: Curculionidae), with special emphasis on atypical behaviour. Journal of Applied Ecology 27: 703-722.

Harris, S. L., Clark, D. A., Jansen, E. B. L 1997. Optimum white clover content for milk production. Proceedings of the New Zealand Society of Animal Production 57: 169 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Hill, R.L., Gourlay, A.H., Fowler, S.V. 2000: The biological control program against gorse in New Zealand. Neal R. Spencer (ed.). Proceedings of the X International Symposium on Biological Control of Weeds, 4-14 July 1999, Montana State University, Bozeman, Montana, USA. Pp. 909-917.

Hyslop, M. G., Fraser, T. J., Smith, D. R., Knight, T. L., Slay, M. W. A., Moffat, C. A 2000.Liveweight gain of young sheep grazing tall fescue or perennial ryegrass swards of different white clover content. Proceedings of the New Zealand Society of Animal Production 60: 51-54. Kelly, D.; McCallum, K. 1995: Evaluating the impact of Rhinocyllus conicus on Carduus nutans in New Zealand. In Delfosse, E.S.; Scott, R.R. eds. Proceedings of the Eighth International Symposium on Biological Control of Weeds. Lincoln University, Canterbury, New Zealand. DSIR/CSIRO, Melbourne. Pp. 205-212.

Iline, I. I., Phillips, C. B 2003. Allozyme variation between European and New Zealand populations of Microctonus aethiopoides . New Zealand Plant Protection 56: 133-137

Jackson, D.J. 1928. The biology of Dinocampus (Perilitus) rutilus Nees, a braconid parasite of Sitona lineata L. Part I. Proceedings of the Zoological Society of London Part 2: 597-630.

Kingsley, P. C., Bryan, M. D., Day, W. H., Burger, T. L., Dysart, R. J., Schwalbe, C. P 1993. Alfalfa weevil (Coleoptera: Curculionidae) biological control: spreading the benefits. Environmental Entomology 22 (6): 1234-1250

Lee, J.M., Woodward, S.L., Waghorn, G.C., Clark, D.A. 2004. Methane emission by dairy cows fed increasing proportions of white clover (Trifolium repens) in pasture. Proceedings of the New Zealand Grasslands Association 66: 151-155.

Leschen, A.B., Lawrence, J.F., Kuschel, G., Thorpe, S., Wang, Q. 2003. Coleoptera genera of New Zealand. New Zealand Entomologist 26: 15-28.

Loan, C (1983) Host and generic relations of the Euphorini (Hymenoptera: Braconidae). Contributions of the American Entomological Institute 20: 388-397.

Loan, C.C. 1967. Studies on the taxonomy and biology of the Euphorinae (Hymenoptera: Braconidae). II. Host relations of six Microctonus species. Annals of the Entomological Society of America 60: 236-240.

Loan, C.C. 1975. A review of Haliday species of Microctonus [Hym.: Braconidae, Euphorinae]. Entomophaga 20: 31-41

Loan, C.C., Holliday, N.J. 1979. Euphorinae parasitic on ground beetles with descriptions of three new species of Microctonus Wesmael (Hymenoptera: Braconidae and Coleoptera: Carabidae). Le Naturaliste Canadian 106: 393-397.

20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Loan, C.C., Holdaway, F.G. 1961. Microctonus aethiops (Nees) Auctt. and Perilitus rutilus (Nees) (Hymenoptera:Braconidae), European parasites of Sitona weevils (Coleoptera: Curculionidae). Canadian Entomologist 93: 1057-1079.

Loan, C.C., Klein, M.G., Coppel, H.C. 1969. Microctonus glyptosceli n.sp., a parasite of Glyptoscelis pubescens (F.) in Winsconsin. Proceedings of the Entomological Society of Washington 71: 230-233.

Loan, C.C., Lloyd, D.C. 1974. Description and field biology of Microctonus hyperodae Loan, n.sp. (Hymenoptera: Braconidae, Euphorinae) a parasite of Hyperodes bonariensis in South America [Coleoptera: Curculionidae]. Entomophaga 19: 7-12.

Luff, M.L. 1976. The biology of Microctonus caudatus (Thomson), a braconid parasite of the ground beetle Harpalus rufipes (Degeer). Ecological Entomology 1: 111-116.

McNeill, M. R., Addison, P.J., Proffitt, J.P., Phillips, C.B., Goldson, S.L. 2002: Microctonus hyperodae: a summary of releases and distribution in New Zealand pasture N.Z. Plant Prot. 56: 272-279.

McNeill, M. R., Goldson, S.L., Proffitt, J.P., Phillips, C.B., Addison, P.J. 2002. A description of the commercial rearing and distribution of Microctonus hyperodae (Hymenoptera: Braconidae) for biological control of Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Biological Control 24: 167-175.

McNeill, M.R., Barratt, B.I.P., Evans, A.A. 2000. Behavioural acceptability of Sitona lepidus (Coleoptera: Curculionidae) to the parasitoid Microctonus aethiopoides (Hymenoptera: Braconidae) using the pathogenic bacterium Serratia marcescens Bizio. Biocontrol Science and Technology 10: 205-214.

McNeill, M.R., Kean, J.M., Goldson, S.L. 2002. Parasitism by Microctonus aethiopoides on a novel host, Listronotus bonariensis, in Canterbury pastures. New Zealand Plant Protection 56: 280-286.

McNeill, M.R., Phillips, C.B., Goldson, S.L. 1993. Diagnostic characteristics and biology of three Microctonus spp. (Hymenoptera: Braconidae, Euphorinae) parasitoids of weevils (Coleoptera: Curculionidae) in New Zealand pasture and lucerne. New Zealand Entomologist 16: 39-44.

Munro, J.A., Post, R.L. 1948. Parasites to aid the control of the sweet clover weevil. Science 108: 609.

Murray, T.J., Barratt, B.I.P., Ferguson, C.M. 2002. Field parasitism of Rhinocyllus conicus Froehlich (Coleoptera: Curculionidae) by Microctonus aethiopoides Loan (Hymenoptera: Braconidae) in Otago and South Canterbury. New Zealand Plant Protection 55: 263-266.

Parliamentary Commissioner for the Environment. 2004. Growing for good: Intensive farming, sustainability and New Zealand's environment. Wellington: Parliamentary Commissioner for the Environment. 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

Phillips, C.B. 1998. Influence of liquid food on fecundity and longevity of Microctonus hyperodae Loan. Proceedings of the 51st New Zealand Weed and Pest Control Conference: 23-27.

Phillips, C.B., Baird, D.B., 1996. A morphometric method to assist in defining the South American origins of Microctonus hyperodae Loan (Hymenoptera: Braconidae) established in New Zealand. Biocontrol Science and Technology 6: 189–205.

Phillips C B, Goldson S L, Emberson R M 1993. Host-associated morphological variation in Canterbury (New Zealand) populations of Microctonus aethiopoides Loan (Hymenoptera: Braconidae, Euphorinae) Proceedings of the 6th Australasian Conference on Grassland Invertebrate Ecology: 405-414

Proffitt J.R., Goldson S.L. 1987. Overwintering biology and survival of the sitona weevil parasitoid Microctonus aethiopoides Proceedings of the 40th New Zealand Weed and Pest Control Conference 23-26.

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Tironi, P., Treuenfels, A.V., Parra, J.R.P. 2004. Biology of Microctonus sp. (Hymenoptera: Braconidae), a parasitoid of Cyrtomon luridus Boh. (Coleoptera: Curculionidae). Scientia Agricola 61:538-541.

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Section Eight - Application Summary Summarise the application in clear, simple language that is able to be understood by the general public. Include a description of the organism(s), the purpose of the conditional release, proposed controls and any associated risks, costs and benefits. Any consultation that was undertaken should be noted. This summary will be used to provide information for those people and agencies who will be notified of the application (e.g. Ministry of Agriculture and Forestry, Ministry for the Environment, Department of Conservation, Regional Councils, etc) and for members of the public who request information. Do not include any commercially sensitive information in this summary – this should be attached as a separate appendix and clearly marked “confidential”.

Since its discovery in 1996, clover root weevil (Sitona lepidus) has become one of the most damaging clover pests found in New Zealand. Both adults and larvae attack clovers all year round, causing significant declines in clover content and quality in pastures. While adults cause significant clover seedling mortality, it is the larval stage that is the most damaging, with the early instars feeding almost exclusively on clover nodules and the older larvae attacking the roots and stolons. This reduces the clover content in pastures, impacting on animal live weight gain and milk production. Perhaps most importantly, the weevil can destroy the nitrogen-fixing capability of white clover in late spring and autumn. Therefore instead of relying on this free, natural source of nitrogen, many farmers in infested regions apply high levels of nitrogen fertiliser (> 200kg N/ha) to maintain soil fertility and farm profitability. However, if 20 Customhouse Quay, Cnr Waring Taylor & Customhouse Quay PO Box 131, Wellington Phone: 04-916 2426 Fax: 04-914 0433 Email: [email protected] Website: www.ermanz.govt.nz

used incorrectly, there is increased risk of nitrogen leaching into our waterways causing undesirable growth of plants and algae and ruining the habitat for our native and introduce freshwater aquatic life.

Control of clover root weevil with conventional insecticides is unacceptable, because of the risks to our environment and exports. Therefore, it is proposed to release a new Irish strain of a small (2.7-3.0 mm) parasitic wasp Microctonus aethiopoides to control this weevil. A Moroccan strain of this parasitoid species is already widespread in New Zealand, having been introduced in 1982 to control the lucerne weevil Sitona discoideus. While ineffective against the clover root weevil, biological control of the lucerne weevil successfully prevents significant losses in lucerne production. The wasp is effective because it has multiple generations per year and parasitized weevils quickly become sterile, which in turn reduces the numbers of larvae attacking plant roots. The advantage of biological control is that it will spread to all New Zealand farmers in clover root weevil-infested regions, giving them the option of maintaining sustainable and highly productive grass/clover pastures without the use of high levels of nitrogen fertiliser.

As the species was already present in New Zealand, the Irish strain could have been released immediately after importation. However, AgResearch scientists pushed to have M. aethiopoides declared an “at risk” species so that ERMA approval must be obtained before the new strain could be released. This was done because the Moroccan strain already in New Zealand attacks a number of native broad-nosed weevils that live in our tussock grasslands and a beneficial weevil introduced to control nodding thistle. The scientists firmly believed the introduction of a new strain must be fully scrutinised and risks and benefits weighed by the community before release.

Because considerable data have been gathered on the impact of the Moroccan strain on non- target species, AgResearch has been able to make comparisons between the two strains and predict the likely impact of the new strain once released. Parasitism by the Moroccan strain has been estimated to give an 8% reduction in numbers of the native weevil species living in ryegrass/white clover pasture. At this level, there is no risk of extinction and at in some localities, native weevil populations reach pest levels. At higher altitudes, higher reductions have been predicted, but field studies have not found this so. There is some evidence to suggest that M. aethiopoides does not survive the winter in high altitude native grassland areas. Our research in quarantine has shown that the Irish strain is less aggressive and more selective in attack on non-target weevil hosts than its Moroccan counterpart. With the exception of one species, it had lower parasitism rates on all non-target hosts tested than has been reported for the Moroccan biotype. Therefore, the ecological impacts of the Irish strain are likely to be less severe than those already imparted by the Moroccan M. aethiopoides.

This biological control programme was initiated after intensive consultation with the pastoral industry and science community and has been supported financially by the dairy, meat, wool and deer sectors since research commenced in 1998. If left unchecked, it has been estimated that the clover root weevil will cost the pastoral community $300 million a year once it infests

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all New Zealand. Consultation with the Māori community was initiated as soon as confirmation was received that release of this organism required HSNO approval.

Checklist

Please check and complete the following before submitting your application:

All sections completed Yes Appendices enclosed Yes Confidential information identified and enclosed separately NA Copies of additional references attached Yes Cheque for initial fee enclosed (incl. GST)† No If “yes”, state amount: $………. Fee direct credited to ERMA bank account: Yes If „yes” give date of DC …/…/… and amount: $………. Application signed and dated Yes Electronic copy of application e-mailed to ERMA New Zealand Yes

*NA – not applicable

† The cost of processing the application will be charged to you in accordance with our pricing policy. A fees and charges schedule, including the initial fee required with the application can be found on our web site under new organism applications. Note that we will be moving to a fixed pricing policy effective from 1 December 2003 – please ask ERMA staff for further details.

Signed: Date:

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